Protein-based coupling vectors

Abstract

The present invention relates to the field of drug delivery and provides molecules comprising or consisting of at least one protein-based carrier building module wherein the protein-based carrier building module comprises at least one, preferably at least two, connection or coupling sites. In particular, the present invention provides a molecule comprising at least one protein-based building module wherein the at least one protein-based building module: a) comprises at least one coupling site or connection point; b) a molecular weight of from 2.5 to 70 kDa; c) having a spherical three-dimensional (3D) structure; d) having a solubility of 10 mg / mL or greater, measured in aqueous solution at room temperature; and does not specifically bind to any human protein, or binds to one or more human proteins at a KD value of greater than 5 x 10 <-4 > mol / L.

Description

Technical Field

[0001] The present invention provides molecules comprising or consisting of at least one protein-based carrier building block, wherein the protein-based carrier building block comprises at least one, preferably at least two, attachment points or coupling sites.

[0002] The present invention also relates to nucleic acids encoding such molecules or portions of such molecules; to host cells comprising such nucleic acids and / or expressing or capable of expressing such molecules or portions of such molecules; to compositions, in particular pharmaceutical compositions, comprising such molecules, nucleic acids and / or host cells; and to the use of such molecules, nucleic acids, host cells and / or compositions, in particular for labeling, prophylactic, therapeutic and / or diagnostic purposes. Background Art

[0003] Protein-based therapeutics (referred to as "biologics") are creating new therapeutic strategies that are difficult to achieve with typical small molecule-based therapeutics. A rapidly developing field includes coupling-based therapeutics, such as antibody-drug conjugates (ADCs). For example, Fatima SW. and Khare SK. ("Benefits and challenges of antibody-drug conjugates as novel form of chemotherapy", J Control Release, 2022, 341: 555-565) reviewed the latest clinical progress of each component (antibody / linker / payload) of ADC and how a single component affects the activity of ADC. This is also shown in, for example, Rader, C., "Chemically programmed antibodies", Trends in Biotechnology, 2014, 32 (4). Antibody-drug conjugates generally rely on the high specificity and affinity of antibodies, their extended circulation half-life, and other effector functions (e.g., complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP)). Thus, antibody-drug conjugates and related molecules (such as chemically programmed antibodies) are selected for at least one of these functions (their targeting properties, half-life extension properties, and / or other effector functions, as described above).

[0004] At present, the manufacture of biological preparations mainly relies on recombinant production, and is therefore limited to the production of polypeptides that can be naturally combined into a functional unit (i.e., IgG). Typical coupling strategies are based on site-specific cysteine or random lysine coupling (see, for example, Zhou et al., 2021, Pharmaceuticals, 14: 672; Sadiki et al., 2020, Antibody Therapeutics, 3: 271). The emergence of atypical or novel amino acids (nAA) further increases the choice of coupling methods (see, for example, Malyshev and Romesberg, 2015, Angew Chem Int Ed Engl., 54 (41); Zhang et al., 2017, PNAS, 114 1317).

[0005] Additional therapeutic strategies that allow for universal production and cargo conjugation are needed. Summary of the Invention

[0006] Existing technologies aim to simplify the production of conjugation-based therapeutics and / or create plug-and-play strategies that can create alternative formats that allow universal conjugation of different cargoes.

[0007] Although classical coupling strategies need to focus on retaining the function of the polypeptide involved (as described above), the present invention adopts a carrier building block based on non-targeted proteins, which is only used as a site-specific coupling medium (protein-based carrier building block). This protein-based carrier building block can be included in a gene construct and is therefore the product of a manufacturing activity, or alternatively can be produced separately (e.g., recombinant production or production by alternative means, such as solid phase peptide synthesis, i.e., SPPS), and then connected to the active and / or targeting moiety (i.e., cargo), as will be explained in detail below. The latter strategy provides greater freedom for cargo coupling conditions of protein-based carrier building blocks. This freedom can be converted into the use of site-specific coupling to common amino acids that are usually used in a random manner (such as lysine), or into coupling conditions that may damage the function / quality of the targeting building block, or into alternative production platforms (such as chemical synthesis). The position and number of coupling sites or connection points can be engineered / adjusted according to the specific application.

[0008] Therefore, the present invention provides molecules comprising at least one protein-based carrier building block or consisting of at least one protein-based carrier building block, and provide such protein-based carrier building blocks (as described below), wherein the protein-based carrier building block comprises at least one connection point or coupling site, preferably at least two connection points or coupling sites. The at least one coupling site or connection point is suitable for coupling or attachment of a load to the protein-based carrier building block. A "load" is any molecule that is attached or coupled to / can be attached or coupled to the protein-based carrier building block by one or more connection points or one or more coupling sites present therein. For example, the load that can be attached or coupled to the protein-based carrier building block of the present invention is a protein, peptide, polyethylene glycol (PEG), a small molecule, a polysaccharide, a lipid, a chelating agent, a fluorophore, a radioisotope, a vitamin (such as folic acid or biotin), a nucleic acid (such as an oligonucleotide or siRNA), etc. Thus, in a further embodiment, the present invention provides a protein-based carrier building block attached to at least one cargo as described herein, i.e. a molecule of the invention comprises (or alternatively consists of) at least one protein-based building block and at least one cargo attached or coupled thereto via said at least one coupling site or connection point.

[0009] The protein-based carrier building blocks of the present invention comprise (and preferably consist of) at least a portion of a protein, preferably an entire protein. Thus, preferably, the protein-based carrier building blocks are polypeptides. The protein-based carrier building blocks have a globular 3D structure and are soluble. Furthermore, the size (molecular mass or molecular weight, MW) of the protein-based carrier building blocks contained in the molecules of the present invention is from about 2.5 to about 70 kDa, preferably from about 2.5 to less than 50 kDa, more preferably from about 2.5 to about 30 kDa, even more preferably from about 2.5 to about 16 kDa, such as about 6 kDa, or about 7 kDa, or about 16 kDa.

[0010] Finally, the protein-based vector building blocks of the present invention do not specifically bind to any human protein, although they may show non-specific binding to one or more human proteins, as explained in detail in this application. In this case, the protein-based vector building blocks may bind to human proteins with low specificity and / or low selectivity, as defined in this application. Preferably, the protein-based vector building blocks also do not specifically bind to any non-protein (preferably human) molecules, such as DNA, RNA, lipids (e.g., phosphatidylserine (PS)) or polysaccharides. The protein-based vector building block can be derived from a target binding protein (e.g., an immunoglobulin single variable domain (ISVD), a DARPin, an affibody or an affitin), as described below. It can also be derived from other proteins that show specific binding to, for example, human proteins (e.g., globular human proteins). This is a so-called "protein-based vector building block precursor." In these cases, preferably, the protein-based building block also does not specifically bind to any molecule (including non-human proteins) to which the protein-based vector building block precursor specifically binds (if any). For example, if the precursor of the protein-based carrier building block is an anti-RSV (respiratory syncytial virus) ISVD, then the protein-based carrier building block preferably does not specifically bind RSV. Therefore, preferably, if the precursor has a target and if the target is a non-human molecule (such as a non-human protein) or a human non-protein molecule (such as human DNA, RNA, polysaccharide, lipid, etc.), then the protein-based carrier building block does not specifically bind the target of the precursor either. In a further preferred embodiment, when the load is coupled to at least one, preferably at least two connection points or coupling sites on the protein-based carrier building block, the protein-based carrier building block does not specifically bind to any human protein, non-human protein and / or non-protein molecule.

[0011] Thus, at least one protein-based vector building block of the present invention:

[0012] a) having at least one attachment point (also referred to as coupling site in this specification), preferably at least two attachment points or coupling sites, wherein the attachment point or coupling site is a reactive group in the side chain of an unnatural or natural amino acid (e.g., Cys, Lys, Tyr, Orn, etc.), preferably located at a solvent-accessible position in the protein-based carrier building block, and / or the N-terminal primary amine and / or C-terminal carboxyl group of the protein-based carrier building block (if these are available). Thus, the at least one, preferably at least two, attachment point or coupling site is preferably located at a solvent-accessible position in the protein-based carrier building block;

[0013] b) has a size (molecular weight) of about 2.5 to about 70 kDa, preferably about 2.5 to about 50 kDa, such as about 2.5 to less than 50 kDa, more preferably about 2.5 to about 30 kDa, even more preferably about 2.5 to about 16 kDa;

[0014] c) has a solubility of about 10 mg / mL or greater, as measured in an aqueous solution at room temperature (RT), preferably in a 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 in a histidine buffer at pH 6.5 comprising histidine (10 mM to 100 mM, such as 10 mM), sucrose (1% to 10%, such as 10%), and optionally Tween 80 (0.001% to 1%, such as 0.01%) at RT, or in a phosphate buffer at pH 7.0 comprising NaH2PO4 / Na2HPO4 (10 and 50 mM, such as 10 mM), sodium chloride (NaCl) (100-150 mM, such as 130 mM NaCl), and optionally Tween 80 (0.001% to 1%, such as 0.01%). 80 (0.001% to 1%, such as 0.01%)) measured at room temperature, preferably wherein the buffer is 5 mM citrate buffer or PBS at a pH of 7.0 or 7.4;

[0015] d) having a spherical 3D structure, as described below;

[0016] e) does not specifically bind to any human protein (or binds to proteins with a specific binding affinity greater than 5x10 -4 mol / L K D (K D value) binds to one or more human proteins), preferably it also does not specifically bind to the target of the precursor (or binds to one or more human proteins at a rate greater than 5x10 -4 mol / L K D (K D value) binds to the precursor target, which may be a non-human protein or non-protein molecule), and preferably it also does not specifically bind to any non-protein molecule such as nucleic acids (e.g. DNA, RNA), lipids or glycans, preferably it does not specifically bind to any human non-protein molecule (or binds to any non-protein molecule at a rate greater than 5x10 -4 mol / L K D (K DPreferably, it also 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 any), as determined, for example, by a cell binding assay or by surface plasmon resonance (SPR), as described, for example, in this application and / or in Ober et al. 2001, Intern. Immunology 13: 1551-1559, or to any human cell or to a concentration greater than 5x10 -4 mol / L K D (K D value) binds to one or more human cells, said binding preferably being determined by a cell binding assay or by SPR;

[0017] f) optionally, does not specifically bind to any (non-human) molecule to which the protein-based vector building block precursor specifically binds, such as protein F of RSV, or to a concentration greater than 5x10 -4 mol / L K D (K D (V) binding to any (non-human) molecule to which a protein-based vector building block precursor specifically binds, such as protein F of RSV, said binding preferably being determined by a cell binding assay or by SPR;

[0018] g) optionally, does not specifically bind to any human cells and / or cell types, or binds to a specific antigen at a concentration greater than 5×10 -4 mol / L K D (K D value) binding to human cells and / or cell types, said binding preferably being determined by a cell binding assay;

[0019] h) optionally, does not specifically bind to any microorganism (such as bacteria, fungi, protists, yeasts and / or any viruses), or binds to a concentration greater than 5×10 -4 mol / L K D (K D value) binds to microorganisms (such as bacteria, fungi, protists, yeasts and / or viruses), said binding preferably being determined by a cell binding assay and / or SPR as described herein;

[0020] i) 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 yeast)), or binds to any biomolecule with a specific binding capacity greater than 5×10 -4 mol / L K D (K Dvalue) binding to biomolecules (including human and non-human biomolecules), said binding preferably being determined by a cell binding assay and / or SPR as described herein;

[0021] j) optionally, when it has at least one cargo attached thereto (via at least one coupling site or connection point contained therein), does not specifically bind to any biomolecule (including human and non-human biomolecules, such as plant biomolecules, viral biomolecules and / or microbial biomolecules (such as bacteria, fungi, protists and / or yeasts)), or binds to any biomolecule at a rate greater than 5×10 -4 mol / L K D (K D value) binding to biomolecules (including human and non-human biomolecules), said binding preferably being determined by a cell binding assay and / or SPR as described herein;

[0022] k) optionally, does not comprise or consist of an amino acid sequence selected from SEQ ID NOs.: 1-34 shown in Table A-1 and Table A-2 of WO 2016 / 055656 and / or SEQ ID NOs.: 1-12 shown in Table A-1 of WO 2010 / 139808; and

[0023] l) Optionally, does not comprise or consists of the amino acid sequence defined by SEQ ID NO.: 214.

[0024] In a first aspect, the present invention relates to a molecule comprising at least one protein-based carrier building block, wherein said at least one protein-based carrier building block:

[0025] a) comprises at least one coupling site or attachment point, preferably at least two attachment sites or coupling sites;

[0026] b) has a molecular weight of about 2.5 to about 70 kDa, preferably about 2.5 to about 50 kDa, such as about 2.5 kDa to less than 50 kDa, more preferably about 2.5 to about 30 kDa, even more preferably about 2.5 to about 16 kDa;

[0027] c) has a spherical 3D structure;

[0028] d) has a solubility of 10 mg / mL or greater, measured in an aqueous solution at room temperature, preferably in a buffer or water at room temperature, more preferably in a buffer such as citrate buffer or phosphate buffered saline (PBS) at pH 7.0 or 7.4 at room temperature, or in a histidine buffer at pH 6.5 comprising histidine (10 mM to 100 mM, such as 10 mM), sucrose (1% to 10%, such as 10%), and optionally Tween 80 (0.001% to 1%, such as 0.01%) at room temperature, or in a phosphate buffer at pH 7.0 comprising NaH2PO4 / Na2HPO4 (10 and 50 mM, such as 10 mM), sodium chloride (NaCl) (100-150 mM, such as 130 mM NaCl), and optionally Tween 80 (0.001% to 1%, such as 0.01%) at room temperature;

[0029] e) does not specifically bind to any human protein or binds to any protein with a specific binding affinity greater than 5x10 -4 mol / L K D (K D value) binds to one or more human proteins, said binding preferably being determined by a cell binding assay or by surface plasmon resonance (SPR), e.g. as described herein and / or in Ober et al. 2001, Intern. Immunology 13: 1551-1559, or does not specifically bind to any human cell or binds to a specific concentration of more than 5x10 -4 mol / L K D (K D value) binds to one or more human cells, said binding preferably being as determined by a cell binding assay or by SPR;

[0030] f) optionally, does not specifically bind to any (non-human) molecule to which the protein-based vector building block precursor specifically binds, such as protein F of RSV, or to a concentration greater than 5x10 -4 mol / L K D (K D (e) binding to any (non-human) molecule to which a protein-based vector building block precursor specifically binds, such as protein F of RSV, said binding preferably as determined by a cell binding assay or by SPR;

[0031] g) optionally, does not specifically bind to any human cells and / or cell types, or binds to a specific antigen at a concentration greater than 5×10 -4 mol / L K D (K D value) binding to human cells and / or cell types, said binding preferably being determined by a cell binding assay;

[0032] h) optionally, does not specifically bind to any microorganism (such as bacteria, fungi, protists, yeasts and / or any viruses), or binds to a concentration greater than 5×10 -4 mol / L K D (K D value) binds to microorganisms (such as bacteria, fungi, protists, yeasts and / or viruses), said binding preferably being determined by a cell binding assay and / or SPR as described herein;

[0033] i) 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 binds to any biomolecule with a specific binding capacity greater than 5×10 -4 mol / L K D (K D value) binding to biomolecules (including human and non-human biomolecules), said binding preferably being determined by a cell binding assay and / or SPR as described herein;

[0034] j) optionally, when it has at least one cargo attached thereto (via at least one coupling site or connection point contained therein), does not specifically bind to any biomolecule (including human and non-human biomolecules, such as plant biomolecules, viral biomolecules and / or microbial biomolecules (such as bacteria, fungi, protists and / or yeasts)), or binds to any biomolecule at a rate greater than 5×10 -4 mol / L K D (K D value) binding to biomolecules (including human and non-human biomolecules), said binding preferably being determined by a cell binding assay and / or SPR as described herein;

[0035] k) optionally, does not comprise or consist of an amino acid sequence selected from SEQ ID NOs.: 1-34 shown in Table A-1 and Table A-2 of WO 2016 / 055656 and / or SEQ ID NOs.: 1-12 shown in Table A-1 of WO 2010 / 139808; and

[0036] a) Optionally, it does not comprise or consists of the amino acid sequence defined by SEQ ID NO.: 214 (EVQLQASGGGLAQPGGSLRLSVTVSGSIDVINNMAWYRQAPGNARELV ATITSGFSTNYASSVKGRFTISRDNAKKAVYLQMNSLKPEDTADYYSKVHLIRLGAARAYDYWGQGTQVTVS).

[0037] Preferably, in the molecules of the invention, the at least one protein-based carrier building block does not specifically bind to any non-protein molecule, for example any human non-protein molecule such as human DNA, human RNA, human lipids or human glycans.

[0038] The molecules of the present invention may comprise more than one protein-based carrier building block, for example, two, three, four, five, six or more protein-based carrier building blocks. These protein-based carrier building blocks may be directly linked to each other, or linked to each other via linkers as described herein.

[0039] Preferably, at least one protein-based carrier building block comprised in a molecule of the invention comprises more than one coupling site or attachment point, preferably at least two coupling sites or attachment points (such as two coupling sites or attachment points), or at least three coupling sites or attachment points (such as three, four, five, six, seven, eight or nine coupling sites or attachment points), which coupling sites or attachment points are preferably reactive groups present in the side chains of natural or unnatural amino acids comprised in the protein-based carrier building block, or may be (additionally or alternatively) the N-terminal primary amine and / or the C-terminal carboxylic acid group of the protein-based building block.

[0040] For example, at least one coupling site or attachment point comprised in the at least one protein-based carrier building block is a free or blocked sulfhydryl group, a free or blocked hydroxyl group and / or a free or blocked primary amine. In a further embodiment, at least one coupling site or attachment point comprised in the at least one protein-based carrier building block 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 a further embodiment, the at least one protein-based building block is comprised in (GG) or (G4S1) 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 -), an N-terminal and / or C-terminal Cys, and / or an N-terminal and / or C-terminal Tyr before or after the N-terminal and / or C-terminal Cys.

[0041] Preferably, at least one protein-based carrier building block present in a molecule of the invention is (i) a small globular non-human protein-based building block, such as an ISVD-based building block, a DARPin-based building block, an affibody-based building block or an affitin-based building block, or (ii) a small globular human protein-based building block, such as cyclin-dependent kinase subunit 1 (CDK-1).

[0042] In one embodiment, the at least one protein-based building block is derived from a heavy chain ISVD, preferably from a V H 、V HH , including camel-derived V H or humanized V HH In another embodiment, the at least one protein-based building block is derived from a protein-based building block belonging to "V H 3" ISVD, preferably wherein the resulting building block comprises 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.

[0043] In another embodiment, the at least one protein-based building block is derived from RSV001A04 (SEQ ID NO.: 179).

[0044] In one embodiment, the at least one protein-based building block is an ISVD-based building block comprising Leu or Gin (preferably Leu) at position 108 according to the Kabat numbering, preferably wherein 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.

