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, toxins, immunomodulators or radiodiagnostics. In some embodiments, the invention involves providing to a subject a compound comprising a payload moiety and a tag (tag conjugate) and a compound binding to the tag of the tag conjugate and a target antigen, e.g., a cell surface antigen on a target cell, (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, toxins, immunomodulators or radiodiagnostics. In some embodiments, the invention involves providing to a subject a compound comprising a payload moiety and a tag (tag conjugate) and a compound binding to the tag of the tag conjugate and a target antigen, e.g., a cell surface antigen on a target cell, (docking compound). In some embodiments, the docking compound comprises a peptide or polypeptide. In some embodiments, the docking compound comprises a binding moiety binding to target cells (primary targeting moiety) and a further binding moiety binding to the tag of the tag conjugate. The tag of the tag conjugate may bind to its binding moiety on the docking compound and the primary targeting moiety of the docking compound may bind to a 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 equipped with a tag which is targeted by the moiety on the docking compound. 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 of 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. 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.

[0006] 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 a docking compound binding to the tag conjugate via its tag, said docking compound comprising a targeting molecule for targeting an antigen on target cells.

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

[0009] (i) a compound comprising a binding moiety binding to a target antigen and a binding moiety for a tag, or a nucleic acid encoding said compound; and

[0010] (ii) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the tag is a peptide tag.

[0011] In some embodiments, the compound under (ii) comprises a moiety comprising a polymer. In some embodiments, the payload moiety and the peptide tag are coupled through a moiety comprising a polymer. In some embodiments, the polymer is not a polymer of proteinogenic amino acids or their D-isomers.

[0012] In one aspect, the invention relates to a kit comprising:

[0013] (i) a compound comprising a binding moiety binding to a target antigen and a binding moiety for a tag, or a nucleic acid encoding said compound; and

[0014] (ii) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the compound under (ii) further comprises a moiety comprising a polymer, wherein the polymer is not a polymer of proteinogenic amino acids or their D-isomers.

[0015] In some embodiments, the payload moiety and the tag are coupled through a moiety comprising a polymer, wherein the polymer is not a polymer of proteinogenic amino acids or their D-isomers.

[0016] In some embodiments, the tag is a peptide tag. In some embodiments, the polymer is selected from the group consisting of polyethylene 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.

[0017] In some embodiments, the polymer comprises at least one poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0018] In some embodiments, the payload moiety and the tag are coupled through a moiety comprising at least one poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0019] In some embodiments, the nucleic acid is RNA.

[0020] In some embodiments, the compound under (ii) comprises one tag.

[0021] In some embodiments, the total number of tags in the compound under (ii) is one.

[0022] In some embodiments, the compound under (ii) comprises one or more payload moieties.

[0023] In some embodiments, the compound under (ii) comprises one payload moiety.

[0024] In some embodiments, the total number of payload moieties in the compound under (ii) is one.

[0025] In some embodiments, the compound under (ii) comprises one tag and one payload moiety.

[0026] In some embodiments, the total number of tags in the compound under (ii) is one and the total number of payload moieties in the compound under (ii) is one.

[0027] In some embodiments, the compound under (ii) comprises one tag and two payload moieties. In some embodiments, the total number of tags in the compound under (ii) is one and the total number of payload moieties in the compound under (ii) is two.

[0028] In some embodiments, the compound under (ii) is SD18317 as disclosed herein.

[0029] In some embodiments, the compound under (ii) is SD17248 as disclosed herein.

[0030] In some embodiments, the compound under (ii) is PIC12 as disclosed herein.

[0031] In some embodiments, the compound under (ii) is PIC13 as disclosed herein.

[0032] In some embodiments, the compound under (ii) is PIC14 as disclosed herein.

[0033] In some embodiments, the compound under (ii) comprises a linking moiety connecting a tag and a payload moiety.

[0034] In some embodiments, the linking moiety is a branched or an unbranched linking moiety. In some embodiments, the linking moiety comprises a continuous or non-continuous poly-2- (2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0035] In some embodiments, the compound under (ii) comprises the formula:

[0036] P-L-T wherein

[0037] P comprises a payload moiety;

[0038] T comprises a tag; and

[0039] L comprises a linking moiety.

[0040] In some embodiments, L comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0041] In some embodiments, L comprises the formula [AEEA]u-[L'-[AEEA]v]w, wherein

[0042] AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid or a derivative thereof;

[0043] L1comprises a linking moiety; u is an integer of 2 or more; each v is an integer of 2 or more; and w is an integer from 1 to 4; wherein the different groups [L'-[AEEA]V] may be identical or different.

[0044] In some embodiments, u and v are each integers from 2 to 10.

[0045] In some embodiments, u and v are each integers from 2 to 8.

[0046] In some embodiments, u and v are each integers of 2, 4 or 6.

[0047] In some embodiments, L or L' comprises an amino acid.

[0048] In some embodiments, Lor L' comprises the D-isomer of an amino acid.

[0049] In some embodiments, Lor L' comprises cysteine or lysine.

[0050] In some embodiments, L or L' is connected to a side chain.

[0051] In some embodiments, a side chain comprises a functional moiety.

[0052] In some embodiments, a functional moiety comprises a solubilizing functional group.

[0053] In some embodiments, a functional moiety comprises a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof. In some embodiments, the payload moiety is selected from the group consisting of radioisotopes, toxins and immunomodulators. In some embodiments, the payload moiety comprises a chelating compound comprising a radioisotope. In some embodiments, a chelating compound in the compound under (ii) is covalently linked with the tag and chelates a radioisotope, such as radionuclides and radiolabels.

[0054] 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.

[0055] 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.

[0056] 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 2, 4 or 6.

[0057] In some embodiments, the compound under (i) comprises a single binding moiety for the tag or at least two, e.g., two, binding moieties for the tag.

[0058] In some embodiments, the tag is an ALFA-tag.

[0059] In some embodiments, the tag is a cyclic ALFA-tag.

[0060] In one aspect, the invention relates to the compound under (ii) described above.

[0061] In one aspect, the invention relates to a compound comprising a payload moiety and a peptide tag.

[0062] In some embodiments, the compound comprises a moiety comprising a polymer.

[0063] In some embodiments, the payload moiety and the peptide tag are coupled through a moiety comprising a polymer. In some embodiments, the polymer is not a polymer of proteinogenic amino acids or their D-isomers.

[0064] In one aspect, the invention relates to a compound comprising a payload moiety and a tag, wherein the compound further comprises a moiety comprising a polymer, wherein the polymer is not a polymer of proteinogenic amino acids or their D-isomers. In some embodiments, the payload moiety and the tag are coupled through a moiety comprising a polymer, wherein the polymer is not a polymer of proteinogenic amino acids or their D-isomers.

[0065] In some embodiments, the tag is a peptide tag.

[0066] In some embodiments, the polymer is selected from the group consisting of polyethylene 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.

[0067] In some embodiments, the polymer comprises at least one poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0068] In some embodiments, the payload moiety and the tag are coupled through a moiety comprising at least one poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0069] In some embodiments, the payload moiety and the tag are coupled through a moiety comprising at least one poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety

[0070] In some embodiments, the compound comprises one tag.

[0071] In some embodiments, the total number of tags in the compound is one.

[0072] In some embodiments, the compound comprises one or more payload moieties.

[0073] In some embodiments, the compound comprises one payload moiety.

[0074] In some embodiments, the total number of payload moieties in the compound is one.

[0075] In some embodiments, the compound comprises one tag and one payload moiety.

[0076] In some embodiments, the total number of tags in the compound is one and the total number of payload moieties in the compound is one.

[0077] In some embodiments, the compound comprises one tag and two payload moieties.

[0078] In some embodiments, the total number of tags in the compound is one and the total number of payload moieties in the compound is two.

[0079] In some embodiments, the compound is SD18317 as disclosed herein.

[0080] In some embodiments, the compound is SD17248 as disclosed herein.

[0081] In some embodiments, the compound is PIC12 as disclosed herein.

[0082] In some embodiments, the compound is PIC13 as disclosed herein. In some embodiments, the compound is PIC14 as disclosed herein.

[0083] In some embodiments, the compound comprises a linking moiety connecting a tag and a payload moiety.

[0084] In some embodiments, the linking moiety is a branched or an unbranched linking moiety.

[0085] In some embodiments, the linking moiety comprises a continuous or non-continuous poly-2- (2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0086] In some embodiments, the compound comprises the formula:

[0087] P-L-T wherein

[0088] P comprises a payload moiety;

[0089] T comprises a tag; and

[0090] L comprises a linking moiety.

[0091] In some embodiments, L comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0092] In some embodiments, Lcomprises the formula [AEEA]u-[L'-[AEEA]v]w, wherein

[0093] AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid or a derivative thereof;

[0094] L' comprises a linking moiety; u is an integer of 2 or more; each v is an integer of 2 or more; and w is an integer from 1 to 4; wherein the different groups [L'-[AEEA]V] may be identical or different.

[0095] In some embodiments, u and v are each integers from 2 to 10.

[0096] In some embodiments, u and v are each integers from 2 to 8.

[0097] In some embodiments, u and v are each integers of 2, 4 or 6.

[0098] In some embodiments, L or L' comprises an amino acid.

[0099] In some embodiments, Lor L' comprises the D-isomer of an amino acid.

[0100] In some embodiments, Lor L' comprises cysteine or lysine.

[0101] In some embodiments, L or L' is connected to a side chain. In some embodiments, a side chain comprises a functional moiety.

[0102] In some embodiments, a functional moiety comprises a solubilizing functional group.

[0103] In some embodiments, a functional moiety comprises a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0104] In some embodiments, the payload moiety is selected from the group consisting of radioisotopes, toxins and immunomodulators. In some embodiments, the payload moiety comprises a chelating compound comprising a radioisotope. In some embodiments, a chelating compound in the compound is covalently linked with the tag and chelates a radioisotope, such as radionuclides and radiolabels.

[0105] 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.

[0106] 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.

[0107] 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 2, 4 or 6.

[0108] In some embodiments, the tag is an ALFA-tag.

[0109] In some embodiments, the tag is a cyclic ALFA-tag.

[0110] In one 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:

[0111] (i) providing to the subject a compound comprising a binding moiety binding to the target antigen and a binding moiety for a tag;

[0112] (ii) allowing the compound comprising a binding moiety binding to the target antigen and a binding moiety for a tag to become associated with cells expressing the target antigen; and

[0113] (iii) administering to the subject a compound described above, e.g., the compound under (ii) described above, comprising a tag to which the binding moiety for a tag binds. In some embodiments, the compound comprising a binding moiety binding to 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 a binding moiety binding to the target antigen and a binding moiety for a tag; and allowing expression of the polypeptide by cells in the subject.

[0114] In some embodiments, the cells expressing the polypeptide are transfected with the RNA.

[0115] In some embodiments, the RNA is administered as particulate formulation such as formulated as lipid nanoparticles.

[0116] In some embodiments, the cells expressing the polypeptide secrete the polypeptide.

[0117] In some embodiments, the cells expressing the polypeptide express the polypeptide such that it is released into the bloodstream.

[0118] In some embodiments, the target antigen is a cell surface antigen.

[0119] In some embodiments, the compound comprising a binding moiety binding to the target antigen and a binding moiety for a tag is a fusion polypeptide comprising the binding moiety binding to the target antigen and the binding moiety for a tag.

[0120] In some embodiments, the binding moiety binding to the target antigen comprises an antibody or an antibody derivative.

[0121] In some embodiments, the binding moiety for a tag comprises an antibody or an antibody derivative.

[0122] In some embodiments, the antibody derivative is an antibody fragment.

[0123] In some embodiments, the disease, disorder or condition is cancer.

[0124] In some embodiments, the cells expressing a target antigen are diseased cells.

[0125] In some embodiments, the cells expressing a target antigen are cancer cells.

[0126] In some embodiments, the target antigen is a tumor antigen.

[0127] In some embodiments, the payload moiety comprises a radioisotope, e.g., a chelating compound comprising a radioisotope.

[0128] In some embodiments, the payload moiety comprises a toxin or immunomodulator.

[0129] In some embodiments, the compound comprising a binding moiety binding to the target antigen and a binding moiety for a tag comprises a single binding moiety for the tag or at least two, e.g., two, binding moieties for the tag. In some embodiments, the tag is an ALFA-tag.

[0130] In some embodiments, the tag is a cyclic ALFA-tag.

[0131] Brief description of the drawings

[0132] Figure 1: Pretrageting approach using monovalent RiboDocker and fluorophore-labeled monovalent ALFA (monALFA) conjugate

[0133] Figure 2: In vitro activity of pre-complexes using a co-culture of two antigen+ tumor cell lines and THP-1 Dual reporter cells indicating IRF and NFB induction as described in the THP-1 co- culture assay protocol. A) The antigen+ tumor cell line used was transfected with the antigen. B) The antigen+ tumor cell line used endogenously expresses the antigen.

[0134] Figure 3: In vitro activity of two STING-targeting small molecule payloads in THP-1 Dual reporter cells.

[0135] Detailed description

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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".

[0140] 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.

[0141] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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 from the 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] As used in the present disclosure, "% w / v" refers to weight by volume percent, which is a unit of concentration measuring the amount of solute in grams (g) expressed as a percent of the total volume of solution in milliliters (mL).

[0151] As used in the present disclosure, "% by weight" refers to weight percent, which is a unit of concentration measuring the amount of a substance in grams (g) expressed as a percent of the total weight of the total composition in grams (g).

[0152] 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.

[0153] As used in the present disclosure, "mol % of the total lipid" is defined as the ratio of the number of moles of one lipid component to the total number of moles of all lipids, multiplied by 100. In this context, in some embodiments, the term "total lipid" includes lipids and lipid- like material. The term "ionic strength" refers to the mathematical relationship between the number of different kinds of ionic species in a particular solution and their respective charges. Thus, ionic strength I is represented mathematically by the formula: in which c is the molar concentration of a particular ionic species and z the absolute value of its charge. The sum Z is taken over all the different kinds of ions (i) in solution.

[0154] According to the disclosure, the term "ionic strength" in some embodiments relates to the presence of monovalent ions. Regarding the presence of divalent ions, in particular divalent cations, their concentration or effective concentration (presence of free ions) due to the presence of chelating agents is, in some embodiments, sufficiently low so as to prevent degradation of a nucleic acid. In some embodiments, the concentration or effective concentration of divalent ions is below the catalytic level for hydrolysis of the phosphodiester bonds between nucleotides such as RNA nucleotides. In some embodiments, the concentration of free divalent ions is 20 pM or less. In some embodiments, there are no or essentially no free divalent ions.

[0155] "Osmolality" refers to the concentration of a particular solute expressed as the number of osmoles of solute per kilogram of solvent.

[0156] The term "lyophilizing" or "lyophilization" refers to the freeze-drying of a substance by freezing it and then reducing the surrounding pressure (e.g., below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or less) to allow the frozen medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms "lyophilizing" and "freeze- drying" are used herein interchangeably.

[0157] The term "spray-drying" refers to spray-drying a substance by mixing (heated) gas with a fluid that is atomized (sprayed) within a vessel (spray dryer), where the solvent from the formed droplets evaporates, leading to a dry powder.

