Protein-based conjugation carriers

Abstract

The present technology relates to the field of drug delivery and provides molecules comprising or consisting of at least one protein-based carrier component, the protein-based carrier component comprising at least one, preferably at least two, attachment or conjugation sites. In particular, the present technology provides molecules comprising at least one protein-based component, the at least one protein-based component a) comprising at least one conjugation site or attachment site, b) having a molecular weight of 2.5 to 70 kDa, c) having a globular three-dimensional (3D) structure, and d) having a solubility of 10 mg / mL or more measured in aqueous solution at room temperature, and not specifically binding to any human protein or having a solubility of 5×10 - K above 4 moles / liter D The present invention provides a molecule that binds to one or more human proteins at a specific value.

Description

[Technical Field]

[0001] The present technology provides molecules that comprise or consist of at least one protein-based carrier component, which protein-based carrier component comprises at least one, and preferably at least two, attachment points or conjugation sites.

[0002] The technology further relates to nucleic acids encoding such molecules or portions of such molecules, host cells containing such nucleic acids and / or expressing or capable of expressing such molecules or portions of such molecules, compositions, particularly pharmaceutical compositions comprising such molecules, nucleic acids and / or host cells and the use of such molecules, nucleic acids, host cells and / or compositions, particularly for labeling, prophylactic, therapeutic and / or diagnostic purposes. [Background technology]

[0003] Protein-based therapeutics (called "biologics") offer new therapeutic strategies that are difficult to achieve with traditional small molecule-based therapeutics. One rapidly growing field involves conjugation-based therapeutics, such as antibody-drug conjugates (ADCs). For example, Fatima SW. and Khare SK. ("Benefits and challenges of antibody drug conjugates as a novel form of chemotherapy," J Control Release, 2022, 341:555-565) outline the recent clinical advances in each component of ADCs (antibody / linker / payload) and how each component affects the activity of ADCs. This is also shown, for example, in Rader, C., "Chemically programmed antibodies," Trends in Biotechnology, 2014, 32(4). Antibody-drug conjugates typically rely on the high specificity and affinity of antibodies, their extended circulating half-lives, and other effector functions (e.g., complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADC), and antibody-dependent cellular phagocytosis (ADCP). Antibody-drug conjugates and related molecules such as chemically programmed antibodies are therefore selected for at least one of these functionalities (their targeting properties, half-life extension properties, and / or other effector functions as described above).

[0004] Currently, biologics production primarily relies on recombinant production and is therefore limited to the production of polypeptides that can naturally combine into a single functional unit (i.e., IgG). Typical conjugation strategies are based on site-specific cysteine ​​or stochastic lysine conjugation (see, e.g., Zhou et al., 2021, Pharmaceuticals, 14:672; Sadiki et al., 2020, Antibody Therapeutics, 3:271). The rise of non-standard or novel amino acids (nAA) further adds options for conjugation methods (see, e.g., Malyshev and Romesberg, 2015, Angew Chem Int Ed Engl., 54(41); Zhang et al., 2017, PNAS, 114 1317). Summary of the Invention [Problem to be solved by the invention]

[0005] Additional therapeutic strategies that allow for versatile production and cargo conjugation are needed. [Means for solving the problem]

[0006] The present technology aims to create plug-and-play strategies that can simplify the generation of conjugation-based therapeutics and / or create alternative formats that allow for the versatile conjugation of different cargoes.

[0007] While traditional conjugation strategies must focus on maintaining the functionality of the polypeptides involved, as described above, the present technology employs a non-targeting protein-based carrier component (protein-based carrier component) that functions solely as a site-specific conjugation vehicle. This protein-based carrier component can be contained within a genetic construct and thus be the product of a single manufacturing campaign, or alternatively, can be produced separately (e.g., recombinantly or by alternative means such as solid-phase peptide synthesis (SPPS)) and subsequently attached to the active moiety and / or targeting moiety (i.e., cargo) (as described in detail below). The latter strategy allows for greater freedom in the conditions for cargo conjugation to the protein-based carrier component. Such freedom can lead to the use of site-specific conjugation to common amino acids (e.g., lysine), which are typically used in a stochastic manner, or to conjugation conditions that may impair the function / quality of the targeting component, or to alternative production platforms (e.g., chemical synthesis). The location and number of conjugation sites or attachment points can be manipulated / tuned to suit specific applications.

[0008] Thus, the present technology provides molecules comprising or consisting of at least one protein-based carrier component, as well as the protein-based carrier component itself, as described below, wherein the protein-based carrier component comprises at least one attachment point or conjugation site, preferably at least two attachment points or conjugation sites. At least one conjugation site or attachment point is suitable for conjugating or binding a cargo to the protein-based carrier component. A "cargo" is any molecule that can be bound or conjugated to a protein-based carrier component via an attachment point or conjugation site present therein. For example, cargoes that can be bound or conjugated to the protein-based carrier component of the present technology include proteins, peptides, polyethylene glycol (PEG), small molecules, glycans, lipids, chelators, fluorophores, radioisotopes, vitamins such as folic acid or biotin, and nucleic acids such as oligonucleotides or siRNAs. Thus, in a further embodiment, the present technology provides a protein-based carrier component, as described herein, bound to at least one cargo, i.e., the molecule of the present technology comprises (or alternatively consists of) at least one protein-based component and at least one cargo bound or conjugated thereto via at least one conjugation site or attachment point.

[0009] The protein-based carrier component of the present technology comprises (and preferably consists of) at least a portion of a protein, preferably the entire protein. Thus, preferably, the protein-based carrier component is a polypeptide. The protein-based carrier component has a globular 3D structure and is soluble. Furthermore, the protein-based carrier component included in the molecule of the present technology has a size (molecular mass or molecular weight, MW) of about 2.5 to about 70 kDa, preferably about 2.5 to less than 50 kDa, more preferably about 2.5 to about 30 kDa, and even more preferably about 2.5 to about 16 kDa, for example, about 6 kDa, about 7 kDa, or about 16 kDa.

[0010] Finally, the protein-based carrier component of the present technology does not specifically bind to any human protein, but may exhibit non-specific binding to one or more human proteins, as described in detail herein. In this case, the protein-based carrier component may bind to human proteins with low specificity and / or low selectivity, as defined herein. Preferably, the protein-based carrier component does not specifically bind to any non-protein (preferably human) molecule, such as DNA, RNA, lipid (e.g., phosphatidylserine (PS)), or glycan. The protein-based carrier component may be derived from a target-binding protein (e.g., immunoglobulin single variable domain (ISVD), DARPin, affibody, or affitin), as described below. It may also be derived from other proteins that exhibit specific binding to human proteins, such as small globular human proteins. This is the so-called "protein-based carrier component precursor." In these cases, preferably, the protein-based component does not specifically bind to any molecule (including non-human proteins) to which the protein-based carrier component precursor (if present) specifically binds. For example, if the precursor of the protein-based carrier component is an anti-RSV (respiratory syncytial virus) ISVD, the protein-based carrier component preferably does not specifically bind to RSV. Thus, preferably, the protein-based carrier component also does not specifically bind to the precursor's target when the precursor has a target, and this is a non-human molecule such as a non-human protein, or a human non-protein molecule such as human DNA, RNA, glycan, lipid, etc. In a further preferred embodiment, the protein-based carrier component does not specifically bind to any human protein, non-human protein, and / or non-protein molecule when the cargo is conjugated to at least one, preferably at least two, attachment points or conjugation sites on the protein-based carrier component.

[0011] Thus, at least one protein-based carrier component of the present technology comprises: a) have at least one point of attachment (also referred to herein as conjugation site), preferably at least two points of attachment or conjugation sites, which are preferably reactive groups in the side chains of unnatural or natural amino acids (e.g., Cys, Lys, Tyr, Orn, etc.) located in solvent-accessible positions on the protein-based carrier component, and / or the N-terminal primary amine and / or C-terminal carboxylic acid group (if available) of the protein-based carrier component. Thus, at least one, preferably at least two points of attachment or conjugation sites are preferably located in solvent-accessible positions on the protein-based carrier component; b) has a size (molecular weight) of about 2.5 to about 70 kDa, preferably about 2.5 to about 50 kDa, for example, about 2.5 to less than 50 kDa, more preferably about 2.5 to about 30 kDa, and even more preferably about 2.5 to about 16 kDa; c) measured in aqueous solution at room temperature (RT), preferably measured in buffer or water at RT, more preferably in buffer, such as citrate buffer or phosphate buffered saline (PBS) at pH 7.0 or 7.4 at RT, or histidine buffer at pH 6.5 at RT (histidine (10 mM to 100 mM, for example 10 mM), sucrose (1% to 10%, for example 10%) and optionally Tween 80 (0.001% to 1%, for example 0.01%)) or phosphate buffer at pH 7.0 at RT (NaH2PO4 / Na2HPO4 (10 and 50 mM, for example 10 mM), sodium chloride (NaCl) (100 to 150 mM, for example 130 mM NaCl) and optionally Tween 80 80 (including 0.001% to 1%, e.g., 0.01%)), preferably the buffer is 5 mM citrate buffer or PBS at pH 7.0 or 7.4; d) have a spherical 3D structure as described below, e) does not specifically bind to any human protein (or 5 x 10 -4 moles / liter K D (K Dvalue) and preferably it does not also specifically bind to the precursor target (or 5×10 -4 moles / liter K D (K D value) and binds to a precursor target, which may be a non-human protein or non-protein molecule), preferably it does not specifically bind to any non-protein molecule, such as a nucleic acid (e.g., DNA, RNA), lipid or glycan, preferably it does not specifically bind to any human non-protein molecule (or 5×10 -4 moles / liter K D (K D value), preferably it does not specifically bind to any non-protein molecule (e.g., nucleic acids (e.g., DNA, RNA), glycans, lipids, etc.) to which the building block precursor specifically binds, if present, or does not specifically bind to any human cell, as determined, for example, by cell binding assays or surface plasmon resonance (SPR), as described herein and / or / or Ober et al. 2001, Intern. Immunology 13:1551-1559, or preferably it does not specifically bind to any non-protein molecule (e.g., nucleic acids (e.g., DNA, RNA), glycans, lipids, etc.) to which the building block precursor specifically binds, if present, or does not specifically bind to any human cell, as determined, for example, by cell binding assays or surface plasmon resonance (SPR), as described, for example, herein and / or / or Ober et al. 2001, Intern. Immunology 13:1551-1559, or preferably it does not specifically bind to any human cell, as determined, for example, by cell binding assays or SPR, as described, for example, by -4 moles / liter K D (K D binds to one or more human cells at f) optionally, does not specifically bind to any (non-human) molecule specifically bound by a protein-based carrier component precursor, such as RSV protein F, or specifically binds to any (non-human) molecule specifically bound by a protein-based carrier component precursor, such as RSV protein F, with a binding affinity of 5×10, preferably as determined by a cell binding assay or SPR. -4 moles / liter K D (K D value), g) optionally, does not specifically bind to any human cells and / or cell types, or preferably binds to less than 5×10 cells / cells as determined by cell binding assays -4 moles / liter K D (KD binds to human cells and / or cell types at h) optionally, do not specifically bind to any microorganism, such as bacteria, fungi, protozoa, yeast, and / or any virus, or bind to less than 5×10 as determined, preferably by cell binding assays and / or SPR, as described herein; -4 moles / liter K D (K D binds to microorganisms such as bacteria, fungi, protozoa, yeast and / or viruses at a specific concentration (value); i) optionally, does not specifically bind to any biomolecules, including human biomolecules and non-human biomolecules, such as plant biomolecules, viral biomolecules, and / or microbial biomolecules (such as bacteria, fungi, protists, and / or yeast), or binds to less than 5×10 as determined, preferably by cell binding assays and / or SPR, as described herein; -4 Super-molar K D (K D value) and binds to biomolecules, including human biomolecules and non-human biomolecules; j) optionally, does not specifically bind to any biomolecule, including human biomolecules and non-human biomolecules, such as plant biomolecules, viral biomolecules, and / or microbial biomolecules (e.g., bacteria, fungi, protists, and / or yeasts), or has at least one cargo attached thereto (via at least one conjugation site or attachment point contained therein), and has at least 5×10 specific binding activity, as determined preferably by cell binding assays and / or SPR, as described herein. -4 moles / liter K D (K D value) and binds to biomolecules, including human biomolecules and non-human biomolecules; k) optionally does not comprise or consist of an amino acid sequence selected from SEQ ID NOs: 1 to 34 set forth in Tables A-1 and A-2 of WO 2016 / 055656 and / or SEQ ID NOs: 1 to 12 set forth in Table A-1 of WO 2010 / 139808; l) optionally, does not comprise or consist of the amino acid sequence defined in SEQ ID NO: 214.

[0012] In a first aspect, the present technology relates to a molecule comprising at least one protein-based carrier block, wherein the at least one protein-based carrier component comprises: a) comprises at least one conjugation site or attachment point, preferably at least two attachment points or conjugation sites; b) has a molecular weight of about 2.5 to about 70 kDa, preferably about 2.5 to about 50 kDa, for example, about 2.5 kDa to less than 50 kDa, more preferably about 2.5 to about 30 kDa, and even more preferably about 2.5 to about 16 kDa; c) has a spherical 3D structure; d) has a solubility of 10 mg / mL or more measured in aqueous solution at RT, preferably measured in buffer or water at RT, more preferably in a buffer, such as citrate buffer or phosphate buffered saline (PBS) at pH 7.0 or 7.4 at RT, or a histidine buffer at pH 6.5 at RT (comprising histidine (10 mM to 100 mM, for example 10 mM), sucrose (1% to 10%, for example 10%) and optionally Tween 80 (0.001% to 1%, for example 0.01%)) or a phosphate buffer at pH 7.0 at RT (comprising NaH2PO4 / Na2HPO4 (10 and 50 mM, for example 10 mM), sodium chloride (NaCl) (100 to 150 mM, for example 130 mM NaCl) and optionally Tween 80 (0.001% to 1%, for example 0.01%)), e) does not specifically bind to any human protein, or binds less than 5×10 specifically to any human protein, preferably as determined by cell binding assays or surface plasmon resonance (SPR), e.g., as described herein and / or in Ober et al. 2001, Intern. Immunology 13:1551-1559. -4 moles / liter K D (K D binds to one or more human proteins with a specific binding activity of 5×10 or less, or does not specifically bind to any human cells, preferably with a specific binding activity of 5×10 or less, as determined by cell binding assays or SPR. -4 moles / liter KD (K D binds to one or more human cells at f) optionally, does not specifically bind to any (non-human) molecule specifically bound by a protein-based carrier component precursor, such as RSV protein F, or specifically binds to any (non-human) molecule specifically bound by a protein-based carrier component precursor, such as RSV protein F, with a binding affinity of 5×10, preferably as determined by a cell binding assay or SPR. -4 moles / liter K D (K D value), g) optionally, does not specifically bind to any human cells and / or cell types, or preferably binds to less than 5×10 cells / cells as determined by cell binding assays -4 moles / liter K D (K D binds to human cells and / or cell types at h) optionally, do not specifically bind to any microorganism, such as bacteria, fungi, protozoa, yeast, and / or any virus, or bind to less than 5×10 as determined, preferably by cell binding assays and / or SPR, as described herein; -4 moles / liter K D (K D binds to microorganisms such as bacteria, fungi, protozoa, yeast and / or viruses at a specific concentration (value); i) optionally, does not specifically bind to any biomolecules, including human biomolecules and non-human biomolecules, such as plant biomolecules, viral biomolecules, and / or microbial biomolecules (such as bacteria, fungi, protists, and / or yeast), or binds to less than 5×10 as determined, preferably by cell binding assays and / or SPR, as described herein; -4 Super-molar K D (K D value) and binds to biomolecules, including human biomolecules and non-human biomolecules; j) optionally, does not specifically bind to any biomolecule, including human biomolecules and non-human biomolecules, such as plant biomolecules, viral biomolecules, and / or microbial biomolecules (such as bacteria, fungi, protists, and / or yeasts), or has at least one cargo attached thereto (via at least one conjugation site or attachment point contained therein), with a binding activity of at least 5×10 as determined preferably by cell binding assays and / or SPR, as described herein. -4 moles / liter K D (K D value) and binds to biomolecules, including human biomolecules and non-human biomolecules; k) optionally does not comprise or consist of an amino acid sequence selected from SEQ ID NOs: 1 to 34 set forth in Tables A-1 and A-2 of WO 2016 / 055656 and / or SEQ ID NOs: 1 to 12 set forth in Table A-1 of WO 2010 / 139808; a) Optionally, it does not comprise or consist of the amino acid sequence defined in SEQ ID NO: 214 (EVQLQASGGGLAQPGGSLRLSVTVSGSIDVINNMAWYRQAPGNARELVATITSGFSTNYASSVKGRFTISRDNAKKAVYLQMNSLKPEDTADYYSKVHLIRLGAARAYDYWGQGTQVTVS).

[0013] Preferably, in the molecules of the present technology, at least one protein-based carrier component does not specifically bind to any non-protein molecule, such as any human non-protein molecule, such as human DNA, human RNA, human lipids, or human glycans.

[0014] The molecules of the present technology may include two or more protein-based carrier components, for example, 2, 3, 4, 5, 6 or more protein-based carrier components, which may be directly linked to each other or linked to each other via linkers, as described herein.

[0015] Preferably, at least one protein-based carrier component comprised in the molecule of the present technology comprises two or more conjugation sites or attachment points, preferably at least two conjugation sites or attachment points, for example two conjugation sites or attachment points or at least three conjugation sites or attachment points, for example 3, 4, 5, 6, 7, 8 or 9 conjugation sites or attachment points, which are preferably reactive groups present in the side chains of natural or unnatural amino acids comprised in the protein-based carrier component, or (in addition or instead) may be the N-terminal primary amine and / or the C-terminal carboxylic acid group of the protein-based component.

[0016] For example, at least one conjugation site or attachment point contained in at least one protein-based carrier component is a free or capped thiol group, a free or capped hydroxyl group, and / or a free or capped primary amine. In a further embodiment, at least one conjugation site or attachment point contained in at least one protein-based carrier component is a reactive group present in the side chain of cysteine ​​and / or the side chain of tyrosine and / or the side chain of lysine and / or the side chain of ornithine. In another further embodiment, at least one protein-based component comprises an N-terminal and / or C-terminal Cys and / or N-terminal and / or C-terminal Tyr, preceded or followed by (GG) or (GS1). 1~3 GG sequences, such as CGG-, -GGC, YGG-, -GGY, -(G4S1) 1~3 GGY, Y(G4S1) 1~3 GG-, YGG(S1G4) 1~3 - or YGG(G4S1) 1~3 -Includes.

[0017] Preferably, at least one protein-based carrier component present in the molecule of the present technology is (i) an ISVD-based component, a DARPin-based component, an affibody-based component, or an affitin-based component, or (ii) a component based on a small globular human protein such as cyclin-dependent kinase subunit 1 (CDK-1).

[0018] In one embodiment, at least one protein-based component is derived from the heavy chain ISVD, preferably a camelized V H or humanized V HH V containing H , V HH In another embodiment, at least one protein-based component is derived from "V H The resulting building blocks are derived from ISVDs belonging to the "Class 3" and preferably contain at least one (preferably engineered) cysteine, at least one (preferably engineered) lysine, at least one unnatural amino acid and / or at least one (preferably engineered) tyrosine at one or more solvent accessible positions of the protein-based building block.

[0019] In another embodiment, at least one protein-based component is derived from RSV001A04, SEQ ID NO:179.

[0020] In one embodiment, at least one protein-based building block is an ISVD-based building block comprising Leu or Gln, preferably Leu, at position 108 according to the Kabat numbering, preferably the ISVD-based building block comprises Val or Leu, preferably Val, at position 11 and / or Val, Thr or Leu, preferably Leu, at position 89 according to the Kabat numbering.

[0021] In another embodiment, at least one protein-based component is SEQ ID NO: 186: [ka] (In the formula, X1 (position 1 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, such as cysteine; X2 (position 3 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, for example, cysteine; X3 (position 5 according to Kabat numbering) can be Val or any amino acid having a reactive group in its side chain, for example cysteine; X4 (position 7 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X5 (position 8 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X6 (position 10 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, e.g., cysteine; X7 (position 11 according to Kabat numbering) can be Leu, Val, Ser, Met, Trp, Phe, Thr, Gln, Glu, Ala, Arg, Gly, Lys, Tyr, Asn, Pro or Ile, preferably Leu or Val or any other amino acid having a reactive group in its side chain, such as cysteine; X8 (position 12 according to Kabat numbering) can be Val or any amino acid having a reactive group in its side chain, for example cysteine; X9 (position 13 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, for example cysteine; X 10 (position 14 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, for example cysteine; X 11(position 15 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, e.g., cysteine; X 12 (position 17 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 13 (position 18 according to Kabat numbering) can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X 14 (position 19 by Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 15 (position 21 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 16 (position 23 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, for example cysteine; X 17 (position 25 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 18 (position 26 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, e.g., cysteine; X 19 (position 27 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, e.g., cysteine; X 20 (position 28 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 21(position 30 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 22 (position 31 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X 23 (position 32 by Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, e.g., cysteine; X 24 (position 39 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, for example cysteine; X 25 (position 41 by Kabat numbering) can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X 26 (position 42 by Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, e.g., cysteine; X 27 (position 43 according to Kabat numbering) can be Lys or any amino acid having a reactive group in its side chain, e.g., cysteine; X 28 (position 44 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, for example cysteine; X 29 (position 45 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X 30 (position 46 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, for example cysteine; X 31(position 52a according to Kabat numbering) can be Trp or any amino acid having a reactive group in its side chain, e.g., cysteine; X 32 (position 53 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X 33 (position 54 by Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, e.g., cysteine; X 34 (position 55 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X 35 (position 56 according to Kabat numbering) can be He or any amino acid having a reactive group in its side chain, for example, cysteine; X 36 (position 57 by Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, e.g., cysteine; X 37 (position 58 according to Kabat numbering) can be He or any amino acid having a reactive group in its side chain, for example, cysteine; X 38 (position 59 by Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, e.g., cysteine; X 39 (position 61 according to Kabat numbering) can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X 40 (position 62 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example cysteine; X 41(position 64 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, for example cysteine; X 42 (position 65 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, e.g., cysteine; X 43 (position 66 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, for example cysteine; X 44 (position 68 by Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, e.g., cysteine; X 45 (position 70 by Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 46 (position 71 by Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X 47 (position 72 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example cysteine; X 48 (position 73 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X 49 (position 74 by Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, for example, cysteine; X 50 (position 75 according to Kabat numbering) can be Lys or any amino acid having a reactive group in its side chain, e.g., cysteine; X 51(position 76 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example cysteine; X 52 (position 79 by Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, e.g., cysteine; X 53 (position 81 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, for example, cysteine; X 54 (position 82a according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X 55 (position 82b according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 56 (position 83 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, for example, cysteine; X 57 (position 84 by Kabat numbering) can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X 58 (position 85 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example cysteine; X 59 (position 87 by Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, e.g., cysteine; X 60(position 89 according to Kabat numbering) can be Leu, Val, Ser, Met, Trp, Phe, Thr, Gln, Glu, Ala, Arg, Gly, Lys, Tyr, Asn, Pro or He, preferably Leu or Val or any other amino acid having a reactive group in its side chain, for example cysteine; X 61 (position 91 by Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, e.g., cysteine; X 62 (position 96 by Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, e.g., cysteine; X 63 (position 98 by Kabat numbering) can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X 64 (position 99 by Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X 65 (position 100 according to Kabat numbering) can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X 66 (position 100a according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X 67 (position 100d according to Kabat numbering) can be He or any amino acid having a reactive group in its side chain, for example, cysteine; X 68 (position 100e according to Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, for example, cysteine; X 69(position 100f according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example cysteine; X 70 (position 100g according to Kabat numbering) can be Trp or any amino acid having a reactive group in its side chain, such as cysteine, X 71 (position 101 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X 72 (position 102 by Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, e.g., cysteine; X 73 (position 103 by Kabat numbering) can be Trp or any amino acid having a reactive group in its side chain, e.g., cysteine; X 74 (position 105 by Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X 75 (position 106 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, e.g., cysteine; X 76 (position 108 according to Kabat numbering) can be Gln, Leu, Arg, Pro, Glu, Lys, Ser, Thr, Met, Ala or His, preferably Gln or Leu or any other amino acid having a reactive group in its side chain, for example cysteine; X 77 (position 110 by Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, e.g., cysteine; X 78(position 112 by Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 79 (position 113 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 80 is absent or is Gly, X 81 is absent or is Gly, X 82 is absent or is Cys) or comprising, or alternatively consisting of, a sequence having 80% or more identity to SEQ ID NO: 186, preferably a sequence having 85% or more, 90% or more, 95% or more, 97% or more, or 99% or more sequence identity to SEQ ID NO: 186, with the proviso that the components have a globular 3D structure, are soluble, and have a size (molecular weight) of about 2.5 to about 70 kDa, for example about 2.5 to about 50 kDa, for example about 2.5 to less than 50 kDa, more preferably about 2.5 to about 30 kDa, for example about 2.5 to about 16 kDa, for example about 5 to about 16 kDa, or about 7 to about 16 kDa, or about 10 to about 16 kDa, and do not specifically bind to any human protein as described herein.

[0022] In another embodiment, at least one protein-based building block is a DARPin-based building block, preferably derived from DARPin K27 as defined in SEQ ID NO: 187. SEQ ID NO:187: [ka]

[0023] In one embodiment, the protein-based building block is SEQ ID NO: 188: [ka] (In the formula, X1 can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X2 can be Leu or any amino acid having a reactive group in its side chain, such as cysteine; X3 can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X4 can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X5 can be Glu or any amino acid having a reactive group in its side chain, such as cysteine; X6 can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X7 can be Ala or any amino acid having a reactive group in its side chain, for example, cysteine; X8 can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X9 can be Gln or any amino acid having a reactive group in its side chain, for example, cysteine; X 10 can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X 11 can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X 12 can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X 13 can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X 14 can be He or any amino acid having a reactive group in its side chain, for example, cysteine; X 15 can be Ala or any amino acid having a reactive group in its side chain, for example, cysteine; X 16 can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X 17 can be Gly or any amino acid having a reactive group in its side chain, for example, cysteine; X 18 can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X 19 can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X 20 can be His or any amino acid having a reactive group in its side chain, for example, cysteine; X 21 can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X 22 can be Thr or any amino acid having a reactive group in its side chain, for example, cysteine; X 23 can be Phe or any amino acid having a reactive group in its side chain, for example, cysteine; X 24 can be Phe or any amino acid having a reactive group in its side chain, for example, cysteine; X 25 can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X 26 can be Tyr or any amino acid having a reactive group in its side chain, for example, cysteine; X 27can be Gly or any amino acid having a reactive group in its side chain, for example, cysteine; X 28 can be His or any amino acid having a reactive group in its side chain, for example, cysteine; X 29 can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X 30 can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X 31 can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X 32 can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X 33 can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X 34 can be Gly or any amino acid having a reactive group in its side chain, for example, cysteine; X 35 can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X 36 can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X 37 can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X 38 can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 39can be Tyr or any amino acid having a reactive group in its side chain, for example, cysteine; X 40 can be Met or any amino acid having a reactive group in its side chain, for example, cysteine; X 41 can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X 42 can be Gly or any amino acid having a reactive group in its side chain, for example, cysteine; X 43 can be His or any amino acid having a reactive group in its side chain, for example, cysteine; X 44 can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X 45 can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X 46 can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X 47 can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X 48 can be Tyr or any amino acid having a reactive group in its side chain, for example, cysteine; X 49 can be Gly or any amino acid having a reactive group in its side chain, for example, cysteine; X 50 can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X 51can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X 52 can be Ala or any amino acid having a reactive group in its side chain, for example, cysteine; X 53 can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X 54 can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X 55 can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X 56 can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X 57 can be Ala or any amino acid having a reactive group in its side chain, for example, cysteine; X 58 can be Gly or any amino acid having a reactive group in its side chain, for example, cysteine; X 59 can be His or any amino acid having a reactive group in its side chain, for example, cysteine; X 60 can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X 61 can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X 62 can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X 63can be Val or any amino acid having a reactive group in its side chain, for example, cysteine; X 64 can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X 65 can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X 66 can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X 67 can be Gly or any amino acid having a reactive group in its side chain, for example, cysteine; X 68 can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X 69 can be Val or any amino acid having a reactive group in its side chain, for example, cysteine; X 70 can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X 71 can be Gln or any amino acid having a reactive group in its side chain, for example, cysteine; X 72 can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X 73 can be Phe or any amino acid having a reactive group in its side chain, for example, cysteine; X 74 can be Gly or any amino acid having a reactive group in its side chain, for example, cysteine; X 75can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X 76 can be Phe or any amino acid having a reactive group in its side chain, for example, cysteine; X 77 can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X 78 can be He or any amino acid having a reactive group in its side chain, for example, cysteine; X 79 can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X 80 can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X 81 can be Gly or any amino acid having a reactive group in its side chain, for example, cysteine; X 82 can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X 83 can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X 84 can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X 85 can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X 86 can be He or any amino acid having a reactive group in its side chain, for example, cysteine; X 87can be Gln or any amino acid having a reactive group in its side chain, for example, cysteine; X 88 can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X 89 is absent or is Leu, X 90 is absent or is Cys) or a DARPin-based building block comprising or alternatively consisting of a sequence having 80% or more identity to SEQ ID NO: 188, preferably a sequence having 85% or more, 90% or more, 95% or more, 97% or more or 99% or more sequence identity to SEQ ID NO: 188, with the proviso that the building block has a globular 3D structure, is soluble, has a size (molecular weight) of about 2.5 to about 70 kDa, such as about 2.5 to about 50 kDa, for example about 2.5 to less than 50 kDa, more preferably about 2.5 to about 30 kDa, for example about 2.5 to about 16 kDa, for example about 5 to about 16 kDa, or about 7 to about 16 kDa, or about 10 to about 16 kDa, and does not specifically bind to human proteins, in particular does not specifically bind to human KRAS protein (GTPase KRas, EC:3.6.5.2, primary accession number P01116, Lim S., et al. al., “Exquisitely specific anti-KRAS biodegraders inform on the cellular prevalence of nucleotide-loaded states”, ACS Cent.Sci.2021,7,2,274-291).