[0045] In another embodiment, the at least one protein-based building block comprises or consists of: SEQ ID NO.: 186:

[0046] X1VX2LX3EX4X5GX6X7X8X9X 10 X 11 GX 12 X 13 X 14 IX 15 CX 16 AX 17 X 18 X 19 X 20 LX 21 X22 X 23 VLGWFRX 24 AX25X 26 X 27 X 28 X 29 X 30 FVAAINX 31 X 32 X 33 X 34 X 35 X 36 X 37 X 38 PX 39 X 40 VX 41 X 42 X 43 FX 44 IX 45 X 46 X 47 X 48 X 49 X 50 X 51 TGX 52 LX 53 MX 54 X 55 LX 56 X 57 X 58 DX 59 AX 60 YX 61 CGAGX 62 PX 63 X 64 X 65 X 66 AYX 67 X 68 X 69 X 70 SYX 71 X 72 X 73 GX 74 X 75 TX 76 VX 77 VX 78 X 79 X 80 X 81 X 82 ,

[0047] in

[0048] 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;

[0049] X2 (position 3 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, such as cysteine;

[0050] X3 (position 5 according to Kabat numbering) can be Val or any amino acid having a reactive group in its side chain, such as cysteine;

[0051] X4 (position 7 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0052] X5 (position 8 according to Kabat numbering) can be Gly or any amino acid with a reactive group in its side chain, such as cysteine;

[0053] X6 (position 10 according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as cysteine;

[0054] 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;

[0055] X8 (position 12 according to Kabat numbering) can be Val or any amino acid having a reactive group in its side chain, such as cysteine;

[0056] X9 (position 13 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, such as cysteine;

[0057] X 10 (position 14 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, such as cysteine;

[0058] X 11 (position 15 according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as cysteine;

[0059] X 12 (position 17 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0060] X 13(position 18 according to Kabat numbering) can be Leu or any amino acid having a reactive group in its side chain, such as cysteine;

[0061] X 14 (position 19 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0062] X 15 : (position 21 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0063] X 16 : (position 23 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, such as cysteine;

[0064] X 17 : (position 25 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0065] X 18 : (position 26 according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as cysteine;

[0066] X 19 : (position 27 according to Kabat numbering) can be Gly or any amino acid with a reactive group in its side chain, such as cysteine;

[0067] X 20 : (position 28 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0068] X 21 : (position 30 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0069] X 22 : (position 31 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine;

[0070] X 23 : (position 32 according to Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, such as cysteine;

[0071] X 24: (position 39 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, such as cysteine;

[0072] X 25 : (position 41 according to Kabat numbering) can be Pro or any amino acid with a reactive group in its side chain, such as cysteine;

[0073] X 26 : (position 42 according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as cysteine;

[0074] X 27 : (position 43 according to Kabat numbering) can be Lys or any amino acid having a reactive group in its side chain, such as cysteine;

[0075] X 28 : (position 44 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, such as cysteine;

[0076] X 29 : (position 45 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, such as cysteine;

[0077] X 30 : (position 46 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, such as cysteine;

[0078] X 31 : (position 52a according to Kabat numbering) can be Trp or any amino acid having a reactive group in its side chain, such as cysteine;

[0079] X 32 : (position 53 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, such as cysteine;

[0080] X 33 : (position 54 according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as cysteine;

[0081] X 34 : (position 55 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as cysteine;

[0082] X 35: (position 56 according to Kabat numbering) can be He or any amino acid having a reactive group in its side chain, such as cysteine;

[0083] X 36 : (position 57 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, such as cysteine;

[0084] X 37 : (position 58 according to Kabat numbering) can be He or any amino acid having a reactive group in its side chain, such as cysteine;

[0085] X 38 : (position 59 according to Kabat numbering) can be Gly or any amino acid with a reactive group in its side chain, such as cysteine;

[0086] X 39 : (position 61 according to Kabat numbering) can be Pro or any amino acid with a reactive group in its side chain, such as cysteine;

[0087] X 40 : (position 62 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine;

[0088] X 41 : (position 64 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, such as cysteine;

[0089] X 42 : (position 65 according to Kabat numbering) can be Gly or any amino acid with a reactive group in its side chain, such as cysteine;

[0090] X 43 : (position 66 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, such as cysteine;

[0091] X 44 : (position 68 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, such as cysteine;

[0092] X 45 : (position 70 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0093] X 46: (position 71 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, such as cysteine;

[0094] X 47 : (position 72 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as cysteine;

[0095] X 48 : (position 73 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine;

[0096] X 49 : (position 74 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, such as cysteine;

[0097] X 50 : (position 75 according to Kabat numbering) can be Lys or any amino acid with a reactive group in its side chain, such as cysteine;

[0098] X 51 : (position 76 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine;

[0099] X 52 : (position 79 according to Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, such as cysteine;

[0100] X 53 : (position 81 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, such as cysteine;

[0101] X 54 : (position 82a according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine;

[0102] X 55 : (position 82b according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0103] X 56 : (position 83 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, such as cysteine;

[0104] X 57: (position 84 according to Kabat numbering) can be Pro or any amino acid with a reactive group in its side chain, such as cysteine;

[0105] X 58 : (position 85 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as cysteine;

[0106] X 59 : (position 87 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, such as cysteine;

[0107] 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 Ile, preferably Leu or Val, or any other amino acid having a reactive group in its side chain, such as cysteine;

[0108] X 61 : (position 91 according to Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, such as cysteine;

[0109] X 62 : (position 96 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, such as cysteine;

[0110] X 63 : (position 98 according to Kabat numbering) can be Leu or any amino acid with a reactive group in its side chain, such as cysteine;

[0111] X 64 : (position 99 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine;

[0112] X 65 : (position 100 according to Kabat numbering) can be Pro or any amino acid with a reactive group in its side chain, such as cysteine;

[0113] X 66 : (position 100a according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as cysteine;

[0114] X 67: (position 100d according to Kabat numbering) can be He or any amino acid having a reactive group in its side chain, such as cysteine;

[0115] X 68 : (position 100e according to Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, such as cysteine;

[0116] X 69 : (position 100f according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as cysteine;

[0117] 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;

[0118] X 71 : (position 101 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as cysteine;

[0119] X 72 : (position 102 according to Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, such as cysteine;

[0120] X 73 : (position 103 according to Kabat numbering) can be Trp or any amino acid having a reactive group in its side chain, such as cysteine;

[0121] X 74 : (position 105 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, such as cysteine;

[0122] X 75 : (position 106 according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as cysteine;

[0123] 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, such as cysteine;

[0124] X 77 : (position 110 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, such as cysteine;

[0125] X 78 : (position 112 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0126] X 79 : (position 113 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0127] X 80 : absent or Gly;

[0128] X 81 : absent or Gly;

[0129] X 82 : does not exist or is Cys,

[0130] or a sequence having 80% or greater identity to SEQ ID NO.: 186, preferably a sequence having 85% or greater, 90% or greater, 95% or greater, 97% or greater or 99% or greater sequence identity to SEQ ID NO.: 186, 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, such as 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, such as 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 as described herein.

[0131] In another embodiment, the 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.

[0132] SEQ ID NO.: 187:

[0133] DLGKKLLEAARAGQDDEVRILMANGADVNAHDTFGFTPLHLAALYGHLEIVEVLLKNGADVNADDSYGRTPLHLAAMRGHLEIVEVLLKYGADVNAADEEGRTPLHLAAKRGHLEIVEVLLKNGADVNAQDKFGKTAFDISIDNGNEDLAEILQKL

[0134] In one embodiment, the protein-based building block is a DARPin-based building block comprising or alternatively consisting of: SEQ ID NO.: 188:

[0135] X1X2GX3X4LLX5AAX6X7X8X9X 10 X 11 X 12 VX 13 X 14 LMX 15 X 16 X 17 AX 18 VX 19 AX 20 X 21 X 22 X 23 GX 24 TPLHLAAX 25 X 26 X 27 X 28 X 29 X 30 IVX 31 VLLX 32 X 33 X 34 AX 35 VX 36 AX 37 DX 38 X 39 GATPLHLAAX 40 X 41 X 42 X 43 X 44 X 45 IVX 46 VLLX 47 X 48 X 49 AX 50 VX 51 AX 52 DX 53 X 54 GATPLHX 55 AAX 56 X 57 X 58 X 59 X 60 X 61 IVX 62 X 63 LX 64 X 65 X 66 X 67 AX 68 X69 X 70 AX 71 DX 72 X 73 X 74 X 75 TAX 76 X 77 ISX 78 X 79 X 80 X 81 X 82 X 83 X 84 LAX 85 X 86 LX 87 X 88 X 89 X 90 ,

[0136] in:

[0137] X1 can be Asp or any amino acid with a reactive group in its side chain, such as cysteine;

[0138] X2 can be Leu or any amino acid with a reactive group in its side chain, such as cysteine;

[0139] X3 can be Lys or any amino acid with a reactive group in its side chain, such as cysteine;

[0140] X4 can be Lys or any amino acid with a reactive group in its side chain, such as cysteine;

[0141] X5 can be Glu or any amino acid with a reactive group in its side chain, such as cysteine;

[0142] X6 can be Arg or any amino acid with a reactive group in its side chain, such as cysteine;

[0143] X7 can be Ala or any amino acid with a reactive group in its side chain, such as cysteine;

[0144] X8 can be Gly or any amino acid with a reactive group in its side chain, such as cysteine;

[0145] X9 can be Gln or any amino acid with a reactive group in its side chain, such as cysteine;

[0146] X 10 It can be Asp or any amino acid with a reactive group in its side chain, such as cysteine;

[0147] X 11It can be Asp or any amino acid with a reactive group in its side chain, such as cysteine;

[0148] X 12 It can be Glu or any amino acid with a reactive group in its side chain, such as cysteine;

[0149] X 13 It can be Arg or any amino acid with a reactive group in its side chain, such as cysteine;

[0150] X 14 It can be Ile or any amino acid with a reactive group in its side chain, such as cysteine;

[0151] X 15 It can be Ala or any amino acid with a reactive group in its side chain, such as cysteine;

[0152] X 16 It can be Asn or any amino acid with a reactive group in its side chain, such as cysteine;

[0153] X 17 It can be Gly or any amino acid with a reactive group in its side chain, such as cysteine;

[0154] X 18 It can be Asp or any amino acid with a reactive group in its side chain, such as cysteine;

[0155] X 19 It can be Asn or any amino acid with a reactive group in its side chain, such as cysteine;

[0156] X 20 It can be His or any amino acid with a reactive group in its side chain, such as cysteine;

[0157] X 21 It can be Asp or any amino acid with a reactive group in its side chain, such as cysteine;

[0158] X 22 It can be Thr or any amino acid with a reactive group in its side chain, such as cysteine;

[0159] X 23 It can be Phe or any amino acid with a reactive group in its side chain, such as cysteine;

[0160] X 24 It can be Phe or any amino acid with a reactive group in its side chain, such as cysteine;

[0161] X 25It can be Leu or any amino acid with a reactive group in its side chain, such as cysteine;

[0162] X 26 It can be Tyr or any amino acid with a reactive group in its side chain, such as cysteine;

[0163] X 27 It can be Gly or any amino acid with a reactive group in its side chain, such as cysteine;

[0164] X 28 Can be His or any amino acid with a reactive group in its side chain, such as cysteine

[0165] X 29 Can be Leu or any amino acid with a reactive group in its side chain, such as cysteine

[0166] X 30 Can be Glu or any amino acid with a reactive group in its side chain, such as cysteine

[0167] X 31 Can be Glu or any amino acid with a reactive group in its side chain, such as cysteine

[0168] X 32 Can be Lys or any amino acid with a reactive group in its side chain, such as cysteine

[0169] X 33 Can be Asn or any amino acid with a reactive group in its side chain, such as cysteine

[0170] X 34 Can be Gly or any amino acid with a reactive group in its side chain, such as cysteine

[0171] X 35 Can be Asp or any amino acid with a reactive group in its side chain, such as cysteine

[0172] X 36 Can be Asn or any amino acid with a reactive group in its side chain, such as cysteine

[0173] X 37 Can be Asp or any amino acid with a reactive group in its side chain, such as cysteine

[0174] X 38 Can be Ser or any amino acid with a reactive group in its side chain, such as cysteine

[0175] X 39It can be Tyr or any amino acid with a reactive group in its side chain, such as cysteine;

[0176] X 40 It can be Met or any amino acid with a reactive group in its side chain, such as cysteine;

[0177] X 41 It can be Arg or any amino acid with a reactive group in its side chain, such as cysteine;

[0178] X 42 It can be Gly or any amino acid with a reactive group in its side chain, such as cysteine;

[0179] X 43 It can be His or any amino acid with a reactive group in its side chain, such as cysteine;

[0180] X 44 It can be Leu or any amino acid with a reactive group in its side chain, such as cysteine;

[0181] X 45 It can be Glu or any amino acid with a reactive group in its side chain, such as cysteine;

[0182] X 46 It can be Glu or any amino acid with a reactive group in its side chain, such as cysteine;

[0183] X 47 It can be Lys or any amino acid with a reactive group in its side chain, such as cysteine;

[0184] X 48 It can be Tyr or any amino acid with a reactive group in its side chain, such as cysteine;

[0185] X 49 It can be Gly or any amino acid with a reactive group in its side chain, such as cysteine;

[0186] X 50 It can be Asp or any amino acid with a reactive group in its side chain, such as cysteine;

[0187] X 51 It can be Asn or any amino acid with a reactive group in its side chain, such as cysteine;

[0188] X 52 It can be Ala or any amino acid with a reactive group in its side chain, such as cysteine;

[0189] X 53It can be Glu or any amino acid with a reactive group in its side chain, such as cysteine;

[0190] X 54 It can be Glu or any amino acid with a reactive group in its side chain, such as cysteine;

[0191] X 55 It can be Leu or any amino acid with a reactive group in its side chain, such as cysteine;

[0192] X 56 It can be Lys or any amino acid with a reactive group in its side chain, such as cysteine;

[0193] X 57 It can be Ala or any amino acid with a reactive group in its side chain, such as cysteine;

[0194] X 58 It can be Gly or any amino acid with a reactive group in its side chain, such as cysteine;

[0195] X 59 It can be His or any amino acid with a reactive group in its side chain, such as cysteine;

[0196] X 60 It can be Leu or any amino acid with a reactive group in its side chain, such as cysteine;

[0197] X 61 It can be Glu or any amino acid with a reactive group in its side chain, such as cysteine;

[0198] X 62 It can be Glu or any amino acid with a reactive group in its side chain, such as cysteine;

[0199] X 63 It can be Val or any amino acid with a reactive group in its side chain, such as cysteine;

[0200] X 64 It can be Leu or any amino acid with a reactive group in its side chain, such as cysteine;

[0201] X 65 It can be Lys or any amino acid with a reactive group in its side chain, such as cysteine;

[0202] X 66 It can be Asn or any amino acid with a reactive group in its side chain, such as cysteine;

[0203] X 67It can be Gly or any amino acid with a reactive group in its side chain, such as cysteine;

[0204] X 68 It can be Asp or any amino acid with a reactive group in its side chain, such as cysteine;

[0205] X 69 It can be Val or any amino acid with a reactive group in its side chain, such as cysteine;

[0206] X 70 It can be Asn or any amino acid with a reactive group in its side chain, such as cysteine;

[0207] X 71 It can be Gln or any amino acid with a reactive group in its side chain, such as cysteine;

[0208] X 72 It can be Lys or any amino acid with a reactive group in its side chain, such as cysteine;

[0209] X 73 It can be Phe or any amino acid with a reactive group in its side chain, such as cysteine;

[0210] X 74 It can be Gly or any amino acid with a reactive group in its side chain, such as cysteine;

[0211] X 75 It can be Lys or any amino acid with a reactive group in its side chain, such as cysteine;

[0212] X 76 It can be Phe or any amino acid with a reactive group in its side chain, such as cysteine;

[0213] X 77 It can be Asp or any amino acid with a reactive group in its side chain, such as cysteine;

[0214] X 78 It can be Ile or any amino acid with a reactive group in its side chain, such as cysteine;

[0215] X 79 It can be Asp or any amino acid with a reactive group in its side chain, such as cysteine;

[0216] X 80 It can be Asn or any amino acid with a reactive group in its side chain, such as cysteine;

[0217] X 81It can be Gly or any amino acid with a reactive group in its side chain, such as cysteine;

[0218] X 82 It can be Asn or any amino acid with a reactive group in its side chain, such as cysteine;

[0219] X 83 It can be Glu or any amino acid with a reactive group in its side chain, such as cysteine;

[0220] X 84 It can be Asp or any amino acid with a reactive group in its side chain, such as cysteine;

[0221] X 85 It can be Glu or any amino acid with a reactive group in its side chain, such as cysteine;

[0222] X 86 It can be Ile or any amino acid with a reactive group in its side chain, such as cysteine;

[0223] X 87 It can be Gln or any amino acid with a reactive group in its side chain, such as cysteine;

[0224] X 88 Can be Lys or any amino acid with a reactive group in its side chain, such as cysteine

[0225] X 89 does not exist or is Leu;

[0226] X 90 Does not exist or is Cys,

[0227] or a sequence having 80% or greater identity to SEQ ID NO.: 188, preferably a sequence having 85% or greater, 90% or greater, 95% or greater, 97% or greater, or 99% or greater sequence identity to SEQ ID NO.: 188, provided 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, such as 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, such as 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 as described herein, in particular does not specifically bind to the human KRAS protein (GTPase KRas, EC: 3.6.5.2, primary accession number P01116, see also Lim S. et al., "Exquisitely specific anti-KRAS biodegraders inform on the cellular prevalence of nucleotide-loaded states”, ACS Cent. Sci. 2021, 7, 2, 274-291).

[0228] In another embodiment, said at least one protein-based building block is a small globular human protein-based building block, preferably derived from the polypeptide defined by SEQ ID NO.:190.

[0229] SEQ ID NO.: 190

[0230] SHKQIYYSDKYDDEEFEYRHVMLPKDIAKLVPKTHLMSESEWRNLGVQQSQGWVHYMIHEPEPHILLFRRPLPKKPKK

[0231] In one embodiment, the protein-based building block is a small globular human protein-based building block comprising or alternatively consisting of: SEQ ID NO.: 191:

[0232] X1X2X3X4IX5X6SX7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 VX 19 LPX 20 X 21 X22 AX 23 X 24 VX 25 X 23b X 24b X 25 b X 26 MX 27 X 28 X 29 X 30 WX 31 X 32 LX 33 VX 34 QX 35 X 36 X 37 WX 38 HX 39 X 40 X 41 X 42 X 43 X 44 X 45 X 46 X 47 ILLFX 48 X 49 X 50 X 51 X 52 X53X 54 X 55 X 56 X 57 ,

[0233] in

[0234] X1 can be Ser or any amino acid with a reactive group in its side chain, such as cysteine;

[0235] X2 can be His or any amino acid with a reactive group in its side chain, such as cysteine;

[0236] X3 can be Lys or any amino acid with a reactive group in its side chain, such as cysteine;

[0237] X4 can be Gln or any amino acid with a reactive group in its side chain, such as cysteine;

[0238] X5 can be Tyr or any amino acid with a reactive group in its side chain, such as cysteine;

[0239] X6 can be Tyr or any amino acid with a reactive group in its side chain, such as cysteine;

[0240] X7 can be Asp or any amino acid with a reactive group in its side chain, such as cysteine;

[0241] X8 can be Lys or any amino acid with a reactive group in its side chain, such as cysteine;

[0242] X9 can be Tyr or any amino acid with a reactive group in its side chain, such as cysteine;

[0243] X 10 Can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X 11 Can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X 12 Can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X 13 Can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X 14 Can be Phe or any amino acid with a reactive group in its side chain, such as cysteine; X 15 Can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X 16 Can be Tyr or any amino acid with a reactive group in its side chain, such as cysteine; X 17 Can be Arg or any amino acid with a reactive group in its side chain, such as cysteine; X 18 Can be His or any amino acid with a reactive group in its side chain, such as cysteine; X 19 Can be Met or any amino acid having a reactive group in its side chain, such as cysteine; X 20 Can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X 21 Can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X 22 Can be He or any amino acid having a reactive group in its side chain, such as cysteine; X 23 Can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X 24 Can be Leu or any amino acid with a reactive group in its side chain, such as cysteine; X 25 Can be Pro or any amino acid with a reactive group in its side chain, such as cysteine; X 23b Can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X 24b Can be Thr or any amino acid with a reactive group in its side chain, such as cysteine; X 25b Can be His or any amino acid with a reactive group in its side chain, such as cysteine; X26 Can be Leu or any amino acid with a reactive group in its side chain, such as cysteine; X 27 Can be Ser or any amino acid with a reactive group in its side chain, such as cysteine; X 28 Can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X 29 Can be Ser or any amino acid with a reactive group in its side chain, such as cysteine; X 30 Can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X 31 Can be Arg or any amino acid with a reactive group in its side chain, such as cysteine; X 32 Can be Asn or any amino acid with a reactive group in its side chain, such as cysteine; X 33 Can be Gly or any amino acid with a reactive group in its side chain, such as cysteine; X 34 Can be Gln or any amino acid with a reactive group in its side chain, such as cysteine; X 35 Can be Ser or any amino acid with a reactive group in its side chain, such as cysteine; X 36 Can be Gln or any amino acid with a reactive group in its side chain, such as cysteine; X 37 Can be Gly or any amino acid with a reactive group in its side chain, such as cysteine; X 38 It can be Val or any amino acid with a reactive group in its side chain, such as cysteine;

[0244] X 39 It can be Tyr or any amino acid with a reactive group in its side chain, such as cysteine;

[0245] X 40 It can be Met or any amino acid with a reactive group in its side chain, such as cysteine;

[0246] X 41 It can be Ile or any amino acid with a reactive group in its side chain, such as cysteine;

[0247] X 42 It can be His or any amino acid with a reactive group in its side chain, such as cysteine;

[0248] X 43 It can be Glu or any amino acid with a reactive group in its side chain, such as cysteine;

[0249] X 44It can be Pro or any amino acid with a reactive group in its side chain, such as cysteine;

[0250] X 45 It can be Glu or any amino acid with a reactive group in its side chain, such as cysteine;

[0251] X 46 It can be Pro or any amino acid with a reactive group in its side chain, such as cysteine;

[0252] X 47 It can be His or any amino acid with a reactive group in its side chain, such as cysteine;

[0253] X 48 It can be Arg or any amino acid with a reactive group in its side chain, such as cysteine;

[0254] X 49 It can be Arg or any amino acid with a reactive group in its side chain, such as cysteine;

[0255] X 50 It can be Pro or any amino acid with a reactive group in its side chain, such as cysteine;

[0256] X 51 It can be Leu or any amino acid with a reactive group in its side chain, such as cysteine;

[0257] X 52 It can be Pro or any amino acid with a reactive group in its side chain, such as cysteine;

[0258] X 53 It can be Lys or any amino acid with a reactive group in its side chain, such as cysteine;

[0259] X 54 It can be Lys or any amino acid with a reactive group in its side chain, such as cysteine;

[0260] X 55 It can be Pro or any amino acid with a reactive group in its side chain, such as cysteine;

[0261] X 56 It can be Lys or any amino acid with a reactive group in its side chain, such as cysteine;

[0262] X 57 It can be Lys or any amino acid with a reactive group in its side chain, such as cysteine;

[0263] or a sequence having 80% or greater identity to SEQ ID NO.: 191, preferably a sequence having 85% or greater, 90% or greater, 95% or greater, 97% or greater or 99% or greater sequence identity to SEQ ID NO.: 191, 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, such as 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, such as 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 as described herein.

[0264] For example, the at least one protein-based building block may be selected from SEQ ID NO.: 80-105, 175, 199, 208, 222-224.