[0158] The term "reconstitute" relates to adding a solvent such as water to a dried product to return it to a liquid state such as its original liquid state.

[0159] 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. The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, 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.

[0160] 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.

[0161] The term "EDTA" refers to ethylenediaminetetraacetic acid disodium salt. All concentrations are given with respect to the EDTA disodium salt.

[0162] The term "cryoprotectant" relates to a substance that is added to a formulation in order to protect the active ingredients during the freezing stages.

[0163] The term "lyoprotectant" relates to a substance that is added to a formulation in order to protect the active ingredients during the drying stages.

[0164] 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.

[0165] Peptides and polypeptides disclosed herein may comprise a linear or a cyclized peptide sequence.

[0166] 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; or a connection between two side chains including sulfur groups of two cysteine amino acids by forming a disulfide bond, or more complicated arrangements.

[0167] 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 peptides and 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.

[0168] 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.

[0169] 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.

[0170] The following table lists the 20 natural amino acids and their abbreviations: 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.

[0171] 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.

[0172] 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 BiotechnoLI3:132-4). 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).

[0173] The term "amide" as used herein, represents a group of formula "-NHC(O)-".

[0174] The term "thioamide" represents a group of formula "-NHC(S)-".

[0175] 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.

[0176] The term "ether" refers to a group or compound having an oxygen between two carbon atoms.

[0177] The term "thioether" refers to a group or compound having a sulfur between two carbon atoms.

[0178] 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-.

[0179] The term "thioester" refers to the group -C(O)S- or -C(S)O-. 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).

[0180] 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.

[0181] 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.

[0182] "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.

[0183] "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.

[0184] 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.

[0185] For the purposes of the present disclosure, "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. 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: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0186] 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 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.

[0187] "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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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 or variant 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 variant molecule 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.

[0194] 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. 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 the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] As used herein, the terms "binding" or "capable of binding" typically is a binding with an affinity corresponding to a KD of about 10-7M or less, such as about 10-8M or less, such as about 109M or less, about 1010M or less, or about 1011M or even less, when determined using 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). The term "kd" (sec1), 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.

[0199] The term "KD" (M), as used herein, refers to the dissociation equilibrium constant of a particular interaction, e.g., antibody-antigen interaction.

[0200] Generally, the terms "bind" or "binding" and "target" or "targeting" are used interchangeably herein.

[0201] 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 or transposon-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 docking compound. RNA can be transfected into cells to transiently express its coded protein.

[0202] 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.

[0203] 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.

[0204] As used herein "endogenous" refers to any material from or produced inside an organism, cell, tissue or system.

[0205] As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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 or polypeptide is a docking compound, the cell secretes the encoded peptide or polypeptide.

[0211] 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.

[0212] The term "expression" as used herein includes the transcription and / or translation of a particular nucleotide sequence.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] The term "average diameter" refers to the mean hydrodynamic diameter of particles as measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Zaverage with the dimension of a length, and the polydispersity index (PDI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here "average diameter", "diameter" or "size" for particles is used synonymously with this value of the Zaverage-

[0217] In some embodiments, the "polydispersity index" is calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the "average diameter". Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of nanoparticles. Docking compound

[0218] According to the disclosure, a payload is delivered specifically to a target cell by providing a docking compound with a moiety that binds to a target on target cells, e.g., an antigen 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".

[0219] 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 a tag for binding by the docking compound which is covalently attached to the payload.

[0220] In some embodiments, a docking compound comprises a "primary targeting moiety", e.g., a moiety targeting a cell surface antigen on target cells, that is 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.

[0221] 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.

[0222] In order to allow specific targeting of primary targets, the 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.

[0223] 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. 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 thereof which still binds to the receptor, e.g., a receptor binding peptide in the case of receptor binding protein ligands.

[0224] 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.

[0225] 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.

[0226] 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 a binding domain binding to a primary target and a binding domain binding to a tag conjugate. In some embodiments, the docking compound comprises an antibody or antibody fragment binding to a primary target and 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 some embodiments, 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 single-domain antibody such as a VHH.

[0227] In some embodiments, the docking compound comprises one binding domain binding to a primary target and one binding domains binding to a tag conjugate. In some embodiments, the docking compound comprises one binding domain binding to a primary target and two binding domains binding to a tag conjugate. In some embodiments, the docking compound comprises two binding domains binding to a primary target and two binding domains binding to a tag conjugate.

[0228] In some embodiments, the docking compound comprises an antibody wherein the Fab fragments of the antibody are replaced by VHHs binding to a primary target and being N- terminally linked to the remaining part of the heavy chains. In some embodiments, the heavy chains are C-terminally linked to VHHs binding to a tag on a tag conjugate.

[0229] In some embodiments, the docking compound comprises a full-length antibody binding to a primary target. In some embodiments, the heavy chains of the full-length antibody are C- terminally linked to VHHs binding to a tag on a tag conjugate.

[0230] In some embodiments, the docking compound comprises a fusion protein which comprises a binding domain binding to a primary target and a binding domain binding to a tag conjugate. In some embodiments, the docking compound comprises a single peptide chain. In some embodiments, the single peptide chain comprises a portion, e.g., antibody, antibody fragment or DARPin, binding to a primary target and a portion, e.g., antibody or antibody fragment, binding to a tag conjugate. In some embodiments, the docking compound comprises two peptide chains which may be covalenty linked, e.g., by one or more disulfide bonds, wherein each peptide chain comprises a portion, e.g., antibody, antibody fragment or DARPin, binding to a primary target and a portion, e.g., antibody or antibody fragment, binding to a tag conjugate. In some embodiments, the antibody fragments are VHH, scFv, or a mixture thereof. In different embodiments, the docking compound or a peptide chain of a docking compound comprises one of the following structures (from N- to C-terminus):

[0231] VHH (a tag conjugatej-optional linker-VHH (a primary target) VHH (a primary targetj-optional linker-VHH (a tag conjugate) VHH (a tag conjugate)-optional linker-scFv (a primary target) scFv (a primary targetj-optional linker-VHH (a tag conjugate) VHH (a primary target)-optional linker-scFv (a tag conjugate) scFv (a tag conjugatej-optional linker-VHH (a primary target) scFv (a tag conjugatej-optional linker-scFv (a primary target) scFv (a primary targetj-optional linker-scFv (a tag conjugate)

[0232] 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, bivalenty or with even more valency) to an epitope tag, e.g., an ALFA-tag and the other specificity binds (monovalently, bivalenty or with even more valency) to a primary target, e.g., a cell surface antigen on target cells. In some embodiments, 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 DARPin, 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 docking compound has a structure selected from the group consisting of NbALFA x anti-primary target DARPin, NbALFA x anti-primary target VHH and NbALFA x anti- primary target scFv. 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 an anti-cancer antigen VHH. 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 an anti-cancer antigen scFv. In some embodiments, the docking compound comprises a bispecific molecule comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-cancer antigen DARPin.

[0233] 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 target cells, 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 preferred embodiments, the nucleic acid encoding the docking compound is RNA. The RNA-encoded docketing compound is also called "RiboDocker" herein.

[0234] Tag conjugate

[0235] The tag conjugate described herein comprises a payload to be delivered and a tag for binding by the docking compound. The tag of the tag conjugate thus is the part of the tag conjugate that forms the binding partner for the docking compound. Generally, the tag of the tag conjugate is covalently attached to the payload moiety in a manner such that it is available for binding to the docking compound.

[0236] In some embodiments, a tag conjugate described herein comprises a single tag, preferably a single ALFA-tag and preferably comprises a single payload moiety, preferably a single immunomodulator moiety such as a single STING agonist. In some embodiments, a tag conjugate described herein comprises a single tag, preferably a single ALFA-tag and comprises two payload moieties, preferably two immunomodulator moiety such as two STING agonists. In some embodiments, a tag conjugate described herein comprises a single tag, preferably a single ALFA-tag and preferably comprises a single payload moiety, preferably a single toxin moiety. In some embodiments, a tag conjugate described herein comprises a single tag, preferably a single ALFA-tag and comprises two payload moieties, preferably two toxin moieties.

[0237] In some embodiments, the tag of the tag conjugate comprises a peptide or protein (e.g., peptide tag).

[0238] In some embodiments, the tag of the tag conjugate comprise a peptide or protein (e.g., peptide tag) and is chemically linked, e.g., through a linker, to the payload moiety.

[0239] 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 tag and the payload moiety may be covalently or non- covalently linked.

[0240] In some embodiments, the tag conjugate comprises a polymer. In some embodiments, the payload moiety of the tag conjugate and the tag of the tag conjugate are connected through a linker comprising the polymer.

[0241] 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.

[0242] 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. 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.

[0243] Modification of the surface of particles with chains of hydrophilic and flexible polymers, e.g., polymers of the polyethylene glycol) type, confers them a steric protection by preventing the opsonins reaching the surface of the particles.

[0244] In some embodiments, the polymer for use herein is selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) (including derivatives thereof).

[0245] 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.

[0246] 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.

[0247] 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.

[0248] In some embodiments, the PEG comprises "PEG2k", also termed "PEG 2000", which has an average molecular weight of about 2000 Daltons.

[0249] In some embodiments, PEG2000, PEG3000 and PEG5000 are used as the polymer.

[0250] 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.

[0251] In some embodiments, a pSar comprises the structure of the following general formula: wherein s is the number of sarcosine units.

[0252] 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.

[0253] In some embodiments, the POX and / or POZ polymer comprises the following general formula: 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.

[0254] In some embodiments, the POX and / or POZ polymer is a polymer of POX and comprises repeating units of the following general formula:

[0255] In some embodiments, the POX and / or POZ polymer is a polymer of POZ and comprises repeating units of the following general formula:

[0256] 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: 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 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 200.

[0257] 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.

[0258] 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.

[0259] 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.

[0260] 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.

[0261] In some embodiments, the polymer comprises the following general formula: wherein

[0262] X2and X1taken together are optionally substituted amide, optionally substituted thioamide ester, or thioester;

[0263] Y is -CH2-, -(CH2)2-, or -(CH2)3-; z is 2 to 24; and n is the number of repeating units, e.g., 1 to 100.

[0264] In some embodiments,

[0265] (i) when X1is -C(O)- then X2is -NR1-;

[0266] (ii) when X1is -NR1- then X2is -C(O)-;

[0267] (iii) when X1is -C(S)- then X2is -NR1-;

[0268] (iv) when X1is -NR1- then X2is -C(S)-;

[0269] (v) when X1is -C(O)- then X2is -O-;

[0270] (vi) when X1is -O- then X2is -C(O)-;

[0271] (vii) when X1is -C(S)- then X2is -O-;

[0272] (viii) when X1is -O- then X2is -C(S)-;

[0273] (ix) when X1is -C(O)- then X2is -S-; or

[0274] (x) when X1is -S- then X2is -C(O)-; wherein R1is hydrogen or Ci-s alkyl; preferably

[0275] (i) when X1is -C(O)- then X2is -NR1-;

[0276] (ii) when X1is -NR1- then X2is -C(O)-;

[0277] (iii) when X1is -C(S)- then X2is -NR1-;

[0278] (iv) when X1is -NR1- then X2is -C(S)-;

[0279] (v) when X1is -C(O)- then X2is -O-; or

[0280] (vi) when X1is -O- then X2is -C(O)-; wherein R1is hydrogen or Ci-s alkyl. In some embodiments, X1is -C(O)- and X2is -NR1-, wherein R1is hydrogen or Ci-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.

[0281] In some embodiments, Y is -CHz- or -(CHz)?-- In some embodiments, Y is -CH2-.

[0282] In some embodiments, the polymer comprises the following general formula: wherein

[0283] R1is hydrogen or Ci-s alkyl; z is 2 to 24; and n is the number of repeating units, e.g., 1 to 100.

[0284] In some embodiments of the above formulas, z is 2 to 10. In some embodiments, z is 2 to 7.

[0285] In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.

[0286] In some embodiments, the polymer comprises the following general formula: wherein

[0287] R1is hydrogen or C1-8 alkyl; and n is the number of repeating units, e.g., 1 to 100.

[0288] In some embodiments of the above formulas, R1is hydrogen or methyl. In some embodiments, R1is hydrogen.

[0289] In some embodiments, the polymer comprises the following general formula: wherein n is the number of repeating units, e.g., 1 to 100.

[0290] 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

[0291] 2, 4, 6, 8, 10, or 12. In some embodiments, n is 2, 4, or 6.

[0292] In some embodiments, a tag conjugate comprises the tag and the payload moiety or payload moieties in an unbranched arrangement.

[0293] In some embodiments, a tag conjugate comprises the formula:

[0294] P-L-T wherein

[0295] P comprises a payload moiety;

[0296] T comprises a tag; and

[0297] L comprises a linking moiety.

[0298] In some embodiments, L comprises a polymer as described herein. In some embodiments, L comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

[0299] 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: wherein n is between 2 and 30, e.g., between 2 and 10, such as 2, 4 or 6.

[0300] In some embodiments, the payload moiety is selected from the group consisting of radioisotopes, toxins and immunomodulators. In some embodiments of all tag conjugates described above, the tag is an ALFA-tag, e.g., a cyclic ALFA-tag.

[0301] The present disclosure provides in one aspect, a tag conjugate as described above.

[0302] Interacting moieties on the tag conjugate and on the docking compound

[0303] In some embodiments, the moiety on the tag conjugate, i.e., the tag, and the moiety on the docking compound interacting with each other non-covalently bind to each other.

[0304] 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.

[0305] In some embodiments, the moieties on the tag conjugate and on the docking compound interacting with each other are antigen / antibody systems.

[0306] 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.

[0307] In some embodiments, the moieties on the tag conjugate and on the docking compound interacting which each other comprise an epitope tag / binder system.

[0308] 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.

[0309] 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.

[0310] 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.

[0311] In preferred embodiments, the compound comprises a high affinity tag, such as a high affinity ALFA-tag.

[0312] For example, in applications which profit from a tight binding between the docking compound and the tag conjugate, for example in applications using a toxin-payload or immunomodulator-payload, a high affinity tag, such as a high affinity ALFA-tag, can be selected. Epitope tag / binder systems with suitable affinities are described herein.

[0313] In other applications, for example in applications using a radiodiagnostic, a comparably lower affinity tag (a medium / low affinity tag), such as a medium / low affinity ALFA-tag, may be suitable. Epitope tag / binder systems with suitable affinities are described herein.

[0314] 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 wild- type 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 t ag) / 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).

[0315] 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.

[0316] Examples of high affinity tags are

[0317] - Ac-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-Arg-Leu-Thr-Glu-NH2,

[0318] - Ac-Ser-Arg-Leu-Glu-(cyclo5)Glu-Glu-Leu-(cyclo8)Lys-Arg-Arg-Leu-Thr-Glu-NH2,

[0319] - 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,

[0320] - Ac-Pro-Ser-Arg-Leu-Glu-(cyclo6)Glu-Glu-Leu-Arg-(cyclolO)Lys-Arg-Leu-Thr-Glu-NH2.

[0321] Examples of medium / low affinity tags are

[0322] 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,

[0323] - Ac-Pro-Ser-Arg-Leu-(cyclo5)Glu-Glu-Glu-Leu-(cyclo9)Lys-Arg-Arg-Leu-Thr-Glu-NH2.