[0024] In another embodiment, at least one protein-based entity is a small, globular human protein-based entity, preferably derived from the polypeptide defined in SEQ ID NO:190. SEQ ID NO: 190 SHKQIYYSDKYDDEEFEYRHVMLPKDIAKLVPKTHLMSESEWRNLGVQQSQGWVHYMIHEPEPHILLFRRPLPKKPKK

[0025] In one embodiment, the protein-based building block is SEQ ID NO: 191: [ka] (In the formula, X1 can be Ser or any amino acid having a reactive group in its side chain, such as cysteine; X2 can be His or any amino acid having a reactive group in its side chain, such as cysteine; X3 can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X4 can be Gln or any amino acid having a reactive group in its side chain, for example, cysteine; X5 can be Tyr or any amino acid having a reactive group in its side chain, for example, cysteine; X6 can be Tyr or any amino acid having a reactive group in its side chain, for example, cysteine; X7 can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X8 can be Lys or any amino acid having a reactive group in its side chain, for example cysteine; X9 can be Tyr or any amino acid having a reactive group in its side chain, such as cysteine; X 10 can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X 11 can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X 12 can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X13 can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X 14 can be Phe or any amino acid having a reactive group in its side chain, for example, cysteine; X 15 can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X 16 can be Tyr or any amino acid having a reactive group in its side chain, for example, cysteine; X 17 can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X 18 can be His or any amino acid having a reactive group in its side chain, for example, cysteine; X 19 can be Met or any amino acid having a reactive group in its side chain, for example, cysteine; X 20 can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X 21 can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X 22 can be He or any amino acid having a reactive group in its side chain, for example, cysteine; X 23 can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X 24 can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X 25can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X 23b can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X 24b can be Thr or any amino acid having a reactive group in its side chain, for example, cysteine; X 25b can be His or any amino acid having a reactive group in its side chain, for example, cysteine; X 26 can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X 27 can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 28 can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X 29 can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 30 can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X 31 can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X 32 can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X 33 can be Gly or any amino acid having a reactive group in its side chain, for example, cysteine; X 34can be Gln or any amino acid having a reactive group in its side chain, for example, cysteine; X 35 can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 36 can be Gln or any amino acid having a reactive group in its side chain, for example, cysteine; X 37 can be Gly or any amino acid having a reactive group in its side chain, for example, cysteine; X 38 can be Val or any amino acid having a reactive group in its side chain, for example, cysteine; X 39 can be Tyr or any amino acid having a reactive group in its side chain, for example, cysteine; X 40 can be Met or any amino acid having a reactive group in its side chain, for example, cysteine; X 41 can be He or any amino acid having a reactive group in its side chain, for example, cysteine; X 42 can be His or any amino acid having a reactive group in its side chain, for example, cysteine; X 43 can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X 44 can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X 45 can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X 46can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X 47 can be His or any amino acid having a reactive group in its side chain, for example, cysteine; X 48 can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X 49 can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X 50 can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X 51 can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X 52 can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X 53 can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X 54 can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X 55 can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X 56 can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X 57 can be Lys or any amino acid having a reactive group in its side chain, e.g., cysteine) or a small, globular, human protein-based entity comprising, or alternatively consisting of, a sequence having 80% or more identity to SEQ ID NO: 191, preferably a sequence having 85% or more, 90% or more, 95% or more, 97% or more, or 99% or more sequence identity to SEQ ID NO: 191, provided that the entity has a globular 3D structure, is soluble, and has a size (molecular weight) of about 2.5 to about 70 kDa, for example about 2.5 to about 50 kDa, for example about 2.5 to less than 50 kDa, more preferably about 2.5 to about 30 kDa, for example about 2.5 to about 16 kDa, for example about 5 to about 16 kDa, or about 7 to about 16 kDa, or about 10 to about 16 kDa, and does not specifically bind to a human protein as described herein.

[0026] For example, at least one protein-based component may be selected from SEQ ID NOs: 80-105, 175, 199, 208, 222-224.

[0027] In one embodiment, the molecule of the present technology comprises at least one protein-based carrier component as defined herein and at least one further moiety or cargo attached to the attachment point or conjugation site, wherein the at least one further moiety or cargo is selected from the following: a) a half-life extending (HLE) moiety such as PEG, and / or b) an EGFR targeting moiety, such as a GE11 peptide or an anti-EGFR VHH, and / or c) a therapeutic moiety or precursor thereof, such as a DR5 binding molecule; d) imaging moieties such as deferoxamine (DFO); e) toxic moieties such as DM4 or cryptophycin; f) nucleic acids such as siRNA; g) Vitamins such as folic acid, h) Toll-like receptor agonists such as resiquimod; i) glycans such as bismannose 6-phosphate (bisM6P) or mannose 6-phosphate (M6P), and / or j) Lipids such as short chain fatty acids.

[0028] In one embodiment, the at least one half-life extending moiety is an albumin binding ISVD, preferably selected from SEQ ID NOs: 50-64 and 106, more preferably SEQ ID NO: 63 or SEQ ID NO: 106 or a sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% identity to SEQ ID NOs: 50-64 and / or 106. In a further embodiment, the at least one half-life extending moiety is a linear or branched polyethylene glycol moiety having a molecular weight of about 1-60 kDa, preferably about 1-15 kDa, such as about 14 or 15 kDa or about 1-10 kDa, such as 5 or 10 kDa.

[0029] In one embodiment, the at least one protein-based carrier component and the at least one further moiety or cargo are directly linked to each other. In a further embodiment, they are linked to each other via a peptide linker, preferably selected from SEQ ID NOs: 158-169 or 193-196, more preferably SEQ ID NO: 163. Other linkers may be used, such as an APN-maleimide linker, as defined below and exemplified in the Examples.

[0030] For example, the molecules of the present technology may comprise or alternatively consist of any one of SEQ ID NOs: 107-127, 170-174, 176, or 200.

[0031] The present technology also provides nucleic acids encoding the molecules of the present technology (or portions of the molecules of the present technology). Additionally, the present technology provides vectors comprising the nucleic acids of the present technology and compositions comprising the molecules of the present technology, such as pharmaceutical compositions.

[0032] Furthermore, the present technology relates to molecules or compositions of the present technology for use in medicine, particularly in the treatment (prophylactic or therapeutic) of diseases and or disorders such as autoimmune / inflammatory diseases, cancer and / or infectious diseases. [Brief explanation of the drawings]

[0033] [Figure 1] Amino acid sequence of ISVD RSV001A04 (SEQ ID NO: 179). [Figure 2] K27m (without C-terminal L), amino acid sequence of SEQ ID NO: 68. [Figure 3] Amino acid sequence of CKS1 component precursor (SEQ ID NO: 190). [Figure 4] Conjugation using an APN-maleimide "bifunctional" linker. The carrier (a protein-based component contained in the molecule) contains at least one attachment point or conjugation site (represented as "-SH" in the diagram). The APN-maleimide "bifunctional" linker can first be attached to a conjugation site present in the carrier. Then, a cargo (represented as "DR5-SH" in the diagram) can be attached to the other side of the APN-maleimide "bifunctional" linker. Thus, the cargo is attached or conjugated to the carrier via the APN-maleimide "bifunctional" linker. [Figure 5A] Binding study of cysteine-engineered ISVD-based carrier components using mass spectrometry deconvolution mass spectra of Mal-APN conjugation to molecules T028100075, which contains an ISVD-based carrier component with one attachment point or conjugation site ("ISVD179-APN," SEQ ID NO: 176, FIG. 5A), and T028100069, which contains an ISVD-based carrier component with three attachment points or conjugation sites ("ISVD107-APN," SEQ ID NO: 107, FIG. 5B). For sample cleanup, mass spectrometry was performed using electrospray ionization (ESI) with an online reversed-phase column (RPC). [Figure 5B]Binding study of cysteine-engineered ISVD-based carrier components using mass spectrometry deconvolution mass spectra of Mal-APN conjugation to molecules T028100075, which contains an ISVD-based carrier component with one attachment point or conjugation site ("ISVD179-APN," SEQ ID NO: 176, FIG. 5A), and T028100069, which contains an ISVD-based carrier component with three attachment points or conjugation sites ("ISVD107-APN," SEQ ID NO: 107, FIG. 5B). For sample cleanup, mass spectrometry was performed using electrospray ionization (ESI) with an online reversed-phase column (RPC). [Figures 6A-6B] Schematic diagram of the molecule of Example 5. The diagram shows a multivalent molecule comprising an HLE moiety ("ALB23," representing SEQ ID NO: 106), a protein-based carrier component ("Carrier," representing SEQ ID NO: 80), and three cargos ("DR5") attached to three conjugation sites, which are three -SH groups of cysteines located in solvent-accessible positions. In Figure 6A, each cargo ("DR5") is a DR5-GGC ISVD ("monovalent," DR5-GGC, SEQ ID NO: 177). In Figure 6B, each cargo ("DR5-DR5-DR5") comprises three DR5 ISVDs ("trivalent," DR5-DR5-DR5-GGC, SEQ ID NO: 178). [Figure 7] SDS-PAGE gel analysis of the conjugates performed in Example 5. Lane 3 showed a 3x conjugation state of a "trivalent" (DR5-DR5-DR5-GGC) cargo attached to three conjugation sites on the building block defined by SEQ ID NO: 176. Lane 5 showed a 3x conjugation state of a "monovalent" (DR5-GGC) cargo attached to three conjugation sites on the building block defined by SEQ ID NO: 176. Control results are not shown in the figure. Lane 4 is a protein ladder. [Figure 8]Caspase 3 / 7 assay. Media containing the indicated concentrations of DR5 agonist was added, followed by 5 μM CellEvent™ Caspase-3 / 7 Green Detection Reagent, as described below. Real-time data acquisition and analysis were performed, and caspase 3 / 7 signals were normalized for cell confluency at 24 hours. "DR5 trivalent form" refers to the trivalent DR5-GGC molecule defined in SEQ ID NO: 178; "DR5 bivalent form" refers to the molecule as set forth in SEQ ID NO: 207; and "cargo nonavalent DR5" refers to a molecule comprising an HLE moiety ("ALB", SEQ ID NO: 106), an ISVD-based carrier component ("carrier", SEQ ID NO: 107) with three conjugation sites (at positions 43, 100f, and 105 according to Kabat), and three cargos linked to the three conjugation sites via APN-maleimide linkers, each cargo conjugating to three DR5 agonists. "Cargo trivalent DR5" refers to a molecule comprising an HLE moiety ("ALB", SEQ ID NO: 106), an ISVD-based carrier component ("Carrier", SEQ ID NO: 107) with three conjugation sites (at positions 43, 100f, and 105 according to Kabat), and three cargoes attached to the three conjugation sites via APN-maleimide linkers, each cargo comprising one DR5 ISVD (SEQ ID NO: 177); and "Cargo control" refers to a control molecule comprising an HLE moiety ("ALB", SEQ ID NO: 106), a control carrier component ("RSV", SEQ ID NO: 176) with a C-terminal Cys to which a molecule comprising three DR5 ISVDs is attached via APN-maleimide linkers. [Figure 9]siRNA conjugation using the molecule defined by SEQ ID NO: 108. "DOL3" refers to the molecule described in SEQ ID NO: 108, with three siRNA molecules bound to three conjugation sites contained in the protein-based component of this molecule. "DOL2" refers to the molecule described in SEQ ID NO: 108, with two siRNA molecules bound to two conjugation sites, and "DOL1" refers to the molecule described in SEQ ID NO: 108, with one siRNA molecule bound to one conjugation site. "T028100070" refers to the molecule described in SEQ ID NO: 108 itself, and "T028100070+TCEP" refers to the molecule described in SEQ ID NO: 108 in its reduced form with TCEP. [Figure 10] Combination of PEGylation (5, 10 or 20 kDa PEG) and radiolabeling (89Zn) of the molecule as set forth in SEQ ID NO: 108 ("ISVD108" in the figure). The concentration of the different molecules (marked as 1 to 4 in the figure) in the blood over time is shown in this figure. [Figure 11A] HPLC / SEC analysis of Crypto / PEG conjugates to molecules with SEQ ID NO: 107 (Panel A) and SEQ ID NO: 113 (Panels B and C). Sample: 5 μL injection; Column: TSK-GEL SW mAb HTP (Tosoh Bioscience) - 4 μm - 4.6 × 150 mm; Solvent: Isocratic with 100% SEC buffer Kpi iPrOH 20% pH = 7 (composition: 200 mL KCl 1 M / 52 mL KH2PO4 1 M / 107 mL K2HPO4 1 M / MQ, 441 mL iPrOH, 200 mL); Run: 15 min at 0.2 mL / min; V0 = excluded volume, Vt = total volume of column. [Figure 11B]HPLC / SEC analysis of Crypto / PEG conjugates to molecules with SEQ ID NO: 107 (Panel A) and SEQ ID NO: 113 (Panels B and C). Sample: 5 μL injection; Column: TSK-GEL SW mAb HTP (Tosoh Bioscience) - 4 μm - 4.6 × 150 mm; Solvent: Isocratic with 100% SEC buffer Kpi iPrOH 20% pH = 7 (composition: 200 mL KCl 1 M / 52 mL KH2PO4 1 M / 107 mL K2HPO4 1 M / MQ, 441 mL iPrOH, 200 mL); Run: 15 min at 0.2 mL / min; V0 = excluded volume, Vt = total volume of column. [Figure 11C] HPLC / SEC analysis of Crypto / PEG conjugates to molecules with SEQ ID NO: 107 (Panel A) and SEQ ID NO: 113 (Panels B and C). Sample: 5 μL injection; Column: TSK-GEL SW mAb HTP (Tosoh Bioscience) - 4 μm - 4.6 × 150 mm; Solvent: Isocratic with 100% SEC buffer Kpi iPrOH 20% pH = 7 (composition: 200 mL KCl 1 M / 52 mL KH2PO4 1 M / 107 mL K2HPO4 1 M / MQ, 441 mL iPrOH, 200 mL); Run: 15 min at 0.2 mL / min; V0 = excluded volume, Vt = total volume of column. [Figure 12] Schematic diagram of the cell internalization assay used in Examples 11-14. [Figure 13] Internalization of ALB-3C_K27m_w1 (SEQ ID NO: 173) and ALB-5C_K27m_w1 (SEQ ID NO: 174) molecules conjugated to GE11 peptide compared to alanine conjugates on NCI-H226 cells using HSA-pHAb as described in Example 11. [Figure 14] Preparation of tert-butyl (2S)-5-[2-(tert-butoxycarbonylamino)ethylamino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)-5-oxo-pentanoate (Intermediate 1). [Figure 15]Preparation of tert-butyl (2S)-2-amino-5-[2-(tert-butoxycarbonylamino)ethylamino]-5-oxo-pentanoate (Intermediate 2). [Figure 16] Preparation of (2S)-5-(2-aminoethylamino)-2-[[4-[(2-amino-4-hydroxy-pteridin-6-yl)methylamino]benzoyl]amino]-5-oxo-pentanoic acid (intermediate 3). [Figure 17] Preparation of (2S)-2-[[4-[(2-amino-4-oxo-1H-pteridin-6-yl)methylamino]benzoyl]amino]-5-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethylamino]-5-oxo-pentanoic acid (folate-aminoethyl-maleimide). [Figure 18] Internalization of HeLa cell line by protein-based carrier molecules conjugated with folic acid. The protein-based carrier components contained in T028100070 and T028100075 were conjugated to either maleimide-folic acid or maleimide-Ala (negative control) as described in Example 12. [Figure 19] BisM6P([(2S,3R,4R,5R)-6-[(3S,4S,5S,6R)-2-[[(2R,3S,4S,5S)-6-[[(2S,3S,4R,5R,6S)-4-[(3R,4R,5R,6S)-4,5-dihydroxy-6-(hydroxymethyl)-3-[(3R,4R,5R,6S)-3,4,5-trihydroxy-6-(phosphonooxymethyl)tetrahydropyran-2-yl]oxy-tetrahydropyran-2-yl]oxy-6-[4-[2-[3-[2-[2-[2-[2 Overview of the synthesis of -(2,5-dioxopyrrol-1-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyl]hydrazine]-4-oxo-butoxy]-3,5-dihydroxy-tetrahydropyran-2-yl]methoxy]-3,4,5-trihydroxy-tetrahydropyran-2-yl]methoxy]-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3-yl]oxy-3,4,5-trihydroxy-tetrahydropyran-2-yl]methyl dihydrogen phosphate). [Figure 20] Schematic of the cell binding assay used in Examples 13 and 16. [Figure 21] Binding of T028100069 (SEQ ID NO: 107), T028100070 (SEQ ID NO: 108), and T028100075 (SEQ ID NO: 176) molecules conjugated with bis-M6P (diM6P) compared to alanine on K-562 cells in flow cytometry using HSA-biotin and streptavidin-phycoerythrin detection as described in Example 13. An ISVD that specifically binds to M6PR ("ISVD specific to M6PR") was used as a positive control, and an ISVD that does not specifically bind to M6PR ("ISVD that does not specifically bind to M6PR") was used as an additional negative control. [Figure 22] Internalization of bis-M6P-conjugated T028100069 (SEQ ID NO: 107), T028100070 (SEQ ID NO: 108), and T028100075 (SEQ ID NO: 176) cargoes compared to alanine on K-562 cells using HSA-pHAb for detection as described in Example 13. An ISVD that specifically binds to M6PR ("ISVD specific to M6PR") was used as a positive control, and an ISVD that does not specifically bind to M6PR ("ISVD that does not specifically bind to M6PR") was used as an additional negative control. [Figure 23] Mass spectrometric lipidation QC of the DOL=0 (mal-ALA) and DOL 6 preparations confirms conjugation of both batches. Near complete conjugation of undecanoic acid and alanine was observed. [Figure 24] Internalization of ALB-RSV_c6 (T028100078, SEQ ID NO: 113) by 11-maleimidoundecanoic acid compared to alanine conjugates on BxPC-3 using HSA-pHAb for detection, as described in Example 14. [Figure 25A](A) SEC analysis of the conjugation of different mal-PEG molecules onto the SH-groups of the side chains of Cys present in the ISVD-derived components of different molecules (6 × 5 kDa, 5 × 5 kDa, 4 × 5 kDa, and 1 × 30 kDa). (B) Size and relative % of the different PEG-conjugated molecules analyzed by SEC. [Figure 25B] (A) SEC analysis of the conjugation of different mal-PEG molecules onto the SH-groups of the side chains of Cys present in the ISVD-derived components of different molecules (6 × 5 kDa, 5 × 5 kDa, 4 × 5 kDa, and 1 × 30 kDa). (B) Size and relative % of the different PEG-conjugated molecules analyzed by SEC. [Figure 26] Measurement of ISVD-based carrier compound concentrations in rat serum. [Figure 27] Non-reduced PAGE analysis of partial CMA1 uploaded CKS-based carriers. DETAILED DESCRIPTION OF THE INVENTION

[0034] definition Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art, which would be apparent to one of ordinary skill in the art. For example, standard handbooks such as Sambrook et al., 1989 (Molecular Cloning: A Laboratory Manual, 2nd Ed., Vols. 1-3, Cold Spring Harbor Laboratory Press), Ausubel et al., 1987 (Current protocols in molecular biology, Green Publishing and Wiley Interscience, New York), Lewin 1985 (Genes II, John Wiley&Sons, New York, NY), Old et al. al., 1981 (Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2nd Ed., University of California Press, Berkeley, CA), Roitt et al., 2001 (Immunology, 6th Ed., Mosby / Elsevier, Edinburgh), Roitt et al., 2001 (Roitt's Essential Immunology, 10th Ed., Blackwell Publishing, UK), and Janeway et al. al., 2005 (Immunobiology, 6th Ed., Garland Science Publishing / Churchill Livingstone, New York) and the general background art cited therein.

[0035] Unless otherwise indicated, all methods, steps, techniques and operations not described in detail herein can be and have been performed in a manner known per se, as will be apparent to those skilled in the art. Reference is made, for example, to the standard handbooks and general background art mentioned herein and the further references cited herein, as well as to the following reviews, for example: Presta 2006 (Adv. Drug Deliv. Rev., 58:640), Levin and Weiss 2006 (Mol. Biosyst., 2:49), Irving et al., 2001 (J. Immunol. Methods, 248:31), Schmitz et al., 2000 (Placenta 21 Suppl. A:S106), Gonzales et al., 2005 (Tumor Biol., 26:31), which describe protein engineering techniques such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins.

[0036] It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "reagent" includes one or more of such different reagents, and reference to a "method" includes reference to equivalent steps and methods known to those skilled in the art that may be modified or substituted for the methods described herein.

[0037] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the technology described herein. Such equivalents are intended to be encompassed by the technology.

[0038] The term "and / or" wherever used in this specification includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0039] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" should be understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprise" can be replaced with the term "containing" or "including," or, as sometimes used herein, the term "having."

[0040] As used herein, the term "sequence" (e.g., "immunoglobulin sequence," "antibody sequence," "variable domain sequence," "V HH "Amino acid sequence" or "protein sequence" should generally be understood to include both related amino acid sequences as well as the nucleic acid or nucleotide sequences encoding them, unless the context requires a more restrictive interpretation. An amino acid sequence is understood to mean a single amino acid or an unbranched sequence of two or more amino acids, depending on the context. A nucleotide sequence is understood to mean an unbranched sequence of three or more nucleotides.

[0041] It is understood that any reference to amino acid sequences is intended to encompass post-translational modifications of these sequences that occur in mammalian cells, such as CHO cells, including, but not limited to, N-glycosylation, O-glycosylation, deamidation, Asp isomerization / fragmentation, pyro-glutamate formation, removal of C-terminal lysine, and Met / Trp oxidation.

[0042] When a nucleotide sequence or amino acid sequence is said to "comprise" or "consist essentially of" another nucleotide sequence or amino acid sequence, respectively, this can mean that the latter nucleotide sequence or amino acid sequence is incorporated into the first-mentioned nucleotide sequence or amino acid sequence, respectively, but more generally means that the first-mentioned nucleotide sequence or amino acid sequence generally includes within its sequence a stretch of nucleotides or amino acid residues, respectively, which has an identical nucleotide sequence or amino acid sequence as the latter sequence, regardless of how the first-mentioned sequence was actually produced or obtained (which may be, for example, by any suitable method described herein).

[0043] Amino acids are amino[a](-NH + 3) and carboxylate (-CO - 2) An organic compound containing a functional group and a side chain (R group) specific to each amino acid. For example, amino acids include L-amino acids, which are commonly found in naturally occurring proteins. In the context of the present technology, "amino acid" also includes D-amino acids and non-natural, unusual, or unnatural amino acids, as described below. Amino acid residues are designated according to the standard three-letter or one-letter amino acid code. See Table A-2 on page 48 of WO 08 / 020079. Examples of amino acids commonly found in proteins and represented in the genetic code are listed in Table 1 below. Other common amino acids (except those listed in Table 1 below) are listed in the table on page 624 of Pure & Appl. Chem., Vol. 56, No. 5, pp. 595-624, 1984, which is reproduced below as Table 2 for convenience.

[0044] [Table 1]

[0045] [Table 2]

[0046] D-amino acids are also included within the definition of “amino acid.” As used herein, the term “D-amino acid” refers to an amino acid in which the stereoisomeric carbon alpha to the amino group has the D-configuration.

[0047] Unusual, unnatural, or non-natural amino acids are also encompassed within the definition of "amino acid." As used herein, the terms "unnatural amino acid" or "non-standard amino acid" or "unnatural amino acid" or "novel amino acid" (and the like) refer to amino acids that are not one of the 20 amino acids commonly found in natural and synthetic peptides, but are known by the single-letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V. Exemplary unnatural amino acids are Young et al., "Beyond the canonical 20 amino acids: expanding the genetic lexicon," J. of Biological Chemistry, 285(15):11039-11044 (2010), the disclosure of which is incorporated herein by reference.

[0048] Alexander R. Noedling et al. (“Using genetically incorporated unnatural amino acids to control protein functions in mammalian cells”, Essays Biochem, 3 July 2019;63(2):237-266), the disclosure of which is incorporated herein by reference, provides an overview of unnatural amino acids that have been successfully incorporated into proteins in mammalian cells (see, e.g., Table 1, beginning on p. 240).

[0049] Non-limiting examples of unnatural amino acids include p-acetyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, L-dopa, p-azido-phenylalanine, N-(propargyloxy)-carbonyl-L-lysine (PrK), and azido-lysine (N-azidoethoxy-carbonyl-L-lysine, AzK). In some embodiments, the unnatural amino acid comprises a selectively reactive group or reactive groups for site-selective labeling or conjugation of a moiety or cargo. In some instances, the chemistry is a bioorthogonal reaction (e.g., a biocompatible and selective reaction). In some cases, the chemistry is a Cu(I)-catalyzed or "copper-free" alkyl azidotriazole-forming reaction, Staudinger ligation, inverse electron demand Diels-Alder (IEDDA) reaction, "photoclick" chemistry, or metal-mediated processes such as olefin metathesis and Suzuki-Miyaura or Sonogashira cross-coupling. For further examples of unnatural amino acids, see WO 2021 / 072167, the disclosure of which is incorporated herein by reference.

[0050] The terms "protein," "peptide," "protein / peptide," and "polypeptide" are used interchangeably throughout this disclosure, and each has the same meaning for purposes of this disclosure. Each term refers to an organic compound made from a linear chain of two or more amino acids. The compound may have 10 or more amino acids, 25 or more amino acids, 50 or more amino acids, 100 or more amino acids, 200 or more amino acids, or even 300 or more amino acids. Those of skill in the art will understand that while polypeptides generally contain fewer amino acids than proteins, there is no art-recognized demarcation line in the number of amino acids that distinguishes proteins from polypeptides; that polypeptides can be made by chemical synthesis or recombinant methods; and that proteins are generally made in vitro or in vivo by recombinant methods as known in the art.

[0051] By convention, the amide bonds in a polypeptide's primary structure are written in the order in which the amino acids are written, with the amine end (N-terminus) of the polypeptide always on the left and the acid end (C-terminus) on the right.

[0052] Any amino acid sequence containing post-translationally modified amino acids may be written as the originally translated amino acid sequence using the symbols shown in Table 1 along with the modified positions, e.g., hydroxylation or glycosylation, but these modifications shall not be explicitly shown in the amino acid sequence. Any peptide or protein that can be represented as a sequence modified by linkages, bridges and end caps, non-peptidyl bonds, etc., is encompassed by this definition.

[0053] In the context of the present technology, the terms "specificity," "specifically binds," or "specific binding" refer to the number of different target molecules, such as antigens, to which a particular binding unit can bind with sufficiently high affinity (see below). "Specificity," "specifically binds," or "specific binding" are used interchangeably herein with "selectivity," "selectively binds," or "selective binding." Generally, binding units, such as binding ISVDs, specifically bind to their designated targets.

[0054] The specificity / selectivity of a binding unit can be determined based on affinity, which indicates the strength or stability of a molecular interaction. Affinity is generally measured as K, which has units of moles / liter (or M). D or dissociation constant. Affinity is given by the binding constant K A This can also be expressed as 1 / K D is equal to (moles / liter) -1 (or M -1 ) units.

[0055] Affinity is a measure of the strength of binding between a moiety and a binding site on a target molecule. D The smaller the value of , the stronger the binding strength between the target molecule and the targeting moiety.

[0056] K DThe value is given by the well-known relationship DG = RT.ln(K D )(Equivalently, DG=-RT.ln(K A It also characterizes the strength of molecular interactions in a thermodynamic sense, as it relates to the change in the free energy of binding (DG) due to ρ, where R is equal to the gas constant, T is equal to the absolute temperature, and ln is the natural logarithm.

[0057] K D is k off and the dissociation rate constant of the complex, denoted as k on It can also be expressed as the ratio of its binding rate (K D =k off / k on and K. A =k on / k off (So ​​that the off-rate k off is in units of s -1 (where s is the SI unit for second). on is in units of M -1 s -1 The on-rate is 10 2 M -1 s -1 ~about 10 7 M -1 s -1 The off-rate can vary with t 1 / 2 =ln(2) / k off The off-rate is related to the half-life of a given molecular interaction by the relationship -6 s -1 (multiple days t 1 / 2 Nearly irreversible complex with ~1s -1 (t 1 / 2 =0.69s).

[0058] If the measurement process has any effect on the intrinsic binding affinity of the molecule implied, for example, due to artifacts associated with coating one molecule on the biosensor, the measured K D is the apparent K DWhen a molecule contains two or more recognition sites for one or more other molecules, the apparent K D In such a situation, the measured affinity may be influenced by the avidity of the interaction by the two molecules.

[0059] Dissociation constant (K D ) can be the actual or apparent dissociation constant, as will be appreciated by those skilled in the art. D Methods for determining 10 will be apparent to those skilled in the art and include, for example, the techniques mentioned below. -4 mol / l or 10 -3 More than 10 mol / l (e.g., -2 It will also be apparent that it may not be possible to measure the dissociation constant in moles / liter (mol / l). Optionally, as will also be apparent to one skilled in the art, it may be possible to measure the (actual or apparent) K D is the (real or apparent) binding constant (K A ) based on the relation (K D =1 / K A ) can be calculated by K A =1 / K D →K A =[AB] / [A]·[B].

[0060] The term "about" when used in connection with a parameter or range of parameters provided herein shall have the following meaning: Unless otherwise indicated, when the term "about" is applied to a particular value or range, that value or range shall be interpreted as being as accurate as the method used to measure it. When a tolerance is not specified in application, the last decimal place of the numerical value indicates its degree of precision. Unless another tolerance is given, the maximum tolerance is ascertained by applying rounding conventions to the last decimal place; for example, for a pH value of approximately pH 2.7, the tolerance is 2.65 to 2.74. However, for the following parameters, special tolerances shall apply: Temperatures specified in °C without decimal places shall have a tolerance of ±1°C (e.g., a temperature value of approximately 50°C means 50°C ±1°C); Durations stated in hours shall have a tolerance of 0.1 hours, regardless of the decimal place (e.g., a time value of approximately 1.0 hour means 1.0 hour ±0.1 hour, and a time value of approximately 0.5 hour means 0.5 hour ±0.1 hour).

[0061] In this application, any parameter designated with the term "about" is also contemplated as being disclosed without the term "about." In other words, an embodiment that refers to a parameter value using the term "about" is also intended to represent an embodiment directed to such a numerical value of said parameter. For example, an embodiment that specifies a pH as "about pH 2.7" is also intended to disclose an embodiment that specifies such a pH as "pH 2.7." An embodiment that specifies a pH range as "about pH 2.7 to about pH 2.1" is also intended to represent an embodiment that specifies a pH range as "pH 2.7 to pH 2.1," etc.

[0062] For purposes of comparing two or more nucleotide sequences, the percentage of "sequence identity" between a first nucleotide sequence and a second nucleotide sequence can be calculated by dividing the number of nucleotides in the first nucleotide sequence that are identical to the nucleotides at corresponding positions in the second nucleotide sequence by the total number of nucleotides in the first nucleotide sequence, and multiplying by 100% (where each deletion, insertion, substitution, or addition of a nucleotide in the second nucleotide sequence compared to the first nucleotide sequence is considered to be a difference at a single nucleotide (position)). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using known computer algorithms for aligning sequences, such as NCBI Blast v2.0 using standard settings. Several other techniques, computer algorithms and settings for determining the degree of sequence identity are described, for example, in WO 04 / 037999, EP 0 967 284, EP 1 085 089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185 and GB 2 357 768. Typically, for the purposes of determining the percentage of "sequence identity" between two nucleotide sequences according to the calculation methods outlined herein above, the nucleotide sequence containing the greatest number of nucleotides is considered to be the "first" nucleotide sequence and the other nucleotide sequence is considered to be the "second" nucleotide sequence.

[0063] For comparing two or more amino acid sequences, the percentage of "sequence identity" (also referred to herein as "amino acid identity") between a first amino acid sequence and a second amino acid sequence can be calculated by dividing the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence by the total number of amino acid residues in the first amino acid sequence and multiplying by 100%, where each deletion, insertion, substitution, or addition of an amino acid residue in the second amino acid sequence is considered a difference in a single amino acid residue (position) compared to the first amino acid sequence, i.e., an "amino acid difference" as defined herein. Alternatively, the degree of sequence identity between two amino acid sequences can be calculated using known computer algorithms, such as those described above for determining the degree of sequence identity of nucleotide sequences, similarly using standard settings. Typically, for purposes of determining the percentage of "sequence identity" between two amino acid sequences according to the calculation method outlined herein above, the amino acid sequence containing the largest number of amino acid residues is considered the "first" amino acid sequence, and the other amino acid sequence is considered the "second" amino acid sequence.