[0265] In one embodiment, a molecule of the invention comprises at least one protein-based carrier building block as defined herein and at least one additional moiety or cargo attached to a point of attachment or coupling site, wherein said at least one additional moiety or cargo is selected from:

[0266] a) half-life extending (HLE) moieties, such as PEG, and / or

[0267] b) a targeting moiety, such as a moiety targeting EGFR, for example a GE11 peptide or an anti-EGFR VHH; and / or

[0268] c) a therapeutic moiety or a precursor therefrom, e.g., a molecule that binds DR5;

[0269] d) imaging moieties, such as deferoxamine (DFO);

[0270] e) toxic moieties, such as DM4 or nostoc;

[0271] f) nucleic acids, such as siRNA;

[0272] g) Vitamins, such as folic acid;

[0273] h) Toll-like receptor agonists, such as resiquimod;

[0274] i) glycans, such as bimannose 6-phosphate (bisM6P) or mannose 6-phosphate (M6P); and / or

[0275] j) Lipids, such as short-chain fatty acids.

[0276] In one embodiment, the at least one half-life extending moiety is an albumin binding ISVD, wherein the albumin binding ISVD is preferably selected from SEQ ID NO.: 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 NO.: 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.

[0277] In one embodiment, the at least one protein-based carrier building block and the at least one additional moiety or cargo are directly linked to each other. In a further embodiment, they are linked to each other via a peptide linker, preferably wherein the peptide linker is 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.

[0278] For example, a molecule of the invention may comprise, or alternatively consist of, any one of SEQ ID NOs.: 107-127, SEQ ID NOs.: 170-174, 176 or 200.

[0279] The present invention also provides nucleic acids encoding the molecules of the present invention (or a portion of the molecules of the present invention). In addition, the present invention provides vectors comprising the nucleic acids of the present invention, and compositions (such as pharmaceutical compositions) comprising the molecules of the present invention.

[0280] Furthermore, the present invention relates to a molecule or composition of the invention for use in medicine, in particular for the (prophylactic or therapeutic) treatment of diseases and or disorders such as autoimmune / inflammatory diseases, cancer and / or infectious diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0281] Figure 1 .Amino acid sequence of ISVD RSV001A04 (SEQ ID NO.: 179).

[0282] Figure 2 .The amino acid sequence of K27m (without the C-terminal L), SEQ ID NO.: 68.

[0283] Figure 3 .Amino acid sequence of the CKS1-building block precursor (SEQ ID NO.: 190).

[0284] Figure 4 . Coupling is performed using the APN-maleimide "bifunctional" linker. The carrier (the protein-based building block contained in the molecule) contains at least one attachment point or coupling site (denoted as "-SH" in the figure). The APN-maleimide "bifunctional" linker can first be attached to the coupling site present in the carrier. Then, the cargo (denoted as "DR5-SH" in the figure) can be attached to the other side of the APN-maleimide "bifunctional" linker. Thus, the cargo has been attached or coupled to the carrier via the APN-maleimide "bifunctional" linker.

[0285] FIG5 . Coupling capability examination of a cysteine engineered ISVD-based vector building block using deconvoluted mass spectra of Mal-APN coupled to molecule T028100075, which includes an ISVD179-APN-based vector building block (“ISVD179-APN,” SEQ ID NO.: 176, Figure 5A ), and the coupling ability of the cysteine engineered ISVD-based vector building block was examined using mass spectrometric deconvolution of Mal-APN coupled to molecule T028100069, which comprises an ISVD-based vector building block with three attachment points or coupling sites, ("ISVD107-APN", SEQ ID NO.: 107, Figure 5B ). Mass spectrometry analysis was performed using electrospray ionization (ESI) with an online reverse phase column (RPC) to clean up the samples.

[0286] Figure 6. Schematic representation of the molecule of Example 5. This figure shows a multivalent molecule comprising an HLE moiety ("ALB23", which represents SEQ ID NO.: 106), a protein-based carrier building block ("Carrier", which represents SEQ ID NO.: 80), and three cargoes ("DR5") attached to three coupling sites, which are three -SH groups of cysteines present in solvent accessible positions. Figure 6A In , each cargo ("DR5") is DR5-GGC ISVD ("monovalent", DR5-GGC, SEQ ID NO.: 177). Figure 6B In the assay, each cargo ("DR5-DR5-DR5") contained three DR5 ISVDs ("trivalent", DR5-DR5-DR5-GGC, SEQ ID NO.: 178).

[0287] Figure 7 SDS-PAGE gel analysis of the coupling performed in Example 5. Lane 3 shows the 3x coupling state of the "trivalent" (DR5-DR5-DR5-GGC) cargo attached to the three coupling sites on the building block defined by SEQ ID NO.: 176. Lane 5 shows the 3x coupling state of the "monovalent" (DR5-GGC) cargo attached to the three coupling sites on the building block defined by SEQ ID NO.: 176. The control results are not shown in the figure. Lane 4 is a protein ladder.

[0288] Figure 8 Caspase 3 / 7 assay. Add medium containing the indicated concentrations of DR5 agonists, as described below, followed by the addition of 5 μM CellEvent TM Caspase-3 / 7 Green Detection Reagent. Real-time data acquisition and analysis were performed, and caspase-3 / 7 signal was normalized to cell confluence at the 24 h time point. "DR5 trivalent form" refers to the trivalent DR5-GGC molecule as defined in SEQ ID NO.: 178; "DR5 divalent form" refers to the molecule described in SEQ ID NO.: 207; "cargo ninevalent DR5" refers to a molecule comprising an HLE portion ("ALB", SEQ ID NO.: 106), an ISVD-based carrier building block ("carrier", SEQ ID NO.: 107) with three coupling sites (at positions 43, 100f, and 105 according to Kabat), and three cargoes attached to the three coupling sites via an APN-maleimide linker, wherein each cargo comprises three DR5 ISVDs (SEQ ID NO.: 178); "cargo trivalent DR5" refers to a molecule comprising an HLE portion ("ALB", SEQ ID NO.: 106), an ISVD-based carrier building block ("carrier", SEQ ID NO.: 107) with three coupling sites (at positions 43, 100f, and 105 according to Kabat), and three cargoes attached to the three coupling sites via an APN-maleimide linker. NO.: 107) and three cargo molecules attached to three coupling sites via an APN-maleimide linker, wherein each cargo comprises one DR5 ISVD (SEQ ID NO.: 177); "Cargo control" refers to a control molecule comprising an HLE portion ("ALB", SEQ ID NO.: 106), a control vector building block ("RSV", SEQ ID NO.: 176) with a C-terminal Cys, wherein a molecule comprising three DR5 ISVDs is attached to the control vector building block via an APN-maleimide linker.

[0289] Figure 9siRNA conjugation using the molecule described in SEQ ID NO.: 108. "DOL3" refers to the molecule described in SEQ ID NO.: 108, wherein three siRNA molecules are attached to the three conjugation sites contained in the protein-based building block of the molecule. "DOL2" refers to the molecule described in SEQ ID NO.: 108, wherein two siRNA molecules are attached to two conjugation sites, and "DOL1" refers to the molecule described in SEQ ID NO.: 108, wherein one siRNA molecule is attached to one conjugation site. "T028100070" refers to the molecule described in SEQ ID NO.: 108 alone, and "T028100070+TCEP" refers to the molecule described in SEQ ID NO.: 108 with the reduced form of TCEP.

[0290] Figure 10 The molecule described in SEQ ID NO.: 108 ("ISVD108" in the figure) was PEGylated (5, 10 or 20 kDa PEG) and radiolabeled ( 89 The figure shows the concentration of different molecules (labeled 1-4 in the figure) in the blood over time.

[0291] Figure 11. HPLC / SEC analysis of a candidin / PEG conjugate coupled to a molecule having SEQ ID NO.: 107 (panel A) and a molecule having SEQ ID NO.: 113 (panels B and C). Sample: 5 μl injection; Column: TSK-GEL SW mAb HTP (Tosoh Bioscience) - 4 μm - 4.6 x 150 mm; Solvent: Isocratic with 100% SEC buffer KpiiPrOH 20% pH = 7 (Composition: KCl 1 M, 200 mL / KH2PO4 1 M, 52 mL / K2HPO4 1 M, 107 mL / MQ, 441 mL / iPrOH, 200 mL); Run: 0.2 mL / min for 15 min; V0 = exclusion volume, V t =Total volume of the column.

[0292] Figure 12 Schematic diagram of the cell internalization assay used in Examples 11-14.

[0293] Figure 13 . Internalization of ALB-3C_K27m_w1 molecules (SEQ ID NO.: 173) and ALB-5C_K27m_w1 molecules (SEQ ID NO.: 174) coupled to GE11 peptide (compared to alanine) on NCI-H226 cells using HSA-pHAb, as described in Example 11.

[0294] Figure 14 Preparation of (2S)-5-[2-(tert-butoxycarbonylamino)ethylamino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)-5-oxo-pentanoic acid tert-butyl ester (Intermediate 1).

[0295] Figure 15 Preparation of (2S)-2-amino-5-[2-(tert-butoxycarbonylamino)ethylamino]-5-oxo-pentanoic acid tert-butyl ester (Intermediate 2).

[0296] 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).

[0297] 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)propionylamino]ethylamino]-5-oxo-pentanoic acid (folic acid-aminoethyl-maleimide).

[0298] Figure 18 Internalization of HeLa cell lines by protein-based carrier molecules conjugated to folic acid. The protein-based carrier building blocks contained in T028100070 and T028100075 have been conjugated to maleimide-folic acid or maleimide-Ala (negative control) as described in Example 12.

[0299] 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-(2,5-dioxopyrrol-1-yl)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) synthetic scheme.

[0300] Figure 20 . Schematic diagram of the cell binding assay used in Examples 13 and 16.

[0301] Figure 21 Binding of T028100069 molecules (SEQ ID NO.: 107), T028100070 molecules (SEQ ID NO.: 108), and T028100075 molecules (SEQ ID NO.: 176) coupled to bisM6P (diM6P) (vs. 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 for M6PR") was used as a positive control, and an ISVD that does not specifically bind to M6PR ("ISVD not specifically binding to M6PR") was used as an additional negative control.

[0302] Figure 22 HSA-pHAb was used to detect the internalization of T028100069 cargo (SEQ ID NO.: 107), T028100070 cargo (SEQ ID NO.: 108), and T028100075 cargo (SEQ ID NO.: 176) coupled to bisM6P (relative to alanine) on K-562 cells as described in Example 13. An ISVD that specifically binds to M6PR ("ISVD specific for M6PR") was used as a positive control, and an ISVD that does not specifically bind to M6PR ("ISVD not specifically binding to M6PR") was used as an additional negative control.

[0303] Figure 23 Lipid QC was performed by mass spectrometry analysis of the DOL=0 (mal-ALA) and DOL 6 preparations to confirm the coupling of both batches. Nearly complete coupling of undecanoic acid and alanine was observed.

[0304] Figure 24 HSA-pHAb was used to detect the internalization of ALB-RSV_c6 (T028100078, SEQ ID NO.: 113) coupled with 11-maleimidoceanoic acid (vs. alanine) on BxPC-3 cells, as described in Example 14.

[0305] Figure 25. (A) SEC analysis of the conjugation of different mal-PEG molecules to the SH-group of the side chain of Cys present in the ISVD-derived building blocks of different molecules (6x5kDa, 5x5kDa, 4x5kDa and 1x30kDa). (B) Size and relative % of different PEG conjugated molecules analyzed by SEC.

[0306] Figure 26 . Measurement of ISVD-based carrier compound concentrations in rat serum.

[0307] Figure 27 : Non-reducing PAGE analysis of CKS-based vector loaded with a portion of CMA1. DETAILED DESCRIPTION

[0308] definition

[0309] Unless otherwise indicated or defined, all terms used have their ordinary meanings in the art, which will be clear to those skilled in the art. Reference is made, for example, to standard manuals, 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., 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., 2005 (Immunobiology, 6th ed., Garland Science Publishing / Churchill Livingstone, New York), and the general background art cited herein.

[0310] Unless otherwise indicated, all methods, steps, techniques and operations not specifically described in detail herein can be carried out and have been carried out in a manner known per se, as will be clear to those skilled in the art. Reference is again made to, for example, standard manuals and the general background art mentioned herein and the other references cited therein; and to, for example, the following reviews: 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 (Tumour Biol., 26: 31), which describe techniques for protein engineering (such as affinity maturation) and other techniques for improving the specificity and other desired properties of proteins (such as immunoglobulins).

[0311] It must 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 "an agent" includes one or more of such different agents, and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that may modify or substitute for the methods described herein.

[0312] 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 many equivalents to the specific embodiments of the technology described herein using only routine experimentation. Such equivalents are intended to be encompassed by the present invention.

[0313] The term "and / or" wherever used in this application includes the meanings of "and," "or," and "all or any other combinations of the elements connected by the term."

[0314] Throughout this specification and the appended claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated 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. When used in this application, the term "comprising" may be replaced by the term "containing" or "including" or, at times when used in this application, by the term "having".

[0315] As used herein, the term "sequence" (e.g., in terms such as "immunoglobulin sequence," "antibody sequence," "variable domain sequence," "V HH References to "amino acid sequence" or "protein sequence" should generally be understood to include the associated amino acid sequence as well as the nucleic acid or nucleotide sequence encoding the amino acid sequence, unless the context requires a more restricted interpretation. "Amino acid sequence" should be interpreted as meaning a single amino acid or an unbranched sequence of two or more amino acids, depending on the context. A nucleotide sequence should be interpreted as meaning an unbranched sequence of three or more nucleotides.

[0316] It will be understood that any reference to amino acid sequences is meant 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, pyroglutamate formation, removal of C-terminal lysine, and Met / Trp oxidation.

[0317] When a nucleotide sequence or amino acid sequence is referred to as "comprising" another nucleotide sequence or amino acid sequence, or as "essentially consisting of" another nucleotide sequence or amino acid sequence, respectively, this may mean that the latter nucleotide sequence or amino acid sequence has been incorporated into the first-mentioned nucleotide sequence or amino acid sequence, respectively, but more usually, this generally means that the first-mentioned nucleotide sequence or amino acid sequence, respectively, contains within its sequence a continuous stretch of nucleotides or amino acid residues having the same nucleotide sequence or amino acid sequence, respectively, as the latter sequence, regardless of how the first-mentioned sequence is actually generated or obtained (for example, it may be generated or obtained by any suitable method described in this application).

[0318] Amino acids contain amino groups [a](-NH + 3) and carboxylate (-CO -2) functional groups together with organic compounds that specifically bind to the side chains (R groups) of each amino acid. For example, amino acids include those L-amino acids commonly found in naturally occurring proteins. In the context of the present invention, "amino acids" also include D-amino acids and non-natural, uncommon or unnatural amino acids, as described below. Amino acid residues will be indicated according to standard three-letter or single-letter amino acid codes. With reference to Table A-2 on page 48 of WO 08 / 020079. Examples of amino acids commonly found in proteins and represented by the genetic code are listed in Table 1 below. Other common amino acids (excluding those listed in Table 1 below) are described 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.

[0319] Table 1: Common amino acids (IUPAC)

[0320]

[0321] Table 2. Other amino acids

[0322]

[0323] D-amino acids are also encompassed by the definition of “amino acid.” As used herein, the term “D-amino acid” refers to an amino acid in which the stereogenic central carbon alpha to the amino group has the D-configuration.

[0324] Uncommon, unnatural, or non-natural amino acids are also encompassed by the definition of "amino acid." As used herein, the term "unnatural amino acid" or "atypical amino acid" or "unnatural amino acid" or "novel amino acid" (or similar terms) refers to an amino acid that is not one of the twenty amino acids commonly found in peptides synthesized in nature and 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 described in 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.

[0325] Alexander R. et al. (“Using genetically incorporated unnatural amino acids to control protein functions in mammalian cells”, Essays Biochem, 2019 Jul 3; 63(2): 237-266, the disclosure of which is incorporated herein by reference) provide an overview of unnatural amino acids that have been successfully incorporated into proteins in mammalian cells, see, for example, Table 1 starting on page 240.

[0326] Non-limiting examples of non-natural amino acids include: p-acetyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, L-DOPA, p-azido-phenylalanine, N6-(propargyloxy)-carbonyl-L-lysine (PrK), azido-lysine (N6-azidoethoxy-carbonyl-L-lysine, AzK). In some embodiments, the non-natural amino acid comprises a selective reactive group or a reactive group for site-selective labeling or coupling of a moiety or payload. In some cases, the chemistry is a biorthogonal reaction (e.g., biocompatible and selective reaction). In some cases, the chemistry is Cu(I)-catalyzed or "copper-free" azide-alkyne triazole formation, Staudinger ligation, inverse-electron-demand Diels-Alder (IEDDA) reaction, "photo-click" chemistry, or metal-mediated processes such as olefin metathesis and Suzuki-Miyaura cross-coupling or Sonogashira cross-coupling. For other examples of unnatural amino acids, we refer to WO 2021 / 072167, the disclosure of which is incorporated herein by reference.

[0327] The terms "protein," "peptide," "protein / peptide," and "polypeptide" are used interchangeably throughout this disclosure and, for purposes of this disclosure, each have the same meaning. Each term refers to an organic compound composed of a linear chain of two or more amino acids. The compound may have ten or more amino acids; twenty-five or more amino acids; fifty or more amino acids; one hundred or more amino acids, two hundred or more amino acids, and even three hundred or more amino acids. Those skilled in the art will understand that polypeptides generally contain fewer amino acids than proteins, although there is no art-recognized dividing line for the number of amino acids that distinguishes polypeptides from proteins; polypeptides can be prepared by chemical synthesis or recombinant methods; and proteins are generally prepared in vitro or in vivo by recombinant methods known in the art.

[0328] By convention, the amide bonds in the primary structure of a polypeptide are arranged 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) always on the right.

[0329] Any amino acid sequence containing post-translationally modified amino acids may be described as the originally translated amino acid sequence using the symbols and modification positions shown in Table 1; for example, hydroxylation or glycosylation, but these modifications should not be explicitly shown in the amino acid sequence. Any peptide or protein that can be represented as a sequence modification, such as linkers, cross-linkers and caps, non-peptidyl bonds, etc., is encompassed by this definition.

[0330] In the context of the present invention, the terms "specificity," "specifically binds," or "specifically binds" refer to the number of different target molecules (e.g., 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 "selectively binds." Typically, a binding unit (e.g., a binding ISVD) specifically binds to its designated target.

[0331] The specificity / selectivity of a binding unit can be determined based on affinity. Affinity indicates the strength or stability of a molecular interaction. Affinity is typically measured by K D Or dissociation constant is given in moles / liter (or M). Affinity can also be expressed as the association constant K A , which is equal to 1 / K D And the unit is (mol / liter) -1 (or M -1 ).

[0332] Affinity is a measure of the strength of binding between a moiety and a binding site on a target molecule: K D The lower the value, the stronger the binding strength between the target molecule and the targeting moiety.

[0333] K D The value also characterizes the strength of molecular interactions in a thermodynamic sense, since it is expressed by the well-known relationship DG = RT.ln(K D )(Equivalently DG = -RT.ln(K A )) is related to the change in binding free energy (DG), where R equals the gas constant, T equals the absolute temperature, and ln denotes the natural logarithm.

[0334] K D It can also be expressed as the dissociation rate constant of the complex (expressed as k off ) and its association rate constant (expressed as k on ) ratio (so K D =k off / kon , and K A =k on / k off ). Dissociation rate k off With unit s -1 (where s is the SI unit symbol for seconds). Association rate k on With unit M -1 s -1 The association rate can be 10 2 M -1 s -1 to about 10 7 M -1 s -1 The dissociation rate is given by the relation t 1 / 2 =ln(2) / k off The dissociation rate can be measured in 10 -6 s -1 (Nearly irreversible complex, t 1 / 2 for multiple days) to 1s -1 (t 1 / 2 =0.69s).

[0335] If the measurement process somehow affects the intrinsic binding affinity of the underlying molecule, for example through artifacts associated with a coating on the biosensor of a molecule, then the measured K D may correspond to the apparent K D In addition, if a molecule contains more than one recognition site for another molecule or molecules, the apparent K D In this case, the measured affinity may be influenced by the avidity with which the two molecules interact.

[0336] As will be clear to those skilled in the art, the dissociation constant (K D ) can be the actual or apparent dissociation constant. D Methods for measuring the effect of the above mentioned methods will be clear to those skilled in the art, including, for example, the techniques mentioned below. In this regard, it will also be clear that it may not be possible to measure the effect of the above mentioned methods. -4 mol / L or 10 -3 mol / L (for example, 10 -2 Optionally, as will be clear to those skilled in the art, the (actual or apparent) K D can be based on the (real or apparent) association constant (K A ) through the relationship (K D =1 / K A ) to calculate. KA =1 / K D -->K A =[AB] / [A].[B].

[0337] The term "about" used in the context of the parameters or parameter ranges provided in this application should have the following meanings. Unless otherwise stated, when the term "about" is applied to a specific value or range, the value or range is interpreted as being as accurate as the method used to measure it. If the error range is not specified in the application, the last decimal place of the numerical value represents its accuracy. In the absence of other error ranges, the maximum range is determined by applying the rounding convention to the last decimal place, for example, for a pH value of about pH 2.7, the error range is 2.65-2.74. However, for the following parameters, a specific range should be used: a temperature expressed in ° C and without decimal places should have an error range of ± 1 ° C (for example, a temperature value of about 50 ° C represents 50 ° C ± 1 ° C); a time expressed in hours should have an error range of 0.1 hours, regardless of the decimal places (for example, a time value of about 1.0 hours represents 1.0 hours ± 0.1 hours; a time value of about 0.5 hours represents 0.5 hours ± 0.1 hours).