[0324] 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.

[0325] In some embodiments, an ALFA-tag comprises the amino acid sequence

[0326] -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,

[0327] AA12, AA13 and AA14 are:

[0328] AAO is Pro or deleted;

[0329] AA1 is Ser, Gly, Thr, or Pro;

[0330] AA2 is Arg, Gly, Ala, Glu, or Pro;

[0331] AA3 is Leu, lie, or Vai;

[0332] AA4 is Glu or Gin;

[0333] AA5 is Glu or Gin;

[0334] AA6 is Glu or Gin;

[0335] AA7 is Leu, lie, or Vai;

[0336] AA8 is Arg, Ala, Gin, or Glu;

[0337] AA9 is Arg, Ala, Gin, or Glu;

[0338] AA10 is Arg;

[0339] AA11 is Leu;

[0340] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;

[0341] AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and

[0342] AA14 is Pro or deleted.

[0343] In some embodiments, an ALFA-tag comprises a sequence selected from the group consisting of SRLEEELRRRLTE, PSRLEEELRRRLTE, SRLEEELRRRLTEP, and PSRLEEELRRRLTEP.

[0344] In some embodiments, an ALFA-tag comprises the cyclized amino acid sequence

[0345] -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; and wherein the amino acids of AAO, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11,

[0346] AA12, AA13 and AA14 which are not XI and X2 are:

[0347] AAO is Pro or deleted;

[0348] AA1 is Ser, Gly, Thr, or Pro;

[0349] AA2 is Arg, Gly, Ala, Glu, or Pro;

[0350] AA3 is Leu, He, or Vai;

[0351] AA4 is Glu or Gin;

[0352] AA5 is Glu or Gin;

[0353] AA6 is Glu or Gin;

[0354] AA7 is Leu, He, or Vai;

[0355] AA8 is Arg, Ala, Gin, or Glu;

[0356] AA9 is Arg, Ala, Gin, or Glu;

[0357] AA10 is Arg;

[0358] AA11 is Leu;

[0359] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;

[0360] AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and

[0361] AA14 is Pro or deleted.

[0362] In some embodiments, XI and X2 are separated by 2 or 3 amino acids.

[0363] In some embodiments, AA5 is XI and AA9 is X2, AA5 is XI and AA8 is X2, AA9 is XI and AA13 is X2, AA6 is XI and AA9 is X2, AA9 is XI and AA12 is X2, AA10 is XI and AA13 is X2, AA6 is XI and AA10 is X2 or AA4 is XI and AA8 is X2.

[0364] In some embodiments, an ALFA-tag comprises a cyclized amino acid sequence selected from the group consisting of 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-, and h. -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;

[0365] AAO is Pro or deleted;

[0366] AA1 is Ser, Gly, Thr, or Pro;

[0367] AA2 is Arg, Gly, Ala, Glu, or Pro;

[0368] AA3 is Leu, lie, or Vai;

[0369] AA4 is Glu or Gin;

[0370] AA5 is Glu or Gin;

[0371] AA6 is Glu or Gin;

[0372] AA7 is Leu, lie, or Vai;

[0373] AA8 is Arg, Ala, Gin, or Glu;

[0374] AA9 is Arg, Ala, Gin, or Glu;

[0375] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;

[0376] AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and

[0377] AA14 is Pro or deleted.

[0378] 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.

[0379] 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.

[0380] 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 side- chain 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.

[0381] 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:

[0382] In some embodiments, Xi is Lys and X2 is Glu. In some embodiments, -cyclo(Lys - Glu)-, - c(Lys - Glu)-, -cyclofK - E)-, -c(K - E)-, -K - E- cyclo, or cycloK - cycloE- comprises the following structure:

[0383] In some embodiments, Xi is Cys and X2 is Cys. In some embodiments, -cyclo(Cys - Cys)-, C)-, -c(C - C)-, -C — C- cyclo, or -cycloC cycloC- comprises the following structure:

[0384] 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<yclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DGlu-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-;

[0385] -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-DGlu)-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-DGlu)-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-z-Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg<eu-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-z-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-z-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<yclo(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-z-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-,

[0386] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-,

[0387] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Cys)-Glu-,

[0388] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-,

[0389] -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-,

[0390] -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-,

[0391] -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-,

[0392] -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-,

[0393] -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-,

[0394] -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-,

[0395] -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-,

[0396] -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-,

[0397] -Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-,

[0398] -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-,

[0399] -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-,

[0400] -Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-,

[0401] -Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-,

[0402] -Pro-Ser-Arg-Leu-Glu-cyclotDCys-Glu-Leu-DCysJ-Arg-Arg-Leu-Thr-Glu-,

[0403] -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-,

[0404] -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-,

[0405] -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-,

[0406] -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-;

[0407] -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-,

[0408] -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-,

[0409] -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-,

[0410] -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-,

[0411] -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-,

[0412] -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-,

[0413] -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-,

[0414] -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)-z-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)-z-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-z-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)-,

[0415] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-Cys)-,

[0416] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-DCys)-,

[0417] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-hCys)-,

[0418] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-,

[0419] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-hCys)-,

[0420] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-,

[0421] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-Cys)-,

[0422] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-DCys)-,

[0423] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-hCys)-,

[0424] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-,

[0425] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DhCys)-,

[0426] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-,

[0427] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-Pen)-,

[0428] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-DPen)-,

[0429] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-Pen)-,

[0430] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-DPen)-,

[0431] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-Cys)-Glu-,

[0432] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-DCys)-Glu-,

[0433] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-DCys)-Glu-,

[0434] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leii-Cys)-Glu-,

[0435] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DCys)-Glu-z

[0436] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-hCys)-Glu-,

[0437] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-hCys)-Glu-,

[0438] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DhCys)-Glu-,

[0439] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DhCys-Arg-Leu-hCys)-Glu-,

[0440] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Asp)-Glu-,

[0441] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Asp-Arg-Leu-Lys)-Glu-,

[0442] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Glu)-Glu-,

[0443] -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-z

[0444] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Asp-Arg-Leu-Lys)-Glu-z

[0445] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Glu)-Glu-,

[0446] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Glu-Arg-Leu-Lys)-Glu-,

[0447] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Thr-Cys)-,

[0448] -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-,

[0449] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-,

[0450] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu-,

[0451] -Ser-Arg-Leu-Glu-cyclo(DGlu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-,

[0452] -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-Pro-,

[0453] -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-Pro-,

[0454] -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-Pro-,

[0455] -Pro-Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-,

[0456] -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-Pro-,

[0457] -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-z-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-,

[0458] -Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-,

[0459] -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-,

[0460] -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-z

[0461] -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-,

[0462] -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-;

[0463] -Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-,

[0464] -Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-,

[0465] -Pro-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-;

[0466] -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-z-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-z-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-DGlu)-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-z

[0467] -Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-,

[0468] -Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-,

[0469] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-,

[0470] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-,

[0471] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-,

[0472] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-,

[0473] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-,

[0474] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-;

[0475] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-,

[0476] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-,

[0477] -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0478] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-Pro-;

[0479] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Cys)-Glu-Pro-,

[0480] -Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leii-Arg-Cys)-Arg-Leu-Thr-Glu-Pro-,

[0481] -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0482] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-Pro-,

[0483] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Cys)-Glu-Pro-z

[0484] -Pro-Ser-Arg<eu-Glu-Glu-cyclo(Cys-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-Pro-,

[0485] -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0486] -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0487] -Ser-Arg-Leu-Glu-cyclofhCys-Glu-Leu-CysJ-Arg-Arg-Leu-Thr-Glu-Pro-,

[0488] -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-z

[0489] -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-z

[0490] -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-z

[0491] -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0492] -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0493] -Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-z

[0494] -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0495] -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-,

[0496] -Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0497] -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0498] -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0499] -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0500] -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0501] -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0502] -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro- /

[0503] -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0504] -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0505] -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0506] -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0507] -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0508] -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0509] -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0510] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DCys)-Pro-,

[0511] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-Cys)-Pro-,

[0512] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DCys)-Pro-,

[0513] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-Cys)-Pro-,

[0514] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-DCys)-Pro-,

[0515] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-hCys)-Pro-,

[0516] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-Pro-,

[0517] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclofDCys-Leu-Thr-hCysJ-Pro-,

[0518] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-Pro-,

[0519] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-Cys)-Pro-,

[0520] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-DCys)-Pro-,

[0521] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-hCys)-Pro-,

[0522] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-Pro-,

[0523] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DhCys)-Pro-,

[0524] -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-,

[0525] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-DPen)-Pro-,

[0526] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-Pen)-Pro-,

[0527] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-DPen)-Pro-;

[0528] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo{DCys-Arg-Leu-Cys)-Glu-Pro-,

[0529] -Ser-Arg-Leu-Glu-Glu-Glii-Leu-Arg-cyclo(DCys-Arg-Leu-DCys)-Glu-Pro-,

[0530] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-DCys)-Glu-Pro-,

[0531] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-Cys)-Glu-Pro-,

[0532] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DCys)-Glu-Pro-,

[0533] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-hCys)-Glu-Pro-,

[0534] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-hCys)-Glu-Pro-,

[0535] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DhCys)-Glu-Pro-7

[0536] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DhCys-Arg-Leu-hCys)-Glu-Pro-,

[0537] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DCys)-Pro-,

[0538] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-Cys)-Pro-,

[0539] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DCys)-Pro-,

[0540] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-Cys)-Pro-,

[0541] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-DCys)-Pro-,

[0542] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclofhCys-Leu-Thr-hCysJ-Pro-,

[0543] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-Pro-,

[0544] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-hCys)-Pro-,

[0545] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclofCys-Leu-Thr-hCysJ-Pro-,

[0546] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-Cys)-Pro-;

[0547] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-DCys)-Pro-;

[0548] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-hCys)-Pro-,

[0549] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-Pro-,

[0550] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DhCys)-Pro-,

[0551] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclofCys-Leu-Thr-DhCysJ-Pro-,

[0552] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-Pen)-Pro-,

[0553] -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-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-Cys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-DCys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-DCys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-Cys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DCys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-hCys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-hCys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DhCys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DhCys-Arg-Leu-hCys)-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-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Asp)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Asp-Arg-Leu-Lys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Glu)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Glu-Arg-Leu-Lys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Thr-Cys)-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-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-cyclo(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-, and -Ser-Arg-Leu-cyclo(Glu-Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-.

[0554] 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.

[0555] 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-, -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)-,

[0556] -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-,

[0557] -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-,

[0558] -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-,

[0559] -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-,

[0560] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Thr-Cys)-,

[0561] -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-,

[0562] -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-,

[0563] -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-,

[0564] -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(Lys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-, and -Ser-Arg-Leu-cyclo(Glu-Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-.

[0565] 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)-.

[0566] 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:

[0567] 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.

[0568] 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.

[0569] 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.

[0570] 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.

[0571] In some embodiments, an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence GVTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.

[0572] 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.

[0573] 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.

[0574] 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.

[0575] The SpyTag / SpyCatcher system is a technology for irreversible conjugation of recombinant proteins. The peptide SpyTag spontaneously reacts with the protein SpyCatcher to form an intermolecular isopeptide bond between the pair. Using the Tag / Catcher pair, bioconjugation can be achieved between two recombinant proteins.

[0576] 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.

[0577] 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.

[0578] 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.

[0579] 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.

[0580] Accordingly, the present disclosure provides in one aspect, a complex comprising:

[0581] (i) a compound comprising a binding moiety binding to a target antigen and a binding moiety for a tag (docking compound), and

[0582] (ii) a compound comprising a payload moiety and a tag, to which the binding moiety for a tag binds (tag conjugate).

[0583] Different embodiments of the tag conjugate and the docking compound which are complexed are described herein.

[0584] In some embodiments, the tag conjugate comprises an ALFA-tag. In these embodiments, the moiety binding to a tag conjugate of the docking compound may be a NbALFA-nanobody (NbALFA). In some embodiments, the docking compound comprises at least two moieties binding to a tag of a tag conjugate, e.g., at least two NbALFA-nanobodies (NbALFA). In some embodiments, the docking compound may have a structure selected from the group consisting of NbALFA x anti-primary target DARPin, NbALFA x anti-primary target VHH and NbALFA x anti-primary target scFv. In some embodiments, the docking compound comprises a full-length anti-primary target antibody comprising two heavy chains and two light chains, wherein NbALFA is linked to the C-terminus of each of the heavy chains.

[0585] Payload

[0586] In some embodiments, a payload comprises a therapeutic or diagnostic moiety.

[0587] 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.

[0588] 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 contrast- providing 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,UC,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;89SG89Zr, 97RlJ / 99^ 110|nlll|n113|n114^ 117^ 120|, 122)^ 123, 124|;125| 166^ 167-j-^169Yb,193Pt,195Pt,201TI, and203Pb. Other elements and isotopes, such as being used for therapy may also be applied for imaging in certain applications.

[0589] 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. The X-ray-responsive moieties include but are not limited to iodine, barium, and barium sulfate.

[0590] 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.

[0591] 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, PCT A, DATA, TRAP, DFO, THP, HBED and DEDPA.

[0592] 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.

[0593] Payloads comprising a detectable radiolabel may be referred to as radiodiagnostic payloads.

[0594] A particularly preferred payload for use herein comprises DOTA chelator for68Ga complexation.

[0595] In some embodiments, a payload comprises DOTA-68Ga.

[0596] 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).

[0597] 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.

[0598] In some embodiments, a pharmaceutically active agent comprises pharmaceutically active RNA or a pharmaceutically active peptide or protein.

[0599] 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.

[0600] 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. 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.

[0601] 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.

[0602] 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 inhibit mitosis. 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.

[0603] 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.

[0604] Chemotherapeutic agents include alkylating agents, antimetabolites, anti-microtubule agents, topoisomerase inhibitors, and cytotoxic antibiotics.

[0605] 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.

[0606] 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 and pemetrexed. 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.

[0607] 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).

[0608] 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.

[0609] In some embodiments, a payload which is useful herein comprises a radiodiagnostic, e.g., a chelator comprising a radiodiagnostic isotope.

[0610] 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.

[0611] 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.

[0612] 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.

[0613] 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.

[0614] 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.

[0615] 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-1 or DM-4.

[0616] 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:

[0617] 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.

[0618] DM1, also known as mertansine, is a maytansinoid having the following formula:

[0619]

[0620] In particular, the term "mertansine" or "DM1" refers to the compound W2'-deacetyl-W2'-(3- mercapto-l-oxopropyl)-maytansine.

[0621] "DM4" refers to the compound A / 2-deacetyl-W2-(4-methyl-4-mercapto-l-oxopentyl)- maytansine.

[0622] 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.

[0623] Exemplary auristatin embodiments include monomethylauristatin drug moieties such as MMAE and MMAF.

[0624] MMAE, also known as Monomethyl auristatin E, has the following formula:

[0625] 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. 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.

[0626] In some embodiments, the payload moiety comprises an exatecan moiety of the following formula: wherein 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.

[0627] In some embodiments, the payload moiety comprises a deruxtecan moiety. In some embodiments, the payload moiety comprises deruxtecan.

[0628] Deruxtecan has the following formula:

[0629] In some embodiments, a deruxtecan moiety comprises the following formula: wherein 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. 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.

