Agents and methods for targeted delivery to cells

Abstract

The invention relates to agents and methods for targeted delivery of payloads to cells. The payload includes a variety of molecules such as therapeutic or diagnostic agents and, in particular, radiodiagnostics, toxins, or immunomodulators. In some embodiments, the invention involves providing to a subject a compound comprising a payload moiety and at least two tags (tag conjugate) and a compound comprising at least two binding moieties for a target antigen and a binding moiety for a tag of the tag conjugate (docking compound).

Description

[0001] AGENTS AND METHODS FOR TARGETED DELIVERY TO CELLS

[0002] The invention relates to agents and methods for targeted delivery of payloads to cells. The payload includes a variety of molecules such as therapeutic or diagnostic agents and, in particular, radiocompounds such as radiodiagnostics and radiotherapeutics, toxins, or immunomodulators. In some embodiments, the invention involves a compound comprising a payload moiety and at least two tags (tag conjugate) and a compound comprising a moiety binding to a tag of the tag conjugate and at least two moieties binding to a target antigen, e.g., a cell surface antigen on a target cell, (docking compound). In some embodiments, the docking compound comprises one or more peptides or polypeptides. In some embodiments, the docking compound comprises at least two binding moieties binding to target cells (primary targeting moiety) and a further binding moiety binding to a tag of the tag conjugate. The tags of the tag conjugate may each bind to a binding moiety for a tag on a docking compound and the primary targeting moieties of the docking compound may bind to target antigen on target cells such as an antigen on cancer cells to thereby precisely deliver a payload to the target cells. In some embodiments, the docking compound is provided to a subject by administering nucleic acid, e.g., RNA, encoding the docking compound. In some embodiments, the docking compound is provided to a subject by administering the docking compound. In some embodiments, a preformed complex wherein the docking compound is bound to the tag conjugate is provided to a subject by administration.

[0003] In many areas of medical therapy and diagnosis, it is desired to selectively deliver an agent, such as a therapeutic agent (a drug) or a diagnostic agent (e.g. an imaging agent), to a specific cell in the body of a subject such as a patient.

[0004] Active targeting of a cell may be achieved by the direct or indirect conjugation of the desired payload to a targeting moiety, which binds to cell surface antigens on the target cell of interest. The targeting moieties are typically constructs that have affinity for cell surface targets, e.g., membrane proteins, and include antibodies or antibody fragments.

[0005] The present invention relates to an approach wherein a docking compound that binds to target cells, e.g., by binding to a cell surface antigen, is used. The docking compound further comprises a moiety, which binds to a compound comprising a payload and being equippedwith at least two tags to be targeted by binding moieties of different docking compounds. In some embodiments, a docking compound which binds to a tag conjugate comprising a payload is administered in the form of nucleic acid encoding the docking compound for expressing the docking compound in a subject. The docking compound may bind to target cells, e.g., by binding to a cell surface antigen, thus resulting in targeting of the payload. Common examples for pairs of interacting moieties on the docking compound and on the tag conjugate are antibody / tag systems.

[0006] The concept described herein allows to use a single type of tag conjugate for targeting a wide range of target cells, i.e., by using a single type of tag conjugate in combination with different docking compounds targeting different primary targets. The concept described herein is of further advantage, as the primary targeting using a single docking compound can be carried out in combination with different tag conjugates comprising different payloads. Also, the format for the docking compound used herein, namely monovalent binding to the tags of the tag conjugate and at least bivalent binding to target antigen, has the potential to be highly target sensitive. Using a tag conjugate with at least two tags and a docking compound that has a single binding moiety for a tag, wherein the tag is preferably a medium / low affinity tag, allows to leverage the avidity effect of the tag conjugates described herein only at the target side, for example in applications using a radiodiagnostic. Furthermore, using a docking compound with at least two moieties binding to a target antigen, e.g., a cell surface antigen on a target cell, also allows to leverage the avidity effect at the target site ensuring highly specific binding of the docking compound at the intended target site with little off-target binding.Summary

[0007] The invention relates to agents and methods for targeted delivery of payloads to cells. In some embodiments, the payload comprises a diagnostic compound, e.g., a radioisotope, or a therapeutic compound, e.g., a toxin or immunomodulator. Targeted delivery of a payload is achieved using a tag conjugate described herein comprising the payload and at least two tags and a docking compound binding to a tag of the tag conjugate, said docking compound comprising targeting molecules for targeting an antigen on target cells.

[0008] In one aspect, the invention relates to a kit comprising:

[0009] (i) a compound comprising at least two binding moieties fora target antigen and a binding moiety for a tag (also referred to herein as "docking compound"), or a nucleic acid encoding said compound; and

[0010] (ii) a compound comprising a payload moiety and at least two tags to which the binding moiety for a tag binds (also referred to herein as "tag conjugate").

[0011] In some embodiments, the nucleic acid is RNA.

[0012] In some embodiments, the compound under (i) comprises two binding moieties for a target antigen.

[0013] In some embodiments, the total number of binding moieties for a target antigen in the compound under (i) is two.

[0014] In some embodiments, the compound under (i) comprises one binding moiety for a tag. In some embodiments, the total number of binding moieties for a tag in the compound under (i) is one.

[0015] In some embodiments, the compound under (i) comprises two binding moieties for a target antigen and one binding moiety for a tag.

[0016] In some embodiments, the total number of binding moieties for a target antigen in the compound under (i) is two and the total number of binding moieties for a tag in the compound under (i) is one.

[0017] In some embodiments, the target antigen is a tumor antigen.

[0018] In some embodiments, the target antigen is claudin 6 (CLDN6).

[0019] In some embodiments, the tag is a peptide tag.In some embodiments, the tag is an ALFA-tag.

[0020] In some embodiments, the tag is a cyclic ALFA-tag.

[0021] In some embodiments, a binding moiety for a target antigen comprises at least one variable domain.

[0022] In some embodiments, a binding moiety for a target antigen comprises an immunoglobulin heavy chain variable region (VH) domain and an immunoglobulin light chain variable region (VL) domain, or a single-domain antibody variable domain (VHH).

[0023] In some embodiments, a binding moiety for a tag comprises at least one variable domain. In some embodiments, a binding moiety for a tag comprises an immunoglobulin heavy chain variable region (VH) domain and an immunoglobulin light chain variable region (VL) domain, or a single-domain antibody variable domain (VHH).

[0024] In some embodiments, a binding moiety for a target antigen comprises a VH domain and a VL domain, or a VHH domain and a binding moiety for a tag comprises a VHH domain.

[0025] In some embodiments, the two binding moieties for a target antigen each comprises a VH domain and a VL domain, or a VHH domain and a binding moiety for a tag comprises a VHH domain.

[0026] In some embodiments, a binding moiety for a target antigen comprises a Fab fragment or a single chain fragment variable (scFv) and a binding moiety for a tag comprises a VHH domain. In some embodiments, the two binding moieties for a target antigen each comprises a Fab fragment or a scFv and a binding moiety for a tag comprises a VHH domain.

[0027] In some embodiments, a VH domain and a VL domain interact to form a binding moiety. In some embodiments, the binding moiety for a target antigen comprises a VH domain of an immunoglobulin with specificity for CLDN6 (VH(CLDN6)) and a VL domain of an immunoglobulin with specificity for CLDN6 (VL(CLDN6)).

[0028] In some embodiments, the binding moiety for a tag comprises an ALFA-tag binding nanobody (NbALFA).

[0029] In some embodiments, the compound under (i) comprises two Fab fragments binding to a target antigen, wherein the Fab fragments are connected via a peptide linker (tandem Fab), and wherein the tandem Fab is linked to a VHH binding to a tag.

[0030] In some embodiments, the compound under (i) comprises:(i) a polypeptide comprising, from N-terminus to C-terminus:

[0031] a first Fab heavy chain (VH-CH1); an optional linker LI; a second Fab heavy chain (VH-CH1); an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag; (ii) a polypeptide comprising a first Fab light chain (VL-CL); and

[0032] (iii) a polypeptide comprising a second Fab light chain (VL-CL),

[0033] wherein the first Fab heavy chain of (i) and the first Fab light chain of (ii) form a Fab fragment binding to a target antigen and the second Fab heavy chain of (i) and the second Fab light chain of (iii) form a Fab fragment binding to a target antigen.

[0034] In some embodiments, the compound under (i) comprises:

[0035] (i) a first polypeptide comprising, from N-terminus to C-terminus:

[0036] an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for the target antigen (VH(TA)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for the target antigen (VH(TA)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag;

[0037] (ii) a second polypeptide comprising, from N-terminus to C-terminus:

[0038] an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for the target antigen (VL(TA)) and an immunoglobulin light chain constant region (CL) domain; and

[0039] (iii) a third polypeptide comprising, from N-terminus to C-terminus:

[0040] an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for the target antigen (VL(TA)) and an immunoglobulin light chain constant region (CL) domain,

[0041] wherein a VH(TA) and a VL(TA) interact to form a binding moiety for the target antigen. In some embodiments, the compound under (i) comprises:

[0042] (i) a first polypeptide comprising, from N-terminus to C-terminus:

[0043] an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for CLDN6 (VH(CLDN6)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; an immunoglobulin heavy chain variable region (VH) domain ofan immunoglobulin with specificity for CLDN6 (VH(CLDN6)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker L2; and an ALFA-tag binding nanobody (NbALFA);

[0044] (ii) a second polypeptide comprising, from N-terminus to C-terminus:

[0045] an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for CLDN6 (VL(CLDN6)) and an immunoglobulin light chain constant region (CL) domain; and

[0046] (iii) a third polypeptide comprising, from N-terminus to C-terminus:

[0047] an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for CLDN6 (VL(CLDN6)) and an immunoglobulin light chain constant region (CL) domain,

[0048] wherein a VH(CLDN6) and a VL(CLDN6) interact to form a binding moiety for CLDN6.

[0049] In some embodiments, the linker LI and the linker L2 is present. In some embodiments, the linker LI and the linker L2 allows formation of disulphide bonds between different Fab polypeptides.

[0050] In some embodiments, the linker LI and / or the linker L2 comprises the amino acid sequence EPKSC or a functional variant thereof.

[0051] In some embodiments, the linker LI and / or the linker L2 comprises a GS sequence.

[0052] In some embodiments, the linker LI comprises the amino acid sequence EPKSCSGPGGGRSGGGGSGGGGS or a functional variant thereof.

[0053] In some embodiments, the linker L2 comprises the amino acid sequence EPKSCGGGGSGGGS or a functional variant thereof.

[0054] In some embodiments, the compound under (i) comprises:

[0055] (i) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a functional variant thereof;

[0056] (ii) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof; and

[0057] (iii) a third polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof,wherein the first polypeptide and the second polypeptide interact to form a binding moiety for CLDN6 and the first polypeptide and the third polypeptide interact to form a binding moiety for CLDN6.

[0058] In some embodiments, the compound under (i) comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first dimerization domain and the second polypeptide comprises a second dimerization domain, said first and second dimerization domains facilitating preferential formation of a heterodimer comprising the first polypeptide and the second polypeptide compared to formation of a homodimer comprising two of the first polypeptides or two of the second polypeptides.

[0059] In some embodiments, the first and second dimerization domains each comprise an immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof.

[0060] In some embodiments, the first and / or second dimerization domain comprises a CH3 domain variant comprising one or more modifications in the CH3 domain that enhance the formation of a heterodimer comprising the first polypeptide and the second polypeptide.

[0061] In some embodiments, the first polypeptide comprises:

[0062] (i) an immunoglobulin heavy chain variable region (VH) domain and an immunoglobulin light chain variable region (VL) domain,

[0063] (ii) an immunoglobulin heavy chain variable region (VH) domain and the compound under (i) comprises a third polypeptide comprising an immunoglobulin light chain variable region (VL) domain, or

[0064] (iii) a single-domain antibody variable domain (VHH),

[0065] wherein the VH and VL form a binding moiety for a target antigen, or the VHH domain binds to a target antigen.

[0066] In some embodiments, the VH domain or the VHH domain, and optionally the VL domain are located N-terminal of the first dimerization domain.

[0067] In some embodiments, the second polypeptide comprises:

[0068] (i) an immunoglobulin heavy chain variable region (VH) domain and an immunoglobulin light chain variable region (VL) domain,(ii) an immunoglobulin heavy chain variable region (VH) domain and the compound under (i) comprises a fourth polypeptide comprising an immunoglobulin light chain variable region (VL) domain, or

[0069] (iii) a single-domain antibody variable domain (VHH),

[0070] wherein the VH domain and the VL domain form a binding moiety for a target antigen, or the VHH domain binds to a target antigen.

[0071] In some embodiments, the VH domain or the VHH domain, and optionally the VL domain are located N-terminal of the second dimerization domain.

[0072] In some embodiments, the first polypeptide or the second polypeptide comprises a singledomain antibody variable domain (VHH) binding to a tag.

[0073] In some embodiments, a binding moiety for a target antigen comprises a Fab fragment and a binding moiety for a tag comprises a VHH domain.

[0074] In some embodiments, the compound under (i) comprises:

[0075] (i) a polypeptide comprising, from N-terminus to C-terminus:

[0076] a first Fab heavy chain (VH-CH1); an optional linker LI; a first dimerization domain; an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag;

[0077] (ii) a polypeptide comprising, from N-terminus to C-terminus:

[0078] a second Fab heavy chain (VH-CH1); an optional linker LI; and a second dimerization domain; (iii) a polypeptide comprising a first Fab light chain (VL-CL); and

[0079] (iv) a polypeptide comprising a second Fab light chain (VL-CL),

[0080] wherein the first Fab heavy chain of (i) and the first Fab light chain of (iii) form a Fab fragment binding to a target antigen and the second Fab heavy chain of (ii) and the second Fab light chain of (iv) form a Fab fragment binding to a target antigen.

[0081] In some embodiments, the compound under (i) comprises:

[0082] (i) a first polypeptide comprising, from N-terminus to C-terminus:

[0083] an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for the target antigen (VH(TA)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; a first immunoglobulin heavy chain constant region 3 (CH3) domain ora variant thereof; an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag;(ii) a second polypeptide comprising, from N-terminus to C-terminus:

[0084] an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for the target antigen (VH(TA)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; and a second immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof;

[0085] (iii) a third polypeptide comprising, from N-terminus to C-terminus:

[0086] an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for the target antigen (VL(TA)) and an immunoglobulin light chain constant region (CL) domain; and

[0087] (iv) a fourth polypeptide comprising, from N-terminus to C-terminus:

[0088] an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for the target antigen (VL(TA)) and an immunoglobulin light chain constant region (CL) domain,

[0089] wherein a VH(TA) and a VL(TA) interact to form a binding moiety for the target antigen. In some embodiments, the compound under (i) comprises:

[0090] (i) a first polypeptide comprising, from N-terminus to C-terminus:

[0091] an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for CLDN6 (VH(CLDN6)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; a first immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof; an optional linker L2; and an ALFA-tag binding nanobody (NbALFA);

[0092] (ii) a second polypeptide comprising, from N-terminus to C-terminus:

[0093] an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for CLDN6 (VH(CLDN6)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; and a second immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof;

[0094] (iii) a third polypeptide comprising, from N-terminus to C-terminus:

[0095] an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for CLDN6 (VL(CLDN6)) and an immunoglobulin light chain constant region (CL) domain; and(iv) a fourth polypeptide comprising, from N-terminus to C-terminus:

[0096] an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for CLDN6 (VL(CLDN6)) and an immunoglobulin light chain constant region (CL) domain,

[0097] wherein a VH(CLDN6) and a VL(CLDN6) interact to form a binding moiety for CLDN6.

[0098] In some embodiments, the first and / or second polypeptides do not comprise an immunoglobulin heavy chain constant region 2 (CH2) domain located N-terminal of the dimerization domain and the CH3 domain or variant thereof, respectively, and C-terminal of the Fab heavy chain and the CHI domain, respectively.

[0099] In some embodiments, the first and / or second polypeptides do not comprise an immunoglobulin heavy chain constant region 2 (CH2) domain.

[0100] In some embodiments, the linker LI and the linker L2 is present. In some embodiments, the linker LI and the linker L2 allows formation of disulphide bonds between different Fab polypeptides and / or heavy chains.

[0101] In some embodiments, the linker LI comprises the amino acid sequence EPKSCDKTHTCPPC or a functional variant thereof.

[0102] In some embodiments, the linker LI and / or the linker L2 comprises a GS sequence.

[0103] In some embodiments, the linker LI comprises the amino acid sequence EPKSCDKTHTCPPCGGGSSGGGSG or a functional variant thereof.

[0104] In some embodiments, the linker L2 comprises the amino acid sequence GGGGSGGGS or a functional variant thereof.

[0105] In some embodiments, the compound under (i) comprises:

[0106] (i) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5 or a functional variant thereof;

[0107] (ii) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6 or a functional variant thereof;

[0108] (iii) a third polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof; and

[0109] (iv) a fourth polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof,wherein the first polypeptide and the third polypeptide interact to form a binding moiety for CLDN6 and the second polypeptide and the fourth polypeptide interact to form a binding moiety for CLDN6.

[0110] In some embodiments, the compound under (i) comprises:

[0111] (i) a first polypeptide which is encoded by a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5 or a functional variant thereof;

[0112] (ii) a second polypeptide which is encoded by a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6 or a functional variant thereof; (iii) a third polypeptide which is encoded by a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof; and

[0113] (iv) a fourth polypeptide which is encoded by a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof, wherein the first polypeptide and the third polypeptide interact to form a binding moiety for CLDN6 and the second polypeptide and the fourth polypeptide interact to form a binding moiety for CLDN6.

[0114] In some embodiments, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 6 or a C-terminal truncation variant thereof, wherein the C-terminal truncation variant of SEQ ID NO: 6 comprises a deletion of lysine at the C-terminus of SEQ ID NO: 6. In some embodiments, the first and / or second polypeptides comprise an immunoglobulin heavy chain constant region 2 (CH2) domain.

[0115] In some embodiments, the first and / or second polypeptides comprise an immunoglobulin heavy chain constant region 2 (CH2) domain located N-terminal of the dimerization domain and the CH3 domain or variant thereof, respectively, and C-terminal of the Fab heavy chain and the CHI domain, respectively.

[0116] In some embodiments, the compound under (i) comprises:

[0117] (i) a first polypeptide comprising, from N-terminus to C-terminus:

[0118] an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for the target antigen (VH(TA)); an immunoglobulin heavy chain constant region 1(CHI) domain; an optional linker LI; an immunoglobulin heavy chain constant region 2 (CH2) domain; a first immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof; an optional linker L2; and a single-domain antibody variable domain (VHH) bindingto a tag;

[0119] (ii) a second polypeptide comprising, from N-terminus to C-terminus:

[0120] an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for the target antigen (VH(TA)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; an immunoglobulin heavy chain constant region 2 (CH2) domain; and a second immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof;

[0121] (iii) a third polypeptide comprising, from N-terminus to C-terminus:

[0122] an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for the target antigen (VL(TA)) and an immunoglobulin light chain constant region (CL) domain; and

[0123] (iv) a fourth polypeptide comprising, from N-terminus to C-terminus:

[0124] an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for the target antigen (VL(TA)) and an immunoglobulin light chain constant region (CL) domain,

[0125] wherein a VH(TA) and a VL(TA) interact to form a binding moiety for the target antigen. In some embodiments, the compound under (i) comprises:

[0126] (i) a first polypeptide comprising, from N-terminus to C-terminus:

[0127] an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for CLDN6 (VH(CLDN6)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; an immunoglobulin heavy chain constant region 2 (CH2) domain; a first immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof; an optional linker L2; and an ALFA-tag binding nanobody (NbALFA);

[0128] (ii) a second polypeptide comprising, from N-terminus to C-terminus:

[0129] an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for CLDN6 (VH(CLDN6)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; an immunoglobulin heavy chain constant region 2 (CH2)domain; and a second immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof;

[0130] (iii) a third polypeptide comprising, from N-terminus to C-terminus:

[0131] an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for CLDN6 (VL(CLDN6)) and an immunoglobulin light chain constant region (CL) domain; and

[0132] (iv) a fourth polypeptide comprising, from N-terminus to C-terminus:

[0133] an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for CLDN6 (VL(CLDN6)) and an immunoglobulin light chain constant region (CL) domain,

[0134] wherein a VH(CLDN6) and a VL(CLDN6) interact to form a binding moiety binding to CLDN6. In some embodiments, the linker LI and the linker L2 is present. In some embodiments, the linker LI and the linker L2 allows formation of disulphide bonds between different Fab polypeptides and / or heavy chains.

[0135] In some embodiments, the linker LI comprises the amino acid sequence EPKSCDKTHTCPPCP or a functional variant thereof.

[0136] In some embodiments, the linker L2 comprises a GS sequence.

[0137] In some embodiments, the linker L2 comprises the amino acid sequence GGGGSGGGS or a functional variant thereof.

[0138] In some embodiments, the compound under (i) comprises:

[0139] (i) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8 or a functional variant thereof;

[0140] (ii) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 9 or a functional variant thereof;

[0141] (iii) a third polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof; and

[0142] (iv) a fourth polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof,wherein the first polypeptide and the third polypeptide interact to form a binding moiety for CLDN6 and the second polypeptide and the fourth polypeptide interact to form a binding moiety for CLDN6.

[0143] In some embodiments, the compound under (i) comprises:

[0144] (i) a first polypeptide which is encoded by a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8 or a functional variant thereof;

[0145] (ii) a second polypeptide which is encoded by a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 9 or a functional variant thereof; (iii) a third polypeptide which is encoded by a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof; and

[0146] (iv) a fourth polypeptide which is encoded by a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof, wherein the first polypeptide and the third polypeptide interact to form a binding moiety for CLDN6 and the second polypeptide and the fourth polypeptide interact to form a binding moiety for CLDN6.

[0147] In some embodiments, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 9 or a C-terminal truncation variant thereof, wherein the C-terminal truncation variant of SEQ ID NO: 9 comprises a deletion of lysine at the C-terminus of SEQ ID NO: 9. In some embodiments, a binding moiety for a target antigen comprises a single chain fragment variable (scFv) and a binding moiety for a tag comprises a single-domain antibody variable domain (VHH).

[0148] In some embodiments, the compound under (i) comprises:

[0149] (i) a polypeptide comprising, from N-terminus to C-terminus:

[0150] (a) a first immunoglobulin heavy chain variable region (VH) domain; an optional linker L3; a first immunoglobulin light chain variable region (VL) domain; an optional linker LI; a first dimerization domain; an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag; or(b) a first immunoglobulin light chain variable region (VL) domain; an optional linker L3; a first immunoglobulin heavy chain variable region (VH) domain; an optional linker LI; a first dimerization domain; an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag;

[0151] (ii) a polypeptide comprising, from N-terminus to C-terminus:

[0152] (a) a second immunoglobulin heavy chain variable region (VH) domain; an optional linker L3; a second immunoglobulin light chain variable region (VL) domain; an optional linker LI; and a second dimerization domain; or

[0153] (b) a second immunoglobulin light chain variable region (VL) domain; an optional linker L3; a second immunoglobulin heavy chain variable region (VH) domain; an optional linker LI; and a second dimerization domain;

[0154] wherein the first VH domain and the first VL domain form a binding moiety for a target antigen and the second VH domain and the second VL domain form a binding moiety for a target antigen.

[0155] In some embodiments, a binding moiety for a target antigen comprises a single-domain antibody variable domain (VHH) and a binding moiety for a tag comprises a single-domain antibody variable domain (VHH).

[0156] In some embodiments, the compound under (i) comprises:

[0157] (i) a polypeptide comprising, from N-terminus to C-terminus:

[0158] a first single-domain antibody variable domain (VHH) binding to a target antigen; an optional linker LI; a first dimerization domain; an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag; and

[0159] (ii) a polypeptide comprising, from N-terminus to C-terminus:

[0160] a second single-domain antibody variable domain (VHH) binding to a target antigen; an optional linker LI; and a second dimerization domain.

[0161] In some embodiments, the compound under (i) comprises:

[0162] (i) a polypeptide comprising, from N-terminus to C-terminus:

[0163] a first single-domain antibody variable domain (VHH) binding to a target antigen; an optional linker L2; a single-domain antibody variable domain (VHH) binding to a tag; an optional linker LI; and a first dimerization domain; and(ii) a polypeptide comprising, from N-terminus to C-terminus:

[0164] a second single-domain antibody variable domain (VHH) binding to a target antigen; an optional linker LI; and a second dimerization domain.

[0165] In some embodiments, the immunoglobulin is a human immunoglobulin.

[0166] In some embodiments, the immunoglobulin is IgGl.

[0167] In some embodiments, the VH(TA) or VH(CLDN6) comprises a CDR1 comprising the amino acid sequence GYSFTGYT or a functional variant thereof, a CDR2 comprising the amino acid sequence INPYNGGT or a functional variant thereof, and a CDR3 comprising the amino acid sequence ARDYGFVLDY ora functional variant thereof and the VL(TA) or VL(CLDN6) comprises a CDR1 comprising the amino acid sequence SSVSY or a functional variant thereof, a CDR2 comprising the amino acid sequence STS or a functional variant thereof, and a CDR3 comprising the amino acid sequence QQRSNYPPWT or a functional variant thereof.

[0168] In some embodiments, the VH(TA) or VH(CLDN6) comprises the amino acid sequence shown in SEQ ID NO: 10 or a functional variant thereof and the VL(TA) or VL(CLDN6) comprises the amino acid sequence shown in SEQ ID NO: 11 or a functional variant thereof.

[0169] In some embodiments, the VHH binding to a tag or NbALFA comprises the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13 or a functional variant thereof.

[0170] In some embodiments, the CHI domain comprises the amino acid sequence shown in SEQ ID NO: 14 or a functional variant thereof.

[0171] In some embodiments, the CL domain comprises the amino acid sequence shown in SEQ ID NO: 15 or a functional variant thereof.

[0172] In some embodiments, the first dimerization domain or first CH3 domain or variant thereof comprises the amino acid sequence shown in SEQ ID NO: 16 or a functional variant thereof and the second dimerization domain or second CH3 domain or variant thereof comprises the amino acid sequence shown in SEQ ID NO: 17 or a functional variant thereof.

[0173] In some embodiments, the CH2 domain comprises the amino acid sequence shown in SEQ ID NO: 18 or a functional variant thereof.

[0174] In some embodiments, a peptide described herein comprises a signal sequence. In some embodiments, a signal sequence comprises the amino acid sequence MDWTWRVFCLLAVAPGAHS.In some embodiments, the compound under (ii) comprises a moiety comprising a polymer. In some embodiments, the payload moiety and the tags are coupled through a moiety comprising a polymer.

[0175] In some embodiments, the polymer is not a polymer of proteinogenic amino acids or their D-isomers.

[0176] In some embodiments, the polymer is selected from the group consisting of poly(ethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine)), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), derivatives and combinations thereof.

[0177] In some embodiments, the polymer comprises at least one poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0178] In some embodiments, the compound under (ii) comprises two of said tags.

[0179] In some embodiments, the total number of tags in the compound under (ii) is two.

[0180] In some embodiments, a tag in the compound under (ii) comprises the amino acid sequence Ser-Arg-Leu-Glu-(cyclo5)Asp-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu, or Pro-Ser-Arg-Leu-(cyclo5)Glu-Glu-Glu-Leu-(cyclo9)Lys-Arg-Arg-Leu-Thr-Glu.

[0181] In some embodiments, the compound under (ii) comprises the following formula (also shown in Figure 10):

[0182]

[0183] In some embodiments, the compound under (ii) comprises the above formula and chelates a radiodiagnostic isotope, e.g.,68Ga. In some embodiments, the DOTA moiety in the above formula chelates a radiodiagnostic isotope, e.g.,68Ga.In some embodiments, the compound under (ii) comprises the following formula (also shown in Figure 11):

[0184]

[0185] In some embodiments, the compound under (ii) comprises the above formula and chelates a radiodiagnostic isotope, e.g.,68Ga. In some embodiments, the DOTA moiety in the above formula chelates a radiodiagnostic isotope, e.g.,68Ga.

[0186] In the above formulas, the DOTA moiety is coupled to a lysine branching group via a linking moiety comprising a [AEEA]i moiety and the tags are coupled to the lysine branching group via a linking moiety comprising a [AEEA]? moiety, wherein AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid.

[0187] In some embodiments, the compound under (ii) comprises payload moieties and tags in a branched (non-linear) configuration or an unbranched (linear) configuration.

[0188] In some embodiments, the payload moiety comprises a radioisotope, e.g., a chelating compound comprising a radioisotope, a toxin or an immunomodulator.

[0189] In a further aspect, the invention relates to a compound corresponding to the compound under (i) as set forth above.

[0190] In a further aspect, the invention relates to a method for treating a subject having a disease, disorder or condition characterized by cells expressing a target antigen, comprising:

[0191] (i) providing to the subject a compound comprising at least two binding moieties for the target antigen and a binding moiety for a tag;(ii) allowing the compound comprising at least two binding moieties for the target antigen and a binding moiety for a tag to become associated with cells expressing the target antigen; and

[0192] (iii) administering to the subject a compound comprising a payload moiety and at least two tags to which the binding moiety for a tag binds.

[0193] Steps (i), (ii) and (iii) of the method described above can be performed in any suitable order. In some embodiments, step (i) is performed first followed by step (iii). In some embodiments, step (iii) is performed first followed by step (i). In some embodiments, steps (i) and (iii) are performed simultanously. Step (ii) can be performed following step (i) and prior to step (iii) or following steps (i) and (iii).

[0194] In some embodiments, the compound comprising at least two binding moieties for the target antigen and a binding moiety for a tag is a compound under (i) as set forth above and / or the compound comprising a payload moiety and at least two tags to which the binding moiety for a tag binds is a compound under (ii) as set forth above.

[0195] In some embodiments, the compound comprising at least two binding moieties for the target antigen and a binding moiety for a tag is provided to the subject by administering to the subject RNA encoding a polypeptide (including polypeptide complexes) comprising at least two binding moieties for the target antigen and a binding moiety for a tag; and allowing expression of the polypeptide by cells in the subject.

[0196] In some embodiments, the cells expressing the polypeptide are transfected with the RNA. In some embodiments, the RNA is administered as particulate formulation such as formulated as lipid nanoparticles.

[0197] In some embodiments, the cells expressing the polypeptide secrete the polypeptide.

[0198] In some embodiments, the cells expressing the polypeptide express the polypeptide such that it is released into the bloodstream.

[0199] In some embodiments, the target antigen is a cell surface antigen.

[0200] In some embodiments, the disease, disorder or condition is cancer.

[0201] In some embodiments, the cells expressing a target antigen are diseased cells.

[0202] In some embodiments, the cells expressing a target antigen are cancer cells.