[0338] In this application, any parameter indicated with the term "about" is also considered to be disclosed without the term "about." In other words, embodiments that use the term "about" to refer to a parameter value should also describe embodiments that refer to the numerical value of the parameter itself. For example, an embodiment that specifies a pH value of "about pH 2.7" should also disclose embodiments that specify a pH value of "pH 2.7" itself; an embodiment that specifies a pH range of "between about pH 2.7 and about pH 2.1" should also describe embodiments that specify a pH range of "between pH 2.7 and pH 2.1," etc.

[0339] For the purpose 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 as follows: [the number of nucleotides in the first nucleotide sequence that are identical to the nucleotides at the corresponding positions in the second nucleotide sequence] / [the total number of nucleotides in the first nucleotide sequence] x [100%], wherein each deletion, insertion, substitution or addition of a nucleotide in the second nucleotide sequence compared to the first nucleotide sequence is considered as a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using a known computer algorithm for sequence alignment (such as NCBI Blast v2.0) using standard settings. For example, other techniques, computer algorithms and settings for determining the degree of sequence identity are described in WO 04 / 037999, EP 0967284, EP 1085089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185 and GB 2357768. Typically, to determine the percentage of "sequence identity" between two nucleotide sequences according to the calculation method outlined above, the nucleotide sequence with the largest number of nucleotides is used as the "first" nucleotide sequence and the other nucleotide sequence is used as the "second" nucleotide sequence.

[0340] For comparison of 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 as follows: ([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] / [the total number of amino acid residues in the first amino acid sequence]) × 100%, wherein each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence compared to the first amino acid sequence is regarded as a difference at a single amino acid residue (position), i.e., is regarded as 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 mentioned above for determining the degree of sequence identity of nucleotide sequences), again using standard settings. Typically, in order to determine the percentage of "sequence identity" between two amino acid sequences according to the calculation method outlined above, the amino acid sequence with the largest number of amino acid residues is taken as the "first" amino acid sequence and the other amino acid sequence is taken as the "second" amino acid sequence.

[0341] In addition, when determining the degree of sequence identity between two amino acid sequences, those skilled in the art may consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue of similar chemical structure and has little or substantially no effect on the 3D structure, function, activity or other biological properties of the 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 relevant teachings of WO 04 / 037999 and WO 98 / 49185 and the additional references cited therein.

[0342] Such conservative substitutions are preferably substitutions in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar 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, nonpolar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp. Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to Ile or to Leu.

[0343] Amino acid sequences and nucleic acid sequences are said to be "identical" 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 a position in the first sequence compared to the second sequence; it is understood that two amino acid sequences may contain one, two, or more such amino acid differences.

[0344] According to the present application, "protein solubility" is a thermodynamic parameter defined as the concentration of a protein in a saturated solution in equilibrium with a solid phase (crystalline or amorphous) under a given set of 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).

[0345] Protein-based building blocks

[0346] The molecules of the invention comprise, or alternatively consist of, at least one protein-based carrier building block as defined herein (also referred to herein as a "carrier building block", "protein-based building block", or simply as a "building block" or "carrier"). For example, a molecule of the invention may comprise, or alternatively consist of, a single protein-based carrier building block. In other embodiments, the molecule comprises more than one protein-based building block, such as two, three, four, five, six or more carrier building blocks. A protein-based carrier building block comprises (and preferably consists of) at least a portion of a protein or an entire structured protein, i.e., a protein-based carrier building block is preferably a polypeptide.

[0347] Protein-based carrier building blocks are designed as "carrier" or "delivery" moieties having at least one attachment point or coupling site, preferably at least two attachment points or coupling sites, for coupling or attaching a cargo as defined in detail below. Suitable cargoes include proteins, peptides, toxic payloads, nucleic acids, oligonucleotides, fluorophores, polysaccharides, chelating agents 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 described below in this specification.

[0348] In the context of the present invention, the connection point or coupling site refers to any group contained in the protein-based building block, which is suitable for attachment or coupling of a load. The connection point or coupling site is preferably present in a solvent accessible position in the protein-based building block, as explained in detail below. The connection point or coupling site can be a reactive group in the side chain of any amino acid present in the protein-based carrier building block (preferably an amino acid present in a solvent accessible position in the protein-based carrier building block), or can be an N-terminal primary amine and / or C-terminal carboxyl group of the protein-based building block. The connection point / coupling site allows the formation of a covalent bond with a group to be coupled and / or attached to a load based on the protein-based carrier building block. In a preferred embodiment, the connection point or coupling site is a reactive group in the side chain of an amino acid present in the protein-based carrier building block (preferably a solvent accessible position in the protein-based carrier building block), which allows the formation of a covalent bond with a group to be coupled and / or attached to a load based on the protein-based carrier building block. In another embodiment, the two coupling sites or attachment points of the protein-based building block are reactive groups present in the side chains of two amino acids present in the protein-based carrier building block (preferably two amino acids present at solvent accessible positions in the protein-based carrier building block). In another embodiment, all coupling sites or attachment points of the protein-based building block are reactive groups present in the side chains of amino acids present in the protein-based carrier building block (preferably amino acids present at solvent accessible positions in the protein-based carrier building block).

[0349] Spherical three-dimensional (3D) structure

[0350] The protein-based carrier building block of the present invention has a globular three-dimensional (3D) structure, that is, it is or contains a structured protein with a globular 3D structure. Globular proteins have an approximately spherical shape. Almost all globular proteins contain a large amount of α-helices and / or β-folds, which fold into a compact structure stabilized by polar and non-polar interactions. The globular 3D structure is naturally formed and generally involves interactions mediated by amino acid side chains. Most commonly, the hydrophobic amino acid side chains are buried and tightly packed inside the globular protein and do not contact water. The hydrophilic amino acid side chains are located on the surface of the globular protein exposed to water. Therefore, globular proteins are generally very soluble in aqueous solution (from "Gene Expression: Translation of the Genetic Code", Chang-Hui Shen, in Diagnostic Molecular Biology, 2019). In the context of the present invention, a protein or a part of a protein with a globular 3D structure can be defined as a protein or a part of a protein comprising at least one α-helix and / or at least one β-fold as part of its secondary structure. From a simple amino acid sequence to its final 3D structure, proteins go through four levels of structuring, called 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. Therefore, the amino acid sequence of a protein is called the " Primary structure ". " Secondary structure " can be defined as the arrangement of polypeptide chains into a more or less regular hydrogen-bonded structure, and it has two essential elements:

[0351] o α-helix - A helical conformation of a polypeptide chain with 3.6 residues (amino acids) per turn. Helices can be left-handed or right-handed, with the latter being more common.

[0352] o Beta strand (or beta-sheet) - two adjacent polypeptide chains bonded together. Two or more chains can interact to form a beta sheet.

[0353] at last," Tertiary structure " can be defined as the level of protein structure at which the entire polypeptide chain folds 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.

[0354] The three-dimensional structure of 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 a common technique for determining 3D protein structure, and NMR (applicable to small proteins) and cryo-EM (applicable to large proteins) can provide information about the tertiary structure of proteins. Circular dichroism is an excellent method for rapidly evaluating 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). Since the CD spectrum of protein is so dependent on their conformation, CD can be used to estimate the structure of unknown protein and monitor the conformational changes caused by temperature, mutation, heat, denaturant or binding interaction. For example, alpha-helical protein has negative bands at 222nm and 208nm and positive bands at 193nm. Proteins with well-defined antiparallel β-pleated sheets (β-helices) have a negative band at 218 nm and a positive band at 195 nm, while disordered proteins have very low ellipticity above 210 nm and a negative band near 195 nm. For further details, see Greenfield NJ., "Using circular dichroismspectra to estimate protein secondary structure", Nat Protoc., 2006, 1(6): 2876-90.

[0355] Thus, the protein-based carrier building blocks of the present invention comprise at least one α-helix and / or at least one β-sheet as part of their secondary structure, preferably more than one α-helix and / or more than one β-sheet as part of their secondary structure, resulting in a globular 3D tertiary structure. This allows for the engineering of site-specific and stereospecific coupling sites or attachment points, as described in detail in this specification. The presence of at least one α-helix and / or at least one β-sheet in a polypeptide or protein can be determined by known techniques (e.g., CD) as explained above.

[0356] Solubility

[0357] The protein-based carrier building blocks of the present invention are soluble. In the context of the present invention, a soluble building block means a building block having a solubility of 10 mg / mL or higher, preferably 20 mg / mL, preferably 50 mg / mL or higher, even more preferably 100 mg / mL or higher, as measured in water or a suitable buffer or solvent (e.g., an aqueous solution or a physiological buffer, such as a buffer suitable for parenteral administration) at room temperature (RT). In a preferred embodiment, the solubility of the protein-based carrier building blocks is measured in water or in a suitable buffer at room temperature, more preferably in a buffer such as citrate buffer (e.g., 5 mM citrate buffer) or PBS at a pH of 7.0 or 7.4 at room temperature. Other preferred suitable buffers for measuring the solubility of protein-based carrier building blocks are Dulbecco's phosphate buffered saline (DPBS, which is a balanced salt solution containing potassium chloride, potassium dihydrogen phosphate, sodium chloride and disodium hydrogen phosphate, such as 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 (Nr14190-094)), preferably at pH 7.0 or 7.3 or 7.4 at room temperature, or histidine buffer (comprising histidine (10 mM to 100 mM, such as 10 mM), sucrose (1% to 10%, such as 10%) and optionally Tween 6 at room temperature. 80 (0.001% to 1%, such as 0.01%)), or a phosphate buffer with a pH of 7.0 (comprising NaH2PO4 / Na2HPO4 (10 and 50 mM, such as 10 mM), sodium chloride (NaCl) (100-150 mM, such as 130 mM NaCl) and optionally Tween 80 (0.001% to 1%, such as 0.01%)).

[0358] Those skilled in the art are aware of methods for measuring the solubility of protein solutions. For example, the supplementary material by 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 solubility measurements of folded proteins.

[0359] Additionally or alternatively, solubility measurements can be performed as follows. A protein solution (e.g., in 5 mM citrate buffer, pH 7.0, or in PBS, pH 7.4, or in water, or in any suitable buffer as described above) is concentrated by ultrafiltration (e.g., via tangential flow filtration (TFF)) until some turbidity appears in the solution. The solution is then spun or filtered through 0.22 μm to remove any insoluble material, and the OD of the supernatant is measured. 280 Using the molar extinction coefficient of the particular protein, the protein concentration of the supernatant (and therefore the concentration of the protein in a saturated solution in equilibrium with the solid phase, i.e., the protein solubility) is obtained.

[0360] For example, in the context of the present invention, physiological buffers suitable for parenteral administration may include 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, e.g., Broadhead J, Gibson M., "Parenteral dosage forms", in: Gibson M., ed., "Pharmaceutical preformulation and formulation", New York: Informa healthcare; 2009, pp. 325-47).

[0361] Preferably, the protein-based carrier building blocks of the present invention are soluble in a reduced state, i.e., they are soluble when the -SH groups present in their amino acid sequence (if any) at solvent-accessible positions (e.g., in the side chains of one or more Cys) are in a reduced form (e.g., "-SH") rather than an oxidized form. For example, a protein-based carrier building block can be reduced when subjected to reducing conditions for a sufficiently long time. For example, reducing conditions can mean the use of β-mercaptoethanol (2-ME), dithiothreitol (DTT), or TCEP (tris(2-carboxyethyl)phosphine).

[0362] Size (Molecular Weight) Protein-based carrier building blocks of the invention have a size (molecular weight) of about 2.5 to about 70 kDa, such as about 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 65 or about 70 kDa. Preferably, the building block is a small building block of a size of about 2.5 to about 50 kDa, such as about 2.5 to about less than 50 kDa, such as about 2.5 to about 40 kDa, or about 2.5 to about 35 kDa; more preferably about 2.5 to about 30 kDa, such as 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, 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 building blocks of the present invention have a size of about 2.5 to about 16 kDa, such as about 5 to about 16 kDa, or about 7 to about 16 kDa, or about 10 to about 16 kDa, or such as about 2.5, 3, 5, 6.5, 7, 10, 11, 12, 13, 14, 15 or 16 kDa. For example, a protein-based building block may have a size (molecular weight) of about 6 kDa, or about 7 kDa, or about 15 kDa, or about 16 kDa. In an even more preferred embodiment, a protein-based vector building block has a size of about 15 kDa.

[0363] Non-functional

[0364] The protein-based carrier building blocks of the present invention do not specifically bind to any human protein. If the building blocks show any interaction with one or more human proteins, such interaction is characterized by low specificity and / or low affinity as defined herein.

[0365] For example, the protein-based carrier building blocks of the present invention do not specifically bind to crystallizable fragment (Fc) receptors (FcRs), Fc-binding proteins, or Fc sensors. For example, the protein-based carrier building blocks do not specifically bind to C-type lectin receptors (CLRs). All antibodies have two functional domains—one that confers antigen specificity, called the antigen-binding fragment (Fab); and another domain that drives antibody function, called the crystallizable fragment (Fc). The specific effector functions triggered by the antibody are determined by the receptors to which the antibody Fc domain binds and the specific innate immune cells that express these FcRs. 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 building blocks of the present invention do not specifically bind. Thus, the protein-based carrier building blocks of the present invention do not exhibit the effector functions of conventional antibodies mediated by the Fc domain. In another embodiment, the protein-based carrier building blocks and / or molecules do not specifically bind to a crystallizable fragment (Fc) receptor (FcR), an Fc binding protein, or an Fc sensor. For example, a protein-based carrier building block and / or molecule does not specifically bind to a C-type lectin receptor (CLR). Thus, in one embodiment, none of the components comprised in the molecules of the present invention (e.g., at least one protein-based carrier building block and / or at least one cargo attached or coupled 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 building blocks and / or molecules of the present invention do not exhibit the effector functions of conventional antibodies mediated by the Fc domain, i.e., none of the components comprised in the molecules of the present invention exhibit the effector functions of conventional antibodies mediated by the Fc domain. In one embodiment, the molecules of the present invention do not include a conventional V H -V L Pairing / interaction and / or excluding C L -C H 1 pairing (such as C L -C H 1 binds to disulfide bridges).

[0366] In another embodiment, the protein-based carrier building blocks of the present invention do not specifically bind to the light chain variable domain (V L ) and / or heavy chain variable domain (V H), such as V of monoclonal antibodies (mAbs) L and / or V H In another embodiment, the protein-based carrier building block does not specifically bind to the first constant domain (C H 1), such as C of mAb H 1. In another embodiment, the protein-based carrier building block does not specifically bind to the constant domain (C L ), such as C of mAb L In another embodiment, the protein-based carrier building block does not specifically bind to the third constant domain (C H 3), such as C of mAb H 3. In another embodiment, the protein-based carrier building block does not specifically bind to the second constant domain (C H 2), such as C of mAb H 2. In one embodiment, the molecules and / or building blocks of the present invention are not derived from the Fab fragment of an antibody (e.g., from a mAb). In one embodiment, the molecules and / or building blocks of the present invention are not derived from the C H , preferably not from an antibody C H 1. Fragments. The molecules and / or building blocks of the present invention are not antibodies (such as mAbs), are not Fc fragments or Fv fragments.

[0367] The protein-based vector building blocks of the invention may be derived from a target binding protein (such as an ISVD, a DARPin, an affibody or an affitin) (a "protein-based vector building block precursor"). In the context of the present invention, a "protein-based vector building block precursor" or "building block precursor" is a protein-based moiety that can be modified to generate a protein-based vector building block comprised in a molecule of the invention.

[0368] In the context of the present invention, a "protein-based vector building block precursor" is a protein that has been modified (e.g., by point mutations and / or by addition / deletion of amino acids to its sequence) to produce a protein-based vector building block of the present invention. For example, a "protein-based vector building block precursor" is modified such that it no longer specifically binds to any human protein, preferably it also does not specifically bind to any (non-human) molecule (including non-human biomolecules) and / or any non-protein (human) molecule (including biomolecules), in particular any molecule (including biomolecules) to which the precursor specifically binds. Furthermore, if desired, a "protein-based vector building block precursor" is modified such that it incorporates one or more attachment points or coupling sites as described herein. A "protein-based vector building block precursor" has at least 60%, such as at least 70% or at least 75%, preferably at least 80% sequence identity with the protein-based vector building block from which it is derived. For example, a "protein-based vector building block precursor" may have at least 85%, such as at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, or greater sequence identity with the protein-based vector building block from which it is derived. For example, a "protein-based vector building block precursor" may share the entire amino acid sequence with the protein-based vector building block from which it is derived, except for at least one, such as one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, twenty or more amino acids. Of course, the protein-based carrier building blocks derived from the protein-based carrier building block precursor have a globular 3D structure; are soluble; have a size (molecular weight) of about 2.5 to about 70 kDa, such as about 2.5 to about 50 kDa, such as 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, such as about 5 to about 16 kDa, or about 7 to about 16 kDa, or about 10 to about 16 kDa; do not specifically bind to any human protein and preferably do not specifically bind to any protein or non-protein molecule to which the precursor specifically binds.

[0369] Preferably, the vector building blocks of the present invention also do not specifically bind to any non-protein molecules (including non-protein biomolecules, such as nucleic acids (e.g. DNA and / or RNA), lipids (e.g. phosphatidylserine (PS)) or glycans), for example any non-protein human molecules (including biomolecules), such as 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 vector building blocks also do not specifically bind to any non-protein molecules (including biomolecules), such as nucleic acids (DNA and / or RNA), lipids (e.g. phosphatidylserine (PS)) or glycans), for example any non-protein human molecules (including biomolecules), such as 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 vector building block precursor specifically binds (i.e., the protein-based building blocks preferably also do not specifically bind to the target of the precursor, such as a non-protein molecule (including biomolecule) or a non-human protein).

[0370] In a further preferred embodiment, the protein-based building blocks of the invention also do not specifically bind to any (non-human) molecule (including biomolecule) to which the protein-based carrier building block precursor specifically binds (i.e., the protein-based building blocks preferably also do not specifically bind to the target of the precursor, such as a non-human protein or non-protein molecule (including biomolecule)), or at a level greater than 5x10 -4 mol / L K D The protein-based vector building block precursor preferably does not specifically bind to any (non-human) molecule to which the protein-based vector building block precursor specifically binds (i.e., the protein-based building block preferably also does not specifically bind to the target of the precursor, such as a non-human protein or non-protein molecule). For example, if the precursor of the protein-based vector building block is an anti-RSV (respiratory syncytial virus) ISVD (i.e., the precursor specifically binds to one or more proteins of RSV, such as protein F of RSV), then the protein-based vector building block derived therefrom preferably does not specifically bind to those RSV proteins (or preferably to a concentration greater than 5x10 as described herein). -4 mol / L K D The value binds to those proteins, such as protein F of RSV).

[0371] For example, if a precursor of a protein-based vector building block specifically binds to a virus (e.g., the precursor is an antiviral ISVD, an antiviral DARPin, an antiviral affitin, an antiviral affibody, etc.) and / or a viral molecule (e.g., the precursor specifically binds to one or more viral biomolecules, such as a viral protein, a viral nucleic acid, a viral lipid, or a viral glycan), then the protein-based vector building block derived therefrom preferably does not specifically bind to those viruses and / or viral molecules (or preferably at a level greater than 5×10 -4 mol / L K D In other embodiments, the protein-based carrier building block specifically binds to viruses (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), just like its precursor, but the specific binding is abolished when at least one cargo is attached to the protein-based building block. Examples of viruses to which protein-based building block precursors (and / or protein-based building blocks of the present invention) can specifically bind include RSV, influenza virus, rabies virus, potato virus Y, bacteriophage, rotavirus, HIV protein, hepatitis B virus, hepatitis C virus, norovirus, Shiga toxin from lambda phage, herpes simplex virus, grapevine leaf virus (GFLV), Ebola virus, Middle East respiratory syndrome (MERS) virus, acute respiratory syndrome (SARS) virus, SARS-CoV2, Vibrio or white spot syndrome virus, cytomegalovirus, parvovirus, Zika virus, 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. If the protein-based building blocks of the present invention show specific binding to one or more of these viruses (or molecules, including biomolecules, contained therein), the specific binding as described herein is lost when at least one cargo is attached to at least one coupling site contained therein.

[0372] For example, if a precursor of a protein-based vector building block specifically binds to protozoa (microorganisms, unicellular eukaryotes) (e.g., the precursor is an antiprotozoan ISVD, an antiprotozoan DARPin, an antiprotozoan affitin, an antiprotozoan affibody, etc.) and / or protozoan molecules (e.g., the precursor specifically binds to one or more protozoan biomolecules, such as protozoan proteins, protozoan nucleic acids, protozoan lipids, or protozoan glycans), then the protein-based vector building block derived therefrom preferably does not specifically bind to those protozoa and / or protozoan molecules (or preferably to a concentration greater than 5×10 5-10 5-10 5-10 5-10 5-10 5-10 5-10 5-10 5-10 5-10 5-10 5-10 5-10 -4 mol / L K D In other embodiments, the protein-based carrier building block specifically binds to protozoa (e.g., it is an antiprotozoan ISVD, an antiprotozoan DARPin, an antiprotozoan affitin, an antiprotozoan affibody, etc.) and / or protozoan molecules (e.g., it specifically binds to one or more protozoan biomolecules, such as protozoan proteins, protozoan nucleic acids, protozoan lipids, or protozoan glycans), just like its precursor, but the specific binding is abolished when at least one cargo is attached to the protein-based building block. Examples of protozoa and protozoan molecules to which protein-based building block precursors (and / or protein-based building blocks of the present invention) can specifically bind include: Trypanosoma evansi, Eimeria stiedae, variant surface glycoprotein (VSG). Thus, in one embodiment, a protein-based building block precursor (e.g., an ISVD) can specifically bind to one or more of these protozoa (or molecules, including biomolecules, contained therein). The resulting protein-based building blocks may not specifically bind to the protozoa (or molecules, including biomolecules, contained therein) to which the precursor binds. If a protein-based building block of the present invention exhibits specific binding to one or more of these protozoa (or molecules, including biomolecules, contained therein), the specific binding as described herein is lost when at least one cargo is attached to at least one coupling site contained therein.