[0630] In some embodiments, the deruxtecan moiety comprises the following formula 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.

[0631] 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.

[0632] In some embodiments, R2comprises one of the following divalent groups: wherein each or - represents a bond.

[0633] 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.

[0634] Embodiments of tag conjugate and docking compound

[0635] In some embodiments, the tag of a tag conjugate is an ALFA-tag.

[0636] In these embodiments, the binding moiety for the tag conjugate on the docking compound comprises an ALFA-specific single-domain antibody (sdAb), NbALFA-nanobody.

[0637] In some embodiments, the docking compound comprises one binding moiety for a tag, e.g., one NbALFA-nanobody. In some embodiments, a docking compound may be monovalent for binding to a tag, e.g., an ALFA-tag, and optionally monovalent for binding to target antigen.

[0638] In some embodiments, the docking compound comprises two binding moieties for a tag, e.g., two NbALFA-nanobodies. In some embodiments, a docking compound may be bivalent for binding to a tag, e.g., an ALFA-tag, and optionally bivalent for binding to target antigen.

[0639] In embodiments, wherein a docking compound is bivalent for binding to an ALFA-tag and bivalent for binding to target antigen, the docking compound may comprise a full-length antibody binding to a primary target, wherein each of the heavy chains of the full-length antibody is C-terminally linked to an NbALFA-nanobody. Thus, the docking compound may comprise the structure IgG x NbALFA. Alternatively, the docking compound comprises the Fc domain of an antibody, e.g., IgG or IgA antibody, wherein each of the N-termini is linked to a binding moiety, e.g., a single-domain antibody such as a VHH, binding to a primary target and each of the C-termini is linked to an NbALFA-nanobody. Thus, the docking compound may comprise the structure VHH - Fc x NbALFA.

[0640] In some embodiments, an ALFA-tag for use herein comprises a cyclized amino acid sequence selected from the group consisting of Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr- Glu), Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu, and Pro-Ser-Arg-Leu- cyclo(Glu-Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu. A particulary preferred ALFA-tag for use herein comprises the cyclized amino acid sequence Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg- cy clo ( Lys- Arg- Le u -Th r-G I u ) .

[0641] In some embodiments, a tag conjugate comprises at least one pAEEA structure. In some embodiments, a payload comprises a toxin and a tag comprises a high affinity tag, such as a high affinity ALFA-tag. In some embodiments, a payload comprises a toxin, a tag comprises a high affinity tag, such as a high affinity ALFA-tag, and the docking compound comprises one or two binding moieties for a tag, e.g., one or two NbALFA-nanobodies. In some embodiments, a payload comprises a toxin, a tag comprises an ALFA-tag selected from the group consisting of:

[0642] Ac-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-Arg-Leu-Thr-Glu-NH2,

[0643] 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, and Ac-Pro-Ser-Arg-Leu-Glu-(cyclo6)Glu-Glu-Leu-Arg-(cyclolO)Lys-Arg-Leu-Thr-Glu-NH2, and optionally the docking compound comprises one or two binding moieties for the tag, e.g., one or two NbALFA-nanobodies. In some of these embodiments, the "Ac" and / or "NH2" moieties may not be present.

[0644] In these embodiments, a pre-formed complex of tag conjugate and docking compound may be administered to a subject.

[0645] In some embodiments, a payload comprises an immunomodulator and a tag comprises a high affinity tag, such as a high affinity ALFA-tag. In some embodiments, a payload comprises an immunomodulator, a tag comprises a high affinity tag, such as a high affinity ALFA-tag, and the docking compound comprises one or two binding moieties for a tag, e.g., one or two NbALFA-nanobodies. In some embodiments, a payload comprises an immunomodulator, a tag comprises an ALFA-tag selected from the group consisting of:

[0646] Ac-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-Arg-Leu-Thr-Glu-NH2,

[0647] 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, and Ac-Pro-Ser-Arg-Leu-Glu-(cyclo6)Glu-Glu-Leu-Arg-(cyclolO)Lys-Arg-Leu-Thr-Glu-NH2, and optionally the docking compound comprises one or two binding moieties for the tag, e.g., one or two NbALFA-nanobodies. In some of these embodiments, the "Ac" and / or "NH2" moieties may not be present. In these embodiments, a pre-formed complex of tag conjugate and docking compound may be administered to a subject.

[0648] In some embodiments, a payload comprises a radiodiagnostic and a tag comprises a medium / low affinity tag, such as a medium / low affinity ALFA-tag. In some embodiments, a payload comprises a radiodiagnostic, a tag comprises a medium / low affinity tag, such as a medium / low affinity ALFA-tag, and the docking compound comprises one or two binding moieties for a tag, e.g., one or two NbALFA-nanobodies . In some embodiments, a payload comprises a radiodiagnostic, a tag comprises an ALFA-tag selected from the group consisting of:

[0649] - Ac-Ser-Arg-Leu-Glu-(cyclo5)Asp-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu-NH2,

[0650] - Ac-Pro-Ser-Arg-Leu-Glu-(cyclo6)Lys-Glu-Leu-Arg-(cyclolO)Glu-Arg-Leu-Thr-Glu-NH2, and

[0651] Ac-Pro-Ser-Arg-Leu-(cyclo5)Glu-Glu-Glu-Leu-(cyclo9)Lys-Arg-Arg-Leu-Thr-Glu-NH2, and optionally the docking compound comprises one or two binding moieties for the tag, e.g., one or two NbALFA-nanobodies. In some of these embodiments, the "Ac" and / or "NH2" moieties may not be present.

[0652] 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.

[0653] In some embodiments, a tag conjugate comprises a payload comprising a toxin, e.g., a toxin comprising an exatecan moiety, and a tag comprising the ALFA-tag Ser-Arg-Leu-Glu-Glu-Glu- Leu-Arg-Arg-Arg-Leu-Thr-Glu, Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr- (cyclol3)Glu or Ser-Arg-Leu-Glu-(cyclo5)Glu-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu.

[0654] In some embodiments, a tag conjugate comprises a payload comprising a toxin, e.g., a toxin comprising an exatecan moiety, and a tag comprising the ALFA-tag Ser-Arg-Leu-Glu-Glu-Glu- Leu-Arg-Arg-Arg-Leu-Thr-Glu.

[0655] In some embodiments, a tag conjugate comprises a payload comprising a toxin, e.g., a toxin comprising an exatecan moiety, and a tag comprising the ALFA-tag Ser-Arg-Leu-Glu- (cyclo5)Glu-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu. In some embodiments, a tag conjugate comprises a payload comprising an immunomodulator, e.g., a STING agonist, and a tag comprising the ALFA-tag Ser-Arg-Leu-Glu- Glu-Glu-Leu-Arg-Arg-Arg-Leu-Thr-Glu, Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu- Thr-(cyclol3)Glu or Ser-Arg-Leu-Glu-(cyclo5)Glu-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu.

[0656] In some embodiments, a tag conjugate comprises a payload comprising an immunomodulator, e.g., a STING agonist, and a tag comprising the ALFA-tag Ser-Arg-Leu-Glu- G I u-G I u- Le u- Arg- Arg- Arg-Le u-Th r-G I u .

[0657] In some embodiments, a tag conjugate comprises a payload comprising an immunomodulator, e.g., a STING agonist, and a tag comprising the ALFA-tag Ser-Arg-Leu-Glu- (cyclo5)Glu-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu.

[0658] In some embodiments, a tag conjugate comprises a payload comprising a chelator comprising a radiodiagnostic isotope, e.g., DOTA-68Ga, and a 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.

[0659] In some embodiments, a tag conjugate comprises a payload comprising a chelator comprising a radiodiagnostic isotope, e.g., DOTA-68Ga, and a tag comprising the ALFA-tag Ser-Arg-Leu- Glu-(cyclo5)Asp-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu.

[0660] In some embodiments, a tag conjugate comprises a payload comprising a chelator comprising a radiodiagnostic isotope, e.g., DOTA-68Ga, and a tag comprising the ALFA-tag Pro-Ser-Arg- Leu-(cyclo5)Glu-Glu-Glu-Leu-(cyclo9)Lys-Arg-Arg-Leu-Thr-Glu.

[0661] Cells for targeted delivery

[0662] 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".

[0663] 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. 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 one embodiment, 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.

[0664] 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 and preferably is accessible from the outside of said cell, e.g., by binding molecules such as antibodies located outside the cell.

[0665] 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.

[0666] 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. 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.

[0667] 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.

[0668] 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 identifies cancer 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 self- protein 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.

[0669] 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).

[0670] 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 certain cell types characterized by the presence or increased amounts to be targeted by the methods and agents described herein.

[0671] 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.

[0672] 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.

[0673] 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, e.g., an immunomodulator, to immune cells close to the diseased cell. For example, if the docking compound comprises Fc sequences, e.g., of the heavy chains of an immunoglobulin, the complex described herein comprising a tag conjugate and a docking compound targeted to diseased cells may by taken up by immune cells located in vicinity to the targeted diseased cells by Fc-mediated internalization. Immune effector cells

[0674] 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, cytotoxic T cells trigger the destruction of target cells by either or both of the following means. First, upon activation T cells release cytotoxins such as perforin, granzymes, and granulysin. Perforin and granulysin create pores in the target cell, and granzymes enter the cell and trigger a caspase cascade in the cytoplasm that induces apoptosis (programmed cell death) of the cell. Second, apoptosis can be induced via Fas-Fas ligand interaction between the T cells and target cells. The cells used in connection with the present disclosure will preferably be autologous cells, although heterologous cells or allogenic cells can be used.

[0675] The term "effector functions" in the context of the present disclosure includes any functions mediated by components of the immune system that result, for example, in the killing of diseased cells such as tumor cells, or in the inhibition of tumor growth and / or inhibition of tumor development, including inhibition of tumor dissemination and metastasis. Preferably, the effector functions in the context of the present disclosure are T cell mediated effector functions. Such functions comprise in the case of a helper T cell (CD4+T cell) the release of cytokines and / or the activation of CD8+lymphocytes (CTLs) and / or B cells, and in the case of CTL the elimination of cells, i.e., cells characterized by expression of an antigen, for example, via apoptosis or perforin-mediated cell lysis, production of cytokines such as IFN-g and TNF-a, and specific cytolytic killing of antigen expressing target cells.

[0676] The term "immune effector cell" or "immunoreactive cell" in the context of the present disclosure relates to a cell which exerts effector functions during an immune reaction. An "immune effector cell" in some embodiments is capable of binding an antigen such as an antigen presented by in the context of MHC on a cell or expressed on the surface of a cell and mediating an immune response. 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. The term "immune effector cell" also includes a cell which can mature into an immune cell (such as T cell, in particular T helper cell, or cytolytic T cell) with suitable stimulation. Immune effector cells comprise CD34+hematopoietic stem cells, immature and mature T cells and immature and mature B cells. The differentiation of T cell precursors into a cytolytic T cell, when exposed to an antigen, is similar to clonal selection of the immune system.

[0677] A "lymphoid cell" is a cell which, is capable of producing an immune response such as a cellular immune response, or a precursor cell of such cell, and includes lymphocytes, preferably T lymphocytes, lymphoblasts, and plasma cells. A lymphoid cell may be an immune effector cell as described herein. A preferred lymphoid cell is a T cell which can be modified to express an antigen receptor on the cell surface. In some embodiments, the lymphoid cell lacks endogenous expression of a T cell receptor.

[0678] 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.

[0679] 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.

[0680] 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 antigen presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response.

[0681] 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. "Regulatory T cells" or "Tregs" are a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. Tregs are immunosuppressive and generally suppress or downregulate induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FoxP3, and CD25.

[0682] As used herein, the term "naive T cell" refers to mature T cells that, unlike activated or memory T cells, have not encountered their cognate antigen within the periphery. Naive T cells are commonly characterized by the surface expression of L-selectin (CD62L), the absence of the activation markers CD25, CD44 or CD69 and the absence of the memory CD45RO isoform.

[0683] As used herein, the term "memory T cells" refers to a subgroup or subpopulation of T cells that have previously encountered and responded to their cognate antigen. At a second encounter with the antigen, memory T cells can reproduce to mount a faster and stronger immune response than the first time the immune system responded to the antigen. Memory T cells may be either CD4+or CD8+and usually express CD45RO.

[0684] As used herein, the term "T cell" also includes a cell which can mature into a T cell with suitable stimulation.

[0685] A majority of T cells have a T cell receptor (TCR) existing as a complex of several proteins. The actual T cell receptor is composed of two separate peptide chains, which are produced from the independent T cell receptor alpha and beta (TCRa and TCRp) genes and are called a- and P-TCR chains. y6 T cells (gamma delta T cells) represent a small subset of T cells that possess a distinct T cell receptor (TCR) on their surface. However, in y6 T cells, the TCR is made up of one y-chain and one 6-chain. This group of T cells is much less common (2% of total T cells) than the ct£ T cells.

[0686] All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitors derived from hematopoietic stem cells populate the thymus and expand by cell division to generate a large population of immature thymocytes. The earliest thymocytes express neither CD4 nor CD8, and are therefore classed as double-negative (CD4 CD8 ) cells. As they progress through their development they become double-positive thymocytes (CD4+CD8+), and finally mature to single-positive (CD4+CD8‘ or CD4 CD8+) thymocytes that are then released from the thymus to peripheral tissues.

[0687] 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 or CD16 and the absence ofthe Tcell receptor. As provided herein, the NK cell can also be differentiated from a stem cell or progenitor cell.

[0688] Targeted delivery of payloads

[0689] 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.

[0690] 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 several parameters, 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.

[0691] 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.

[0692] In some embodiments, the above-described transient expression is achieved without integration of the nucleic acid into the target cell genome of the host.

[0693] Binding moieties and agents

[0694] The present disclosure describes binding moieties or agents such as antibodies or antibody derivatives. Moreover, the disclosure describes bispecific or multispecific binding agents such as 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.

[0695] 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. 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.

[0696] 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 / or T- lymphocytes. An antigen may have one or more epitopes (B-cell and T-cell epitopes).

[0697] 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.

[0698] 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. 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 the CHI 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 VL 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)).

[0699] The term "antibody" (Ab) as used herein refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a 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. As indicated above, 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, or a monovalent antibody as described in W02007059782 (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, they collectively 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.

[0700] 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. 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 into single-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.

[0701] 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.

[0702] 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).

[0703] The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. The human monoclonal antibodies may be generated by a hybridoma which includes a B cell obtained from a transgenic or transchromosomal non-human animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene, fused to an immortalized cell. The term "chimeric antibody" as used herein, refers to an antibody wherein the variable region is derived from a non-human species (e.g. derived from rodents) and the constant region is derived from a different species, such as human. Chimeric monoclonal antibodies for therapeutic applications are developed to reduce antibody immunogenicity. The terms "variable region" or "variable domain" as used in the context of chimeric antibodies, refer to a region which comprises the CDRs and framework regions of both the heavy and light chains of the immunoglobulin. Chimeric antibodies may be generated by using standard DNA techniques as described in Sambrook et al., 1989, Molecular Cloning: A laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch. 15. The chimeric antibody may be a genetically or an enzymatically engineered recombinant antibody. It is within the knowledge of the skilled person to generate a chimeric antibody, and thus, generation of the chimeric antibody may be performed by other methods than described herein.