[0203] In some embodiments, the target antigen is a tumor antigen.In some embodiments, the payload moiety comprises a radioisotope, e.g., a chelating compound comprising a radioisotope, toxin or immunomodulator.

[0204] In a further aspect, the invention relates to a compound described herein, e.g., a compound under (i) as set forth above and / or a compound under (ii) as set forth above, for use in a method for treating a subject described herein.

[0205] Sequences described above are as follows:

[0206]

[0207]

[0208]

[0209]

[0210] Brief description of the drawings

[0211] Figure 1: Molecular design of the docking compound RD1. The Fd fragments of two CLDN6-specific Fabs were fused using a linker in a head-to-tail fashion. The C-terminal end of the second Fd-fragment was fused to the NbALFA VHH. Light chains are encoded as separate polypeptide chains.

[0212] Figure 2: Molecular design of the docking compound RD3. This molecule exhibits a Knob-into-hole-based asymmetric ACH2 IgG design. The Knob-mutation-containing CH3 domain was C-terminally fused to the NbALFA VHH, giving raise to the RD3 design.

[0213] Figure 3: Molecular design of the docking compound RD5. While the heterodimerization of the heavy chains was enforced using the Knob-into-Hole mutation, the C-terminus of the Knob-containing CH3 domain was fused to a single NbALFA VHH moiety.

[0214] Figure 4: PK study comparison of different formats (RD1, RD3 and RD5)

[0215] Figure 5: Comparison of cell binding of RD1 and RD3 on tumor cells

[0216] Figure 6: Comparison of binding kinetics of RD1 and RD3 on tumor cells

[0217] Figure 7: Ex vivo analysis of Tmax of RD1 and RD3 in OV-90 tumor

[0218] Figure 8: Analysis of RD1 and RD3 integrity and purity

[0219] Figure 9: Biodistribution of RD1 and RD3 in combination with Ga68-DOTA-bisALFA

[0220] Figures 10 and 11: Structures of tag conjugatesDetailed description

[0221] Although the present disclosure is further described in more detail below, it is to be understood that this disclosure is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0222] In the following, the elements of the present disclosure will be described in more detail. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present disclosure to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0223] The practice of the present disclosure will employ, unless otherwise indicated, conventional chemistry, biochemistry, pharmaceutical, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field.

[0224] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated feature, element, member, integer or step or group of features, elements, members, integers or steps but not the exclusion of any other feature, element, member, integer or step or group of features, elements, members, integers or steps. The term "consisting essentially of" limits the scope of a claim or disclosure to the specified features, elements, members, integers, or steps and those that do not materially affect the basic and novel characteristic(s) of the claim or disclosure. The term "consisting of" limits the scope of a claim or disclosure to the specified features, elements, members,integers, or steps. The term "comprising" encompasses the term "consisting essentially of" which, in turn, encompasses the term "consisting of". Thus, at each occurrence in the present application, the term "comprising" may be replaced with the term "consisting essentially of" or "consisting of". Likewise, at each occurrence in the present application, the term "consisting essentially of" may be replaced with the term "consisting of".

[0225] The terms "a", "an" and "the" and similar references used in the context of describing the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context.

[0226] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context.

[0227] The use of any and all examples, or exemplary language (e.g., "such as"), provided herein is intended merely to better illustrate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0228] The term "optional" or "optionally" as used herein means that the subsequently described event, circumstance or condition may or may not occur, and that the description includes instances where said event, circumstance, or condition occurs and instances in which it does not occur.

[0229] Where used herein, "and / or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "X and / or Y" is to be taken as specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, just as if each is set out individually herein.

[0230] In the context of the present disclosure, the term "about" denotes an interval of accuracy that the person of ordinary skill will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, and for example ±0.01%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±10%. In some embodiments, "about" indicates deviation fromthe indicated numerical value by ±5%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±4%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±3%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±2%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±1%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.9%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.8%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.7%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.6%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.5%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.4%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.3%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.2%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.1%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.05%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.01%. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect.

[0231] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0232] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.In the following, definitions and embodiments will be provided which apply to all aspects of the present disclosure. Terms which are defined in the following have the meanings as defined unless otherwise indicated. Any undefined terms have their art recognized meanings.

[0233] Terms such as "reduce" or "inhibit" as used herein means the ability to cause an overall decrease, for example, of about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 40% or greater, about 50% or greater, or about 75% or greater, in the level. The term "inhibit" or similar phrases includes a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero.

[0234] Terms such as "enhance" as used herein means the ability to cause an overall increase, or enhancement, for example, by at least about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 40% or greater, about 50% or greater, about 75% or greater, or about 100% or greater in the level. "Physiological pH" as used herein refers to a pH of about 7.4. In some embodiments, physiological pH is from 7.3 to 7.5. In some embodiments, physiological pH is from 7.35 to 7.45. In some embodiments, physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0235] As used in the present disclosure, "mol %" is defined as the ratio of the number of moles of one component to the total number of moles of all components, multiplied by 100.

[0236] The term "recombinant" in the context of the present disclosure means "made through genetic engineering". In some embodiments, a "recombinant object" in the context of the present disclosure is not occurring naturally.

[0237] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. Forexample, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring. The term "found in nature" means "present in nature" and includes known objects as well as objects that have not yet been discovered and / or isolated from nature, but that may be discovered and / or isolated in the future from a natural source.As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably herein and refer to temperatures from at least about 15°C, e.g., from about 15°C to about 35°C, from about 15°C to about 30°C, from about 15°C to about 25°C, or from about 17°C to about 22°C. Such temperatures will include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C and 22°C.

[0238] The term "EDTA" refers to ethylenediaminetetraacetic acid disodium salt. All concentrations are given with respect to the EDTA disodium salt.

[0239] The term "cryoprotectant" relates to a substance that is added to a formulation in order to protect the active ingredients during the freezing stages.

[0240] The term "lyoprotectant" relates to a substance that is added to a formulation in order to protect the active ingredients during the drying stages.

[0241] According to the present disclosure, the term "peptide" refers to substances which comprise about two or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150, consecutive amino acids linked to one another via peptide bonds. The term "polypeptide" refers to large peptides, in particular peptides having at least about 151 amino acids. "Peptides" and "polypeptides" are both protein molecules. Thus, the terms "peptide", "protein" and "polypeptide" are used herein usually as synonyms.

[0242] Peptides and polypeptides disclosed herein may comprise a linear or a cyclized peptide sequence.

[0243] In some embodiments, the peptides disclosed herein comprises at least one cyclic portion, i.e., a polypeptide chain that contains a circular sequence of bonds that is referred to herein as a "cyclic peptide." The circular sequence can occur through a connection between the amino and carboxyl ends of the peptide; a connection between the amino end and a side chain; a connection between the carboxyl end and a side chain; ora connection between two side chains including sulfur groups of two cysteine amino acids by forming a disulfide bond, or more complicated arrangements.

[0244] In some embodiments, the peptides and polypeptides disclosed herein are composed of naturally occurring amino acids, non-naturally occurring amino acids, amino acid derivatives and non-amino acid components, or a mixture thereof. In some embodiments, the peptidesand polypeptides disclosed herein comprise amino acid mimetics and amino acid analogs. In some embodiments, the peptides and polypeptides disclosed herein comprise non-naturally occurring amino acid sequences that are resistant to enzymatic cleavage.

[0245] In some embodiments, one or more positions of a peptide or polypeptide disclosed herein are substituted with a non-naturally occurring amino acid. In some embodiments, the substituted amino acid is chemically related to the original residue (e.g., aliphatic, charged, basic, acidic, aromatic, hydrophilic) or an isostere of the original residue.

[0246] In its broadest sense, as used herein, the term "amino acid" refers to a compound and / or substance that can be, is, or has been incorporated into a peptide, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid. In some embodiments, an amino acid is a D-amino acid. In some embodiments, an amino acid is an L-amino acid. "Standard amino acid" or "proteinogenic amino acid" refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides and polypeptides. "Nonstandard amino acid" refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino-terminal amino acid in a peptide or polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a peptide or polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a peptide or polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid. In some embodiments it may be used to refer to an amino acid residue of a peptide or polypeptide.The following table lists the 20 natural amino acids and their abbreviations:

[0247]

[0248] Generally, amino acids are L-amino acids while D-amino acids are denoted by the prefix "D". The prefix "homo" or "h" designates an a-amino acid that is otherwise similar to one of the common ones, but that contains one more methylene group in the carbon chain.

[0249] As used herein, "Orn" means ornithine or 2,5-diaminopentanoic acid, "Dab" means 2,4-diaminobutanoic acid, "Dap" means 2,3-diaminopropanoic acid, "hLys" means 2,7-diaminoheptanoic acid, "hCys" means 2-amino-4-mercaptobutanoic acid, and "Pen" means penicillamine or 2-amino-3-methyl-3-sulfanylbutanoic acid.It may also be possible to include non-peptide linkages and other chemical modification. For example, part or all of the peptide or polypeptide may be synthesized as a peptidomimetic, e.g., a peptoid (see, e.g., Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89:9367-71 and Horwell (1995) Trends Biotechnol.l3:132-4). A peptide or polypeptide may include one or more (e.g., all) non-hydrolyzable bonds. Many non-hydrolyzable peptide bonds are known in the art, along with procedures for synthesis of peptides containing such bonds. Exemplary non-hydrolyzable bonds include -[CH2NH]- reduced amide peptide bonds, -[COCH2]-ketomethylene peptide bonds, -[CH(CN)NH]- (cyanomethylene)amino peptide bonds, -[CH2CH(OH)]- hydroxyethylene peptide bonds, -[CH2O]- oxymethylene peptide bonds, and -[CH2S]- thiomethylene peptide bonds (see e.g., U.S. Pat. No. 6,172,043).

[0250] The term "amide" as used herein, represents a group of formula "-NHC(O)-".

[0251] The term "thioamide" represents a group of formula "-NHC(S)-".

[0252] As used herein the term "disulfide bond", "disulfide bridge" or "disulfide" includes the covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group.

[0253] The term "ether" refers to a group or compound having an oxygen between two carbon atoms.

[0254] The term "thioether" refers to a group or compound having a sulfur between two carbon atoms.

[0255] The term "ester" refers a compound derived from an carboxylic acid and an alcohol by linking with formal loss of water the hydroxyl group of the -C(=O)OH group in the former and a hydroxy group of the latter. Thus, the term refers to the group -C(O)O-.

[0256] The term "thioester" refers to the group -C(O)S- or -C(S)O-.

[0257] The term "triazole" refers to chemical compounds that incorporate in their structure any heterocyclic structure having a five-membered ring of two carbon atoms and three nitrogen atoms (e.g., 1,2,3-triazole).

[0258] The term "portion" refers to a fraction. With respect to a particular structure such as an amino acid sequence or protein the term "portion" thereof may designate a continuous or a discontinuous fraction of said structure.The terms "part" and "fragment" are used interchangeably herein and refer to a continuous element. For example, a part of a structure such as an amino acid sequence or protein refers to a continuous element of said structure. When used in context of a composition, the term "part" means a portion of the composition. For example, a part of a composition may be any portion from 0.1% to 99.9% (such as 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%) of said composition.

[0259] "Fragment", with reference to an amino acid sequence (peptide or polypeptide), relates to a part of an amino acid sequence, i.e., a sequence which represents the amino acid sequence shortened at the N-terminus and / or C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 3'-end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 5'-end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation. A fragment of an amino acid sequence comprises, e.g., at least 50 %, at least 60 %, at least 70 %, at least 80%, at least 90% of the amino acid residues from an amino acid sequence. A fragment of an amino acid sequence comprises, e.g., at least 6, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from an amino acid sequence. A fragment of an amino acid sequence comprises, e.g., a sequence of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55, consecutive amino acids of the amino acid sequence.

[0260] "Variant," as used herein and with reference to an amino acid sequence (peptide or polypeptide), is meant an amino acid sequence that differs from a parent amino acid sequence by virtue of at least one amino acid (e.g., a different amino acid, or a modification of the same amino acid). The parent amino acid sequence may be a naturally occurring or wild type (WT) amino acid sequence, or may be a modified version of a wild type amino acid sequence. In some embodiments, the variant amino acid sequence has at least one amino acid difference as compared to the parent amino acid sequence, e.g., from 1 to about 20 amino acid differences, such as from 1 to about 10 or from 1 to about 5 amino acid differences compared to the parent.By "wild type" or "WT" or "native" as used herein and with reference to an amino acid sequence (peptide or polypeptide) is meant an amino acid sequence that is found in nature, including allelic variations. A wild type amino acid sequence, peptide or polypeptide has an amino acid sequence that has not been intentionally modified.

[0261] For the purposes of the present disclosure, "variants" (including functional variants) of an amino acid sequence (peptide or polypeptide) may comprise amino acid insertion variants, amino acid addition variants, amino acid deletion variants and / or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, in particular those which are naturally occurring. The term "variant" includes, in particular, fragments of an amino acid sequence.

[0262] Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid addition variants comprise amino- and / or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein. Amino acid deletion variants that comprise the deletion at the N-terminal and / or C-terminal end of the protein are also called N-terminal and / or C-terminal truncation variants. Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous peptides or polypeptides and / or to replacing amino acids with other ones having similar properties. In some embodiments, amino acid changes in peptide and polypeptide variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate),basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In some embodiments, conservative amino acid substitutions include substitutions within the following groups:

[0263] glycine, alanine;

[0264] valine, isoleucine, leucine;

[0265] aspartic acid, glutamic acid;

[0266] asparagine, glutamine;

[0267] serine, threonine;

[0268] lysine, arginine; and

[0269] phenylalanine, tyrosine.

[0270] In some embodiments, the degree of similarity, such as identity between a given amino acid sequence and an amino acid sequence which is a variant (including functional variant) of said given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the degree of similarity or identity is given for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given, e.g., for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments continuous amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. The alignment for determining sequence similarity, such as sequence identity can be done with art known tools, such as using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5."Sequence similarity" indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.

[0271] The terms "% identical" and "% identity" or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or "window of comparison", in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of algorithms, e.g., the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website (e.g., at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC =align2seq). In some embodiments, the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match / Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. In some embodiments, the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment.Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.

[0272] In some embodiments, the degree of similarity or identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments continuous nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence. Homologous amino acid sequences exhibit according to the disclosure at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and, e.g., at least 95%, at least 98 or at least 99% identity of the amino acid residues.

[0273] The amino acid sequence variants described herein may readily be prepared by the skilled person, for example, by recombinant DNA manipulation. The manipulation of DNA sequences for preparing peptides or polypeptides having substitutions, additions, insertions or deletions, is described in detail in Molecular Cloning: A Laboratory Manual, 4th Edition, M.R. Green and J. Sambrook et al. (1989), eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012, for example. Furthermore, the peptides, polypeptides and amino acid variants described herein may be readily prepared with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis and similar methods.

[0274] In some embodiments, a fragment or variant of an amino acid sequence (peptide or polypeptide) is a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment orva riant exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent. With respect to sequences of binding agents such as antibodies, one particular function is one or more binding activities displayed by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant", as used herein, in particular refers to a variantmolecule or sequence that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that is still capable of fulfilling one or more of the functions of the parent molecule or sequence, e.g., binding to a target molecule. In some embodiments, the modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present, e.g., function of the functional fragment or functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence. However, in other embodiments, function of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0275] In some embodiments, a polypeptide described herein, e.g., one or more of the polypeptide chains of a docking compound, comprises one or more posttranslational modifications. Included herein are polypeptides which are derived from one or more posttranslational modifications of a polypeptide described herein. In some embodiments, the one or more posttranslational modifications are selected from pyroglutamylation, deamidation, e.g., asparagine deamidation, isomerisation, e.g., aspartate isomerisation, glycation, e.g., lysine glycation, and lysine clipping. Particular posttranslational modifications are the deletion of lysine at the C-terminal end of a polypeptide such as at the C-terminal end of a CH3 domain and pyroglutamylation of an N-terminal glutamine or glutamic acid.

[0276] An amino acid sequence (peptide or polypeptide) "derived from" a designated amino acid sequence (peptide or polypeptide) refers to the origin of the first amino acid sequence. In some embodiments, the amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof. Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof, preferably functional variants thereof as described herein, including functional fragments. For example, it will be understood by one of ordinary skill in the art that the sequences suitable for use herein may be altered such that they vary in sequence from thenaturally occurring or native sequences from which they were derived, while retaining or essentially retaining the desirable activity of the native sequences.

[0277] In some embodiments, "isolated" means removed (e.g., purified) from the natural state or from an artificial composition, such as a composition from a production process. For example, a nucleic acid, peptide or polypeptide naturally present in a living animal is not "isolated", but the same nucleic acid, peptide or polypeptide partially or completely separated from the coexisting materials of its natural state is "isolated". An isolated nucleic acid, peptide or polypeptide can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0278] The term "bind" or "binding" relates to the non-covalent interaction with a target. In some embodiments, the term "bind" or "binding" relates to a specific binding. By the term "specific binding" or "specifically binds", as used herein, is meant a molecule such as an antibody which recognizes a specific target molecule, but does not substantially recognize or bind other molecules in a sample or in a subject. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.

[0279] In some instances, the terms "specific binding" or "specifically binds", can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A" and the antibody, will reduce the amount of labeled A bound to the antibody.

[0280] As used herein, the terms "binding" or "capable of binding" typically is a binding with an affinity corresponding to a KDof about 10'7M or less, such as about 10'8M or less, such as about 10'9M or less, about 1010M or less, or about 1011M or even less, when determinedusing Bio-Layer Interferometry (BLI), or, for instance, when determined using surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument. In some embodiments, a binding moiety or agent binds to a predetermined target with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its affinity for binding to a non-specific target (e.g., BSA, casein).

[0281] The term "kd" (sec-1), as used herein, refers to the dissociation rate constant of a particular interaction, e.g., antibody-antigen interaction. Said value is also referred to as the kOff value. The term "KD" (M), as used herein, refers to the dissociation equilibrium constant of a particular interaction, e.g., antibody-antigen interaction.

[0282] Generally, the terms "bind" or "binding" and "target" or "targeting" are used interchangeably herein.

[0283] The term "genetic modification" or simply "modification" includes the transfection of cells with nucleic acid. The term "transfection" relates to the introduction of nucleic acids, e.g., DNA and / or RNA, into a cell. For purposes of the present disclosure, the term "transfection" also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such cell, wherein the cell may be present in a subject, e.g., a patient, or the cell may be in vitro, e.g., outside of a patient. Thus, according to the present disclosure, a cell for transfection of a nucleic acid described herein can be present in vitro or in vivo, e.g. the cell can form part of an organ, a tissue and / or the body of a patient. According to the disclosure, transfection can be transient or stable. For some applications of transfection, it is sufficient if the transfected genetic material is only transiently expressed. RNA can be transfected into cells to transiently express its coded protein. Since the nucleic acid introduced in the transfection process is usually not integrated into the nuclear genome, the foreign nucleic acid will be diluted through mitosis or degraded. Cells allowing episomal amplification of nucleic acids greatly reduce the rate of dilution. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, a stable transfection must occur. Such stable transfection can be achieved by using virus-based systems ortransposon-based systems for transfection, for example. Generally, cells that are transfected with nucleic acid encoding a docking compound are transiently transfected with nucleic acid encoding the dockingcompound. RNA can be transfected into cells to transiently express its coded protein.

[0284] As used herein, the terms "linked", "fused", or "fusion" are used interchangeably. These terms refer to the joining together of two or more elements or components or domains.

[0285] The term "fusion protein" as used herein refers to a polypeptide or protein comprising two or more subunits. Preferably, the fusion protein is a translational fusion between the two or more subunits. The translational fusion may be generated by genetically engineering the coding nucleotide sequence for one subunit in a reading frame with the coding nucleotide sequence of a further subunit. Subunits may be interspersed by a linker.

[0286] As used herein "endogenous" refers to any material from or produced inside an organism, cell, tissue or system.

[0287] As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0288] The term "autologous" is used to describe anything that is derived from the same subject. For example, "autologous transplant" refers to a transplant of tissue or organs derived from the same subject. Such procedures are advantageous because they overcome the immunological barrier which otherwise results in rejection.

[0289] The term "allogeneic" is used to describe anything that is derived from different individuals of the same species. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical.

[0290] The term "syngeneic" is used to describe anything that is derived from individuals or tissues having identical genotypes, i.e., identical twins or animals of the same inbred strain, or their tissues.

[0291] The term "heterologous" is used to describe something consisting of multiple different elements. As an example, the transfer of one individual's bone marrow into a different individual constitutes a heterologous transplant. A heterologous gene is a gene derived from a source other than the subject.

[0292] According to various embodiments of the present disclosure, a nucleic acid encoding a peptide or polypeptide is taken up by or introduced, i.e. transfected or transduced, into a cell which cell may be present in vitro or in a subject, resulting in expression of said peptide or polypeptide. The cell may, e.g., express the encoded peptide or polypeptide intracellularly(e.g. in the cytoplasm and / or in the nucleus), may secrete the encoded peptide or polypeptide, and / or may express it on the surface. In some embodiments, if the encoded peptide(s) or polypeptide(s) is / are or form a docking compound, the cell secretes the encoded peptide(s) or polypeptide(s).

[0293] According to the present disclosure, terms such as "nucleic acid expressing" and "nucleic acid encoding" or similar terms are used interchangeably herein and with respect to a particular peptide or polypeptide mean that the nucleic acid, if present in the appropriate environment, e.g. within a cell, can be expressed to produce said peptide or polypeptide.

[0294] The term "expression" as used herein includes the transcription and / or translation of a particular nucleotide sequence.

[0295] In the context of the present disclosure, the term "transcription" relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA (especially mRNA). Subsequently, the RNA may be translated into peptide or polypeptide.

[0296] With respect to RNA, the term "expression" or "translation" relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or polypeptide.

[0297] A medical preparation, in particular kit, described herein may comprise instructional material or instructions. As used herein, "instructional material" or "instructions" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the present disclosure. The instructional material of the kit of the present disclosure may, for example, be affixed to a container which contains the compositions / formulations of the present disclosure or be shipped together with a container which contains the compositions / formulations. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compositions be used cooperatively by the recipient.

[0298] Docking compound

[0299] According to the disclosure, a payload is delivered specifically to a target cell by providing a docking compound with at least two moieties that bind to a target on target cells, e.g., anantigen on target cells, and a moiety that binds to a compound which carries the payload (tag conjugate). The target on target cells is also referred to herein as "primary target".

[0300] A "docking compound" is used to form a connection, such as a non-covalent connection, between a primary target, e.g., a target cell or an antigen on target cells, and the docking compound. The docking compound may form a connection, such as a non-covalent or covalent connection, to a compound comprising a payload to be delivered to a target cell (tag conjugate). The tag conjugate comprises tags for binding by the docking compound which are covalently attached to the payload.

[0301] In some embodiments, a docking compound comprises at least two "primary targeting moieties", e.g., moieties targeting a cell surface antigen on target cells, that are capable of binding to the primary target of interest, e.g., a cell surface antigen on target cells. A "primary targeting moiety" as used herein relates to the part of the docking compound which binds to a primary target. Such targeting moieties are typically moieties that have affinity for cell surface targets. These moieties can be any peptide or protein (e.g. antibodies or antibody fragments) binding to the primary target. Particular embodiments of suitable primary targeting moieties for use herein include cell surface antigen binding moieties, such as antibodies, antibody fragments and DARPins. Other examples of primary targeting moieties are peptides or proteins which bind to a receptor.

[0302] A primary targeting moiety preferably binds with high specificity and / or high affinity and the bond with the primary target is preferably stable within the body.

[0303] In order to allow specific targeting of primary targets, a primary targeting moiety of the docking compound can comprise compounds including but not limited to antibodies, antibody fragments, e.g. F(ab')2, Fab, scFV, VHH domains, and other proteins or peptides.

[0304] According to some embodiments, the primary target is a cell surface antigen such as a cancer antigen, and suitable primary targeting moieties include but are not limited to, peptides and polypeptides targeting the cell surface antigen, e.g., antibodies, antibody fragments and DARPins.

[0305] According to some embodiments, the primary target is a receptor and suitable primary targeting moieties include but are not limited to, the ligand of such a receptor or a part thereofwhich still binds to the receptor, e.g., a receptor binding peptide in the case of receptor binding protein ligands.

[0306] According to some embodiments, the primary target and primary targeting moiety are selected so as to result in the specific or increased targeting of certain cells, e.g., diseased cells, such as cells involved in and characteristic for a disease such as cancer, an inflammation, an infection, a cardiovascular disease, e.g. thrombus, atherosclerotic lesion, hypoxic site, e.g. stroke, tumor, cardiovascular disorder, brain disorder, apoptosis, and angiogenesis. This can be achieved by selecting primary targets with cell-specific expression. For example, cancer antigens, e.g., those described herein, may be expressed in cancer cells while they are not expressed or expressed in a lower amount in normal non-cancerous cells.

[0307] The docking compound further comprises a group which serves as a binding moiety for a respective tag of a tag conjugate. The moiety of the docking compound binding to the tag conjugate and the primary targeting moiety are linked to each other, preferably by a covalent linkage.

[0308] According to some embodiments, the docking compound comprises a bispecific molecule, such as a bispecific polypeptide, e.g., a bispecific antibody. In some embodiments, the docking compound comprises at least two binding domains binding to a primary target and a binding domain binding to a tag conjugate. In some embodiments, a binding domain binding to a primary target comprises an antibody or antibody fragment binding to a primary target and the binding domain binding to a tag conjugate comprises an antibody or antibody fragment binding to a tag conjugate. In some embodiments, at least one binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. In some embodiments, each binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. In some embodiments, at least one binding domain comprises a single-domain antibody such as a VHH. In some embodiments, each binding domain comprises a single-domain antibody such as a VHH. In some embodiments, one binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody and the other binding domain comprises a single-domain antibody such as a VHH. In some embodiments, the binding domain binding to a primary target comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. In someembodiments, the binding domain binding to a primary target comprises a single-domain antibody such as a VHH. In some embodiments, the binding domain binding to a tag conjugate comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. In some embodiments, the binding domain binding to a tag conjugate comprises a single-domain antibody such as a VHH. In some embodiments, the binding domain binding to a primary target comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody and the binding domain binding to a tag conjugate comprises a singledomain antibody such as a VHH.

[0309] In some embodiments, the docking compound comprises two binding domains binding to a primary target and one binding domain binding to a tag conjugate.

[0310] In some embodiments, the docking compound comprises a full-length antibody binding to a primary target. In some embodiments, one of the heavy chains of the full-length antibody is C-terminally linked to a VHH binding to a tag on a tag conjugate.

[0311] In some embodiments, the docking compound does not comprise a full-length antibody. For example, in some embodiments, the docking compound comprises an antibody wherein the Fab fragments of the antibody are replaced by scFv or VHHs binding to a primary target and being N-terminally linked to the remaining part of the heavy chains. In some embodiments, one of the heavy chains is C-terminally linked to a VHH binding to a tag on a tag conjugate. In these and other embodiments, the docking compound may comprise an antibody or antibodylike molecule which does not comprise an immunoglobulin heavy chain constant region 2 (CH2) domain. In some embodiments, a target antigen binding moiety, e.g., Fab, scFV, or VHH domain, is fused to an immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof. The CH3 domain or variant thereof mediates the dimerization of the target antigen binding moiety, resulting in a bivalent antibody fragment. One of the CH3 domains or variants thereof may be linked to a VHH binding to a tag on a tag conjugate.

[0312] The present disclosure provides in one aspect, a docking compound as described herein. In some embodiments, the docking compound comprises a bispecific molecule, such as a bispecific polypeptide, e.g., a bispecific antibody, wherein one specificity binds (monovalently) to an epitope tag, e.g., an ALFA-tag and the other specificity binds (bivalenty or with even more valency) to a primary target, e.g., a cell surface antigen on target cells. In someembodiments, the specificity which binds to an epitope tag is an antibody or antibody fragment such as an NbALFA-nanobody (NbALFA). In some embodiments, the specificity which binds to a primary target is an antibody, antibody fragment or DARPin. In some embodiments, the moiety targeting a primary target of the docking compound is selected from the group consisting of an anti-primary target Fab, an anti-primary target VHH and an anti-primary target scFv and / or the moiety binding to a tag conjugate of the docking compound is an NbALFA-nanobody (NbALFA). In some embodiments, the primary target is a cancer antigen. In some embodiments, the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and two anti-cancer antigen Fabs. In some embodiments, the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and two anti-cancer antigen VHHs. In some embodiments, the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and two anti-cancer antigen scFvs.

[0313] In some embodiments, the docking compound comprises two Fab fragments binding to target antigen and a VHH binding to a tag. The Fab fragments and VHH may be connected in a linear or non-linear arrangement.

[0314] In some embodiments, the Fab fragments are connected via a peptide linker (tandem Fab), and the tandem Fab is linked to the VHH.

[0315] In some embodiments, the Fab fragments are each connected to a pair of dimerization domains (e.g., CH3 domains or variants thereof) allowing heterodimer formation. In some embodiments, one of the monomers of a heterodimer is linked to the VHH. In some embodiments, a first dimerization domain (e.g., CH3 domain or variant thereof) forms a disulphide bond with a second dimerization domain (e.g., CH3 domain or variant thereof). Generally, the Fab fragments binding to target antigen are connected to the N-terminus of the dimerization domains (e.g., CH3 domains or variants thereof) and the VHH domain binding to a tag is connected to the C-terminus of one dimerization domain (e.g., CH3 domain or variant thereof).

[0316] In some embodiments, the docking compound comprises two scFvs or VHHs binding to target antigen and a VHH binding to a tag. In some embodiments, the scFvs or VHHs are each connected to a pair of dimerization domains (e.g., CH3 domains or variants thereof) allowingheterodimer formation. In some embodiments, one of the monomers of a heterodimer is linked to the VHH. In some embodiments, a first dimerization domain (e.g., CH3 domain or variant thereof) forms a disulphide bond with a second dimerization domain (e.g., CH3 domain or variant thereof). Generally, the scFvs or VHHs binding to target antigen are connected to the N-terminus of the dimerization domains (e.g., CH3 domains or variants thereof) and the VHH domain binding to a tag is connected to the C-terminus of one dimerization domain (e.g., CH3 domain or variant thereof).

[0317] In some embodiments, the docking compound comprises a first and a second polypeptide, wherein the first and the second polypeptide each comprise and / or are connected to one target antigen binding domain (e.g., Fab, scFv or VHH) N-terminal of a dimerization domain (e.g., CH3 domain or variant thereof), wherein the target antigen binding domains N-terminal of the dimerization domain (e.g., CH3 domain or variant thereof) of the first and the second polypeptide have the same or different binding specificities, and the first or the second polypeptide comprises and / or is connected to one tag binding moiety (e.g. a VHH domain) C-terminal of the dimerization domain (e.g., CH3 domain or variant thereof).