[0064] When determining the degree of sequence identity between two amino acid sequences, those skilled in the art can also take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and has little or no effect on the 3D structure, function, activity or other biological properties of a polypeptide. Such conservative amino acid substitutions are well known in the art, for example, from WO 04 / 037999, GB 335768, WO 98 / 49185, WO 00 / 46383 and WO 01 / 09300, and (preferred) types and / or combinations of such substitutions can be selected based on the appropriate teachings from WO 04 / 037999 and WO 98 / 49185 and from the further documents cited therein.

[0065] Such conservative substitutions are preferably those in which one amino acid residue within the following groups (a) to (e) is substituted for another amino acid residue within the same group: (a) small, aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large, aliphatic, non-polar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp. Particularly preferred conservative substitutions are: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln, or Glu; Met to Leu, Tyr, or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile, or Leu.

[0066] Amino acid and nucleic acid sequences are said to be "exactly the same" if they have 100% sequence identity (as defined herein) over their entire length. When comparing two amino acid sequences, the term "amino acid difference" refers to the insertion, deletion, or substitution of a single amino acid residue at the position in the first sequence that is compared with the second sequence, and it will be understood that two amino acid sequences may contain one, two, or more such amino acid differences.

[0067] According to the present specification, "protein solubility" is a thermodynamic parameter defined as the concentration of a protein in a saturated solution that is in equilibrium with either a crystalline or amorphous solid phase under given conditions (see, for example, Kramer RM. et al., "Toward a molecular understanding of protein solubility: increased negative surface charge correlates with increased solubility", Biophys J., 2012, 102(8):1907-15).

[0068] Protein-based building blocks The molecules of the present technology comprise or alternatively consist of at least one protein-based carrier component (also referred to herein as a "carrier component," "protein-based component," or simply "component" or "carrier"), as defined herein. For example, the molecules of the present technology may comprise or alternatively consist of a single protein-based carrier component. In other embodiments, the molecules comprise two or more protein-based carrier components, e.g., two, three, four, five, six, or more carrier components. The protein-based carrier component comprises (and preferably consists of) at least a portion of a protein or an entire structured protein, i.e., the protein-based carrier component is preferably a polypeptide.

[0069] Protein-based carrier components are designed as "carriers" or "delivery" moieties having at least one attachment point or conjugation site, preferably at least two attachment points or conjugation sites, for conjugation or attachment of a cargo, as defined in detail below. Suitable cargoes include proteins, peptides, toxic payloads, nucleic acids, oligonucleotides, fluorophores, glycans, radioisotope chelators and / or radioisotopes, polyethylene glycol (PEG) molecules, vitamins (such as biotin or folic acid), and the like. Specific, non-limiting examples of suitable cargoes are provided herein below.

[0070] In the context of the present technology, a point of attachment or conjugation site refers to any group contained in a protein-based component that is suitable for attaching or conjugating a cargo thereto. The point of attachment or conjugation site is preferably located at a solvent-accessible position within the protein-based component, as described in detail below. The point of attachment or conjugation site may be a reactive group located in the side chain of any amino acid within the protein-based carrier component, preferably an amino acid located at a solvent-accessible position within the protein-based carrier component, or may be the N-terminal primary amine and / or C-terminal carboxylic acid group of the protein-based component. The point of attachment / conjugation site allows for the formation of a covalent bond with a group present within the cargo for conjugation and / or attachment to the protein-based carrier component. In a preferred embodiment, the point of attachment or conjugation site is a reactive group located in the side chain of an amino acid within the protein-based carrier component, preferably located at a solvent-accessible position within the protein-based carrier component, thereby allowing for the formation of a covalent bond with a group present within the cargo for conjugation and / or attachment to the protein-based carrier component. In another embodiment, two of the conjugation sites or attachment points of the protein-based component are reactive groups present in the side chains of two amino acids present in the protein-based carrier component, preferably two amino acids present in solvent-accessible positions within the protein-based carrier component, hi another embodiment, all of the conjugation sites or attachment points of the protein-based carrier component are reactive groups present in the side chains of amino acids present in the protein-based carrier component, preferably amino acids present in solvent-accessible positions within the protein-based carrier component.

[0071] Spherical three-dimensional (3D) structure The protein-based carrier component of the present technology is or includes a structured protein having a globular three-dimensional (3D) structure, i.e., a spherical 3D structure. Globular proteins have a roughly spherical shape. Almost all globular proteins contain a significant number of α-helices and / or β-sheets folded into a compact structure stabilized by both polar and non-polar interactions. Globular 3D structures are formed naturally and often involve interactions mediated by amino acid side chains. In most cases, hydrophobic amino acid side chains are buried and tightly packed inside the globular protein to avoid contact with water. Hydrophilic amino acid side chains are present on the surface of the globular protein exposed to water. As a result, globular proteins are usually highly soluble in aqueous solutions (from "Gene Expression: Translation of the Genetic Code," Chang-Hui Shen, in Diagnostic Molecular Biology, 2019). In the context of the present technology, a protein or portion of a protein having a globular 3D structure can be defined as a protein or portion thereof that contains at least one α-helix and / or at least one β-sheet as part of its secondary structure. From a simple sequence of amino acids to its final 3D structure, a protein passes through four levels of structuring, known as primary, secondary, tertiary, and quaternary. At the end of these stages, the protein begins to fold into a stable 3D structure that allows it to perform its proper function. Thus, the amino acid sequence of a protein is known as the "primary structure" of that protein. "Secondary structure" can be defined as the arrangement of polypeptide chains into a more or less regular hydrogen-bonded configuration, which has two basic elements: Alpha helix - a helical structure of a polypeptide chain with 3.6 residues (amino acids) per turn. The helix can be left-handed or right-handed, the latter being more common. Beta strand (or β-sheet) - two adjacent polypeptide chains joined together. Two or more chains may interact to form a beta sheet.

[0072] Finally, "tertiary structure" can be defined as the level of protein structure at which the entire polypeptide chain is folded into a 3D structure. In multi-chain proteins, the term tertiary structure applies to the individual chains. See Smith, AD, et al., eds. 1997, Oxford Dictionary of Biochemistry and Molecular Biology, New York: Oxford University Press.

[0073] The three-dimensional structure of a protein can be determined by techniques such as X-ray crystallography, nuclear magnetic resonance (NMR), cryo-electron microscopy (EM), or circular dichroism (CD). X-ray crystallography is the common technique used to determine 3D protein structures, but NMR (suitable for small proteins) and cryo-EM (suitable for larger proteins) can also provide information about the tertiary structure of a protein. Circular dichroism is an excellent method for rapidly assessing the secondary structure, folding, and binding properties of proteins (see, for example, Jones, C. ("Circular dichroism of biopharmaceutical proteins in a quality-regulated environment", J Pharm Biomed Anal., 2022, 219:114945). Because the CD spectra of proteins are highly dependent on their conformation, CD can be used to predict the structure of unknown proteins and monitor conformational changes due to temperature, mutations, heat, denaturants, or binding interactions. For example, α-helical proteins have negative bands at 222 nm and 208 nm and a positive band at 193 nm. Proteins with well-defined antiparallel β-pleated sheets (β-helices) have negative bands at 218 nm and a positive band at 195 nm, while amorphous proteins have very low ellipticity above 210 nm and a negative band around 195 nm. For further details, see Greenfield NJ, "Using circular dichroism See "Dichroism spectra to estimate protein secondary structure", Nat Protoc., 2006, 1(6):2876-90.

[0074] Thus, the protein-based carrier component of the present technology comprises at least one α-helix and / or at least one β-sheet as part of its secondary structure, and preferably comprises two or more α-helices and / or two or more β-sheets as part of its secondary structure, resulting in a globular 3D tertiary structure. This allows for site- and stereospecific engineering of conjugation sites or attachment points, as described in detail herein. The presence of at least one α-helix and / or at least one β-sheet in a particular polypeptide or protein can be determined by known techniques, such as CD, as described above.

[0075] solubility The protein-based carrier component of the present technology is soluble. In the context of the present technology, a soluble component means that the component has a solubility of 10 mg / mL or more, preferably 20 mg / mL or more, preferably 50 mg / mL or more, and even more preferably 100 mg / mL or more, measured at room temperature (RT) in water or a suitable buffer or solvent (e.g., a physiological buffer such as an aqueous solution or a buffer suitable for parenteral administration). In a preferred embodiment, the solubility of the protein-based carrier component is measured in water or a suitable buffer at RT, more preferably in a buffer such as a citrate buffer (e.g., 5 mM citrate buffer) or PBS at pH 7.0 or 7.4 at RT. Other preferred buffers suitable for measuring the solubility of protein-based carrier components are preferably Dulbecco's phosphate buffered saline (DPBS, a balanced salt solution containing potassium chloride, monobasic potassium phosphate, sodium chloride and dibasic sodium phosphate, e.g., 2.7 mM KCl, 1.5 mM KH2PO4, 136.9 mM NaCl, 8.9 mM Na2HPO4·7H2O, pH 7.0-7.3, commercially available from GIBCO (Nr 14190-094)) at pH 7.0 or 7.3 or 7.4 at RT or histidine buffer (histidine (10 mM to 100 mM, e.g., 10 mM), sucrose (1% to 10%, e.g., 10%) and optionally Tween 100, preferably ... 80 (0.001% to 1%, for example 0.01%) or phosphate buffer (NaH2PO4 / Na2HPO4 (10 and 50 mM, for example 10 mM), sodium chloride (NaCl) (100 to 150 mM, for example 130 mM NaCl) and optionally Tween 80 (0.001% to 1%, for example 0.01%)) at pH 7.0 at RT.

[0076] Those skilled in the art are aware of methods for measuring the solubility of protein solutions. For example, the supplementary material to Kramer RM. et al., "Toward a molecular understanding of protein solubility: increased negative surface charge correlates with increased solubility", Biophys J., 2012, 102(8):1907-15, describes the solubility measurement of folded proteins.

[0077] Additionally or alternatively, solubility measurements can be performed as follows: A protein solution (e.g., in citrate buffer 5 mM, pH 7.0, or PBS pH 7.4, or in water, or any of the suitable buffers mentioned above) is concentrated by ultrafiltration (e.g., via tangential flow filtration (TFF)) until some turbidity appears in the solution. The solution is then spun at high speed or filtered through a 0.22 μm filter to remove insoluble material, and the OD of the supernatant is measured. 280 The molar extinction coefficient of a particular protein is used to obtain the protein concentration in the supernatant (and therefore the concentration of the protein in a saturated solution in equilibrium with the solid phase, i.e., the protein solubility).

[0078] For example, in the context of the present technology, physiological buffers suitable for parenteral administration may contain the following components: glutamate, tartrate, lactate, citrate, malate, gluconate, ascorbate, maleate, phosphate, succinate, acetate, bicarbonate, aspartate, histidine, benzoate, tromethamine, diethanolamine, ammonium or glycine. The most common buffers used in parenteral formulations are based on histidine, citrate, phosphate and acetate (see, for example, Broadhead J, Gibson M., "Parenteral dosage forms", in: Gibson M., editor, "Pharmaceutical preformulation and formulation", New York: Informa healthcare; 2009, pp. 325-47).

[0079] Preferably, the protein-based carrier component of the present technology is soluble in a reduced state, i.e., it is soluble when -SH groups present in solvent-accessible positions in its amino acid sequence (e.g., in the side chains of one or more Cys), if present, are in reduced form (as "-SH") and not oxidized. For example, the protein-based carrier component can be reduced when subjected to reducing conditions for a sufficient period of time. For example, reducing conditions can mean using β-mercaptoethanol (2-ME), dithiothreitol (DTT), or TCEP (tris(2-carboxyethyl)phosphine).

[0080] Size (Molecular Weight) The protein-based carrier component of the present technology has a size (molecular weight) of about 2.5 to about 70 kDa, for example, about 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 65, or about 70 kDa. Preferably, the component is about 2.5 to about 50 kDa, for example, about 2.5 to less than about 50 kDa, for example, about 2.5 to about 40 kDa or about 2.5 to about 35 kDa, more preferably about 2.5 to about 30 kDa, for example, about 5 to about 30 kDa, or about 7 to about 30 kDa, or about 10 to about 30 kDa, or about 2.5 to about 25 kDa, or about 5 to about 25 kDa. The components of the present technology are small components having a size of about 2.5 to about 16 kDa, for example, about 5 to about 16 kDa, or about 7 to about 25 kDa, or about 10 to about 25 kDa, or about 2.5 to about 20 kDa, or about 5 to about 20 kDa, or about 7 to about 20 kDa, or about 10 to about 20 kDa, or about 2.5 to about 18 kDa, or about 5 to about 18 kDa, or about 7 to about 18 kDa, or about 10 to about 18 kDa. More preferably, the components of the present technology have a size of about 2.5 to about 16 kDa, for example, about 5 to about 16 kDa, or about 7 to about 16 kDa, or about 10 to about 16 kDa, or for example, about 2.5, 3, 5, 6.5, 7, 10, 11, 12, 13, 14, 15, or 16 kDa. For example, the protein-based component may have a size (molecular weight) of about 6 kDa, or about 7 kDa, or about 15 kDa, or about 16 kDa. In a more preferred embodiment, the protein-based carrier component has a size of about 15 kDa.

[0081] non-functional The protein-based carrier components of the present technology do not specifically bind to any human proteins, and if the components exhibit interactions with one or more human proteins, such interactions are characterized by low specificity and / or low affinity, as defined herein.

[0082] For example, the protein-based carrier components of the present technology do not specifically bind to crystallizable fragment (Fc) receptors (FcRs), Fc-binding proteins, or Fc sensors. For example, the protein-based carrier components do not specifically bind to C-type lectin receptors (CLRs). All antibodies have two functional domains: one domain that confers antigen specificity, known as the antigen-binding fragment (Fab), and the other domain that drives antibody function, known as the crystallizable fragment (Fc). The specific effector functions elicited by antibodies are determined by the receptors to which the antibody Fc domain binds and the specific innate immune cells on which these FcRs are expressed. These sensors include both classical FcRs and non-classical C-type lectin receptors (CLRs) (see Lu, L. et al., "Beyond binding: antibody effector functions in infectious diseases," Nat Rev Immunol, 2018, 18, 46-61). Table 1 of Lu, L. et al. provides non-limiting examples of Fc domain sensors (e.g., Fcγ or FcRn) to which the protein-based carrier components of the present technology do not specifically bind. Thus, the protein-based carrier components of the present technology do not exhibit the effector functions of conventional antibodies mediated by the Fc domain. In another embodiment, the protein-based carrier components and / or molecules do not specifically bind to a crystallizable fragment (Fc) receptor (FcR), an Fc-binding protein, or an Fc sensor. For example, the protein-based carrier components and / or molecules do not specifically bind to a C-type lectin receptor (CLR). Thus, in one embodiment, none of the components included in the molecules of the present technology (e.g., at least one protein-based carrier component and / or at least one cargo bound or conjugated thereto) specifically bind to a crystallizable fragment (Fc) receptor (FcR), an Fc-binding protein, an Fc sensor, and / or a CLR.In another embodiment, the protein-based components and / or molecules of the present technology do not exhibit the effector functions of conventional antibodies mediated by the Fc domain, i.e., none of the components included in the molecules of the present technology exhibit the effector functions of conventional antibodies mediated by the Fc domain. H -V L No pairing / interaction and / or C L -C H C as a 1-bond disulfide bridge L -C H Does not include a pair.

[0083] In another embodiment, the protein-based carrier component of the present technology comprises a V L and / or V H The variable domain of the antibody light chain (V L ) and / or the variable domain of the heavy chain (V H In another embodiment, the protein-based carrier component does not specifically bind to the first constant domain of the heavy chain of an antibody (C H 1), e.g., C of mAb H In another embodiment, the protein-based carrier component does not specifically bind to the constant domain of the light chain of an antibody (C L ), e.g., C for mAb L In another embodiment, the protein-based carrier component does not specifically bind to the third constant domain of the heavy chain of an antibody (C H 3), e.g., C of mAb H In another embodiment, the protein-based carrier component does not specifically bind to the second constant domain of the heavy chain of an antibody (C H 2), e.g., C of mAb H In one embodiment, the molecules and / or components of the present technology are not Fab fragments derived from antibodies such as mAbs. In one embodiment, the molecules and / or components of the present technology are not C H Preferably, C derived from an antibody such as a mAb is used instead of HThe molecules and / or components of the present technology are not antibodies such as mAbs, and are not Fc or Fv fragments.

[0084] The protein-based carrier components of the present technology may be derived from target-binding proteins (e.g., ISVDs, DARPins, affibodies, or affitins) ("protein-based carrier component precursors"). In the context of the present technology, a "protein-based carrier component precursor" or "component precursor" is a protein-based moiety that can be modified to generate a protein-based carrier component included in a molecule of the present technology.

[0085] In the context of the present technology, a "protein-based carrier component precursor" is a protein that has been modified (e.g., by point mutation and / or addition / deletion of amino acids to its sequence) to generate a protein-based carrier component of the present technology. For example, the "protein-based carrier component precursor" is modified so that it no longer specifically binds to any human protein, preferably so that it no longer specifically binds to any (non-human) molecule (including non-human biomolecules) and / or any non-protein (human) molecule (including biomolecules), particularly any molecule (including biomolecules) to which the precursor specifically binds. Furthermore, if necessary, the "protein-based carrier component precursor" is modified to incorporate one or more attachment points or conjugation sites described herein. The "protein-based carrier component precursor" has at least 60%, for example at least 70% or at least 75%, preferably at least 80% sequence identity with the protein-based carrier component from which it is derived. For example, a "protein-based carrier component precursor" has at least 85%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99% or more sequence identity with the protein-based carrier component from which it is derived. For example, a "protein-based carrier component precursor" may share its entire amino acid sequence with the protein-based carrier component from which it is derived, except for at least one, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or more amino acids.It will be appreciated that the protein-based carrier component derived from the protein-based carrier component precursor has a spherical 3D structure, is soluble, has a size (molecular weight) of about 2.5 to about 70 kDa, for example about 2.5 to about 50 kDa, for example about 2.5 to less than 50 kDa, more preferably about 2.5 to about 30 kDa, for example about 2.5 to about 16 kDa, for example about 5 to about 16 kDa, or about 7 to about 16 kDa, or about 10 to about 16 kDa, and does not specifically bind to any human protein, and preferably does not specifically bind to any protein or non-protein molecule to which the precursor specifically binds.

[0086] Preferably, the carrier component of the present technology does not specifically bind to any non-protein molecule (including non-protein biomolecules such as nucleic acids, e.g., DNA and / or RNA, lipids (e.g., phosphatidylserine (PS)), or glycans), e.g., any non-protein human molecule (including biomolecules), e.g., human nucleic acids, e.g., human DNA and / or human RNA, human lipids (e.g., phosphatidylserine (PS)), or human glycans, e.g., human glycolipids. In particular, preferably, the carrier component does not specifically bind to any non-protein molecule (including biomolecules) (such as nucleic acids, e.g., DNA and / or RNA, lipids (e.g., phosphatidylserine (PS)), or glycans), e.g., any non-protein human molecule (including biomolecules), e.g., human nucleic acids, e.g., human DNA and / or human RNA, human lipids (e.g., phosphatidylserine (PS)), or human glycans, e.g., human glycolipids, to which the protein-based carrier component precursor specifically binds (i.e., the protein-based component preferably does not specifically bind to the target of the precursor, e.g., a non-protein molecule (including biomolecules) or a non-human protein).

[0087] In another preferred embodiment, the protein-based component of the present technology does not specifically bind to any (non-human) molecule (including biomolecules) to which the protein-based carrier component precursor specifically binds (i.e., the protein-based component preferably does not specifically bind to the target of the precursor, e.g., a non-human protein or non-protein molecule (including biomolecules)), or as described herein, ... to which the protein-based carrier component precursor specifically binds). -4 moles / liter K D At a value of 0.01, the protein-based carrier component precursor binds to any (non-human) molecule to which it specifically binds (i.e., the protein-based component does not also specifically bind to the precursor's target, e.g., a non-human protein or non-protein molecule). For example, if the protein-based carrier component precursor is an anti-RSV (respiratory syncytial virus) ISVD (i.e., it specifically binds to one or more proteins of RSV, such as RSV protein F), the protein-based carrier component derived therefrom preferably does not specifically bind to these RSV proteins (or, as described herein, binds to those proteins, such as RSV protein F, preferably at a value of 0.01, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 10.10, 11.11, 12.12, 13.13, 14.14, 15.15, 16.16, 17.17, 18.18, 19.19, 20.19, 21.19, 22.19, 23.19, 24.19, 25.19, 26.19, 27.19, 28.19, 29.19, 30.19, 31.19, 32.19, 33.19, 34.19, 35.19, 36.19, 37.19, 38.19, 39.19, 40.19, 41.19, 42.19, 43.19, 44.19, 45.19, 46.19, 47.19, 48.19, 49.19 -4 moles / liter K D join by value).

[0088] For example, if a precursor of a protein-based carrier component specifically binds to a virus (e.g., it is an antiviral ISVD, an antiviral DARPin, an antiviral affitin, an antiviral affibody, etc.) and / or viral molecules (e.g., it specifically binds to one or more viral biomolecules, such as viral proteins, viral nucleic acids, viral lipids, or viral glycans), then a protein-based carrier component derived therefrom preferably does not specifically bind to these viruses and / or viral molecules (or, as described herein, binds to these viruses and / or viral molecules at a concentration of, preferably, 5×10 -4 moles / liter K DIn other embodiments, the protein-based carrier component specifically binds to a virus (e.g., it is an antiviral ISVD, an antiviral DARPin, an antiviral affitin, an antiviral affibody, etc.) and / or a viral molecule (e.g., it specifically binds to one or more viral biomolecules, such as a viral protein, viral nucleic acid, viral lipid, or viral glycan) when its precursor specifically binds, but the specific binding is removed once at least the cargo is conjugated to the protein-based component. Examples of viruses to which a protein-based building block precursor (and / or protein-based building blocks of the present technology) can specifically bind include RSV, influenza virus, rabies virus, potyvirus, bacteriophage, rotavirus, HIV proteins, hepatitis B virus, hepatitis C virus, norovirus, Shiga toxin derived from lambdoid prophage, herpes simplex virus, grapevine fanleaf virus (GFLV), Ebola, Middle East Respiratory Syndrome (MERS) virus, acute respiratory syndrome (SARS) virus, SARS-COV2, vibrio or white spot syndrome virus, cytomegalovirus, parvovirus, Zika virus, and chikungunya virus (CHIKV). Thus, in one embodiment, a protein-based building block precursor, e.g., ISVD, can specifically bind to one or more of these viruses (or molecules, including biomolecules, contained therein). The resulting protein-based building block may not specifically bind to the virus (or molecules, including biomolecules, contained therein) to which the precursor binds. Where the protein-based components of the present technology exhibit specific binding to one or more of these viruses (or molecules, including biomolecules, contained therein), that specific binding as described herein is lost when at least one cargo is attached to at least one conjugation site contained therein.

[0089] For example, if the precursor of a protein-based carrier component specifically binds to a protozoan (microorganism, unicellular eukaryote) (e.g., it is an antiprotozoan ISVD, an antiprotozoan DARPin, an antiprotozoan affitin, an antiprotozoan affibody, etc.) and / or a protozoan molecule (e.g., it specifically binds to one or more protozoan biomolecules, such as a protozoan protein, a protozoan nucleic acid, a protozoan lipid, or a protozoan glycan), then a protein-based carrier component derived therefrom preferably does not specifically bind to those protozoans and / or protozoan molecules (or to these protozoans and / or protozoan molecules, as described herein, preferably at least 5×10 -4 moles / liter K DIn other embodiments, a protein-based carrier component specifically binds to a protozoan (e.g., it is an antiprotozoan ISVD, an antiprotozoan DARPin, an antiprotozoan affitin, an antiprotozoan affibody, etc.) and / or a protozoan molecule (e.g., it specifically binds to one or more protozoan biomolecules, such as a protozoan protein, a protozoan nucleic acid, a protozoan lipid, or a protozoan glycan) when its precursor specifically binds, but the specific binding is removed once at least the cargo is conjugated to the protein-based component. Examples of protozoans and protozoan molecules to which a protein-based component precursor (and / or a protein-based component of the present technology) can specifically bind are: Trypanosoma evansi, Eimeria stiedae, and variable surface glycoprotein (VSG). Thus, in one embodiment, a protein-based component precursor, e.g., an ISVD, can specifically bind to one or more of these protozoans (or molecules, including biomolecules contained therein). The resulting protein-based building blocks may not specifically bind to the protozoan (or molecules, including biomolecules, contained therein) to which the precursor binds. If the protein-based building blocks of the present technology exhibit specific binding to one or more of these protozoans (or molecules, including biomolecules, contained therein), the specific binding described herein is lost when at least one cargo is attached to at least one conjugation site contained therein.

[0090] For example, if the precursor of a protein-based carrier component specifically binds to mammalian proteins (e.g., it is an anti-mammalian protein ISVD, an anti-mammalian protein DARPin, an anti-mammalian protein affitin, an anti-mammalian protein affibody, etc.), then the protein-based carrier component derived therefrom preferably does not specifically bind to these mammalian proteins (or, as described herein, binds to those mammalian proteins at a concentration of preferably 5×10 -4 moles / liter K D(binding at a specific value). In other embodiments, a protein-based carrier component specifically binds to a mammalian protein when its precursor specifically binds, but the specific binding is removed once at least a cargo is conjugated to the protein-based component. An example of a mammalian protein to which a protein-based component precursor (and / or a protein-based component of the present technology) can specifically bind is bovine serum albumin. Thus, in one embodiment, a protein-based component precursor, e.g., an ISVD, can specifically bind to this mammalian protein. The resulting protein-based component may not specifically bind to the mammalian protein to which the precursor binds. If the protein-based component of the present technology exhibits specific binding to this mammalian protein, the specific binding described herein is lost once at least a cargo is conjugated to at least one conjugation site contained therein.

[0091] For example, if the precursor of a protein-based carrier component specifically binds to avian proteins (e.g., it is an anti-avian protein ISVD, an anti-avian protein DARPin, an anti-avian protein affitin, an anti-avian protein affibody, etc.), then the protein-based carrier component derived therefrom preferably does not specifically bind to these avian proteins (or, as described herein, preferably does not specifically bind to avian proteins at least 5×10 -4 moles / liter K D(The protein-based carrier component binds to the avian protein at a specific value). In other embodiments, the protein-based carrier component specifically binds to the avian protein when its precursor specifically binds, but the specific binding is removed once at least a cargo is conjugated to the protein-based component. An example of an avian protein to which a protein-based component precursor (and / or a protein-based component of the present technology) can specifically bind is ovalbumin (chicken). Thus, in one embodiment, a protein-based component precursor, e.g., ISVD, can specifically bind to this avian protein. The resulting protein-based component may not specifically bind to the avian protein to which the precursor binds. If the protein-based component of the present technology exhibits specific binding to this avian protein, the specific binding described herein is lost once at least a cargo is conjugated to at least one conjugation site contained therein.

[0092] For example, if the precursor of a protein-based carrier component specifically binds to yeast and / or fungal proteins (e.g., it is an anti-yeast and / or anti-fungal protein ISVD, an anti-yeast and / or fungal protein DARPin, an anti-yeast and / or fungal protein affitin, an anti-yeast and / or fungal protein affibody, etc.), then the protein-based carrier component derived therefrom preferably does not specifically bind to those yeast and / or fungal proteins (or, as described herein, preferably binds to less than 5×10 -4 moles / liter K D(The protein-based carrier component binds to these yeast and / or mold proteins at a specific binding value.) In other embodiments, the protein-based carrier component specifically binds to yeast and / or mold proteins when its precursor specifically binds, but the specific binding is removed once at least a cargo is conjugated to the protein-based component. Examples of yeast and mold proteins to which a protein-based component precursor (and / or protein-based component of the present technology) can specifically bind are yeast extract, inactivated yeast, and Candida. Thus, in one embodiment, a protein-based component precursor, e.g., an ISVD, can specifically bind to one or more of these yeast and / or mold proteins. The resulting protein-based component may not specifically bind to at least one of these yeast and / or mold proteins to which the precursor binds. If the protein-based component of the present technology exhibits specific binding to these yeast and / or mold proteins, that specific binding, as described herein, is lost once at least a cargo is conjugated to at least one conjugation site contained therein.

[0093] For example, if a precursor of a protein-based carrier component specifically binds to a plant protein (e.g., it is an anti-plant protein ISVD, an anti-plant protein DARPin, an anti-plant protein affitin, an anti-plant protein affibody, etc.), then the protein-based carrier component derived therefrom preferably does not specifically bind to those plant proteins (or, as described herein, preferably does not bind to 5×10 -4 moles / liter K D(It binds to those plant proteins at a specific value). In other embodiments, a protein-based carrier component specifically binds to a plant protein when its precursor specifically binds, but the specific binding is removed once at least a cargo is conjugated to the protein-based component. Examples of plant proteins to which a protein-based component precursor (and / or a protein-based component of the present technology) can specifically bind are starch branching enzyme II (corn), polyphenols, linoleic acid (sunflower, corn), and plant seeds. Thus, in one embodiment, a protein-based component precursor, such as an ISVD, can specifically bind to one or more of these plant proteins. The resulting protein-based component may not specifically bind to at least one of the plant proteins to which the precursor binds. If the protein-based component of the present technology exhibits specific binding to these plant proteins, the specific binding described herein is lost once at least a cargo is conjugated to at least one conjugation site contained therein.

[0094] For example, if the precursor of a protein-based carrier component specifically binds to fungal proteins (e.g., it is an antifungal protein ISVD, an antifungal protein DARPin, an antifungal protein affitin, an antifungal protein affibody, etc.), then the protein-based carrier component derived therefrom preferably does not specifically bind to these fungal proteins (or, as described herein, preferably does not specifically bind to these fungal proteins). -4 moles / liter K D(The protein-based carrier component binds to these fungal proteins at a specific binding value.) In other embodiments, the protein-based carrier component specifically binds to a fungal protein when its precursor specifically binds, but the specific binding is removed once at least one cargo is conjugated to the protein-based component. Examples of fungal proteins to which a protein-based component precursor (and / or a protein-based component of the present technology) can specifically bind are cutinase, chitin, and fungal sphingolipids. Thus, in one embodiment, a protein-based component precursor, e.g., an ISVD, can specifically bind to at least one of these fungal proteins. The resulting protein-based component may not specifically bind to at least one of these fungal proteins to which the precursor binds. If the protein-based component of the present technology exhibits specific binding to at least one of these fungal proteins, its specific binding as described herein is lost once at least one cargo is conjugated to at least one conjugation site contained therein.

[0095] For example, if a precursor of a protein-based carrier component specifically binds to bacteria (e.g., it is an antimicrobial ISVD, an antimicrobial DARPin, an antimicrobial affitin, an antimicrobial affibody, etc.) and / or bacterial molecules (e.g., it specifically binds to one or more bacterial biomolecules, such as bacterial proteins, bacterial nucleic acids, bacterial lipids, or bacterial glycans), then a protein-based carrier component derived therefrom preferably does not specifically bind to these bacteria and / or bacterial molecules (or, as described herein, binds to these bacteria and / or bacterial molecules at a density of, preferably, 5×10 -4 moles / liter K DIn other embodiments, the protein-based carrier component specifically binds to bacteria (e.g., it is an antimicrobial ISVD, an antimicrobial DARPin, an antimicrobial affitin, an antimicrobial affibody, etc.) and / or bacterial molecules (e.g., it specifically binds to one or more bacterial biomolecules, such as bacterial proteins, bacterial nucleic acids, bacterial lipids, or bacterial glycans) when its precursor specifically binds, but the specific binding is removed once at least the cargo is conjugated to the protein-based component. Examples of bacteria and bacterial molecules to which the protein-based building block precursors (and / or protein-based building blocks of the present technology) can specifically bind include: β-lactamase, tetanus toxin, lactate oxidase, Salmonella typhimurium, Helicobacter pylori, Mycobacterium tuberculosis, Clostridium difficile (toxins A and B), Pseudomonas aeruginosa, Bacillus anthracis, botulinum neurotoxin, Treponema pallidum, Chlamydia trachomatis, Escherichia coli, Campylobacter jejuni, and the like. jejuni (flagellum), Salmonella enterica, Bordetella pertussis (toxin), Shigella spp., Streptomyces venezuelae, chloramphenicol. Thus, in one embodiment, a protein-based building block precursor, e.g., an ISVD, may specifically bind to one or more of these bacteria (or molecules thereof, including biomolecules). The resulting protein-based building block may not specifically bind to the bacteria (or molecules thereof, including biomolecules) to which the precursor binds.Where the protein-based components of the present technology exhibit specific binding to one or more of these bacteria (or molecules, including biomolecules, contained therein), that specific binding as described herein is lost when at least one cargo is attached to at least one conjugation site contained therein.