[0704] The term "humanized antibody" as used herein, refers to a genetically engineered non-human antibody, which contains human antibody constant domains and non-human variable domains modified to contain a high level of sequence homology to human variable domains. This can be achieved by grafting of the six non-human antibody complementarity-determining regions (CDRs), which together form the antigen binding site, onto a homologous human acceptor framework region (FR) (see WO92 / 22653 and EP0629240). In order to fully reconstitute the binding affinity and specificity of the parental antibody, the substitution of framework residues from the parental antibody (i.e. the non-human antibody) into the human framework regions (back-mutations) may be required. Structural homology modeling may help to identify the amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and fully human constant regions. Optionally, additional amino acid modifications, which are not necessarily back- mutations, may be applied to obtain a humanized antibody with preferred characteristics, such as affinity and biochemical properties.

[0705] The term "human antibody" as used herein, refers to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse or rat, have been grafted onto human framework sequences. Human monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibody can be employed, e.g., viral or oncogenic transformation of B-lymphocytes or phage display techniques using libraries of human antibody genes. A suitable animal system for preparing hybridomas that secrete human monoclonal antibodies is the murine system. Hybridoma production in the mouse is a very well established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known. Human monoclonal antibodies can thus e.g. be generated using transgenic or transchromosomal mice or rats carrying parts of the human immune system rather than the mouse or rat system. Accordingly, in some embodiments, a human antibody is obtained from a transgenic animal, such as a mouse or a rat, carrying human germline immunoglobulin sequences instead of animal immunoglobulin sequences. In such embodiments, the antibody originates from human germline immunoglobulin sequences introduced in the animal, but the final antibody sequence is the result of said human germline immunoglobulin sequences being further modified by somatic hypermutations and affinity maturation by the endogeneous animal antibody machinery, see e.g. Mendez et al. 1997 Nat Genet. 15(2):146-56.

[0706] 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 "half- molecule" herein.

[0707] 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.

[0708] 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.

[0709] 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.

[0710] 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. Thus, the binding agent may comprise two or more antibodies as described herein or fragments thereof. In particular, a binding agent described herein may be an artificial protein that is composed of two different antibodies, an antibody and a fragment of a different antibody, and fragments of two different antibodies (said fragments of two different antibodies forming two binding domains).

[0711] 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 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.

[0712] The term "bispecific" as used herein refers to an agent having two different antigen-binding regions binding to different epitopes.

[0713] "Multispecific binding agents" are molecules which have more than two different binding specificities.

[0714] 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. A bispecific binding agent according to the present disclosure is not limited to any particular bispecific format or method of producing it.

[0715] Examples of bispecific antibody molecules which may be used herein comprise (i) a single antibody that has two arms comprising different antigen-binding regions; (ii) a single chain antibody that has specificity to two different epitopes, e.g., via two scFvs linked in tandem by an extra peptide linker; (iii) a dual-variable-domain antibody (DVD-lg), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD- lg™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (iv) a chemically- linked bispecific (Fab')2 fragment; (v) a Tandab, which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (vi) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (vii) a so-called "dock and lock" molecule, based on the "dimerization and docking domain" in Protein Kinase A, which, when applied to Fabs, can yield a trivalent bispecific binding protein consisting of two identical Fab fragments linked to a different Fab fragment; (viii) a so-called Scorpion molecule, comprising, e.g., two scFvs fused to both termini of a human Fab-arm; and (ix) a diabody.

[0716] The term "bispecific antibody" includes diabodies. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g. , Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444- 6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123). Bispecific antibodies also include bispecific single chain antibodies. The term "bispecific single chain antibody" denotes a single polypeptide chain comprising two binding domains. In particular, the term "bispecific single chain antibody" or "single chain bispecific antibody" or related terms as used herein preferably mean antibody constructs resulting from joining at least two antibody variable regions in a single polypeptide chain devoid of the constant and / or Fc portion(s) present in full immunoglobulins. For example, a bispecific single chain antibody may be a construct with a total of two antibody variable regions, for example two VH regions, each capable of specifically binding to a separate epitope, and connected with one another through a short polypeptide spacer such that the two antibody variable regions with their interposed spacer exist as a single contiguous polypeptide chain. Another example of a bispecific single chain antibody may be a single polypeptide chain with three antibody variable regions. Here, two antibody variable regions, for example one VH and one VL, may make up an scFv, wherein the two antibody variable regions are connected to one another via a synthetic polypeptide linker, the latter often being genetically engineered so as to be minimally immunogenic while remaining maximally resistant to proteolysis. This scFv is capable of specifically binding to a particular epitope, and is connected to a further antibody variable region, for example a VH region, capable of binding to a different epitope than that bound by the scFv. Yet another example of a bispecific single chain antibody may be a single polypeptide chain with four antibody variable regions. Here, the first two antibody variable regions, for example a VH region and a VL region, may form one scFv capable of binding to one epitope, whereas the second VH region and VL region may form a second scFv capable of binding to another epitope. Within a single contiguous polypeptide chain, individual antibody variable regions of one specificity may advantageously be separated by a synthetic polypeptide linker, whereas the respective scFvs may advantageously be separated by a short polypeptide spacer as described above. According to some embodiments, the first binding domain of the bispecific antibody comprises one antibody variable domain, preferably a VHH domain. According to some embodiments, the first binding domain of the bispecific antibody comprises two antibody variable domains, preferably a scFv, i.e. VH-VL or VL-VH. According to some embodiments, the second binding domain of the bispecific antibody comprises one antibody variable domain, preferably a VHH domain. According to some embodiments, the second binding domain of the bispecific antibody comprises two antibody variable domains, preferably a scFv, i.e. VH-VL or VL-VH. In its minimal form, the total number of antibody variable regions in the bispecific antibody is thus only two. For example, such an antibody could comprise two VH or two VHH domains. According to some embodiments, the first binding domain and the second binding domain of the bispecific antibody each comprise one antibody variable domain, preferably a VHH domain. According to some embodiments, the first binding domain and the second binding domain of the bispecific antibody each comprise two antibody variable domains, preferably a scFv, i.e. VH-VL or VL-VH. In this embodiment, the binding agent preferably comprises (i) a heavy chain variable domain (VH) of a first antibody, (ii) a light chain variable domain (VL) of a first antibody, (iii) a heavy chain variable domain (VH) of a second antibody and (iv) a light chain variable domain (VL) of a second antibody.

[0717] In some embodiments, the bispecific molecules comprise two Fab regions, each being directed against different epitopes. In some embodiments, the molecule of the disclosure is an antigen binding fragment (Fab)2 complex. The Fab2 complex is composed of two Fab fragments, one Fab fragment comprising a Fv domain, i.e. VH and VL domains, specific for one epitope, and the other Fab fragment comprising a Fv domain specific for another epitope. Each of the Fab fragments may be composed of two single chains, a VL-CL module and a VH-CH module. Alternatively, each of the individual Fab fragments may be arranged in a single chain, preferably, VL-CL-CH-VH, and the individual variable and constant domains may be connected with a peptide linker.

[0718] In some embodiments, the binding agent according to the disclosure includes various types of bivalent and trivalent single-chain variable fragments (scFvs), fusion proteins mimicking the variable domains of two antibodies. Divalent (or bivalent) single-chain variable fragments (di- scFvs, bi-scFvs) can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two VH and two VL regions, yielding tandem scFvs. The disclosure also includes multispecific molecules comprising more than two scFvs binding domains.

[0719] Another possibility is the creation of scFvs with linker peptides that are too short for the two variable regions to fold together (about five amino acids), forcing scFvs to dimerize. This type is known as diabodies. Still shorter linkers (one or two amino acids) lead to the formation of trimers, so-called triabodies or tribodies. Tetrabodies have also been produced. They exhibit an even higher affinity to their targets than diabodies.

[0720] A particularly preferred example of a bispecific antibody fragment is a diabody (Kipriyanov, Int. J. Cancer 77 (1998), 763-772), which is a small bivalent and bispecific antibody fragment. Diabodies comprise a heavy chain variable domain (VH) and a light chain variable domain (VL) on the same polypeptide chain (VH-VL) connected by a peptide linker that is too short to allow pairing between the two domains on the same chain. This forces pairing with the complementary domains of another chain and promotes the assembly of a dimeric molecule with two functional antigen binding sites.

[0721] In some embodiments, the bispecific or multispecific molecule according to the disclosure comprises variable (VH, VL) and constant domains (C) of immunoglobulins. In some embodiments the bispecific molecule is a minibody, preferably, a minibody comprising two single VH-VL-C chains that are connected with each other via the constant domains (C) of each chain. According to this aspect, the corresponding variable heavy chain regions (VH), corresponding variable light chain regions (VL) and constant domains (C) are arranged, from N-terminus to C-terminus, in the order VH(Epitope l)-VL(Epitope l)-(C) and VH(Epitope 2)- VL(Epitope 2)-C, wherein C is preferably a CH3 domain, Epitope 1 refers to a first epitope and Epitope 2 refers to a second epitope. Pairing of the constant domains results in formation of the minibody.

[0722] According to another aspect, the bispecific binding agent of the disclosure is in the format of a bispecific single chain antibody construct, whereby said construct comprises or consists of at least two binding domains. In some embodiments, each binding domain comprises one variable region from an antibody heavy chain ("VH region"), wherein the VH region of the first binding domain specifically binds to Epitope 1, and the VH region of the second binding domain specifically binds to Epitope 2. The two binding domains are optionally linked to one another by a short polypeptide spacer. Each binding domain may additionally comprise one variable region from an antibody light chain ("VL region"), the VH region and VL region within each of the first and second binding domains being linked to one another via a polypeptide linker long enough to allow the VH region and VL region of the first binding domain and the VH region and VL region of the second binding domain to pair with one another.

[0723] In some embodiments, the binding agent described herein comprises an antibody, e.g., a full- length antibody, comprising the first binding domain. In some embodiments, the binding agent described herein comprises an antibody fragment such as scFv or VHH comprising the second binding domain which is covalently linked to the antibody comprising the first binding domain. In some embodiments, the binding agent comprises the antibody fragment such as scFv or VHH covalently linked to the N-terminus or C-terminus of the light chain or heavy chain of the antibody. In some embodiments, a binding moiety described herein, e.g., a binding moiety comprised in a docking compound binding to a primary target, comprises a DARPin. In some embodiments, the binding moiety directs a payload to target cells, such as cancer cells.

[0724] 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.

[0725] Ankyrin repeat proteins have been identified in 1987 through sequence comparisons between four such proteins in Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans. Breeden and Nasmyth reported multiple copies of a repeat unit of approximately 33 residues in the sequences of swi6p, cddOp, notch and lin-12 (Breeden et al., Nature 329, 651- 654 (1987)). The subsequent discovery of 24 copies of this repeat unit in the ankyrin protein led to the naming of this repeat unit as the ankyrin repeat (Lux et aL, Nature 344, 36-42 (1990)). Later, this repeat unit has been identified in several hundreds of proteins of different organisms and viruses (Bork, Proteins 17(4), 363-74 (1993)). These proteins are located in the nucleus, the cytoplasm or the extracellular space. This is consistent with the fact that the ankyrin repeat domain of these proteins is independent of disulfide bridges and thus independent of the oxidation state of the environment. The number of repeat units per protein varies from two to more than twenty. Tertiary structures of ankyrin repeat units share a characteristic fold (Sedgwick and Smerdon, Trends Biochem Sci. 24(8), 311-6 (1999)) composed of a (3-hairpin followed by two antiparallel a-helices and ending with a loop connecting the repeat unit with the next one. Domains built of ankyrin repeat units are formed by stacking the repeat units to an extended and curved structure. Proteins containing ankyrin repeat domains often contain additional domains. While the latter domains have variable functions, the function of the ankyrin repeat domain is most often the binding of other proteins. When analysing the repeat units of these proteins, the target interaction residues are mainly found in the P-hairpin and the exposed part of the first a-helix. These target interaction residues are hence forming a large contact surface on the ankyrin repeat domain. This contact surface is exposed on a framework built of stacked units of a-helix 1, a-helix 2 and the loop.

[0726] DARPins that bind to specific targets can be identified by screening combinatorial libraries of DARPins and selecting those with desired binding properties for the target. Such screening methods are described in, e.g., Muench et al., Molecular Therapy, 16(4), 686-693, 2011. For example, ribosomal display or phage display methods can be used to select target-specific DARPins from diverse libraries.

[0727] The term "repeat protein" refers to a (poly)peptide / protein comprising one or more repeat domains. In one embodiment, a repeat protein comprises up to four repeat domains. In one embodiment, a repeat protein comprises up to three repeat domains. In one embodiment, a repeat protein comprises up to two repeat domains. In the most preferred embodiment, a repeat protein comprises one repeat domain.

[0728] The individual domains of a repeat protein may be connected to each other directly or via (poly)peptide linkers. The term "(poly)peptide linker" refers to an amino acid sequence which is able to link two protein domains. Such linkers include, for example, glycine-serine-linkers of variable lengths and are known to the person skilled in the relevant art.

[0729] The term "repeat domain" refers to a protein domain comprising two or more consecutive repeat units (modules). In one embodiment, said repeat units are structural units having the same or a similar folding structure, and preferably stack tightly to preferably create a superhelical structure having a joint hydrophobic core.

[0730] The term "structural unit" refers to a locally ordered part of a (poly)peptide, formed by three- dimensional interactions between two or more segments of secondary structure that are near one another along the (poly)peptide chain. Such a structural unit comprises a structural motif. The term "structural motif" refers to a three-dimensional arrangement of secondary structure elements present in at least one structural unit. Structural motifs are well known to the person skilled in the relevant art. Said structural units may alone not be able to acquire a defined three-dimensional arrangement; however, their consecutive arrangement as repeat modules in a repeat domain leads to a mutual stabilization of neighbouring units which may result in a superhelical structure. The term "repeat modules" refers to the repeated amino acid sequences of the repeat proteins, which are derived from the repeat units of naturally occurring proteins. Each repeat module comprised in a repeat domain is derived from one or more repeat units of a family of naturally occurring repeat proteins, e.g., ankyrin repeat proteins.

[0731] The term "set of repeat modules" refers to the total number of repeat modules present in a repeat domain. Such "set of repeat modules" present in a repeat domain comprises two or more consecutive repeat modules, and may comprise just one type of repeat module in two or more copies, or two or more different types of modules, each present in one or more copies. Such set of repeat modules comprising, for example, 3 repeat modules may comprise consecutively, form N- to C-terminus, repeat module 1, repeat module 2, and repeat module 3.

[0732] Different repeat domains may have an identical number of repeat modules per repeat domain or may differ in the number of repeat modules per repeat domain.

[0733] Preferably, the repeat modules comprised in a set are homologous repeat modules. In the context of the present disclosure, the term "homologous repeat modules" refers to repeat modules, wherein more than 70% of the framework residues of said repeat modules are homologous. Preferably, more than 80% of the framework residues of said repeat modules are homologous. Most preferably, more than 90% of the framework residues of said repeat modules are homologous. Computer programs to determine the percentage of homology between polypeptides, such as Fasta, Blast or Gap, are known to the person skilled in the relevant art.