[0318] By "N-terminal of a dimerization domain" it is meant that an entity, e.g., a target antigen binding domain, is located N-terminal of the dimerization domain by any means known in the art. In some embodiments, the entity, e.g., a target antigen binding domain, is fused directly to the N-terminus of the dimerization domain, i.e. the entity, e.g., a target antigen binding domain, may be immediately adjacent to the dimerization domain. However, in some embodiments, the entity, e.g., a target antigen binding domain, is connected to the N-terminus of the dimerization domain by a linking sequence, e.g., a linking sequence as described herein. Such linking sequence may or may not comprise a CH2 domain.

[0319] By "C-terminal of a dimerization domain" it is meant that an entity, e.g., a tag binding moiety, is located C-terminal of the dimerization domain by any means known in the art. In some embodiments, the entity, e.g., a tag binding moiety, is fused directly to the C-terminus of the dimerization domain, i.e. the entity, e.g., a tag binding moiety, may be immediately adjacent to the dimerization domain. However, in some embodiments, the entity, e.g., a tag binding moiety, is connected to the C-terminus of the dimerization domain by a linking sequence, e.g., a linking sequence as described herein.In some embodiments:

[0320] a) the sequence of the first polypeptide between the dimerization domain (e.g., CH3 domain or variant thereof) of the first polypeptide and the target antigen binding domain located N-terminal of the dimerization domain (e.g., CH3 domain or variant thereof) in the first polypeptide comprises a linking sequence LI; and

[0321] b) the sequence of the second polypeptide between the dimerization domain (e.g., CH3 domain or variant thereof) of the second polypeptide and the target antigen binding domain located N-terminal of the dimerization domain (e.g., CH3 domain or variant thereof) in the second polypeptide comprises a linking sequence LI,

[0322] wherein LI of the first polypeptide and LI of the second polypeptide may be the same or different.

[0323] The linking sequence may comprise one or more linkers (e.g. GS linkers) and / or an antibody hinge region sequence, such as those linkers and antibody hinge regions that are widely known in the art.

[0324] The linking sequence may include cysteine residues or be devoid of cysteine residues. Thus, the linking sequences LI of the first polypeptide and of the second polypeptide may form inter-domain disulphides or may not form any inter-domain disulphide bonds.

[0325] An illustrative linking sequence comprises the sequence EPKSCDKTHTCPPCGGGSSGGGSG (SEQ ID NO: 23).

[0326] In some embodiments, the first polypeptide and the second polypeptide do not contain any constant immunoglobulin domain(s) other than a CHI domain and a CH3 domain or variant thereof. In some embodiments, the first polypeptide and the second polypeptide do not contain a CH2 domain.

[0327] In some embodiments, the first polypeptide comprises a single CH3 domain or variant thereof and the second polypeptide comprises a single CH3 domain or variant thereof.

[0328] In some embodiments, a docking compound may be provided by administering to a subject nucleic acid encoding the docking compound and allowing expression of the docking compound by cells of the subject. Delivery of nucleic acid encoding a docking compound to target cells for expression may be effected by using particles comprising the nucleic acid. The particles may comprise a targeting molecule that binds to a target, e.g., an antigen on targetcells, for expression. In some embodiments, the docking compound is secreted from the cells expressing the nucleic acid. In some embodiments, the docking compound comprises a signal peptide, e.g., an N-terminal signal peptide, which allows secretion of the docking compound from the cell expressing the nucleic acid. In some embodiments, the cells expressing the nucleic acid are the same cells as those to which a payload is to be delivered herein. In some embodiments, the cells expressing the nucleic acid are different to the cells to which a payload is to be delivered herein. In some embodiments, the cells expressing the nucleic acid are liver cells. In some embodiments, the cells expressing the nucleic acid secrete the docking compound into the bloodstream. In some embodiments, the signal peptide is cleavable and is removed from the mature polypeptide. In some preferred embodiments, the nucleic acid encoding the docking compound is RNA. The RNA-encoded docking compound is also called "RiboDocker" herein.

[0329] Tag conjugate

[0330] The tag conjugate described herein comprises a payload to be delivered and tags for binding by the docking compound. The tags of the tag conjugate thus are the part of the tag conjugate that forms the binding partner for the docking compound. Generally, the tags of the tag conjugate are covalently attached to the payload moiety in a manner such that they are available for binding to the docking compound.

[0331] A tag conjugate described herein comprises at least two tags. In some embodiments, a tag conjugate described herein comprises 2, 3, 4, 5, 6, 7, 8 or even more tags, which tags may be identical or different. In some embodiments, the tags of a tag conjugate described herein are identical. In some embodiments, a tag conjugate described herein comprises partially or completely different tags, but the binding moiety for a tag of a docking compound binds to the different tags. In some embodiments, each of the tags of a tag conjugate described herein comprises an ALFA-tag. In some embodiments, a tag conjugate described herein comprises two tags, preferably two ALFA-tags. In some embodiments, a tag conjugate described herein comprises two identical tags, preferably two identical ALFA-tags.

[0332] In some embodiments, the tags of the tag conjugate comprise a peptide or protein (e.g., peptide tags).In some embodiments, the tags of the tag conjugate comprise a peptide or protein (e.g., peptide tags) and are chemically linked, e.g., through a linker, to the payload moiety.

[0333] A tag conjugate further comprises a moiety, termed "payload moiety" or "payload" herein, that is capable of attracting, providing or bringing about the desired diagnostic, imaging, and / or therapeutic effect. The tags and the payload moiety may be covalently or non-covalently linked.

[0334] In some embodiments, the payload moiety is selected from the group consisting of radioisotopes, toxins and immunomodulators.

[0335] In some embodiments, the tag conjugate comprises a polymer. In some embodiments, the payload moiety of the tag conjugate and the tags of the tag conjugate are connected through a linker comprising the polymer.

[0336] In some embodiments, the polymer is not a polymer of proteinogenic amino acids or their D-isomers. In some embodiments, the polymer is a hydrophilic polymer. In some embodiments, the polymer portion of the tag conjugate contributes to conferring stealth properties on the tag conjugate. In some embodiments, the plasmatic half-life of the tag conjugate described herein is greater than 2 hours, e.g., between 3 and 10 hours. This characteristic advantageously allows the tag conjugate to accumulate at the target cells and to liberate therein their contents (payload) within reasonable amounts of time. The effectiveness of the targeted delivery described herein therefore increases as a result.

[0337] The term "stealth" is used herein to describe the ability of the particles described herein not to be detected and then sequestered and / or degraded, or to be hardly detected and then sequestered and / or degraded, and / or to be detected and then sequestered and / or degraded late, by the immune system of the host to which they are administered.

[0338] Macrophages constitute one of the most important components of the immune system and play a predominant role in eliminating foreign particles, including liposomes and other colloidal particles, from the blood circulation. At the molecular level, the clearance of particles takes place in two steps: opsonization by the depositing of serum proteins (or "opsonins") at the surface of the particles followed by recognition and capture of the opsonized particles by macrophages.Modification of the surface of particles with chains of hydrophilic and flexible polymers, e.g., polymers of the poly(ethylene glycol) type, confers them a steric protection by preventing the opsonins reaching the surface of the particles.

[0339] In some embodiments, the polymer for use herein is selected from the group consisting of poly(ethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) (including derivatives and combinations thereof).

[0340] In some embodiments, a polymer is designed to sterically stabilize a tag conjugate by forming a protective hydrophilic layer. In some embodiments, a polymer can reduce association of a tag conjugate with serum proteins and / or the resulting uptake by the reticuloendothelial system when such tag conjugates are administered in vivo.

[0341] In some embodiments, the PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some embodiments, the PEG is unsubstituted. In some embodiments, the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy or aryl groups. In some embodiments, the PEG has a molecular weight of from about 130 to about 50,000, in another embodiment about 150 to about 30,000, in another embodiment about 150 to about 20,000, in another embodiment about 150 to about 15,000, in another embodiment about 150 to about 10,000, in another embodiment about 150 to about 6000, in another embodiment about 150 to about 5000, in another embodiment about 150 to about 4000, in another embodiment about 150 to about 3000, in another embodiment about 300 to about 3000, in another embodiment about 1000 to about 3000, and in still another embodiment about 1500 to about 2500.

[0342] In some embodiments, the PEG moiety has a molecular weight of 1000 or more. In some embodiments, the PEG moiety comprises 10 units or more of formula (O-CHz-CHzJn. In some embodiments, the PEG comprises from 20 to 200 ethylene oxide units, such as about 45 ethylene oxide units.

[0343] In some embodiments, the PEG comprises "PEG2k", also termed "PEG 2000", which has an average molecular weight of about 2000 Daltons.

[0344] In some embodiments, PEG2000, PEG3000 and PEG5000 are used as the polymer.In some embodiments, a pSar comprises between 2 and 200 sarcosine units, such as between 5 and 100 sarcosine units, between 10 and 50 sarcosine units, between 15 and 40 sarcosine units, e.g., about 23 sarcosine units.

[0345] In some embodiments, a pSar comprises the structure of the following general formula:

[0346]

[0347] wherein s is the number of sarcosine units.

[0348] In some embodiments, the POX and / or POZ polymer comprises between 2 and 200, between 2 and 190, between 2 and 180, between 2 and 170, between 2 and 160, between 2 and 150, between 2 and 140, between 2 and 130, between 2 and 120, between 2 and 110, between 2 and 100, between 2 and 90, between 2 and 80, between 2 and 70, between 5 and 200, between 5 and 190, between 5 and 180, between 5 and 170, between 5 and 160, between 5 and 150, between 5 and 140, between 5 and 130, between 5 and 120, between 5 and 110, between 5 and 100, between 5 and 90, between 5 and 80, between 5 and 70, between 10 and 200, between 10 and 190, between 10 and 180, between 10 and 170, between 10 and 160, between 10 and 150, between 10 and 140, between 10 and 130, between 10 and 120, between 10 and 110, between 10 and 100, between 10 and 90, between 10 and 80, or between 10 and 70 POX and / or POZ repeating units.

[0349] In some embodiments, the POX and / or POZ polymer comprises the following general formula:

[0350]

[0351] wherein a is an integer between 1 and 2; Rn is alkyl, in particular C1-3 alkyl, such as methyl, ethyl, iso-propyl, or n-propyl, and is independently selected for each repeating unit; and m refers to the number of POX and / or POZ repeating units.

[0352] In some embodiments, the POX and / or POZ polymer is a polymer of POX and comprises repeating units of the following general formula:

[0353]

[0354] In some embodiments, the POX and / or POZ polymer is a polymer of POZ and comprises repeating units of the following general formula:

[0355]

[0356] In any of the above embodiments of formulas, m (i.e., the number of repeating units in the polymer) preferably is between 2 and 190, such as between 2 and 180, between 2 and 170, between 2 and 160, between 2 and 150, between 2 and 140, between 2 and 130, between 2 and 120, between 2 and 110, between 2 and 100, between 2 and 90, between 2 and 80, between 2 and 70, between 5 and 200, between 5 and 190, between 5 and 180, between 5 and 170, between 5 and 160, between 5 and 150, between 5 and 140, between 5 and 130, between 5 and 120, between 5 and 110, between 5 and 100, between 5 and 90, between 5 and 80, between 5 and 70, between 10 and 200, between 10 and 190, between 10 and 180, between 10 and 170, between 10 and 160, between 10 and 150, between 10 and 140, between 10 and 130, between 10 and 120, between 10 and 110, between 10 and 100, between 10 and 90, between 10 and 80, or between 10 and 70. In certain embodiments, m is 2 to 180, such as 4 to 160, 6 to 140, 8 to 120 or 10 to 100, e.g., 20 to 80, 30 to 70, or 40 to 50. In some embodiments, the POX and / or POZ polymer is a copolymer comprising repeating units of the following general formulas:

[0357]

[0358] wherein the number of repeating units shown on the left in the copolymer is 1 to 199; the number of repeating units of formula on the right in the copolymer is 1 to 199; and the sumof the number of repeating units of formula on the left and the number of repeating units of formula on the right in the copolymer is 2 to 200.

[0359] In some embodiments, the number of repeating units of formula on the left in the copolymer is 1 to 179, such as 1 to 159, 1 to 139, 1 to 119 or 1 to 99; the number of repeating units of formula on the right in the copolymer is 1 to 179, such as 1 to 159, 1 to 139, 1 to 119 or 1 to 99; and the sum of the number of repeating units of formula on the left and the number of repeating units of formula on the right in the copolymer is 2 to 180, such as 4 to 160, 6 to 140, 8 to 120 or 10 to 100, e.g., 20 to 80, 30 to 70, or 40 to 50.

[0360] In some of the above embodiments, Rn at each occurrence (i.e., in each repeating unit) may be the same alkyl group (e.g., Rn may be methyl in each repeating unit). In some alternative embodiments, Rn in at least one repeating unit differs from Rn in another repeating unit (e.g., for at least one repeating unit Rn is one specific alkyl (such as ethyl), and for at least one different repeating unit Rn is a different specific alkyl (such as methyl)). For example, each Rn may be selected from two different alkyl groups (such as methyl and ethyl) and not all Rn are the same alkyl.

[0361] In any of the above embodiments, Rn preferably is methyl or ethyl, more preferably methyl. Thus, in some embodiments, each Rn is methyl or each Rn is ethyl. In some alternative embodiments, Rn is independently selected from methyl and ethyl for each repeating unit, wherein in at least one repeating unit Rn is methyl, and in at least one repeating unit Rn is ethyl.

[0362] In some embodiments, the polymer comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof.

[0363] In some embodiments, the polymer comprises the following general formula:

[0364]

[0365] wherein

[0366] X2and X1taken together are optionally substituted amide, optionally substituted thioamide ester, or thioester;Y is -CH2-, -(CH2)2-, or -(CH2)3-;

[0367] z is 2 to 24; and

[0368] n is the number of repeating units, e.g., 1 to 100.

[0369] In some embodiments,

[0370] (i) when X1is -C(O)- then X2is -NR1-;

[0371] (ii) when X1is -NR1- then X2is -C(O)-;

[0372] (iii) when X1is -C(S)- then X2is -NR1-;

[0373] (iv) when X1is -NR1- then X2is -C(S)-;

[0374] (v) when X1is -C(O)- then X2is -O-;

[0375] (vi) when X1is -O- then X2is -C(O)-;

[0376] (vii) when X1is -C(S)- then X2is -O-;

[0377] (viii) when X1is -O- then X2is -C(S)-;

[0378] (ix) when X1is -C(O)- then X2is -S-; or

[0379] (x) when X1is -S- then X2is -C(O)-;

[0380] wherein R1is hydrogen or C1-8 alkyl; preferably

[0381] (i) when X1is -C(O)- then X2is -NR1-;

[0382] (ii) when X1is -NR1- then X2is -C(O)-;

[0383] (iii) when X1is -C(S)- then X2is -NR1-;

[0384] (iv) when X1is -NR1- then X2is -C(S)-;

[0385] (v) when X1is -C(O)- then X2is -O-; or

[0386] (vi) when X1is -O- then X2is -C(O)-;

[0387] wherein R1is hydrogen or C1-8 alkyl.

[0388] In some embodiments, X1is -C(O)- and X2is -NR1-, wherein R1is hydrogen or C1-8 alkyl. In some embodiments, X1is -C(O)- and X2is -NR1-, wherein R1is hydrogen or methyl. In some embodiments, X1is -C(O)- and X2is -NR1-, wherein R1is hydrogen.

[0389] In some embodiments, Y is -CH2- or -(CH2)2-. In some embodiments, Y is -CH2-.

[0390] In some embodiments, the polymer comprises the following general formula:

[0391]

[0392] wherein

[0393] R1is hydrogen or Ci-8 alkyl;

[0394] z is 2 to 24; and

[0395] n is the number of repeating units, e.g., 1 to 100.

[0396] In some embodiments of the above formulas, z is 2 to 10. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.

[0397] In some embodiments, the polymer comprises the following general formula:

[0398]

[0399] wherein

[0400] R1is hydrogen or Ci-8 alkyl; and

[0401] n is the number of repeating units, e.g., 1 to 100.

[0402] In some embodiments of the above formulas, R1is hydrogen or methyl. In some embodiments, R1is hydrogen.

[0403] In some embodiments, the polymer comprises the following general formula:

[0404]

[0405] wherein

[0406] n is the number of repeating units, e.g., 1 to 100.

[0407] In some embodiments of the above formulas, n is 2 to 50. In some embodiments, n is 4 to 25. In some embodiments, n is 6 to 20. In some embodiments, n is 8 to 16. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, n is 2, 4, 6, 8, 10, or 12. In some embodiments, n is 2, 4, or 6.

[0408] In some embodiments, the tag conjugate comprises more than one payload moiety.

[0409] In some embodiments, the total number of payload moieties in the tag conjugate is one. In some embodiments, the tag conjugate comprises two or more payload moieties.

[0410] In some embodiments, the total number of payload moieties in the tag conjugate is two.In some embodiments, the tag conjugate comprises payload moieties and tags in an unbranched (linear) configuration.

[0411] In some embodiments, the tag conjugate comprises at least two tags and at least one payload moiety in an unbranched (linear) configuration.

[0412] In some embodiments, the tag conjugate comprises at least two tags and at least two payload moieties in an unbranched (linear) configuration.

[0413] In some embodiments, the tag conjugate comprises two tags and one or two payload moieties in an unbranched (linear) configuration.

[0414] In some embodiments, the tag conjugate comprises one or more linking moieties connecting tags and payload moieties in an unbranched (linear) configuration.

[0415] In some embodiments, a linking moiety is a branched or unbranched linking moiety.

[0416] In some embodiments, at least one linking moiety comprises a continuous or non-continuous poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0417] In some embodiments, the tag conjugate comprises two tags which are connected by a linking moiety

[0418] In some embodiments, the linking moiety connecting the tags comprises a continuous or non-continuous poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0419] In some embodiments, a payload moiety is connected to at least one of the tags.

[0420] In some embodiments, a payload moiety is connected to a tag by a linking moiety.

[0421] In some embodiments, the linking moiety connecting a payload moiety and a tag comprises a continuous or non-continuous poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0422] In some embodiments, the linking moiety connecting a payload moiety and a tag comprises an enzymatic cleavage site.

[0423] In some embodiments, the tag conjugate comprises the formula:

[0424] P-LA-T-LB-T

[0425] or

[0426] P-LA-T-LB-T-LC-P

[0427] whereinP comprises a payload moiety;

[0428] T comprises a tag;

[0429] LA comprises a linking moiety;

[0430] LB comprises a linking moiety; and

[0431] Lc comprises a linking moiety.

[0432] In some embodiments, one or more of LA, LB, and Lc comprises a polymer as described herein. In some embodiments, one or more of LA, LB, and Lc comprises a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0433] In some embodiments, LB comprises a polymer as described herein. In some embodiments, LB comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0434] In some embodiments, LA and / or Lc comprise a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0435] In some embodiments, LAand / or Ledo not comprise a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0436] In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 30, e.g., between 2 and 10, such as 2, 4 or 6. In some embodiments, the poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2- (2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof. In some embodiments, the poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof comprises the following general formula:

[0437]

[0438] wherein n is between 2 and 30, e.g., between 2 and 10, such as 2, 4 or 6.

[0439] In some embodiments, LA and / or Lc comprise an enzymatic cleavage site.

[0440] In some embodiments, one or more of LA, LB, and Lc comprises the formula [AEEA]U-[LD- [AEEA]V]W,wherein

[0441] AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid or a derivative thereof;

[0442] LD comprises a linking moiety;

[0443] u is an integer of 2 or more;

[0444] each v is an integer of 2 or more; and

[0445] w is an integer from 1 to 4;

[0446] wherein

[0447] the different groups [LD-[AEEA]V] may be identical or different.

[0448] In some embodiments, LB comprises the formula [AEEA]U-[LD-[AEEA]V]W,

[0449] wherein

[0450] AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid or a derivative thereof;

[0451] LD comprises a linking moiety;

[0452] u is an integer of 2 or more;

[0453] each v is an integer of 2 or more; and

[0454] w is an integer from 1 to 4;

[0455] wherein

[0456] the different groups [LD-[AEEA]V] may be identical or different.

[0457] In some embodiments, u and v are each integers from 2 to 10.

[0458] In some embodiments, u and v are each integers from 2 to 8.

[0459] In some embodiments, u and v are each integers of 2, 4 or 6.

[0460] In some embodiments, LD comprises an amino acid.

[0461] In some embodiments, LD comprises the D-isomer of an amino acid.

[0462] In some embodiments, LD comprises cysteine or lysine.

[0463] In some embodiments, LD is connected to a side chain.

[0464] In some embodiments, a side chain comprises a functional moiety.

[0465] In some embodiments, a functional moiety comprises a solubilizing functional group.

[0466] In some embodiments, a tag conjugate comprises a branching moiety to which the tags and the payload moieties are connected through a linking moiety.

[0467] The term "branching moiety" refers to a chemical moiety, e.g., an amino acid or polymer such as a peptide, that provides the functional groups for linking to the tags, e.g., via a linkingmoiety comprising a polymer as described herein, and provides the functional group(s) for linking to the payload moiety / moieties, e.g., via a linking moiety which may comprise a polymer as described herein. Thus, a tag conjugate described herein may comprise several arms which are connected by a branching moiety. In some embodiments, a tag conjugate described herein comprises two arms ending in a tag, and one or more arms ending in a payload moiety.

[0468] In some embodiments, the term "branching moiety" refers to a chemical moiety in an arm ending in a payload moiety which provides the functional groups for linking, e.g., via a linking moiety, more than one payload moiety and / or further functional groups to the arm.

[0469] In some embodiments, at least one of the linking moieties connecting a tag to the branching moiety comprises a polymer as described above. In some embodiments, each of the linking moieties connecting a tag to the branching moiety comprises a polymer as described above. In some embodiments, at least one of the linking moieties connecting a payload moiety to the branching moiety comprises a polymer as described above. In some embodiments, each of the linking moieties connecting a payload moiety to the branching moiety comprises a polymer as described above.

[0470] In some embodiments, the tag conjugate comprises payload moieties and tags in a branched (non-linear) configuration.

[0471] In some embodiments, the tag conjugate comprises at least two tags and at least one payload moiety in a branched (non-linear) configuration.

[0472] In some embodiments, the tag conjugate comprises at least two tags and at least two payload moieties in a branched (non-linear) configuration.

[0473] In some embodiments, the tag conjugate comprises two tags and one or two payload moieties in a branched (non-linear) configuration.

[0474] In some embodiments, the tag conjugate comprises one or more linking moieties connecting tags and payload moieties in a branched (non-linear) configuration.

[0475] In some embodiments, at least one linking moiety is a branched linking moiety.

[0476] In some embodiments, at least one linking moiety comprises a continuous or non-continuous poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.In some embodiments, the tag conjugate comprises two tags which are connected by a linking moiety

[0477] In some embodiments, the linking moiety connecting the tags comprises a continuous or non-continuous poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0478] In some embodiments, a payload moiety is connected to the linking moiety connecting the tags.

[0479] In some embodiments, a payload moiety is connected to the linking moiety connecting the tags by a linking moiety.

[0480] In some embodiments, the linking moiety connecting a payload moiety and a linking moiety connecting the tags comprises a continuous or non-continuous poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0481] In some embodiments, the tag conjugate comprises a main chain connecting two tags and one or more side chains branching from the main chain and connecting one or more payload moieties to the main chain.

[0482] In some embodiments, one or more payload moieties are connected to the main chain through a moiety comprising a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0483] In some embodiments, a side chain is connected to the main chain through a branching moiety in the main chain.

[0484] In some embodiments, the main chain comprises a continuous or non-continuous poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0485] In some embodiments, the main chain comprises two or more poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moieties or derivatives thereof.

[0486] In some embodiments, poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moieties or derivatives thereof of the main chain are connected through a moiety comprising a branching moiety.

[0487] In some embodiments, the tag conjugate comprises the formula:

[0488] [[P]m-Ll]n-Bi-[L2-T]o

[0489] whereinP comprises a payload moiety;

[0490] Bi comprises a branching moiety;

[0491] T comprises a tag;

[0492] Li comprises a linking moiety;

[0493] l_2 comprises a linking moiety;

[0494] m is an integer from 1 to 4;

[0495] n is an integer from 1 to 4; and

[0496] o is an integer from 2 to 4; wherein

[0497] the different groups [L2-T] may be identical or different, the different groups P may be identical or different, and the different groups [[ P]m-Li] may be identical or different.

[0498] In some embodiments, the valency of Bi corresponds to or is greater than the sum of n and o. In some embodiments, Bi comprises an amino acid or bis-amino acid.

[0499] In some embodiments, Bi comprises the D-isomer of an amino acid.

[0500] In some embodiments, Bi comprises cysteine or lysine.

[0501] In some embodiments, Li comprises a 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, or a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0502] In some embodiments, Li comprises an enzymatic cleavage site.

[0503] In some embodiments, L2 comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0504] In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 30, e.g., between 2 and 10, such as 2, 4 or 6. In some embodiments, the poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof. In some embodiments, the poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof comprises the following general formula:

[0505]

[0506] wherein n is between 2 and 30, e.g., between 2 and 10, such as 2, 4 or 6.

[0507] In some embodiments, L2 comprises one or more selected from the group consisting of a moiety constraining conformation, a moiety for albumin binding, a moiety which increases circulation time, a moiety which reduces renal retention or uptake and an enzymatic cleavage site.

[0508] In some embodiments, L2 comprises (i) a moiety comprising cysteine and (ii) a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof, wherein the poly-2- (2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or derivative thereof and T are linked by the moiety comprising cysteine.

[0509] In some embodiments, [[P]m-Li] comprises the formula [[P-Lr]m-B2-Li"],

[0510] wherein

[0511] B2 comprises a branching moiety;

[0512] Lr comprises a linking moiety;

[0513] Li" comprises a linking moiety; and

[0514] m is an integer from 1 to 4; wherein

[0515] the different groups [[P-Lr]m-B2-Li"] may be identical or different, and wherein in a group [[P- Lr]m-B2-Li"] the different groups [P-Lr] may be identical or different.

[0516] In some embodiments, the valency of B2 corresponds to or is greater than the value of m plus 1.

[0517] In some embodiments, B2 comprises an amino acid.

[0518] In some embodiments, B2 comprises the D-isomer of an amino acid.

[0519] In some embodiments, B2 comprises cysteine or lysine.

[0520] In some embodiments, Lr comprises a 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof, cysteine, or a combination thereof.

[0521] In some embodiments, Lr- comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.In some embodiments, L comprises a moiety which is substituted by a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0522] In some embodiments, the moiety which is substituted by a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is an amino acid. In some embodiments, the amino acid which is substituted by a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is lysine.

[0523] In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof in Lr is between 2 and 30, e.g., between 2 and 10, such as 2, 4 or 6.

[0524] In some embodiments, the poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof. In some embodiments, the poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof comprises the following general formula:

[0525]

[0526] wherein n is between 2 and 30, e.g., between 2 and 10, such as 2, 4 or 6.

[0527] In some embodiments, Lr and / or L is substituted with a solubilizing functional group.

[0528] In some embodiments, Lr and / or Lr comprises an amino acid which is substituted with a solubilizing functional group.

[0529] In some embodiments, the amino acid which is substituted with a solubilizing functional group is lysine or cysteine.

[0530] In some embodiments, m is an integer from 1 to 3, n is an integer from 1 to 3, and o is 2 or 3. In some embodiments, o is 2.

[0531] In some embodiments, n is 1 or 2.

[0532] In some embodiments, m is 1 or 2.

[0533] In some embodiments, o is 2, n is 1 and m is 1.In some embodiments, the payload moiety comprises a radioisotope, e.g., a chelating compound comprising a radioisotope. In some embodiments, a chelating compound in the tag conjugate is covalently linked with the tags and chelates a radioisotope, such as radionuclides and radiolabels. In some embodiments, the tag conjugate comprises 1, 2, 3, 4, 5, or 6 molecules of a radioisotope, e.g., a chelating compound comprising a radioisotope, which molecules of a radioisotope and / or chelating compound are preferably covalently attached to the tag conjugate. In some embodiments, the tag conjugate comprises 1 molecule of a radioisotope, e.g., a chelating compound comprising a radioisotope.

[0534] In some embodiments, o is 2, n is 2 and m is 2.

[0535] In some embodiments, the payload moiety comprises a toxin or immunomodulator. In some embodiments, the tag conjugate comprises 1, 2, 3, 4, 5, or 6 molecules of a toxin or immunomodulator, which molecules of a toxin or immunomodulator are preferably covalently attached to the tag conjugate. In some embodiments, the tag conjugate comprises 4 molecules of a toxin or immunomodulator.

[0536] In some embodiments, the tag conjugate comprises the formula:

[0537] P-LI-BI-[L2-T]2

[0538] wherein

[0539] P comprises a payload moiety;

[0540] Bi comprises a branching moiety;

[0541] T comprises a tag;

[0542] Li comprises a linking moiety; and

[0543] l_2 comprises a linking moiety; wherein

[0544] the different groups [ L2-T] may be identical or different.

[0545] In some embodiments, the tag conjugate comprises the formula:

[0546] P-[AEEA]p-Bi-[[AEEA]q-R-[AEEA]r-C-T]2

[0547] wherein

[0548] P comprises a payload moiety;

[0549] Bi comprises a branching moiety;

[0550] R is optional and comprises a moiety constraining conformation;

[0551] C is optional and comprises a connecting moiety;T comprises a tag;

[0552] p is an integer from 0 to 6;

[0553] q is an integer from 1 to 4; and

[0554] r is an integer from 1 to 4; wherein

[0555] the different groups [[AEEA]q-R-[AEEA]r-C-T] may be identical or different.

[0556] In some embodiments, the tag conjugate comprises the formula:

[0557] [[P-L1']2-B2-L1"]2-B1-[I_2-T]2

[0558] wherein

[0559] P comprises a payload moiety;

[0560] Bi comprises a branching moiety;

[0561] B2 comprises a branching moiety;

[0562] T comprises a tag;

[0563] Lr comprises a linking moiety;

[0564] Li" comprises a linking moiety; and

[0565] L2 comprises a linking moiety; wherein

[0566] the different groups [[P-L1 2-B2-L1"] may be identical or different, wherein in a group [[P-Lrh-B2-L1"] the different groups [P-Lr] may be identical or different, and the different groups [L2-T] may be identical or different.

[0567] In some embodiments, the tag conjugate comprises the formula:

[0568] [[P-Lr]2-B2-[AEEA]s]2-Bi-[[AEEA]t-T]2

[0569] wherein

[0570] P comprises a payload moiety;

[0571] Bi comprises a branching moiety;

[0572] B2 comprises a branching moiety;

[0573] T comprises a tag;

[0574] Lr comprises a linking moiety;

[0575] s is an integer from 2 to 8; and

[0576] t is an integer from 2 to 8; whereinthe different groups [ [P-Lr]2-B2-[AEE A]s] may be identical or different, wherein in a group [[P-Li']2-B2-[AEEA]S] the different groups [P-Lr] may be identical or different, and the different groups [[AEEA]t-T] may be identical or different.