[0096] For example, if the precursor of a protein-based carrier component specifically binds to a non-human animal protein such as a snake protein (e.g., it is an anti-snake protein ISVD, an anti-snake protein DARPin, an anti-snake protein affitin, an anti-snake protein affibody, etc.), then the protein-based carrier component derived therefrom preferably does not specifically bind to these snake proteins (or, as described herein, preferably does not bind to more than 5×10 -4 moles / liter K D (The protein-based carrier component specifically binds to those snake proteins at a specific binding site.) In other embodiments, the protein-based carrier component specifically binds to the snake protein when its precursor specifically binds, but the specific binding is removed once at least a cargo is conjugated to the protein-based component. An example of a snake protein to which a protein-based component precursor (and / or a protein-based component of the present technology) can specifically bind is cobra toxin. Thus, in one embodiment, a protein-based component precursor, e.g., ISVD, can specifically bind to this snake protein. The resulting protein-based component may not specifically bind to the snake protein to which the precursor binds. If the protein-based component of the present technology exhibits specific binding to this snake protein, the specific binding described herein is lost once at least a cargo is conjugated to at least one conjugation site contained therein.

[0097] For example, if a precursor of a protein-based carrier component specifically binds to green fluorescent protein (GFP), a protein derived from jellyfish (sea jellies) and corals, sea anemones, zooplankton, copepods, and amphioxus (e.g., it is an anti-GFP ISVD, anti-GFP DARPin, anti-GFP affitin, anti-GFP affibody, etc.), then the protein-based carrier component derived therefrom preferably does not specifically bind to GFP (or, as described herein, does not specifically bind to GFP at least 5×10 -4 moles / liter K D (The protein-based carrier component binds to GFP at a specific binding value). In other embodiments, the protein-based carrier component specifically binds to GFP when its precursor specifically binds, but the specific binding is removed when at least a cargo is conjugated to the protein-based component. Thus, in one embodiment, a protein-based component precursor, e.g., an ISVD, can specifically bind to GFP. The resulting protein-based component may not specifically bind to the GFP to which the precursor binds. When the protein-based component of the present technology exhibits specific binding to GFP, the specific binding described herein is lost when at least a cargo is conjugated to at least one conjugation site contained therein.

[0098] For example, if the precursor of a protein-based carrier component specifically binds to insect proteins (e.g., it is an anti-insect protein ISVD, an anti-insect protein DARPin, an anti-insect protein affitin, an anti-insect protein affibody, etc.), then the protein-based carrier component derived therefrom preferably does not specifically bind to those insect proteins (or, as described herein, preferably does not bind to more than 5×10 -4 moles / liter K D(These proteins bind to these insect proteins at a specific binding site.) In other embodiments, a protein-based carrier component specifically binds to an insect protein when its precursor specifically binds, but the specific binding is removed once at least a cargo is conjugated to the protein-based component. Examples of insect proteins to which a protein-based component precursor (and / or a protein-based component of the present technology) can specifically bind are Androctonus autralis hector toxin, chitin, chitin-binding domain (CBD), V-ATPase subunit C, trehalase, cytochrome p450 monooxygenase, chitin deacetylase, chitin synthase, and NPC1 sterol transporter. Thus, in one embodiment, a protein-based component precursor, e.g., an ISVD, can specifically bind to at least one of these insect proteins. The resulting protein-based component may not specifically bind to at least one of these insect proteins to which the precursor binds. Where the protein-based components of the present technology exhibit specific binding to at least one of these insect proteins, that specific binding as described herein is lost when at least one cargo is attached to at least one conjugation site contained therein.

[0099] For example, if a precursor of a protein-based carrier component specifically binds to the crustacean protein chitin (e.g., it is an anti-chitin ISVD, anti-chitin DARPin, anti-chitin affitin, anti-chitin affibody, etc.), then a protein-based carrier component derived therefrom preferably does not specifically bind to chitin (or, as described herein, preferably does not bind to chitin at least 5×10 -4 moles / liter K D(The protein-based carrier component binds to chitin at a specific binding site.) In other embodiments, the protein-based carrier component specifically binds to chitin when its precursor specifically binds, but the specific binding is removed once at least a cargo is conjugated to the protein-based component. Thus, in one embodiment, a protein-based component precursor, e.g., an ISVD, can specifically bind to chitin. The resulting protein-based component may not specifically bind to the chitin to which the precursor binds. If the protein-based component of the present technology exhibits specific binding to chitin, the specific binding described herein is lost once at least a cargo is conjugated to at least one conjugation site contained in the chitin.

[0100] Therefore, preferably, the protein-based carrier component does not specifically bind to the precursor target of the protein-based carrier component precursor, if the precursor has a target, and the target is a non-human molecule (including a biomolecule), such as a non-human protein. Thus, in one embodiment, at least one protein-based component included in the molecule of the present technology does not specifically bind to any RSV protein, such as RSV protein F, as described herein, or binds to any RSV protein, such as RSV protein F, at a concentration of 5x10 -4 moles / liter K D (K D The F protein is linked via a nucleotide sequence (a nucleotide sequence, a ...

[0101] In a further preferred embodiment, the protein-based component of the present technology, when having at least one cargo (such as a "model cargo", e.g., maleimide-modified alanine) attached thereto (via at least one conjugation site or attachment point contained therein), does not specifically bind to any molecule (including a biomolecule) to which the protein-based carrier component precursor specifically binds (i.e., the protein-based component to which at least the cargo is attached preferably does not specifically bind to the precursor's target, e.g., a non-human protein or non-protein molecule (including a biomolecule)), or, as described herein, does not specifically bind to any molecule (including a biomolecule) to which the protein-based carrier component precursor specifically binds (i.e., the protein-based component to which at least the cargo is attached preferably does not specifically bind to the precursor's target, e.g., a non-human protein or non-protein molecule (including a biomolecule)). -4 moles / liter K D and binds to any (non-human) molecule (including a biomolecule) to which the protein-based carrier component precursor specifically binds (i.e., the protein-based component to which the cargo is attached preferably also does not specifically bind to the precursor's target, e.g., a non-human protein or non-protein molecule). Thus, in a preferred embodiment, if the protein-based carrier component of the present technology exhibits any specific binding to a molecule (including a biomolecule, e.g., a human, non-human animal, plant, microorganism, virus, etc.) or cell (e.g., an animal, human, or plant cell), microorganism, virus, etc., that specific binding is removed once at least the cargo is attached to at least one attachment point or conjugation site included in the protein-based component. In this particular embodiment, the cargo attached to the protein-based component may, of course, exhibit specific binding to a target (including a biomolecule as described herein), but the protein-based component no longer specifically binds to that target.

[0102] In a further preferred embodiment, the protein-based carrier components of the present technology do not specifically bind to any human or non-human (e.g., non-human animal, plant, yeast, etc.) cells and / or cell types. If the components exhibit interactions with one or more human or non-human cells and / or cell types, such interactions are characterized by low specificity and / or low affinity, as defined herein. In particular, preferably, the carrier components do not specifically bind to any human or non-human cells and / or cell types to which the protein-based carrier component precursor specifically binds (i.e., the protein-based components preferably do not specifically bind to the precursor's target, e.g., a non-protein molecule or protein present on the surface of a human cell). The lack of binding to any human or non-human cells and / or cell types can be assessed, for example, by a "cell binding assay" as described below (see, for example, Hunter SA and Cochran JR, "Cell-binding assays for determining the affinity of protein-protein interactions: technologies and considerations," Methods Enzymol., 2016, 580:21-44).

[0103] In another embodiment, the protein-based carrier components of the present technology do not specifically bind to any microorganisms, such as bacteria, fungi, protists, yeast, and / or viruses, or any microbial or viral molecules (including biomolecules). If the components exhibit interactions with one or more microorganisms and / or viruses or any microbial or viral molecules (including biomolecules), such interactions are characterized by low specificity and / or low affinity, as defined herein. In particular, preferably, the carrier components do not specifically bind to any microorganisms and / or viruses (or any microbial or viral molecules (including biomolecules)) to which the protein-based carrier component precursor specifically binds (i.e., the protein-based components do not specifically bind to the precursor's target, e.g., a virus, a microorganism, a non-protein molecule (including biomolecules), or a protein present on the surface of a microorganism and / or virus). The lack of binding to any microorganism, or virus, or microbial molecule, or viral molecule can be assessed, for example, by a "cell binding assay" and / or SPR, as described herein.

[0104] In another embodiment, the protein-based carrier component of the present technology, when having at least one cargo (a "model cargo," such as a maleimide-modified alanine) attached or conjugated thereto (via at least one attachment point or conjugation site contained therein), does not specifically bind to any microorganism, such as a bacterium, fungus, protist, yeast, and / or virus (and / or any microbial or viral molecule or biomolecule, such as a microbial or viral protein, nucleic acid, lipid, glycan, etc.). If the component with the cargo attached thereto exhibits any interaction with one or more microorganisms and / or viruses (or with any microbial or viral protein, nucleic acid, lipid, glycan, etc., any microbial or viral molecule or biomolecule, etc.), such interaction is characterized by low specificity and / or low affinity, as defined herein. In particular, preferably, the carrier component does not specifically bind to any microorganism and / or virus (or any microorganism or viral molecule or biomolecule, such as a microorganism or viral protein, nucleic acid, lipid, glycan, etc.) to which the protein-based carrier component precursor specifically binds when the protein-based carrier component has at least one cargo bound or conjugated thereto (i.e., the protein-based component, preferably, when having at least one cargo bound or conjugated thereto, does not specifically bind to the precursor's target, such as a non-protein molecule or biomolecule or protein present on the surface of or present in the microorganism and / or virus). For example, the protein-based component of the present technology does not specifically bind to any virus and / or viral protein, such as RSV and / or one or more proteins of RSV, such as RSV protein F, when the component has at least one cargo bound or conjugated thereto.Thus, for example, a protein-based carrier component (e.g., a DARPin-based carrier component) may exhibit specific binding to a microorganism and / or virus, such as RSV and / or a RSV protein, such as RSV protein F, but when at least one cargo is bound or conjugated to the protein-based component, the specific binding (as defined herein), if present, is lost.

[0105] The lack of specific binding to any microorganism can be assessed, for example, in a "cell binding assay" as described herein. The lack of specific binding to viruses, microorganisms and / or viral molecules or biomolecules can be assessed, for example, by surface plasmon resonance as described herein.

[0106] In another embodiment, the protein-based carrier component of the present technology does not specifically bind to any molecule, including biomolecules, including human and non-human molecules (including human and non-human biomolecules, e.g., human and / or non-human proteins, human and / or non-human nucleic acids such as DNA and / or RNA, human and / or non-human lipids (e.g., phosphatidylserine (PS)) or non-human glycans, etc.), or binds to any molecule, including biomolecules, including human and non-human molecules (including human and non-human biomolecules, e.g., human and / or non-human proteins, nucleic acids such as DNA and / or RNA, lipids (e.g., phosphatidylserine (PS)) or glycans, etc.), as described herein, at a concentration of at least 5×10 -4 moles / liter K D (K D For example, the protein-based carrier component may not specifically bind to any human and / or non-human animal biomolecules (e.g., human and / or non-human animal proteins, human and / or non-human nucleic acids such as DNA and / or RNA, human and / or non-human lipids (e.g., phosphatidylserine (PS)), or human and / or non-human glycans), or may bind to any human and / or non-human biomolecules (e.g., human and / or non-human animal proteins, human and / or non-human nucleic acids such as DNA and / or RNA, human and / or non-human lipids (e.g., phosphatidylserine (PS)), or human and / or non-human glycans) at a binding affinity of at least 5×10, as described herein. -4 moles / liter K D (K DFor example, the protein-based carrier component may not specifically bind to any bacterial molecule (e.g., including bacterial proteins, nucleic acids such as DNA and / or RNA, lipids (e.g., phosphatidylserine (PS)), or glycans), or may bind to any bacterial molecule as defined above with a binding affinity of at least 5×10, as described herein. -4 moles / liter K D (K D For example, the protein-based carrier components of the present technology may not specifically bind to any viral molecule (including biomolecules, e.g., viral proteins, nucleic acids such as DNA and / or RNA, lipids (e.g., phosphatidylserine (PS)), or glycans), or may bind to any viral molecule as defined herein at a binding affinity of more than 5×10, as described herein. -4 moles / liter K D (K D For example, the protein-based carrier component may not specifically bind to any fungal molecule (including biomolecules, e.g., fungal proteins, nucleic acids such as DNA and / or RNA, lipids (e.g., phosphatidylserine (PS)), or glycans), or may bind to any fungal molecule as defined herein at a binding affinity of at least 5×10, as described herein. -4 moles / liter K D (K D For example, the protein-based carrier component may not specifically bind to any yeast molecule (including biomolecules, e.g., yeast proteins, nucleic acids such as DNA and / or RNA, lipids (e.g., phosphatidylserine (PS)), or glycans), or may bind to any yeast molecule as described herein at a binding affinity of at least 5×10, as described herein. -4 moles / liter K D (K DFor example, the protein-based carrier component may not specifically bind to any plant molecule (including biomolecules, e.g., plant proteins, nucleic acids such as DNA and / or RNA, lipids (e.g., phosphatidylserine (PS)), or glycans), or may bind to any plant molecule as defined herein at a binding affinity of at least 5×10, as described herein. -4 moles / liter K D (K D For example, the protein-based carrier component may not specifically bind to any mammalian molecule (including mammalian biomolecules, e.g., mammalian proteins, nucleic acids such as DNA and / or RNA, lipids (e.g., phosphatidylserine (PS)), or glycans), or may bind to any mammalian molecule as described herein at a binding affinity of at least 5×10, as described herein. -4 moles / liter K D (K D value).

[0107] In the context of the present technology, the term "biomolecule" or "biological molecule" refers to a molecule present in living organisms, including animals, plants, and microorganisms, that plays a role in one or more biological processes, such as cell division, morphogenesis, or development. Biomolecules are the building blocks of life and perform important functions in living organisms. Biomolecules include primary metabolites, which are large macromolecules such as proteins, carbohydrates (glycans), lipids (e.g., PS), and nucleic acids (DNA, RNA, etc.), as well as small molecules such as vitamins and hormones. The four major types of biomolecules are carbohydrates (glycans), lipids, nucleic acids, and proteins.

[0108] In a further preferred embodiment, the protein-based carrier component of the present technology does not specifically bind to any non-human protein and / or any non-protein molecule (including biomolecules) when at least one cargo (a "model cargo", e.g., maleimide-modified alanine) is conjugated to at least one attachment point or conjugation site on the protein-based carrier component, and preferably it does not bind to any non-human protein and / or any non-protein molecule (including biomolecules) to which the protein-based carrier component precursor specifically binds, or as described herein, does not specifically bind to any non-human protein and / or any non-protein molecule (including biomolecules). -4 moles / liter K D (K D value).

[0109] Thus, in one embodiment, the present technology provides a molecule comprising at least one protein-based carrier component described in the present technology, wherein the protein-based carrier component has at least a cargo (a "model cargo," e.g., maleimide-modified alanine) attached or conjugated thereto (via at least one attachment point or conjugation site included in the protein-based carrier component), and the protein-based carrier component does not specifically bind to any molecule (including a biomolecule) and / or organism (cell, microorganism, virus, etc.). Thus, in one embodiment, the protein-based component loses its target binding specificity once at least a cargo is conjugated thereto. For example, the protein-based component comprising a cargo attached thereto does not specifically bind to any molecule (including a biomolecule) and / or organism (cell, microorganism, virus, etc.) to which the protein-based carrier component precursor specifically binds (i.e., the protein-based component to which at least a cargo is attached preferably does not specifically bind to the target of the precursor, or to any (non-human) molecule (including a biomolecule) and / or organism (cell, microorganism, virus, etc.) to which the protein-based carrier component precursor specifically binds, as described herein, at a binding specificity of at least 5×10 -4 moles / liter K D (K Dvalue) (i.e., the protein-based entity to which the cargo is attached preferably does not also specifically bind to the precursor target).

[0110] Those skilled in the art will recognize means to reduce and / or eliminate specific binding of protein-based carrier component precursors to proteins and / or non-protein molecules (including biomolecules). For example, mutations can be made in the amino acid sequence of the precursor component so that it no longer specifically binds to human proteins, or any non-human protein, or non-protein molecule (including biomolecules), or as described herein, reduces the specific binding of the precursor component to 5×10 -4 moles / liter K D (K D value).

[0111] The affinity of a molecular interaction between two molecules (e.g., two biomolecules) can be measured via various techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technique (see, e.g., Ober et al. 2001, Intern. Immunology 13:1551-1559, in particular the section "Surface plasmon resonance (SPR) experiments" beginning at p. 1552, which describes the conditions for measuring the affinity of a molecular interaction between two molecules or the explanations provided herein). The term "surface plasmon resonance" as used herein refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by the detection of changes in protein concentration within a biosensor matrix, where one molecule is immobilized on a biosensor chip and the other molecule passes over the immobilized molecule under flow conditions, resulting in k on , k off measurements, hence K D (or K A) value. Surface plasmon resonance can be performed, for example, using the well-known BIAcore® system (BIAcore International AB, Cytiva lifesciences company, Uppsala, Sweden and Piscataway, NJ). For further description, see Jonsson et al. (1993, Ann. Biol. Clin. 51:19-26), Jonsson et al. (1991 Biotechniques 11:620-627), Johnson et al. (1995, J. Mol. Recognit. 8:125-131), and Johnson et al. (1991, Anal. Biochem. 198:268-277). For example, the affinity (K) of a molecular interaction between two molecules can be determined. D) can be determined via SPR on a ProteOn XPR36 instrument (Bio-Rad Laboratories). Experiments can be performed at 25°C, using assay buffer PBS pH 7.4 (Bio-Rad Laboratories) containing 0.005% Tween 20. Targets such as human proteins or non-protein molecules (biomolecules) as described herein or non-human biomolecules, such as nucleic acids (e.g., DNA, RNA), lipids (e.g., phosphatidylserine (PS)), or glycans, can be immobilized on different ligand lanes from a GLC sensor chip (Bio-Rad Laboratories) according to the manufacturer's instructions, for example, using the ProteOn Amine Coupling Kit (Bio-Rad Laboratories). Protein-based components of the technology can be captured on the target-immobilized ligand lane. One ligand lane can serve as a reference surface, with no protein-based components captured on the surface. Different concentrations (e.g., ranging from 300 nM to 1.2 nM) diluted in running buffer can be flowed over each protein-based component and reference surface in a multi-cycle kinetic for 2 minutes, followed by a constant flow of assay buffer for 15 minutes. Between different injections, the surfaces can be regenerated with 3 M MgCl2 (Cytiva) or 10 mM glycine pH 1.5 (Cytiva). For double referencing, several buffer blanks can be injected. Data can be analyzed, for example, using ProteOn Manager 3.1.0 software (Bio-Rad Laboratories). Rate constants (ka and kd) can be calculated by fitting the sensorgrams via a Langmuir 1:1 interaction ligand binding model. The equilibrium dissociation constant, K, is D can be calculated as the ratio kd / ka. See also, for example, https: / / nicoyalife.com / wp-content / uploads / 2023 / 02 / characterization-of-Influenza-using-Alto.pdf.

[0112] Another well-known biosensor technique for determining the affinity of biomolecular interactions is biolayer interferometry (BLI) (see, e.g., Abdiche et al. 2008, Anal. Biochem. 377:209-217). The term "biolayer interferometry" or "BLI," as used herein, refers to a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized proteins on a biosensor chip (signal beam). Changes in the number of molecules bound to the biosensor tip cause a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the biosensor tip surface. Interactions can be measured in real time, allowing the association and dissociation rates and affinity to be determined. BLI can be performed, for example, using the well-known Octet® Systems (ForteBio, a division of Pall Life Sciences, Menlo Park, USA).

[0113] Alternatively, affinity can be measured by the KinExA (KindexA) equilibrium exclusion method (see, e.g., Drake et al., "Characterizing high-affinity antigen / antibody complexes by kinetic- and equilibrium-based methods," Anal. Biochem., 2004, 328:35-43) using the KinExA® platform (Sapidyne Instruments Inc, Boise, USA). As used herein, the term "KinExA" refers to a solution-based method for measuring the true equilibrium binding affinity and kinetics of unmodified molecules. An equilibrium solution of a binding entity / target complex, such as an antibody / antigen complex, is passed through a column containing beads precoated with the antigen (or antibody), allowing free antibody (or antigen) to bind to the coated molecule. Detection of the antibody (or antigen) captured in this way is performed by a fluorescently labeled protein conjugated to the antibody (or antigen).

[0114] Additionally, the GYROLAB® immunoassay system provides a platform for automated bioanalysis and rapid sample turnaround (Fraley et al., “The Gyrolab™ immunoassay system: a platform for automated bioanalysis and rapid sample turnaround”, Bioanalysis 2013, 5:1765-74).

[0115] Furthermore, the affinity of molecular interactions between two molecules (e.g., between two biomolecules, such as between two proteins) or between one biomolecule, such as one protein and one cell, can be measured using flow cytometry to analyze ligand binding to antigens, such as proteins, lipids (e.g., phosphatidylserine (PS)), or sugars, displayed on the surface of cells ("cell-binding assay"). Cell-binding assays for determining the affinity of a particular soluble molecule (such as a molecule of the present technology) and a binding partner present on the surface of cells, such as human cells, are well known to those skilled in the art. For example, Hunter SA and Cochran JR ("Cell-binding assays for determining the affinity of protein-protein interactions: technologies and considerations," Methods Enzymol. 2016, 580:21-44) provide practical guidelines for measuring binding events between a soluble ligand and a binding partner, particularly one expressed on the surface of mammalian cells. For example, as shown in the Examples, a cell-binding assay can be performed as follows: a. Adding a fixed number of (human or non-human) cells to a 96-well V-bottom plate (e.g., 50 μL of human cell suspension (e.g., 5E+04 / 96 well) or tubes such as Eppendorf tubes in cold fluorescence-activated cell sorting (FACS) buffer (e.g., consisting of D-PBS, 2% heat-inactivated fetal bovine serum (HI FBS) and 0.05% sodium azide); b. optionally performing a washing step; c. adding a soluble molecule, such as a molecule of the present technology or a protein-based component of the present technology, preferably marked with a fluorescent label or epitope tag, and incubating with shaking at low temperature, typically 4°C, for a period of time, generally several hours, for example about 3 hours, until the reaction reaches equilibrium; d. Assessing binding of soluble molecules to human cells by flow cytometry, eg, FACS.

[0116] Thus, a cell binding assay may be performed by adding some cells (human or non-human, e.g., non-human animal, plant, microorganism, etc.) to a recipient (e.g., a 96-well V-bottom plate or tube, as described above), preferably in a physiological buffer, adding a preferably marked molecule whose binding is to be assessed (e.g., a molecule of the present technology or a protein-based component of the present technology), incubating it with the cells for a period of time, e.g., when the reaction has reached equilibrium, generally several hours, e.g., about 3 hours, preferably at low temperature, typically 4°C, preferably with shaking, and finally assessing the binding of the soluble molecule to the human cells by flow cytometry, e.g., FACS.

[0117] The binding affinity (e.g., K) between soluble molecules, e.g., molecules of the present technology, and human cells D and / or EC 50 ), the soluble molecules in step c above are calculated based on the expected K D and / or EC 50Various concentrations, spanning two orders of magnitude above and below EC , can be added to each well / tube. Binding values ​​can be determined from the mean signal value (e.g., mean fluorescence value) of each sample, plotting the bound fraction versus ligand concentration (logarithmic scale), and fitting a sigmoidal curve using nonlinear regression analysis. The ligand concentration at half the bound fraction is known as the EC 50 Also known as the equilibrium dissociation constant (K D ) is a first approximation of

[0118] Those skilled in the art can determine whether a molecule can specifically bind to a human protein, as defined in the context of the present technology. For example, those skilled in the art can use commercially available protein arrays to determine the binding affinity of a particular molecule (protein) to a human protein. For example, those skilled in the art can use commercially available Proteome Profiler™ antibody arrays, which allow semi-quantitative measurement of more than 100 proteins in a single sample. Alternatively or additionally, those skilled in the art can use HuProt™ assays, such as version v4.0, which consist of more than 21,000 unique human proteins, isoform variants, and protein fragments, covering 16,794 unique genes. This includes 15,889 of the 19,613 standard human proteins listed in the Human Protein Atlas, providing broad coverage across protein subclasses. Those skilled in the art can use commercially available cell arrays, such as arrays of humans, non-human animals, plants, bacteria, yeast, etc., to determine the binding affinity (e.g., K) of a particular molecule (protein) to human cells. D and / or EC 50 ) can also be determined. See also, e.g., Example 16.

[0119] Similarly, one skilled in the art can determine whether a molecule can specifically bind to a non-human protein, such as a bacterial protein or a viral protein. For example, one skilled in the art can use a protein binding assay to determine the binding affinity of a particular molecule (e.g., a protein) to a non-human (e.g., bacterial or viral) protein. Similarly, one skilled in the art can determine whether a molecule (e.g., a protein) can specifically bind to a human non-protein molecule, such as human DNA, human RNA, human lipids (e.g., phosphatidylserine (PS)), or human glycans (see, for example, Campanero-Rhodes MA et al., "Microarray strategies for exploring bacterial surface glycans and their interactions with glycan-binding proteins," Front Microbiol. 2020, 10:2909). For example, as described above, the binding affinity of a molecular interaction between two molecules (e.g., two proteins or a protein and a non-protein molecule) can be measured by SPR. SPR, as described in detail above, can be used to measure the K of a potential interaction between two molecules. D This allows for the determination of

[0120] As will be apparent to those skilled in the art, at least one carrier component included in the molecules of the present technology may exhibit non-specific binding to one or more human proteins (and / or one or more non-human proteins, as described above, and / or one or more non-protein molecules, such as human non-protein molecules, and / or one or more human cell types). This is because there may be molecular forces in the form of, for example, hydrophobic interactions, hydrogen bonds, van der Waals interactions, and other non-specific interactions between at least one carrier component of the present technology and one or more human proteins (and / or one or more non-human proteins and / or one or more human non-protein molecules, e.g., human non-protein molecules and / or one or more human cells). Thus, when this occurs, at least one carrier component included in the molecules of the present technology may non-specifically bind to one or more human proteins (and / or one or more non-protein molecules, such as one or more non-human proteins and / or human non-protein molecules, as described above, and / or one or more human cells in this case). In the context of the present technology, 5×10 -4 Any K above moles / liter D value (or 2 x 10 3 Any K less than liters / mole A The 5×10 component is generally considered to represent nonspecific binding. -4 moles / liter K D (K D value) (or 2 x 10 3 K less than liter / mole A value), e.g., 5.5×10 -4 moles / liter K D (K D value) (or 1.8 x 10 3 K less than liter / mole A value) or 6 x 10 -4 moles / liter K D (K D value) (or 1.7 x 10 3 K less than liter / mole Avalue) of any human protein (or non-human protein and / or in this case any human cell, as explained above). Furthermore, in the context of the present technology, in a preferred embodiment, the carrier component is capable of binding to any human protein (or non-human protein and / or in this case any human cell) with a -4 moles / liter K D (K D value) (or 2 x 10 3 K less than liter / mole A value), e.g., 5.5×10 -4 moles / liter K D (K D value) (or 1.8 x 10 3 K less than liter / mole A value) or 6 x 10 -4 moles / liter K D (K D value) (or 1.7 x 10 3 K less than liter / mole A The building blocks can bind to any non-protein molecule, for example, any human non-protein molecule (e.g., DNA, RNA, lipids (e.g., phosphatidylserine (PS)), glycans) with a 5×10 -4 KD (KD value) in moles / liter (or 2 x 10 3 liters / mol less than the KA value), e.g., 5.5 x 10 -4 KD (KD value) in moles / liter (or 1.8 x 10 3 liters / mol less than KA value) or 6 x 10 -4 KD (KD value) in moles / liter (or 1.7 x 10 3 It can bind to any human cell with a K A value of less than 1 / mol / liter. In the context of the present technology, this binding affinity is considered to be "non-specific binding."

[0121] In one embodiment, the protein-based carrier component of the present technology is an antibody (two Cs), such as the Fc fragment of a monoclonal antibody (mAb). H 2 domains and 2 C H In another embodiment, the protein-based carrier component of the present technology is not derived from a crystallizable fragment of the Fc fragment (Fc containing three domains). H 2 and / or CH In another embodiment, the protein-based carrier component of the present technology is not derived from the C3 domain contained in the antigen-binding fragment (Fab) of an antibody. H 1 and / or C L domains, such as the C in the Fab of mAb H 1 and / or C L In one embodiment, the molecule of the present technology is not (or is not derived from) a crystallizable fragment (Fc) of an antibody such as a mAb. In another embodiment, the molecule of the present technology is not (or is not derived from) a Fab of an antibody such as a mAb.

[0122] In one embodiment, the molecule of the present technology is V H -V L at least one V that does not contain a pair or that interacts (is bound to) each other, e.g., by an antibody H and at least one V L In another embodiment, the molecule of the present technology does not contain C L ~C H 1 conjugate, and at least one C linked to each other, for example, via a disulfide bridge. L and at least one C H Does not include 1.

[0123] Attachment or conjugation site As mentioned above, the carrier component present in the molecule of the present technology preferably has at least one attachment point (also referred to as a conjugation site in the present disclosure), as further defined below, in a solvent-accessible position. Preferably, at least one protein-based carrier component comprises two or more attachment points or conjugation sites, preferably in solvent-accessible positions. In a preferred embodiment, the protein-based carrier component comprises at least two attachment points or conjugation sites. In another embodiment, the protein-based component comprises three or more conjugation sites, for example, six or nine conjugation sites. For example, the protein-based carrier component may have 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conjugation sites. In one embodiment, if two or more, the conjugation sites present in the carrier component are different from each other. For example, if a carrier component contains two conjugation sites, these conjugation sites can be functionally / chemically distinct from one another; i.e., each conjugation site or point of attachment is chemically distinct from the other (e.g., if there are two conjugation sites, one conjugation site can be an -SH group present in the side chain of a cysteine ​​located in a solvent-accessible position, and the other conjugation site can be an -NH2 group present in the side chain of a lysine located in a solvent-accessible position). If a component has three or more conjugation sites (e.g., at least three conjugation sites, such as 3, 4, 5, 6, 7, 8, 9, 10, etc.), there can be at least two types of conjugation sites among the at least three conjugation sites present in the component. In another embodiment, if a component has three conjugation sites, each conjugation site is functionally distinct from the other. In another embodiment, when a component has three conjugation sites, two conjugation sites are the same and one conjugation site is functionally different from the other two conjugation sites, hi another embodiment, all conjugation sites present in a component are functionally different from each other.In another embodiment, all conjugation sites present in the carrier component are the same, e.g., a protein-based component has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conjugation sites that are all the same, e.g., they are all -SH groups present in the side chains of cysteines located in solvent-accessible positions of the protein-based component.