[0373] For example, if a precursor of a protein-based carrier building block specifically binds to mammalian proteins (e.g., the precursor 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 building block derived therefrom preferably does not specifically bind to those mammalian proteins (or preferably does not specifically bind to those mammalian proteins at a level greater than 5×10 -4 mol / L KD In other embodiments, the protein-based carrier building block specifically binds to a mammalian protein, just as its precursor does, but the specific binding is abolished when at least one cargo is attached to the protein-based building block. An example of a mammalian protein to which a protein-based building block precursor (and / or a protein-based building block of the present invention) can specifically bind is bovine serum albumin. Thus, in one embodiment, a protein-based building block precursor (e.g., an ISVD) can specifically bind to such a mammalian protein. The resulting protein-based building block may not specifically bind to the mammalian protein to which the precursor binds. If a protein-based building block of the present invention exhibits specific binding to such a mammalian protein, the specific binding as described herein is lost when at least one cargo is attached to at least one coupling site contained therein.

[0374] For example, if a precursor of a protein-based carrier building block specifically binds to avian proteins (e.g., the precursor 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 building block derived therefrom preferably does not specifically bind to those avian proteins (or preferably at a level greater than 5×10 -4 mol / L K D In other embodiments, the protein-based carrier building block specifically binds to an avian protein, just as its precursor does, but the specific binding is abolished when at least one cargo is attached to the protein-based building block. An example of an avian protein to which a protein-based building block precursor (and / or a protein-based building block of the present invention) can specifically bind is ovalbumin (chicken). Thus, in one embodiment, a protein-based building block precursor (e.g., an ISVD) can specifically bind to this avian protein. The resulting protein-based building block may not specifically bind to the avian protein to which the precursor binds. If a protein-based building block of the present invention exhibits specific binding to this avian protein, the specific binding as described herein is lost when at least one cargo is attached to at least one coupling site contained therein.

[0375] For example, if a precursor of a protein-based carrier building block specifically binds to yeast and / or mycoproteins (e.g., the precursor is an anti-yeast and / or mycoprotein ISVD, an anti-yeast and / or mycoprotein DARPin, an anti-yeast and / or mycoprotein affitin, an anti-yeast and / or mycoprotein affibody, etc.), then the protein-based carrier building block derived therefrom preferably does not specifically bind to those yeast and / or mycoproteins (or preferably at a level greater than 5×10 -4 mol / L K D In other embodiments, the protein-based carrier building block specifically binds to yeast and / or mycoproteins, just as its precursor does, but the specific binding is abolished when at least one cargo is attached to the protein-based building block. Examples of yeast and mycoproteins to which protein-based building block precursors (and / or protein-based building blocks of the present invention) can specifically bind include yeast extracts, inactivated yeast, and Candida species. Thus, in one embodiment, a protein-based building block precursor (e.g., an ISVD) can specifically bind to one or more of these yeast and / or mycoproteins. The resulting protein-based building block may not specifically bind to at least one of the yeast and / or mycoproteins to which the precursor binds. If a protein-based building block of the present invention exhibits specific binding to these yeast and / or mycoproteins, the specific binding, as described herein, is lost when at least one cargo is attached to at least one coupling site contained therein.

[0376] For example, if a precursor of a protein-based carrier building block specifically binds to plant proteins (e.g., the precursor 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 building block derived therefrom preferably does not specifically bind to those plant proteins (or preferably does not bind to those plant proteins at a level greater than 5×10 -4 mol / L K DIn other embodiments, the protein-based carrier building block specifically binds to plant proteins, just like its precursor, but the specific binding is abolished when at least one cargo is attached to the protein-based building block. Examples of plant proteins to which the protein-based building block precursor (and / or the protein-based building block of the present invention) can specifically bind are starch branching enzyme II (maize), polyphenols, linoleic acid (sunflower, maize), and plant seeds. Thus, in one embodiment, the protein-based building block precursor (e.g., ISVD) can specifically bind to one or more of these plant proteins. The resulting protein-based building block may not specifically bind to at least one of the plant proteins to which the precursor binds. If the protein-based building block of the present invention exhibits specific binding to these plant proteins, the specific binding as described herein is lost when at least one cargo is attached to at least one coupling site contained therein.

[0377] For example, if a precursor of a protein-based carrier building block specifically binds to a fungal protein (e.g., the precursor is an antifungal protein ISVD, an antifungal protein DARPin, an antifungal protein affitin, an antifungal protein affibody, etc.), then the protein-based carrier building block derived therefrom preferably does not specifically bind to those fungal proteins (or preferably does not specifically bind to those fungal proteins at a level greater than 5x10 as described herein). -4 mol / L K D In other embodiments, the protein-based carrier building block specifically binds to a fungal protein, just as its precursor does, but the specific binding is abolished when at least one cargo is attached to the protein-based building block. Examples of fungal proteins to which the protein-based building block precursor (and / or the protein-based building blocks of the present invention) can specifically bind are cutinases, chitin, and fungal sphingolipids. Thus, in one embodiment, the protein-based building block precursor (e.g., ISVD) can specifically bind to at least one of these fungal proteins. The resulting protein-based building block may not specifically bind to at least one of these fungal proteins to which the precursor binds. If the protein-based building block of the present invention exhibits specific binding to at least one of these fungal proteins, the specific binding as described herein is lost when at least one cargo is attached to at least one coupling site contained therein.

[0378] For example, if a precursor of a protein-based carrier building block specifically binds to bacteria (e.g., the precursor is an antibacterial ISVD, an antibacterial DARPin, an antibacterial affitin, an antibacterial affibody, etc.) and / or bacterial molecules (e.g., the precursor specifically binds to one or more bacterial biomolecules, such as bacterial proteins, bacterial nucleic acids, bacterial lipids, or bacterial glycans), then the protein-based carrier building block derived therefrom preferably does not specifically bind to those bacteria and / or bacterial molecules (or preferably at a level greater than 5×10 -4 mol / L K DIn other embodiments, the protein-based carrier building block specifically binds bacteria (e.g., it is an antibacterial ISVD, an antibacterial DARPin, an antibacterial affitin, an antibacterial affibody, etc.) and / or bacterial molecules (e.g., it specifically binds one or more bacterial biomolecules, such as bacterial proteins, bacterial nucleic acids, bacterial lipids, or bacterial glycans), just like its precursor, but the specific binding is abolished when at least one cargo is attached to the protein-based building block. Examples of bacteria and bacterial molecules to which protein-based building block precursors (and / or protein-based building blocks of the invention) can specifically bind are as follows: 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 (flagellum), Salmonella enterica, Bordetella pertussis (toxin), Shigella spp. spp), Streptomyces venezuelae, and chloramphenicol. Thus, in one embodiment, a protein-based building block precursor (e.g., ISVD) can 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. If a protein-based building block of the present invention exhibits specific binding to one or more of these bacteria (or molecules thereof, including biomolecules), then when at least one cargo is attached to at least one coupling site contained therein, the specific binding as described herein is lost.

[0379] For example, if a precursor of a protein-based vector building block specifically binds to a non-human animal protein, such as a snake protein (e.g., the precursor is an antivenom ISVD, antivenom DARPin, antivenom affitin, antivenom affibody, etc.), then the protein-based vector building block derived therefrom preferably does not specifically bind to those snake proteins (or preferably at a level greater than 5x10 as described herein). -4 mol / L K D (The value of binding to those snake proteins is unclear). In other embodiments, the protein-based carrier building block specifically binds to a snake protein, just as its precursor does, but when at least one cargo is attached to the protein-based building block, the specific binding is abolished. An example of a snake protein to which a protein-based building block precursor (and / or a protein-based building block of the present invention) can specifically bind is cobra toxin. Thus, in one embodiment, a protein-based building block precursor (e.g., an ISVD) can specifically bind to such a snake protein. The resulting protein-based building block may not specifically bind to the snake protein to which the precursor binds. If a protein-based building block of the present invention exhibits specific binding to such a snake protein, the specific binding, as described herein, is lost when at least one cargo is attached to at least one coupling site contained therein.

[0380] For example, if a precursor of a protein-based vector building block specifically binds to green fluorescent protein (GFP, a protein from jellyfish (sea jellies) and corals, anemones, zoanthids, copepods and amphioxus) (e.g., the precursor is an anti-GFP ISVD, an anti-GFP DARPin, an anti-GFP affitin, an anti-GFP affibody, etc.), then the protein-based vector building block derived therefrom preferably does not specifically bind to GFP (or preferably does not bind to GFP at a level greater than 5x10 as described herein). -4 mol / L K D In other embodiments, a protein-based carrier building block specifically binds to GFP, just as its precursor does, but the specific binding is abolished when at least one cargo is attached to the protein-based building block. Thus, in one embodiment, a protein-based building block precursor (e.g., an ISVD) can specifically bind to GFP. The resulting protein-based building block may not specifically bind to the GFP bound by the precursor. If a protein-based building block of the present invention exhibits specific binding to GFP, the specific binding as described herein is lost when at least one cargo is attached to at least one coupling site contained therein.

[0381] For example, if a precursor of a protein-based carrier building block specifically binds to an insect protein (e.g., the precursor 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 building block derived therefrom preferably does not specifically bind to those insect proteins (or preferably does not bind to those insect proteins at a level greater than 5x10 as described herein). -4 mol / L K D In other embodiments, the protein-based carrier building block specifically binds to insect proteins, just as its precursor does, but the specific binding is abolished when at least one cargo is attached to the protein-based building block. Examples of insect proteins to which the protein-based building block precursor (and / or the protein-based building blocks of the present invention) can specifically bind include Androctonus autralis hecor 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, the protein-based building block precursor (e.g., ISVD) can specifically bind to at least one of these insect proteins. The resulting protein-based building block may not specifically bind to at least one of the insect proteins to which the precursor binds. If the protein-based building block of the present invention exhibits specific binding to at least one of these insect proteins, the specific binding, as described herein, is lost when at least one cargo is attached to at least one coupling site contained therein.

[0382] For example, if a precursor of a protein-based carrier building block specifically binds to chitin (belonging to crustacean proteins) (e.g., the precursor is an anti-chitin ISVD, an anti-chitin DARPin, an anti-chitin affitin, an anti-chitin affibody, etc.), then the protein-based carrier building block derived therefrom preferably does not specifically bind to chitin (or preferably does not specifically bind to chitin at a level greater than 5×10 -4 mol / L K DIn other embodiments, the protein-based carrier building block specifically binds to chitin, just as its precursor does, but the specific binding is abolished when at least one cargo is attached to the protein-based building block. Thus, in one embodiment, a protein-based building block precursor (e.g., an ISVD) can specifically bind to chitin. The resulting protein-based building block may not specifically bind to the chitin to which the precursor was bound. If a protein-based building block of the present invention exhibits specific binding to chitin, the specific binding as described herein is lost when at least one cargo is attached to at least one coupling site contained therein.

[0383] Thus, preferably, if the protein-based vector building block precursor has a target and if the target is a non-human molecule (including a biomolecule), such as a non-human protein, the protein-based vector building block does not specifically bind to the target of the precursor. Thus, in one embodiment, at least one protein-based building block comprised in a molecule of the invention does not specifically bind to any RSV protein, such as protein F of RSV, or to a concentration greater than 5x10 -4 mol / L K D (K D (value) in conjunction with any RSV protein, such as RSV protein F. Table A-1 and Table A-2 of WO 2009 / 147248 provide examples of F protein sequences. In one embodiment, at least one protein-based building block included in a molecule of the invention does not comprise or consist of an amino acid sequence selected from SEQ ID NO.: 1-34 as shown in Table A-1 and Table A-2 of WO 2016 / 055656. In another embodiment, at least one protein-based building block included in a molecule of the invention does not comprise or consist of an amino acid sequence as defined in SEQ ID NO.: 214.

[0384] In further preferred embodiments, the protein-based building blocks of the present invention, when they have at least one cargo attached thereto (via at least one coupling site or connection point contained therein) (e.g., a "model cargo," such as a maleimide-modified alanine), do not specifically bind to any molecule (including a biomolecule) to which the protein-based carrier building block precursor specifically binds (i.e., the protein-based building block preferably does not specifically bind to the target of the precursor, such as a non-human protein or non-protein molecule (including a biomolecule) when at least one cargo is attached thereto), or at a concentration greater than 5x10 -4 mol / L K DThe value does not specifically bind to any (non-human) molecule (including biomolecule) to which the protein-based carrier building block precursor specifically binds (i.e., the protein-based building block preferably also does not specifically bind to the target of the precursor, such as a non-human protein or non-protein molecule, when a cargo is attached thereto). Thus, in a preferred embodiment, if a protein-based carrier building block of the present invention shows any specific binding to a particular target (such as to a molecule (including biomolecules, such as humans, non-human animals, plants, microorganisms, viruses, etc.), or to a cell (e.g., animal, human, plant cell), microorganism, virus, etc.), the specific binding is abolished when at least one cargo is attached to at least one connection point or coupling site contained in the protein-based building block. In this embodiment, the cargo attached to the protein-based building block may of course show specific binding to the target (including biomolecules as described herein), but the protein-based building block no longer specifically binds to its target.

[0385] In a further preferred embodiment, the protein-based carrier building blocks of the present invention do not specifically bind to any human or non-human (for example, non-human animals, plants, yeast, etc.) cells and / or cell types. If building blocks show any interaction with one or more people or non-human cells and / or cell types, this interaction is characterized by low specificity and / or low affinity as defined in the application. Especially, preferably, the carrier building blocks do not specifically bind to any human or non-human cells and / or cell types specifically bound by the protein-based carrier building block precursor (that is, the target of the protein-based building blocks is preferably not specifically bound to precursors, such as non-protein molecules or proteins present on human cell surfaces). The loss of the combination with any human or non-human cells and / or cell types can be, for example, assessed with " cell binding assay " as described below (see also, 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).

[0386] In another embodiment, the protein-based carrier building blocks of the present invention do not specifically bind to any microorganism (such as bacteria, fungi, protozoa, yeast and / or viruses) or any microorganism or viral molecule (including biomolecules). If the building block shows any interaction with one or more microorganisms and / or viruses or with any microorganism or viral molecule (including biomolecules), the interaction is characterized by low specificity and / or low affinity as defined herein. In particular, preferably, the carrier building block does not specifically bind to any microorganism and / or virus (or any microorganism or viral molecule (including biomolecules)) to which the protein-based carrier building block precursor specifically binds (that is, the protein-based building blocks preferably do not specifically bind to the target of the precursor, such as a virus, microorganism, non-protein molecule (including biomolecules) or a protein present on the surface of a microorganism and / or virus). The loss of binding to any microorganism, or virus, or microbial molecule, or viral molecule can be assessed, for example, using a "cell binding assay" and / or SPR as described herein.

[0387] In another embodiment, the protein-based carrier building blocks of the present invention do not specifically bind to any microorganism, such as bacteria, fungi, protists, yeasts and / or viruses (and / or any microbial or viral molecules or biomolecules, such as microbial or viral proteins, nucleic acids, lipids, glycans, etc.) when they have at least one cargo attached or coupled thereto (via at least one attachment point or coupling site contained therein) (e.g., a "model cargo," such as a maleimide-modified alanine). If the building block comprising the cargo attached thereto shows any interaction with one or more microorganisms and / or viruses (or with any microbial or viral molecules or biomolecules, such as microbial or viral proteins, nucleic acids, lipids, glycans, etc.), such interaction is characterized by low specificity and / or low affinity as defined herein. In particular, preferably, when the protein-based carrier building block has at least one cargo attached or coupled thereto, the carrier building block also does not specifically bind to any microorganism and / or virus (or any microorganism or virus molecule or biomolecule, such as a microorganism or viral protein, nucleic acid, lipid, glycan, etc.) to which the protein-based carrier building block precursor specifically binds (i.e., when it has at least one cargo attached or coupled thereto, the protein-based building block preferably also does not specifically bind to the target of the precursor, such as a non-protein molecule or biomolecule, or a protein present on the surface of or in a microorganism and / or virus). For example, when the building block has at least one cargo attached or coupled thereto, the protein-based building block of the present invention does not specifically bind to any virus and / or viral protein, such as RSV and / or one or more proteins of RSV, such as protein F of RSV. Thus, for example, a protein-based carrier building block (e.g., a DARPin-based carrier building block) can exhibit specific binding to a microorganism and / or virus (such as RSV and / or an RSV protein, e.g., protein F of RSV), but, when at least one cargo is attached or coupled to the protein-based building block, the specific binding (as defined herein), if any, is lost.

[0388] The loss of specific binding to any microorganism can be assessed, for example, using a "cell binding assay" as described herein. The loss of specific binding to viruses, microorganisms and / or viral molecules or biomolecules can be assessed, for example, by surface plasmon resonance as described herein.

[0389] In another embodiment, the protein-based carrier building blocks of the present invention do not specifically bind to any molecule, including biomolecules, including human molecules and non-human molecules (including human and non-human biomolecules, such as 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 (such as phosphatidylserine (PS)) or non-human glycans); or at a concentration greater than 5x10 -4 mol / L K D (K D The protein-based vector building blocks do 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 (e.g., DNA and / or RNA), human and / or non-human lipids (e.g., phosphatidylserine (PS)), or human and / or non-human glycans), or to any molecule, including biomolecules, including human and non-human molecules (including human and non-human biomolecules, such as human and / or non-human proteins, nucleic acids (e.g., DNA and / or RNA), lipids (e.g., phosphatidylserine (PS)), or glycans), or to any molecule, including biomolecules, including human and non-human molecules (including human and non-human biomolecules, such as human and / or non-human proteins, nucleic acids (e.g., DNA and / or RNA), human and / or non-human lipids (e.g., phosphatidylserine (PS)), or human and / or non-human ... -4 mol / L K D (K D For example, a protein-based vector building block does not specifically bind to any bacterial molecule (including bacterial biomolecules, such as bacterial proteins, nucleic acids (such as DNA and / or RNA), lipids (such as phosphatidylserine (PS)) or glycans), or binds to any human and / or non-human animal biomolecule at a concentration greater than 5x10 as described herein. -4 mol / L K D (K D For example, the protein-based vector building blocks of the present invention do not specifically bind to any viral molecule (including biomolecules such as viral proteins, nucleic acids (such as DNA and / or RNA), lipids (such as phosphatidylserine (PS)) or glycans), or bind to any bacterial molecule as defined above at a concentration greater than 5x10 -4 mol / L K D (K D For example, a protein-based vector building block does not specifically bind to any fungal molecule (including biomolecules such as fungal proteins, nucleic acids (such as DNA and / or RNA), lipids (such as phosphatidylserine (PS)) or glycans), or binds to any viral molecule as defined herein at a concentration greater than 5x10 -4 mol / L K D (K DFor example, a protein-based vector building block does not specifically bind to any yeast molecule (including biomolecules such as yeast proteins, nucleic acids (such as DNA and / or RNA), lipids (such as phosphatidylserine (PS)), or glycans), or binds to any fungal molecule as defined herein at a concentration greater than 5x10 -4 mol / L K D (K D For example, a protein-based vector building block does not specifically bind to any plant molecule (including biomolecules such as plant proteins, nucleic acids (such as DNA and / or RNA), lipids (such as phosphatidylserine (PS)), or glycans), or binds to any yeast molecule as described herein at a concentration greater than 5x10 -4 mol / L K D (K D For example, a protein-based vector building block does not specifically bind to any mammalian molecule (including mammalian biomolecules, such as mammalian proteins, nucleic acids (such as DNA and / or RNA), lipids (such as phosphatidylserine (PS)), or glycans), or binds to any plant molecule as defined herein at a concentration greater than 5x10 -4 mol / L K D (K D value) in combination with any mammalian molecule as defined herein.

[0390] In the context of the present invention, the term "biomolecule" refers to a molecule that is present in an organism (including animals, plants, microorganisms) and plays a role in one or more biological processes (such as cell division, morphogenesis or development). Biomolecules are the building blocks of life and play an important role in living organisms. Biomolecules include primary metabolites, which are macromolecules such as proteins, carbohydrates (polysaccharides), lipids (e.g., PS) and nucleic acids (such as DNA, RNA), as well as small molecules such as vitamins and hormones. The four main types of biomolecules are carbohydrates (polysaccharides), lipids, nucleic acids and proteins.

[0391] In a further preferred embodiment, when at least one cargo (e.g., a "model cargo," such as a maleimide-modified alanine) is coupled to at least one attachment point or coupling site on a protein-based carrier building block, the protein-based carrier building block of the present invention does not specifically bind to any non-human protein and / or any non-protein molecule (including biomolecules), preferably the protein-based carrier building block does not specifically bind to any non-human protein and / or any non-protein molecule (including biomolecules) to which a protein-based carrier building block precursor specifically binds, or at a concentration greater than 5x10 -4 mol / L K D (K D value) to combine them.