[0577] In some embodiments, [AEEA] is 2-(2-(2-aminoethoxy)ethoxy)acetic acid or a derivative thereof.

[0578] In some embodiments, the tag conjugate comprises the formula:

[0579] P-[AEEA]-BI-[[AEEA]2-C-T]2

[0580] wherein

[0581] P comprises a payload moiety;

[0582] Bi comprises a branching moiety;

[0583] C is optional and comprises a connecting moiety; and

[0584] T comprises an ALFA-tag; wherein

[0585] the different groups [[AEEA]2-C-T] may be identical or different.

[0586] In some embodiments, P comprises a radioisotope, a chelating compound comprising a radioisotope, or a chelating compound for a radioisotope. In some embodiments, Bi comprises an amino acid. In some embodiments, C comprises an amino acid.

[0587] In some embodiments, the tag conjugate comprises the formula:

[0588] P-[AEEA]-BI-[[AEEA]2-C-T]2

[0589] wherein

[0590] P comprises a radioisotope, a chelating compound comprising a radioisotope, or a chelating compound fora radioisotope;

[0591] Bi comprises an amino acid, preferably lysine;

[0592] C comprises a cysteine moiety; and

[0593] T comprises an ALFA-tag; wherein

[0594] the different groups [[AEEA]2-C-T] may be identical or different.

[0595] In some embodiments, T comprises an ALFA-tag of the formula -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In some embodiments, T comprises an ALFA-tag of the formula -Pro-Ser-Arg-Leu-cyclo(Glu-Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-.In some embodiments, the tag conjugate comprises at least two chains, wherein each of said chains comprises a tag, and wherein the at least two chains are covalently connected. In some embodiments, the tag conjugate comprises two chains, wherein each of said chains comprises a tag, and wherein the two chains are covalently connected. In some embodiments, at least one of the chains comprises at least one payload moiety. In some embodiments, each of the chains comprises at least one payload moiety. In some embodiments, the tag conjugate comprises two chains, wherein each of said chains comprises a tag and a payload moiety, and wherein the two chains are covalently connected. In some embodiments, the tag conjugate comprises two chains, wherein each of said chains comprises a tag and two payload moieties, and wherein the two chains are covalently connected.

[0596] In some embodiments, the tag comprises an ALFA-tag.

[0597] In some embodiments, the tag conjugate comprises two chains, wherein each of said chains comprises an ALFA-tag and two payload moieties, and wherein the two chains are covalently connected.

[0598] In some embodiments, the covalent connection comprises a triazole. In some embodiments, the covalent connection comprises a 1,2,3-triazole. In some embodiments, the covalent connection is formed through an intermolecular cycloaddition ("click") reaction between azides and alkynes comprised in the chains to be connected.

[0599] In some embodiments, the tag conjugate comprises two chains, wherein each of said chains comprises an ALFA-tag and two payload moieties, and wherein the two chains are covalently connected through a moiety comprising a 1,2,3-triazole.

[0600] In some embodiments, each chain comprises a moiety of the formula:

[0601] *-[[AEEA]2-C-T]

[0602] wherein

[0603] C is optional and comprises a connecting moiety;

[0604] T comprises a tag; and

[0605] * is the attachment point to a moiety that forms a covalent connection to another of the chains.

[0606] In some embodiments, the tag conjugate comprises two chains wherein each chain comprises a moiety of the formula:*-[[AEEA]2-C-T]

[0607] wherein

[0608] C is optional and comprises a connecting moiety;

[0609] T comprises a tag; and

[0610] * is the attachment point to a moiety that forms a covalent connection to the other chain. In some embodiments, the moiety that forms a covalent connection to another of the chains or the moiety that forms a covalent connection to the other chain comprises one or more payload moieties. In some embodiments, the moiety that forms a covalent connection to another of the chains or the moiety that forms a covalent connection to the other chain comprises two payload moieties.

[0611] In some embodiments, the moiety that forms a covalent connection to another of the chains or the moiety that forms a covalent connection to the other chain comprises a 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, or a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof. In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 10 such as 2, 4 or 6, in particular 2.

[0612] In some embodiments, the moiety that forms a covalent connection to another of the chains or the moiety that forms a covalent connection to the other chain comprises a main chain extending from the attachment point * and comprising two amino acids, wherein the two amino acids are connected through a moiety comprising a 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, or a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof, and wherein the two amino acids are each connected to a payload moiety through their side chains. In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 10 such as 2, 4 or 6, in particular 2. In some embodiments, the amino acid comprises cysteine. In some embodiments, the tag conjugate comprises two chains wherein each chain comprises a moiety of the formula:

[0613] Cys-[AEEA]U-Cys-A-[AEEA]2-C-Twherein

[0614] A is optional and comprises a moiety attaching the moiety Cys-[AEEA]U-Cys to the moiety [AEEA]2-C-T;

[0615] C is optional and comprises a connecting moiety;

[0616] T comprises a tag;

[0617] Cys is cysteine connected to a payload moiety through its side chain; and

[0618] u is 1 or 2.

[0619] In some embodiments, C comprises an amino acid.

[0620] In some embodiments, A comprises a 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, or a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof. In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 10 such as 2, 4 or 6, in particular 2. In some embodiments, A forms a covalent connection to the other chain. In some embodiments, the covalent connection comprises a triazole. In some embodiments, the covalent connection comprises a 1,2,3-triazole. In some embodiments, the covalent connection is formed through an intermolecular cycloaddition ("click") reaction between azides and alkynes comprised in the chains to be connected.

[0621] In some embodiments, T comprises an ALFA-tag of the formula -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In some embodiments, T comprises an ALFA-tag of the formula -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-Arg-Leu-Thr-Glu-.

[0622] In some embodiments of all aspects and embodiments described herein, the tag is an ALFA-tag.

[0623] In some embodiments of all aspects and embodiments described herein, the tag is a cyclic ALFA-tag.

[0624] Interacting moieties on the tag conjugate and on the docking compound

[0625] In some embodiments, the moiety on the tag conjugate, i.e., the tags, and the moiety on the docking compound interacting with each other non-covalently bind to each other.In some embodiments, the moieties on the tag conjugate and on the docking compound interacting with each other bind to each other under physiological conditions.

[0626] In some embodiments, the moieties on the tag conjugate and on the docking compound interacting with each other are antigen / antibody systems.

[0627] In some embodiments, the moiety of the tag conjugate binding to the docking compound comprises a peptide or protein, e.g., a peptide tag, and the moiety of the docking compound binding to the tag conjugate comprises a binder, e.g., an antibody or antibody fragment, binding to the peptide or protein.

[0628] In some embodiments, the moieties on the tag conjugate and on the docking compound interacting which each other comprise an epitope tag / binder system.

[0629] As used herein, an "epitope tag" refers to a stretch of amino acids to which an antibody or proteinaceous molecule with antibody-like function can bind.

[0630] In some embodiments, the epitope tag comprises an ALFA-tag. In some embodiments, the epitope tag / binder system comprises an ALFA-tag and an ALFA-specific single-domain antibody (sdAb), NbALFA-nanobody.

[0631] The use of an ALFA-tag / ALFA-specific sdAb system is particularly advantageous in the context of the present disclosure. It will be possible to select an ALFA-tag with a suitable affinity to the sdAb for the respective application.

[0632] In some embodiments, a comparably lower affinity tag (a medium / low affinity tag), such as a medium / low affinity ALFA-tag, is used to increase the avidity effect at the target site, for example at a tumor cell. In such embodiments, a target site (e.g., target cells) with a high density of target antigen will lead to binding of docking compounds (with one tag-binding moiety) in close proximity to each other. Due to this proximity, the tag conjugates comprising two tags will preferably bind to the docking compounds at the target site, due to an avidity effect resulting from the proximity of two tag-binding moieties of two docking compounds at the target site or on the target cells. Binding to single docking compounds at sites of lower docking compound proximity (e.g. in circulation or at sides of target-unspecific docking compound deposition) is less likely.

[0633] One main advantage of this avidity effect is that off-target binding of free tag-conjugates comprising two tags can be minimized while a target-sepcific accumulation of the tag-conjugates occurs on target cells with a high density of bound docking compounds comprising one tag-binding moiety. This is particularly advantageous for radiodiagnostic applications, where off-target binding can disturb the imaging readount. However, this effect can also be advantageous to reduce off-target effects of tag-conjugtes with other payload classes, such as toxins, therapeutic radionuclides or immunomodulators.

[0634] In some embodiments, the affinity of WT ALFA (Ac-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-Arg-Leu-Thr-Glu-NH2) to NbALFA is used as a reference point. In some embodiments, a high affinity tag (e.g., high affinity Alfa-tag) is characterized by having a value of dissociation from a particular binding partner (e.g., NbAlfa), such as measured by kdis, that, relative to the corresponding value of the corresponding wild-type tag (e.g., Alfa-tag), is 5 or less, e.g., 4 or less, 3 or less, 2 or less, or 1 or less. In other words, in some embodiments, the ratio kdis (high affinity tag) / kdis (wild-type tag) is 5 or less, e.g., 4 or less, 3 or less, 2 or less, or 1 or less. In some embodiments, a medium / low affinity tag (e.g., medium / low affinity Alfa-tag) is characterized by having a value of dissociation from a particular binding partner (e.g., NbAlfa), such as measured by kdis, that, relative to the corresponding value of the corresponding wildtype tag (e.g., Alfa-tag), is more than 5, e.g., more than 6, more than 7, or more than 8. In other words, in some embodiments, the ratio kdis (medium / low affinity tag) / kdis (wild-type tag) is more than 5, e.g., more than 6, more than 7, or more than 8. In some embodiments, affinities are determined by biolayer interferometry (BLI).

[0635] In some embodiments it is also possible to select a tag / binder system with suitable binding properties, such as suitable kdis, that is different from the ALFA-tag / ALFA-specific sdAb system by using WT ALFA as a reference.

[0636] Examples of high affinity tags are

[0637] Ac-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-Arg-Leu-Thr-Glu-NH2,

[0638] Ac-Ser-Arg-Leu-Glu-(cyclo5)Glu-Glu-Leu-(cyclo8)Lys-Arg-Arg-Leu-Thr-Glu-NH2, Ac-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-(cyclol3)Glu-NH2, Ac-Ser-Arg-Leu-Glu-(cyclo5)Glu-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu-NH2, Ac-Pro-Ser-Arg-Leu-Glu-(cyclo6)Glu-Glu-Leu-Arg-(cyclolO)Lys-Arg-Leu-Thr-Glu-NH2. Examples of medium / low affinity tags are

[0639] Ac-Ser-Arg-Leu-Glu-(cyclo5)Asp-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu-NH2,Ac-Pro-Ser-Arg-Leu-Glu-(cyclo6)Lys-Glu-Leu-Arg-(cyclolO)Glu-Arg-Leu-Thr-Glu-NH2, Ac-Pro-Ser-Arg-Leu-(cyclo5)Glu-Glu-Glu-Leu-(cyclo9)Lys-Arg-Arg-Leu-Thr-Glu-NH2. Based on the provided examples of high and medium / low affinity tags and the provided method of determining the suitability of an epitope tag / binder system for the respective application a skilled person can select a suitable system.

[0640] In some embodiments, an ALFA-tag comprises the amino acid sequence

[0641] -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13-AA14-, wherein the amino acids of AAO, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13 and AA14 are:

[0642] AAO is Pro or deleted;

[0643] AA1 is Ser, Gly, Thr, or Pro;

[0644] AA2 is Arg, Gly, Ala, Glu, or Pro;

[0645] AA3 is Leu, lie, or Vai;

[0646] AA4 is Glu or Gin;

[0647] AA5 is Glu or Gin;

[0648] AA6 is Glu or Gin;

[0649] AA7 is Leu, lie, or Vai;

[0650] AA8 is Arg, Ala, Gin, or Glu;

[0651] AA9 is Arg, Ala, Gin, or Glu;

[0652] AA10 is Arg;

[0653] AA11 is Leu;

[0654] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;

[0655] AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and

[0656] AA14 is Pro or deleted.

[0657] In some embodiments, an ALFA-tag comprises a sequence selected from the group consisting of SRLEEELRRRLTE, PSRLEEELRRRLTE, SRLEEELRRRLTEP, and PSRLEEELRRRLTEP.

[0658] In some embodiments, an ALFA-tag comprises the cyclized amino acid sequence

[0659] -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13-AA14-, wherein the side-chains of any two of the amino acids of AAO, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13 and AA14 (XI, X2) are connected covalently; andwherein the amino acids of AAO, AA1, AA2, AA3, AA4, AA5, AAO, AA7, AA8, AA9, AA10, AA11, AA12, AA13 and AA14 which are not XI and X2 are:

[0660] AAO is Pro or deleted;

[0661] AA1 is Ser, Gly, Thr, or Pro;

[0662] AA2 is Arg, Gly, Ala, Glu, or Pro;

[0663] AA3 is Leu, lie, or Vai;

[0664] AA4 is Glu or Gin;

[0665] AA5 is Glu or Gin;

[0666] AAO is Glu or Gin;

[0667] AA7 is Leu, lie, or Vai;

[0668] AA8 is Arg, Ala, Gin, or Glu;

[0669] AA9 is Arg, Ala, Gin, or Glu;

[0670] AA10 is Arg;

[0671] AA11 is Leu;

[0672] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;

[0673] AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and

[0674] AA14 is Pro or deleted.

[0675] In some embodiments, XI and X2 are separated by 2 or 3 amino acids.

[0676] In some embodiments, AA5 is XI and AA9 is X2, AA5 is XI and AA8 is X2, AA9 is XI and AA13 is X2, AAO is XI and AA9 is X2, AA9 is XI and AA12 is X2, AA10 is XI and AA13 is X2, AAO is XI and AA10 is X2 or AA4 is XI and AA8 is X2.

[0677] In some embodiments, an ALFA-tag comprises a cyclized amino acid sequence selected from the group consisting of

[0678] a. -AA0-AAl-AA2-AA3-AA4-cyclo(Xl-AA6-AA7-AA8-X2)-Arg-Leu-AA12-AA13-AA14-, b. -AA0-AAl-AA2-AA3-AA4-cyclo(Xl-AA6-AA7-X2)-AA9-Arg-Leu-AA12-AA13-AA14-, c. -AA0-AAl-AA2-AA3-AA4-AA5-AA6-AA7-AA8-cyclo(Xl-Arg-Leu-AA12-X2)-AA14-, d. -AA0-AAl-AA2-AA3-AA4-AA5-cyclo(Xl-AA7-AA8-X2)-Arg-Leu-AA12-AA13-AA14-, e. -AA0-AAl-AA2-AA3-AA4-AA5-AA6-AA7-AA8-cyclo(Xl-Arg-Leu-X2)-AA13-AA14-, f. -AA0-AAl-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-cyclo(Xl-Leu-AA12-X2)-AA14-, g. -AA0-AAl-AA2-AA3-AA4-AA5-cyclo(Xl-AA7-AA8-AA9-X2)-Leu-AA12-AA13-AA14-, andh. -AA0-AAl-AA2-AA3-cyclo(Xl-AA5-AA6-AA7-X2)-AA9-Arg-Leu-AA12-AA13-AA14-, wherein the side-chains of Xi and X2 amino acid residues are connected covalently;

[0679] AAO is Pro or deleted;

[0680] AA1 is Ser, Gly, Thr, or Pro;

[0681] AA2 is Arg, Gly, Ala, Glu, or Pro;

[0682] AA3 is Leu, lie, or Vai;

[0683] AA4 is Glu or Gin;

[0684] AA5 is Glu or Gin;

[0685] AA6 is Glu or Gin;

[0686] AA7 is Leu, lie, or Vai;

[0687] AA8 is Arg, Ala, Gin, or Glu;

[0688] AA9 is Arg, Ala, Gin, or Glu;

[0689] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;

[0690] AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and

[0691] AA14 is Pro or deleted.

[0692] In some embodiments, Xi and X2 in the peptides disclosed herein are connected covalently via an amide, disulfide, thioether, ether, ester, thioester, thioamide, alkylene, alkenylene, alkynylene, and / or 1,2,3-triazole.

[0693] In some embodiments, a cyclized amino acid sequence described herein is generated by linking an amino group of a side-chain of one of Xi and X2 to the carboxyl group of a side-chain of the other of Xi and X2 via an amide bond. The amino group of the side chain of an amino acid that possesses a pendant amine group, e.g., lysine or a lysine derivative, and the carboxyl group of the side chain of an acidic amino acid, e.g., aspartic acid, glutamic acid or a derivative thereof, can be used to generate a cyclized amino acid sequence via an amide bond.

[0694] In some embodiments, a cyclized amino acid sequence described herein is generated by linking a sulfhydryl group of a side-chain of one of Xi and X2 to the sulfhydryl group of a sidechain of the other of Xi and X2 via a disulfide bond. Sulfhydryl group-containing amino acids include cysteine and other sulfhydryl-containing amino acids as Pen.In some embodiments, Xi and X2 are, independently, selected from the group consisting of Glu, DGIu, Asp, DAsp, Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, DDap, Cys, DCys, hCys, DhCys, Pen, and DPen, with the proviso that when Xi is Glu, DGIu, Asp, or DAsp, X2 is Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, or DDap; when XI is Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, or DDap, X2 is Glu, DGIu, Asp, or DAsp; and when XI is Cys, DCys, hCys, DhCys, Pen, or DPen, X2 is Cys, DCys, hCys, DhCys, Pen, or DPen.

[0695] In some embodiments, Xi is Glu and X2 is Lys. In some embodiments, -cyclo(Glu - Lys)-, -c(Glu - Lys)-, -cyclo(E - K)-, -c(E - K)-, -E - K- cyclo, or -cycloE- — cycloK- comprises the following structure:

[0696]

[0697] In some embodiments, Xi is Lys and X2 is Glu. In some embodiments, -cyclo(Lys - Glu)-, -c(Lys - Glu)-, -cyclo(K - E)-, -c(K - E)-, -K - E- cyclo, or cycloK - cycloE- comprises the following structure:

[0698]

[0699] In some embodiments, Xi is Cys and X2 is Cys. In some embodiments, -cyclo(Cys - Cys)-, c(Cys - Cys)-, -cyclo(C - C)-, -c(C - C)-, -C- — C- cyclo, or -cycloC - cycloC- comprises the following structure:

[0700]

[0701] Particular cyclized amino acid sequences of the above-identified generic formulas include, for example,-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Glu)-, -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Asp)-, -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-cyclo(Glu-Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Thr-Cys)-, -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Thr-Cys)-, -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-, -Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-,-Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Glu)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DGIu)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DGIu)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu- -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Glu)-Arg-Arg-Leu-Thr-Glu-,-Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DGIu)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DGIu)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Cys)-Glu-, -Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-,-Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Cys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-,-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-Cys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-Cys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-DCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-hCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-hCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-Cys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-DCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-hCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DhCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-Pen)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-DPen)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-Pen)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-DPen)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-Cys)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-DCys)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-DCys)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-Cys)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DCys)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-hCys)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-hCys)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DhCys)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DhCys-Arg-Leu-hCys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-Cys)-,-Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-Cys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-DCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-hCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-hCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-Cys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-DCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-hCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DhCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-Pen)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-DPen)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-Pen)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-DPen)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-Cys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-DCys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-DCys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-Cys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DCys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-hCys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-hCys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DhCys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DhCys-Arg-Leu-hCys)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Asp)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Asp-Arg-Leu-Lys)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Glu)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Glu-Arg-Leu-Lys)-Glu-,-Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Asp)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Asp-Arg-Leu-Lys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Glu)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Glu-Arg-Leu-Lys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Thr-Cys)-, -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-,-Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Glu)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DGIu)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DGIu)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Glu)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DGIu)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DGIu)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-,-Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Cys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Cys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-,-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DCys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-Cys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DCys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-Cys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-DCys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-hCys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-hCys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-Cys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-DCys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-hCys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DhCys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-Pro-,-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-Pen)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-DPen)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-Pen)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-DPen)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-Cys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-DCys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-DCys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-Cys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DCys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-hCys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-hCys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DhCys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DhCys-Arg-Leu-hCys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-Cys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-Cys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-DCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-hCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-hCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-Cys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-DCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-hCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DhCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-Pen)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-DPen)-Pro-,-Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-Pen)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-DPen)-Pro-,

[0702] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-Cys)-Glu-Pro-,

[0703] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-DCys)-Glu-Pro-,

[0704] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-DCys)-Glu-Pro-,

[0705] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-Cys)-Glu-Pro-,

[0706] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DCys)-Glu-Pro-,

[0707] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-hCys)-Glu-Pro-,

[0708] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-hCys)-Glu-Pro-,

[0709] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DhCys)-Glu-Pro-,

[0710] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DhCys-Arg-Leu-hCys)-Glu-Pro-,

[0711] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Asp)-Glu-Pro-,

[0712] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Asp-Arg-Leu-Lys)-Glu-Pro-,

[0713] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Glu)-Glu-Pro-,

[0714] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Glu-Arg-Leu-Lys)-Glu-Pro-,

[0715] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Asp)-Glu-Pro-,

[0716] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Asp-Arg-Leu-Lys)-Glu-Pro-,

[0717] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Glu)-Glu-Pro-,

[0718] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Glu-Arg-Leu-Lys)-Glu-Pro-,

[0719] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Thr-Cys)-Pro-,

[0720] -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-Pro-,

[0721] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-Pro-,

[0722] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu-Pro-,

[0723] -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-, and

[0724] -Ser-Arg-Leu-cyclo(Glu-Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-.

[0725] In some embodiments, the cyclic peptide is attached to a 3-mercaptopropionyl moiety through an a-amine moiety of the leftmost amino acid in the cyclic peptide. In some embodiments, the rightmost amino acid in the cyclic peptide comprises an amide.

[0726] In some embodiments, the cyclized amino acid sequence is one selected from the group consisting of-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-,

[0727] -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-,

[0728] -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-,

[0729] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Glu)-,

[0730] -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-,

[0731] -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-,

[0732] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Asp)-,

[0733] -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu- -Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-,

[0734] -Pro-Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu--Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu--Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu- -Pro-Ser-Arg-Leu-cyclo(Glu-Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-,

[0735] -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-,

[0736] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Thr-Cys)-,

[0737] -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-,

[0738] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-,

[0739] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu-,

[0740] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Thr-Cys)-,

[0741] -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-,

[0742] -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-,

[0743] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-,

[0744] -Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu-,

[0745] -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-, and

[0746] -Ser-Arg-Leu-cyclo(Glu-Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-.

[0747] In some embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In some other embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In yet some other embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-. In still some other embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Glu)-.

[0748] The cyclic peptides may have different cyclic bridging moieties forming the ring structure. Preferably, chemically stable bridging moieties are included in the ring structure such as, for example, an amide group, a lactone group, an ether group, a thioether group, a disulfide group, an alkylene group, an alkenyl group, or a 1,2,3-triazole. The following are examples illustrating the variability of bridging moieties in a peptide:

[0749]

[0750] In some embodiments, an ALFA-tag binding moiety comprises an antibody or antibody fragment, e.g., a camelid VHH domain. In some embodiments, an ALFA-tag binding moiety comprises a single-domain antibody (sdAb), NbALFA-nanobody.

[0751] In some embodiments, an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence VTXiSALNAMAMG, wherein Xi is I or V,the CDR2 sequence AVSX2RGNAM, wherein X2 is E, H, N, D, or S, and the CDR3 sequence LEDRVDSFHDY.

[0752] In some embodiments, an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence GVTXiSALNAMAMG, wherein Xi is I or V, the CDR2 sequence AVSX2RGNAM, wherein X2 is E, H, N, D, or S, and the CDR3 sequence LEDRVDSFHDY.

[0753] In some embodiments, an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence VTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.

[0754] In some embodiments, an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence G VTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.

[0755] In some embodiments, an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the amino acid sequence EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSERGNAMYRESV QGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence, or a fragment of said amino acid sequence or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence. In some embodiments, the amino acid sequence comprises CDR1, CDR2 and CDR3 sequences as described above.

[0756] In some embodiments, the epitope tag / binder system comprises an epitope tag comprising the sequence PDRVRAVSHWSS (Spot-tag) and the binder comprises a single-domain antibody (sdAb, or nanobody) (Spot-nanobody (14.7 kD)) that specifically binds to the Spot-tag.

[0757] In some embodiments, following binding of the moieties on the tag conjugate and on the docking compound interacting which each other, a covalent connection is formed. In these embodiments, the system used herein may comprise a Tag / Catcher system forming a covalent bond, e.g., SpyTag / SpyCatcher forming an isopeptide bond.

[0758] The SpyTag / SpyCatcher system is a technology for irreversible conjugation of recombinant proteins. The peptide SpyTag spontaneously reacts with the protein SpyCatcher to form anintermolecular isopeptide bond between the pair. Using the Tag / Catcher pair, bioconjugation can be achieved between two recombinant proteins.

[0759] In some embodiments, the interacting moieties on the tag conjugate and on the docking compound comprise Digoxigenin and an antibody, antibody fragment or derivative, e.g., scFv, or any protein binding to Digoxigenin.

[0760] In some embodiments, the interacting moieties on the tag conjugate and on the docking compound comprise caffeine and an antibody, antibody fragment or derivative, e.g., nanobody, binding to caffeine.

[0761] In some embodiments, the interacting moieties on the tag conjugate and on the docking compound comprise GFP and an antibody, antibody fragment or derivative, e.g., nanobody, binding to GFP.

[0762] In some embodiments, the interacting moieties on the tag conjugate and on the docking compound comprise biotin and an antibody, antibody fragment or derivative binding to biotin. The present disclosure provides in one aspect, a complex wherein a tag conjugate is bound to a docking compound. Thus, the tag conjugate and the docking compound comprise moieties interacting which each other.

[0763] Accordingly, the present disclosure provides in one aspect, a complex comprising:

[0764] (i) a compound comprising at least two binding moieties binding to a target antigen and a binding moiety for a tag (docking compound), and

[0765] (ii) a compound comprising a payload moiety and at least two tags to which the binding moiety for a tag binds (tag conjugate).

[0766] Different embodiments of the tag conjugate and the docking compound which are complexed are described herein.

[0767] In some embodiments, the tag conjugate comprises at least two ALFA-tags. In some embodiments, the at least two ALFA-tags may be identical or different. In some embodiments, the tag conjugate comprises at least two identical ALFA-tags. In these embodiments, the moiety binding to a tag conjugate of the docking compound may be a NbALFA-nanobody (NbALFA).Payload

[0768] In some embodiments, a payload comprises a therapeutic or diagnostic moiety.

[0769] The payload can, e.g., be a detectable label. A "detectable label" as used herein relates to a compound which allows its detection, e.g., when present in a cell, tissue or organism. One type of detectable label envisaged within the context of the present disclosure is a contrast providing agent. Different types of detectable labels are envisaged within the context of the present disclosure and are described herein below.

[0770] Thus, according to some embodiments of the present disclosure, the agents and methods of the present disclosure are used in imaging, especially medical imaging. In order to identify the primary target, use may be made, as the payload, of an imaging probe comprising one or more detectable labels. Particular examples of detectable labels of the imaging probe are contrastproviding moieties used in traditional imaging systems such as MRI-imageable constructs, spin labels, optical labels, ultrasound- responsive constructs, X-ray-responsive moieties, radionuclides, (bio)luminescent and FRET-type dyes. Exemplary detectable labels envisaged within the context of the present disclosure include, but are not limited to, fluorescent molecules, e.g. autofluorescent molecules, molecules that fluoresce upon contact with a reagent, etc., radioactive labels; biotin, e.g., to be detected through binding of biotin by avidin; fluorescent tags, imaging constructs for MRI comprising paramagnetic metal, imaging reagents and the like. The radionuclide used for imaging can be, for example, an isotope selected from the group consisting of3H,nC,13N,150,18F,19F,51Cr,52Fe,52Mn,55Co,60Cu,61Cu,62Zn,62Cu,63Zn,64Cu,66Ga,67Ga,68Ga,70As,71As,72As,74As,75Se,75Br,76Br,77Br,80Br,82Br,82Rb,86Y,88Y,89Sr,89Zr,97Ru,99Tc,110ln,inln,113ln,114ln,117Sn,120l,122Xe,123l,124l,125l,166Ho,167Tm,169Yb,193Pt,195Pt,201TI, and203Pb. Other elements and isotopes, such as being used for therapy may also be applied for imaging in certain applications.

[0771] The MRI-imageable moiety can be a paramagnetic ion or a superparamagnetic particle. The paramagnetic ion can be an element selected from the group consisting of Gd, Fe, Mn, Cr, Co, Ni, Cu, Pr, Nd, Yb, Tb, Dy, Ho, Er, Sm, Eu, Ti, Pa, La, Sc, V, Mo, Ru, Ce, Dy, Tl.

[0772] The X-ray-responsive moieties include but are not limited to iodine, barium, and barium sulfate.Moreover, detectable labels envisaged within the context of the present disclosure also include peptides or polypeptides that can be detected by antibody binding, e.g., by binding of a detectable labeled antibody. In some embodiments the detectable labels are small size organic PET and SPECT labels, such as18F,nC or123L

[0773] Many of the radiolabels described herein are preferably provided as a chelate. Chelating groups are well known to those of skill in the art. In certain embodiments, chelating groups are derived from ethylene diamine tetra-acetic acid (EDTA), diethylene triamine penta-acetic acid (DTPA), cyclohexyl 1,2-diamine tetra-acetic acid (CDTA), ethyleneglycol-O,O'-bis(2-aminoethyl)-N,N,N',N'-tetra-acetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), triethylene tetramine hexa-acetic acid (TTHA), 1,4,7,10-tetraazacyclododecane-N,N'-,N",N"' -tetra-acetic acid (DOTA), hydroxyethyldiamine triacetic acid (HEDTA), l,4,8,ll-tetra-azacyclotetradecane-N,N',N",N"'-tetra-acetic acid (TETA), substituted DTPA, substituted EDTA, and the like. In some embodiments, a chelating group is selected from the group consisting of DOTA, DOTAM, NOTA, NOTP, PCTA, DATA, TRAP, DFO, THP, HBED and DEDPA.

[0774] The chelator 1,4, 7, 10-tetraazacyclododecane-N,N,N",N"' -tetraacetic acid (DOTA), is of particular interest because of its ability to chelate a number of diagnostically and therapeutically important metals, such as radionuclides and radiolabels.

[0775] Payloads comprising a detectable radiolabel may be referred to as radiodiagnostic payloads. A particularly preferred payload for use herein comprises DOTA chelator for68Ga complexation.

[0776] In some embodiments, a payload comprises DOTA-68Ga.