[0124] In another embodiment, instead or in addition, when there are two or more conjugation sites, the conjugation sites are spatially separated from one another (spatially separated from one another). Those skilled in the art will understand that the minimum distance between conjugation sites is determined by the nature of the cargo (and linker, if used) attached or conjugated to the attachment point or conjugation site in the protein-based carrier component. For larger cargoes (e.g., ISVDs), the minimum distance can still be kept small when used in combination with a long linker that adds the necessary flexibility and intended target binding. A short distance between conjugation sites, combined with a short linker, if present, is likely to limit target binding of larger cargoes, limiting engagement (e.g., increasing cell specificity). Furthermore, the solubility of the molecule may be reduced (i.e., the molecule may be more prone to aggregation). On the other hand, if the cargo to be attached is quite small (e.g., a radioisotope), the minimum distance can be kept small even without a linker (see, e.g., Example 5 below). Thus, one skilled in the art will be able to select the location of a particular conjugation site and the length and flexibility of the linker, if any, depending on the nature of the cargo to be attached or conjugated to the protein-based carrier component.

[0125] The "conjugation site" or "attachment point" may be a reactive group in the side chain of a natural or unnatural (also referred to as "non-standard," "unnatural," or "unusual," as described above) amino acid, preferably located at a solvent-accessible position within the protein-based carrier component. It may also be the C-terminal and / or N-terminal reactive groups (-COOH and -NH groups, respectively) of the protein-based carrier component. In the context of the present technology, a "reactive group in the side chain of an amino acid" (either natural or unnatural, as defined above) refers to any chemical group present in the side chain of an amino acid that can form a covalent bond. For example, if the amino acid is lysine (or ornithine (Orn), or diaminopropionic acid (Dap), or diaminobutyric acid (Dab)), the reactive group present on its side chain is a primary amine. For example, if the amino acid is cysteine, the reactive group present on its side chain is a thiol group. For example, if the amino acid is aspartic acid or glutamic acid, the reactive group present in their side chain is a carboxylic acid group. For example, if the amino acid is tyrosine, the reactive group present on its side chain is a phenolic hydroxyl group. For example, if the amino acid is arginine, the reactive group present on its side chain is a guanidino group. For example, if the amino acid is methionine, the reactive group present on its side chain is a thioether group.

[0126] In the context of the present technology, "C-terminal or N-terminal reactive group of a protein-based carrier component" refers to the -COOH and -NH reactive groups present in the C-terminal and N-terminal amino acids of a protein-based carrier component. If a carrier component does not have a free C-terminus and / or N-terminus (e.g., because the C-terminus and / or N-terminus of the carrier component are linked to another protein-based component or another peptide or protein, or because the N-terminus is amidated, or because the C-terminus is acetylated), the N-terminus and C-terminus of the carrier component are not suitable as attachment points or conjugation sites as defined herein. In some embodiments, the "conjugation site" or "attachment point" is not the C-terminal or N-terminal reactive group of the protein-based carrier component.

[0127] At least one conjugation site or attachment point present in a component of the present technology may already be present in the component precursor (e.g., an -NH group in the side chain of a lysine present in the component precursor) or may be engineered. Preferably, at least one or more of the attachment points or conjugation sites of a protein-based component are engineered. In the context of the present technology, an "engineered" attachment point or conjugation site refers to a conjugation site or attachment point that is present in the protein-based carrier component but was not present in its precursor at the same or corresponding position. For example, a protein-based component precursor can be modified to introduce one or more attachment points or conjugation sites, as described in detail below. A non-limiting example of an engineered attachment point or conjugation site is a reactive group present in the side chain of an amino acid in the protein-based carrier component that was not present in the same or equivalent position in the component precursor. For example, if a component precursor has a serine at a particular position X (preferably a solvent-accessible position) in the component precursor and that serine is mutated to a cysteine ​​in the carrier component, the -SH group of that cysteine ​​would be the engineered attachment point or conjugation site. For example, if an amino acid (e.g., Cys or Tyr) is added to the N- or C-terminus of a building block precursor, the reactive group present in the side chain of that newly added amino acid in the carrier building block will be the engineered point of attachment or conjugation site.

[0128] Thus, in a preferred embodiment, the protein-based carrier component of the present technology has at least two conjugation sites or attachment points, at least one of which, preferably at least two of which, are engineered attachment points or conjugation sites, i.e., they were not present in the component precursor at the same or corresponding positions. In another preferred embodiment, all of the conjugation sites or attachment points present in the protein-based component are engineered attachment points or conjugation sites, i.e., they were not present in the component precursor at the same or corresponding positions. In one embodiment, the carrier component has two or more engineered attachment points or conjugation sites, such as 3, 4, 5, 6, 7, 8, 9, 10 or more engineered attachment points or conjugation sites.

[0129] As used herein, a residue position in one polypeptide sequence "corresponds" to a residue position in another polypeptide sequence if it occurs at an equivalent position in the polypeptide sequence, for example, as indicated by primary sequence homology or functional equivalence or Kabat numbering. Corresponding positions can be identified by aligning two polypeptide sequences. The alignment used to identify corresponding positions or corresponding regions can be obtained using a conventional alignment algorithm such as Blast (Altschul et al., "Basic local alignment search tool", J. Mol. Biol., 1990, 215(3):403-10).

[0130] At least one conjugation site present in the carrier component of the present technology can be free (i.e., ready for reaction) or capped / protected. Thus, the α-amino group, carboxylic acid terminus, or reactive group (e.g., amine, carboxylic acid, alcohol, thiol) present in the side chain of one or more amino acids of the carrier component can be capped or protected with a protecting group, for example, to prevent polymerization of the amino acid, minimize undesired side reactions during synthesis of the component, or selectively attach different cargoes. Of course, if at least one conjugation site is capped or protected, it must be uncapped or deprotected before attaching or conjugating a cargo, as described in detail below.

[0131] Thus, at least one conjugation site present in the protein-based carrier component of the present technology can be (without limitation) a primary amine, a thiol group, a hydroxyl group, a guanidino group, a carboxyl group, or a thioether group. For example, the conjugation site can be a free or capped (protected) thiol group.

[0132] Thus, in some embodiments, at least one conjugation site present in the protein-based carrier component of the present technology may be a primary amine present in the side chain of lysine (or ornithine (Orn), or diaminopropionic acid (Dap), or diaminobutyric acid (Dab)) in the protein-based component, preferably located at a solvent-accessible position. In other embodiments, the conjugation site is a thiol group present in the side chain of cysteine ​​in the protein-based component, preferably located at a solvent-accessible position in the protein-based component. In other embodiments, the conjugation site is a carboxylic acid group present in the side chain of aspartic acid or glutamic acid in the protein-based component, preferably located at a solvent-accessible position in the protein-based component. In other embodiments, the conjugation site is a guanidino group present in the side chain of arginine in the protein-based component, preferably located at a solvent-accessible position in the protein-based component. In other embodiments, the conjugation site is a thioether group present in the side chain of methionine in the protein-based component, preferably located at a solvent-accessible position in the protein-based component. In another embodiment, the conjugation site is the phenolic OH group of a tyrosine in the protein-based component, preferably located at a solvent-accessible position in the protein-based component. In one embodiment, the tyrosine is preferably located at the N-terminus or C-terminus of the protein-based carrier component of the molecule. In another embodiment, the conjugation site is the N-terminal primary amine of the carrier component, when it is free and preferably solvent-accessible. In another embodiment, the conjugation site is the C-terminal carboxyl group of the carrier component, when it is free and preferably solvent-accessible.

[0133] As mentioned above, the conjugation site can be free or protected. For example, as already mentioned above, if the conjugation site is a thiol group (e.g., from a cysteine ​​in a protein-based building block, preferably located at a solvent-accessible position in the protein-based building block), the thiol group can be free (-SH) or protected / capped. A capped thiol group refers to a thiol group that has been (reversibly) protected with a protecting group (e.g., another cysteine, glutathione (GSH), cysteamine, or benzyl (Bzl, Bn), trityl (Trt), diphenylmethyl (Dpm, Bzh, Bh), tetrahydropyranyl (Thp), tert-butyl (tBu), etc.). Spears, R., et al. ("Cysteine ​​protecting groups: applications in peptide and protein science", Chem. Soc. Rev., 2021, 50, 11098) provides an overview of different cysteine ​​protecting groups. Furthermore, Isidro-Llobet, A., et al., ("Amino acid-protecting groups", Chem Rev., 2009, 109(6):2455-504) provides an overview of different amino acid protecting groups.

[0134] In one embodiment, the protein-based carrier component of the present technology comprises at least two attachment points or conjugation sites, which are two reactive groups present in the side chains of two amino acids (which may be natural or non-natural) within the protein-based component, preferably located in solvent-accessible positions within the protein-based carrier component. For example, in one embodiment, the protein-based carrier component comprises at least two attachment points or conjugation sites, which are two reactive groups present in the side chains of two natural amino acids (e.g., two Cys) within the protein-based component, preferably located in solvent-accessible positions within the protein-based carrier component.

[0135] In one embodiment, at least one of the attachment points or conjugation sites present in the protein-based component is linked (directly or via a linker) to a cargo, as defined herein. In a preferred embodiment, the molecule of the present technology comprises at least one protein-based carrier component and at least one cargo, and the at least one cargo is attached or conjugated to the at least one protein-based carrier component via at least one attachment point or conjugation site. A "cargo" can be any molecule that can be attached or conjugated to a protein-based carrier component via an attachment point or conjugation site present therein. For example, cargoes that can be attached or conjugated to the protein-based carrier component of the present technology include proteins, peptides, ISVDs (e.g., V HH , V L or V H ), polyethylene glycol (PEG), small molecules (e.g., cryptophycin, DM4), glycans (e.g., M6P), lipids, chelators, fluorophores, radioisotopes, vitamins (e.g., folic acid or biotin), nucleic acids (e.g., oligonucleotides or siRNA), etc. The cargoes can have different functionalities. For example, at least one cargo can be a half-life extension (HLE) molecule, a targeting molecule, a therapeutic molecule or a precursor thereof, an imaging molecule, a toxic molecule, an agonist (e.g., Toll-like receptor (TLR) agonist), a T-cell engagement molecule, a sweeping / degradation molecule, a cell-permeable molecule, a nuclear localization molecule, a blood-brain barrier (BBB) ​​shuttle, a radiotherapeutic molecule, or an imaging probe.

[0136] Thus, in another embodiment, the molecule of the present technology comprises at least one protein-based carrier component and at least one cargo, wherein the cargo is bound or conjugated to the at least one protein-based carrier component through at least one attachment point or conjugation site, and the cargo is an HLE molecule such as an albumin-binding ISVD (as described herein, for example, SEQ ID NO: 63 or 106 as defined in Table 8), or a PEG molecule described herein, or an ELNN polypeptide. In another embodiment, the molecule of the present technology comprises at least one protein-based carrier component and at least one cargo, wherein the cargo is bound or conjugated to the at least one protein-based carrier component through at least one attachment point or conjugation site, and the cargo is a targeting moiety and / or therapeutic moiety described herein. In a further embodiment, the molecule of the present technology comprises at least one protein-based carrier component and at least two cargoes, wherein the cargoes are attached or conjugated to the at least one protein-based carrier component via at least two attachment points or conjugation sites, and the at least two cargoes are one HLE molecule as described herein and one therapeutic and / or targeting moiety as described herein.

[0137] In one embodiment, at least one protein-based carrier component included in the molecule of the present technology preferably contains at least two cysteines located in solvent-accessible positions, for example, three, six, or nine cysteines, preferably located in solvent-accessible positions, which have at least two, for example, three, six, or nine conjugation sites, free or capped thiol groups, as defined herein. In one embodiment, at least one protein-based carrier component included in the molecule of the present technology preferably contains three cysteines located in solvent-accessible positions, which have three conjugation sites, free or capped thiol groups, as defined herein. In one embodiment, the protein-based carrier component does not contain any other cysteines in solvent-accessible positions other than the three cysteines located in solvent-accessible positions with three conjugation sites (free or capped thiol groups) (although it may contain one or more cysteines in solvent-inaccessible positions). In another embodiment, at least one protein-based carrier component included in a molecule of the present technology comprises 4, 5, 6, 7, 8, 9, 10 or more cysteines, preferably located in solvent-accessible positions, with free or capped thiol groups that are 4, 5, 6, 7, 8, 9, 10 or more conjugation sites as defined herein. In one embodiment, the protein-based carrier component has 4, 5, 6, 7, 8, 9, 10 or more conjugation sites (free or capped thiol groups) and does not contain any other cysteines in solvent-accessible positions other than the 4, 5, 6, 7, 8, 9, 10 or more cysteines located in solvent-accessible positions within the component. In other embodiments, at least one protein-based component included in a molecule of the present technology comprises at least one amino acid, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, which may be natural or non-natural, preferably located in a solvent-accessible position, and which comprises a reactive group on a side chain that is a conjugation site as defined herein.In another embodiment, at least one protein-based component included in the molecule of the present technology comprises at least two conjugation sites, one of which is a cysteine-derived thiol group (free or protected) preferably located at a solvent-accessible position in the protein-based carrier component, and the other is a tyrosine-derived -OH group, preferably an N- or C-terminally exposed tyrosine-derived -OH group, preferably located at a solvent-accessible position in the protein-based carrier component. In another embodiment, at least one protein-based component included in the molecule of the present technology comprises at least two conjugation sites, one of which is a cysteine-derived thiol group (free or protected) preferably located at a solvent-accessible position in the protein-based carrier component, and the other is a non-natural amino acid-derived reactive group, preferably located at a solvent-accessible position in the protein-based carrier component.

[0138] In one embodiment, the conjugation site or attachment point in the protein-based building block is a selenol (-HSe) group derived from selenocysteine ​​(Sec or U), which may be located, for example, at the C-terminus of the protein-based carrier building block. In another embodiment, the conjugation site or attachment point in the protein-based building block is the keto group of p-acetylphenylalanine (pAcPhe), which can be selectively attached to an alkoxyamine-derivatized cargo (see, e.g., Jun Y. Axup et al., "Synthesis of site-specific antibody-drug conjugates using unnatural amino acids," PNAS, 2012, 109(40)16101-16106).

[0139] Those skilled in the art will then recognize methods for incorporating one or more unnatural amino acids into at least one protein-based component of the molecules of the present technology. For example, WO 2021 / 050554, the contents of which are incorporated herein by reference, describes in detail methods for incorporating one or more unnatural amino acids into proteins.

[0140] In one embodiment, the conjugation site is a free or capped thiol group in the side chain of a cysteine, preferably located in a solvent-accessible position within the building block. Due to its favorable properties (thiol nucleophilic profile at neutral / near-neutral pH, low natural abundance, and general ease of incorporation into proteins by site-directed mutagenesis), cysteine ​​is often the site of choice for site-specific modification of proteins, also known as bioconjugates (Spears RJ et al., "Cysteine ​​protecting groups: applications in peptide and protein science", Chem. Soc. Rev., 2021, 50, 11098-11155).

[0141] In a preferred embodiment, at least one conjugation site or attachment point is selected from a thiol group (-SH, free or capped) present in the side chain of a cysteine, preferably located in a solvent-accessible position within the protein-based carrier component, -NH (a primary amine from the N-terminus of the protein-based component or present in the side chain of an amino acid such as lysine or ornithine), -OH present in the side chain of a tyrosine (either a C-terminal tyrosine, an N-terminal tyrosine, or a tyrosine preferably located in any other solvent-accessible position within the protein-based carrier component), a C-terminal -COOH present in the side chain of an unnatural amino acid (such as azidolysine), and an azide group. More preferably, at least one conjugation site or attachment point is a thiol group (free or capped) present in the side chain of a cysteine, preferably located in a solvent-accessible position within the protein-based component.

[0142] In one embodiment, the protein-based building blocks of the present technology comprise six attachment points or conjugation sites, three of which are present in the side chains of three Cys, preferably located in solvent-accessible positions, and three of which are -NH2, preferably located in the side chains of three Lys, preferably located in solvent-accessible positions of the protein-based building blocks.

[0143] Thus, at least one conjugation site present in at least one component of the molecule of the present technology allows for the conjugation of different cargoes (directly or via a linker, as will be apparent to those skilled in the art and described in detail below). Those skilled in the art will recognize how to link cargoes to the conjugation sites present in the components. For example, Spicer CD et al. ("Achieving controlled biomolecule-biomaterial conjugation", Chem Rev. 2018, 118(16):7702-7743) (the contents of which are incorporated herein by reference) provides an overview of the chemistry of biomolecular conjugation and provides a comprehensive overview of key strategies for achieving controlled functionalization.

[0144] For example, if the conjugation site is a -SH group (free or capped) present in the side chain of a cysteine, preferably located in a solvent-accessible position within the protein-based carrier component, the cargo can be attached or conjugated to the component (directly or via a linker) by alkylation, metal-assisted arylation, disulfide exchange, or addition to a maleimide Michael acceptor. It can also be attached or conjugated using so-called "PODS-based conjugation" (see, e.g., Davydova M. et al., "Synthesis and bioconjugation of thiol-reactive reagents for the creation of site-selectively modified immunoconjugates," J Vis Exp., 2019, 145:10.3791 / 59063). These different methods offer a high level of chemoselectivity for cysteines (see, e.g., D. Alvarez Dorta, ... (See, e.g., et al., Chem. Eur. J. 2020, 26, 14257). When at least one conjugation site is a -SH group (free or capped) present in the side chain of a cysteine, preferably located in a solvent-accessible position within the protein-based carrier component, a cargo can be attached or conjugated thereto by addition to a maleimide Michael acceptor. The maleimide present in the cargo reacts specifically with at least one free thiol to form a thioether bond, generally at pH 6.5-7.5. Of course, if the -SH group is capped or protected, it must first be decapped or deprotected (e.g., reduced with a reducing reagent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP)) before the cargo can be attached thereto.For example, the APN-maleimide "bifunctional" linker (see Formula I in the Examples), also known as 3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propiolonitrile), can be used to attach or conjugate cargoes to -SH attachment points present in the side chains of cysteines, preferably located in solvent-accessible positions within the protein-based building blocks. For example, the bis-maleimide-PEG3-linker (1,11-bismaleimide-triethylene glycol) can be used to attach or conjugate cargoes to -SH attachment points present in the side chains of cysteines, preferably located in solvent-accessible positions within the protein-based carrier building blocks. Additionally, maleimide-modified cargoes (see, e.g., PEG-maleimide, N-ethylmaleimide, maleimide-PEG-acid, resiquimod (R-848)-maleimide, cryptophycin-PEG-maleimide) can be attached to -SH attachment points present in the side chains of cysteines, preferably located in solvent-accessible positions within protein-based carrier components (see also Examples).

[0145] For example, if the conjugation site is the -OH group of a tyrosine, preferably located in a solvent-accessible position within the protein-based carrier component, the cargo can be attached or conjugated to the component (directly or via a linker) by several chemical methods, such as cross-linking via catalytic tyrosine single-electron oxidation, three-component Mannich-type tyrosine conjugation, conjugation by sulfur-fluorine exchange chemistry (SuFEx), transition metal complexes for tyrosine conjugation, diazonium coupling reactions, reaction with triazolinediones, etc. (For a review, see, e.g., D. Alvarez Dorta et al., Chem. Eur. J., 2020, 26, 14257).

[0146] Alternatively or additionally, when the conjugation site is the -OH group of the N- and / or C-terminal tyrosine, the cargo may be enzymatically linked or conjugated to the building block (directly or via a linker), as described, for example, in Alan M. Marmelstein et al., Journal of the American Chemical Society, 2020, 142(11), 5078-5086. When conjugation of at least one cargo to the N- and / or C-terminal tyrosine is performed as described herein, the protein-based building block may be provided with a flexible (GG) or (GS) linker to facilitate enzymatic addition, as described in Alan M. Marmelstein et al., cited above. 1~3 Preferably, the enzyme is extended with a GG tag. In this case, tyrosinase from mushroom (Agaricus bisporus) (abTYR), a copper-dependent enzyme that converts tyrosine to melanin via an o-quinone intermediate, can be used. Alternatively, the much smaller Bacillus megaterium tyrosinase (bmTYR) can be used to catalyze the reaction.

[0147] For example, if the conjugation site is the N-terminal primary amine of the protein-based carrier component and / or a primary amine present in the side chain of an amino acid preferably located in a solvent-accessible position of the protein-based carrier component (e.g., Lys, Orn, or any unnatural amino acid with a primary amine on its side chain), the cargo can be attached or conjugated to the carrier component (directly or via a linker) by reaction of a group present in the cargo / linker (e.g., isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, or fluorophenyl ester) and the primary amine. See, e.g., Bioconjugate Techniques (Third edition), 2013, Chapter 3—“The reactions of bioconjugation,” Greg T. Hermanson.

[0148] For example, if a component contains at least two conjugation sites, including a tyrosine-derived -OH and a cysteine-derived -SH, preferably located in a solvent-accessible position within the protein-based carrier component, the thiol nucleophile can be conveniently capped through disulfide formation with Ellman's reagent. After the coupling reaction in the tyrosine-derived -OH, the thiol group can be decapped by brief exposure to an appropriate reducing agent, as described by Alan M. Marmelstein et al., supra.

[0149] Another option for attaching or conjugating cargoes (directly or via a linker) to attachment or conjugation sites within protein-based building blocks is the use of sortase-mediated transpeptidation reactions. Sortases allow for N-terminal, C-terminal functionalization and the generation of non-native fusions (i.e., N-N or C-C chimeras) through the installation of click handles (see, for example, Guimaraes CP et al. ("Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions," Nature Protocols, 2013, 8(9):1787-1799)). As described in this protocol, sortase-mediated reactions are applicable to any protein of interest (e.g., the protein-based carrier building blocks of the present technology), provided that it contains either (i) an LPXTG motif as a sortase target (where X can be any amino acid, and glycine cannot be a free carboxylate) or (ii) a suitably exposed glycine residue to serve as the incoming nucleophile. The natural nucleophile for sortase is an oligoglycine (Gly) at the N-terminus. 1~5 The peptide can then be substituted with any peptide / protein bearing a nucleotide sequence (a single glycine is often sufficient). The peptide can then be substituted with any cargo molecule (e.g., fluorophore, biotin, crosslinker, lipid, carbohydrate, nucleic acid), provided that a free N-terminal glycine remains available on the peptide to be used as the incoming nucleophile. Thus, incubation of sortase, LPXTG-containing protein, and nucleophile results in the covalent attachment of that nucleophile to the protein of interest in a site-specific manner. Guimaraes CP et al., supra, provides a protocol that allows for the functionalization of any given protein at its C-terminus. The target protein is engineered with a sortase recognition motif (LPXTG). Upon recognition, sortase cleaves the protein between the threonine and glycine residues and attaches an exogenously added oligoglycine (Gly) modified with an optimal functional group (e.g., a cargo to be attached to a protein-based carrier component). 1~5) facilitates the attachment of peptides. Theile CSet et al. ("Site-specific N-terminal labeling of proteins using sortase-mediated reactions", Nature Protocols, 2013, 8(9):1800-1807) describe the use of sortase-mediated reactions to label the N-terminus of any given protein of interest. As described in this protocol, the protein to be labeled is engineered with an exposed stretch of glycine or alanine at its N-terminus when using sortase A from Staphylococcus aureus (S. aureus) or Streptococcus pyogenes (S. pyogenes), respectively. Then, a peptide decorated with a suitable functional group (fluorophore, biotin, lipid, nucleic acid, carbohydrate, etc.) and containing the sortase recognition motif LPXTG / A sequence (where X is any amino acid as described above) at its C-terminus (e.g., cargo) is added to the reaction with sortase. Sortase A cleaves between the threonine and glycine / alanine residues to form a thioester intermediate with the peptide probe. Nucleophilic attack by the N-terminally modified protein of interest decomposes the intermediate, resulting in the formation of a covalent bond between the peptide probe (e.g., cargo) and the N-terminus of the protein (see Figure 1 in Theile CSet et al., supra). Alternatively, depsipeptides can be used for N-terminal labeling (see Theile CSet et al., supra). Finally, Witte MD et al. ("Production of unnaturally linked chimeric proteins using a combination of sortase-catalyzed transpeptidation and click chemistry," Nature Protocols, 2013, 8(9):1808-1819) describe a procedure for the production of N- and C-terminal fusion proteins. Unnatural N- and C-terminal fusion proteins are established by equipping the N- or C-terminus of the protein of interest with a series of click handles using sortase A, followed by strain-promoted click reactions.As described by Witte MD et al., peptides for creating CC-linked proteins are synthesized with an N-terminal triglycine motif and an azide or cyclooctyne (DIBAC) at the C-terminus (see also Figure 2 herein). The protein of interest is engineered with a C-terminal LPXTG sequence. To prepare NN-linked proteins, the authors of this protocol synthesize a peptide containing an LPXTGG sortase A recognition sequence at the C-terminus (X can be any residue, but the authors prefer polar residues such as glutamic acid to facilitate peptide precipitation after cleavage from the resin and increase the peptide's solubility in water) and an azide or cyclooctyne group at the N-terminus of the probe. The protein to be linked should contain one to five Glys at the N-terminus. The final step of the procedure is to fuse the click handle-containing protein (see Figure 1 in Witte MD et al.).

[0150] In view of the above, it is possible to use sortase to attach or conjugate (directly or via a linker) cargo to a protein-based carrier component, as described in detail in Guimaraes CP et al., Theile CS et al., and Witte MD et al., the contents of which are incorporated herein by reference. Using the sortase method described above, cargo can be attached or conjugated (directly or via a linker) to a conjugation site or attachment point within the protein-based carrier component that is at either the N-terminus or C-terminus of the protein-based component. Thus, when the conjugation site or attachment point of a protein-based carrier component is at the C-terminus of the component, the C-terminus of the component contains a sortase recognition motif (LPXTG) and the cargo is N-terminally conjugated to an oligoglycine ((Gly) 1~5) modified peptide, cargo can be attached or conjugated thereto using a sortase (see Figure 2 in Guimaraes CP et al.). When the conjugation site or attachment point of a protein-based carrier component is the N-terminus of the component, the N-terminus of the component can be modified by (Gly) 1~5 Cargos can be attached or conjugated to it using a sortase, provided that it contains a tag sequence and the cargo contains a sortase recognition motif (LPXTG / A) at the C-terminus (see Figure 1 in Theile CSet al.). Additionally, protein or peptide cargos can be attached to the N / C-terminus of a protein-based carrier component as NN and / or CC, as described in detail in Witte MD et al.

[0151] Thus, by choosing an appropriate (possibly initially capped) conjugation site, one skilled in the art can attach or conjugate different cargoes to the component.

[0152] Solvent accessible location As noted above, at least one conjugation site or attachment point present in the protein-based carrier component is preferably located at a solvent-accessible position within the component.

[0153] Those skilled in the art can identify "solvent-accessible positions" in carrier component precursors. This can be done in silico by computer modeling. For example, those skilled in the art can use readily available software tools such as MAESTRO (Schroedinger, LLC, New York, NY, 2021), a multi-agent prediction system based on statistical scoring functions (SSFs) and different machine learning approaches (see, for example, Laimer et al. BMC Bioinformatics (2015) 16:116). Furthermore, those skilled in the art can also use readily available software tools such as YASARA (www.yasara.org) to identify at least potential solvent-accessible positions of at least one conjugation site of a component. Using in silico tools such as MAESTRO or YASARA, those skilled in the art can identify solvent-accessible positions that are potentially suitable for manipulating the conjugation site as defined above. Thus, using tools such as MAESTRO or YASARA, potentially suitable conjugation sites are identified. Examples of methods for identifying solvent-accessible positions potentially suitable for engineering conjugation sites as defined above are provided in the Examples of the present application (e.g., Examples 1-3). As described herein, a protein is selected as a starting point for developing a protein-based carrier building block (a so-called "building block precursor"). For example, using MAESTRO, residues in the building block precursor with a solvent-accessible surface area (SASA) of, for example, 27 Å (square angstroms) or greater can be considered solvent-accessible. The stability (ΔG in solvent) of each mutation of the identified residues (e.g., relative to a cysteine ​​residue) can then be calculated. For further details, see, for example, Laimer J. et al., "MAESTRO—multiagent stability prediction upon point mutations," BMC Bioinformatics, 2015, 16:116.Destabilizing mutations (e.g., mutations with higher ΔG calculated in solvent) are generally not further considered as potential locations for conjugation sites or attachment points. Thus, once potentially suitable conjugation sites are identified using tools such as MAESTRO or YASARA, the stability (ΔG in solvent) of each mutation of the identified residues (e.g., for cysteine ​​residues) is calculated. Residues with lower calculated ΔG in solvent are preferably further selected as potential locations for conjugation sites or attachment points. For example, ΔG values ​​in the range of -20 to +5 can be considered non-destabilizing mutations. Those skilled in the art will understand that the ΔG value of each of the identified residue mutations may vary depending on the particular protein and / or the particular mutation being considered. Those skilled in the art will also understand that the preferred mutation is the mutation with the lowest ΔG value. Depending on these ΔG values, the number of conjugation sites, and the type of cargo to be conjugated, those skilled in the art will further select specific positions from those initially identified as solvent-accessible using tools such as MAESTRO or YASARA.

[0154] Alternatively or additionally, one skilled in the art can use hydrogen / deuterium exchange mass spectrometry (HDX-MS) to determine the least potentially solvent-accessible positions in a protein. HDX-MS reports on the local chemical environment and solvent accessibility of the protein backbone by monitoring the exchange of peptide bond amide protons with deuterium from DO solvent. The rate of hydrogen-deuterium exchange depends on the solvent accessibility and folded state of the protein (see Englander SW. et al., "Hydrogen exchange: the modern legacy of Linderstroem-Lang," Protein Sci., 1997, 6(5):1101-9).

[0155] If an identified solvent accessible position is occupied by a particular amino acid having a reactive group in its side chain (e.g., by cysteine), the in silico modeling (e.g., by MAESTRO) also takes into account potential interactions of the reactive group of that amino acid (e.g., the -SH present in the side chain of cysteine) along with other reactive groups present in the side chains of other amino acids present within the protein-based carrier component (e.g., other -SH groups present in the protein, if any).

[0156] Additionally or alternatively, "solvent-accessible positions" can be empirically identified and / or confirmed. For example, as described above, "solvent-accessible positions" theoretically identified using available in silico software tools such as MAESTRO can preferably be empirically confirmed by manufacturability. The formulation and process stability of potential building block candidates can help narrow down the likely candidates at an early stage before large-scale manufacturing (see the Examples and further, e.g., Ramachander, R., Rathore, N. (2013) "Molecule and manufacturability assessment leading to robust commercial formulation for therapeutic proteins" in: Kolhe, P., Shah, M., Rathore, N. (eds) Sterile Product Development, AAPS Advances in the Pharmaceutical Sciences Series, vol. 6. Springer, New York, NY). Therefore, after potential suitable solvent-accessible positions are theoretically identified in a protein-based building block precursor, the expression level, conjugation efficiency, formulation, quality control, solubility, process stability, etc. of the resulting protein-based carrier building block should preferably be evaluated. Solvent accessible locations that result in building blocks with superior expression yield, manufacturability, solubility and / or stability are preferred. See the Examples for further details.