[0392] Thus, in one embodiment, the present invention provides a molecule comprising at least one protein-based carrier building block as described herein, wherein the protein-based carrier building block has at least one cargo (e.g., a "model cargo," such as a maleimide-modified alanine) attached or coupled thereto (via at least one attachment point or coupling site contained in the protein-based carrier building block), and wherein the protein-based carrier building block does not specifically bind to any molecule (including biomolecules) and / or organism (e.g., cell, microorganism, virus, etc.). Thus, in one embodiment, the protein-based building block loses its target binding specificity when at least one cargo is coupled thereto. For example, the protein-based building block comprising a cargo attached thereto does not specifically bind to any molecule (including biomolecules) and / or organism (e.g., cell, microorganism, virus, etc.) to which a precursor of the protein-based carrier building block specifically binds (i.e., the protein-based building block preferably does not specifically bind to the target of the precursor when at least one cargo is attached thereto), or at a concentration greater than 5x10 -4 mol / L K D The value binds to any (non-human) molecule (including biomolecule) and / or organism (such as cell, microorganism, virus etc.) to which the protein-based carrier building block precursor specifically binds (i.e. the protein-based building block preferably also does not specifically bind to the target of the precursor when a cargo is attached thereto).

[0393] Those skilled in the art are aware of ways to reduce and / or eliminate specific binding of protein-based carrier building block precursors to proteins and / or non-protein molecules (including biomolecules). For example, mutations can be made in the amino acid sequence of the precursor building block so that it no longer specifically binds to human proteins, or any non-human proteins, or non-protein molecules (including biomolecules), or at a concentration greater than 5×10 -4 mol / L K D (KD value) to combine them.

[0394] The affinity of a molecular interaction between two molecules can be measured via different techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technology (see, for example, Ober et al. 2001, Intern. Immunology 13: 1551-1559, in particular the section "Surface plasmon resonance (SPR) experiments" starting on page 1552, which describes conditions for measuring the affinity of a molecular interaction between two molecules; or the explanations provided herein). As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows the analysis of real-time biospecific interactions by detecting changes in protein concentration in a biosensor matrix, in which one molecule is immobilized on a biosensor chip and another molecule passes over the immobilized molecule under flow conditions, thereby generating a k on 、k off The measured value, and thus the K D (or K A ) value. For example, this can be done using the well-known System (BIAcore International AB, Cytiva lifesciences, Uppsala, Sweden and Piscataway, New Jersey). For further description, see Jonsson et al. (1993, Ann.Biol.Clin.51:19-26); Jonsson et al. (1991 Biotechniques 11:620-627); Johnsson et al. (1995, J.Mol.Recognit.8:125-131); and Johnnson et al. (1991, Anal.Biochem.198:268-277). For example, the affinity (K) of a molecular interaction between two molecules can be determined via SPR on a ProteOn XPR36 instrument (Bio-Rad Laboratories). D). Experiments can be performed at 25°C and PBS pH 7.4 containing 0.005% Tween 20 (Bio-Rad Laboratories) can be used as the assay buffer. For example, targets (such as human proteins described herein or non-protein molecules (biomolecules) 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) using the ProteOn amine coupling kit (Bio-Rad Laboratories) according to the manufacturer's instructions. The protein-based building blocks of the present invention can be captured on the target-immobilized ligand lane. One ligand lane can be used as a reference surface, and no protein-based building blocks are captured on the surface. Different concentrations (e.g., ranging from 300 nM to 1.2 nM) diluted in running buffer can be flowed over the corresponding protein-based building blocks and the reference surface in multi-cycle kinetics for 2 minutes, followed by constant flow of assay buffer for 15 minutes. Between different injections, the surface can be regenerated with 3 M MgCl2 (Cytiva) or 10 mM glycine pH 1.5 (Cytiva). Several buffer blanks can be injected for double referencing. The data can be analyzed, for example, using ProteOn Manager 3.1.0 software (Bio-Rad Laboratories). The kinetic 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 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.

[0395] Another well-known biosensor technology for determining the affinity of biomolecular interactions is biolayer interferometry (BLI) (see, e.g., Abdiche et al. 2008, Anal. Biochem. 377:209-217). As used herein, the term "biolayer interferometry" or "BLI" 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 protein on the biosensor tip (signal beam). A change in the number of molecules bound to the biosensor tip results in a shift in the interference pattern, reported as a wavelength shift (nm), which is a direct measure of the number of molecules bound to the biosensor tip surface. Because the interactions can be measured in real time, association and dissociation rates as well as affinity can be determined. For example, BLI can be used to measure the affinity of the binding site. The system was performed (ForteBio, a division of Pall Life Sciences, Menlo Park, USA).

[0396] Alternatively, you can use Affinity is measured in a kinetic exclusion assay (KinExA) using a platform (Sapidyne Instruments Inc, Boise, USA) (see, for example, Drake et al., "Characterizing high-affinity antigen / antibody complexes by kinetic-and equilibrium-based methods", Anal. Biochem., 2004, 328: 35-43). As used herein, the term "KinExA" refers to a solution-based method for measuring the true equilibrium binding affinity and kinetics of unmodified molecules. The equilibrium solution of the binding unit / target complex (such as the antibody / antigen complex) is passed through a column with beads pre-coated with the antigen (or antibody), thereby allowing free antibody (or antigen) to bind to the coated molecule. Detection of the thus captured antibody (or antigen) is completed with a fluorescently labeled protein that binds to the antibody (or antigen).

[0397] also, Immunoassay systems provide a platform for automated bioanalysis and rapid sample turnover (Fraley et al., “The Gyrolab TM immunoassay system: a platform for automated bioanalysis and rapid sample turnaround", Bioanalysis 2013, 5: 1765-74).

[0398] In addition, the affinity of the molecular interaction between two molecules (e.g., between two biomolecules, such as between two proteins) or between a biomolecule (e.g., a protein) and a cell can be measured using flow cytometry to analyze the binding of a ligand to an antigen (e.g., protein, lipid (e.g., phosphatidylserine (PS)), sugar, etc.) present on the cell surface ("cell binding assay"). Those skilled in the art are familiar with cell binding assays to determine the affinity of a soluble molecule (e.g., a molecule of the present invention) to a binding partner present on the surface of a cell (e.g., a human cell). 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) propose a practical guide for measuring binding events between soluble ligands expressed, particularly on the surface of mammalian cells, and binding partners. For example, as shown in the examples, cell binding assays can be performed as follows:

[0399] a. Add a fixed number of (human or non-human) cells in cold fluorescence activated cell sorting (FACS) buffer (e.g., composed of D-PBS, 2% heat-inactivated fetal bovine serum (HI FBS), and 0.05% sodium azide) to a 96-well V-bottom plate (e.g., 50 μL of human cell suspension (e.g., 5E+04 / 96 well) or to a test tube (e.g., an Eppendorf tube);

[0400] b. optionally performing a washing step;

[0401] c. adding a soluble molecule (e.g., a molecule of the invention), or a protein-based building block of the invention, preferably labeled with a fluorescent marker or epitope tag, and incubating for a certain amount of time until the reaction reaches equilibrium, typically at low temperature (typically 4° C.) with shaking for several hours, for example for about 3 h;

[0402] d. Assessing binding of soluble molecules to human cells by flow cytometry, e.g., by FACS.

[0403] Thus, a cell binding assay can be performed by adding a plurality of cells (human or non-human cells, such as non-human animal, plant, microbial cells, etc.) to a recipient (e.g., a 96-well V-bottom plate or test tube as described above), preferably in a physiological buffer, adding the molecule to be assessed for binding (e.g., a molecule of the invention, or a protein-based building block of the invention), which molecule is preferably labeled, and incubating it with the cells for a certain amount of time, such as typically several hours (e.g., for about 3 h) while the reaction reaches equilibrium, preferably at low temperature (typically 4° C.), preferably with shaking, and finally evaluating the binding of the soluble molecule to the human cells by flow cytometry (e.g., by FACS).

[0404] To calculate the binding affinity (e.g., K) between a soluble molecule (e.g., a molecule of the invention) and human cells D and / or EC 50 ), different concentrations (spanning above and below the expected K D and / or EC 50 The soluble molecule from step c above (two orders of magnitude) is added to each well / test tube. The binding value can be determined from the average signal value (e.g., average fluorescence value) of each sample, and the bound fraction is plotted against the ligand concentration (logarithmic scale) and a sigmoidal curve is fitted using nonlinear regression analysis. The ligand concentration at half the bound fraction (also known as EC 50 ) will be the equilibrium dissociation constant (K D ) is a first approximation of .

[0405] A person skilled in the art can determine whether a molecule is capable of specifically binding to a human protein, as defined in the context of the present invention. For example, a person skilled in the art can use commercially available protein arrays to determine the binding affinity of a molecule (protein) to a human protein. For example, a person skilled in the art can use commercially available proteome profilers TM Antibody arrays, which allow semi-quantitative measurement of more than 100 proteins in a single sample. Alternatively or additionally, one skilled in the art can utilize, for example, HuProt TM The assay (e.g., version v4.0) consists of >21,000 unique human proteins, isomeric variants, and protein fragments, covering 16,794 unique genes. This includes 15,889 of the 19,613 canonical human proteins described in the Human Protein Atlas, which provides extensive coverage of protein subclasses. One skilled in the art can also use commercially available cell arrays, such as human, non-human animal, plant, bacterial, yeast, etc. arrays to determine the binding affinity (e.g., K) of a molecule (protein) to human cells. D and / or EC 50 ). See also, e.g., Example 16.

[0406] Similarly, one skilled in the art can determine whether a molecule can specifically bind to a non-human protein, such as a bacterial or viral protein. For example, one skilled in the art can use protein binding assays to determine the binding affinity of a 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 non-protein molecule, such as 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 proteins and non-protein molecules) can be measured by SPR. SPR allows the determination of the K of a potential interaction between two molecules. D , as described in detail above.

[0407] As will be apparent to the skilled person, at least one carrier building block comprised in a molecule of the invention may exhibit non-specific binding to one or more human proteins (and / or to one or more non-human proteins, and / or to one or more non-protein molecules (such as human non-protein molecules), and / or to one or more human cell types, as described above). This is because there are molecular forces, e.g. in the form of hydrophobic interactions, hydrogen bonding, van der Waals interactions and other non-specific interactions, between at least one carrier building block of the invention and one or more human proteins (and / or to one or more non-human proteins, and / or to one or more non-protein molecules (such as human non-protein molecules), and / or to one or more human cells, as described above). Thus, if this occurs, at least one carrier building block comprised in a molecule of the invention may non-specifically bind to one or more human proteins (and / or to one or more non-human proteins, and / or to one or more non-protein molecules (such as human non-protein molecules), and / or to one or more human cells, as described above, if this is the case). In the context of the present invention, greater than 5x10 -4 mol / L of any K D value (or less than 2x10 3 Liter / mol of any K AValues) are generally considered to represent "non-specific binding". Thus, a building block can bind to any human protein (or non-human protein, and / or any human cell, if this is the case, as described above) with an affinity greater than 5x10 -4 mol / L K D (K D value) (or less than 2x10 3 Liter / mol K A value), such as greater than 5.5x10 -4 mol / L K D (K D value) (or less than 1.8x10 3 Liter / mol K A value), or greater than 6x10 -4 mol / L K D (K D value) (or less than 1.7x10 3 Liter / mol K A Furthermore, in the context of the present invention, in a preferred embodiment, the carrier building block can bind to any non-protein molecule, such as any human non-protein molecule (e.g., DNA, RNA, lipids (e.g., phosphatidylserine (PS)), glycans) with an affinity greater than 5x10 -4 mol / L K D (K D value) (or less than 2x10 3 Liter / mol K A value), such as greater than 5.5x10 -4 mol / L K D (K D value) (or less than 1.8x10 3 Liter / mol K A value), or greater than 6x10 -4 mol / L K D (K D value) (or less than 1.7x10 3 Liter / mol K A Furthermore, in the context of the present invention, the building blocks can bind to any human cell with an affinity greater than 5x10 -4 mol / L K D (K D value) (or less than 2x10 3 Liter / mol K A value), such as greater than 5.5x10 -4 mol / L K D (K D value) (or less than 1.8x10 3 Liter / mol K Avalue), or greater than 6x10 -4 mol / L K D (K D value) (or less than 1.7x10 3 Liter / mol K A In the context of the present invention, this binding affinity is considered to be "non-specific binding".

[0408] In one embodiment, the protein-based carrier building blocks of the present invention are not derived from the crystallizable fragment of an antibody (Fc, which contains two C H 2 and two Cs H 3 domains), such as the Fc fragment of a monoclonal antibody (mAb). In another embodiment, the protein-based carrier building blocks of the present invention are not derived from the C H 2 and / or C H In another embodiment, the protein-based carrier building blocks of the present invention are not derived from C domains contained in the antigen-binding fragment (Fab) of an antibody. H 1 and / or C L domains, such as the C H 1 and / or C L In one embodiment, the molecule of the invention 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 invention is not (or is not derived from) a Fab of an antibody (such as a mAb).

[0409] In one embodiment, the molecules of the invention do not comprise V H -V L or, for example, it does not comprise at least one V that interact (bind) with each other as in an antibody H and at least one V L In another embodiment, the molecules of the invention do not comprise C L -C H 1 conjugate, for example, which does not contain at least one C L and at least one C H 1.

[0410] Attachment point or coupling site

[0411] As mentioned above, the carrier building blocks present in the molecules of the present invention have at least one attachment point (also referred to as coupling site in this application), preferably at a solvent accessible position, as further defined below. Preferably, the at least one protein-based carrier building block comprises more than one attachment point or coupling site, preferably at a solvent accessible position. In a preferred embodiment, the protein-based carrier building block comprises at least two attachment points or coupling sites. In another embodiment, the protein-based building block comprises three or more coupling sites, such as six or nine coupling sites. For example, the protein-based carrier building block can have two, three, four, five, six, seven, eight, nine, ten or more coupling sites. In one embodiment, if there are more than one coupling site, the coupling sites present in the carrier building block are different from each other. For example, if a carrier building block comprises two coupling sites, these coupling sites can be functionally / chemically different from each other, i.e., each coupling site or point of attachment is chemically different from each other (e.g., if there are two coupling sites, one coupling site can be an -SH group present in the side chain of a cysteine located at a solvent accessible position, and the other coupling site can be an -NH2 group present in the side chain of a lysine located at a solvent accessible position). If a building block has more than two coupling sites (e.g., at least three coupling sites, such as three, four, five, six, seven, eight, nine, ten, etc.), at least two types of coupling sites can be present in the at least three coupling sites present in the building block. In another embodiment, if a building block has three coupling sites, each coupling site is functionally different from each other. In another embodiment, if a building block has three coupling sites, two coupling sites are identical and one coupling site is functionally different from the other two coupling sites. In another embodiment, all coupling sites present in the building block are functionally different from each other. In another embodiment, all coupling sites present in a carrier building block are identical. For example, a protein-based building block may contain one, two, three, four, five, six, seven, eight, nine, ten or more identical coupling sites, for example, all -SH groups present in the side chains of cysteines located at solvent accessible positions in the protein-based building block.

[0412] In another embodiment, alternatively or additionally, if there is more than one coupling site, the coupling sites are spatially separated from each other (spaced apart from each other). It will be understood by those skilled in the art that the minimum distance between the coupling sites will be determined by the properties of the cargo (and linker, if used), which is to be attached or coupled to the attachment point or coupling site in the protein-based carrier building block. For larger cargos (e.g., ISVDs), the minimum distance can still be kept small when used in combination with long linkers, which increase the required flexibility and target binding envisioned. The short distance between the coupling sites combined with short linkers (if any) may limit the target binding of the larger cargo and result in limited engagement (e.g., increased cell specificity). In addition, the solubility of the molecule may be reduced (i.e., the molecule may be more susceptible to aggregation). On the other hand, if the cargo to be attached is quite small (e.g., a radioisotope), the minimum distance may also be kept very small even in the absence of a linker, as shown in Example 5 below. Thus, one skilled in the art will be able to select the location of a particular conjugation site, as well as 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 building block.

[0413] A "coupling site" or "point of attachment" can be a reactive group in the side chain of a natural or non-natural (also referred to as "atypical," "unnatural," or "uncommon," as described above) amino acid, preferably located at a solvent accessible position in the protein-based carrier building block. It can also be a C-terminal and / or N-terminal reactive group (a -COOH and -NH2 group, respectively) of the protein-based carrier building block. In the context of the present invention, a "reactive group in the side chain of an amino acid (natural or non-natural, as defined above)" refers to any chemical group present in the side chain of an amino acid that is capable of forming 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 in its side chain is a primary amine. For example, if the amino acid is cysteine, the reactive group present in its side chain is a thiol group. For example, if the amino acid is aspartic acid or glutamic acid, the reactive group present in its side chain is a carboxyl group. For example, if the amino acid is tyrosine, the reactive group present in 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 guanidine group.For example, if the amino acid is methionine, the reactive group present on its side chain is a thioether group.

[0414] In the context of the present invention, "the C-terminal or N-terminal reactive group of a protein-based carrier building block" refers to the -COOH and -NH2 reactive groups present in the C-terminal and N-terminal amino acids of a protein-based carrier building block. If the carrier building block does not have a free C-terminus and / or N-terminus (e.g., because the carrier building block is C-terminally and / or N-terminally connected to another protein-based building block or to another peptide or protein, or because the N-terminus is amidated, or because the C-terminus is acetylated, etc.), then the N-terminus and C-terminus of the carrier building block are not suitable as attachment points or coupling sites as defined herein. In some embodiments, a "coupling site" or "attachment point" is not a C-terminal or N-terminal reactive group of a protein-based carrier building block.

[0415] At least one coupling site or connection point present in the building block of the present invention may already be present in the building block precursor (e.g., an -NH2 group in the side chain of a lysine present in the building block precursor, preferably in a solvent accessible position) or may be engineered. Preferably, at least one or more connection points or coupling sites of a protein-based building block are engineered. In the context of the present invention, an "engineered" connection point or coupling site means a coupling site or connection point that is present in the protein-based carrier building block but not in the same or corresponding position in its precursor. For example, a protein-based building block precursor may be modified to introduce one or more connection points or coupling sites, as described in detail below. A non-limiting example of an engineered connection point or coupling site is a reactive group present in the side chain of an amino acid in a protein-based carrier building block that is not present in the same or equivalent position in the building block precursor. For example, if a building block precursor has a serine at a certain position X (preferably a solvent accessible position) in the building block precursor, and this serine is mutated to a cysteine in the carrier building block, then the -SH group of the cysteine will be an engineered attachment point or coupling site. For example, if an amino acid (e.g., Cys or Tyr) is added to the N-terminus or C-terminus of the building block precursor, then the reactive group present in the side chain of the newly added amino acid in the carrier building block will be an engineered attachment point or coupling site.

[0416] Thus, in a preferred embodiment, the protein-based carrier building blocks of the present invention have at least two coupling sites or attachment points, at least one of which, preferably at least two, are engineered attachment sites or coupling sites, i.e., they are not present at the same or corresponding positions in the building block precursor. In another preferred embodiment, all coupling sites or attachment points present in the protein-based carrier building blocks are engineered attachment sites or coupling sites, i.e., they are not present at the same or corresponding positions in the building block precursor. In one embodiment, the carrier building block has two or more engineered attachment sites or coupling sites, such as three, four, five, six, seven, eight, nine, ten or more engineered attachment sites or coupling sites.

[0417] As used herein, a residue position in one polypeptide sequence "corresponds to" a residue position in another polypeptide sequence if it is present at the equivalent position in the polypeptide sequence, as indicated, for example, by primary sequence homology or functional equivalence or Kabat numbering. Corresponding positions can be identified by alignment of the two polypeptide sequences. Alignments for identifying corresponding positions or corresponding regions can be obtained using conventional alignment algorithms such as Blast (Altschul et al., "Basic local alignment search tool", J Mol Biol., 1990, 215(3):403-10).

[0418] At least one coupling site present in a carrier building block of the present invention may be free (i.e., ready to react) or blocked / protected. Thus, the α-amino group, the carboxylic acid terminus, or a reactive group (e.g., an amine, carboxylic acid, alcohol, thiol) present in the side chain of one or more amino acids of the carrier building block may be blocked or protected with a protecting group (e.g., a protecting group), for example, to prevent polymerization of amino acids, to minimize undesirable side reactions during synthesis of the building block, or to selectively attach different cargoes. Of course, if at least one coupling site is blocked or protected, it must be unblocked or deprotected prior to attaching or coupling the cargo, as described in detail below.

[0419] Thus, at least one coupling site present in the protein-based carrier building blocks of the present invention may be (without limitation) a primary amine, a thiol group, a hydroxyl group, a guanidinium group, a carboxyl group or a thioether group. For example, the coupling site may be a free or blocked (protected) thiol group.

[0420] Thus, in some embodiments, at least one coupling site present in a protein-based carrier building block of the invention can be a primary amine present in the side chain of a lysine (or ornithine (Orn), or diaminopropionic acid (Dap), or diaminobutyric acid (Dab)) present in the protein-based building block, preferably located at a solvent accessible position. In other embodiments, the coupling site is a thiol group present in the side chain of a cysteine present in the protein-based building block, preferably located at a solvent accessible position in the protein-based building block. In other embodiments, the coupling site is a carboxyl group present in the side chain of an aspartic acid or glutamic acid present in the protein-based building block, preferably located at a solvent accessible position in the protein-based building block. In other embodiments, the coupling site is a guanidinium group present in the side chain of an arginine present in the protein-based building block, preferably located at a solvent accessible position in the protein-based building block. In other embodiments, the coupling site is a thioether group present in the side chain of a methionine present in the protein-based building block, preferably located at a solvent accessible position in the protein-based building block. In other embodiments, the coupling site is a phenolic OH-group of a tyrosine in a protein-based building block, preferably located at a solvent accessible position in the protein-based building block. In one embodiment, the tyrosine is preferably located at the N-terminus or C-terminus of the protein-based carrier building block of the molecule. In other embodiments, the coupling site is the N-terminal primary amine of the carrier building block, if it is free and preferably solvent accessible. In other embodiments, the coupling site is the C-terminal carboxyl group of the carrier building block, if it is free and preferably solvent accessible.