[0777] The payload can also be a therapeutic agent such as a pharmaceutically active agent. Examples of pharmaceutically active agents are known to the skilled person and provided herein. A therapeutic agent can optionally also comprise a detectable label. In some embodiments, a detectable label as described herein, e.g., a radioactive label, is used for therapeutic purposes. Thus, according to some embodiments, the agents and methods described herein are used for targeted therapy. This is achieved by making use of a payload comprising one or more pharmaceutically active agents (e.g., a drug or a radioactive isotope for radiation therapy).The term "pharmaceutically active agent" relates to any agent such as compound or cell being therapeutically effective when administered to an individual. The term "pharmaceutically active agent" further relates to any agent that changes, preferably cures, alleviates or partially arrests the clinical manifestations of a given disease and its complications in a therapeutic intervention comprising the administration of said agent.

[0778] In some embodiments, a pharmaceutically active agent comprises pharmaceutically active RNA or a pharmaceutically active peptide or protein.

[0779] A "pharmaceutically active RNA" is RNA that encodes a pharmaceutically active peptide or protein or is pharmaceutically active in its own, e.g., it has one or more pharmaceutical activities such as those described for pharmaceutically active proteins. For example, the RNA may be one or more strands of RNA interference (RNAi). Such agents include short interfering RNAs (siRNAs), or short hairpin RNAs (shRNAs), or precursor of a siRNA or microRNA-like RNA, targeted to a target transcript, e.g., a transcript of an endogenous disease-related transcript of a subject.

[0780] A "pharmaceutically active peptide or protein" has a positive or advantageous effect on the condition or disease state of a subject when administered to the subject in a therapeutically effective amount. Preferably, a pharmaceutically active peptide or protein has curative or palliative properties and may be administered to ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease or disorder. A pharmaceutically active peptide or protein may have prophylactic properties and may be used to delay the onset of a disease or to lessen the severity of such disease or pathological condition. The term "pharmaceutically active peptide or protein" includes entire proteins or polypeptides, and can also refer to pharmaceutically active fragments thereof. It can also include pharmaceutically active analogs of a peptide or protein. The term "pharmaceutically active peptide or protein" includes peptides and proteins that are antigens, i.e., administration of the peptide or protein to a subject elicits an immune response in a subject which may be therapeutic or partially or fully protective.

[0781] Examples of pharmaceutically active proteins include, but are not limited to, cytokines and immune system proteins such as immunologically active compounds (e.g., interleukins, colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, addressins, seletins, homing receptors, T cell receptors, immunoglobulins, soluble major histocompatibility complex antigens, immunologically active antigens such as bacterial, parasitic, or viral antigens, allergens, autoantigens, antibodies), hormones (insulin, thyroid hormone, catecholamines, gonadotrophines, trophic hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptins and the like), growth hormones (e.g., human grown hormone), growth factors (e.g., epidermal growth factor, nerve growth factor, insulin-like growth factor and the like), growth factor receptors, enzymes (tissue plasminogen activator, streptokinase, cholesterol biosynthestic or degradative, steriodogenic enzymes, kinases, phosphodiesterases, methylases, de-methylases, dehydrogenases, cellulases, proteases, lipases, phospholipases, aromatases, cytochromes, adenylate or guanylaste cyclases, neuramidases and the like), receptors (steroid hormone receptors, peptide receptors), binding proteins (growth hormone or growth factor binding proteins and the like), transcription and translation factors, tumor growth suppressing proteins (e.g., proteins which inhibit angiogenesis), structural proteins (such as collagen, fibroin, fibrinogen, elastin, tubulin, actin, and myosin), blood proteins (thrombin, serum albumin, Factor VII, Factor VIII, insulin, Factor IX, Factor X, tissue plasminogen activator, protein C, von Wilebrand factor, antithrombin III, glucocerebrosidase, erythropoietin granulocyte colony stimulating factor (GCSF) or modified Factor VIII, anticoagulants and the like.

[0782] In some embodiments, the pharmaceutically active protein is a cytokine which is involved in regulating lymphoid homeostasis, preferably a cytokine which is involved in and preferably induces or enhances development, priming, expansion, differentiation and / or survival of T cells. In some embodiments, the cytokine is an interleukin. In some embodiments, the pharmaceutically active protein according to the disclosure is an interleukin selected from the group consisting of IL-2, IL-7, IL-12, IL-15, and IL-21.

[0783] In some embodiments, a payload comprises a compound useful in radiation therapy and / or chemotherapy. In some embodiments, a payload comprises a chemotherapeutic compound. Chemotherapy is a type of cancer treatment that uses one or more anti-cancer drugs (chemotherapeutic agents), usually as part of a standardized chemotherapy regimen. The term chemotherapy has come to connote non-specific usage of intracellular poisons to inhibitmitosis. The connotation excludes more selective agents that block extracellular signals (signal transduction). The development of therapies with specific molecular or genetic targets, which inhibit growth-promoting signals from classic endocrine hormones (primarily estrogens for breast cancer and androgens for prostate cancer) are now called hormonal therapies. By contrast, other inhibitions of growth-signals like those associated with receptor tyrosine kinases are referred to as targeted therapy.

[0784] Traditional chemotherapeutic agents are cytotoxic by means of interfering with cell division (mitosis) but cancer cells vary widely in their susceptibility to these agents. To a large extent, chemotherapy can be thought of as a way to damage or stress cells, which may then lead to cell death if apoptosis is initiated.

[0785] Chemotherapeutic agents include alkylating agents, antimetabolites, anti-microtubule agents, topoisomerase inhibitors, and cytotoxic antibiotics.

[0786] Alkylating agents have the ability to alkylate many molecules, including proteins, RNA and DNA. The subtypes of alkylating agents are the nitrogen mustards, nitrosoureas, tetrazines, aziridines, cisplatins and derivatives, and non-classical alkylating agents. Nitrogen mustards include mechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide and busulfan. Nitrosoureas include N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU) and semustine (MeCCNU), fotemustine and streptozotocin. Tetrazines include dacarbazine, mitozolomide and temozolomide. Aziridines include thiotepa, mytomycin and diaziquone (AZQ). Cisplatin and derivatives include cisplatin, carboplatin and oxaliplatin. They impair cell function by forming covalent bonds with the amino, carboxyl, sulfhydryl, and phosphate groups in biologically important molecules. Non-classical alkylating agents include procarbazine and hexamethylmelamine. In one particularly preferred embodiment, the alkylating agent is cyclophosphamide.

[0787] Anti-metabolites are a group of molecules that impede DNA and RNA synthesis. Many of them have a similar structure to the building blocks of DNA and RNA. Anti-metabolites resemble either nucleobases or nucleosides, but have altered chemical groups. These drugs exert their effect by either blocking the enzymes required for DNA synthesis or becoming incorporated into DNA or RNA. Subtypes of the anti-metabolites are the anti-folates, fluoropyrimidines, deoxynucleoside analogues and thiopurines. The anti-folates include methotrexate andpemetrexed. The fluoropyrimidines include fluorouracil and capecitabine. The deoxynucleoside analogues include cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, and pentostatin. The thiopurines include thioguanine and mercaptopurine.

[0788] Anti-microtubule agents block cell division by preventing microtubule function. The vinca alkaloids prevent the formation of the microtubules, whereas the taxanes prevent the microtubule disassembly. Vinca alkaloids include vinorelbine, vindesine, and vinflunine. Taxanes include docetaxel (Taxotere) and paclitaxel (Taxol).

[0789] Topoisomerase inhibitors are drugs that affect the activity of two enzymes: topoisomerase I and topoisomerase II and include, but not limited to, irinotecan, topotecan, camptothecin, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, and aclarubicin. The cytotoxic antibiotics are a varied group of drugs that have various mechanisms of action. The common theme that they share in their chemotherapy indication is that they interrupt cell division. The most important subgroup is the anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin pirarubicin, and aclarubicin) and the bleomycins; other prominent examples include mitomycin C, mitoxantrone, and actinomycin.

[0790] In some embodiments, a payload which is useful herein comprises a radiodiagnostic, e.g., a chelator comprising a radiodiagnostic isotope.

[0791] In some embodiments, a payload which is useful herein comprises an immunomodulator, e.g., a Stimulator of Interferon Genes (STING) agonist or TLR, e.g., TLR7 / 8, agonist. In some embodiments, a payload comprises a STING agonist.

[0792] STING is expressed broadly in numerous tissue types, of both immune and non-immune origin, and is required for the type 1 interferon response in both immune and non-immune cells. STING has been shown to directly bind to a variety of different cyclic-di-nucleotides. The substantial pre-clinical anti-tumor activity of STING agonists has led to the development of multiple pharmacologic classes of agents at various stages of being translated into the clinic. In some embodiments, a payload which is useful herein comprises a toxin. In some embodiments, a payload comprises a cytotoxic or cytostatic agent. A cytotoxin or cytotoxic agent includes any agent that is detrimental to and, in particular, kills cells.Useful classes of cytotoxic agents include, for example, antitubulin agents, DNA minor groove binders (e.g., enediynes and lexitropsins), DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, nitrosoureas, platinols, pre-forming compounds, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes (e.g., paclitaxel and docetaxel), topoisomerase inhibitors, vinca alkaloids, or the like.

[0793] Individual cytotoxic agents include, for example, an androgen, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, buthionine sulfoximine, camptothecin, carboplatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin (formerly actinomycin), daunorubicin, decarbazine, docetaxel, doxorubicin, an estrogen, 5-fluordeoxyuridine, 5-fluorouracil, gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU), mechlorethamine, melphalan, 6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone, nitroimidazole, paclitaxel, plicamycin, procarbizine, streptozotocin, tenoposide, 6-thioguanine, thioTEPA, topotecan, vinblastine, vincristine, vinorelbine, VP-16 and VM-26.

[0794] Examples of anti-tubulin agents include, but are not limited to, dolastatins (e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB), maytansinoids, taxanes (e.g., paclitaxel, docetaxel), T67 (Tularik), vinca alkyloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine), baccatin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colcimid, estramustine, cryptophysins, cemadotin, combretastatins, discodermolide, and eleutherobin. In specific embodiments, the cytotoxic or cytostatic agent is auristatin E (also known in the art as dolastatin-10) or a derivative thereof. Typically, the auristatin E derivative is, e.g., an ester formed between auristatin E and a keto acid. For example, auristatin E can be reacted with paraacetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatin derivatives include AFP, MMAF, and MMAE.In certain embodiments, the cytotoxic or cytostatic agent is a maytansinoid, another group of anti-tubulin agents. For example, in specific embodiments, the maytansinoid is maytansine, DM-l or DM-4.

[0795] Maytansinoids are potent microtubule-targeted compounds that inhibit proliferation of cells at mitosis. Maytansinoids are derivatives of maytansine which is a 19-membered ansa macrolide structure attached to a chlorinated benzene ring. Maytansine has the following formula:

[0796]

[0797] Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Particularly preferred maytansinoids are the thiol-containing derivatives of maytansine, such as DM1 and DM4. Such thiol-containing derivatives of maytansine include compounds wherein the methyl group bound to the carbonyl group is replaced by a group containg a free sulfhydryl group such as the group -R-SH where R represents an alkylene group or other carbon-containing group of atoms.

[0798] DM1, also known as mertansine, is a maytansinoid having the following formula:

[0799]

[0800] In particular, the term "mertansine" or "DM1" refers to the compound / \ / 2'-deacetyl-A / 2'-(3-mercapto-l-oxopropyl)-maytansine.

[0801] "DM4" refers to the compound / V2-deacetyl- / \ / 2'-(4-methyl-4-mercapto-l-oxopentyl)-maytansine.

[0802] In some embodiments, a payload comprises a compound selected from the group consisting of dolastatins or dolostatin peptidic analogs and derivatives, the auristatins. Auristatins are synthetic analogs of dolostatin 10, a natural product derived from a marine mollusk, Dolabela auricularia. Like the maytansinoids, auristatins are microtubule disruptors.

[0803] Exemplary auristatin embodiments include monomethylauristatin drug moieties such as MMAE and MMAF.

[0804] MMAE, also known as Monomethyl auristatin E, has the following formula:

[0805]

[0806] In particular, the term "MMAE" refers to the compound (S)-N-((3R,4S,5S)-l-((S)-2-((lR,2R)-3-(((lS,2R)-l-hydroxy-l-phenylpropan-2-yl)amino)-l-methoxy-2-methyl-3-oxopropyl)pyrrolidin-l-yl)-3-methoxy-5-methyl-l-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamide. MMAE is actually desmethyl-auristatin E, i.e., the N-terminal amino group has only one methyl substituent instead of two as in auristatin E itself.

[0807] MMAF, also known as Monomethyl auristatin F, refers to the compound (S)-2-((2R,3R)-3-((S)-l-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid.

[0808] In some embodiments, the payload moiety comprises an exatecan moiety of the following formula:

[0809]

[0810] wherein ww represents the bond by which the exatecan moiety may be attached to a different molecule (such as a tag conjugate), optionally via a linking moiety, which linking moiety may comprise a cleavage site, such as an enzymatic cleavage site. Examples of such cleavage sites are described herein.

[0811] In some embodiments, the payload moiety comprises a deruxtecan moiety. In some embodiments, the payload moiety comprises deruxtecan.

[0812] Deruxtecan has the following formula:

[0813]

[0814] In some embodiments, a deruxtecan moiety comprises the following formula:

[0815]

[0816] wherein ww represents the bond by which the deruxtecan moiety may be attached to a different molecule (such as a tag conjugate), optionally via a linking moiety.

[0817] In some embodiments, the linking moiety comprises a functional group resulting from the reaction of one of the following coupling pairs: maleimide / thiol; azide / alkyne; thiol / bromoacetamide; carboxylate / amine. Optionally, the linking moiety may further comprise an alkylene moiety.

[0818] In some embodiments, the deruxtecan moiety comprises the following formula

[0819]

[0820] wherein R1is selected from the group consisting of *-alkylene-R2, wherein * represents the attachment point to the remainder of the deruxtecan moiety; alkylene is optionally substituted with 1, 2, or 3 R11, wherein each R11is independently selected from the group consisting of OH, halogen, Ci-6 alkyl, and =0; and R2is a divalent functional group resulting from the reaction of one of the coupling pairs.

[0821] In some embodiments, the alkylene moiety is a C1-12 alkylene moiety, such as a C2-6 alkylene moiety, wherein the alkylene moiety is optionally substituted with 1, 2, or 3 R11, wherein each R11is independently selected from the group consisting of OH, halogen, C1-6 alkyl, and =0. In some embodiments, R2comprises one of the following divalent groups:

[0822]

[0823] wherein each ww or - represents a bond.

[0824] In some embodiments, a payload to be delivered to a target cell is taken up by the target cell. In some embodiments, a payload is released from the tag conjugate at the target cell, e.g., by extracellular cleavage. In some embodiments, a tag conjugate is cleavable. In some embodiments, a tag conjugate is cleavable in an intracellular environment. In some embodiments, a tag conjugate is cleavable in an extracellular environment. In some embodiments, cleavage results in the release of the payload moiety from the tag conjugate. In some embodiments, a tag conjugate comprises an enzymatic cleavage site for cleaving the payload moiety from the tag conjugate, e.g., a cathepsin B linker, an MMP linker, a legumain linker, a glucosidase linker, or an ester bond.Embodiments of tag conjugate and docking compound

[0825] In some embodiments, the tags of a tag conjugate are ALFA-tags.

[0826] In some embodiments, the tag conjugate comprises two ALFA-tags. In some embodiments, the ALFA-tags are identical. In these embodiments, the binding moiety for the tag conjugate on the docking compound comprises an ALFA-specific single-domain antibody (sdAb), NbALFA-nanobody.

[0827] In some embodiments, the docking compound comprises one binding moiety for a tag, e.g., one NbALFA-nanobody and two binding moieties for a target antigen. In some embodiments, the tag conjugate comprises two, preferably identical tags, e.g., ALFA-tags, and the docking compound comprises one binding moiety for a tag, e.g., one NbALFA-nanobody and two binding moieties for a target antigen. In these embodiments, a binding moiety for a tag, e.g., a NbALFA-nanobody, of two different docking compounds may bind to one of the tags, e.g., ALFA-tags, of a tag conjugate. In some embodiments, a docking compound may be monovalent for binding to a tag, e.g., an ALFA-tag, and bivalent for binding to target antigen. In these embodiments, the payload preferably comprises a chelator comprising a radiodiagnostic isotope, e.g., DOTA-68Ga. In these embodiments, the payload preferably comprises a radiodiagnostic.

[0828] In some embodiments, a tag comprises a medium / low affinity tag, such as a medium / low affinity ALFA-tag. In some embodiments, a tag comprises a medium / low affinity tag, such as a medium / low affinity ALFA-tag, and the docking compound comprises a single binding moiety for a tag, e.g., a single NbALFA-nanobody, for binding monovalently to the tag, and comprises at least two binding moieties for a target antigen, for binding at least bivalently to the target antigen. In some embodiments, a medium / low affinity ALFA-tag is selected from the group consisting of:

[0829] Ac-Ser-Arg-Leu-Glu-(cyclo5)Asp-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu-NH2, Ac-Pro-Ser-Arg-Leu-Glu-(cyclo6)Lys-Glu-Leu-Arg-(cyclolO)Glu-Arg-Leu-Thr-Glu-NH2, and

[0830] Ac-Pro-Ser-Arg-Leu-(cyclo5)Glu-Glu-Glu-Leu-(cyclo9)Lys-Arg-Arg-Leu-Thr-Glu-NH2.In some of these embodiments, the "Ac" and / or "NH2" moieties may not be present. In some of these embodiments, a tag conjugate comprises two tags. In some of these embodiments, a payload comprises a radiodiagnostic.

[0831] In these embodiments, a complex of tag conjugate and docking compound may be formed in the body of a subject. For example, following administration of RNA encoding docking compound, docking compound is produced in a subject's body for binding to target antigen and tag conjugate.

[0832] In some embodiments, a tag conjugate comprises a payload and two tags each tag comprising the ALFA-tag Ser-Arg-Leu-Glu-(cyclo5)Asp-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu, or Pro-Ser-Arg-Leu-(cyclo5)Glu-Glu-Glu-Leu-(cyclo9)Lys-Arg-Arg-Leu-Thr-Glu, and the docking compound comprises a single binding moiety for the tag, e.g., a single NbALFA-nanobody. In some embodiments, a tag conjugate comprises a payload and two tags each tag comprising the ALFA-tag Ser-Arg-Leu-Glu-(cyclo5)Asp-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu, and the docking compound comprises a single binding moiety for the tag, e.g., a single NbALFA-nanobody.

[0833] In some embodiments, a tag conjugate comprises a payload and two tags each tag comprising the ALFA-tag Pro-Ser-Arg-Leu-(cyclo5)Glu-Glu-Glu-Leu-(cyclo9)Lys-Arg-Arg-Leu-Thr-Glu, and the docking compound comprises a single binding moiety for the tag, e.g., a single NbALFA-nanobody.

[0834] In some of these embodiments, a payload comprises a chelator comprising a radiodiagnostic isotope, e.g., DOTA-68Ga.

[0835] In some embodiments, a tag conjugate comprises the formula shown in Figure 10 or Figure 11. In some embodiments, a tag conjugate comprises the formula shown in Figure 11.

[0836] In some embodiments, a tag conjugate comprises the formula shown in Figure 10 or Figure 11, wherein the DOTA moiety in the formula chelates a radiodiagnostic isotope. In some embodiments, a tag conjugate comprises the formula shown in Figure 11, wherein the DOTA moiety in the formula chelates a radiodiagnostic isotope.

[0837] In some embodiments, a tag conjugate comprises the formula shown in Figure 10 or Figure 11, wherein the DOTA moiety in the formula chelates68Ga. In some embodiments, a tagconjugate comprises the formula shown in Figure 11, wherein the DOTA moiety in the formula chelates68Ga.

[0838] In some embodiments, a tag conjugate comprises the formula shown in Figure 10 or Figure 11 and the docking compound comprises a format designated herein as "RD3 format". In some embodiments, a tag conjugate comprises the formula shown in Figure 11 and the docking compound comprises a format designated herein as "RD3 format".

[0839] In some embodiments, a tag conjugate comprises the formula shown in Figure 10 or Figure 11, wherein the DOTA moiety in the formula chelates a radiodiagnostic isotope, and the docking compound comprises a format designated herein as "RD3 format". In some embodiments, a tag conjugate comprises the formula shown in Figure 11, wherein the DOTA moiety in the formula chelates a radiodiagnostic isotope, and the docking compound comprises a format designated herein as "RD3 format".

[0840] In some embodiments, a tag conjugate comprises the formula shown in Figure 10 or Figure 11, wherein the DOTA moiety in the formula chelates68Ga, and the docking compound comprises a format designated herein as "RD3 format". In some embodiments, a tag conjugate comprises the formula shown in Figure 11, wherein the DOTA moiety in the formula chelates68Ga, and the docking compound comprises a format designated herein as "RD3 format".

[0841] In some embodiments, a docking compound comprising a format designated herein as "RD3 format" comprises:

[0842] (i) a polypeptide comprising, from N-terminus to C-terminus:

[0843] a first Fab heavy chain (VH-CH1); an optional linker LI; a first dimerization domain; an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag;

[0844] (ii) a polypeptide comprising, from N-terminus to C-terminus:

[0845] a second Fab heavy chain (VH-CH1); an optional linker LI; and a second dimerization domain; (iii) a polypeptide comprising a first Fab light chain (VL-CL); and

[0846] (iv) a polypeptide comprising a second Fab light chain (VL-CL),

[0847] wherein the first Fab heavy chain of (i) and the first Fab light chain of (iii) form a Fab fragment binding to a target antigen and the second Fab heavy chain of (ii) and the second Fab light chain of (iv) form a Fab fragment binding to a target antigen.In some embodiments, a docking compound comprising a format designated herein as "RD3 format" comprises:

[0848] (i) a first polypeptide comprising, from N-terminus to C-terminus:

[0849] an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for the target antigen (VH(TA)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; a first immunoglobulin heavy chain constant region 3 (CH3) domain ora variant thereof; an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag;

[0850] (ii) a second polypeptide comprising, from N-terminus to C-terminus:

[0851] an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for the target antigen (VH(TA)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; and a second immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof;

[0852] (iii) a third polypeptide comprising, from N-terminus to C-terminus:

[0853] an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for the target antigen (VL(TA)) and an immunoglobulin light chain constant region (CL) domain; and

[0854] (iv) a fourth polypeptide comprising, from N-terminus to C-terminus:

[0855] an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for the target antigen (VL(TA)) and an immunoglobulin light chain constant region (CL) domain,

[0856] wherein a VH(TA) and a VL(TA) interact to form a binding moiety for the target antigen. In some embodiments, a docking compound comprising a format designated herein as "RD3 format" comprises:

[0857] (i) a first polypeptide comprising, from N-terminus to C-terminus:

[0858] an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for CLDN6 (VH(CLDN6)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; a first immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof; an optional linker L2; and an ALFA-tag binding nanobody (NbALFA);(ii) a second polypeptide comprising, from N-terminus to C-terminus:

[0859] an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for CLDN6 (VH(CLDN6)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; and a second immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof;

[0860] (iii) a third polypeptide comprising, from N-terminus to C-terminus:

[0861] an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for CLDN6 (VL(CLDN6)) and an immunoglobulin light chain constant region (CL) domain; and

[0862] (iv) a fourth polypeptide comprising, from N-terminus to C-terminus:

[0863] an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for CLDN6 (VL(CLDN6)) and an immunoglobulin light chain constant region (CL) domain,

[0864] wherein a VH(CLDN6) and a VL(CLDN6) interact to form a binding moiety for CLDN6.

[0865] In some embodiments of a docking compound comprising a format designated herein as "RD3 format", the first and / or second polypeptides do not comprise an immunoglobulin heavy chain constant region 2 (CH2) domain located N-terminal of the dimerization domain and the CH3 domain or variant thereof, respectively, and C-terminal of the Fab heavy chain and the CHI domain, respectively.

[0866] In some embodiments of a docking compound comprising a format designated herein as "RD3 format", the first and / or second polypeptides do not comprise an immunoglobulin heavy chain constant region 2 (CH2) domain.

[0867] In some embodiments of a docking compound comprising a format designated herein as "RD3 format", the linker LI and the linker L2 is present. In some embodiments, the linker LI and the linker L2 allows formation of disulphide bonds between different Fab polypeptides and / or heavy chains. In some embodiments, the linker LI comprises the amino acid sequence EPKSCDKTHTCPPC or a functional variant thereof. In some embodiments, the linker LI and / or the linker L2 comprises a GS sequence. In some embodiments, the linker LI comprises the amino acid sequence EPKSCDKTHTCPPCGGGSSGGGSG or a functional variant thereof. In someembodiments, the linker L2 comprises the amino acid sequence GGGGSGGGS or a functional variant thereof.

[0868] In some embodiments, a docking compound comprising a format designated herein as "RD3 format" comprises:

[0869] (i) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5 or a functional variant thereof;

[0870] (ii) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6 or a functional variant thereof;

[0871] (iii) a third polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof; and

[0872] (iv) a fourth polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof,

[0873] wherein the first polypeptide and the third polypeptide interact to form a binding moiety for CLDN6 and the second polypeptide and the fourth polypeptide interact to form a binding moiety for CLDN6.

[0874] In some embodiments, a docking compound comprising a format designated herein as "RD3 format" comprises:

[0875] (i) a first polypeptide which is encoded by a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5 or a functional variant thereof;

[0876] (ii) a second polypeptide which is encoded by a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6 or a functional variant thereof; (iii) a third polypeptide which is encoded by a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof; and

[0877] (iv) a fourth polypeptide which is encoded by a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof, wherein the first polypeptide and the third polypeptide interact to form a binding moiety for CLDN6 and the second polypeptide and the fourth polypeptide interact to form a binding moiety for CLDN6.In some embodiments, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 6 or a C-terminal truncation variant thereof, wherein the C-terminal truncation variant of SEQ ID NO: 6 comprises a deletion of lysine at the C-terminus of SEQ ID NO: 6.

[0878] Cells for targeted delivery

[0879] According to the disclosure, a payload is delivered specifically to a target cell by targeting a target on target cells, e.g., an antigen on target cells, also referred to herein as "primary target".

[0880] In some embodiments, the primary target is a structure such as a protein present on the surface of a target cell such as a cell surface antigen including a cell surface receptor.

[0881] Terms such as "expressed on the cell surface", "associated with the cell surface" or "cell surface molecule" mean that a molecule such as a receptor or antigen is associated with and located at the plasma membrane of a cell, wherein at least a part of the molecule faces the extracellular space of said cell and is accessible from the outside of said cell, e.g., by a binding molecule such as an antibody located outside the cell. In this context, a part is preferably at least 4, preferably at least 8, preferably at least 12, more preferably at least 20 amino acids. The association may be direct or indirect. For example, the association may be by one or more transmembrane domains, one or more lipid anchors, or by the interaction with any other protein, lipid, saccharide, or other structure that can be found on the outer leaflet of the plasma membrane of a cell. For example, a molecule associated with the surface of a cell may be a transmembrane protein having an extracellular portion or may be a protein associated with the surface of a cell by interacting with another protein that is a transmembrane protein. "Cell surface" or "surface of a cell" is used in accordance with its normal meaning in the art, and thus includes the outside of the cell which is accessible to binding by proteins and other molecules. An antigen is expressed on the surface of cells if it is located at the surface of said cells and is accessible to binding by e.g. antigen-specific antibodies added to the cells. In some embodiments, an antigen expressed on the surface of cells is an integral membrane protein having an extracellular portion recognized by a binding molecule such as an antibody.

[0882] The term "extracellular portion" or "exodomain" in the context of the present invention refers to a part of a molecule such as a protein that is facing the extracellular space of a cell andpreferably is accessible from the outside of said cell, e.g., by binding molecules such as antibodies located outside the cell.

[0883] In some embodiments, a primary target may be present on a diseased cell. In these embodiments, the agents and methods described herein may deliver a payload which is a toxin or radiodiagnostic.

[0884] The primary target may be upregulated during a disease, e.g. infection or cancer. In diseased tissues, markers can differ from healthy tissue and offer unique possibilities for therapy, especially targeted therapy.

[0885] In some embodiments, the primary target is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen. This allows diseased cells to be targeted by the methods and agents described herein, e.g., for delivering a pharmaceutically active agent. The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. Disease-associated antigens may be associated with infection by microbes, typically microbial antigens, or associated with cancer, typically tumors.

[0886] In some embodiments, the primary target is a tumor antigen. In the context of the present disclosure, the term "tumor antigen" or "tumor-associated antigen" relates to proteins that are under normal conditions specifically expressed in a limited number of tissues and / or organs or in specific developmental stages, for example, the tumor antigen may be under normal conditions specifically expressed in stomach tissue, preferably in the gastric mucosa, in reproductive organs, e.g., in testis, in trophoblastic tissue, e.g., in placenta, or in germ line cells, and are expressed or aberrantly expressed in one or more tumor or cancer tissues. In this context, "a limited number" preferably means not more than 3, more preferably not more than 2. The tumor antigens in the context of the present disclosure include, for example, differentiation antigens, preferably cell type specific differentiation antigens, i.e., proteins that are under normal conditions specifically expressed in a certain cell type at a certain differentiation stage, cancer / testis antigens, i.e., proteins that are under normal conditions specifically expressed in testis and sometimes in placenta, and germ line specific antigens. In the context of the present disclosure, the tumor antigen is preferably associated with the cell surface of a cancer cell and is preferably not or only rarely expressed in normal tissues. Preferably, the tumor antigen or the aberrant expression of the tumor antigen identifiescancer cells. In the context of the present disclosure, the tumor antigen that is expressed by a cancer cell in a subject, e.g., a patient suffering from a cancer disease, is preferably a selfprotein in said subject. In preferred embodiments, the tumor antigen in the context of the present disclosure is expressed under normal conditions specifically in a tissue or organ that is non-essential, i.e., tissues or organs which when damaged by the immune system do not lead to death of the subject, or in organs or structures of the body which are not or only hardly accessible by the immune system. Preferably, the amino acid sequence of the tumor antigen is identical between the tumor antigen which is expressed in normal tissues and the tumor antigen which is expressed in cancer tissues.

[0887] Examples for tumor antigens include p53, ART-4, BAGE, beta-catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, the cell surface proteins of the claudin family, such as CLAUDIN-6, CLAUDIN-18.2 and CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, GaplOO, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-l / Melan-A, MC1R, Myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 minor BCR-abL, Pml / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE and WT. Particularly preferred tumor antigens include CLAUDIN-18.2 (CLDN18.2) and CLAUDIN-6 (CLDN6).