[0157] For example, once suitable solvent-accessible positions have been theoretically identified in the building block precursor, protein expression of the selected mutants (i.e., the resulting protein-based building blocks with amino acids that have conjugation sites at the theoretically selected solvent-accessible positions) can be performed. This step allows for determining whether the introduction of specific amino acids at the theoretically identified solvent-accessible positions (e.g., point mutations, addition of amino acids at the N- and / or C-termini of the protein, etc.) adversely affects, for example, the synthesis, expression level, conjugation efficiency, or 3D globular structure of each specific mutant. Furthermore, as described in detail above, the minimum required solubility and lack of specific binding to human proteins (and optionally, non-protein molecules and / or non-human proteins, preferably the precursor target) can be evaluated. Possible changes in the 3D structure can be evaluated, for example, by CD (circular dichroism) spectroscopy, as described in detail above. Furthermore, the stability of the resulting mutants can also be confirmed by thermal shift assays. This assay can be used to detect the protein melting temperature (Tm) and thus check the stability of the protein. It can be used to characterize the stability / folding of a protein's 3D structure. SYPRO® Orange is a naturally quenched dye that interacts with the hydrophobic core of proteins, becoming visible after thermal denaturation. As a result, the temperature during the thermal denaturation process is expressed as the melting temperature, Tm. This is a way to assess the stability of the resulting mutants or variants.

[0158] Furthermore, a "model cargo" can be attached or conjugated to the selected mutant to quantify the degree of conjugation (conjugation efficiency), i.e., to confirm whether the resulting protein-based component with a conjugation site at a selected solvent-accessible position is actually suitable for binding or conjugating the desired cargo. A "model cargo" can be, for example, any molecule with a molecular weight higher than 100 Da. For example, if the potential conjugation site is a thiol group, the "model cargo" can be maleimide-modified alanine (e.g., N-maleoyl-β-alanine) or biotin-alanine, as described in Junutula, J. et al. ("Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index," Nat Biotechnol, 26, 925-932 (2008)). For example, if the conjugation of a "model cargo" results in a stable conjugate (a protein-based component to which one or more model cargoes are conjugated) with an acceptable degree of conjugation (which should be determined, depending on the individual case, for example, at a conjugation efficiency of ≧90%, e.g., 90% conjugation efficiency, or 95% conjugation efficiency, or 97% conjugation efficiency, or 99% conjugation efficiency or higher), thereby allowing standard PK in vivo, retaining its globular 3D structure and in vivo conjugation state, etc., then those solvent-accessible positions should be favorable for cargo conjugation, and conjugation of the desired cargo can be carried out (see also examples below).

[0159] point mutation In one embodiment, at least one conjugation site present in a building block can be generated by introducing a specific point mutation at a solvent-accessible position in the peptide sequence of the building block precursor. For example, a point mutation can be introduced at a solvent-accessible position in a building block precursor to generate a protein-based building block included in the molecules of the present technology that includes at least one conjugation site or attachment point at a defined solvent-accessible position, as described herein.

[0160] For example, conjugation sites can be generated by mutating specific amino acids, preferably at solvent-accessible positions of building block precursors, to cysteine ​​("Cys-mutations"). Alternatively or additionally, conjugation sites can be generated by mutating specific amino acids, preferably at solvent-accessible positions of precursors to natural or unnatural amino acids bearing reactive groups in their side chains. Amino acid distribution data of occurrence at specific positions (e.g., Cys, Ser) in building block precursors can also be used to guide the design and introduction of conjugation sites.

[0161] Additionally or alternatively, as described herein, the building block precursors may be modified by adding one or more amino acids to the N- and / or C-terminus of the protein sequence to introduce at least one conjugation site or attachment point, preferably at a solvent-accessible location, to generate the protein-based building blocks of the present technology.

[0162] In another embodiment, at least one conjugation site may already be present in a solvent-accessible position in the protein-based building block precursor, and does not need to be generated, such as a primary amine at the N-terminus of the building block, a -COOH at the C-terminus or in a side chain of the building block, for example a primary amine in the side chain of a lysine that is preferably already present in a solvent-accessible position in the building block precursor, or a thiol group in the side chain of a cysteine ​​that is preferably already present in a solvent-accessible position in the building block precursor.

[0163] Where a building block comprises two or more conjugation sites, these may be generated, for example, by introducing specific point mutations, preferably at solvent-accessible positions in the peptide sequence of the building block precursor. Additionally or alternatively, other suitable conjugation sites or attachment points may already exist at solvent-accessible positions in the building block precursor, i.e., it is not necessary to generate these conjugation sites, for example, by introducing specific point mutations and / or adding one or more amino acids to the N-terminus and / or C-terminus of the building block precursor. Those skilled in the art will determine the number and locations of attachment points or conjugation sites based on the protein-based building block and the cargo to be attached thereto, either directly or via a linker, as described herein.

[0164] As described in detail above, preferably, the point mutation is a non-destabilizing point mutation. The stability of the mutant can be calculated using different methods, for example, artificial intelligence (AI)-based methods that predict the effect of the mutation on protein stability. For example, the stability of the mutant can be calculated using MAESTRO, as defined above and described in detail in the Examples, or can be empirically confirmed by manufacturability (including, but not limited to, expression level and stability assessment, as described above).

[0165] In a preferred embodiment, the point mutation is a mutation of an amino acid, preferably located at a solvent-accessible position of the building block precursor, to cysteine. In another embodiment, the point mutation consists of substituting a serine residue, preferably located at a solvent-accessible position of the building block precursor, with cysteine. In another embodiment, the point mutation is a mutation of a solvent-accessible amino acid, preferably in the building block precursor, to lysine. In another embodiment, the point mutation is a mutation of a solvent-accessible amino acid, preferably in the building block precursor, to tyrosine. In another embodiment, the point mutation is a mutation of a solvent-accessible amino acid, preferably in the building block precursor, to a natural or unnatural amino acid, as described above.

[0166] Addition of C- or N-natural and / or unnatural amino acids with reactive groups in the side chain For example, conjugation sites can be generated in the building block precursor by adding one or more C- or N-terminal natural amino acids and / or one or more C- or N-terminal unnatural amino acids bearing reactive groups in their side chains. Preferably, if present, the one or more terminal natural or unnatural amino acids are added to the C-terminus of the building block precursor. For example, one or more of the conjugation sites are generated by adding an N- or C-terminal cysteine, an N- or C-terminal tyrosine, and / or an N- or C-terminal unnatural amino acid to the protein-based building block precursor. Preferably, at least one of the conjugation sites is generated by adding an N- or C-terminal tyrosine, preferably a C-terminal tyrosine, to the protein-based building block precursor. In a preferred embodiment, the N- and / or C-terminal Tyr is a flexible (GG) or ((G4S1)) amino acid, as described in detail in Alan M. Marmelstein et al., Journal of the American Chemical Society, 2020, 142(11), 5078-5086. 1~3 GG) sequences (e.g., -GGY, -(G4S1) 1~3 GGY, YGG-, Y(G4S1) 1~3 GG-, YGG(S1G4) 1~3 - or YGG(G4S1) 1~3 -) is in front / back.

[0167] Thus, at least one protein-based carrier component included in the molecules of the present technology is -GGY or -(G4S1) 1~3 Like the GGY tag (sequence), it may contain N- and / or C-terminal Cys, Tyr and / or unnatural amino acids, for example C-terminal Tyr.

[0168] Additionally, at least one protein-based carrier component of the present technology may comprise an N-terminal and / or C-terminal conjugation site or attachment point suitable for conjugation with a sortase, as described above. In such cases, the protein-based carrier component may comprise a C-terminal sortase recognition motif (LPXTG, where X can be any amino acid), an N-terminal polyglycoside (Gly), or a C-terminal polyglycoside (Gly). 1~5 Additionally, if N-N and / or C-C attachment or conjugation is required, the protein-based carrier component should be engineered to contain a C-terminal sortase recognition motif (for C-C attachment) or an N-terminal polyglycoside (Gly) tag, as described in detail above. 1~5 ) tag (in the case of NN binding). See, in particular, Guimaraes CP et al., Theile CS et al., and Witte MD et al., listed above.

[0169] Finally, as noted above, conjugation sites may be generated by a combination of the above mechanisms, for example, at least one conjugation site may be generated by a point mutation (e.g., Ser-Cys at a solvent accessible position of the component, as described above), or by the addition of a cysteine ​​or tyrosine or an unnatural amino acid or a sortase recognition motif or polyglycol (Gly) as described above. 1~5 ) tags can be added to protein-based building block precursors.

[0170] Example of a component Small, globular, non-human protein-based constructs The protein-based carrier component of the present technology may be based on a small, globular, non-human protein. In the context of the present technology, a "small, globular, non-human protein" refers to a non-human protein having a size (molecular weight) of about 2.5 to about 70 kDa, preferably about 2.5 to about 50 kDa, e.g., about 2.5 to less than 50 kDa, more preferably about 2.5 to about 30 kDa, and even more preferably about 2.5 to about 16 kDa, as described herein, and having a globular, three-dimensional (3D) structure, as described herein. Furthermore, at least one non-human protein-based carrier component does not specifically bind to a human protein, as defined herein; preferably, it does not specifically bind to any non-protein molecule (such as a nucleic acid (e.g., DNA, RNA), glycan, lipid (e.g., phosphatidylserine (PS)), etc.), such as any human non-protein molecule (biomolecule) (human DNA, human RNA, human glycan, human lipid (e.g., phosphatidylserine (PS)), etc.), preferably, it does not specifically bind to any non-protein molecule (e.g., a nucleic acid (DNA, RNA), glycan, lipid (e.g., phosphatidylserine (PS)), etc.) to which a component precursor, if present, specifically binds; preferably, it does not specifically bind to any non-human protein (e.g., a bacterial and / or viral protein) to which a component precursor, if present, specifically binds. Furthermore, preferably, at least one non-human protein-based carrier component (i) does not specifically bind to any human cell and / or cell type, or, preferably, binds to less than 5×10 human cells and / or cell types, as determined by a cell binding assay. -4 moles / liter K D (K D (ii) bind to human cells and / or cell types with a specific binding activity of 5×10 or less as determined by cell binding assays and / or SPR, as described herein, and (iii) do not specifically bind to bacteria, fungi, protozoa, yeast, and / or any virus, or preferably bind to human cells and / or cell types with a specific binding activity of 5×10 or less as determined by cell binding assays and / or SPR, as described herein. -4 moles / liter K D (K Dand / or (iii) does not specifically bind to any biomolecules, including human biomolecules and non-human biomolecules, such as plant biomolecules, viral biomolecules, and / or microbial biomolecules (such as bacteria, fungi, protists, and / or yeast), at a binding affinity of 5×10 or less, as determined preferably by cell binding assays and / or SPR, as described herein. -4 moles / liter K D (K D value) and bind to biomolecules, including human and non-human biomolecules.

[0171] In a preferred embodiment, the protein-based carrier component does not specifically bind to any non-human protein and / or non-protein molecule, preferably a precursor target, when a cargo is conjugated to at least one attachment point or conjugation site on the protein-based carrier component, as described above. Thus, in a preferred embodiment, the molecule of the present technology comprises at least one protein-based component and at least one cargo attached to the at least one protein-based component via at least one conjugation site or attachment point, and does not specifically bind to any non-human protein or non-protein molecule, such as any human non-protein molecule, as described herein, and in particular does not specifically bind to any protein or non-protein molecule to which the component precursor binds, if present.

[0172] As mentioned above, in the context of the present technology, if the protein-based components or molecules of the present technology exhibit any interaction with one or more human proteins (or non-human proteins or non-protein molecules, as described above), such interaction is characterized by low specificity and / or low affinity, as explained in detail above.

[0173] Human proteins are proteins present in the human body. The Human Protein Atlas (HPA, https: / / www.proteinatlas.org) is a Swedish program launched in 2003 with the aim of mapping all human proteins in cells, tissues, and organs.

[0174] Small globular non-human proteins relevant to the present technology include proteins that are derived from human proteins but have been modified so that they are no longer human proteins. An example of a small globular non-human protein is an ISVD, such as a "human ISVD" (e.g., V H , V L ) and "non-human ISVDs" (e.g., V HH , non-human V H , V L or engineered ISVD), DARPin (derived from ankyrin repeat protein), affibody or affitin.

[0175] Small globular non-human proteins may have therapeutic or targeting activity.

[0176] Immunoglobulin Single Variable Domain (ISVD)-Based Constructs In one embodiment, at least one protein-based carrier component of the present technology is V H or V HH The present invention is based on a polypeptide comprising, or alternatively consisting of, at least one immunoglobulin single variable domain (ISVD), such as an ISVD (heavy chain ISVD) derived from

[0177] As described above, the protein-based carrier components of the present technology have a spherical 3D structure, are soluble, and have a size (molecular weight) of about 2.5 to about 70 kDa, for example, about 2.5 to about 50 kDa, for example, about 2.5 to less than about 50 kDa, more preferably about 2.5 to about 30 kDa, for example, about 2.5 to about 16 kDa, for example, about 5 to about 16 kDa, or about 7 to about 16 kDa, or about 10 to about 16 kDa. Furthermore, at least one component included in the molecule of the present technology does not specifically bind to a human protein, as defined herein, and preferably it does not specifically bind to any non-protein molecule (such as human DNA, human RNA, glycans, lipids (e.g., phosphatidylserine (PS)), etc.), for example, any human non-protein molecule (such as human DNA, human RNA, human glycans, human lipids (e.g., phosphatidylserine (PS)), etc.), preferably it does not specifically bind to any non-protein molecule (such as DNA, glycans, lipids (e.g., phosphatidylserine (PS)), etc.) to which the component precursor, if present, specifically binds, and preferably it does not specifically bind to any non-human protein (e.g., bacterial and / or viral protein) to which the component precursor, if present, specifically binds.

[0178] In a preferred embodiment, the protein-based carrier component does not specifically bind to any non-human protein and / or non-protein molecule, preferably a precursor target, when a cargo is conjugated to at least one attachment point or conjugation site on the protein-based carrier component, as described above. Thus, in a preferred embodiment, the molecule of the present technology comprises at least one protein-based component and at least one cargo attached to the at least one protein-based component via at least one conjugation site or attachment point, and does not specifically bind to any non-human protein or non-protein molecule, such as any human non-protein molecule, as described herein, and in particular does not specifically bind to any protein or non-protein molecule to which the component precursor binds, if present.

[0179] As mentioned above, in the context of the present technology, if the protein-based components or molecules of the present technology exhibit any interaction with one or more human proteins (or non-human proteins or non-protein molecules, as described above), such interaction is characterized by low specificity and / or low affinity, as explained in detail above.

[0180] Thus, in certain embodiments where at least one protein-based component is based on an ISVD, preferably a heavy chain ISVD, the resulting ISVD-based component does not specifically bind to any human protein. Moreover, as explained above, it is preferred that the ISVD-based component does not specifically bind to any non-protein molecule, such as any human non-protein molecule. Furthermore, it is also preferred that the ISVD-based component does not specifically bind to any non-human protein or non-protein molecule to which the protein-based carrier component precursor, if present, specifically binds, as described above.

[0181] In the context of the present technology, an "ISVD-based component" refers to a protein-based component that is derived from an ISVD, i.e., structurally similar to an ISVD, but does not specifically bind to any human protein, and preferably does not specifically bind to any target to which an ISVD specifically binds. For example, an ISVD-based component has at least 60%, or 70%, or 80% sequence identity with an ISVD, such as its ISVD precursor. For example, an ISVD-based component has at least 85%, such as at least 90%, for example at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99% or more sequence identity with an ISVD, such as its ISVD precursor. For example, an ISVD-based component can share an entire amino acid sequence with its ISVD precursor, except for at least one amino acid, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 20, 25, 30, or more. Furthermore, the ISVD-based component has a globular 3D structure, is soluble, has a size (molecular weight) of about 2.5 to about 70 kDa, e.g., about 2.5 to about 50 kDa or about 2.5 to about less than 50 kDa, more preferably about 2.5 to about 30 kDa, e.g., about 2.5 to about 16 kDa, e.g., about 5 to about 16 kDa, or about 7 to about 16 kDa, or about 10 to about 16 kDa, and does not specifically bind to any human protein, preferably any protein or non-protein molecule to which the precursor specifically binds.

[0182] Those skilled in the art will recognize means to eliminate specific binding properties of a particular ISVD precursor, for example, by making mutations in amino acids involved in binding of the ISVD to the target (e.g., in one or more of the amino acids that fit into the CDRs of the ISVD), adding amino acids, and / or deleting amino acids from the precursor sequence.

[0183] The term "immunoglobulin single variable domain" (ISVD), used interchangeably with "single variable domain," defines an immunoglobulin molecule in which the antigen-binding site is present on and formed by a single immunoglobulin domain. This distinguishes ISVDs from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (e.g., Fab, Fab', F(ab')2, scFv, di-scFv), in which two immunoglobulin domains, in particular two variable domains, interact to form the antigen-binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (V H ) and the light chain variable domain (V L ) interact to form the antigen-binding site. In this case, V H and V L Both complementarity-determining regions (CDRs) of the nucleotides contribute to the antigen-binding site, i.e., a total of six CDRs are involved in forming the antigen-binding site.

[0184] In view of the above definition, fragments of conventional four-chain antibodies (e.g., IgG, IgM, IgA, IgD or IgE molecules known in the art), or Fv fragments such as Fab fragments, F(ab')2 fragments, disulfide-linked Fv or scFv fragments or diabodies derived from such conventional four-chain antibodies (all known in the art) are not generally considered to be ISVDs, as in these cases binding to each epitope of an antigen is typically not by one (single) immunoglobulin domain, but by a pair of (related) immunoglobulin domains, such as light and heavy chain variable domains, i.e., the V of immunoglobulin domains which jointly bind to the respective epitopes of the antigen. H -V L This is because it occurs in pairs.

[0185] In contrast, ISVDs are generally capable of specifically binding to an epitope of an antigen without pairing with an additional immunoglobulin variable domain. The binding site of an ISVD consists of a single V H , a single V HH or a single V L Formed by domains.

[0186] In the context of the present technology, in certain embodiments where at least one protein-based building block is based on an ISVD, the ISVD building block precursor comprises a light chain variable domain sequence (e.g., a V L sequence) or a suitable fragment thereof or a heavy chain variable domain sequence (e.g., V H Array or V HH The ISVD may be a protein-based building block (e.g., a nucleotide sequence) or a suitable fragment thereof, as long as the resulting building block has a globular 3D structure, has a size (molecular weight) of about 2.5 to about 70 kDa, for example, about 2.5 to about 50 kDa or about 2.5 to about less than 50 kDa, more preferably about 2.5 to about 30 kDa, for example, about 2.5 to about 16 kDa, for example, about 5 to about 16 kDa, or about 7 to about 16 kDa, or about 10 to about 16 kDa, and is soluble as defined above in detail. An ISVD that may be a precursor of the protein-based building block contained in the molecule of the present technology is, for example, camelized V. H or humanized V HH heavy chain ISVDs, e.g., V H , V HH In one embodiment, the protein-based building block precursor may be a camelized V protein, as long as the resulting protein-based building block is soluble. H or humanized V HH V containing HH The heavy chain ISVD has a spherical 3D structure and a size (molecular weight) of about 2.5 to about 70 kDa, e.g., about 2.5 to about 50 kDa or about 2.5 to less than 50 kDa, more preferably about 2.5 to about 30 kDa, e.g., about 2.5 to about 16 kDa, e.g., about 5 to about 16 kDa, about 7 to about 16 kDa, or about 10 to about 16 kDa, and does not specifically bind to human proteins. Furthermore, preferably, the resulting building block does not specifically bind to any non-protein molecules, such as DNA, RNA, lipids (e.g., phosphatidylserine (PS)), or glycans, e.g., glycolipids. Furthermore, preferably, the resulting building block does not specifically bind to any non-human proteins to which the protein-based carrier building block precursor, if present, specifically binds, as described above. The heavy chain ISVD can be derived from a conventional four-chain antibody or a heavy chain antibody.

[0187] For example, an ISVD precursor may be a single domain antibody (or an amino acid sequence suitable for use as a single domain antibody), a "dAb" or dAb (or an amino acid sequence suitable for use as a dAb), or a Nanobody® ISVD (as defined herein, HH and the other single variable domain or any suitable fragment of any one thereof, as long as the resulting protein-based component is soluble, has a globular 3D structure, does not specifically bind to human proteins, and preferably does not specifically bind to any non-protein (human) molecules, such as DNA, RNA, lipids (such as phosphatidylserine (PS)) or glycans, such as glycolipids, and preferably does not specifically bind to any non-human proteins to which the protein-based carrier component precursor specifically binds, as described above.

[0188] Preferably, the ISVD precursor is V H , humanized V H , Human V H , V HH , humanized V HH or Camelization V H More preferably, the ISVD precursor is a Nanobody® ISVD (humanized VSVD), insofar as the protein-based component is soluble, has a globular 3D structure, and specifically binds to human proteins. HH or Camelization V H V containing HH ) or a suitable fragment thereof, which preferably does not specifically bind to any non-protein (human) molecule such as DNA, RNA, lipids (such as phosphatidylserine (PS)) or glycans, e.g. glycolipids, and preferably does not specifically bind to any non-human protein to which the protein-based carrier component precursor specifically binds when present as described above. Nanobody® is a registered trademark of Ablynx NV.

[0189] "V HH Domain" is V HH , V HH Antibody fragments and VHH Also known as antibodies, they were originally described as the antigen-binding immunoglobulin variable domains of "heavy chain antibodies" (i.e., "antibodies without light chains"; see Hamers-Casterman et al., Nature 363:446-448, 1993). HH The term "variable domain" refers to these variable domains, as compared to the heavy chain variable domains (herein referred to as "V" domains) present in conventional four-chain antibodies. H domain) present in conventional four-chain antibodies and the light chain variable domain (referred to herein as "V L The domain name was chosen to distinguish it from the domains referred to as "domains." HH For further description, see the review article by Muyldermans ("Single domain camel antibodies: current status", J Biotechnol., 2001, 74:277-302). VHH domains can be obtained from heavy chain-only antibodies (HCAbs) circulating in Camelidae (see, for example, Muyldermans S., "A guide to: generation and design of nanobodies", FEBS J., 2021, 288(7):2084-2102). Thus, in a preferred embodiment, the ISVD-based building blocks are V HH (Humanized V HH or Camelization V H etc.), for example, that V HH It has at least 80% sequence identity with the precursor. For example, an ISVD-based component is V HH , for example, that V HHFor example, the ISVD-based component has at least 60%, or at least 70%, or 80%, or at least 85%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, such as at least 99% or more sequence identity with the precursor. For example, the ISVD-based component has at least one, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 20, 25, 30 or more amino acids in its V that differ from the protein-based carrier component. HH It may share the complete amino acid sequence with the precursor.

[0190] Typically, producing immunoglobulins involves immunizing laboratory animals, fusing immunoglobulin-producing cells to create hybridomas, and screening for the desired specificity. Alternatively, immunoglobulins can be made by screening naive, immune, or synthetic libraries, e.g., phage display.

[0191] V HHs The generation of immunoglobulin sequences such as these has been widely described in various published documents, among which WO 94 / 04678, Hamers-Casterman et al. 1993 ("Naturally occurring antibodies devoid of light chains", Nature, 363:446-448, 1993) and Muyldermans et al. 2001 ("Single domain camel antibodies: current status", J. Biotechnol., 2001, 74:277-302). In these methods, camelids are immunized with a target antigen to induce an immune response against said target antigen. The V obtained from said immunization is HH The repertoire of V HH are screened for binding (or not) to the target antigen.

[0192] Immunoglobulin sequences of different origins may be used in the context of the present technology, including mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences, including fully human, humanized or chimeric sequences. Also included in the context of this technology are camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences or camelized domain antibodies, such as camelized dAbs as described by Ward et al. (Nature, 341:544, 1989) (see, for example, WO 94 / 04678 and Davies and Riechmann, "'Camelizing' human antibody fragments: NMR studies on VH domains", Febs Lett., 339:285-290, 1994 and "Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability", Prot. Eng., 1996, 9(6):531-537).

[0193] "Humanized V HH " is a naturally occurring V HH corresponding to the amino acid sequence of the naturally occurring V HH One or more amino acid residues in the amino acid sequence (and particularly in the framework sequences) of the V H The term "humanized" includes amino acid sequences that have been "humanized" by substituting one or more amino acid residues (e.g., as shown above) present at the corresponding positions in the domain. This can be done in a manner known per se and will be clear to those skilled in the art, for example, based on the further explanations herein and the prior art (e.g., WO 2008 / 020079). Again, such humanized V HHcan be obtained by any suitable method known per se and therefore is not a naturally occurring V HH It should be noted that the present invention is not strictly limited to polypeptides obtained using a polypeptide containing a V domain as a starting material. HH If V HH is a humanized V HH is.

[0194] "Camelization V H " is a naturally occurring V H The naturally occurring V domain corresponds to the amino acid sequence of the V domain but is "camelized", i.e., derived from a conventional four-chain antibody. H One or more amino acid residues in the amino acid sequence of the V domain of a heavy chain antibody HH The term "camelized" includes amino acid sequences that have been "camelized" by substituting one or more amino acid residues occurring at the corresponding positions in the V domain. This can be done in a manner known per se and will be clear to the skilled artisan, for example, based on the further explanations herein and the prior art (e.g., WO 2008 / 020079). Such "camelized" substitutions are usually made at the V domain, as defined herein. H -V L The amino acids are inserted at positions that form and / or are present at interfaces and / or so-called camelid hallmark residues (see, e.g., WO 94 / 04678 and Davies and Riechmann, 1994 and 1996, supra). H V, which is used as a starting material or starting point for generating or designing H The sequence is V from mammals H sequence or human-derived V H Array, e.g. V H 3 sequence. However, such camelized V H can be obtained in any suitable manner known per se, and thus can be used without the naturally occurring V as starting material. H It should be noted that the present invention is not strictly limited to polypeptides obtained using polypeptides containing the domain.

[0195] The structure of an ISVD sequence can be considered to consist of four framework regions ("FRs"), which are referred to in the art and herein as "framework region 1" ("FR1"), "framework region 2" ("FR2"), "framework region 3" ("FR3"), and "framework region 4" ("FR4"), respectively; these framework regions are interrupted by three complementarity-determining regions ("CDRs"), which are referred to in the art and herein as "complementarity-determining region 1" ("CDR1"), "complementarity-determining region 2" ("CDR2"), and "complementarity-determining region 3" ("CDR3"), respectively.

[0196] As further described in paragraph q) on pages 58 and 59 of WO 2008 / 020079, the amino acid residues of the ISVD are determined by the V sequence assigned by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH, Bethesda, MD, Publication No. 91). H Following the common numbering of domains, V from camelids in the article by Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240(1-2); 185-195; see e.g., Figure 2 of this publication) HH The numbering is also applied to the domain. H Domain and V HHIt should be noted that, as is well known in the art for domains, the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering. That is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering. This generally means that the Kabat numbering may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. H Domain and V HH The total number of amino acid residues in a domain will typically be in the range of 110 to 120, often 112 to 115. However, it should be noted that shorter and longer sequences may also be suitable for the purposes described herein.

[0197] In the present application, the CDR sequences were determined according to the Kabat numbering system with AbM CDR annotations as described in Kontermann and Duebel (Eds. 2010, Antibody Engineering, Vol. 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51). According to this system, FR1 contains amino acid residues 1-25, CDR1 contains amino acid residues 26-35, FR2 contains amino acid residues 36-49, CDR2 contains amino acid residues 50-58, FR3 contains amino acid residues 59-94, CDR3 contains amino acid residues 95-102, and FR4 contains amino acid residues 103-113.

[0198] The CDR regions can be determined according to different methods: In the CDR determination according to Kabat, FR1 of the ISVD comprises amino acid residues 1 to 30, CDR1 of the ISVD comprises amino acid residues 31 to 35, FR2 of the ISVD comprises amino acid residues 36 to 49, CDR2 of the ISVD comprises amino acid residues 50 to 65, FR3 of the ISVD comprises amino acid residues 66 to 94, CDR3 of the ISVD comprises amino acid residues 95 to 102, and FR4 of the ISVD comprises amino acid residues 103 to 113.

[0199] In such immunoglobulin sequences, the framework regions may be any suitable framework sequence, and examples of suitable framework sequences will be clear to the skilled person based on, for example, standard handbooks and the further disclosure and prior art referred to herein.

[0200] The framework sequences are suitable combinations of immunoglobulin framework sequences or framework sequences derived from immunoglobulin framework sequences, for example, by humanization or camelization. For example, the framework sequences may be those of the light chain variable domain (e.g., V L sequence) and / or heavy chain variable domain (e.g., V H Array or V HH In one aspect, the framework sequences may be derived from V HH -framework sequences derived from conventional V sequences (in which said framework sequences may optionally be partially or fully humanized), or camelized (as defined herein) H It can be either an array.

[0201] In particular, the framework sequences present in the ISVD sequences referred to in the present technology may be used in conjunction with the ISVD sequences, e.g., humanized V HH or Camelization V H V containing HHand the like. Some non-limiting examples of suitable combinations of such framework sequences will be apparent from the disclosure herein.

[0202] However, it should be noted that in the context of the present technology, there is no limitation as to the origin of the ISVD sequence or the origin of the nucleotide sequence used to express it, nor as to the manner in which the ISVD sequence or nucleotide sequence is produced or obtained, or produced or obtained. Thus, the ISVD sequence may be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In certain, but non-limiting, aspects, the ISVD sequence may be a "humanized" immunoglobulin sequence (as defined herein) (e.g., a partially or fully humanized mouse or rabbit immunoglobulin sequence, particularly a partially or fully humanized V HH immunoglobulin sequences), "camelized" immunoglobulin sequences (as defined herein), as well as immunoglobulin sequences obtained by affinity maturation (e.g., starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments from different immunoglobulin sequences, techniques such as PCR assembly using overlapping primers and similar techniques for engineering immunoglobulin sequences that are well known to those skilled in the art, or any suitable combination of any of the foregoing.

[0203] Likewise, the nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, and may be, for example, a sequence isolated by PCR from a suitable naturally occurring template, for example DNA or RNA isolated from a cell, a nucleotide sequence isolated from a library (and in particular an expression library), a nucleotide sequence prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), a nucleotide sequence prepared by PCR using overlapping primers or a nucleotide sequence prepared using techniques for DNA synthesis known per se.

[0204] As mentioned above, the ISVD precursor is preferably a humanized V HH Or Camelization V H V containing HH or a suitable fragment thereof, more preferably a humanized V HH or a suitable fragment thereof. The resulting protein-based entity should be soluble, have a globular 3D structure, not specifically bind to human proteins, and preferably not specifically bind to any non-protein molecules, and preferably is a V-type molecule as described above. HH It should not specifically bind to any non-human protein to which the precursor specifically binds. Preferably, as described above, the molecule contains at least one V HH (Humanized V HH or Camelization V H and at least one cargo attached thereto via at least one conjugation site or attachment point, and does not specifically bind to any non-protein molecule, and / or HH (Humanized V HH or Camelization V H It does not specifically bind to any non-human protein to which the precursor (including

[0205] Furthermore, preferably, at least one ISVD-based carrier component (i) does not specifically bind to any human cells and / or cell types, or preferably binds to less than 5×10 cells / cells as determined by cell binding assays. -4 moles / liter K D (K D (ii) bind to human cells and / or cell types with a specific binding activity of 5×10 or less as determined by cell binding assays and / or SPR, as described herein, and (iii) do not specifically bind to bacteria, fungi, protozoa, yeast, and / or any virus, or preferably bind to human cells and / or cell types with a specific binding activity of 5×10 or less as determined by cell binding assays and / or SPR, as described herein. -4 moles / liter K D (K D and / or (iii) does not specifically bind to any biomolecules, including human biomolecules and non-human biomolecules, such as plant biomolecules, viral biomolecules, microbial biomolecules (such as bacteria, fungi, protists, and / or yeast), at a binding affinity of 5×10 or less, as determined preferably by cell binding assays and / or SPR, as described herein. -4 moles / liter K D (K D value) and bind to biomolecules, including human and non-human biomolecules.