[0734] The term "repeat unit" refers to amino acid sequences comprising sequence motifs of one or more naturally occurring proteins, wherein said "repeat units" are found in multiple copies, and which exhibit a defined folding topology common to all said motifs determining the fold of the protein. Such repeat units comprise framework residues and interaction residues.

[0735] One example of such repeat units is an ankyrin repeat unit. Naturally occurring proteins containing two or more such repeat units are referred to as "naturally occurring repeat proteins". The amino acid sequences of the individual repeat units of a repeat protein may have a significant number of mutations, substitutions, additions and / or deletions when compared to each other, while still substantially retaining the general pattern, or motif, of the repeat units.

[0736] The term "repeat sequence motif" or "repeat consensus sequence" refers to an amino acid sequence, which is deduced from one or more repeat units. Such repeat sequence motifs comprise framework residue positions and target interaction residue positions. Said framework residue positions correspond to the positions of framework residues of said repeat units. Said target interaction residue positions correspond to the positions of target interaction residues of said repeat units. Such repeat sequence motifs comprise fixed positions and randomized positions. The term "fixed position" refers to an amino acid position in a repeat sequence motif, wherein said position is set to a particular amino acid. Frequently, such fixed positions correspond to the positions of framework residues.

[0737] The term "randomized position" refers to an amino acid position in a repeat sequence motif, wherein two or more amino acids are allowed at said amino acid position. Frequently, such randomized positions correspond to the positions of target target interaction residues. However, some positions of framework residues may also be randomized.

[0738] The term "folding topology" refers to the tertiary structure of said repeat units. The folding topology will be determined by stretches of amino acids forming at least parts of a-helices or p-sheets, or amino acid stretches forming linear polypeptides or loops, or any combination of a-helices, p-sheets and / or linear polypeptides / loops.

[0739] The term "consecutive" refers to an arrangement, wherein said modules are arranged in tandem.

[0740] In repeat proteins, there are at least 2, frequently 6 or more, 10 or more, or 20 or more repeat units, usually about 2 to 6 repeat units. For the most part, the repeat proteins are structural proteins and / or adhesive proteins, being present in prokaryotes and eukaryotes, including vertebrates and non-vertebrates.

[0741] In most cases, said repeat units will exhibit a high degree of sequence identity (same amino acid residues at corresponding positions) or sequence similarity (amino acid residues being different, but having similar physicochemical properties), and some of the amino acid residues might be key residues being strongly conserved in the different repeat units found in naturally occurring proteins. However, a high degree of sequence variability by amino acid insertions and / or deletions, and / or substitutions between the different repeat units found in naturally occurring proteins will be possible as long as the common folding topology is maintained.

[0742] The term "framework residues" relates to amino acid residues of the repeat units, or the corresponding amino acid residues of the repeat modules, which contribute to the folding topology, i.e. which contribute to the fold of said repeat unit (or module) or which contribute to the interaction with a neighboring unit (or module). Such contribution might be the interaction with other residues in the repeat unit (module), or the influence on the polypeptide backbone conformation as found in a-helices or 3-sheets, or amino acid stretches forming linear polypeptides or loops.

[0743] The term "target interaction residues" refers to amino acid residues of the repeat units, or the corresponding amino acid residues of the repeat modules, which contribute to the interaction with target substances. Such contribution might be the direct interaction with the target substances, or the influence on other directly interacting residues, e.g. by stabilising the conformation of the (poly)peptide of said repeat unit (module) to allow or enhance the interaction of said directly interacting residues with said target.

[0744] A "target" may be an individual molecule such as a nucleic acid molecule, a (poly)peptide protein, a carbohydrate, or any other naturally occurring molecule, including any part of such individual molecule, or complexes of two or more of such molecules. The target may be, in particular, a molecule on immune effector cells, in particular CD8.

[0745] In one embodiment, the repeat modules are directly connected. In the context of the present invention, the term "directly connected" refers to repeat modules, which are arranged as direct repeats in a repeat protein without an intervening amino acid sequence.

[0746] In another embodiment, the repeat modules are connected by a (poly)peptide linker. Thus, the repeat modules may be linked indirectly via a (poly)peptide linker as intervening sequence separating the individual modules. An "intervening sequence" may be any amino acid sequence, which allows to connect the individual modules without interfering with the folding topology or the stacking of the modules. Preferentially, said intervening sequences are short (poly)peptide linkers of less than 10, and even more preferably, of less than 5 amino acid residues. In one embodiment, a repeat protein further comprises an N- and / or a C-terminal capping module having an amino acid sequence different from any one of said repeat modules. The term "capping module" refers to a polypeptide fused to the N- or C- terminal repeat module of a repeat domain, wherein said capping module forms tight tertiary interactions with said repeat module thereby providing a cap that shields the hydrophobic core of said repeat module at the side not in contact with the consecutive repeat module from the solvent.

[0747] Said N- and / or C-terminal capping module may be, or may be derived from, a capping unit or other domain found in a naturally occurring repeat protein adjacent to a repeat unit.

[0748] The term "capping unit" refers to a naturally occurring folded (poly)peptide, wherein said (poly)peptide defines a particular structural unit which is N- or C-terminally fused to a repeat unit, wherein said (poly)peptide forms tight tertiary interactions with said repeat unit thereby providing a cap that shields the hydrophobic core of said repeat unit at one side from the solvent. Such capping units may have sequence similarities to said repeat sequence motif.

[0749] Nucleic acids

[0750] 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.

[0751] 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.

[0752] 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, such as N7-C1-4alkyl-guanine, N6-C1-4alkyl-adenine, 5-C1-4 alkyl-cytosine, 5-C1-4 alkyl-u racil, and N(l)- C1-4 alkyl-uracil, preferably N7-methyl-guanine, N6-methyl-adenine, 5-methyl-cytosine, 5- methyl-uracil, and N(l)-methyl-uracil.

[0753] DNA

[0754] Herein, the term "DNA" relates to a nucleic acid molecule which includes deoxyribonucleotide residues. In preferred embodiments, the DNA contains all or a majority of deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide which lacks a hydroxyl group at the 2'-position of a (J-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 non- nucleotide 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).

[0755] 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.

[0756] 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 artificial chromosomes (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.

[0757] RNA

[0758] 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 refer to 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 (i.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).

[0759] "RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self- amplifying 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.

[0760] The term "in vitro transcription" or "IVT" as used herein means that the transcription (i.e., the generation of RNA) is conducted in a cell-free manner. I.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)).

[0761] 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. 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. According to the present disclosure, "dsRNA" means double-stranded RNA and is RNA with two partially or completely complementary strands.

[0762] 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.

[0763] 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: A Laboratory 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.

[0764] 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 promoter for 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.

[0765] 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) are typically 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).

[0766] 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.

[0767] 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 truncation of 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).

[0768] 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 (i.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 p-phosphate (such as m27,2 OG(5')ppSp(5')G (referred to as beta-S-ARCA or P-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.

[0769] 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 OG(5')ppp(5')G, and m27'3'0Gppp(mi2'°)ApG.

[0770] 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 21.

[0771] The 5'-cap analog beta-S-ARCA (P-S-ARCA) has the following structure: 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.

[0772] The 5'-cap analog m27,3%ppp(mi2'0)ApG (also referred to as m27'3'oG(5,)ppp(5,)m2'oApG) which is a building block of a capl has the following structure:

[0773] An exemplary capO mRNA comprising p-S-ARCA and mRNA has the following structure: An exemplary capO mRNA comprising m27'3 OG(5')ppp(5')G and mRNA has the following structure:

[0774] An exemplary capl mRNA comprising m27,3 OGppp(mi2 O)ApG and mRNA has the following structure:

[0775] 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.

[0776] 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).

[0777] 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 A nucleotides, 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.

[0778] 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.

[0779] 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. coli 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.

[0780] 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.

[0781] 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.

[0782] 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 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 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.

[0783] 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 31- 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. 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 enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA.

[0784] 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.

[0785] In some embodiments, the modified uridine replacing uridine is selected from the group consisting of pseudouridine (4>), Nl-methyl-pseudouridine (mlip), 5-methyl-uridine (m5U), and combinations thereof.

[0786] 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 (tm5U), 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 (cmnmSUm), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)- 2'-0-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.

[0787] 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 "m lip-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 MJ- or mlUJ- 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.

[0788] 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 corresponding sequence regions of the wild type coding sequence. In some embodiments, the codon- optimization and / or the increase in the G / C content preferably does not change the sequence of the encoded amino acid sequence.

[0789] 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".

[0790] 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.

[0791] 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.

[0792] 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 (MJ) or N(l)-methylpseudouridine (mlUJ) 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 51- 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 (4J) or N(l)-methylpseudouridine (mlip) or 5-methyluridine (m5U) for uridine), and (v) codon optimization. Particles

[0793] A nucleic acid such as RNA may be administered with one or more delivery vehicles that protect the nucleic acid from degradation, maximize delivery to on-target cells and minimize exposure to off-target cells. Such delivery vehicles may complex or encapsulate the nucleic acid and include a range of materials, including polymers, lipids and mixtures thereof. In some embodiments, such delivery vehicles may form particles with the nucleic acid.

[0794] In the context of the present disclosure, the term "particle" relates to a structured entity formed by molecules or molecule complexes, in particular particle forming compounds. In some embodiments, a particle is a nucleic acid containing particle such as a particle comprising DNA, RNA or a mixture thereof. In some embodiments, the particle contains an envelope (e.g., one or more layers or lamellas) made of one or more types of amphiphilic substances (e.g., amphiphilic lipids). In this context, the expression "amphiphilic substance" means that the substance possesses both hydrophilic and lipophilic properties. The envelope may also comprise additional substances (e.g., additional lipids) which do not have to be amphiphilic. Thus, the particle may be a monolameliar or multilamellar structure, wherein the substances constituting the one or more layers or lamellas comprise one or more types of amphiphilic substances (in particular selected from the group consisting of amphiphilic lipids) optionally in combination with additional substances (e.g., additional lipids) which do not have to be amphiphilic. In some embodiments, the term "particle" relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure. According to the present disclosure, the term "particle" includes nanoparticles.

[0795] The term "nanoparticle" relates to a nano-sized particle comprising at least one particle forming agent, e.g., at least one cationic or cationically ionizable lipid, wherein all three external dimensions of the particle are in the nanoscale, i.e., at least about 1 nm and below about 1000 nm. Preferably, the size of a particle is its diameter.

[0796] In some embodiments, the particles described herein have a size (such as a diameter) in the range of about 10 to about 2000 nm, such as in the range of about 20 to about 1500 nm, such as about 30 to about 1200 nm, about 40 to about 1100 nm, about 50 to about 1000 nm, about 60 to about 900 nm, about 70 to about 800 nm, about 80 to about 700 nm, about 90 to about 600 nm, or about 50 to about 500 nm or about 100 to about 500 nm, such as in the range of 10 to 1000 nm, 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, 50 to 250 nm, 60 to 200 nm, 70 to 150 nm, or 80 to 150 nm. In some embodiments, the particles described herein have a size (such as a diameter) in the range of from about 40 nm to about 200 nm, such as from about 50 nm to about 180 nm, from about 60 nm to about 160 nm, from about 80 nm to about 150 nm or from about 80 nm to about 120 nm.

[0797] Particles described herein may exhibit a polydispersity index (PDI) less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, or less than about 0.05. By way of example, the particles can exhibit a polydispersity index in a range of about 0.01 to about 0.4 or about 0.1 to about 0.3.

[0798] A "nucleic acid particle" can be used to deliver nucleic acid to a target site of interest (e.g., cell, tissue, organ, and the like). A nucleic acid particle may be formed from at least one cationic or cationically ionizable compound such as a polymer or lipid complexing the nucleic acid. Without intending to be bound by any theory, it is believed that the cationic or cationically ionizable compound combines together with the nucleic acid to form aggregates, and this aggregation results in colloidally stable particles.

[0799] In some embodiments, nucleic acid may be noncovalently associated with a particle. In some embodiments, the nucleic acid may be adhered to the outer surface of the particle (surface nucleic acid) and / or may be contained in the particle (encapsulated nucleic acid).

[0800] The N / P ratio gives the ratio of the nitrogen groups in the lipid to the number of phosphate groups in the nucleic acid. It is correlated to the charge ratio, as the nitrogen atoms (depending on the pH) are usually positively charged and the phosphate groups are negatively charged. The N / P ratio, where a charge equilibrium exists, depends on the pH. Lipid formulations are frequently formed at N / P ratios larger than four up to twelve, because positively charged nanoparticles are considered favorable for transfection. In that case, nucleic acid is considered to be completely bound to nanoparticles.

[0801] The term "particle forming components" or "particle forming agents" relates to any components which form particles, e.g., by associating with nucleic acid. Nucleic acid delivery vehicles such as particle forming agents useful herein include polymers, polymer derivatives, lipids, and mixtures thereof. Such components include any component which can be part of nucleic acid particles, e.g., cationic or cationically ionizable lipids. A "polymer," as used herein, is given its ordinary meaning, i.e., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. The repeat units can all be identical, or in some cases, there can be more than one type of repeat unit present within the polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer such as a protein. In some cases, additional moieties can also be present in the polymer, for example targeting moieties.

[0802] If more than one type of repeat unit is present within the polymer, then the polymer is said to be a "copolymer." It is to be understood that the polymer being employed herein can be a copolymer. The repeat units forming the copolymer can be arranged in any fashion. For example, the repeat units can be arranged in a random order, in an alternating order, or as a "block" copolymer, i.e., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc. Block copolymers can have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.

[0803] In certain embodiments, the polymer is biocompatible. Biocompatible polymers are polymers that typically do not result in significant cell death at moderate concentrations. In certain embodiments, the biocompatible polymer is biodegradable, i.e., the polymer is able to degrade, chemically and / or biologically, within a physiological environment, such as within the body.

[0804] In certain embodiments, polymer may be protamine or polyalkyleneimine.

[0805] The term "protamine" refers to any of various strongly basic proteins of relatively low molecular weight that are rich in arginine and are found associated especially with DNA in place of somatic histones in the sperm cells of various animals (as fish). In particular, the term "protamine" refers to proteins found in fish sperm that are strongly basic, are soluble in water, are not coagulated by heat, and yield chiefly arginine upon hydrolysis. In purified form, they are used in a long-acting formulation of insulin and to neutralize the anticoagulant effects of heparin.

[0806] According to the disclosure, the term "protamine" as used herein is meant to comprise any protamine amino acid sequence obtained or derived from natural or biological sources including fragments thereof and multimeric forms of said amino acid sequence or fragment thereof as well as (synthesized) polypeptides which are artificial and specifically designed for specific purposes and cannot be isolated from native or biological sources.

[0807] In some embodiments, the polyalkyleneimine comprises polyethylenimine and / or polypropylenimine, preferably polyethyleneimine. A preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75-102to 107Da, preferably 1000 to 105Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, even more preferably 20000 to 25000 Da.

[0808] Preferred according to the disclosure is linear polyalkyleneimine such as linear polyethyleneimine (PEI).

[0809] Cationic polymers (including polycationic polymers) contemplated for use herein include any cationic polymers which are able to electrostatically bind nucleic acid. In some embodiments, cationic polymers contemplated for use herein include any cationic polymers with which nucleic acid can be associated, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.