[0421] As mentioned above, the coupling site can be free or protected.For example, as described above, if the coupling site is a thiol group (for example, from the cysteine in the building block based on protein, preferably located at the solvent accessible position in the building block based on protein), the thiol group can be free (-SH) or protected / end-capped. The end-capped thiol group refers to the thiol group (for example, with another cysteine, with glutathione (GSH), with cysteamine or with such as benzyl (Bzl, Bn), trityl (Trt), diphenylmethyl (Dpm, Bzh, Bh), tetrahydropyranyl (Thp), tert-butyl (tBu) and other protecting groups) (reversibly) protected. Spears, R. et al. (" Cysteine protecting groups: applications in peptide and protein science ", Chem.Soc.Rev., 2021, 50, 11098) provide a review on different cysteine protecting groups. In addition, Isidro-Llobet, A. et al. (“Aminoacid-protecting groups”, Chem Rev., 2009, 109(6): 2455-504) provide a review on different amino acid protecting groups.

[0422] In one embodiment, the protein-based carrier building blocks of the present invention comprise at least two attachment points or coupling sites, which are two reactive groups present in the side chains of two amino acids (which may be natural or non-natural) in the protein-based carrier building block, preferably located at solvent accessible positions in the protein-based carrier building block. For example, in one embodiment, the protein-based carrier building block comprises at least two attachment points or coupling sites, which are two reactive groups present in the side chains of two natural amino acids (e.g., two Cys) in the protein-based carrier building block, preferably located at solvent accessible positions in the protein-based carrier building block.

[0423] In one embodiment, at least one of the attachment points or coupling sites present in the protein-based building block is linked (directly or via a linker) to a cargo as defined herein. In a preferred embodiment, the molecule of the invention comprises at least one protein-based carrier building block and at least one cargo, wherein said at least one cargo is attached or coupled to said at least one protein-based carrier building block via said at least one attachment point or coupling site. A "cargo" can be any molecule that is attached or coupled to / can be attached or coupled to a protein-based carrier building block via one or more attachment points or one or more coupling sites present therein. For example, cargo that can be attached or coupled to a protein-based carrier building block of the invention is a protein, a peptide, an ISVD (such as a V HH 、V L or V H ), polyethylene glycol (PEG), small molecules (e.g., nostoc, DM4), polysaccharides (e.g., M6P), lipids, chelating agents, fluorophores, radioisotopes, vitamins (e.g., folic acid or biotin), nucleic acids (e.g., oligonucleotides or siRNA), etc. The cargo may have different functions. For example, at least one cargo may be a half-life extending (HLE) molecule, a targeting molecule, a therapeutic molecule or a precursor thereof, an imaging molecule, a toxic molecule, an agonist (e.g., a Toll-like receptor (TLR) agonist), a T cell engaging molecule, a scavenging / degrading molecule, a cell penetrating molecule, a nuclear localization molecule, a blood-brain barrier (BBB) shuttle, a radiotherapeutic molecule, or an imaging probe.

[0424] Thus, in a further embodiment, the molecules of the invention comprise at least one protein-based carrier building block and at least one cargo, wherein the cargo is attached or coupled to the at least one protein-based carrier building block via the at least one attachment point or coupling site, and wherein the cargo is an HLE molecule (such as an albumin-binding ISVD (as described herein, e.g., as defined in Table 8, such as SEQ ID NO.: 63 or 106)), or a PEG molecule, or an ELNN polypeptide, as described herein. In a further embodiment, the molecules of the invention comprise at least one protein-based carrier building block and at least one cargo, wherein the cargo is attached or coupled to the at least one protein-based carrier building block via the at least one attachment point or coupling site, and wherein the cargo is a targeting moiety and / or therapeutic moiety as described herein. In a further embodiment, the molecules of the invention comprise at least one protein-based carrier building block and at least two cargos, wherein the cargo is attached or coupled to the at least one protein-based carrier building block via at least two attachment points or coupling sites, wherein the at least two cargos are one HLE molecule as described herein and one therapeutic and / or targeting moiety as described herein.

[0425] In one embodiment, at least one protein-based carrier building block comprised in a molecule of the invention comprises at least two cysteines, preferably in solvent accessible positions, such as preferably three cysteines, or six cysteines, or nine cysteines, preferably in solvent accessible positions, said cysteines having free or blocked thiol groups, said thiol groups being at least two (such as three, or six, or nine) coupling sites as defined herein. In one embodiment, at least one protein-based carrier building block comprised in a molecule of the invention comprises three cysteines, preferably in solvent accessible positions, said cysteines having free or blocked thiol groups, said thiol groups being three coupling sites as defined herein. In one embodiment, apart from the three cysteines with three coupling sites (free or blocked thiol groups) in solvent accessible positions, the protein-based carrier building block does not comprise any other cysteines in solvent accessible positions (but may comprise one or more cysteines that are not in solvent accessible positions). In another embodiment, at least one protein-based carrier building block comprised in a molecule of the invention comprises four, five, six, seven, eight, nine, ten or more cysteines with free or blocked thiol groups, preferably located in solvent accessible positions, said cysteines being four, five, six, seven, eight, nine, ten or more coupling sites as defined herein. In one embodiment, in addition to the four, five, six, seven, eight, nine, ten or more cysteines with four, five, six, seven, nine, ten or more coupling sites (free or blocked thiol groups) located in solvent accessible positions in the building block, the protein-based carrier building block does not comprise any other cysteines in solvent accessible positions. In other embodiments, at least one protein-based building block comprised in a molecule of the invention comprises at least one amino acid, such as one, two, three, four, five, six, seven, eight, nine, ten or more amino acids, which may be natural or non-natural, preferably located in a solvent accessible position, comprising a reactive group on its side chain, said reactive group being a coupling site as defined herein. In another embodiment, at least one protein-based building block comprised in a molecule of the invention comprises at least two coupling sites, one of which is a (free or protected) thiol group from a cysteine, preferably located in a solvent accessible position in the protein-based carrier building block, and the other is a -OH group from a tyrosine, preferably from an N-terminally or C-terminally exposed tyrosine, preferably located in a solvent accessible position in the protein-based carrier building block.In another embodiment, at least one of the protein-based building blocks comprised in the molecules of the invention comprises at least two coupling sites, one of which is a (free or protected) thiol group from a cysteine, preferably located at a solvent accessible position in the protein-based carrier building block, and the other is a reactive group from an unnatural amino acid, preferably located at a solvent accessible position in the protein-based carrier building block.

[0426] In one embodiment, the coupling site or point of attachment in the protein-based building block is a selenol (-HSe) group from selenocysteine (Sec or U), which can be located, for example, in the C-terminus of the protein-based carrier building block. In another embodiment, the coupling site or point of attachment in the protein-based building block is the keto group of p-acetylphenylalanine (pAcPhe), which can be selectively coupled to alkoxyamine-derived cargoes, see, for example, Jun Y. Axup et al., "Synthesis of site-specific antibody-drug conjugates using unnatural amino acids", PNAS, 2012, 109(40)16101-16106.

[0427] If this is the case, those skilled in the art will know how to incorporate one or more unnatural amino acids into at least one protein-based building block included in the molecules of the invention. For example, WO 2021 / 050554 (the contents of which are hereby incorporated by reference) describes in detail how to incorporate one or more unnatural amino acids into proteins.

[0428] In one embodiment, the coupling site is a free or blocked thiol group in the cysteine side chain, preferably present in a solvent accessible position in the building block. When it comes to site-specific modification of proteins (also known as bioconjugation), cysteine is typically the site of choice due to its favorable properties (nucleophilic characteristics of thiols at neutral / near-neutral pH, low natural abundance, and general ease of incorporation into proteins via site-directed mutagenesis) (from Spears RJ et al., "Cysteine protecting groups: applications in peptide and protein science", Chem. Soc. Rev., 2021, 50, 11098-11155).

[0429] In a preferred embodiment, the at least one coupling site or attachment point is selected from: a thiol group (-SH, free or blocked) present in the side chain of a cysteine, preferably located at a solvent accessible position in the protein-based carrier building block; -NH2 (primary amine, from the N-terminus of the protein-based building block or present in the side chain of an amino acid such as lysine or ornithine); -OH present in the side chain of a tyrosine (C-terminal tyrosine, N-terminal tyrosine, or preferably a tyrosine present at any other solvent accessible position in the protein-based carrier building block); C-terminal -COOH; and an azido group present in the side chain of an unnatural amino acid such as azidolysine. More preferably, the at least one coupling site or attachment point is a thiol group (free or blocked) present in the side chain of a cysteine, preferably located at a solvent accessible position in the protein-based building block.

[0430] In one embodiment, the protein-based building blocks of the invention comprise six attachment points or coupling sites, wherein three of them are -SH groups present in the side chains of the three Cys, preferably located at solvent accessible positions, and wherein three of them are -NH2 present in the side chains of the three Lys, preferably located at solvent accessible positions in the protein-based building blocks.

[0431] Thus, at least one coupling site present in at least one building block included in the molecule of the present invention allows coupling of different payloads (directly or through a linker, as will be clear to those skilled in the art and described in detail below). Those skilled in the art know how to attach a payload to one or more coupling sites present in a building block. For example, Spicer CD et al. ("Achieving controlled biomolecule-biomaterial conjugation", Chem Rev. 2018, 118 (16): 7702-7743, the contents of which are hereby incorporated by reference) provide a review of biomolecule coupling chemistry and provide a comprehensive overview of key strategies for achieving controlled functionalization.

[0432] For example, if the coupling site is present in the side chain of cysteine -SH group (free or blocked), the cysteine is preferably located at a solvent accessible position in a protein-based carrier building block, then the load can be attached or coupled to the building block (directly or through a linker) by alkylation, metal-assisted arylation, disulfide exchange or addition to maleimide Michael acceptor. It can also be attached or coupled using so-called "coupling based on PODS", see, for example, 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 provide a high level of chemical selectivity for cysteine (see, for example, D. Alvarez Dorta et al., Chem. Eur. J. 2020, 26, 14257). If at least one coupling site is an -SH group (free or blocked) present in the side chain of a cysteine, which is preferably located at a solvent-accessible position in the protein-based carrier building block, the cargo can be attached or coupled to the building block by addition to a maleimide Michael acceptor. Typically at pH 6.5 to 7.5, the maleimide present in the cargo will react specifically with the at least one free thiol to form a thioether bond. Of course, if the -SH group is blocked or protected, it should first be unblocked or deprotected (e.g., reduced with a reducing agent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP)) before the cargo can be attached to it. 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)propiolenitrile) can be used to attach or couple a cargo to a -SH attachment point present in the side chain of a cysteine, preferably located at a solvent accessible position in a protein-based carrier building block. For example, the bis-maleimido-PEG3-linker (1,11-bismaleimido-triethylene glycol) can be used to attach or couple a cargo to a -SH attachment point present in the side chain of a cysteine, preferably located at a solvent accessible position in a protein-based carrier building block.In addition, maleimide-modified cargoes (see, e.g., PEG-maleimide, N-ethylmaleimide, maleimido-PEG-acid, resiquimod (R-848)-maleimide, nostoc-PEG-maleimide) can be attached to -SH attachment points present in the side chains of cysteines, which are preferably located at solvent accessible positions in protein-based carrier building blocks, see also the Examples.

[0433] For example, if the coupling site is the -OH group of tyrosine, which is preferably located at a solvent-accessible position in the protein-based carrier building block, the cargo can be attached or coupled to the building block (directly or through a linker) by several chemical methods, such as cross-linking via catalytic one-electron oxidation of tyrosine, three-component Mannich-type tyrosine coupling, coupling via sulfur hexafluoride exchange chemistry (SuFEx), transition metal complexes for tyrosine coupling, diazo coupling reactions, reactions with triazolinediones, etc. (for review, see, for example, D. Alvarez Dorta et al., Chem. Eur. J., 2020, 26, 14257).

[0434] Alternatively or additionally, if the coupling site is the -OH group of the N-terminal and / or C-terminal tyrosine, the cargo can be enzymatically attached or coupled 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. As described therein, if coupling of at least one cargo to the N-terminal and / or C-terminal tyrosine is to be performed, the protein-based building block can preferably be formed with a flexible (GG) or (G4S1) 1-3 The GG tag (sequence) is extended to facilitate enzymatic addition, as described by Alan M. Marmelstein et al. cited above. In this case, tyrosinase (abTYR) from Agaricus bisporus can be used, which is a copper-dependent enzyme that converts tyrosine to melanin via an o-quinone intermediate. Alternatively, the much smaller Bacillus megaterium tyrosinase (bmTYR) can be used to catalyze the reaction.

[0435] For example, if the coupling site is an N-terminal primary amine of a protein-based carrier building block and / or a primary amine present in an amino acid side chain, preferably located at a solvent accessible position in the protein-based carrier building block (e.g., Lys, Orn, or any unnatural amino acid having a primary amine on its side chain), the cargo can be attached or coupled to the carrier building block (directly or through a linker) by reaction of a group present in the cargo / linker (e.g., an isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidate, carbodiimide, anhydride, or fluorophenyl ester) with the primary amine. See, for example, Bioconjugate Techniques (3rd edition), 2013, Chapter 3 - "The reactions of bioconjugation", Greg T. Hermanson.

[0436] For example, if the building block contains at least two coupling sites, including -OH from tyrosine and -SH from cysteine (the coupling sites are preferably located at solvent-accessible positions in the protein-based carrier building block), the thiol nucleophile can be conveniently blocked by forming a disulfide with Ellman's reagent. After the coupling reaction of the -OH from tyrosine, the thiol group can be deblocked by brief exposure to an appropriate reducing agent, as described by Alan M. Marmelstein et al., mentioned above.

[0437] Another option for attaching or coupling a cargo to a connection point or coupling site in a protein-based building block (directly or through a linker) is to use a sortase-mediated transpeptidation reaction. Sorting enzymes allow functionalization of the N-terminus, C-terminus, and the creation of non-natural fusions (i.e., NN or CC chimeras) via the installation of click handles, see, for example, Guimaraes CP et al. (“Site-specific C-terminal and internal loop labelling of proteins using sortase-mediated reactions”, Nature Protocols, 2013, 8(9): 1787-1799). As described in this scheme, sortase-mediated reactions are applicable to any protein of interest (e.g., a protein-based carrier building block of the present invention) provided that it contains (i) an LPXTG motif (where X can be any amino acid and glycine cannot be a free carboxylate) as a sortase target, or (ii) an appropriately exposed glycine residue to act as an introduced nucleophile. This natural nucleophile can be replaced by an oligoglycine residue (Gly) at the N-terminus.1-5 )(In many cases a single glycine is sufficient) can be substituted for any peptide / protein. In turn, the peptides can be decorated with any cargo molecule (e.g., fluorophores, biotin, cross-linkers, lipids, carbohydrates, nucleic acids), provided that a free N-terminal glycine remains available on the peptide for use as the introduced nucleophile. Thus, incubation of the sortase, the LPXTG-containing protein and the nucleophile results in the covalent attachment of the nucleophile to the protein of interest in a site-specific manner. Guimaraes CP et al., mentioned above, provide a scheme that allows any given protein to be functionalized at its C-terminus. The target protein is engineered with a sortase recognition motif (LPXTG). Upon recognition, the sortase cleaves the protein between the threonine and glycine residues, thereby facilitating the exogenously added oligoglycine (Gly) modified with the selected functional group. 1-5 ) peptide (e.g., cargo to be attached to a protein-based carrier building block). Theile CS 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 the protocol, the protein to be labeled is engineered at its N-terminus with an exposed segment of glycine or alanine (when sortase A from Staphylococcus aureus (S. aureus) or Streptococcus pyogenes (S. pyogenes), respectively). A peptide (e.g., cargo) decorated with a selected functional group (fluorophore, biotin, lipid, nucleic acid, carbohydrate, etc.) and containing the sortase recognition motif LPXTG / A sequence (X is any amino acid, as described above) at its C-terminus is then added to the reaction along with the sortase. Sortase A cleaves between threonine and glycine / alanine residues, forming 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 Theile CS et al., supra). Figure 1). Alternatively, depsi peptides can be used for N-terminal tagging, see Theile CS et al. mentioned above. 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 producing N- to -N and C- to -C fusion proteins. By equipping the N- or C-terminus of the target protein with a set of click handles using sortase A, and then performing a strain-promoted click reaction, unnatural N- to -N and C- to -C linked (heterologous) fusion proteins are created. As described by Witte MD et al., peptides for creating C- to -C linked proteins are synthesized with an N-terminal triglycine motif and an azide or a C-terminal cyclooctyne (DIBAC) (see also the present application). Figure 2 ). The protein of interest is engineered with a C-terminal LPXTG sequence. To prepare N-to-N linked proteins, the authors of this protocol synthesized peptides containing the 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 aid precipitation of the peptide after cleavage from the resin and to increase the solubility of the peptide in water), and an azide group, or a cyclooctyne group at the N-terminus of the probe. The protein to be linked should contain 1-5 Gly at the N-terminus. The final step of the procedure is to fuse the protein containing the click handle, see Witte MD et al. Figure 1 .

[0438] In view of the above, cargo can be attached or coupled to a protein-based carrier building block (directly or through a linker) using a sortase, as described in detail by Guimaraes CP et al., Theile CS et al., and Witte MD et al., the contents of which are hereby incorporated by reference. Using the above-described sortase method, cargo can be attached or coupled (directly or through a linker) at a coupling site or connection point in a protein-based carrier building block (which is at the N-terminus or C-terminus of the protein-based building block). Thus, if the coupling site or connection point of a protein-based carrier building block is the C-terminus of the building block, cargo can be attached or coupled thereto using a sortase, provided that the C-terminus of the building block contains a sortase recognition motif (LPXTG) and the cargo contains a cargo that is N-terminally separated by an oligoglycine ((Gly) 1-5) modified peptides (see Guimaraes CP et al. Figure 2 If the coupling site or point of attachment of a protein-based carrier building block is the N-terminus of the building block, the cargo can be attached or coupled thereto using a sortase, provided that the N-terminus of the building block includes (Gly) 1-5 The tag sequence and the cargo contains a sortase recognition motif (LPXTG / A) at the C-terminus (see Theile CS et al. Figure 1 ). In addition, protein or peptide cargo can be attached to the N / C termini of protein-based carrier building blocks in an N-to-N and / or C-to-C manner as described by Witte MD et al.

[0439] Thus, by selecting appropriate (possibly initially blocked) coupling sites, one skilled in the art is able to attach or couple different cargoes to the building block.

[0440] Solvent accessible locations

[0441] As stated above, at least one coupling site or attachment point present in a protein-based carrier building block is preferably located at a solvent accessible position in the building block.

[0442] One skilled in the art is able to identify "solvent accessible positions" in a carrier building block precursor. This can be done in silico by computer modeling. For example, one skilled in the art can utilize readily available software tools such as MAESTRO ( LLC, New York, NY, 2021)), a multi-agent prediction system based on a statistical scoring function (SSF) and different machine learning methods, see, for example, Laimer et al. BMC Bioinformatics (2015) 16: 116. In addition, 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 coupling site of a building block. With the help of computer tools such as MAESTRO or YASARA, those skilled in the art are able to identify solvent accessible positions that may be suitable for engineering coupling sites as defined above. Therefore, with the help of tools such as MAESTRO or YASARA, potentially suitable coupling sites are identified. Examples of how to identify solvent accessible positions that are potentially suitable for engineering coupling sites as defined above are provided in the examples of the present application (e.g., Examples 1-3). As described therein, a protein is selected as a starting point for developing a protein-based carrier building block (a so-called "building block precursor"). Using, for example, MAESTRO, the solvent accessible surface area (SASA) in the building block precursor is greater than or equal to, for example (square angstrom) residue can be considered as solvent accessible.Then the stability (Δ G in solvent) of the mutation (for example, mutation to cysteine residues) of each identified residue can be calculated, for other details, see, for example, Laimer J. et al. " MAESTRO--multi agent stability prediction upon point mutations ", BMC Bioinformatics, 2015, 16: 116. Destabilizing mutations (for example, mutations with a higher Δ G calculated in a solvent) are generally not further considered as potential positions of coupling sites or connection points. Therefore, once potential suitable coupling sites are identified with the help of tools (such as MAESTRO or YASARA), the stability (Δ G in solvent) of the mutation (for example, mutation to cysteine residues) of each identified residue is calculated. Those residues with the lower Δ G calculated in a solvent will preferably be further selected as potential positions of coupling sites or connection points. For example, the Δ G values in the range of -20 to +5 can be considered as non-destabilizing mutations. Those skilled in the art will appreciate that the ΔG values for each mutation of the identified residues can vary depending on the specific protein and / or specific mutation under consideration. Those skilled in the art will also appreciate that preferred mutations are those with the lowest ΔG values. Based on these ΔG values, the number of coupling sites, and the type of cargo to be coupled, those skilled in the art will further select certain positions with the help of tools such as MAESTRO or YASARA over other positions initially identified as potentially solvent accessible.

[0443] Alternatively or additionally, one skilled in the art can use hydrogen / deuterium exchange mass spectrometry (HDX-MS) to determine at least potentially solvent accessible positions in a protein. By monitoring the exchange of peptide bond amide protons with deuterons of the D2O solvent, HDX-MS reports on the local chemical environment and solvent accessibility of the protein backbone. The rate of hydrogen-deuterium exchange depends on the solvent accessibility and folding state of the protein (see Englander SW. et al., "Hydrogen exchange: the modern legacy of Protein Sci.,1997,6(5):1101-9).