[0206] As mentioned above, the ISVD precursor is preferably a VHH, humanized VHH or camelized VH, such as a Nanobody® ISVD or a suitable fragment thereof, more preferably a humanized Nanobody® ISVD or a suitable fragment thereof. The resulting protein-based entity should be soluble, have a globular 3D structure and not specifically bind to human proteins, and preferably not specifically bind to any non-protein molecule, and preferably not specifically bind to any non-human protein to which the VHH, humanized VHH or camelized VH specifically binds when present as a precursor, such as a Nanobody® ISVD, as described above. Preferably, as mentioned above, a molecule comprising at least one VHH, humanized VHH or camelized VH, such as a protein-based component derived from a Nanobody® ISVD, and at least one cargo attached thereto via at least one conjugation site or attachment point, does not specifically bind to any non-protein molecule, and / or the VHH, humanized VHH or camelized VH, e.g., a Nanobody® ISVD precursor, specifically binds. For a general description of Nanobody® ISVDs, reference is made to this specification and the prior art cited therein. However, in this respect, this description and the prior art do not specifically bind to so-called "VHHs" or "VHs." H 3 Class”, i.e. V H It should be noted that the present disclosure primarily describes Nanobody® ISVDs of three classes of human germline sequences (e.g., Nanobody® ISVDs with a high degree of sequence homology to DP-47, DP-51, or DP-29). However, the present technology, in its broadest sense, can generally be used with any type of Nanobody® ISVD, including, for example, so-called "V" ISVDs, as described, for example, in WO 2007 / 118670. H 4 class" i.e. V such as DP-78 H It should be noted that we also use Nanobody® ISVDs that belong to four classes of Nanobody® ISVDs with a high degree of sequence homology to human germline sequences.

[0207] In one embodiment, at least one protein-based carrier component included in the molecule of the present technology is a so-called "V H Nanobody® ISVDs belonging to the "3 class" of nanobodies, i.e., V such as DP-47, DP-51, or DP-29. H Derived from Nanobody® ISVDs with a high degree of sequence homology to three classes of human germline sequences, the protein-based components are soluble, have a globular 3D structure, and preferably do not specifically bind to any non-protein human molecules unless they specifically bind to human proteins, and preferably do not specifically bind to any non-human proteins to which the ISVD precursors specifically bind as described above.

[0208] Generally, Nanobody® ISVDs (especially (partially) humanized V HH Sequence and camelized V H V containing arrays HH A Nanobody® ISVD may be characterized by the presence of one or more "hallmark residues" (also as further described herein) in one or more of the framework sequences (as described herein). Generally, a Nanobody® ISVD has the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 wherein FR1-FR4 refer to framework regions 1-4, respectively, and CDR1-CDR3 refer to complementarity determining regions 1-3, respectively, and one or more of the hallmark residues are as further defined herein.

[0209] Specifically, Nanobody® ISVD has the following (generic) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 where FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and the framework sequences are as further defined herein.

[0210] More specifically, Nanobody® ISVD has the following (generic) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and wherein one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, 108 according to the Kabat numbering are selected from the hallmark residues set forth in Table 3 below.

[0211] [Table 3]

[0212] [Table 4]

[0213] Thus, Nanobody® ISVD has the following (generic) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively, and wherein one or more amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 according to the Kabat numbering are selected from the hallmark residues referred to in Table 3.

[0214] For example, if the protein-based component of the present technology is based on an ISVD, it may be derived from an antiviral ISVD, such as an antiviral VHH or Nanobody® ISVD. For example, the component of the present technology may be derived from a functional ISVD (i.e., an ISVD that specifically binds to a human protein and / or a non-human protein, such as a viral protein and / or a bacterial protein and / or a non-protein molecule, such as a human non-protein molecule), as described in detail above, that has been engineered / modified so that it no longer specifically binds to any human protein, preferably so that it no longer specifically binds to any non-human protein, e.g., bacterial and / or viral protein, to which it was originally bound, and / or preferably so that it no longer specifically binds to any non-protein molecule, if present, to which it was originally bound. In a further preferred embodiment, the ISVD-based component of the present technology is derived from an ISVD, such as a heavy chain ISVD, preferably a Nanobody® ISVD, that has been further engineered / modified to include mutations that prevent / eliminate binding by existing antibodies / factors. Examples of such mutations are described, for example, in WO 2012 / 175741 and WO 2015 / 173325. For example, to prevent / eliminate binding by existing antibodies / factors, the amino acid at position 11 (according to Kabat) may be Val or Leu, preferably Val, and / or the amino acid at position 89 (according to Kabat) may be preferably Val, Thr or Leu, preferably Leu, and / or the amino acid at position 110 (according to Kabat) may be preferably Thr, Lys or Gln, preferably Thr, and / or the amino acid at position 112 (according to Kabat) may be Ser, Lys or Gln, preferably Ser, and / or the ISVD-based building block may comprise a C-terminal extension of 1 to 5 amino acids selected from any naturally occurring amino acid.

[0215] The resulting ISVD-based building blocks can be derived from variants from anti-hRSV ISVDs, such as those shown in Table A-2 of WO 2018 / 099968, beginning on page 69. In a preferred embodiment, the resulting ISVD-based building block is derived from a variant from ISVD RSV001A04 herein, SEQ ID NO: 179, also referred to as RSV001A04, and detailed in Table A-1 of WO 2010 / 139808, page 388, SEQ ID NO: 5 (referred to herein as NC41). In this particular embodiment, the protein-based carrier building block derived from RSV001A04 does not specifically bind to any human protein. In this embodiment, the "building block precursor" (or "ISVD precursor") is derived from RSV001A04, SEQ ID NO: 179: EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSS is.

[0216] Once an ISVD is selected as the starting point (see above as an "ISVD precursor"), residues preferably located in solvent-accessible positions should be identified to generate at least one conjugation site, as detailed herein above. Additionally or alternatively, conjugation sites may already be present in the ISVD precursor, preferably as reactive groups in the side chains of amino acids located in solvent-accessible positions, or as free N-terminal primary amines and / or free C-terminal carboxylic acids.

[0217] For example, one or more of the identified residues, preferably located at solvent accessible positions in the amino acid sequence of the ISVD precursor, are substituted with cysteine, lysine, tyrosine and / or a non-natural amino acid.

[0218] In one embodiment, at least one protein-based component included in the molecule of the present technology is an ISVD, e.g., a so-called "V H In another embodiment, at least one protein-based component included in the molecule of the present technology is derived from an ISVD, such as a so-called "V 3 class" ISVD, and the resulting component preferably comprises at least one cysteine, at least one lysine, at least one unnatural amino acid, and / or at least one tyrosine located in one or more solvent-accessible positions. H The resulting components derived from ISVDs belonging to the "Class 3" preferably contain at least one engineered cysteine, at least one engineered lysine, at least one unnatural amino acid, and / or at least one engineered tyrosine located in one or more solvent accessible positions.

[0219] Preferably, the molecular components of the present technology, as described above, when derived from an ISVD, are preferably V HH (Humanized V HH or Camelization V H or, if derived from the Nanobody® ISVD, comprises a leucine at position 108 (according to Kabat numbering). In other embodiments, the molecular components of the present technology, when derived from the ISVD as described above, comprise a valine at position 11, a leucine at position 89, and / or a leucine at position 108 (according to Kabat numbering).

[0220] In one embodiment, at least one protein-based carrier component present in the molecule of the present technology is SEQ ID NO: 186: [ka] (In the formula, X1 (position 1 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, such as cysteine; X2 (position 3 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, for example, cysteine; X3 (position 5 according to Kabat numbering) can be Val or any amino acid having a reactive group in its side chain, for example cysteine; X4 (position 7 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X5 (position 8 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X6 (position 10 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, e.g., cysteine; X7 (position 11 according to Kabat numbering) can be Leu, Val, Ser, Met, Trp, Phe, Thr, Gln, Glu, Ala, Arg, Gly, Lys, Tyr, Asn, Pro or Ile, preferably Leu or Val or any amino acid having a reactive group in its side chain, for example cysteine; X8 (position 12 according to Kabat numbering) can be Val or any amino acid having a reactive group in its side chain, for example cysteine; X9 (position 13 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, for example cysteine; X 10 (position 14 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, for example cysteine; X 11 (position 15 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, e.g., cysteine; X 12 (position 17 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 13(position 18 according to Kabat numbering) can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X 14 (position 19 by Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 15 (position 21 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 16 (position 23 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, for example cysteine; X 17 (position 25 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 18 (position 26 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, e.g., cysteine; X 19 (position 27 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, e.g., cysteine; X 20 (position 28 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 21 (position 30 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 22 (position 31 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X 23(position 32 by Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, e.g., cysteine; X 24 (position 39 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, for example cysteine; X 25 (position 41 by Kabat numbering) can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X 26 (position 42 by Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, e.g., cysteine; X 27 (position 43 according to Kabat numbering) can be Lys or any amino acid having a reactive group in its side chain, e.g., cysteine; X 28 (position 44 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, for example cysteine; X 29 (position 45 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X 30 (position 46 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, for example cysteine; X 31 (position 52a according to Kabat numbering) can be Trp or any amino acid having a reactive group in its side chain, e.g., cysteine; X 32 (position 53 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X 33(position 54 by Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, e.g., cysteine; X 34 (position 55 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X 35 (position 56 according to Kabat numbering) can be He or any amino acid having a reactive group in its side chain, for example, cysteine; X 36 (position 57 by Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, e.g., cysteine; X 37 (position 58 according to Kabat numbering) can be He or any amino acid having a reactive group in its side chain, for example, cysteine; X 38 (position 59 by Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, e.g., cysteine; X 39 (position 61 according to Kabat numbering) can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X 40 (position 62 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example cysteine; X 41 (position 64 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, for example cysteine; X 42 (position 65 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, e.g., cysteine; X 43(position 66 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, for example cysteine; X 44 (position 68 by Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, e.g., cysteine; X 45 (position 70 by Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 46 (position 71 by Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X 47 (position 72 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example cysteine; X 48 (position 73 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X 49 (position 74 by Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, for example, cysteine; X 50 (position 75 according to Kabat numbering) can be Lys or any amino acid having a reactive group in its side chain, e.g., cysteine; X 51 (position 76 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example cysteine; X 52 (position 79 by Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, e.g., cysteine; X 53(position 81 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, for example, cysteine; X 54 (position 82a according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X 55 (position 82b according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 56 (position 83 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, for example, cysteine; X 57 (position 84 by Kabat numbering) can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X 58 (position 85 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example cysteine; X 59 (position 87 by Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, e.g., cysteine; X 60 (position 89 according to Kabat numbering) can be Leu, Val, Ser, Met, Trp, Phe, Thr, Gln, Glu, Ala, Arg, Gly, Lys, Tyr, Asn, Pro or He, preferably Leu, Val, Ser or Glu, more preferably Leu or Val or any other amino acid having a reactive group in its side chain, for example cysteine; X 61 (position 91 by Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, e.g., cysteine; X 62(position 96 by Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, e.g., cysteine; X 63 (position 98 by Kabat numbering) can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X 64 (position 99 by Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X 65 (position 100 according to Kabat numbering) can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X 66 (position 100a according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X 67 (position 100d according to Kabat numbering) can be He or any amino acid having a reactive group in its side chain, for example, cysteine; X 68 (position 100e according to Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, for example, cysteine; X 69 (position 100f according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example cysteine; X 70 (position 100g according to Kabat numbering) can be Trp or any amino acid having a reactive group in its side chain, such as cysteine, X 71 (position 101 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X 72(position 102 by Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, e.g., cysteine; X 73 (position 103 by Kabat numbering) can be Trp or any amino acid having a reactive group in its side chain, e.g., cysteine; X 74 (position 105 by Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X 75 (position 106 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, e.g., cysteine; X 76 (position 108 according to Kabat numbering) can be Gln, Leu, Arg, Pro, Glu, Lys, Ser, Thr, Met, Ala or His, preferably Gln or Leu or any other amino acid having a reactive group in its side chain, for example cysteine; X 77 (position 110 by Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, e.g., cysteine; X 78 (position 112 by Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 79 (position 113 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 80 is absent or is Gly, X 81 is absent or is Gly, X 82 is absent or is Cys) or comprising, or alternatively consisting of, a sequence having 80% or more identity to SEQ ID NO: 186, preferably a sequence having 85% or more, 90% or more, 95% or more, 97% or more or 99% or more sequence identity to SEQ ID NO: 186, with the proviso that the component has a globular 3D structure, is soluble, has a size (molecular weight) of about 2.5 to about 70 kDa, such as about 2.5 to about 50 kDa or about 2.5 to less than about 50 kDa, more preferably about 2.5 to about 30 kDa, for example about 2.5 to about 16 kDa, for example about 5 to about 16 kDa, or about 7 to about 16 kDa, or about 10 to about 16 kDa, and does not specifically bind to any human protein, and preferably does not specifically bind to any non-human protein, such as a bacterial and / or viral protein, to which it is originally bound, as described in detail above, and / or preferably does not specifically bind to any non-protein molecule, if present, to which it is originally bound, as described in detail above. Preferably, as described above, a molecule comprising at least one such ISVD-derived protein-based component and at least one cargo attached thereto via at least one conjugation site or attachment point does not specifically bind to any non-protein molecule and / or does not specifically bind to any non-human protein to which the ISVD precursor specifically binds.

[0221] Preferably, the protein-based carrier component of the present technology comprises or alternatively consists of SEQ ID NO: 186 as defined above, wherein one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are selected from the hallmark residues mentioned in Table 3 above.

[0222] In a further preferred embodiment, in addition or alternatively, the protein-based carrier component of the present technology comprises or alternatively consists of SEQ ID NO: 186 as defined above, which has been further engineered / modified to include mutations that prevent / eliminate binding by existing antibodies / factors. Examples of such mutations are described, for example, in WO 2012 / 175741 and WO 2015 / 173325. For example, to prevent / remove binding by existing antibodies / factors, the amino acid at position 11 (according to Kabat) in SEQ ID NO: 186 is preferably Val, and / or the amino acid at position 89 (according to Kabat) in SEQ ID NO: 186 is preferably Thr or Leu, and / or the amino acid at position 110 (according to Kabat) in SEQ ID NO: 186 is preferably Lys or Gln, and / or the amino acid at position 112 (according to Kabat) in SEQ ID NO: 186 is preferably Lys or Gln, and / or SEQ ID NO: 186 contains a C-terminal extension of 1 to 5 amino acids selected from any naturally occurring amino acid.

[0223] Thus, the present technology provides a polypeptide comprising or alternatively consisting of SEQ ID NO: 186 as defined above. Preferably, the polypeptide comprises or alternatively consists of SEQ ID NO: 186 as defined above, wherein one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 according to the Kabat numbering are selected from the hallmark residues mentioned in Table 3 above. In a further embodiment, additionally or alternatively, the polypeptide comprises or alternatively consists of SEQ ID NO: 186, which has been further engineered / modified to include mutations that prevent / eliminate binding by existing antibodies / factors. Examples of such mutations are described, for example, in WO 2012 / 175741 and WO 2015 / 173325. For example, to prevent / remove binding by existing antibodies / factors, the amino acid at position 11 (according to Kabat) in SEQ ID NO: 186 is preferably Val, and / or the amino acid at position 89 (according to Kabat) in SEQ ID NO: 186 is preferably Thr or Leu, and / or the amino acid at position 110 (according to Kabat) in SEQ ID NO: 186 is preferably Lys or Gln, and / or the amino acid at position 112 (according to Kabat) in SEQ ID NO: 186 is preferably Lys or Gln, and / or SEQ ID NO: 186 contains a C-terminal extension of 1 to 5 amino acids selected from any naturally occurring amino acid.

[0224] In one embodiment, at least one protein-based carrier component present in the molecule of the present technology is SEQ ID NO: 206: [ka] (In the formula, X 1a (position 1 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X1 (position 7 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; Z1 (position 11 according to Kabat numbering) can be Leu, Val, Ser, Met, Trp, Phe, Thr, Gln, Glu, Ala, Arg, Gly, Lys, Tyr, Asn, Pro or Ile, preferably Leu, Val, Ser or Glu, more preferably Leu or Val; X2 (position 13 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, for example, cysteine; X3 (position 17 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X4 (position 19 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X5 (position 21 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X6 (position 23 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, for example, cysteine; X7 (position 25 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 7b (position 26 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, e.g., cysteine; X 7c (position 28 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X8 (position 31 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example cysteine; X9 (position 43 according to Kabat numbering) can be Lys or any amino acid having a reactive group in its side chain, for example cysteine; X 10 (position 44 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, for example cysteine; X 11 (position 55 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X 12 (position 62 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example cysteine; X 13 (position 65 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, e.g., cysteine; X 14 (position 68 by Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, e.g., cysteine; X 15 (position 70 by Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 16 (position 72 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example cysteine; X 17 (position 74 by Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, for example, cysteine; X 18(position 75 according to Kabat numbering) can be Lys or any amino acid having a reactive group in its side chain, e.g., cysteine; X 19 (position 82b according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 19b (position 85 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example cysteine; Z2 (position 89 according to Kabat numbering) can be Leu, Val, Ser, Met, Trp, Phe, Thr, Gln, Glu, Ala, Arg, Gly, Lys, Tyr, Asn, Pro or Ile, preferably Leu, Val, Ser or Glu, more preferably Leu or Val; X 20 (position 100a according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X 21 (position 100f according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example cysteine; X 22 (position 105 by Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; Z3 (position 108 according to Kabat numbering) can be Gln, Leu, Arg, Pro, Glu, Lys, Ser, Thr, Met, Ala or His, preferably Gln or Leu; X 23 (position 112 by Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 24 is absent or is Gly, X 25 is absent or is Gly, X 26 is absent or is Cys) or comprising, or alternatively consisting of, a sequence having 80% or more identity to SEQ ID NO: 206, preferably a sequence having 85% or more, 90% or more, 95% or more, 97% or more or 99% or more sequence identity to SEQ ID NO: 206, with the proviso that the component has a globular 3D structure, is soluble, has a size (molecular weight) of about 2.5 to about 70 kDa, such as about 2.5 to about 50 kDa or about 2.5 to less than about 50 kDa, more preferably about 2.5 to about 30 kDa, such as about 2.5 to about 16 kDa, for example about 5 to about 16 kDa, or about 7 to about 16 kDa, or about 10 to about 16 kDa, and does not specifically bind to any human protein, and preferably does not specifically bind to any non-human protein, such as a bacterial and / or viral protein, to which it is originally bound, as described in detail above, and / or preferably does not specifically bind to any non-protein molecule, if present, to which it is originally bound, as described in detail above. Preferably, as described above, a molecule comprising at least one such ISVD-derived protein-based component and at least one cargo attached thereto via at least one conjugation site or attachment point does not specifically bind to any non-protein molecule and / or does not specifically bind to any non-human protein to which the ISVD precursor specifically binds.

[0225] Preferably, the protein-based carrier component of the present technology comprises or alternatively consists of SEQ ID NO: 206 as defined above, wherein one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are selected from the hallmark residues mentioned in Table 3 above.

[0226] In a further preferred embodiment, the protein-based carrier component of the present technology additionally or alternatively comprises or alternatively consists of SEQ ID NO: 206, which has been further engineered / modified to include mutations that prevent / eliminate binding by existing antibodies / factors. Examples of such mutations are described, for example, in WO 2012 / 175741 and WO 2015 / 173325. For example, to prevent / remove binding by existing antibodies / factors, the amino acid at position 11 (according to Kabat) in SEQ ID NO:206 is preferably Val, and / or the amino acid at position 89 (according to Kabat) in SEQ ID NO:206 is preferably Thr or Leu, and / or the amino acid at position 110 (according to Kabat) in SEQ ID NO:206 is preferably Lys or Gln, and / or the amino acid at position 112 (according to Kabat) in SEQ ID NO:206 is preferably Lys or Gln, and / or SEQ ID NO:206 contains a C-terminal extension of 1 to 5 amino acids selected from any naturally occurring amino acid.

[0227] Thus, the present technology provides a polypeptide comprising or alternatively consisting of SEQ ID NO: 206 as defined above. Preferably, the polypeptide comprises or alternatively consists of SEQ ID NO: 206 as defined above, wherein one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 according to the Kabat numbering are selected from the hallmark residues mentioned in Table 3 above. In a further embodiment, additionally or alternatively, the polypeptide comprises or alternatively consists of SEQ ID NO: 206, which has been further engineered / modified to include mutations that prevent / eliminate binding by existing antibodies / factors. Examples of such mutations are described, for example, in WO 2012 / 175741 and WO 2015 / 173325. For example, to prevent / remove binding by existing antibodies / factors, the amino acid at position 11 (according to Kabat) in SEQ ID NO:206 is preferably Val, and / or the amino acid at position 89 (according to Kabat) in SEQ ID NO:206 is preferably Thr or Leu, and / or the amino acid at position 110 (according to Kabat) in SEQ ID NO:206 is preferably Lys or Gln, and / or the amino acid at position 112 (according to Kabat) in SEQ ID NO:206 is preferably Lys or Gln, and / or SEQ ID NO:206 contains a C-terminal extension of 1 to 5 amino acids selected from any naturally occurring amino acid.

[0228] In one embodiment, at least one protein-based carrier component present in the molecule of the present technology is SEQ ID NO: 185: [ka] (In the formula, X1 (position 7 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; Z1 (position 11 according to Kabat numbering) can be Leu, Val, Ser, Met, Trp, Phe, Thr, Gln, Glu, Ala, Arg, Gly, Lys, Tyr, Asn, Pro or Ile, preferably Leu, Val, Ser or Glu, more preferably Leu or Val; X2 (position 13 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, for example, cysteine; X3 (position 17 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X4 (position 19 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X5 (position 21 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X6 (position 23 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, for example, cysteine; X7 (position 25 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X8 (position 31 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example cysteine; X9 (position 43 according to Kabat numbering) can be Lys or any amino acid having a reactive group in its side chain, for example cysteine; X 10 (position 44 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, for example cysteine; X 11(position 55 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X 12 (position 62 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example cysteine; X 13 (position 65 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, e.g., cysteine; X 14 (position 68 by Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, e.g., cysteine; X 15 (position 70 by Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 16 (position 72 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example cysteine; X 17 (position 74 by Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, for example, cysteine; X 18 (position 75 according to Kabat numbering) can be Lys or any amino acid having a reactive group in its side chain, e.g., cysteine; X 19 (position 82b according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; Z2 (position 89 according to Kabat numbering) can be Leu, Val, Ser, Met, Trp, Phe, Thr, Gln, Glu, Ala, Arg, Gly, Lys, Tyr, Asn, Pro or Ile, preferably Leu, Val, Ser or Glu, more preferably Leu or Val; X 20 (position 100a according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X 21 (position 100f according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example cysteine; X 22 (position 105 by Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; Z3 (position 108 according to Kabat numbering) can be Gln, Leu, Arg, Pro, Glu, Lys, Ser, Thr, Met, Ala or His, preferably Gln or Leu; X 23 (position 112 by Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X 24 is absent or is Gly, X 25 is absent or is Gly, X 26 is absent or is Cys) or comprising, or alternatively consisting of, a sequence having 80% or more identity to SEQ ID NO: 185, preferably a sequence having 85% or more, 90% or more, 95% or more, 97% or more or 99% or more sequence identity to SEQ ID NO: 185, with the proviso that the component has a globular 3D structure, is soluble, has a size (molecular weight) of about 2.5 to about 70 kDa, such as about 2.5 to about 50 kDa or about 2.5 to less than about 50 kDa, more preferably about 2.5 to about 30 kDa, for example about 2.5 to about 16 kDa, for example about 5 to about 16 kDa, or about 7 to about 16 kDa, or about 10 to about 16 kDa, and does not specifically bind to any human protein, and preferably does not specifically bind to any non-human protein, such as a bacterial and / or viral protein, to which it is originally bound, as described in detail above, and / or preferably does not specifically bind to any non-protein molecule, if present, to which it is originally bound, as described in detail above. Preferably, as described above, a molecule comprising at least one such ISVD-derived protein-based component and at least one cargo attached thereto via at least one conjugation site or attachment point does not specifically bind to any non-protein molecule and / or does not specifically bind to any non-human protein to which the ISVD precursor specifically binds.

[0229] Preferably, the protein-based carrier component of the present technology comprises or alternatively consists of SEQ ID NO: 185 as defined above, wherein one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are selected from the hallmark residues mentioned in Table 3 above.

[0230] In a further preferred embodiment, in addition or alternatively, the protein-based carrier component of the present technology comprises or alternatively consists of SEQ ID NO: 185 as defined above, which has been further engineered / modified to include mutations that prevent / eliminate binding by existing antibodies / factors. Examples of such mutations are described, for example, in WO 2012 / 175741 and WO 2015 / 173325. For example, to prevent / remove binding by existing antibodies / factors, the amino acid at position 11 (according to Kabat) in SEQ ID NO: 185 is preferably Val, and / or the amino acid at position 89 (according to Kabat) in SEQ ID NO: 185 is preferably Thr or Leu, and / or the amino acid at position 110 (according to Kabat) in SEQ ID NO: 185 is preferably Lys or Gln, and / or the amino acid at position 112 (according to Kabat) in SEQ ID NO: 185 is preferably Lys or Gln, and / or SEQ ID NO: 185 contains a C-terminal extension of 1 to 5 amino acids selected from any naturally occurring amino acid.

[0231] Thus, the present technology provides a polypeptide comprising or alternatively consisting of SEQ ID NO: 185 as defined above. Preferably, the polypeptide comprises or alternatively consists of SEQ ID NO: 185 as defined above, wherein one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 according to the Kabat numbering are selected from the hallmark residues mentioned in Table 3 above. In a further embodiment, additionally or alternatively, the polypeptide comprises or alternatively consists of SEQ ID NO: 185, which has been further engineered / modified to include mutations that prevent / eliminate binding by existing antibodies / factors. Examples of such mutations are described, for example, in WO 2012 / 175741 and WO 2015 / 173325. For example, to prevent / remove binding by existing antibodies / factors, the amino acid at position 11 (according to Kabat) in SEQ ID NO: 185 is preferably Val, and / or the amino acid at position 89 (according to Kabat) in SEQ ID NO: 185 is preferably Thr or Leu, and / or the amino acid at position 110 (according to Kabat) in SEQ ID NO: 185 is preferably Lys or Gln, and / or the amino acid at position 112 (according to Kabat) in SEQ ID NO: 185 is preferably Lys or Gln, and / or SEQ ID NO: 18 contains a C-terminal extension of 1 to 5 amino acids selected from any naturally occurring amino acid.

[0232] In one embodiment, the protein-based carrier component comprises at least one amino acid having a reactive group in its side chain, such as a cysteine, or a lysine, or a tyrosine, or an unnatural amino acid, preferably a cysteine, in at least one of the following solvent accessible positions: * 43, 100f, 105, or * 43, 75, 100a, or * 21, 68, 100f, or * 7, 44, 55, or * 13, 72, 100a, * 13, 31, 100f, or * C-terminal Cys(-GGC), More preferably, at least one of the following solvent accessible positions in SEQ ID NO: 179 (according to Kabat numbering): * 43, 100f, 105, or * 43, 75, 100a.