[0888] In some embodiments, CLDN6 comprises the following sequence (SEQ ID NO: 33) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto: MASAGMQILGVVLTLLGWVNGLVSCALPMWKVTAFIGNSIVVAQVVWEGLWMSCVVQSTGQMQCK VYDSLLALPQDLQAARALCVIALLVALFGLLVYLAGAKCTTCVEEKDSKARLVLTSGIVFVISGVLTLIPVCWT AHAIIRDFYNPLVAEAQKRELGASLYLGWAASGLLLLGGGLLCCTCPSGGSQGPSHYMARYSTSAPAISRG PSEYPTKNYV

[0889] In some embodiments, the primary target is a structure such as a protein present on the surface of a target cell such as a cell surface antigen or cell surface receptor the presence or amount of which is characteristic for certain cell types compared to others. This allows certaincell types characterized by the presence or increased amounts to be targeted by the methods and agents described herein.

[0890] In some embodiments, a primary target may be present on a diseased cell, such as a tumor cells, and the agents and methods described herein may deliver a payload to the diseased cell. In some embodiments, the cells for targeted delivery are immune effector cells and the primary target is a cell surface antigen that is characteristic for immune effector cells.

[0891] In some embodiments, a primary target, e.g., CD3, such CD3e, CD4 or CD8, may be present on an immune cell, such as an immune effector cell. In these embodiments, the agents and methods described herein may deliver a payload which is an immunomodulator.

[0892] The immune cells used in connection with the methods and agents described herein include, in particular, immune effector cells such as cells with lytic potential, in particular lymphoid cells, and are preferably T cells, in particular cytotoxic lymphocytes, preferably selected from cytotoxic T cells, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells. Upon activation, each of these cytotoxic lymphocytes triggers the destruction of target cells. For example, immune effector cells comprise T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the context of the present disclosure, "immune effector cells" are T cells, preferably CD4+and / or CD8+T cells, most preferably CD8+T cells.

[0893] The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+T cells) and cytotoxic T cells (CTLs, CD8+T cells) which comprise cytolytic T cells. T cells belong to a group of white blood cells known as lymphocytes, and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors (TCR). The thymus is the principal organ responsible for the maturation of T cells. Several different subsets of T cells have been discovered, each with a distinct function. T helper cells assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and activation of cytotoxic T cells and macrophages, among other functions. These cells are also known as CD4+T cells because they express the CD4 glycoprotein on their surface. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules that are expressed on the surface of antigenpresenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response.

[0894] Cytotoxic T cells destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8+T cells since they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of nearly every cell of the body. As used herein, the term "T cell" also includes a cell which can mature into a T cell with suitable stimulation.

[0895] As used herein, the term "NK cell" or "Natural Killer cell" refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 orCD16 and the absence of the T cell receptor. As provided herein, the NK cell can also be differentiated from a stem cell or progenitor cell.

[0896] Targeted delivery of payloads

[0897] The agents and methods described herein find use in a variety of applications in which it is desired to deliver a payload, e.g., a therapeutic or diagnostic compound, to a target cell. The agents described herein may be administered by in vitro or in vivo protocols.

[0898] Delivery of payloads using the methods and agents described herein can be used with a variety of target cells such that the payload is delivered to the target cells and optionally introduced into the target cells (or cells in vicinity to the target cells). The present disclosure may provide for in vitro or in vivo delivery of the payload to the target cell, depending on the location of the target cell. For example, where the target cell is an isolated cell, the payload may be delivered directly to the cell under cell culture conditions permissive of viability of the target cell. Alternatively, where the target cell or cells are part of a multicellular organism, the targeting compounds described herein (tag conjugate / docking compound) may be provided to the organism or host in a manner such that the targeting compounds are able to reach and optionally enter the target cell(s) (or cells in vicinity to the target cells). By "in vivo" it is meant that the targeting compounds (or a nucleic acid encoding therefor) are administered to a living body of an animal. By "ex vivo" it is meant that cells are modified outside of the body. Such cells may be returned to a living body. The route of administration of the targeting compounds (or a nucleic acid encoding therefor) to the multicellular organism depends on severalparameters, including the nature of the targeting compounds. Of particular interest as systemic routes are vascular routes, by which the targeting compounds (or a nucleic acid encoding therefor) are introduced into the vascular system of the host, e.g., an artery or vein, where intravenous routes of administration are of particular interest in many embodiments. For administration, targeting compounds (or a nucleic acid encoding therefor) typically are present in a pharmaceutical preparation, e.g., comprising a pharmaceutically acceptable carrier, diluent and / or adjuvant, and include an effective amount of the payload. In certain embodiments, the targeting compounds (or a nucleic acid encoding therefor) are administered in an aqueous delivery vehicle, e.g., a saline solution. As such, in many embodiments, the targeting compounds (or a nucleic acid encoding therefor) are administered intravascularly, e.g., intraarterially or intravenously, employing an aqueous based delivery vehicle, e.g., a saline solution.

[0899] In many embodiments, the targeting compounds (or a nucleic acid encoding therefor) are administered to a multicellular organism in an in vivo manner such that the payload is introduced into a target cell of the multicellular organism. In the case of nucleic acid, administration is typically under conditions sufficient for expression of the nucleic acid to occur. In some embodiments, the agents and methods described herein result in transient expression of the nucleic acid, as opposed to persistent expression, as indicated above. By transient expression is meant that the expression of nucleic acid at a detectable level does not persist for an extended period of time, following administration of the nucleic acid. By extended period of time is meant at least 1 week, usually at least 2 months and more usually at least 6 months. By detectable level is meant that the expression of the nucleic acid is at a level such that one can detect the encoded protein in the mammal, e.g., in the serum of the mammal, at a therapeutic concentration.

[0900] In some embodiments, the above-described transient expression is achieved without integration of the nucleic acid into the target cell genome of the host.

[0901] Binding moieties and agents

[0902] The present disclosure describes binding moieties or agents such as antibodies or antibody derivatives. Moreover, the disclosure describes bispecific or multispecific binding agents suchas bispecific antibodies comprising a first and a second binding domain, wherein the first binding domain is capable of binding to a primary target and the second binding domain is capable of binding to a tag conjugate.

[0903] The term "binding agent" as used herein refers to any agent capable of binding to desired antigens. In certain embodiments, the binding agent is or comprises an antibody, antibody fragment, or any other binding protein, or any combination thereof.

[0904] The term "binding moiety" as used herein refers to any moiety, group or domain capable of binding to desired antigens. In certain embodiments, the binding moiety is or comprises an antibody, antibody fragment, or any other binding protein, or any combination thereof. As used herein, the term "antigen" is a molecule capable of being bound by a binding moiety or agent, such as an antibody. An antigen may additionally be capable of inducing a humoral immune response and / or cellular immune response leading to the production of B- and / orT-lymphocytes. An antigen may have one or more epitopes (B-cell and T-cell epitopes).

[0905] The term "epitope" refers to a part or fragment of a molecule or antigen that is recognized by a binding agent. For example, the epitope may be recognized by an antibody or any other binding protein. An epitope may include a continuous or discontinuous portion of the antigen and may be between about 5 and about 100, such as between about 5 and about 50, more preferably between about 8 and about 30, most preferably between about 8 and about 25 amino acids in length, for example, the epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some embodiments, an epitope is between about 10 and about 25 amino acids in length. The term "epitope" includes structural epitopes.

[0906] The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized. See for instance Fundamental Immunology Ch.

[0907] 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as VH or VH) and a heavy chain constant region (abbreviated herein as CH or CH). The heavy chain constant region typically is comprised of three domains, CHI, CH2, and CH3. The hinge region is the region between theCHI and CH2 domains of the heavy chain and is highly flexible. Disulphide bonds in the hinge region are part of the interactions between two heavy chains in an IgG molecule. Each light chain typically is comprised of a light chain variable region (abbreviated herein as Vi.or VL) and a light chain constant region (abbreviated herein as CL or CL). The light chain constant region typically is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and / or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)).

[0908] The term "antibody" (Ab) as used herein refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, ora derivative of either thereof, which has the ability to bind, preferably specifically bind to an antigen. In some embodiments, binding takes place under typical physiological conditions with a half-life of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and / or modulate a physiological response associated with antibody binding to the antigen). The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The term "antigen-binding region", "binding region" or "binding domain", as used herein, refers to the region or domain which interacts with the antigen and typically comprises both a VH region and a VL region. The term antibody when used herein comprises not only monospecific antibodies, but also multispecific antibodies which comprise multiple, such as two or more, e.g. three or more, different antigen-binding regions. The constant regions of the antibodies (Abs) may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as Clq, the first component in the classical pathway of complement activation.CH3 domains are commonly understood in the art as the third constant immunoglobulin domain in an antibody heavy polypeptide chain, starting from the N terminus. CH3 domains can also be distinguished as the constant immunoglobulin domains that form an interface between the polypeptide chains within an antibody construct comprising an Fc region having two constant domains, i.e. an IgG, IgD and IgA antibody construct, thereby contributing to the dimerisation of the polypeptide chains. This also applies in other antibody-derived formats. Further still, CH3 domains can be recognised by comparison with known CH3 domain sequences, such as by reference to the CH3 domain sequences described herein.

[0909] In contrast, CH2 domains are commonly understood in the art as the second constant immunoglobulin domain in an antibody heavy polypeptide chain, starting from the N terminus. CH2 domains can also be distinguished as the constant immunoglobulin domains which (in the absence of specific identifiable modifications for achieving this) do not generally form an interface contributing to the dimerisation of the polypeptide chains within an IgG, IgD and IgA antibody construct. CH2 domains also commonly contain a glycosylated residue, such as asparagine at position 297 relative to the IgG Fc region. Further still, CH2 domains can be recognised by comparison with known CH2 domain sequences.

[0910] In a preferred embodiment, the CH3 domain is an IgGl constant domain.

[0911] An illustrative human CH3 sequence is as follows:

[0912] GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 34).

[0913] In some embodiments, a CH3 domain comprises or consists of a sequence as set forth in SEQ ID NO: 34, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the sequences of the CH3 domains further comprise a modification that enhances the formation of a heterodimer comprising the first and second polypeptides as defined herein.

[0914] In some embodiments, the first and second polypeptides comprise modifications that enhance the formation of a dimer, preferably a heterodimer, comprising one monomer of the first polypeptide and one monomer of the second polypeptide. In some embodiments, the modifications can be those modifications which are widely known in the art of antibody technology for the purpose of (hetero)dimerisation between antibody chains (e.g. "knobs-into-holes" ( Ki H ) modifications). In a typical embodiment, the modifications are present in the CH3 domains of the first and second polypeptides.

[0915] As used herein, the term "enhances the formation of the construct comprising the first and second polypeptide" includes promoting dimerisation of the first and second polypeptides. Thus, in some embodiments, the modifications promote dimerisation of the first and second polypeptides. In some embodiments, the modifications promote heterodimerisation of the first and second polypeptides. By heterodimerisation it is meant that the first polypeptide is different to the second polypeptide, and the first polypeptide will form a dimer with the second polypeptide, but the first polypeptide will not form a dimer with the first polypeptide and the second polypeptide will not form a dimer with the second polypeptide. In some embodiments, the modifications promote homodimerisation of the first and second polypeptides. By homodimerisation it is meant that the first polypeptide is the same as the second polypeptide, and the first polypeptide will form a dimer with the second polypeptide. Various means of promoting dimerisation of two domains (e.g. homo- or hetero-dimerisation) are known in the art. In some embodiments, the two domains may comprise a modification that enhances formation of dimer, e.g. a modification that increases the affinity of the two domains, promotes and / or enables the formation of one or more disulphide bonds, reduces steric hindrance to the dimerisation, promotes electrostatic and / or hydrophilic / hydrophobic interactions between the two domains, or any combination thereof.

[0916] In some embodiments, one or more of the CH3 domains of the first polypeptide and / or one or more of the CH3 domains of the second polypeptide comprise a modification that enhances the affinity between the first and second polypeptides. In some embodiments, the modification enhances the affinity between the CH3 domains of the first and second polypeptides.

[0917] In some embodiments, one or more of the CH3 domains of the first polypeptide and / or one or more of the CH3 domains of the second polypeptide comprise a modification that reduces steric hindrance to the formation of the construct comprising the first and second polypeptide. In some embodiments, the modification reduces steric hindrance between the CH3 domains of the first and second polypeptides. In some embodiments, one or more of the CH3 domains of the first polypeptide and one or more of the CH3 domains of the secondpolypeptide comprise a 'knobs-into-holes' modification. In some embodiments, one or more of the CH3 domains of the first polypeptide and one or more of the CH3 domains of the second polypeptide are associated via a 'knobs-into-holes' modification.

[0918] In some embodiments, one or more of the CH3 domains of the first polypeptide and / or one or more of the CH3 domains of the second polypeptide comprise a modification that promotes electrostatic interactions between the first and second polypeptide. In some embodiments, the modification promotes electrostatic interactions between the CH3 domains of the first and second polypeptides. In some embodiments, the electrostatic interactions that are promoted are favourable to the formation of a dimer (e.g. a heterodimer) comprising the first and second polypeptide.

[0919] In some embodiments, one or more of the CH3 domains of the first polypeptide and / or one or more of the CH3 domains of the second polypeptide comprise a modification that promotes hydrophilic / hydrophobic interactions between the first and second polypeptide. In some embodiments, the modification promotes hydrophilic / hydrophobic interactions between the CH3 domains of the first and second polypeptides. In some embodiments, the hydrophilic / hydrophobic interactions that are promoted are favourable to the formation of a dimer comprising the first and second polypeptide.

[0920] In some embodiments, a CH3 domain of the first polypeptide forms a disulphide bond with a CH3 domain of the second polypeptide. In a preferred embodiment, a CH3 domain of the first polypeptide forms a disulphide bond with a CH3 domain of the second polypeptide, and in addition one or more of the CH3 domains of the first polypeptide and / or one or more of the CH3 domains of the second polypeptide comprise a modification that enhances the affinity between the first and second polypeptides.

[0921] Any suitable technology for promoting the dimerisation of CH3 domains, i.e. of at least one of the CH3 domains of the first polypeptide and at least one of the CH3 domains of the second polypeptide, may be employed in the practice of the invention.

[0922] Preferably, the modification that enhances the formation of a dimer between the CH3 domains is Ki H technology (Ridgeway et al., Protein Engineering, Design and Selection, 1996, 9: 617-621 and Merchant et al., Nat Biotechnol, 1998, 16: 677-681). The KiH technology is based on an engineered pairof CH3 domains that heterodimerises (CH3 heterodimer) in whichasymmetric hydrophobic mutations are introduced between the homodimeric CH3 domain. KiH involves introducing mutations that create a protuberance ("knob") in the interface of the first CH3 domain and a corresponding cavity ("hole") in the interface of the second CH3 domain, such that the protuberance can be positioned in the cavity to promote heterodimer assembly and hinder homodimer formation. KiH variants therefore thermodynamically favour the formation of heterodimers rather than homodimers.

[0923] By way of further example, the present invention may employ: the strand-exchange engineered domain (SEED) CH3 dimers (Davis et al., Protein Eng Des Sei., 2010, 23:195-202); electrostatic steering employing DD-KK variants with asymmetric electrostatic interactions (Gunasekaran et al., J Biol Chem., 2010 , 285: 19637-46): Azymetric technologies by Zymeworks (https: / / www.zymeworks.com / technologies / azymetric / ): Bispecific Engagement by Antibodies based on the T-cell receptor (BEAT) (Skegro etal., J Biol Chem., 2017, 292: 9745-9759): Fast-lg and ART-lg (https: / / www.chuqaipharm. co.jp / english / profile / rd / technologies.html): DEKK dimerisation technology (https: / / merus.nl / technology / multiclonics-platform / and Nardis et al, J Biol Chem., 2017, 292: 14706-14717): HA-TF variants with asymmetric hydrophobic interactions (Moore etal., MAbs, 2011, 3: 546-57): computationally designed CH3 interfaces, such as the 7.8.60 design (Leaver-Fay et al., Structure, 2016, 24: 641-651): EW-RVT variants, which were designed to replace the conserved electrostatic interactions with asymmetric hydrophobic interactions and to add asymmetric long-range electrostatic interactions at the rim of the heterodimeric CH3 interface (Choi etal., Mol CancerTher., 2013, 12: 2748-59): and the K370ECH3A-E357NCH3B mutations employed in the "A107" variant which replaced the homodimer-favouring electrostatic interactions with heterodimer-stabilizing hydrogen bonds.

[0924] Herein, EU numbering is used to denote the residues of an immunoglobulin domain (Edelman et al., Proc Natl Acad Sci U S A., 1969, 63: 78-85).

[0925] In some embodiments, one or more of the CH3 domains of the first polypeptide and one or more of the CH3 domains of the second polypeptide are associated via a KiH modification. In some embodiments, the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide, where present, each comprise a KiH modification.Any suitable KiH modifications known in the art may be employed in the practice of the present invention. For example, the T366W ("knob") modification and the T366S, L368A and Y407V ("hole") modifications may be used.

[0926] In some embodiments, the first polypeptide comprises the T366W modification within the CH3 domain. In some embodiments, the second polypeptide comprises the T366S, L368A and Y407V modifications within the CH3 domain. In some embodiments, the first polypeptide comprises the T366W modification within the CH3 domain and the second polypeptide comprises the T366S, L368A and Y407V modifications within the CH3 domain. In some embodiments, these modifications are according to EU residue numbering.

[0927] In some embodiments, one or more of the CH3 domains of the first polypeptide and one or more of the CH3 domains of the second polypeptide, where present, comprise a modification to introduce a cysteine residue to promote the formation of a disulphide bond between the CH3 domains of the first and second polypeptides.

[0928] In some embodiments, the modifications to introduce a cysteine residue to promote the formation of a disulphide bond between the CH3 domains of the first and second polypeptides are configured to provide an asymmetric disulphide bond.

[0929] Suitably, the S354C mutation and the Y349C mutation described herein may be used to provide an asymmetric disulphide bond between the CH3 domains of the first and second peptide.

[0930] Accordingly, in some embodiments, the first polypeptide comprises the S354C modification within the CH3 domain. In some embodiments, the second polypeptide comprises the Y349C modification within the CH3 domain. In some embodiments, the first polypeptide comprises the S354C modification within the CH3 domain and the second polypeptide comprises the Y349C modification within the CH3 domain.

[0931] In a preferred embodiment, the modification that promotes the dimerisation of the first and second polypeptides (e.g. the KiH modification) is compatible with the modification to provide a disulphide bond (e.g. an asymmetric disulphide bond) between the CH3 domains of the first and second polypeptides as described herein.

[0932] In some embodiments, one or more of the CH3 domains of the first polypeptide and one or more of the CH3 domains of the second polypeptide are associated via a KiH modification andone or more of the CH3 domains of the first polypeptide and one or more of the CH3 domains of the second polypeptide comprise a modification to introduce a cysteine residue to promote the formation of a disulphide bond between the CH3 domains of the first and second polypeptides. In some embodiments, the modifications to introduce a cysteine residue to promote the formation of a disulphide bond between the CH3 domains of the first and second polypeptides are configured to provide an asymmetric disulphide bond.

[0933] In some embodiments, the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide, where present, each comprise a KiH modification and a modification to introduce a cysteine residue to promote the formation of a disulphide bond between the CH3 domains of the first and second polypeptides. In some embodiments, the modifications to introduce a cysteine residue to promote the formation of a disulphide bond between the CH3 domains of the first and second polypeptides are configured to provide an asymmetric disulphide bond.

[0934] In some embodiments, the first polypeptide comprises the S354C and T366W modifications within the CH3 domain. In some embodiments, the second polypeptide comprises the Y349C, T366S, L368A and Y407V modifications within the CH3 domain. In some embodiments, the first polypeptide comprises the S354C and T366W modifications within the CH3 domain and the second polypeptide comprises the Y349C, T366S, L368A and Y407V modifications within the CH3 domain. In some embodiments, these modifications are according to EU residue numbering.

[0935] An illustrative CH3 domain comprising the S354C modification and T366W ("knob") modification is provided in SEQ ID NO: 16.

[0936] An illustrative CH3 domain comprising the Y349C modification and the T366S, L368A and Y407V ("hole") modifications is provided in SEQ ID NO: 17.

[0937] In some embodiments, the first polypeptide comprises a sequence as set forth in SEQ ID NO: 16, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the second polypeptide comprises a sequence as set forth in SEQ ID NO: 17, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.In some embodiments, the first polypeptide comprises a sequence as set forth in SEQ ID NO: 16, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and the second polypeptide comprises a sequence as set forth in SEQ ID NO: 17, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In a more specific embodiment, the CH3 domains of the first and / or second polypeptides comprise or consist of the respective sequences as defined above.

[0938] As used herein, the term "variable immunoglobulin domain" may refer to a variable domain of an immunoglobulin, e.g. a VHH domain.

[0939] The term antibody as used herein, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that are antigen-binding fragments, i.e., retain the ability to specifically bind to the antigen, and antibody derivatives, i.e., constructs that are derived from an antibody. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed within the term "antibody" include (i) a Fab' or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains, ora monovalent antibody as described in WQ2007059782 (Genmab); (ii) F(ab’)2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CHI domains; (iv) a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341, 544-546 (1989)), which consists essentially of a VH domain and also called domain antibodies (Holt et al; Trends Biotechnol. 2003 Nov;21(ll):484-90); (vi) camelid or Nanobody molecules (Revets et al; Expert Opin Biol Ther. 2005 Jan;5(l):lll-24) and (vii) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), see for instance Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. Although such fragments are generally included within the meaning of antibody, theycollectively and each independently are unique features of the present disclosure, exhibiting different biological properties and utility. These and other useful antibody fragments in the context of the present disclosure, as well as bispecific formats of such fragments, are discussed further herein. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques.

[0940] The phrase "Fragment antigen binding" or "Fab" refers to polypeptides comprising monovalent antigen-binding domains of an antibody composed of a polypeptide consisting of a heavy chain variable region (VH) and heavy chain constant region 1 (CHI) portion and a polypeptide consisting of a light chain variable region (VL) and a light chain constant region (in which the CL and CHI portions may be bound together, for example by a disulfide bond between Cys residues). The part of a Fab fragment comprising VH and CHI is also referred to as "Fab heavy chain" or "Fd" herein. The part of a Fab fragment comprising VL and CL is also referred to as "Fab light chain" herein.

[0941] The phrase "single chain Fv" or "scFv" refers to an antibody in which the variable domains of the heavy chain and of the light chain (VH and VL) of a traditional two chain antibody have been joined to form one chain. Optionally, a linker (usually a peptide) is inserted between the two chains to allow for proper folding and creation of an active binding site.

[0942] A single-domain antibody, also known as a nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain. In some embodiments, a single-domain antibody is a variable domain (VH) of a heavy-chain antibody. These are called VHH fragments. Like a whole antibody, a single-domain antibody is able to bind selectively to a specific antigen. The first single-domain antibodies were engineered from heavy-chain antibodies found in camelids. Cartilaginous fishes also have heavy-chain antibodies (IgNAR, 'immunoglobulin new antigen receptor'), from which single-domain antibodies called VNAR fragments can be obtained. An alternative approach is to split the dimeric variable domains from common immunoglobulin G (IgG) from humans or mice into monomers. Although most research intosingle-domain antibodies is currently based on heavy chain variable domains, nanobodies derived from light chains have also been shown to bind specifically to target epitopes.

[0943] An antibody can possess any isotype. As used herein, the term "isotype" refers to the immunoglobulin class (for instance IgGl, lgG2, lgG3, lgG4, IgD, IgA, IgE, or IgM) that is encoded by heavy chain constant region genes. When a particular isotype, e.g. IgGl, is mentioned herein, the term is not limited to a specific isotype sequence, e.g. a particular IgGl sequence, but is used to indicate that the antibody is closer in sequence to that isotype, e.g. IgGl, than to other isotypes. Thus, e.g. an IgGl antibody may be a sequence variant of a naturally-occurring IgGl antibody, including variations in the constant regions.

[0944] In various embodiments, an antibody is an IgGl antibody, more particularly an IgGl, kappa or IgGl, lambda isotype (i.e. IgGl, K, X), an lgG2a antibody (e.g. lgG2a, K, X), an lgG2b antibody (e.g. lgG2b, K, X), an lgG3 antibody (e.g. lgG3, K, X) or an lgG4 antibody (e.g. lgG4, K, X).

[0945] When used herein, unless contradicted by context, the term "Fab-arm", "binding arm" or "arm" includes one heavy chain-light chain pair and is used interchangeably with "halfmolecule" herein.

[0946] The term "full-length" when used in the context of an antibody indicates that the antibody is not a fragment, but contains all of the domains of the particular isotype normally found for that isotype in nature, e.g. the VH, CHI, CH2, CH3, hinge, VL and CL domains for an IgGl antibody.

[0947] When used herein, unless contradicted by context, the term "Fc region" refers to an antibody region consisting of the two Fc sequences of the heavy chains of an immunoglobulin, wherein said Fc sequences comprise at least a hinge region, a CH2 domain, and a CH3 domain.

[0948] The term "specificity" as used herein is intended to have the following meaning unless contradicted by context. Two antibodies have the "same specificity" if they bind to the same antigen and the same epitope.

[0949] Naturally occurring antibodies are generally monospecific, i.e. they bind to a single antigen. Described herein are binding agents, e.g., docking compounds, binding to different epitopes on e.g. a primary target and a tag conjugate. Such binding agents are at least bispecific or multispecific such as trispecific, tetraspecific and so on.According to the disclosure, a bispecific binding agent, in particular a bispecific protein, such as a bispecific antibody is a molecule that has two different binding specificities and thus may bind to two epitopes. Particularly, the term "bispecific antibody" as used herein refers to an antibody comprising at least two antigen-binding sites, a first binding site having affinity for a first epitope and a second binding site having binding affinity for a second epitope distinct from the first.

[0950] The term "bispecific" as used herein refers to an agent having at least two different antigenbinding regions binding to different epitopes.

[0951] "Multispecific binding agents" are molecules which have more than two different binding specificities.

[0952] Many different formats and uses of bispecific antibodies are known in the art, and were reviewed by Kontermann; Drug Discov Today, 2015 Jul;20(7):838-47 and; MAbs, 2012 Mar-Apr;4(2):182-97.

[0953] A bispecific binding agent according to the present disclosure is not limited to any particular bispecific format or method of producing it.

[0954] A docking compound described herein generally binds monovalently to the tags of the tag conjugate and at least bivalently to target antigen (either to the same or different epitopes of a target antigen). In some embodiments, a docking compound described is trivalent. A docking compound described herein may also have valencies of 4 or of higher than 4.

[0955] As used herein, "valent", "valence", "valencies", or other grammatical variations thereof, mean the number of antigen binding sites or binding domains in a binding agent. Antigen binding sites binding to the same antigen may recognize different epitopes or preferably the same epitope.

[0956] The term "DARPin" refers to designed ankyrin repeat proteins. DARPins are based on naturally occurring ankyrin repeat proteins, yet contain one or more amino acid mutations that can affect, for example, their binding affinity to a target molecule, their cell surface expression, and the like. DARPins preferably include 2 to 3 ankyrin repeat modules flanked by N- and C-capping repeats. Each ankyrin repeat module includes about 33 amino acid residues.Nucleic acids

[0957] The term "nucleic acid" comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. In some embodiments, a nucleic acid is DNA. In some embodiments, a nucleic acid is RNA. In some embodiments, a nucleic acid is a mixture of DNA and RNA. A nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule. A nucleic acid can be isolated. The term "isolated nucleic acid" means, according to the present disclosure, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis. The term "nucleoside" (abbreviated herein as "N") relates to compounds which can be thought of as nucleotides without a phosphate group. While a nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[0958] The five standard nucleosides which usually make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine and guanosine. The five nucleosides are commonly abbreviated to their one letter codes U, A, T, C and G, respectively. However, thymidine is more commonly written as "dT" ("d" represents "deoxy") as it contains a 2'-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) and not ribonucleic acid (RNA). Conversely, uridine is found in RNA and not DNA. The remaining three nucleosides may be found in both RNA and DNA. In RNA, they would be represented as A, C and G, whereas in DNA they would be represented as dA, dC and dG.

[0959] A modified purine (A or G) or pyrimidine (C, T, or U) base moiety is, in some embodiments, modified by one or more alkyl groups, e.g., one or more C1-4 alkyl groups, e.g., one or more methyl groups. Particular examples of modified purine or pyrimidine base moieties include N7-alkyl-guanine, N6-alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil, and N(l)-alkyl-uracil, suchas N7-CI-4alkyl-guanine, N6-CI-4 alkyl-adenine, 5-C1-4 alkyl-cytosine, 5-C1-4 alkyl-uracil, and N ( 1)-Ci-4 alkyl-uracil, preferably N7-methyl-guanine, N6-methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil, and N(l)-methyl-uracil.

[0960] DNA

[0961] Herein, the term "DNA" relates to a nucleic acid molecule which includes deoxyribonucleotide residues. In preferred embodiments, the DNA contains all ora majority of deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide which lacks a hydroxyl group at the 2'-position of a P-D-ribofuranosyl group. DNA encompasses without limitation, double stranded DNA, single stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of nonnucleotide material to internal DNA nucleotides or to the end(s) of DNA. It is also contemplated herein that nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, these altered DNAs are considered analogs of naturally-occurring DNA. A molecule contains "a majority of deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard ( / .e., naturally occurring) nucleotide residues or analogs thereof).

[0962] DNA may be recombinant DNA and may be obtained by cloning of a nucleic acid, in particular cDNA. The cDNA may be obtained by reverse transcription of RNA.

[0963] Nucleic acids may be comprised in a vector. The term "vector" as used herein includes any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retroviral, adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificialchromosomes (YAC), or Pl artificial chromosomes (PAC). Said vectors include expression as well as cloning vectors. Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments.

[0964] RNA

[0965] The term "RNA" relates to a nucleic acid molecule which includes ribonucleotide residues. In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2'-position of a [3-D-ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may referto addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered / modified nucleotides can be referred to as analogs of naturally occurring nucleotides, and the corresponding RNAs containing such altered / modified nucleotides ( / .e., altered / modified RNAs) can be referred to as analogs of naturally occurring RNAs. A molecule contains "a majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard ( / .e., naturally occurring) nucleotide residues or analogs thereof)."RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), selfamplifying RNA (saRNA), trans-amplifying RNA (taRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA) and immunostimulatory RNA (isRNA). In some embodiments, "RNA" refers to mRNA.

[0966] The term "in vitro transcription" or "IVT" as used herein means that the transcription ( / .e., the generation of RNA) is conducted in a cell-free manner, / .e., IVT does not use living / cultured cells but rather the transcription machinery extracted from cells (e.g., cell lysates or the isolated components thereof, including an RNA polymerase (preferably T7, T3 or SP6 polymerase)).

[0967] According to the present disclosure, the term '"RNA" includes "mRNA". According to the present disclosure, the term "mRNA" means "messenger-RNA" and includes a "transcript" which may be generated by using a DNA template. Generally, mRNA encodes a peptide or polypeptide.