[0810] Particles described herein may also comprise polymers other than cationic polymers, i.e., non- cationic polymers and / or anionic polymers. Collectively, anionic and neutral polymers are referred to herein as non-cationic polymers.

[0811] In some embodiments of the present disclosure, the nucleic acid such as RNA described herein may be present in lipoplex particles.

[0812] Lipoplexes (LPX) are electrostatic complexes which are generally formed by mixing preformed cationic lipid liposomes with anionic nucleic acid. Formed lipoplexes possess distinct internal arrangements of molecules that arise due to the transformation from liposomal structure into compact nucleic acid lipoplexes.

[0813] Generally, lipoplex particles are obtainable by adding nucleic acid to a colloidal liposome dispersion. Using the ethanol injection technique, such colloidal liposome dispersion is, in some embodiments, formed as follows: an ethanol solution comprising lipids, such as cationic or cationically ionizable lipids and additional lipids, is injected into an aqueous solution under stirring.

[0814] In some embodiments, liposomes are self-closed unilamellar or multilamellar vesicular particles wherein the lamellae comprise lipid bilayers and the encapsulated lumen comprises an aqueous phase. A prerequisite for using liposomes for nanoparticle formation is that the lipids in the mixture as required are able to form lamellar (bilayer) phases in the applied aqueous environment.

[0815] In certain embodiments, the nucleic acid lipoplex particles include both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

[0816] In some embodiments, nucleic acid described herein is present in the form of lipid nanoparticles (LNPs).

[0817] In general, lipid nanoparticles are obtainable from direct mixing of nucleic acid, e.g., RNA, in an aqueous phase with lipids in a phase comprising an organic solvent, such as ethanol. In that case, lipids or lipid mixtures can be used for particle formation, which do not form lamellar (bilayer) phases in water.

[0818] LNPs typically comprise four components: cationically ionizable lipid, neutral lipids such as phospholipids, a steroid such as cholesterol, and a polymer-conjugated lipid such as PEG-lipid. In some embodiments, the LNP comprises from 40 to 60 mol percent, 40 to 55 mol percent, from 45 to 55 mol percent, or from 45 to 50 mol percent of the cationically ionizable lipid.

[0819] In some embodiments, the neutral lipid is present in a concentration ranging from 5 to 15 mol percent, from 7 to 13 mol percent, or from 9 to 11 mol percent.

[0820] In some embodiments, the steroid is present in a concentration ranging from 30 to 50 mol percent, from 30 to 45 mol percent, from 35 to 45 mol percent or from 35 to 43 mol percent. In some embodiments, the LNP comprises from 1 to 10 mol percent, from 1 to 5 mol percent, or from 1 to 2.5 mol percent of the polymer-conjugated lipid.

[0821] In some embodiments, the LNP comprises from 45 to 55 mol percent of a cationically ionizable lipid; from 5 to 15 mol percent of a neutral lipid; from 30 to 45 mol percent of a steroid; from 1 to 5 mol percent of a polymer-conjugated lipid; and the nucleic acid, encapsulated within or associated with the lipid nanoparticle.

[0822] In some embodiments, the mol percent is determined based on total mol of lipid present in the lipid nanoparticle. In some embodiments, the mol percent is determined based on total mol of cationically ionizable lipid, neutral lipid, steroid and polymer-conjugated lipid present in the lipid nanoparticle. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC.

[0823] In some embodiments, the steroid is cholesterol.

[0824] In some embodiments, the polymer conjugated lipid is a pegylated lipid.

[0825] In some embodiments, the cationically ionizable lipid component of the LNPs has one of the following structures:

[0826]

[0827] Pharmaceutical compositions

[0828] The agents described herein, e.g., tag conjugates, docking compounds, complexes comprising tag conjugate and docking compound, and nucleic acid encoding docking compound, may be administered in pharmaceutical compositions and may be administered in the form of any suitable pharmaceutical composition. Accordingly, described herein are pharmaceutical compositions comprising the agents described herein, e.g., tag conjugates, docking compounds, complexes comprising tag conjugate and docking compound, and nucleic acid encoding docking compound.

[0829] In some embodiments, the agents described herein may be administered in a pharmaceutical composition which may comprise a pharmaceutically acceptable carrier and may optionally comprise one or more stabilizers etc. In some embodiments, the pharmaceutical composition is for therapeutic or prophylactic treatments, e.g., for use in treating or preventing a disease. The term "pharmaceutical composition" relates to a formulation comprising a therapeutically effective agent, preferably together with pharmaceutically acceptable carriers, diluents and / or excipients. Said pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease or disorder by administration of said pharmaceutical composition to a subject. A pharmaceutical composition is also known in the art as a pharmaceutical formulation.

[0830] The pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and in "a pharmaceutically acceptable preparation".

[0831] The term "pharmaceutically acceptable" refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition.

[0832] The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses and / or agents. In the case of the treatment of a particular disease, the desired reaction preferably relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting or reversing the progress of the disease. The desired reaction in a treatment of a disease may also be delay of the onset or a prevention of the onset of said disease or said condition. An effective amount of the compositions described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the compositions described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[0833] The pharmaceutical compositions of the present disclosure may contain salts, buffers, preservatives, and optionally other therapeutic agents. In some embodiments, the pharmaceutical compositions of the present disclosure comprise one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0834] Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, paraben and thimerosal. The term "excipient" as used herein refers to a substance which may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient. Examples of excipients, include without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants.

[0835] The term "diluent" relates a diluting and / or thinning agent. Moreover, the term "diluent" includes any one or more of fluid, liquid or solid suspension and / or mixing media. Examples of suitable diluents include ethanol, glycerol and water.

[0836] The term "carrier" refers to a component which may be natural, synthetic, organic, inorganic in which the active component is combined in order to facilitate, enhance or enable administration of the pharmaceutical composition. A carrier as used herein may be one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to subject. Suitable carriers include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxy-propylene copolymers. In some embodiments, the pharmaceutical composition of the present disclosure includes isotonic saline.

[0837] Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).

[0838] Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0839] In some embodiments, pharmaceutical compositions described herein may be administered intravenously, intraarterially, subcutaneously, intradermally or intramuscularly. In certain embodiments, the pharmaceutical composition is formulated for local administration or systemic administration. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to the administration in any manner other than through the gastrointestinal tract, such as by intravenous injection. In some embodiments, the pharmaceutical composition is formulated for systemic administration, e.g., for intravenous administration.

[0840] Treatments

[0841] The agents, compositions and methods described herein can be used to treat a subject with a disease, e.g., a disease characterized by the presence of diseased cells expressing an antigen. The agents, compositions and methods described herein may be used in the therapeutic or prophylactic treatment of various diseases. Particularly preferred diseases are cancer diseases. In some embodiments, the agents, compositions and methods described herein are useful in a prophylactic and / or therapeutic treatment of a disease involving an antigen.

[0842] For example, if the antigen is derived from a virus, the agents, compositions and methods may be useful in the treatment of a viral disease caused by said virus. If the antigen is a tumor antigen, the agents, compositions and methods may be useful in the treatment of a cancer disease wherein cancer cells express said tumor antigen.

[0843] The term "disease" refers to an abnormal condition that affects the body of an individual. A disease is often construed as a medical condition associated with specific symptoms and signs. A disease may be caused by factors originally from an external source, such as infectious disease, or it may be caused by internal dysfunctions, such as autoimmune diseases. In humans, "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, or death to the individual afflicted, or similar problems for those in contact with the individual. In this broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behaviors, and atypical variations of structure and function, while in other contexts and for other purposes these may be considered distinguishable categories. Diseases usually affect individuals not only physically, but also emotionally, as contracting and living with many diseases can alter one's perspective on life, and one's personality.

[0844] In the present context, the term "treatment", "treating" or "therapeutic intervention" relates to the management and care of a subject for the purpose of combating a condition such as a disease or disorder. The term is intended to include the full spectrum of treatments for a given condition from which the subject is suffering, such as administration of the therapeutically effective compound to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate or relief the symptoms and complications, and / or to cure or eliminate the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of an individual for the purpose of combating the disease, condition or disorder and includes the administration of the active compounds to prevent the onset of the symptoms or complications.

[0845] The term "therapeutic treatment" relates to any treatment which improves the health status and / or prolongs (increases) the lifespan of an individual. Said treatment may eliminate the disease in an individual, arrest or slow the development of a disease in an individual, inhibit or slow the development of a disease in an individual, decrease the frequency or severity of symptoms in an individual, and / or decrease the recurrence in an individual who currently has or who previously has had a disease.

[0846] The terms "prophylactic treatment" or "preventive treatment" relate to any treatment that is intended to prevent a disease from occurring in an individual. The terms "prophylactic treatment" or "preventive treatment" are used herein interchangeably.

[0847] The terms "individual" and "subject" are used herein interchangeably. They refer to a human or another mammal (e.g. mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate) that can be afflicted with or is susceptible to a disease or disorder (e.g., cancer) but may or may not have the disease or disorder. In many embodiments, the individual is a human being. Unless otherwise stated, the terms "individual" and "subject" do not denote a particular age, and thus encompass adults, elderlies, children, and newborns. In embodiments of the present disclosure, the "individual" or "subject" is a "patient".

[0848] The term "patient" means an individual or subject for treatment, in particular a diseased individual or subject.

[0849] In some embodiments of the disclosure, the aim is to deliver a detectable label to diseased cells expressing an antigen such as cancer cells expressing a tumor antigen, and to diagnose a disease such as a cancer disease involving cells expressing an antigen such as a tumor antigen. In some embodiments of the disclosure, the aim is to deliver a pharmaceutically active agent to diseased cells expressing an antigen such as cancer cells expressing a tumor antigen, and to treat a disease such as a cancer disease involving cells expressing an antigen such as a tumor antigen.

[0850] In some embodiments of the disclosure, the aim is to deliver a compound producing radiation which kills or damages cells, e.g., ionizing radiation, e.g., radionuclides and radiolabels, to diseased cells expressing an antigen such as cancer cells expressing a tumor antigen, and to treat a disease such as a cancer disease involving cells expressing an antigen such as a tumor antigen.

[0851] In some embodiments of the disclosure, the aim is to deliver a payload, e.g., an immunomodulator, to immune cells to modulate, e.g., induce or enhance development, priming, expansion, differentiation and / or survival of, immune cells. In some embodiments, immune cells may target diseased cells expressing an antigen such as cancer cells expressing a tumor antigen for treating a disease such as a cancer disease involving cells expressing an antigen such as a tumor antigen. In some embodiments, immune cells exert one or more immune effector functions on diseased cells, e.g., kill diseased cells by means of a cellular immune response.

[0852] The term "disease involving an antigen", "disease involving cells expressing an antigen" or similar terms refer to any disease which implicates an antigen, e.g. a disease which is characterized by the presence of an antigen. The disease involving an antigen can be an infectious disease, or a cancer disease or simply cancer. As mentioned above, the antigen may be a disease-associated antigen, such as a tumor-associated antigen, a viral antigen, or a bacterial antigen. In some embodiments, a disease involving an antigen is a disease involving cells expressing an antigen, preferably on the cell surface.

[0853] The term "infectious disease" refers to any disease which can be transmitted from individual to individual or from organism to organism, and is caused by a microbial agent (e.g. common cold). Infectious diseases are known in the art and include, for example, a viral disease, a bacterial disease, or a parasitic disease, which diseases are caused by a virus, a bacterium, and a parasite, respectively. In this regard, the infectious disease can be, for example, hepatitis, sexually transmitted diseases (e.g. chlamydia or gonorrhea), tuberculosis, HIV / acquired immune deficiency syndrome (AIDS), diphtheria, hepatitis B, hepatitis C, cholera, severe acute respiratory syndrome (SARS), the bird flu, and influenza.

[0854] The terms "cancer disease" or "cancer" refer to or describe the physiological condition in an individual that is typically characterized by unregulated cell growth. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particularly, examples of such cancers include bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, carcinoma of the sexual and reproductive organs, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the bladder, cancer of the kidney, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spinal axis tumors, glioma, meningioma, and pituitary adenoma. The term "cancer" according to the disclosure also comprises cancer metastases.

[0855] The term "solid tumor" or "solid cancer" as used herein refers to the manifestation of a cancerous mass, as is well known in the art for example in Harrison's Principles of Internal Medicine, 14th edition. Preferably, the term refers to a cancer or carcinoma of body tissues other than blood, preferably other than blood, bone marrow, and lymphoid system. For example, but not by way of limitation, solid tumors include cancers of the prostate, lung cancer, colorectal tissue, bladder, oropharyngeal / laryngeal tissue, kidney, breast, endometrium, ovary, cervix, stomach, pancrease, brain, and central nervous system.

[0856] The methods and agents described herein are, in particular, useful for the treatment of cancers, e.g., solid cancers, characterized by diseased cells expressing an antigen a docking compound is directed to.

[0857] Citation of documents and studies referenced herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the contents of these documents.

[0858] The following description is presented to enable a person of ordinary skill in the art to make and use the various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those of ordinary skill in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Thus, the various embodiments are not intended to be limited to the examples described herein and shown, but are to be accorded the scope consistent with the claims.

[0859] Abbreviations

[0860] Some abbreviations that may appear in this application are defined hereinafter:

[0861] Examples

[0862] Example 1 - Examples of ALFA STING conjugates

[0863]

[0864]

[0865]

[0866]

[0867]

[0868]

[0869] Materials and methods

[0870] UPLC-MS Instrument and Methods

[0871] Instrument: Waters H-Class UPLC with Q.SM, sample organizer, column heater, PDA UV detector and QDa mass spectrometer.

[0872] Column: Waters BEH C18 Column, 100 x 2.1 mm, 1.7 pm, 130 A pore size

[0873] Lcmsjong method: 40°C column temperature. UV absorption wavelength: 214 nm. MS range: 200-1250 Da. Mobile phase A: 0.1% TFA in water. Mobile phase B: 0.085% TFA in acetonitrile. Flowrate: 0.5 mL / min. Gradient: Column: Waters BEH Cis Column, 50x 2.1 mm, 1.7 pm, 130 A pore size

[0874] Lcms_short method: 40°C column temperature. UV absorption wavelength: 214 nm. MS range: 200-1250 Da. Mobile phase A: 0.1% TFA in water. Mobile phase B: 0.085% TFA in acetonitrile. Flowrate: 0.5 mL / min. Gradient:

[0875] Lcms_long_C4_60C method: Same as Lcms_long_C4 method but with 60°C column temperature.

[0876] SEC Instrument and Methods

[0877] Preparative size-exclusion chromatography was performed on a JAI Preparative Recycling HPLC (LaboACE LC-7080) system equipped with 2.5HR Plus and 2HR Plus columns connected in series (20x600 mm) using 10% MeOH / CHCI3 as eluent.