[0233] Thus, in one embodiment, the protein-based component of the technology has the following sequence: * SEQ ID NO: 185 (wherein: X1 (position 7 according to Kabat numbering) is Ser; Z1 (position 11 according to Kabat numbering) is Leu or Val, X2 (position 13 according to Kabat numbering) is Gln; X3 (position 17 by Kabat numbering) is Ser; X4 (position 19 by Kabat numbering) is Ser; X5 (position 21 by Kabat numbering) is Ser; X6 (position 23 by Kabat numbering) is Ala; X7 (position 25 by Kabat numbering) is Ser; X8 (position 31 by Kabat numbering) is Asn; X9 (position 43 according to Kabat numbering) can be any amino acid having a reactive group in the side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine; X 10(position 44 by Kabat numbering) is Glu, X 11 (position 55 by Kabat numbering) is Asp, X 12 (position 62 by Kabat numbering) is Asn, X 13 (position 65 by Kabat numbering) is Gly; X 14 (position 68 by Kabat numbering) is Thr; X 15 (70th position by Kabat numbering) is Ser, X 16 (position 72 by Kabat numbering) is Asp, X 17 (74th position by Kabat numbering) is Ala, X 18 (position 75 by Kabat numbering) is Lys, X 19 (position 82b according to Kabat numbering) is Ser, Z2 (position 89 according to Kabat numbering) is Val or Leu; X 20 (position 100a according to Kabat numbering) is Gly, X 21 (position 100f according to Kabat numbering) can be any amino acid having a reactive group in the side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine; X 22 (position 105 according to Kabat numbering) can be any amino acid with a reactive group in the side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine; Z3 (position 108 according to Kabat numbering) is Gln or Leu; X 23 (position 112 by Kabat numbering) is Ser, X 24 does not exist, X 25 does not exist, X 26 does not exist), or * SEQ ID NO: 185 (wherein: X1 (position 7 according to Kabat numbering) is Ser; Z1 (position 11 according to Kabat numbering) is Leu or Val, X2 (position 13 according to Kabat numbering) is Gln; X3 (position 17 by Kabat numbering) is Ser; X4 (position 19 by Kabat numbering) is Ser; X5 (position 21 by Kabat numbering) is Ser; X6 (position 23 by Kabat numbering) is Ala; X7 (position 25 by Kabat numbering) is Ser; X8 (position 31 by Kabat numbering) is Asn; X9 (position 43 according to Kabat numbering) can be any amino acid having a reactive group in the side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine; X 10 (position 44 by Kabat numbering) is Glu, X 11 (position 55 by Kabat numbering) is Asp, X 12 (position 62 by Kabat numbering) is Asn, X 13 (position 65 by Kabat numbering) is Gly; X 14 (position 68 by Kabat numbering) is Thr; X 15 (70th position by Kabat numbering) is Ser, X 16 (position 72 by Kabat numbering) is Asp, X 17 (74th position by Kabat numbering) is Ala, X 18(position 75 according to Kabat numbering) can be any amino acid with a reactive group in the side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine; X 19 (position 82b according to Kabat numbering) is Ser, Z2 (position 89 according to Kabat numbering) is Val or Leu; X 20 (position 100a according to Kabat numbering) can be any amino acid having a reactive group in the side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine; X 21 (position 100f according to Kabat numbering) is Asp, X 22 (position 105 by Kabat numbering) is Arg, Z3 (position 108 according to Kabat numbering) is Gln or Leu; X 23 (position 112 by Kabat numbering) is Ser, X 24 does not exist, X 25 does not exist, X 26 does not exist), or * SEQ ID NO: 185 (wherein: X1 (position 7 according to Kabat numbering) is Ser; Z1 (position 11 according to Kabat numbering) is Leu or Val, X2 (position 13 according to Kabat numbering) is Gln; X3 (position 17 by Kabat numbering) is Ser; X4 (position 19 by Kabat numbering) is Ser; X5 (position 21 according to Kabat numbering) can be any amino acid having a reactive group in the side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine; X6 (position 23 by Kabat numbering) is Ala; X7 (position 25 by Kabat numbering) is Ser; X8 (position 31 by Kabat numbering) is Asn; X9 (position 43 according to Kabat numbering) is Lys; X 10 (position 44 by Kabat numbering) is Glu, X 11 (position 55 by Kabat numbering) is Asp, X 12 (position 62 by Kabat numbering) is Asn, X 13 (position 65 by Kabat numbering) is Gly; X 14 (position 68 according to Kabat numbering) can be any amino acid with a reactive group in the side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine; X 15 (70th position by Kabat numbering) is Ser, X 16 (position 72 by Kabat numbering) is Asp, X 17 (74th position by Kabat numbering) is Ala, X 18 (position 75 by Kabat numbering) is Lys, X 19 (position 82b according to Kabat numbering) is Ser, Z2 (position 89 according to Kabat numbering) is Val or Leu; X 20 (position 100a according to Kabat numbering) is Gly, X 21 (position 100f according to Kabat numbering) can be any amino acid having a reactive group in the side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine; X 22(position 105 by Kabat numbering) is Arg, Z3 (position 108 according to Kabat numbering) is Gln or Leu; X 23 (position 112 by Kabat numbering) is Ser, X 24 does not exist, X 25 does not exist, X 26 does not exist), or * SEQ ID NO: 185 (wherein: X1 (position 7 according to Kabat numbering) can be any amino acid having a reactive group in the side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine; Z1 (position 11 according to Kabat numbering) is Leu or Val, X2 (position 13 according to Kabat numbering) is Gln; X3 (position 17 by Kabat numbering) is Ser; X4 (position 19 by Kabat numbering) is Ser; X5 (position 21 by Kabat numbering) is Ser; X6 (position 23 by Kabat numbering) is Ala; X7 (position 25 by Kabat numbering) is Ser; X8 (position 31 by Kabat numbering) is Asn; X9 (position 43 according to Kabat numbering) is Lys; X 10 (position 44 according to Kabat numbering) can be any amino acid with a reactive group in the side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine; X 11 (position 55 according to Kabat numbering) can be any amino acid having a reactive group in the side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine; X 12(position 62 by Kabat numbering) is Asn, X 13 (position 65 by Kabat numbering) is Gly; X 14 (position 68 by Kabat numbering) is Thr; X 15 (70th position by Kabat numbering) is Ser, X 16 (position 72 by Kabat numbering) is Asp, X 17 (74th position by Kabat numbering) is Ala, X 18 (position 75 by Kabat numbering) is Lys, X 19 (position 82b according to Kabat numbering) is Ser, Z2 (position 89 according to Kabat numbering) is Val or Leu; X 20 (position 100a according to Kabat numbering) is Gly, X 21 (position 100f according to Kabat numbering) is Asp, X 22 (position 105 by Kabat numbering) is Arg, Z3 (position 108 according to Kabat numbering) is Gln or Leu; X 23 (position 112 by Kabat numbering) is Ser, X 24 does not exist, X 25 does not exist, X 26 does not exist), or * SEQ ID NO: 185 (wherein: X1 (position 7 according to Kabat numbering) is Ser; Z1 (position 11 according to Kabat numbering) is Leu or Val, X2 (position 13 according to Kabat numbering) can be any amino acid having a reactive group in the side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine; X3 (position 17 by Kabat numbering) is Ser; X4 (position 19 by Kabat numbering) is Ser; X5 (position 21 by Kabat numbering) is Ser; X6 (position 23 by Kabat numbering) is Ala; X7 (position 25 by Kabat numbering) is Ser; X8 (position 31 by Kabat numbering) is Asn; X9 (position 43 according to Kabat numbering) is Lys; X 10 (position 44 by Kabat numbering) is Glu, X 11 (position 55 by Kabat numbering) is Asp, X 12 (position 62 by Kabat numbering) is Asn, X 13 (position 65 by Kabat numbering) is Gly; X 14 (position 68 by Kabat numbering) is Thr; X 15 (70th position by Kabat numbering) is Ser, X 16 (position 72 according to Kabat numbering) can be any amino acid having a reactive group in the side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine; X 17 (74th position by Kabat numbering) is Ala, X 18 (position 75 by Kabat numbering) is Lys, X 19 (position 82b according to Kabat numbering) is Ser, Z2 (position 89 according to Kabat numbering) is Val or Leu; X 20 (position 100a according to Kabat numbering) can be any amino acid having a reactive group in the side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine; X 21 (position 100f according to Kabat numbering) is Asp, X 22 (position 105 by Kabat numbering) is Arg, Z3 (position 108 according to Kabat numbering) is Gln or Leu; X 23 (position 112 by Kabat numbering) is Ser, X 24 does not exist, X 25 does not exist, X 26 does not exist), or * SEQ ID NO: 185 (wherein: X1 (position 7 according to Kabat numbering) is Ser; Z1 (position 11 according to Kabat numbering) is Leu or Val, X2 (position 13 according to Kabat numbering) can be any amino acid having a reactive group in the side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine; X3 (position 17 by Kabat numbering) is Ser; X4 (position 19 by Kabat numbering) is Ser; X5 (position 21 by Kabat numbering) is Ser; X6 (position 23 by Kabat numbering) is Ala; X7 (position 25 by Kabat numbering) is Ser; X8 (position 31 according to Kabat numbering) can be any amino acid having a reactive group in the side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine; X9 (position 43 according to Kabat numbering) is Lys; X 10 (position 44 by Kabat numbering) is Glu, X 11 (position 55 by Kabat numbering) is Asp, X 12 (position 62 by Kabat numbering) is Asn, X 13 (position 65 by Kabat numbering) is Gly; X 14 (position 68 by Kabat numbering) is Thr; X 15 (70th position by Kabat numbering) is Ser, X 16 (position 72 by Kabat numbering) is Asp, X 17 (74th position by Kabat numbering) is Ala, X 18 (position 75 by Kabat numbering) is Lys, X 19 (position 82b according to Kabat numbering) is Ser, Z2 (position 89 according to Kabat numbering) is Val or Leu; X 20 (position 100a according to Kabat numbering) is Gly, X 21 (position 100f according to Kabat numbering) can be any amino acid having a reactive group in the side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine; X 22 (position 105 by Kabat numbering) is Arg, Z3 (position 108 according to Kabat numbering) is Gln or Leu; X 23 (position 112 by Kabat numbering) is Ser, X 24 does not exist, X 25does not exist, X 26 does not exist), or * SEQ ID NO: 185 (wherein: X1 (position 7 according to Kabat numbering) is Ser; Z1 (position 11 according to Kabat numbering) is Leu or Val, X2 (position 13 according to Ka...

Claims

1. A molecule comprising at least one protein-based component, said at least one protein-based component comprising: a) comprises at least two conjugation sites or attachment points; b) has a molecular weight of about 2.5 to about 70 kDa; c) has a spherical three-dimensional (3D) structure; d) has a solubility of 10 mg / mL or more at room temperature as measured in an aqueous solution of citrate buffer or PBS at pH 7.0 or 7.4; e) does not specifically bind to any human protein or has a specific binding affinity of less than 5×10 as determined by surface plasmon resonance, e.g., as described in Ober et al. 2001, Intern. Immunology 13:1551-1559. －４ More than moles / liter of K Ｄ binds to one or more human proteins with a value f) A molecule that does not contain or consist of an amino acid sequence selected from SEQ ID NOs: 1 to 34 as set out in Tables A-1 and A-2 of WO 2016 / 055656 and / or SEQ ID NOs: 1 to 12 as set out in Table A-1 of WO 2010 / 139808.

2. The molecule of claim 1 , wherein one of the at least two, preferably the at least two, conjugation sites or attachment points is located at a solvent-accessible position within the protein-based entity.

3. 3. The molecule of claim 1 or 2, wherein at least one of the attachment points or conjugation sites, preferably at least two of the attachment points or conjugation sites, more preferably all of the attachment points or conjugation sites, is an engineered attachment point or conjugation site.

4. 4. The molecule of any one of claims 1 to 3, wherein the at least two attachment points or conjugation sites are reactive groups present in the side chains of any amino acids within the protein-based carrier component, preferably reactive groups present in the side chains of cysteine, and / or tyrosine, and / or lysine, and / or unnatural amino acids.

5. 5. The molecule according to any one of claims 1 to 4, wherein the at least two conjugation sites are selected from primary amines, thiol groups, hydroxyl groups, guanidino groups, carboxyl groups and / or thioether groups, preferably primary amines and / or thiol groups, more preferably thiol groups.

6. The at least one protein-based entity does not specifically bind to any non-protein molecule, such as DNA, RNA, lipids, or glycans, or ... －４ More than moles / liter of K Ｄ The molecule according to any one of claims 1 to 5, which binds to one or more non-protein molecules at a value.

7. The molecule of any one of claims 1 to 6, wherein the at least one protein-based entity comprises at least one cargo attached to at least one of the attachment points or conjugation sites.

8. 8. The molecule of any one of claims 1 to 7, wherein the protein-based entity is a small globular non-human protein-based entity or a small globular human protein-based entity, preferably the small globular non-human protein-based entity is an immunoglobulin single variable domain (ISVD)-based entity, a DARP-in-based entity, an affibody-based entity or an affitin-based entity, and the small globular human protein-based entity is a cyclin-dependent kinase subunit 1 (CKS1) protein-based entity.

9. The ISVD-based components are Ｈ , humanized V Ｈ , Human V Ｈ , V ＨＨ , humanized V ＨＨ or Camelization V Ｈ (derived from heavy chain ISVD), preferably derived from "V Ｈ The molecule of claim 8, derived from an ISVD belonging to "Class 3".

10. 10. The molecule of claim 8 or 9, wherein the ISVD-derived component is derived from RSV001A04 (SEQ ID NO: 179).

11. The ISVD derived component is SEQ ID NO: 186: 【Chemistry 1】 (In the formula, X １ (position 1 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X ２ (position 3 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, for example cysteine; X ３ (position 5 according to Kabat numbering) can be Val or any amino acid having a reactive group in its side chain, for example cysteine; X ４ (position 7 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example cysteine; X ５ (position 8 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X ６ (position 10 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X ７ (position 11 according to Kabat numbering) can be Leu, Val, Ser, Met, Trp, Phe, Thr, Gln, Glu, Ala, Arg, Gly, Lys, Tyr, Asn, Pro or He, preferably Leu or Val or any amino acid having a reactive group in its side chain, for example cysteine; X ８ (position 12 according to Kabat numbering) can be Val or any amino acid having a reactive group in its side chain, for example, cysteine; X ９ (position 13 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, for example, cysteine; X １０ (position 14 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, for example cysteine; X １１ (position 15 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X １２ (position 17 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X １３ (position 18 according to Kabat numbering) can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X １４ (position 19 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X １５ (position 21 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X １６ (position 23 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, for example cysteine; X １７ (position 25 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X １８ (position 26 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X １９ (position 27 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X ２０ (position 28 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X ２１ (position 30 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X ２２ (position 31 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example cysteine; X ２３ (position 32 according to Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, for example, cysteine; X ２４ (position 39 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, for example, cysteine; X ２５ (position 41 according to Kabat numbering) can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X ２６ (position 42 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X ２７ (position 43 according to Kabat numbering) can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X ２８ (position 44 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X ２９ (position 45 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X ３０ (position 46 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X ３１ (position 52a according to Kabat numbering) can be Trp or any amino acid having a reactive group in its side chain, for example, cysteine; X ３２ (position 53 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X ３３ (position 54 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, for example, cysteine; X ３４ (position 55 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X ３５ (position 56 according to Kabat numbering) can be He or any amino acid having a reactive group in its side chain, for example cysteine; X ３６ (position 57 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, for example, cysteine; X ３７ (position 58 according to Kabat numbering) can be He or any amino acid having a reactive group in its side chain, for example, cysteine; X ３８ (position 59 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, for example, cysteine; X ３９ (position 61 according to Kabat numbering) can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X ４０ (position 62 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example cysteine; X ４１ (position 64 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X ４２ (position 65 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X ４３ (position 66 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X ４４ (position 68 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, for example, cysteine; X ４５ (position 70 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X ４６ (position 71 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X ４７ (position 72 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X ４８ (position 73 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example cysteine; X ４９ (position 74 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, for example cysteine; X ５０ (position 75 according to Kabat numbering) can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X ５１ (position 76 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example cysteine; X ５２ (position 79 according to Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, for example, cysteine; X ５３ (position 81 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, for example, cysteine; X ５４ (position 82a according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X ５５ (position 82b according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X ５６ (position 83 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, for example, cysteine; X ５７ (position 84 according to Kabat numbering) can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X ５８ (position 85 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X ５９ (position 87 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, for example, cysteine; X ６０ (position 89 according to Kabat numbering) can be Leu, Val, Ser, Met, Trp, Phe, Thr, Gln, Glu, Ala, Arg, Gly, Lys, Tyr, Asn, Pro or He, preferably Leu, Val, Ser or Glu, more preferably Leu or Val or any other amino acid having a reactive group in its side chain, for example cysteine; X ６１ (position 91 according to Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, for example, cysteine; X ６２ (position 96 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, for example, cysteine; X ６３ (position 98 according to Kabat numbering) can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X ６４ (position 99 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X ６５ (position 100 according to Kabat numbering) can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X ６６ (position 100a according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, for example, cysteine; X ６７ (position 100d according to Kabat numbering) can be He or any amino acid having a reactive group in its side chain, for example, cysteine; X ６８ (position 100e according to Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, for example, cysteine; X ６９ (position 100f according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X ７０ (position 100g according to Kabat numbering) can be Trp or any amino acid having a reactive group in its side chain, for example, cysteine; X ７１ (position 101 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X ７２ (position 102 according to Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, for example, cysteine; X ７３ (position 103 according to Kabat numbering) can be Trp or any amino acid having a reactive group in its side chain, e.g., cysteine; X ７４ (position 105 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X ７５ (position 106 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, for example, cysteine; X ７６ (position 108 according to Kabat numbering) can be Gln, Leu, Arg, Pro, Glu, Lys, Ser, Thr, Met, Ala or His, preferably Gln or Leu or any other amino acid having a reactive group in its side chain, for example cysteine; X ７７ (position 110 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, for example, cysteine; X ７８ (position 112 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X ７９ (position 113 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X ８０ is absent or is Gly, X ８１ is absent or is Gly, X ８２ is absent or is Cys) or comprising, or alternatively consisting of, a sequence having 80% or more identity to SEQ ID NO: 186, preferably a sequence having 85% or more, 90% or more, 95% or more, 97% or more or 99% or more sequence identity to SEQ ID NO: 186, with the proviso that said entity has a globular 3D structure, is soluble, and has a size (molecular weight) of about 2.5 to about 70 kDa, such as about 2.5 to about 50 kDa or about 2.5 to less than 50 kDa, more preferably about 2.5 to about 30 kDa, such as about 2.5 to about 16 kDa, for example about 5 to about 16 kDa, or about 7 to about 16 kDa, or about 10 to about 16 kDa, and does not specifically bind to any human protein.

12. The ISVD-derived component is SEQ ID NO:206: 【Chemistry 2】 (In the formula, X １ａ (position 1 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X １ (position 7 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example cysteine; Z １ (position 11 according to Kabat numbering) can be Leu, Val, Ser, Met, Trp, Phe, Thr, Gln, Glu, Ala, Arg, Gly, Lys, Tyr, Asn, Pro or He, preferably Leu, Val, Ser or Glu, more preferably Leu or Val; X ２ (position 13 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, for example, cysteine; X ３ (position 17 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X ４ (position 19 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X ５ (position 21 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X ６ (position 23 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, for example cysteine; X ７ (position 25 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X ７ｂ (position 26 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X ７ｃ (position 28 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X ８ (position 31 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example cysteine; X ９ (position 43 according to Kabat numbering) can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X １０ (position 44 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X １１ (position 55 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X １２ (position 62 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, for example cysteine; X １３ (position 65 according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X １４ (position 68 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, for example, cysteine; X １５ (position 70 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X １６ (position 72 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X １７ (position 74 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, for example cysteine; X １８ (position 75 according to Kabat numbering) can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X １９ (position 82b according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X １９ｂ (position 85 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; Z ２ (position 89 according to Kabat numbering) can be Leu, Val, Ser, Met, Trp, Phe, Thr, Gln, Glu, Ala, Arg, Gly, Lys, Tyr, Asn, Pro or He, preferably Leu, Val, Ser or Glu, more preferably Leu or Val; X ２０ (position 100a according to Kabat numbering) can be GIy or any amino acid having a reactive group in its side chain, for example, cysteine; X ２１ (position 100f according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X ２２ (position 105 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; Z ３ (position 108 according to Kabat numbering) can be Gln, Leu, Arg, Pro, Glu, Lys, Ser, Thr, Met, Ala, or His, preferably Gln or Leu; X ２３ (position 112 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X ２４ is absent or is Gly, X ２５ is absent or is Gly, X ２６ is absent or is Cys) or comprising, or alternatively consisting of, a sequence having 80% or more identity to SEQ ID NO:206, preferably a sequence having 85% or more, 90% or more, 95% or more, 97% or more or 99% or more sequence identity to SEQ ID NO:206, with the proviso that said entity has a globular 3D structure, is soluble, and has a size (molecular weight) of about 2.5 to about 70 kDa, such as about 2.5 to about 50 kDa or about 2.5 to less than 50 kDa, more preferably about 2.5 to about 30 kDa, such as about 2.5 to about 16 kDa, for example about 5 to about 16 kDa, or about 7 to about 16 kDa, or about 10 to about 16 kDa, and does not specifically bind to any human protein.

13. The molecule of claim 8, wherein the DARPin-based component is derived from the polypeptide defined in SEQ ID NO:

187.

14. At least one protein-based component is set forth in SEQ ID NO: 188: 【Transformation 3】 (In the formula, X １ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X ２ can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X ３ can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X ４ can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X ５ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X ６ can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X ７ can be Ala or any amino acid having a reactive group in its side chain, for example cysteine; X ８ can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X ９ can be Gln or any amino acid having a reactive group in its side chain, for example, cysteine; X １０ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X １１ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X １２ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X １３ can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X １４ can be He or any amino acid having a reactive group in its side chain, for example, cysteine; X １５ can be Ala or any amino acid having a reactive group in its side chain, for example cysteine; X １６ can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X １７ can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X １８ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X １９ can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X ２０ can be His or any amino acid having a reactive group in its side chain, for example, cysteine; X ２１ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X ２２ can be Thr or any amino acid having a reactive group in its side chain, for example, cysteine; X ２３ can be Phe or any amino acid having a reactive group in its side chain, for example, cysteine; X ２４ can be Phe or any amino acid having a reactive group in its side chain, for example, cysteine; X ２５ can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X ２６ can be Tyr or any amino acid having a reactive group in its side chain, for example, cysteine; X ２７ can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X ２８ can be His or any amino acid having a reactive group in its side chain, for example, cysteine; X ２９ can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X ３０ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X ３１ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X ３２ can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X ３３ can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X ３４ can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X ３５ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X ３６ can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X ３７ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X ３８ can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X ３９ can be Tyr or any amino acid having a reactive group in its side chain, for example, cysteine; X ４０ can be Met or any amino acid having a reactive group in its side chain, for example, cysteine; X ４１ can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X ４２ can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X ４３ can be His or any amino acid having a reactive group in its side chain, for example, cysteine; X ４４ can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X ４５ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X ４６ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X ４７ can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X ４８ can be Tyr or any amino acid having a reactive group in its side chain, for example, cysteine; X ４９ can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X ５０ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X ５１ can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X ５２ can be Ala or any amino acid having a reactive group in its side chain, for example cysteine; X ５３ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X ５４ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X ５５ can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X ５６ can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X ５７ can be Ala or any amino acid having a reactive group in its side chain, for example cysteine; X ５８ can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X ５９ can be His or any amino acid having a reactive group in its side chain, for example, cysteine; X ６０ can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X ６１ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X ６２ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X ６３ can be Val or any amino acid having a reactive group in its side chain, for example, cysteine; X ６４ can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X ６５ can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X ６６ can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X ６７ can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X ６８ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X ６９ can be Val or any amino acid having a reactive group in its side chain, for example, cysteine; X ７０ can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X ７１ can be Gln or any amino acid having a reactive group in its side chain, for example, cysteine; X ７２ can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X ７３ can be Phe or any amino acid having a reactive group in its side chain, for example, cysteine; X ７４ can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X ７５ can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X ７６ can be Phe or any amino acid having a reactive group in its side chain, for example, cysteine; X ７７ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X ７８ can be He or any amino acid having a reactive group in its side chain, for example, cysteine; X ７９ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X ８０ can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X ８１ can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X ８２ can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X ８３ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X ８４ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X ８５ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X ８６ can be He or any amino acid having a reactive group in its side chain, for example, cysteine; X ８７ can be Gln or any amino acid having a reactive group in its side chain, for example, cysteine; X ８８ can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X ８９ can be absent or Leu, X ９０ may be absent or Cys) or comprising, or alternatively consisting of, a sequence having 80% or more identity to SEQ ID NO: 188, preferably a sequence having 85% or more, 90% or more, 95% or more, 97% or more or 99% or more sequence identity to SEQ ID NO: 188, with the proviso that said entity has a globular 3D structure, is soluble, and has a size (molecular weight) of about 2.5 to about 70 kDa, such as about 2.5 to about 50 kDa or about 2.5 to less than 50 kDa, more preferably about 2.5 to about 30 kDa, such as about 2.5 to about 16 kDa, for example about 5 to about 16 kDa, or about 7 to about 16 kDa, or about 10 to about 16 kDa, and does not specifically bind to any human protein.

15. The at least one protein-based component is selected from the group consisting of SEQ ID NO: 189 【Chemistry 4】 (In the formula, X １ can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X ２ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X ３ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X ４ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X ５ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X ６ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X ７ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X ８ can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X ９ can be Ala or any amino acid having a reactive group in its side chain, for example cysteine; X １０ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X １１ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X １２ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X １３ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X １４ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X １５ can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X １６ can be absent or Leu, X １７ may be absent or Cys) or comprising, or alternatively consisting of, a sequence having 80% or more identity to SEQ ID NO: 189, preferably a sequence having 85% or more, 90% or more, 95% or more, 97% or more or 99% or more sequence identity to SEQ ID NO: 189, with the proviso that said entity has a globular 3D structure, is soluble, and has a size (molecular weight) of about 2.5 to about 70 kDa, such as about 2.5 to about 50 kDa or about 2.5 to less than 50 kDa, more preferably about 2.5 to about 30 kDa, such as about 2.5 to about 16 kDa, for example about 5 to about 16 kDa, or about 7 to about 16 kDa, or about 10 to about 16 kDa, and does not specifically bind to any human protein.

16. The molecule of claim 8, wherein the CSK1-derived component is derived from the polypeptide defined in SEQ ID NO:

190.

17. The at least one protein-based component is SEQ ID NO: 191: 【Transformation 5】 (In the formula, X １ can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X ２ can be His or any amino acid having a reactive group in its side chain, for example, cysteine; X ３ can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X ４ can be Gln or any amino acid having a reactive group in its side chain, for example, cysteine; X ５ can be Tyr or any amino acid having a reactive group in its side chain, for example, cysteine; X ６ can be Tyr or any amino acid having a reactive group in its side chain, for example, cysteine; X ７ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X ８ can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X ９ can be Tyr or any amino acid having a reactive group in its side chain, for example, cysteine; X １０ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X １１ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X １２ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X １３ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X １４ can be Phe or any amino acid having a reactive group in its side chain, for example, cysteine; X １５ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X １６ can be Tyr or any amino acid having a reactive group in its side chain, for example, cysteine; X １７ can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X １８ can be His or any amino acid having a reactive group in its side chain, for example, cysteine; X １９ can be Met or any amino acid having a reactive group in its side chain, for example, cysteine; X ２０ can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X ２１ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X ２２ can be He or any amino acid having a reactive group in its side chain, for example, cysteine; X ２３ can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X ２４ can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X ２５ can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X ２３ｂ can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X ２４ｂ can be Thr or any amino acid having a reactive group in its side chain, for example, cysteine; X ２５ｂ can be His or any amino acid having a reactive group in its side chain, for example, cysteine; X ２６ can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X ２７ can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X ２８ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X ２９ can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X ３０ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X ３１ can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X ３２ can be Asn or any amino acid having a reactive group in its side chain, for example, cysteine; X ３３ can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X ３４ can be Gln or any amino acid having a reactive group in its side chain, for example, cysteine; X ３５ can be Ser or any amino acid having a reactive group in its side chain, for example, cysteine; X ３６ can be Gln or any amino acid having a reactive group in its side chain, for example, cysteine; X ３７ can be GIy or any amino acid having a reactive group in its side chain, for example cysteine; X ３８ can be Val or any amino acid having a reactive group in its side chain, for example, cysteine; X ３９ can be Tyr or any amino acid having a reactive group in its side chain, for example, cysteine; X ４０ can be Met or any amino acid having a reactive group in its side chain, for example, cysteine; X ４１ can be He or any amino acid having a reactive group in its side chain, for example, cysteine; X ４２ can be His or any amino acid having a reactive group in its side chain, for example, cysteine; X ４３ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X ４４ can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X ４５ can be Glu or any amino acid having a reactive group in its side chain, for example, cysteine; X ４６ can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X ４７ can be His or any amino acid having a reactive group in its side chain, for example, cysteine; X ４８ can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X ４９ can be Arg or any amino acid having a reactive group in its side chain, for example, cysteine; X ５０ can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X ５１ can be Leu or any amino acid having a reactive group in its side chain, for example, cysteine; X ５２ can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X ５３ can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X ５４ can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X ５５ can be Pro or any amino acid having a reactive group in its side chain, for example, cysteine; X ５６ can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X ５７ can be Lys or any amino acid having a reactive group in its side chain, e.g., cysteine) or comprising, or alternatively consisting of, a sequence having 80% or more identity to SEQ ID NO: 191, preferably a sequence having 85% or more, 90% or more, 95% or more, 97% or more or 99% or more sequence identity to SEQ ID NO: 191, with the proviso that said entity has a globular 3D structure, is soluble, and has a size (molecular weight) of about 2.5 to about 70 kDa, such as about 2.5 to about 50 kDa or about 2.5 to less than 50 kDa, more preferably about 2.5 to about 30 kDa, such as about 2.5 to about 16 kDa, for example about 5 to about 16 kDa, or about 7 to about 16 kDa, or about 10 to about 16 kDa, and does not specifically bind to any human protein.

18. The at least one protein-based component is SEQ ID NO:205: SHKQIYYSX １ X ２ X ３ X ４ X ５ EEFEYRHVX ６ LPKDIAKLVPX ７ THLMSESEWRNLGVQQSX ８ GWVHYX ９ IHEPEPHILLFRRPLPKKPKX １０ (In the formula, X １ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X ２ can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X ３ can be Tyr or any amino acid having a reactive group in its side chain, for example, cysteine; X ４ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X ５ can be Asp or any amino acid having a reactive group in its side chain, for example, cysteine; X ６ can be Met or any amino acid having a reactive group in its side chain, for example, cysteine; X ７ can be Lys or any amino acid having a reactive group in its side chain, for example, cysteine; X ８ can be Gln or any amino acid having a reactive group in its side chain, for example, cysteine; X ９ can be Met or any amino acid having a reactive group in its side chain, for example, cysteine; X １０ can be Lys or any amino acid having a reactive group in its side chain, e.g., cysteine) or comprising, or alternatively consisting of, a sequence having 80% or more identity to SEQ ID NO:205, preferably a sequence having 85% or more, 90% or more, 95% or more, 97% or more or 99% or more sequence identity to SEQ ID NO:205, with the proviso that said entity has a globular 3D structure, is soluble, and has a size (molecular weight) of about 2.5 to about 70 kDa, such as about 2.5 to about 50 kDa or about 2.5 to less than 50 kDa, more preferably about 2.5 to about 30 kDa, such as about 2.5 to about 16 kDa, for example about 5 to about 16 kDa, or about 7 to about 16 kDa, or about 10 to about 16 kDa, and does not specifically bind to any human protein.

19. 19. The molecule of any one of claims 1 to 18, wherein said at least one protein-based component comprises or consists of a polypeptide selected from SEQ ID NOs: 80-105, 175, 199, 208 and / or 222-224.

20. at least one protein-based component and at least one further moiety or cargo, preferably said at least one further moiety or cargo comprising: a) a half-life extending (HLE) moiety, and / or b) a targeting moiety, preferably an EGFR targeting moiety such as the GE11 peptide, and / or c) a therapeutic moiety or a precursor thereof; d) an imaging moiety; e) toxic moiety; f) siRNA; g) vitamins, preferably folic acid; h) Toll-like receptor agonists; i) a glycan, preferably bismannose 6-phosphate (bisM6P) or mannose 6-phosphate (M6P), and / or j) lipids, preferably short chain fatty acids The molecule according to any one of claims 1 to 19, selected from:

21. The molecule of claim 20, wherein the at least one cargo is directly bound to the at least one protein-based component, or the at least one cargo is bound to the at least one protein-based component via a linker.

22. 22. The molecule of claim 20 or 21, wherein the cargo is an (in vivo) half-life extending moiety, preferably a PEG molecule, preferably a PEG molecule of 1-20 kDa, more preferably a PEG molecule of 1-10 kDa, even more preferably a PEG molecule of 1-5 kDa, an ELNN polypeptide or an albumin binding polypeptide.

23. 23. The molecule of claim 22, wherein said albumin binding polypeptide is an albumin binding ISVD, preferably said albumin binding ISVD comprises or alternatively consists of a polypeptide as defined in any one of SEQ ID NOs: 50 to 64 or 106, preferably SEQ ID NO: 63 or 106.

24. 24. A molecule according to any one of claims 1 to 23, comprising or alternatively consisting of a polypeptide as defined in any one of SEQ ID NOs: 107-127, 170-174, 176 or 200.

25. A nucleic acid encoding a molecule according to any one of claims 1 to 24, a part of a molecule according to any one of claims 1 to 24 and / or a protein-based building block according to any one of claims 1 to 19.

26. A vector comprising the nucleic acid of claim 25.

27. A composition, such as a pharmaceutical composition, comprising a molecule according to any one of claims 1 to 24.

28. A method for producing a molecule according to any one of claims 1 to 24, comprising the steps of: a) expressing in a suitable host cell or host organism or another suitable expression system at least one protein-based carrier component and / or molecule or a nucleic acid sequence encoding a part of said molecule according to any one of claims 1 to 24, b) optionally isolating and / or purifying said at least one protein-based carrier component expressed in a) and / or said molecule or part of said molecule, c) optionally conjugating one or more (further) cargoes to said attachment points or conjugation sites of said protein-based carrier component; A method comprising:

29. A method for producing a molecule according to any one of claims 1 to 24, comprising the steps of: a) chemically synthesizing at least one protein-based carrier component and / or molecule or part of said molecule according to any one of claims 1 to 24, preferably by using solid phase peptide synthesis, b) optionally isolating and / or purifying said at least one protein-based carrier component synthesized in a) and / or said molecule or part of said molecule; c) optionally conjugating one or more (further) cargoes to said attachment points or conjugation sites of said protein-based carrier component; A method comprising:

30. A molecule according to any one of claims 1 to 24 or a composition according to claim 27 for use in medicine.

31. A molecule according to any one of claims 1 to 24 or a composition according to claim 27 for use in the prophylactic and / or therapeutic treatment of autoimmune / inflammatory diseases, infectious diseases and / or cancer, such as hematological (blood) and solid tumour cancer diseases.

32. A molecule according to any one of claims 1 to 24 or a composition according to claim 27 for use as a vaccine.

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