[0968] mRNA is single-stranded but may contain self-complementary sequences that allow parts of the mRNA to fold and pair with itself to form double helices.

[0969] According to the present disclosure, "dsRNA" means double-stranded RNA and is RNA with two partially or completely complementary strands.

[0970] In some embodiments, the mRNA which preferably encodes a peptide or polypeptide has a length of at least 45 nucleotides (such as at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), preferably up to 15,000, such as up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides or up to 10,000 nucleotides.

[0971] As established in the art, mRNA generally contains a 5' untranslated region (5'-UTR), a peptide / polypeptide coding region and a 3' untranslated region (3'-UTR). In some embodiments, the mRNA is produced by in vitro transcription or chemical synthesis. In some embodiments, the mRNA is produced by in vitro transcription using a DNA template. The in vitro transcription methodology is known to the skilled person; cf., e.g., Molecular Cloning: ALaboratory Manual, 4thEdition, M.R. Green and J. Sambrook eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012. Furthermore, a variety of in vitro transcription kits is commercially available, e.g., from Thermo Fisher Scientific (such as TranscriptAid™ T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc. (such as HiScribe™ T7 kit, HiScribe™ T7 ARCA mRNA kit), Promega (such as RiboMAX™, HeLaScribe®, Riboprobe® systems), Jena Bioscience (such as SP6 or T7 transcription kits), and Epicentre (such as AmpliScribe™). For providing modified mRNA, correspondingly modified nucleotides, such as modified naturally occurring nucleotides, non-naturally occurring nucleotides and / or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or modifications can be effected in and / or added to the mRNA after transcription.

[0972] In some embodiments, RNA is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoterfor controlling transcription can be any promoter for any RNA polymerase. Particular examples of RNA polymerases are the T7, T3, and SP6 RNA polymerases. Preferably, the in vitro transcription is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.

[0973] In some embodiments of the present disclosure, the RNA is "replicon RNA" or simply a "replicon", in particular "self-replicating RNA" or "self-amplifying RNA". In certain embodiments, the replicon or self-replicating RNA is derived from or comprises elements derived from an ssRNA virus, in particular a positive-stranded ssRNA virus such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5'-cap, and a 3' poly(A) tail. The genome of alphaviruses encodes non-structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome. The four non-structural proteins (nsPl-nsP4) aretypically encoded together by a first ORF beginning near the 5' terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3' terminus of the genome. Typically, the first ORF is larger than the second ORF, the ratio being roughly 2:1. In cells infected by an alphavirus, only the nucleic acid sequence encoding non-structural proteins is translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from a subgenomic transcript, which is an RNA molecule that resembles eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111-124). Following infection, i.e. at early stages of the viral life cycle, the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non-structural poly-protein (nsP1234).

[0974] Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms. In simple approaches, the open reading frame encoding alphaviral structural proteins is replaced by an open reading frame encoding a protein of interest. Alphavirus-based trans-replication (trans-amplification) systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans-replication system). Trans-replication requires the presence of both these nucleic acid molecules in a given host cell. The nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase.

[0975] In some embodiments of the present disclosure, the RNA (in particular, mRNA) described herein contains one or more modifications, e.g., in order to increase its stability and / or increase translation efficiency and / or decrease immunogenicity and / or decrease cytotoxicity. For example, in order to increase expression of the RNA (in particular, mRNA), it may be modified within the coding region, i.e., the sequence encoding the expressed peptide or polypeptide, preferably without altering the sequence of the expressed peptide or polypeptide. Such modifications are described, for example, in WO 2007 / 036366 and PCT / EP2019 / 056502, and include the following: a 5’-cap structure; an extension or truncationof the naturally occurring poly(A) tail; an alteration of the 5'- and / or 3'-untranslated regions (UTR) such as introduction of a UTR which is not related to the coding region of said RNA; the replacement of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (e.g., to alter, preferably increase, the GC content of the RNA).

[0976] In some embodiments, the RNA (in particular, mRNA) described herein comprises a 5'-cap structure. In some embodiments, the RNA does not have uncapped 5'-triphosphates. In some embodiments, the RNA (in particular, mRNA) may comprise a conventional 5'-cap and / or a 5'-cap analog. The term "conventional 5'-cap" refers to a cap structure found on the 5'-end of an RNA molecule and generally comprises a guanosine 5'-triphosphate (Gppp) which is connected via its triphosphate moiety to the 5'-end of the next nucleotide of the RNA ( / .e., the guanosine is connected via a 5' to 5' triphosphate linkage to the rest of the RNA). The guanosine may be methylated at position N7(resulting in the cap structure m7Gppp). The term "5'-cap analog" includes a 5'-cap which is based on a conventional 5'-cap but which has been modified at either the 2'- or 3'-position of the m7guanosine structure in order to avoid an integration of the 5'-cap analog in the reverse orientation (such 5'-cap analogs are also called anti-reverse cap analogs (ARCAs)). Particularly preferred 5'-cap analogs are those having one or more substitutions at the bridging and non-bridging oxygen in the phosphate bridge, such as phosphorothioate modified 5'-cap analogs at the (3-phosphate (such as m27'2OG(5')ppSp(5')G (referred to as beta-S-ARCA or (3-S-ARCA)), as described in PCT / EP2019 / 056502. Providing an RNA (in particular, mRNA) with a 5'-cap structure as described herein may be achieved by in vitro transcription of a DNA template in presence of a corresponding 5'-cap compound, wherein said 5'-cap structure is co-transcriptionally incorporated into the generated RNA (in particular, mRNA) strand, or the RNA (in particular, mRNA) may be generated, for example, by in vitro transcription, and the 5'-cap structure may be attached to the RNA post-transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus.

[0977] In some embodiments, the RNA (in particular, mRNA) comprises a 5'-cap structure selected from the group consisting of m27'2 OG(5')ppSp(5')G (in particular its DI diastereomer), m27-3'°G(5')ppp(5')G, and m27-3'0Gppp(mi2'0)ApG.In some embodiments, the RNA (in particular, mRNA) comprises a capO, capl, or cap2, preferably capl or cap2. According to the present disclosure, the term "capO" means the structure "m7GpppN", wherein N is any nucleoside bearing an OH moiety at position 2'. According to the present disclosure, the term "capl" means the structure "m7GpppNm", wherein Nm is any nucleoside bearing an OCH3 moiety at position 2'. According to the present disclosure, the term "cap2" means the structure "m7GpppNmNm", wherein each Nm is independently any nucleoside bearing an OCH3 moiety at position 2'.

[0978] The 5'-cap analog beta-S-ARCA ((3-S-ARCA) has the following structure:

[0979]

[0980] The "DI diastereomer of beta-S-ARCA" or "beta-S-ARCA(Dl)" is the diastereomer of beta-S- ARCA which elutes first on an HPLC column compared to the D2 diastereomer of beta-S-ARCA (beta-S-ARCA(D2)) and thus exhibits a shorter retention time. The HPLC preferably is an analytical HPLC. In some embodiments, a Supelcosil LC-18-T RP column, preferably of the format: 5 pm, 4.6 x 250 mm is used for separation, whereby a flow rate of 1.3 ml / min can be applied. In some embodiments, a gradient of methanol in ammonium acetate, for example, a 0-25% linear gradient of methanol in 0.05 M ammonium acetate, pH = 5.9, within 15 min is used. UV-detection (VWD) can be performed at 260 nm and fluorescence detection (FLD) can be performed with excitation at 280 nm and detection at 337 nm.

[0981] The 5'-cap analog m27'3'°Gppp(mi2'°)ApG (also referred to as m27'30G(5')ppp(5')m2'0ApG) which is a building block of a capl has the following structure:

[0982]

[0983] An exemplary capO mRNA comprising P-S-ARCA and mRNA has the following structure:

[0984]

[0985] An exemplary capO mRNA comprising m27,3 OG(5')ppp(5')G and mRNA has the following

[0986]

[0987] An exemplary capl mRNA comprising m27,3 OGppp(mi2' °)ApG and mRNA has the following structure:

[0988]

[0989] As used herein, the term "poly-A tail" or "poly-A sequence" refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3'-end of an RNA (in particular, mRNA) molecule. Poly-A tails or poly-A sequences are known to those of skill in the art and may follow the 3'-UTR in the RNAs (in particular, mRNAs) described herein. An uninterrupted poly-A tail is characterized by consecutive adenylate residues. In nature, an uninterrupted poly-A tail is typical. RNAs (in particular, mRNAs) disclosed herein can have a poly-A tail attached to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription or a poly-A tail encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0990] It has been demonstrated that a poly-A tail of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5') of the poly-A tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0991] The poly-A tail may be of any length. In some embodiments, a poly-A tail comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides. In this context, "essentially consists of" means that most nucleotides in the poly-A tail, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly-A tail are A nucleotides, but permits that remaining nucleotides are nucleotides other than Anucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consists of" means that all nucleotides in the poly-A tail, i.e., 100% by number of nucleotides in the poly-A tail, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate.

[0992] In some embodiments, a poly-A tail is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding a poly-A tail (coding strand) is referred to as poly(A) cassette.

[0993] In some embodiments, the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such a cassette is disclosed in WO 2016 / 005324 Al, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016 / 005324 Al may be used in the present disclosure. A poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. co / / and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed. Consequently, in some embodiments, the poly-A tail contained in an RNA (in particular, mRNA) molecule described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.

[0994] In some embodiments, the poly(A) tail comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence of 10 nucleotides.

[0995] In some embodiments, no nucleotides other than A nucleotides flank a poly-A tail at its 3'-end, i.e., the poly-A tail is not masked or followed at its 3'-end by a nucleotide other than A. In some embodiments, a poly-A tail may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may essentially consist of at least 20, at least 30, at least40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail comprises at least 100 nucleotides. In some embodiments, the poly-A tail comprises about 150 nucleotides. In some embodiments, the poly-A tail comprises about 120 nucleotides.

[0996] In some embodiments, RNA (in particular, mRNA) described in present disclosure comprises a 5'-UTR and / or a 3'-UTR. The term "untranslated region" or "UTR" relates to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5'-UTR) and / or 3' (downstream) of an open reading frame (3'-UTR). A 5'-UTR, if present, is located at the 5'-end, upstream of the start codon of a protein-encoding region. A 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap. A 3'-UTR, if present, is located at the 3'-end, downstream of the termination codon of a protein-encoding region, but the term "3'-UTR" does generally not include the poly-A sequence. Thus, the 3'-UTR is upstream of the poly-A sequence (if present), e.g., directly adjacent to the poly-A sequence. Incorporation of a 3'-UTR into the 3'-non translated region of an RNA (preferably mRNA) molecule can result in an enhancement in translation efficiency. A synergistic effect may be achieved by incorporating two or more of such 3'-UTRs (which are preferably arranged in a head-to-tail orientation; cf., e.g., Holtkamp et al., Blood 108, 4009-4017 (2006)). The 3'-UTRs may be autologous or heterologous to the RNA (e.g., mRNA) into which they are introduced. In certain embodiments, the 3'-UTR is derived from a globin gene or mRNA, such as a gene or mRNA of alpha2-globin, alphal-globin, or beta-globin, e.g., beta-globin, e.g., human beta-globin. For example, the RNA (e.g., mRNA) may be modified by the replacement of the existing 3'-UTR with or the insertion of one or more, e.g., two copies of a 3'-UTR derived from a globin gene, such as alpha2-globin, alphal-globin, beta-globin, e.g., beta-globin, e.g., human beta-globin.

[0997] In some embodiments, a 5'-UTR is or comprises a modified human alpha-globin 5'-UTR. In some embodiments, a 3'-UTR comprises a first sequence from the amino terminal enhancerof split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA.

[0998] The RNA (in particular, mRNA) described herein may have modified ribonucleotides in order to increase its stability and / or decrease immunogenicity and / or decrease cytotoxicity. For example, in some embodiments, uridine in the RNA (in particular, mRNA) described herein is replaced (partially or completely, preferably completely) by a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

[0999] In some embodiments, the modified uridine replacing uridine is selected from the group consisting of pseudouridine (<JJ), Nl-methyl-pseudouridine (mlip), 5-methyl-uridine (m5U), and combinations thereof.

[1000] In some embodiments, the modified nucleoside replacing (partially or completely, preferably completely) uridine in the RNA may be any one or more of 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5-bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (TITI5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(Tm5s2U), l-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m5s2U), l-methyl-4-thio-pseudouridine (mls4ip), 4-thio-l-methyl-pseudouridine, 3-methyl-pseudouridine (m3ip), 2-thio-l-methyl-pseudouridine, 1-methyl-l-deaza-pseudouridine, 2-thio-l-methyl-l-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, Nl-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), l-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 ip), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (ipm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-(l-E-propenylamino)uridine, or any other modified uridine known in the art.

[1001] An RNA (preferably mRNA) which is modified by pseudouridine (replacing partially or completely, preferably completely, uridine) is referred to herein as "IP-modified", whereas the term "mliP-modified" means that the RNA (preferably mRNA) contains N(l)-methylpseudouridine (replacing partially or completely, preferably completely, uridine). Furthermore, the term "m5U-modified" means that the RNA (preferably mRNA) contains 5-methyluridine (replacing partially or completely, preferably completely, uridine). Such J- or mlM-1- or m5U-modified RNAs usually exhibit decreased immunogenicity compared to their unmodified forms and, thus, are preferred in applications where the induction of an immune response is to be avoided or minimized. In some embodiments, the RNA (preferably mRNA) contains N(l)-methylpseudouridine replacing completely uridine.

[1002] The codons of the RNA (in particular, mRNA) described in the present disclosure may further be optimized, e.g., to increase the GC content of the RNA and / or to replace codons which are rare in the cell (or subject) in which the peptide or polypeptide of interest is to be expressed by codons which are synonymous frequent codons in said cell (or subject). In some embodiments, the amino acid sequence encoded by the RNA (in particular, mRNA) described in the present disclosure is encoded by a coding sequence which is codon-optimized and / or the G / C content of which is increased compared to wild type coding sequence. This also includes embodiments, wherein one or more sequence regions of the coding sequence are codon-optimized and / or increased in the G / C content compared to the correspondingsequence regions of the wild type coding sequence. In some embodiments, the codonoptimization and / or the increase in the G / C content preferably does not change the sequence of the encoded amino acid sequence.

[1003] The term "codon-optimized" refers to the alteration of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism without preferably altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, coding regions may be codon-optimized for optimal expression in a subject to be treated using the RNA (in particular, mRNA) described herein. Codon-optimization is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells. Thus, the sequence of RNA (in particular, mRNA) may be modified such that codons for which frequently occurring tRNAs are available are inserted in place of "rare codons".

[1004] In some embodiments, the guanosine / cytosine (G / C) content of the coding region of the RNA (in particular, mRNA) described herein is increased compared to the G / C content of the corresponding coding sequence of the wild type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence encoded by the wild type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that RNA. Sequences having an increased G (guanosine) / C (cytosine) content are more stable than sequences having an increased A (adenosine) / U (uracil) content. In respect to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), the most favorable codons for the stability can be determined (so-called alternative codon usage). Depending on the amino acid to be encoded by the RNA, there are various possibilities for modification of the RNA sequence, compared to its wild type sequence. In particular, codons which contain A and / or U nucleotides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and / or U or contain a lower content of A and / or U nucleotides.

[1005] In various embodiments, the G / C content of the coding region of the RNA (in particular, mRNA) described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%,at least 50%, at least 55%, or even more compared to the G / C content of the coding region of the wild type RNA.

[1006] A combination of the above described modifications, i.e., incorporation of a 5'-cap structure, incorporation of a poly-A sequence, unmasking of a poly-A sequence, alteration of the 5'-and / or 3'-UTR (such as incorporation of one or more 3'-UTRs), replacing one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine and / or pseudouridine (if) or N(l)-methyl pseudouridine (mlM-1) or 5-methyluridine (m5U) for uridine), and codon optimization, has a synergistic influence on the stability of RNA (preferably mRNA) and increase in translation efficiency. Thus, in some embodiments, the RNA (in particular, mRNA) described in the present disclosure contains a combination of at least two, at least three, at least four or all five of the above-mentioned modifications, i.e., (i) incorporation of a 5'-cap structure, (ii) incorporation of a poly-A sequence, unmasking of a poly-A sequence; (iii) alteration of the 5'- and / or 3'-UTR (such as incorporation of one or more 3'-UTRs); (iv) replacing one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine and / or pseudouridine (if) or N(l)-methylpseudouridine (mlM-1) or 5-methyluridine (m5U) for uridine), and (v) codon optimization.

[1007] Particles

[1008] In some embodiments, an agent to be delivered to a subject, e.g., a nucleic acid, in particular RNA, is encapsulated in a particle. In some embodiments, the agent is formulated in (e.g., encapsulated in) a particle, as further described herein. In some embodiments, a particle is a nucleic acid particle wherein the nucleic acid particle comprises a nucleic acid (e.g., mRNA), and a cationic lipid, a cationical ly ionizable lipid, or a cationic polymer.

[1009] A "nucleic acid particle", as used herein, refers to a particle that encompasses or contains a nucleic acid, and, is part of a composition (e.g., a pharmaceutical composition) comprising multiple nucleic acid particles, that is useful for (i) enhancing nucleic acid stability, e.g., during storage, (ii) improving biodistribution of the nucleic acid or delivering a nucleic acid to a target site of interest (e.g., cell, tissue, organ, and the like), and / or (iii) facilitating cell uptake of the nucleic acid. As described herein, a nucleic acid particle may be formed from i) at least one cationic or cationically ionizable lipid or lipid-like m...

Claims

1. Claims1. A kit comprising:(i) a compound comprising at least two binding moieties fora target antigen and a binding moiety for a tag, or a nucleic acid encoding said compound; and(ii) a compound comprising a payload moiety and at least two tags to which the binding moiety for a tag binds.

2. The kit of claim 1, wherein the nucleic acid is RNA.

3. The kit of claim 1 or 2, wherein the compound under (i) comprises two binding moieties for a target antigen.

4. The kit of any one of claims 1 to 3, wherein the total number of binding moieties for a target antigen in the compound under (i) is two.

5. The kit of any one of claims 1 to 4, wherein the compound under (i) comprises one binding moiety for a tag.

6. The kit of any one of claims 1 to 5, wherein the total number of binding moieties for a tag in the compound under (i) is one.

7. The kit of any one of claims 1 to 6, wherein the compound under (i) comprises two binding moieties for a target antigen and one binding moiety for a tag.

8. The kit of any one of claims 1 to 7 , wherein the total number of binding moieties for a target antigen in the compound under (i) is two and the total number of binding moieties for a tag in the compound under (i) is one.

9. The kit of any one of claims 1 to 8, wherein the target antigen is a tumor antigen.

10. The kit of any one of claims 1 to 9, wherein the target antigen is claudin 6 (CLDN6).

11. The kit of any one of claims 1 to 10, wherein the tag is a peptide tag.

12. The kit of any one of claims 1 to 11, wherein the tag is an ALFA-tag.

13. The kit of any one of claims 1 to 12, wherein the tag is a cyclic ALFA-tag.

14. The kit of any one of claims 1 to 13, wherein a binding moiety for a target antigen comprises at least one variable domain.

15. The kit of any one of claims 1 to 14, wherein a binding moiety for a target antigen comprises an immunoglobulin heavy chain variable region (VH) domain and an immunoglobulin light chain variable region (VL) domain, or a single-domain antibody variable domain (VHH).

16. The kit of any one of claims 1 to 15, wherein a binding moiety for a tag comprises at least one variable domain.

17. The kit of any one of claims 1 to 16, wherein a binding moiety for a tag comprises an immunoglobulin heavy chain variable region (VH) domain and an immunoglobulin light chain variable region (VL) domain, or a single-domain antibody variable domain (VHH).

18. The kit of any one of claims 1 to 17, wherein a binding moiety for a target antigen comprises a VH domain and a VL domain, or a VHH domain and a binding moiety for a tag comprises a VHH domain.

19. The kit of any one of claims 1 to 18, wherein the two binding moieties for a target antigen each comprises a VH domain and a VL domain, or a VHH domain and a binding moiety for a tag comprises a VHH domain.

20. The kit of any one of claims 1 to 19, wherein a binding moiety for a target antigen comprises a Fab fragment or a single chain fragment variable (scFv) and a binding moiety for a tag comprises a VHH domain.

21. The kit of any one of claims 1 to 20, wherein the two binding moieties for a target antigen each comprises a Fab fragment or a scFv and a binding moiety for a tag comprises a VHH domain.

22. The kit of any one of claims 15 to 21, wherein a VH domain and a VL domain interact to form a binding moiety.

23. The kit of any one of claims 1 to 22, wherein the binding moiety for a target antigen comprises a VH domain of an immunoglobulin with specificity for CLDN6 (VH(CLDN6)) and a VL domain of an immunoglobulin with specificity for CLDN6 (VL(CLDN6)).

24. The kit of any one of claims 1 to 23, wherein the binding moiety for a tag comprises an ALFA-tag binding nanobody (NbALFA).

25. The kit of any one of claims 1 to 24, wherein the compound under (i) comprises two Fab fragments binding to a target antigen, wherein the Fab fragments are connected via a peptide linker (tandem Fab), and wherein the tandem Fab is linked to a VHH binding to a tag.

26. The kit of any one of claims 1 to 25, wherein the compound under (i) comprises: (i) a polypeptide comprising, from N-terminus to C-terminus:a first Fab heavy chain (VH-CH1); an optional linker LI; a second Fab heavy chain (VH-CH1); an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag; (ii) a polypeptide comprising a first Fab light chain (VL-CL); and(iii) a polypeptide comprising a second Fab light chain (VL-CL),wherein the first Fab heavy chain of (i) and the first Fab light chain of (ii) form a Fab fragment binding to a target antigen and the second Fab heavy chain of (i) and the second Fab light chain of (iii) form a Fab fragment binding to a target antigen.18427. The kit of any one of claims 1 to 26, wherein the compound under (i) comprises: (i) a first polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for the target antigen (VH(TA)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for the target antigen (VH(TA)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag;(ii) a second polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for the target antigen (VL(TA)) and an immunoglobulin light chain constant region (CL) domain; and(iii) a third polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for the target antigen (VL(TA)) and an immunoglobulin light chain constant region (CL) domain,wherein a VH(TA) and a VL(TA) interact to form a binding moiety for the target antigen.

28. The kit of any one of claims 1 to 27, wherein the compound under (i) comprises: (i) a first polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for CLDN6 (VH(CLDN6)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for CLDN6 (VH(CLDN6)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker L2; and an ALFA-tag binding nanobody (NbALFA);(ii) a second polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for CLDN6 (VL(CLDN6)) and an immunoglobulin light chain constant region (CL) domain; and(iii) a third polypeptide comprising, from N-terminus to C-terminus:185an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for CLDN6 (VL(CLDN6)) and an immunoglobulin light chain constant region (CL) domain,wherein a VH(CLDN6) and a VL(CLDN6) interact to form a binding moiety for CLDN6.

29. The kit of any one of claims 26 to 28, wherein the linker LI and / or the linker L2 comprises the amino acid sequence EPKSC or a functional variant thereof.

30. The kit of any one of claims 26 to 29, wherein the linker LI and / or the linker L2 comprises a GS sequence.

31. The kit of any one of claims 26 to 30, wherein the linker LI comprises the amino acid sequence EPKSCSGPGGGRSGGGGSGGGGS or a functional variant thereof.

32. The kit of any one of claims 26 to 31, wherein the linker L2 comprises the amino acid sequence EPKSCGGGGSGGGS or a functional variant thereof.

33. The kit of any one of claims 1 to 32, wherein the compound under (i) comprises: (i) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a functional variant thereof;(ii) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof; and(iii) a third polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof,wherein the first polypeptide and the second polypeptide interact to form a binding moiety for CLDN6 and the first polypeptide and the third polypeptide interact to form a binding moiety for CLDN6.

34. The kit of any one of claims 1 to 24, wherein the compound under (i) comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first dimerization domain and the second polypeptide comprises a second dimerization domain, said first and second dimerization domains facilitating preferential formation of a heterodimer186comprising the first polypeptide and the second polypeptide compared to formation of a homodimer comprising two of the first polypeptides or two of the second polypeptides.

35. The kit of claim 34, wherein the first and second dimerization domains each comprise an immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof.

36. The kit of claim 34 or 35, wherein the first and / or second dimerization domain comprises a CH3 domain variant comprising one or more modifications in the CH3 domain that enhance the formation of a heterodimer comprising the first polypeptide and the second polypeptide.

37. The kit of any one of claims 34 to 36, wherein the first polypeptide comprises:(i) an immunoglobulin heavy chain variable region (VH) domain and an immunoglobulin light chain variable region (VL) domain,(ii) an immunoglobulin heavy chain variable region (VH) domain and the compound under (i) comprises a third polypeptide comprising an immunoglobulin light chain variable region (VL) domain, or(iii) a single-domain antibody variable domain (VHH),wherein the VH and VL form a binding moiety for a target antigen, or the VHH domain binds to a target antigen.

38. The kit of claim 37, wherein the VH domain or the VHH domain, and optionally the VL domain are located N-terminal of the first dimerization domain.

39. The kit of any one of claims 34 to 38, wherein the second polypeptide comprises: (i) an immunoglobulin heavy chain variable region (VH) domain and an immunoglobulin light chain variable region (VL) domain,(ii) an immunoglobulin heavy chain variable region (VH) domain and the compound under (i) comprises a fourth polypeptide comprising an immunoglobulin light chain variable region (VL) domain, or(iii) a single-domain antibody variable domain (VHH),187wherein the VH domain and the VL domain form a binding moiety for a target antigen, or the VHH domain binds to a target antigen.

40. The kit of claim 39, wherein the VH domain or the VHH domain, and optionally the VL domain are located N-terminal of the second dimerization domain.

41. The kit of any one of claims 34 to 40, wherein the first polypeptide or the second polypeptide comprises a single-domain antibody variable domain (VHH) binding to a tag.

42. The kit of any one of claims 1 to 24 and 34 to 41, wherein a binding moiety for a target antigen comprises a Fab fragment and a binding moiety for a tag comprises a VHH domain.

43. The kit of any one of claims 1 to 24 and 34 to 42, wherein the compound under (i) comprises:(i) a polypeptide comprising, from N-terminus to C-terminus:a first Fab heavy chain (VH-CH1); an optional linker LI; a first dimerization domain; an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag;(ii) a polypeptide comprising, from N-terminus to C-terminus:a second Fab heavy chain (VH-CH1); an optional linker LI; and a second dimerization domain; (iii) a polypeptide comprising a first Fab light chain (VL-CL); and(iv) a polypeptide comprising a second Fab light chain (VL-CL),wherein the first Fab heavy chain of (i) and the first Fab light chain of (iii) form a Fab fragment binding to a target antigen and the second Fab heavy chain of (ii) and the second Fab light chain of (iv) form a Fab fragment binding to a target antigen.

44. The kit of any one of claims 1 to 24 and 34 to 43, wherein the compound under (i) comprises:(i) a first polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for the target antigen (VH(TA)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; a first immunoglobulin heavy chain constant region 3188(CH3) domain ora variant thereof; an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag;(ii) a second polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for the target antigen (VH(TA)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; and a second immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof;(iii) a third polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for the target antigen (VL(TA)) and an immunoglobulin light chain constant region (CL) domain; and(iv) a fourth polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for the target antigen (VL(TA)) and an immunoglobulin light chain constant region (CL) domain,wherein a VH(TA) and a VL(TA) interact to form a binding moiety for the target antigen.

45. The kit of any one of claims 1 to 24 and 34 to 44, wherein the compound under (i) comprises:(i) a first polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for CLDN6 (VH(CLDN6)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; a first immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof; an optional linker L2; and an ALFA-tag binding nanobody (NbALFA);(ii) a second polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for CLDN6 (VH(CLDN6)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; and a second immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof;(iii) a third polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for CLDN6 (VL(CLDN6)) and an immunoglobulin light chain constant region (CL) domain; and(iv) a fourth polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for CLDN6 (VL(CLDN6)) and an immunoglobulin light chain constant region (CL) domain,wherein a VH(CLDN6) and a VL(CLDN6) interact to form a binding moiety for CLDN6.

46. The kit of any one of claims 43 to 45, wherein the first and / or second polypeptides do not comprise an immunoglobulin heavy chain constant region 2 (CH2) domain located N-terminal of the dimerization domain and the CH3 domain or variant thereof, respectively, and C-terminal of the Fab heavy chain and the CHI domain, respectively.

47. The kit of any one of claims 34 to 46, wherein the first and / or second polypeptides do not comprise an immunoglobulin heavy chain constant region 2 (CH2) domain.

48. The kit of any one of claims 43 to 47, wherein the linker LI comprises the amino acid sequence EPKSCDKTHTCPPC or a functional variant thereof.

49. The kit of any one of claims 43 to 48, wherein the linker LI and / or the linker L2 comprises a GS sequence.

50. The kit of any one of claims 43 to 49, wherein the linker LI comprises the amino acid sequence EPKSCDKTHTCPPCGGGSSGGGSG or a functional variant thereof.

51. The kit of any one of claims 43 to 50, wherein the linker L2 comprises the amino acid sequence GGGGSGGGS or a functional variant thereof.

52. The kit of any one of claims 1 to 24 and 34 to 51, wherein the compound under (i) comprises:(i) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5 or a functional variant thereof;(ii) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6 or a functional variant thereof;(iii) a third polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof; and(iv) a fourth polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof,wherein the first polypeptide and the third polypeptide interact to form a binding moiety for CLDN6 and the second polypeptide and the fourth polypeptide interact to form a binding moiety for CLDN6.

53. The kit of any one of claims 34 to 45 and 48 to 52, wherein the first and / or second polypeptides comprise an immunoglobulin heavy chain constant region 2 (CH2) domain.

54. The kit of any one of claims 43 to 45 and 48 to 53, wherein the first and / or second polypeptides comprise an immunoglobulin heavy chain constant region 2 (CH2) domain located N-terminal of the dimerization domain and the CH3 domain or variant thereof, respectively, and C-terminal of the Fab heavy chain and the CHI domain, respectively.

55. The kit of any one of claims 1 to 24, 34 to 45 and 48 to 54, wherein the compound under (i) comprises:(i) a first polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for the target antigen (VH(TA)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; an immunoglobulin heavy chain constant region 2 (CH2) domain; a first immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof; an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag;(ii) a second polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for the target antigen (VH(TA)); an immunoglobulin heavy chain constant region 1(CHI) domain; an optional linker LI; an immunoglobulin heavy chain constant region 2 (CH2) domain; and a second immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof;(iii) a third polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for the target antigen (VL(TA)) and an immunoglobulin light chain constant region (CL) domain; and(iv) a fourth polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for the target antigen (VL(TA)) and an immunoglobulin light chain constant region (CL) domain,wherein a VH(TA) and a VL(TA) interact to form a binding moiety for the target antigen.