[0878] Synthesis of monoALFA intermediates

[0879] Synthesis of SD17248-A

[0880] SD17248-A1 SD17248-A

[0881] Step 1: Synthesis of cyclic ALFA peptide intermediate, SD17248-A3

[0882] The cyclic ALFA peptide intermediate is synthesized using Fmoc solid phase peptide synthesis chemistry. Rink amide AM resin is loaded onto an automated peptide synthesizer suspended in 10 mL of 1:1 dichloromethane / dimethylformamide (DCM:DMF) for pre-swelling and resin transfer. The peptide synthesis is initiated by the deprotection of the N-terminal a Fmoc protecting group using 4 mL of 20% piperidine in DMF heated by microwave for 3 min at 50 °C. After draining, the resin is washed three times with 5 mL DMF at five seconds per wash. Then the coupling reaction is performed by the addition of 2.5 mL of the next 0.3 M amino acid (7.5 eq.) in DMF, 1 mL of a 1 M DIC (10 eq.) solution in DMF, and 0.5 mL 1 M Oxyma (5 eq.) with 0.1M DIEA solution in DMF to the RV. The coupling reaction is heated to 60 °C for 10 min. The reaction solution is drained, and the coupling reaction is repeated. After the second coupling, the resin is drained and washed four times with 4 mL of DMF. Then 4 mL of 20% piperidine in DMF is added to the RV to deprotect the Fmoc group and the next amino acid is coupled. This cycle of Fmoc removal and coupling is repeated for every amino acid sequentially. Fmoc-Glu(O-2-PhiPr)-OH and Fmoc-Lys(Mmt)-OH amino acids are incorporated into the peptide intermediate sequence to allow for selective cyclization. The peptide- containing resin is transferred to a 20 mL fritted syringe after solid phase peptide synthesis is completed. To the resin is added 7 mL of capping solution (81.25% DMF, 12.5% 2 M DIEA in NMP, 6.25% acetic anhydride) and the reaction is agitated for 15 min. The capping solution is removed, and another 7 mL of capping solution is added and the reaction is agitated for another 15 min. Then the resin is washed thoroughly with DCM and DMF.

[0883] Step 2: Selective deprotection of Lys(Mmt) and Glu(O-2-PhiPr), SD17248-A2

[0884] To the resin containing SD17248-A3 in a fritted syringe is added 5 mL of partial deprotection cocktail (2% TFA, 2% TIS in DCM). The reaction is agitated for 5 min, then the deprotection cocktail is removed from the resin via filtration. Another 5 mL of partial deprotection cocktail is added, agitated for 5 minutes, and removed. This process is repeated six times until the yellow / orange color of the flowthrough disappears (30 minutes total of Mmt and 2-PhiPr removal). The resin is rinsed thoroughly with DCM and DMF to afford SD17248-A2.

[0885] Step 3: Lactam cyclization via side chain amide bond formation, SD17248-A1 Cyclization is carried out in the same fritted syringe as the partial deprotection from Step 2. To the syringe is added 5 eq. of PyAOP and 5 eq. of HOAt as a solution in DMF. The resin is allowed to swell and then 10 eq. of DIEA is added. The reaction is agitated for 2-3 hours. The reaction mixture is removed from the resin via filtration and the remaining resin is rinsed thoroughly with DCM and DMF to afford SD17248-A1.

[0886] Step 4: Cleavage and global deprotection, SD17248-A

[0887] To the resin containing SD17248-A1 in a fritted syringe is added 10 mL of cleavage cocktail (87.5% TFA, 5% thioanisole, 2.5% water, 2.5% TIS, 2.5% EDT). The reaction is agitated for 2 h at room temp followed by 1 h at 40 °C. The resulting peptide intermediate is then precipitated out of solution by adding the reaction solution to ~25 mL cold diethyl ether which is then centrifuged. The supernatant is removed and the resulting pellet is redissolved in 50:50 MeCNrwater. The resulting crude peptide intermediated is purified by RP HPLC to afford SD17248-A. Isolated 159.2 mg from 0.3-mmol scale synthesis (25% yield, >95% pure).

[0888] [M+2H]2+= 1038.99

[0889] Synthesis of SD18317-A / PIC8-A

[0890] PIC8A

[0891] The title compound was prepared following analogous protocol to the synthesis of SD17248A.

[0892] [M+2H]2+= 1184.23

[0893] Synthesis of PIC12-A

[0894] PIC12A

[0895] The title compound was prepared following analogous protocol to the synthesis of SD17248A using Fmoc-L-Cysteic acid. [M+2H]2+= 1114.18

[0896] Synthesis of PIC13-A

[0897] PIC13A

[0898] The title compound was prepared following analogous protocol to the synthesis of SD17248A using Fmoc-AEPA-OH.

[0899] [...

Claims

Claims1. A kit comprising:(i) a compound comprising a binding moiety binding to a 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 a tag to which the binding moiety for a tag binds, wherein the tag is a peptide tag.

2. The kit of claim 1, wherein the compound under (ii) comprises a moiety comprising a polymer.

3. The kit of claim 1 or 2, wherein the payload moiety and the peptide tag are coupled through a moiety comprising a polymer.

4. The kit of claim 2 or 3, wherein the polymer is not a polymer of proteinogenic amino acids or their D-isomers.

5. A kit comprising:(i) a compound comprising a binding moiety binding to a 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 a tag to which the binding moiety for a tag binds, wherein the compound under (ii) further comprises a moiety comprising a polymer, wherein the polymer is not a polymer of proteinogenic amino acids or their D-isomers.

6. The kit of claim 5, wherein the payload moiety and the tag are coupled through a moiety comprising a polymer, wherein the polymer is not a polymer of proteinogenic amino acids or their D-isomers.

7. The kit of claim 5 or 6, wherein the tag is a peptide tag.

8. The kit of any one of claims 2 to 7, wherein the polymer is selected from the group consisting of polyethylene 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.

9. The kit of any one of claims 2 to 8, wherein the polymer comprises at least one poly- 2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

10. The kit of any one of claims 1 to 9, wherein the payload moiety and the tag are coupled through a moiety comprising at least one poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

11. The kit of any one of claims 1 to 10, wherein the nucleic acid is RNA.

12. The kit of any one of claims 1 to 11, wherein the compound under (ii) comprises one tag.

13. The kit of any one of claims 1 to 12, wherein the total number of tags in the compound under (ii) is one.

14. The kit of any one of claims 1 to 13, wherein the compound under (ii) comprises one or more payload moieties.

15. The kit of any one of claims 1 to 14, wherein the compound under (ii) comprises one payload moiety.

16. The kit of any one of claims 1 to 15, wherein the total number of payload moieties in the compound under (ii) is one.

17. The kit of any one of claims 1 to 16, wherein the compound under (ii) comprises a linking moiety connecting a tag and a payload moiety.

18. The kit of claim 17, wherein the linking moiety is a branched or an unbranched linking moiety.

19. The kit of claim 17 or 18, wherein the linking moiety comprises a continuous or non- continuous poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

20. The kit of any one of claims 1 to 19, wherein the compound under (ii) comprises the formula:P-L-T whereinP comprises a payload moiety;T comprises a tag; andL comprises a linking moiety.

21. The kit of claim 20, wherein L comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

22. The kit of claim 20 or 21, wherein L comprises the formula [AEEA]u-[L'-[AEEA]v]w, whereinAEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid or a derivative thereof;L1comprises a linking moiety; u is an integer of 2 or more; each v is an integer of 2 or more; and w is an integer from 1 to 4; wherein the different groups [L'-[AEEA]V] may be identical or different.

23. The kit of claim 22, wherein u and v are each integers from 2 to 10.

24. The kit of claim 22 or 23, wherein u and v are each integers from 2 to 8.

25. The kit of any one of claims 22 to 24, wherein u and v are each integers of 2, 4 or 6.

26. The kit of any one of claims 20 to 25, wherein L or L' comprises an amino acid.

27. The kit of any one of claims 20 to 26, wherein L or L' comprises the D-isomer of an amino acid.

28. The kit of any one of claims 20 to 27, wherein L or L' comprises cysteine or lysine.

29. The kit of any one of claims 20 to 28, wherein L or L' is connected to a side chain.

30. The kit of claim 29, wherein a side chain comprises a functional moiety.

31. The kit of claim 30, wherein a functional moiety comprises a solubilizing functional group.

32. The kit of claim 30 or 31, wherein a functional moiety comprises a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

33. The kit of any one of claims 1 to 32, wherein the payload moiety is selected from the group consisting of radioisotopes, toxins and immunomodulators.

34. The kit of any one of claims 8 to 33, wherein 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.

35. The kit of any one of claims 8 to 34, wherein 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.

36. The kit of any one of claims 8 to 35, wherein 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 2, 4 or 6.

37. The kit of any one of claims 1 to 36, wherein the compound under (i) comprises a single binding moiety for the tag.

38. The kit of any one of claims I to 36, wherein the compound under (i) comprises at least two binding moieties for the tag.

39. The kit of any one of claims 1 to 36, wherein the compound under (i) comprises two binding moieties for the tag.

40. The kit of any one of claims 1 to 39, wherein the tag is an ALFA-tag.

41. The kit of any one of claims 1 to 40, wherein the tag is a cyclic ALFA-tag.

42. A compound comprising a payload moiety and a peptide tag.

43. The compound of claim 42, which comprises a moiety comprising a polymer.

44. The compound of claim 42 or 43, wherein the payload moiety and the peptide tag are coupled through a moiety comprising a polymer.

45. The compound of claim 43 or 44, wherein the polymer is not a polymer of proteinogenic amino acids or their D-isomers.

46. A compound comprising a payload moiety and a tag, wherein the compound further comprises a moiety comprising a polymer, wherein the polymer is not a polymer of proteinogenic amino acids or their D-isomers.

47. The compound of claim 46, wherein the payload moiety and the tag are coupled through a moiety comprising a polymer, wherein the polymer is not a polymer of proteinogenic amino acids or their D-isomers.

48. The compound of claim 46 or 47, wherein the tag is a peptide tag.

49. The compound of any one of claims 43 to 48, wherein the polymer is selected from the group consisting of polyethylene 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.

50. The compound of any one of claims 43 to 49, wherein the polymer comprises at least one poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

51. The compound of any one of claims 42 to 50, wherein the payload moiety and the tag are coupled through a moiety comprising at least one poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

52. The compound of any one of claims 42 to 51, wherein the payload moiety and the tag are coupled through a moiety comprising at least one poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety.

53. The compound of any one of claims 42 to 52, which comprises one tag.

54. The compound of any one of claims 42 to 53, wherein the total number of tags in the compound is one.

55. The compound of any one of claims 42 to 54, which comprises one or more payload moieties.

56. The compound of any one of claims 42 to 55, which comprises one payload moiety.

57. The compound of any one of claims 42 to 56, wherein the total number of payload moieties in the compound is one.

58. The compound of any one of claims 42 to 57, which comprises a linking moiety connecting a tag and a payload moiety.

59. The compound of claim 58, wherein the linking moiety is a branched or an unbranched linking moiety.

60. The compound of claim 58 or 59, wherein the linking moiety comprises a continuous or non-continuous poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

61. The compound of any one of claims 42 to 60, which comprises the formula:P-L-T whereinP comprises a payload moiety;T comprises a tag; andL comprises a linking moiety.

62. The compound of claim 61, wherein L comprises a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

63. The compound of claim 61 or 62, wherein L comprises the formula [AEEA]U-[L'- [AEEA]v]w, whereinAEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid or a derivative thereof;L1comprises a linking moiety; u is an integer of 2 or more; each v is an integer of 2 or more; and w is an integer from 1 to 4; whereinthe different groups [L'-[AEEA]V] may be identical or different.

64. The compound of claim 63, wherein u and v are each integers from 2 to 10.

65. The compound of claim 63 or 64, wherein u and v are each integers from 2 to 8.

66. The compound of any one of claims 63 to 65, wherein u and v are each integers of 2, 4 or 6.

67. The compound of any one of claims 61 to 66, wherein L or L' comprises an amino acid.

68. The compound of any one of claims 61 to 67, wherein L or L' comprises the D-isomer of an amino acid.

69. The compound of any one of claims 61 to 68, wherein L or L' comprises cysteine or lysine.

70. The compound of any one of claims 61 to 69, wherein L or L' is connected to a side chain.

71. The compound of claim 70, wherein a side chain comprises a functional moiety.

72. The compound of claim 71, wherein a functional moiety comprises a solubilizing functional group.

73. The compound of claim 71 or 72, wherein a functional moiety comprises a poly-2-(2- (2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.

74. The compound of any one of claims 42 to 73, wherein the payload moiety is selected from the group consisting of radioisotopes, toxins and immunomodulators.

75. The compound of any one of claims 49 to 74, wherein 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.

76. The compound of any one of claims 49 to 75, wherein 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.

77. The compound of any one of claims 49 to 76, wherein 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 2, 4 or 6.

78. The compound of any one of claims 42 to 77, wherein the tag is an ALFA-tag.

79. The compound of any one of claims 42 to 78, wherein the tag is a cyclic ALFA-tag.

80. 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 a binding moiety binding to the target antigen and a binding moiety for a tag;(ii) allowing the compound comprising a binding moiety binding to 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 of any one of claims 42 to 79 comprising a tag to which the binding moiety for a tag binds.

81. The method of claim 80, wherein the compound comprising a binding moiety binding to 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 a binding moiety binding to the target antigen and a binding moiety for a tag; and allowing expression of the polypeptide by cells in the subject.

82. The method of claim 81, wherein the cells expressing the polypeptide are transfected with the RNA.

83. The method of claim 81 or 82, wherein the RNA is administered as particulate formulation such as formulated as lipid nanoparticles.

84. The method of any one of claims 81 to 83, wherein the cells expressing the polypeptide secrete the polypeptide.

85. The method of any one of claims 81 to 84, wherein the cells expressing the polypeptide express the polypeptide such that it is released into the bloodstream.

86. The method of any one of claims 80 to 85, wherein the target antigen is a cell surface antigen.

87. The method of any one of claims 80 to 86, wherein the compound comprising a binding moiety binding to the target antigen and a binding moiety for a tag is a fusion polypeptide comprising the binding moiety binding to the target antigen and the binding moiety for a tag.

88. The method of any one of claims 80 to 87, wherein the binding moiety binding to the target antigen comprises an antibody or an antibody derivative.

89. The method of any one of claims 80 to 88, wherein the binding moiety binding to a tag comprises an antibody or an antibody derivative.

90. The method of claim 88 or 89, wherein the antibody derivative is an antibody fragment.

91. The method of any one of claims 80 to 90, wherein the disease, disorder or condition is cancer.

92. The method of any one of claims 80 to 91, wherein the cells expressing a target antigen are diseased cells.

93. The method of any one of claims 80 to 92, wherein the cells expressing a target antigen are cancer cells.

94. The method of any one of claims 80 to 93, wherein the target antigen is a tumor antigen.

95. The method of any one of claims 80 to 94, wherein the payload moiety comprises a toxin, an immunomodulator or a radioisotope, e.g., a chelating compound comprising a radioisotope.

96. The method of any one of claims 80 to 95, wherein the compound under (i) comprises a single binding moiety for the tag.

97. The method of any one of claims 80 to 95, wherein the compound under (i) comprises at least two binding moieties for the tag.

98. The method of any one of claims 80 to 95, wherein the compound under (i) comprises two binding moieties for the tag.

99. The method of any one of claims 80 to 98, wherein the tag is an ALFA-tag.

100. The method of any one of claims 80 to 99, wherein the tag is a cyclic ALFA-tag.

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