56. The kit of any one of claims 1 to 24, 34 to 45 and 48 to 55, wherein the compound under (i) comprises:(i) a first polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for CLDN6 (VH(CLDN6)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; an immunoglobulin heavy chain constant region 2 (CH2) domain; a first immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof; an optional linker L2; and an ALFA-tag binding nanobody (NbALFA);(ii) a second polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for CLDN6 (VH(CLDN6)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; an immunoglobulin heavy chain constant region 2 (CH2) domain; and a second immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof;(iii) a third polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for CLDN6 (VL(CLDN6)) and an immunoglobulin light chain constant region (CL) domain; and(iv) a fourth polypeptide comprising, from N-terminus to C-terminus:192an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for CLDN6 (VL(CLDN6)) and an immunoglobulin light chain constant region (CL) domain,wherein a VH(CLDN6) and a VL(CLDN6) interact to form a binding moiety binding to CLDN6.

57. The kit of any one of claims 43 to 56, wherein the linker LI comprises the amino acid sequence EPKSCDKTHTCPPCP or a functional variant thereof.

58. The kit of any one of claims 43 to 57, wherein the linker L2 comprises a GS sequence.

59. The kit of any one of claims 43 to 58, wherein the linker L2 comprises the amino acid sequence GGGGSGGGS or a functional variant thereof.

60. The kit of any one of claims 1 to 24, 34 to 45 and 48 to 59, wherein the compound under (i) comprises:(i) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8 or a functional variant thereof;(ii) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 9 or a functional variant thereof;(iii) a third polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof; and(iv) a fourth polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof,wherein the first polypeptide and the third polypeptide interact to form a binding moiety for CLDN6 and the second polypeptide and the fourth polypeptide interact to form a binding moiety for CLDN6.

61. The kit of any one of claims 1 to 24 and 34 to 41, wherein a binding moiety for a target antigen comprises a single chain fragment variable (scFv) and a binding moiety for a tag comprises a single-domain antibody variable domain (VHH).19362. The kit of any one of claims 1 to 24, 34 to 41 and 61, wherein the compound under (i) comprises:(i) a polypeptide comprising, from N-terminus to C-terminus:(a) a first immunoglobulin heavy chain variable region (VH) domain; an optional linker L3; a first immunoglobulin light chain variable region (VL) domain; an optional linker LI; a first dimerization domain; an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag; or(b) a first immunoglobulin light chain variable region (VL) domain; an optional linker L3; a first immunoglobulin heavy chain variable region (VH) domain; an optional linker LI; a first dimerization domain; an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag;(ii) a polypeptide comprising, from N-terminus to C-terminus:(a) a second immunoglobulin heavy chain variable region (VH) domain; an optional linker L3; a second immunoglobulin light chain variable region (VL) domain; an optional linker LI; and a second dimerization domain; or(b) a second immunoglobulin light chain variable region (VL) domain; an optional linker L3; a second immunoglobulin heavy chain variable region (VH) domain; an optional linker LI; and a second dimerization domain;wherein the first VH domain and the first VL domain form a binding moiety for a target antigen and the second VH domain and the second VL domain form a binding moiety for a target antigen.

63. The kit of any one of claims 1 to 19, 24 and 34 to 41, wherein a binding moiety for a target antigen comprises a single-domain antibody variable domain (VHH) and a binding moiety for a tag comprises a single-domain antibody variable domain (VHH).

64. The kit of any one of claims 1 to 19, 24, 34 to 41 and 63, wherein the compound under (i) comprises:(i) a polypeptide comprising, from N-terminus to C-terminus:a first single-domain antibody variable domain (VHH) binding to a target antigen; an optional linker LI; a first dimerization domain; an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag; and194(ii) a polypeptide comprising, from N-terminus to C-terminus:a second single-domain antibody variable domain (VHH) binding to a target antigen; an optional linker LI; and a second dimerization domain.

65. The kit of any one of claims 1 to 19, 24, 34 to 41 and 63, wherein the compound under (i) comprises:(i) a polypeptide comprising, from N-terminus to C-terminus:a first single-domain antibody variable domain (VHH) binding to a target antigen; an optional linker L2; a single-domain antibody variable domain (VHH) binding to a tag; an optional linker LI; and a first dimerization domain; and(ii) a polypeptide comprising, from N-terminus to C-terminus:a second single-domain antibody variable domain (VHH) binding to a target antigen; an optional linker LI; and a second dimerization domain.

66. The kit of any one of claims 15 to 65, wherein the immunoglobulin is a human immunoglobulin.

67. The kit of any one of claims 15 to 66, wherein the immunoglobulin is IgGl.

68. The kit of any one of claims 15 to 62, 66 and 67, wherein the VH(TA) or VH(CLDN6) comprises a CDR1 comprising the amino acid sequence GYSFTGYT or a functional variant thereof, a CDR2 comprising the amino acid sequence INPYNGGT or a functional variant thereof, and a CDR3 comprising the amino acid sequence ARDYGFVLDY or a functional variant thereof and the VL(TA) or VL(CLDN6) comprises a CDR1 comprising the amino acid sequence SSVSY or a functional variant thereof, a CDR2 comprising the amino acid sequence STS or a functional variant thereof, and a CDR3 comprising the amino acid sequence QQRSNYPPWT or a functional variant thereof.

69. The kit of any one of claims 15 to 62, and 66 to 68, wherein the VH(TA) or VH(CLDN6) comprises the amino acid sequence shown in SEQ ID NO: 10 or a functional variant thereof and the VL(TA) or VL(CLDN6) comprises the amino acid sequence shown in SEQ ID NO: 11 or a functional variant thereof.19570. The kit of any one of claims 17 to 69, wherein the VHH binding to a tag or NbALFA comprises the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13 or a functional variant thereof.

71. The kit of any one of claims 26 to 33, 43 to 60 and 66 to 70, wherein the CHI domain comprises the amino acid sequence shown in SEQ ID NO: 14 or a functional variant thereof.

72. The kit of any one of claims 26 to 33, 43 to 60 and 66 to 71, wherein the CL domain comprises the amino acid sequence shown in SEQ ID NO: 15 or a functional variant thereof.

73. The kit of any one of claims 34 to 72, wherein the first dimerization domain or first CH3 domain or variant thereof comprises the amino acid sequence shown in SEQ ID NO: 16 or a functional variant thereof and the second dimerization domain or second CH3 domain or variant thereof comprises the amino acid sequence shown in SEQ ID NO: 17 or a functional variant thereof.

74. The kit of any one of claims 46 to 60 and 66 to 73, wherein the CH2 domain comprises the amino acid sequence shown in SEQ ID NO: 18 or a functional variant thereof.

75. The kit of any one of claims 1 to 74, wherein the compound under (ii) comprises a moiety comprising a polymer.

76. The kit of any one of claims 1 to 75, wherein the payload moiety and the tags are coupled through a moiety comprising a polymer.

77. The kit of claim 75 or 76, wherein the polymer is not a polymer of proteinogenic amino acids or their D-isomers.

78. The kit of any one of claims 75 to 77, wherein the polymer is selected from the group consisting of poly(ethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine)), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), derivatives and combinations thereof.19679. The kit of any one of claims 75 to 78, wherein the polymer comprises at least one poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

80. The kit of any one of claims 1 to 79, wherein the compound under (ii) comprises two of said tags.

81. The kit of any one of claims 1 to 80, wherein the total number of tags in the compound under (ii) is two.

82. The kit of any one of claims 1 to 81, wherein a tag in the compound under (ii) comprises the amino acid sequence Ser-Arg-Leu-Glu-(cyclo5)Asp-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu, or Pro-Ser-Arg-Leu-(cyclo5)Glu-Glu-Glu-Leu-(cyclo9)Lys-Arg-Arg-Leu-Thr-Glu, in particular, wherein the compound under (ii) comprises the formula shown in Figure 10 or Figure 11, wherein, in particular, the compound under (ii) comprises the formula shown in Figure 11,in particular, wherein the compound under (ii) comprises the formula shown in Figure 10 or Figure 11, wherein the DOTA moiety in the formula chelates a radiodiagnostic isotope, wherein, in particular, the compound under (ii) comprises the formula shown in Figure 11, wherein the DOTA moiety in the formula chelates a radiodiagnostic isotope,in particular, wherein the compound under (ii) comprises the formula shown in Figure 10 or Figure 11, wherein the DOTA moiety in the formula chelates68Ga, wherein, in particular, the compound under (ii) comprises the formula shown in Figure 11, wherein the DOTA moiety in the formula chelates68Ga.

83. The kit of any one of claims 1 to 82, wherein the compound under (ii) comprises payload moieties and tags in a branched (non-linear) configuration or an unbranched (linear) configuration.

84. The kit of any one of claims 1 to 83, wherein the payload moiety comprises a radioisotope, e.g., a chelating compound comprising a radioisotope, a toxin or an immunomodulator.19785. A compound comprising at least two binding moieties for a target antigen and a binding moiety for a tag, or a nucleic acid encoding said compound.

86. The compound of claim 85, wherein the nucleic acid is RNA.

87. The compound of claim 85 or 86, wherein the compound comprises two binding moieties for a target antigen.

88. The compound of any one of claims 85 to 87, wherein the total number of binding moieties for a target antigen in the compound is two.

89. The compound of any one of claims 85 to 88, wherein the compound comprises one binding moiety for a tag.

90. The compound of any one of claims 85 to 89, wherein the total number of binding moieties for a tag in the compound is one.

91. The compound of any one of claims 85 to 90, wherein the compound comprises two binding moieties for a target antigen and one binding moiety for a tag.

92. The compound of any one of claims 85 to 91, wherein the total number of binding moieties for a target antigen in the compound is two and the total number of binding moieties for a tag in the compound is one.

93. The compound of any one of claims 85 to 92, wherein the target antigen is a tumor antigen.

94. The compound of any one of claims 85 to 93, wherein the target antigen is claudin 6 (CLDN6).

95. The compound of any one of claims 85 to 94, wherein the tag is a peptide tag.19896. The compound of any one of claims 85 to 95, wherein the tag is an ALFA-tag.

97. The compound of any one of claims 85 to 96, wherein the tag is a cyclic ALFA-tag.

98. The compound of any one of claims 85 to 97, wherein a binding moiety for a target antigen comprises at least one variable domain.

99. The compound of any one of claims 85 to 98, wherein a binding moiety for a target antigen comprises an immunoglobulin heavy chain variable region (VH) domain and an immunoglobulin light chain variable region (VL) domain, or a single-domain antibody variable domain (VHH).

100. The compound of any one of claims 85 to 99, wherein a binding moiety for a tag comprises at least one variable domain.

101. The compound of any one of claims 85 to 100, wherein a binding moiety for a tag comprises an immunoglobulin heavy chain variable region (VH) domain and an immunoglobulin light chain variable region (VL) domain, or a single-domain antibody variable domain (VHH).

102. The compound of any one of claims 85 to 101, wherein a binding moiety for a target antigen comprises a VH domain and a VL domain, or a VHH domain and a binding moiety for a tag comprises a VHH domain.

103. The compound of any one of claims 85 to 102, wherein the two binding moieties for a target antigen each comprises a VH domain and a VL domain, or a VHH domain and a binding moiety for a tag comprises a VHH domain.

104. The compound of any one of claims 85 to 103, wherein a binding moiety for a target antigen comprises a Fab fragment or a single chain fragment variable (scFv) and a binding moiety for a tag comprises a VHH domain.199105. The compound of any one of claims 85 to 104, wherein the two binding moieties for a target antigen each comprises a Fab fragment or a scFv and a binding moiety for a tag comprises a VHH domain.

106. The compound of any one of claims 99 to 105, wherein a VH domain and a VL domain interact to form a binding moiety.

107. The compound of any one of claims 85 to 106, wherein the binding moiety for a target antigen comprises a VH domain of an immunoglobulin with specificity for CLDN6 (VH(CLDN6)) and a VL domain of an immunoglobulin with specificity for CLDN6 (VL(CLDN6)).

108. The compound of any one of claims 85 to 107, wherein the binding moiety for a tag comprises an ALFA-tag binding nanobody (NbALFA).

109. The compound of any one of claims 85 to 108, wherein the compound comprises two Fab fragments binding to a target antigen, wherein the Fab fragments are connected via a peptide linker (tandem Fab), and wherein the tandem Fab is linked to a VHH binding to a tag.

110. The compound of any one of claims 85 to 109, wherein the compound comprises: (i) a polypeptide comprising, from N-terminus to C-terminus:a first Fab heavy chain (VH-CH1); an optional linker LI; a second Fab heavy chain (VH-CH1); an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag; (ii) a polypeptide comprising a first Fab light chain (VL-CL); and(iii) a polypeptide comprising a second Fab light chain (VL-CL),wherein the first Fab heavy chain of (i) and the first Fab light chain of (ii) form a Fab fragment binding to a target antigen and the second Fab heavy chain of (i) and the second Fab light chain of (iii) form a Fab fragment binding to a target antigen.

111. The compound of any one of claims 85 to 110, wherein the compound comprises: (i) a first polypeptide comprising, from N-terminus to C-terminus:200an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for the target antigen (VH(TA)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for the target antigen (VH(TA)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag;(ii) a second polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for the target antigen (VL(TA)) and an immunoglobulin light chain constant region (CL) domain; and(iii) a third polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for the target antigen (VL(TA)) and an immunoglobulin light chain constant region (CL) domain,wherein a VH(TA) and a VL(TA) interact to form a binding moiety for the target antigen.

112. The compound of any one of claims 85 to 111, wherein the compound comprises: (i) a first polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for CLDN6 (VH(CLDN6)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for CLDN6 (VH(CLDN6)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker L2; and an ALFA-tag binding nanobody (NbALFA);(ii) a second polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for CLDN6 (VL(CLDN6)) and an immunoglobulin light chain constant region (CL) domain; and(iii) a third polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for CLDN6 (VL(CLDN6)) and an immunoglobulin light chain constant region (CL) domain,wherein a VH(CLDN6) and a VL(CLDN6) interact to form a binding moiety for CLDN6.

113. The compound of any one of claims 110 to 112, wherein the linker LI and / orthe linker L2 comprises the amino acid sequence EPKSC or a functional variant thereof.

114. The compound of any one of claims 110 to 113, wherein the linker LI and / orthe linker L2 comprises a GS sequence.

115. The compound of any one of claims 110 to 114, wherein the linker LI comprises the amino acid sequence EPKSCSGPGGGRSGGGGSGGGGS or a functional variant thereof.

116. The compound of any one of claims 110 to 115, wherein the linker L2 comprises the amino acid sequence EPKSCGGGGSGGGS or a functional variant thereof.

117. The compound of any one of claims 85 to 116, wherein the compound comprises: (i) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a functional variant thereof;(ii) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof; and(iii) a third polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof,wherein the first polypeptide and the second polypeptide interact to form a binding moiety for CLDN6 and the first polypeptide and the third polypeptide interact to form a binding moiety for CLDN6.

118. The compound of any one of claims 85 to 108, wherein the compound comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first dimerization domain and the second polypeptide comprises a second dimerization domain, said first and second dimerization domains facilitating preferential formation of a heterodimer comprising the first polypeptide and the second polypeptide compared to formation of a homodimer comprising two of the first polypeptides or two of the second polypeptides.

119. The compound of claim 118, wherein the first and second dimerization domains each comprise an immunoglobulin heavy chain constant region 3 (CH3) domain ora variant thereof.

120. The compound of claim 118 or 119, wherein the first and / or second dimerization domain comprises a CH3 domain variant comprising one or more modifications in the CH3 domain that enhance the formation of a heterodimer comprising the first polypeptide and the second polypeptide.

121. The compound of any one of claims 118 to 120, wherein the first polypeptide comprises:(i) an immunoglobulin heavy chain variable region (VH) domain and an immunoglobulin light chain variable region (VL) domain,(ii) an immunoglobulin heavy chain variable region (VH) domain and the compound comprises a third polypeptide comprising an immunoglobulin light chain variable region (VL) domain, or (iii) a single-domain antibody variable domain (VHH),wherein the VH and VL form a binding moiety for a target antigen, or the VHH domain binds to a target antigen.

122. The compound of claim 121, wherein the VH domain or the VHH domain, and optionally the VL domain are located N-terminal of the first dimerization domain.

123. The compound of any one of claims 118 to 122, wherein the second polypeptide comprises:(i) an immunoglobulin heavy chain variable region (VH) domain and an immunoglobulin light chain variable region (VL) domain,(ii) an immunoglobulin heavy chain variable region (VH) domain and the compound comprises a fourth polypeptide comprising an immunoglobulin light chain variable region (VL) domain, or(iii) a single-domain antibody variable domain (VHH),wherein the VH domain and the VL domain form a binding moiety for a target antigen, or the VHH domain binds to a target antigen.203124. The compound of claim 123, wherein the VH domain or the VHH domain, and optionally the VL domain are located N-terminal of the second dimerization domain.

125. The compound of any one of claims 118 to 124, wherein the first polypeptide or the second polypeptide comprises a single-domain antibody variable domain (VHH) binding to a tag.

126. The compound of any one of claims 85 to 108 and 118 to 125, wherein a binding moiety for a target antigen comprises a Fab fragment and a binding moiety for a tag comprises a VHH domain.

127. The compound of any one of claims 85 to 108 and 118 to 126, wherein the compound comprises:(i) a polypeptide comprising, from N-terminus to C-terminus:a first Fab heavy chain (VH-CH1); an optional linker LI; a first dimerization domain; an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag;(ii) a polypeptide comprising, from N-terminus to C-terminus:a second Fab heavy chain (VH-CH1); an optional linker LI; and a second dimerization domain; (iii) a polypeptide comprising a first Fab light chain (VL-CL); and(iv) a polypeptide comprising a second Fab light chain (VL-CL),wherein the first Fab heavy chain of (i) and the first Fab light chain of (iii) form a Fab fragment binding to a target antigen and the second Fab heavy chain of (ii) and the second Fab light chain of (iv) form a Fab fragment binding to a target antigen.

128. The compound of any one of claims 85 to 108 and 118 to 127, wherein the compound comprises:(i) a first polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for the target antigen (VH(TA)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; a first immunoglobulin heavy chain constant region 3 (CH3) domain ora variant thereof; an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag;204(ii) a second polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for the target antigen (VH(TA)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; and a second immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof;(iii) a third polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for the target antigen (VL(TA)) and an immunoglobulin light chain constant region (CL) domain; and(iv) a fourth polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for the target antigen (VL(TA)) and an immunoglobulin light chain constant region (CL) domain,wherein a VH(TA) and a VL(TA) interact to form a binding moiety for the target antigen.

129. The compound of any one of claims 85 to 108 and 118 to 128, wherein the compound comprises:(i) a first polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for CLDN6 (VH(CLDN6)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; a first immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof; an optional linker L2; and an ALFA-tag binding nanobody (NbALFA);(ii) a second polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for CLDN6 (VH(CLDN6)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; and a second immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof;(iii) a third polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for CLDN6 (VL(CLDN6)) and an immunoglobulin light chain constant region (CL) domain; and205(iv) a fourth polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for CLDN6 (VL(CLDN6)) and an immunoglobulin light chain constant region (CL) domain,wherein a VH(CLDN6) and a VL(CLDN6) interact to form a binding moiety for CLDN6.

130. The compound of any one of claims 127 to 129, wherein the first and / or second polypeptides do not comprise an immunoglobulin heavy chain constant region 2 (CH2) domain located N-terminal of the dimerization domain and the CH3 domain or variant thereof, respectively, and C-terminal of the Fab heavy chain and the CHI domain, respectively.

131. The compound of any one of claims 118 to 130, wherein the first and / or second polypeptides do not comprise an immunoglobulin heavy chain constant region 2 (CH2) domain.

132. The compound of any one of claims 127 to 131, wherein the linker LI comprises the amino acid sequence EPKSCDKTHTCPPC or a functional variant thereof.

133. The compound of any one of claims 127 to 132, wherein the linker LI and / orthe linker L2 comprises a GS sequence.

134. The compound of any one of claims 127 to 133, wherein the linker LI comprises the amino acid sequence EPKSCDKTHTCPPCGGGSSGGGSG or a functional variant thereof.

135. The compound of any one of claims 127 to 134, wherein the linker L2 comprises the amino acid sequence GGGGSGGGS or a functional variant thereof.

136. The compound of any one of claims 85 to 108 and 118 to 135, wherein the compound comprises:(i) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5 or a functional variant thereof;206(ii) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6 or a functional variant thereof;(iii) a third polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof; and(iv) a fourth polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof,wherein the first polypeptide and the third polypeptide interact to form a binding moiety for CLDN6 and the second polypeptide and the fourth polypeptide interact to form a binding moiety for CLDN6.

137. The compound of any one of claims 118 to 129 and 132 to 136, wherein the first and / or second polypeptides comprise an immunoglobulin heavy chain constant region 2 (CH2) domain.

138. The compound of any one of claims 127 to 129 and 132 to 137, wherein the first and / or second polypeptides comprise an immunoglobulin heavy chain constant region 2 (CH2) domain located N-terminal of the dimerization domain and the CH3 domain or variant thereof, respectively, and C-terminal of the Fab heavy chain and the CHI domain, respectively.

139. The compound of any one of claims 85 to 108, 118 to 129 and 132 to 138, wherein the compound comprises:(i) a first polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for the target antigen (VH(TA)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; an immunoglobulin heavy chain constant region 2 (CH2) domain; a first immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof; an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag;(ii) a second polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for the target antigen (VH(TA)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; an immunoglobulin heavy chain constant region 2 (CH2)207domain; and a second immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof;(iii) a third polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for the target antigen (VL(TA)) and an immunoglobulin light chain constant region (CL) domain; and(iv) a fourth polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for the target antigen (VL(TA)) and an immunoglobulin light chain constant region (CL) domain,wherein a VH(TA) and a VL(TA) interact to form a binding moiety for the target antigen.

140. The compound of any one of claims 85 to 108, 118 to 129 and 132 to 139, wherein the compound comprises:(i) a first polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for CLDN6 (VH(CLDN6)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; an immunoglobulin heavy chain constant region 2 (CH2) domain; a first immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof; an optional linker L2; and an ALFA-tag binding nanobody (NbALFA);(ii) a second polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin heavy chain variable region (VH) domain of an immunoglobulin with specificity for CLDN6 (VH(CLDN6)); an immunoglobulin heavy chain constant region 1 (CHI) domain; an optional linker LI; an immunoglobulin heavy chain constant region 2 (CH2) domain; and a second immunoglobulin heavy chain constant region 3 (CH3) domain or a variant thereof;(iii) a third polypeptide comprising, from N-terminus to C-terminus:an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for CLDN6 (VL(CLDN6)) and an immunoglobulin light chain constant region (CL) domain; and(iv) a fourth polypeptide comprising, from N-terminus to C-terminus:208an immunoglobulin light chain variable region (VL) domain of an immunoglobulin with specificity for CLDN6 (VL(CLDN6)) and an immunoglobulin light chain constant region (CL) domain,wherein a VH(CLDN6) and a VL(CLDN6) interact to form a binding moiety binding to CLDN6.

141. The compound of any one of claims 127 to 140, wherein the linker LI comprises the amino acid sequence EPKSCDKTHTCPPCP or a functional variant thereof.

142. The compound of any one of claims 127 to 141, wherein the linker L2 comprises a GS sequence.

143. The compound of any one of claims 127 to 142, wherein the linker L2 comprises the amino acid sequence GGGGSGGGS or a functional variant thereof.

144. The compound of any one of claims 85 to 108, 118 to 129 and 132 to 143, wherein the compound comprises:(i) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8 or a functional variant thereof;(ii) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 9 or a functional variant thereof;(iii) a third polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof; and(iv) a fourth polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof,wherein the first polypeptide and the third polypeptide interact to form a binding moiety for CLDN6 and the second polypeptide and the fourth polypeptide interact to form a binding moiety for CLDN6.

145. The compound of any one of claims 85 to 108 and 118 to 125, wherein a binding moiety for a target antigen comprises a single chain fragment variable (scFv) and a binding moiety for a tag comprises a single-domain antibody variable domain (VHH).209146. The compound of any one of claims 85 to 108, 118 to 125 and 145, wherein the compound comprises:(i) a polypeptide comprising, from N-terminus to C-terminus:(a) a first immunoglobulin heavy chain variable region (VH) domain; an optional linker L3; a first immunoglobulin light chain variable region (VL) domain; an optional linker LI; a first dimerization domain; an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag; or(b) a first immunoglobulin light chain variable region (VL) domain; an optional linker L3; a first immunoglobulin heavy chain variable region (VH) domain; an optional linker LI; a first dimerization domain; an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag;(ii) a polypeptide comprising, from N-terminus to C-terminus:(a) a second immunoglobulin heavy chain variable region (VH) domain; an optional linker L3; a second immunoglobulin light chain variable region (VL) domain; an optional linker LI; and a second dimerization domain; or(b) a second immunoglobulin light chain variable region (VL) domain; an optional linker L3; a second immunoglobulin heavy chain variable region (VH) domain; an optional linker LI; and a second dimerization domain;wherein the first VH domain and the first VL domain form a binding moiety for a target antigen and the second VH domain and the second VL domain form a binding moiety for a target antigen.

147. The compound of any one of claims 85 to 103, 108 and 118 to 125, wherein a binding moiety for a target antigen comprises a single-domain antibody variable domain (VHH) and a binding moiety for a tag comprises a single-domain antibody variable domain (VHH).

148. The compound of any one of claims 85 to 103, 108, 118 to 125 and 147, wherein the compound comprises:(i) a polypeptide comprising, from N-terminus to C-terminus:a first single-domain antibody variable domain (VHH) binding to a target antigen; an optional linker LI; a first dimerization domain; an optional linker L2; and a single-domain antibody variable domain (VHH) binding to a tag; and210(ii) a polypeptide comprising, from N-terminus to C-terminus:a second single-domain antibody variable domain (VHH) binding to a target antigen; an optional linker LI; and a second dimerization domain.

149. The compound of any one of claims 85 to 103, 108, 118 to 125 and 147, wherein the compound comprises:(i) a polypeptide comprising, from N-terminus to C-terminus:a first single-domain antibody variable domain (VHH) binding to a target antigen; an optional linker L2; a single-domain antibody variable domain (VHH) binding to a tag; an optional linker LI; and a first dimerization domain; and(ii) a polypeptide comprising, from N-terminus to C-terminus:a second single-domain antibody variable domain (VHH) binding to a target antigen; an optional linker LI; and a second dimerization domain.

150. The compound of any one of claims 99 to 149, wherein the immunoglobulin is a human immunoglobulin.

151. The compound of any one of claims 99 to 150, wherein the immunoglobulin is IgGl.

152. The compound of any one of claims 99 to 146, 150 and 151, wherein the VH(TA) or VH(CLDN6) comprises a CDR1 comprising the amino acid sequence GYSFTGYT or a functional variant thereof, a CDR2 comprising the amino acid sequence INPYNGGT or a functional variant thereof, and a CDR3 comprising the amino acid sequence ARDYGFVLDY or a functional variant thereof and the VL(TA) or VL(CLDN6) comprises a CDR1 comprising the amino acid sequence SSVSY or a functional variant thereof, a CDR2 comprising the amino acid sequence STS or a functional variant thereof, and a CDR3 comprising the amino acid sequence QQRSNYPPWT or a functional variant thereof.

153. The compound of any one of claims 99 to 146, and 150 to 152, wherein the VH(TA) or VH(CLDN6) comprises the amino acid sequence shown in SEQ ID NO: 10 or a functional variant thereof and the VL(TA) or VL(CLDN6) comprises the amino acid sequence shown in SEQ ID NO: 11 or a functional variant thereof.211154. The compound of any one of claims 101 to 153, wherein the VHH binding to a tag or NbALFA comprises the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13 or a functional variant thereof.

155. The compound of any one of claims 110 to 117, 127 to 144 and 150 to 154, wherein the CHI domain comprises the amino acid sequence shown in SEQ ID NO: 14 or a functional variant thereof.

156. The compound of any one of claims 110 to 117, 127 to 144 and 150 to 155, wherein the CL domain comprises the amino acid sequence shown in SEQ ID NO: 15 or a functional variant thereof.

157. The compound of any one of claims 118 to 156, wherein the first dimerization domain or first CH3 domain or variant thereof comprises the amino acid sequence shown in SEQ ID NO: 16 or a functional variant thereof and the second dimerization domain or second CH3 domain or variant thereof comprises the amino acid sequence shown in SEQ ID NO: 17 or a functional variant thereof.

158. The compound of any one of claims 130 to 144 and 150 to 157, wherein the CH2 domain comprises the amino acid sequence shown in SEQ ID NO: 18 or a functional variant thereof.

159. A method for treating a subject having a disease, disorder or condition characterized by cells expressing a target antigen, comprising:(i) providing to the subject a compound comprising at least two binding moieties for the target antigen and a binding moiety for a tag;(ii) allowing the compound comprising at least two binding moieties for the target antigen and a binding moiety for a tag to become associated with cells expressing the target antigen; and(iii) administering to the subject a compound comprising a payload moiety and at least two tags to which the binding moiety for a tag binds.212160. The method of claim 159, wherein the compound comprising at least two binding moieties for the target antigen and a binding moiety for a tag is a compound under (i) as set forth in any one of claims 1 to 84 and / or the compound comprising a payload moiety and at least two tags to which the binding moiety for a tag binds is a compound under (ii) as set forth in any one of claims 1 to 84.

161. The method of claim 159 or 160, wherein the compound comprising at least two binding moieties for the target antigen and a binding moiety for a tag is provided to the subject by administering to the subject RNA encoding a polypeptide comprising at least two binding moieties for the target antigen and a binding moiety for a tag; and allowing expression of the polypeptide by cells in the subject.

162. The method of claim 161, wherein the cells expressing the polypeptide are transfected with the RNA.

163. The method of claim 161 or 162, wherein the cells expressing the polypeptide secrete the polypeptide.

164. The method of any one of claims 161 to 163, wherein the cells expressing the polypeptide express the polypeptide such that it is released into the bloodstream.

165. The method of any one of claims 159 to 164, wherein the target antigen is a cell surface antigen.

166. The method of any one of claims 159 to 165, wherein the disease, disorder or condition is cancer.

167. The method of any one of claims 159 to 166, wherein the cells expressing a target antigen are diseased cells.

168. The method of any one of claims 159 to 167, wherein the cells expressing a target antigen are cancer cells.213169. The method of any one of claims 159 to 168, wherein the target antigen is a tumor antigen.

170. The method of any one of claims 159 to 169, wherein the payload moiety comprises a radioisotope, e.g., a chelating compound comprising a radioisotope, toxin or immunomodulator.214

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