[0444] If the identified solvent accessible position would be occupied by an amino acid having a reactive group on its side chain (e.g., occupied by cysteine), computer modeling (e.g., using MAESTRO) would also consider potential interactions of the reactive group of that amino acid (e.g., the -SH present in the side chain of cysteine) with other reactive groups present in the side chains of other amino acids in the protein-based carrier building block (e.g., with other -SH groups present in the protein, if any).

[0445] Additionally or alternatively, "solvent accessible positions" can be empirically identified and / or verified. For example, as described above, "solvent accessible positions" theoretically identified using available computer software tools (such as MAESTRO described above) can preferably be empirically confirmed by manufacturability. The formulation and process stability of potential building block candidates help to narrow the scope of example candidates in the early stages before large-scale manufacturing (see Examples, also see, for example, Ramachander, R., Rathore, N. (2013), "Molecule and manufacturability assessment leading to robust commercial formulation for therapeutic proteins" in: Kolhe, P., Shah, M., Rathore, N. (ed.) Sterile Product Development, AAPS Advances in the Pharmaceutical Sciences Series, Vol. 6. Springer, New York, NY). Therefore, once a potential suitable solvent accessible position in a protein-based building block precursor has been theoretically identified, the resulting protein-based carrier building block should preferably be evaluated for expression level, coupling efficiency, formulation, quality control, solubility, process stability, etc. Solvent accessible positions that result in building blocks that excel in expression yield, manufacturability, solubility, and / or stability are preferred; see the Examples for additional details.

[0446] For example, once the appropriate solvent accessible position in the building block precursor has been theoretically identified, protein expression of the selected variant (i.e., the resulting protein-based building block having an amino acid with one or more coupling sites at one or more solvent accessible positions selected in theory) can be performed. In this step, it can be asserted whether the introduction of specific amino acids (e.g., point mutations, addition of amino acids at the N-terminus and / or C-terminus of the protein, etc.) at the solvent accessible position identified in theory has a negative impact on the synthesis, expression level, coupling efficiency or 3D globular structure of, for example, each specific variant. In addition, as described in detail above, the required minimum solubility and the loss of specific binding to human proteins (and optionally to non-protein molecules and / or non-human proteins, preferably to the target of the precursor) can be assessed. As described in detail above, possible changes in the 3D structure can be assessed, for example, by CD (circular dichroism) spectral analysis. In addition, the stability of the resulting variant can also be confirmed by thermal shift assay. This assay detects protein melting temperature (Tm) and can therefore be used to check protein stability. It can be used to characterize the stability / folding of the 3D structure of a protein. Orange is a naturally quenching dye that interacts with the hydrophobic core of proteins (which becomes visible after thermal denaturation). Therefore, the temperature in the middle of the thermal denaturation process is labeled the melting temperature, Tm. This is one way to assess the stability of the resulting variants or mutants.

[0447] In addition, "model cargo " can be attached or coupled to selected variants to quantify the degree of coupling (coupling efficiency), that is, to determine whether the resulting protein-based building blocks with coupling sites at selected solvent accessible positions are suitable for attachment or coupling of desired cargo in practice. "Model cargo " can be any molecule with a molecular weight higher than, for example, 100Da. For example, if the potential coupling site is a thiol group, "model cargo " can be maleimide-modified alanine (e.g., N-maleyl-β-alanine) or biotin-maleimide, 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 conjugation of "one or more model cargoes" results in a stable conjugate (protein-based building block having one or more model cargoes conjugated thereto) with an acceptable degree of conjugation (determined on a case-by-case basis, e.g., ≥90% conjugation efficiency, such as 90% conjugation efficiency, or 95% conjugation efficiency, or 97% conjugation efficiency, or 99% conjugation efficiency or higher) while allowing for standard in vivo PK, retention of globular 3D structure and in vivo conjugation status, etc., then those solvent accessible positions should be preferred for cargo conjugation and conjugation of the desired cargoe(s) can occur, see also the Examples below.

[0448] point mutation

[0449] In one embodiment, at least one coupling 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 a 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 contained in a molecule of the invention comprising at least one coupling site or attachment point at a defined solvent accessible position as described herein.

[0450] For example, coupling sites can be generated by mutating a specific amino acid, preferably at a solvent accessible position in a building block precursor, to cysteine ("Cys mutation"). Alternatively or additionally, coupling sites can be generated by mutating a specific amino acid, preferably at a solvent accessible position in a building block precursor, to a natural or unnatural amino acid having a reactive group in its side chain. The distribution data of amino acids occurring at certain positions in a building block precursor (e.g., Cys, Ser) can also be used to guide the design and introduction of coupling sites.

[0451] Additionally or alternatively, the building block precursors may be modified by adding one or more amino acids to the N-terminus and / or C-terminus of the protein sequence to introduce at least one coupling site or attachment point, preferably at a solvent accessible position as described herein, to produce the protein-based building blocks of the invention.

[0452] In another embodiment, the at least one coupling site may preferably already be present at a solvent accessible position in the protein-based building block precursor and does not need to be generated. This is the case for 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 which is preferably already present at a solvent accessible position in the building block precursor, or a thiol group of a cysteine which is preferably already present at a solvent accessible position in the building block precursor.

[0453] If a building block comprises more than one coupling site, these coupling sites can be generated by introducing, for example, specific point mutations, preferably at solvent accessible positions in the peptide sequence of the building block precursor. Additionally or alternatively, other suitable coupling sites or connection points may preferably already be present at solvent accessible positions in the building block precursor, i.e., without the need for introducing, for example, specific point mutations and / or adding one or more amino acids at the N-terminus and / or C-terminus of the building block precursor. A person skilled in the art will determine the number and position of connection points or coupling sites based on the protein-based building block and the one or more cargoes to be attached thereto (directly or via a linker as described herein).

[0454] 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 predicting the effect of mutations on protein stability (e.g., based on artificial intelligence (AI)). For example, the stability of the mutant can be calculated using MAESTRO (as defined above and explained in detail in the Examples), and can also be confirmed empirically by manufacturability (including but not limited to expression level and stability assessments as described above).

[0455] In a preferred embodiment, the point mutation is the mutation of an amino acid, preferably located at a solvent accessible position in the building block precursor, to cysteine. In another embodiment, the point mutation consists of replacing a serine residue, preferably at a solvent accessible position in the building block precursor, with cysteine. In another embodiment, the point mutation is the mutation of a solvent accessible amino acid, preferably in the building block precursor, to lysine. In another embodiment, the point mutation is the mutation of a solvent accessible amino acid, preferably in the building block precursor, to tyrosine. In another embodiment, the point mutation is the mutation of a solvent accessible amino acid, preferably in the building block precursor, to a natural or unnatural amino acid as described above.

[0456] Addition of C- or N-natural and / or unnatural amino acids having reactive groups in their side chains

[0457] For example, a coupling site can be generated by adding one or more C-terminal or N-terminal natural amino acids and / or one or more C-terminal or N-terminal non-natural amino acids having a reactive group in their side chain to a building block precursor. Preferably, one or more terminal natural or non-natural amino acids (if present) are added to the C-terminus of the building block precursor. For example, one or more coupling sites are generated by adding an N-terminal or C-terminal cysteine, an N-terminal or C-terminal tyrosine and / or an N-terminal or C-terminal non-natural amino acid to a protein-based building block precursor. Preferably, at least one coupling site is generated by adding an N-terminal or C-terminal tyrosine (preferably a C-terminal tyrosine) to a protein-based building block precursor. In a preferred embodiment, the N-terminal and / or C-terminal Tyr is preceded / followed by a flexible (GG) or ((G4S1) 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 -), as described in detail in Alan M. Marmelstein et al., Journal of the American Chemical Society, 2020, 142(11), 5078-5086.

[0458] Thus, at least one protein-based carrier building block comprised in a molecule of the invention may comprise an N-terminal and / or C-terminal Cys, Tyr and / or an unnatural amino acid, such as a C-terminal Tyr, as in -GGY or -(G4S1) 1-3 GGY tag (sequence).

[0459] In addition, at least one protein-based carrier building block of the present invention may comprise an N-terminal and / or C-terminal coupling site or connection point suitable for coupling to a sortase as described above. In these cases, the protein-based carrier building block should be engineered to comprise a C-terminal sortase recognition motif (LPXTG, where X can be any amino acid), an N-terminal polyglycine ((Gly) 1-5 ) tag or both. In addition, if N-to-N- and / or C-to-C- attachment or coupling is desired, the protein-based carrier building block should be engineered to contain a C-terminal sortase recognition motif (for C-to-C- attachment) or an N-terminal polyglycine ((Gly) 1-5) tags (for N-to-N-attachment), as described in detail above. See in particular Guimaraes CP et al., Theile CS et al., and Witte MD et al., listed above.

[0460] Finally, as described above, the coupling site may be generated by a combination of the above mechanisms, for example, the at least one coupling site may be obtained by making a point mutation (e.g., a Ser to Cys mutation at a solvent accessible position of the building block, as described above), or by replacing the C-terminal and / or N-terminal amino acid (e.g., cysteine, or tyrosine, or an unnatural amino acid), or a sortase recognition motif, or a polyglycine ((Gly)) 1-5 ) tags are added to the protein-based building block precursors as described above.

[0461] Building Block Examples

[0462] Building blocks based on small, globular non-human proteins

[0463] One or more protein-based carrier building blocks of the present invention can be based on a small globular non-human protein. In the context of the present invention, 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 as described herein, preferably about 2.5 to about 50 kDa, such as about 2.5 to less than 50 kDa, more preferably about 2.5 to about 30 kDa, even more preferably about 2.5 to about 16 kDa, and having a globular three-dimensional (3D) structure as described herein. Furthermore, the at least one non-human protein-based vector building block does not specifically bind to any human protein, as defined herein, and preferably does not specifically bind to any non-protein molecule (such as nucleic acids (such as DNA, RNA), glycans, lipids (such as phosphatidylserine (PS)), etc.), such as any human non-protein molecule (biomolecule) (such as human DNA, human RNA, human glycans, human lipids (such as phosphatidylserine (PS)), etc.), preferably does not specifically bind to any non-protein molecule (such as nucleic acids (DNA, RNA), glycans, lipids (such as phosphatidylserine (PS)), etc.) to which the building block precursor (if any) specifically binds, and preferably does not specifically bind to any non-human protein (such as bacterial and / or viral proteins) to which the building block precursor (if any) specifically binds. Furthermore, preferably, the at least one non-human protein-based vector building block (i) does not specifically bind to any human cell and / or cell type, or binds to any non-human cell type at a rate greater than 5x10 -4 mol / L K D (K Dvalue) (preferably as determined by a cell binding assay) binds to human cells and / or cell types, (ii) does not specifically bind to any microorganism (such as bacteria, fungi, protists, yeasts) and / or any virus, or binds to a specific concentration greater than 5×10 -4 K in mol / L (preferably as determined by cell binding assays and / or SPR as described herein) D (K D value) binds to microorganisms (e.g., bacteria, fungi, protists, yeasts) and / or viruses), and / or (iii) 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 with a binding capacity greater than 5x10 -4 K in mol / L (preferably as determined by cell binding assays and / or SPR as described herein) D (K D value) binds to biomolecules (including human and non-human biomolecules).

[0464] In a preferred embodiment, when a cargo is coupled to at least one attachment point or coupling site on a protein-based carrier building block as described above, the protein-based carrier building block does not specifically bind to any non-human protein and / or non-protein molecule, preferably the target of the precursor. Thus, in a preferred embodiment, the molecule of the invention (which comprises at least one protein-based building block and at least one cargo attached to said at least one protein-based building block via at least one coupling site or attachment point) does not specifically bind to any non-human protein or non-protein molecule (such as any human non-protein molecule) as described herein, in particular it does not specifically bind to any protein or non-protein molecule to which the building block precursor (if any) is bound.

[0465] As mentioned above, in the context of the present invention, if a protein-based building block or molecule of the invention shows 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 described in detail above.

[0466] 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 that aims to map all human proteins in cells, tissues, and organs.

[0467] In the context of the present invention, small globular non-human proteins include proteins that are derived from human proteins but have been modified so that they are no longer human proteins. Examples of small globular non-human proteins are ISVDs (e.g., "human ISVDs" (e.g., V H 、V L ) and “non-human ISVD” (e.g. V HH 、Non-Human V H 、V L or engineered ISVD)), DARPin (derived from ankyrin repeat protein), affibody or affitin.

[0468] The globular non-human protein may have therapeutic or targeting activity.

[0469] Immunoglobulin single variable domain (ISVD)-based building blocks

[0470] In one embodiment, at least one protein-based carrier building block of the invention is based on a polypeptide comprising at least one immunoglobulin single variable domain (ISVD) (e.g., derived from V H or V HH or alternatively consisting of an ISVD (heavy chain ISVD)).

[0471] As described above, the protein-based carrier building blocks of the present invention have a globular 3D structure, are soluble, and have a size (molecular weight) of about 2.5 to about 70 kDa, such as about 2.5 to about 50 kDa, such as 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, such as about 5 to about 16 kDa, or about 7 to about 16 kDa, or about 10 to about 16 kDa. Furthermore, at least one building block comprised in a molecule of the invention does not specifically bind to any human protein, as defined in this specification, and preferably it also does not specifically bind to any non-protein molecule (such as DNA, RNA, glycans, lipids (such as phosphatidylserine (PS)) etc.), such as any human non-protein molecule (such as human DNA, human RNA, human glycans, human lipids (such as phosphatidylserine (PS)) etc.), and preferably it also does not specifically bind to any non-protein molecule (such as DNA, RNA, glycans, lipids (such as phosphatidylserine (PS)) etc.) to which the building block precursor (if any) is specifically bound, and preferably it also does not specifically bind to any non-human protein (such as bacterial and / or viral proteins) to which the building block precursor (if any) is specifically bound.

[0472] In a preferred embodiment, when a cargo is coupled to at least one attachment point or coupling site on a protein-based carrier building block as described above, the protein-based carrier building block does not specifically bind to any non-human protein and / or non-protein molecule, preferably the target of the precursor. Thus, in a preferred embodiment, the molecule of the invention (which comprises at least one protein-based building block and at least one cargo attached to said at least one protein-based building block via at least one coupling site or attachment point) does not specifically bind to any non-human protein or non-protein molecule (such as any human non-protein molecule) as described herein, in particular it does not specifically bind to any protein or non-protein molecule to which the building block precursor (if any) is bound.

[0473] As mentioned above, in the context of the present invention, if a protein-based building block or molecule of the invention shows 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 described in detail above.

[0474] Thus, in the embodiment wherein the at least one protein-based building block is based on an ISVD (preferably a heavy chain ISVD), the resulting ISVD-based building block does not specifically bind to any human protein. Furthermore, as explained above, it is preferred that the ISVD-based building block does not specifically bind to any non-protein molecules, such as any human non-protein molecules. Furthermore, it is also preferred that the ISVD-based building block does not specifically bind to any non-human protein or non-protein molecule to which the protein-based vector building block precursor (if any) as described above specifically binds.

[0475] In the context of the present invention, an "ISVD-based building block" refers to a protein-based building block derived from an ISVD, i.e., it is structurally similar to an ISVD but does not specifically bind to any human protein, preferably does not specifically bind to any target to which an ISVD specifically binds. For example, an ISVD-based building block has at least 60%, 70%, or 80% sequence identity with an ISVD (e.g., with its ISVD precursor). For example, an ISVD-based building block has at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, or more sequence identity with an ISVD (e.g., with its ISVD precursor). For example, an ISVD-based building block can share the entire amino acid sequence with its ISVD precursor, except for at least one, such as one, two, three, four, five, six, seven, eight, nine, ten, fifteen, eighteen, twenty, twenty-five, thirty or more amino acids. Furthermore, the ISVD-based 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, 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, such as 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.

[0476] Those skilled in the art are aware of ways to eliminate the specific binding properties of a certain ISVD precursor, for example, by making mutations in the amino acids responsible for binding of the ISVD to the target (e.g., in one or more amino acids corresponding to the CDRs of the ISVD), by adding amino acids and / or by deleting amino acids from the sequence of the precursor.

[0477] The term "immunoglobulin single variable domain" (ISVD) is used interchangeably with "single variable domain" to define 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, the heavy chain variable domain (V H ) and light chain variable domain (V L ) interact to form an antigen binding site. In this case, V H and VL The complementarity determining regions (CDRs) of the two will contribute to the antigen binding site, that is, a total of 6 CDRs will participate in the formation of the antigen binding site.

[0478] In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or the antigen-binding domain of a Fab fragment, F(ab')2 fragment, Fv fragment (such as a disulfide-linked Fv or scFv fragment) or diabody (all known in the art) derived from such a conventional 4-chain antibody would not generally be considered an ISVD because, in these cases, binding to the corresponding epitope of the antigen generally does not occur through a single immunoglobulin domain, but rather through a pair of associated immunoglobulin domains (such as a light chain and a heavy chain variable domain), i.e., through the V domains of the immunoglobulin domains that jointly bind to the epitope of the corresponding antigen. H -V L To happen.

[0479] In contrast, ISVDs are typically able to bind specifically to antigenic epitopes without pairing with additional immunoglobulin variable domains. The binding site of an ISVD consists of a single V H , Single V HH or a single V L Domain formation.

[0480] In the context of the present invention, in embodiments wherein the at least one protein-based building block is based on an ISVD, the ISVD building block precursor may be a light chain variable domain sequence (e.g., V L -sequence) or a suitable fragment thereof, or a heavy chain variable domain sequence (e.g., V H -Sequence or V HH sequence) or a suitable fragment thereof; provided that the resulting building block has a globular 3D structure, 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, such as 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 in detail above. The ISVD (which may preferably be a precursor of a protein-based building block comprised in a molecule of the invention) may for example be a heavy chain ISVD, such as a V H 、V HH , including camel-derived V H or humanized V HH In one embodiment, the protein-based building block precursor is V HH (Including camel-derived V H or humanized V HH), as long as the resulting protein-based building block is soluble, has a globular 3D structure, 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, such as 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 addition, 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, such as glycolipids. In addition, preferably, the resulting building block also does not specifically bind to any non-human protein to which the protein-based carrier building block precursor (if any) as described above is specifically bound. The heavy chain ISVD can be derived from a conventional four-chain antibody or a heavy chain antibody.

[0481] For example, the ISVD precursor can be a single domain antibody (or an amino acid sequence suitable for use as a single domain antibody), a "dAb" or a dAb (or an amino acid sequence suitable for use as a dAb), or ISVD (as defined in this application and including but not limited to V HH ); is any other single variable domain, or any suitable fragment of any of them, as long as the resulting protein-based building block is soluble, has a globular 3D structure and does not specifically bind to human proteins, preferably does not specifically bind to any non-protein (human) molecule such as DNA, RNA, lipids (e.g. phosphatidylserine (PS)) or glycans, such as glycolipids, and preferably also does not specifically bind to any non-human protein to which the protein-based carrier building block precursor (if any) as described above is specifically bound.

[0482] Preferably, the ISVD precursor is V H , humanized V H 、People V H 、V HH , humanized V HH or camel-derived V H More preferably, the ISVD precursor is ISVD (such as V HH , including humanized V HH or camel-derived V H ) or a suitable fragment thereof, as long as the protein-based building block is soluble, has a globular 3D structure and does not specifically bind to human proteins, preferably does not specifically bind to any non-protein (human) molecules such as DNA, RNA, lipids (e.g. phosphatidylserine (PS)) or glycans, such as glycolipids, and preferably also does not specifically bind to any non-human protein to which the protein-based carrier building block precursor (if any) as described above is specifically bound. is a registered trademark of Ablynx NV.

[0483] “V HH domain", also known as V HH 、V HH Antibody fragments and V HH Antibodies, originally described as antigen-binding immunoglobulin variable domains of "heavy chain antibodies" (i.e., "antibodies without light chains"), are described by Hamers-Casterman et al. Nature 363:446-448, 1993. The term "V HH domains” in order to combine these variable domains with the heavy chain variable domains present in conventional 4-chain antibodies (referred to in this application as “V H domains") and the light chain variable domains present in conventional 4-chain antibodies (referred to herein as "V L domain”) to distinguish them. HH For further description, please refer to the review article by Muyldermans ("Singledomain camel antibodies: current status", J Biotechnol., 2001, 74: 277-302). HH The domain can be obtained from heavy chain antibodies (HCAbs) circulating in camelids, see for example, Muyldermans S., "A guide to: generation and design of nanobodies", FEBS J., 2021, 288(7): 2084-2102. Therefore, in a preferred embodiment, the building blocks based on ISVD are combined with V HH (such as humanized V HH or camel-derived V H )(For example, its V HH For example, the building blocks based on ISVD and V HH (For example, its V HH Precursor) has at least 60%, or at least 70%, or 80%, or at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, or greater sequence identity. For example, a building block based on an ISVD can be similar to its V HHThe precursors share the entire amino acid sequence except for at least one, such as one, two, three, four, five, six, seven, eight, nine, ten, fifteen, eighteen, twenty, twenty-five, thirty or more amino acids that differ in the protein-based carrier building block.

[0484] Typically, the production of immunoglobulins involves immunization of experimental animals, fusion of immunoglobulin-producing cells to produce hybridomas, and screening for the desired specificity. Alternatively, immunoglobulins can be produced by screening naive, immune or synthetic libraries, for example by phage display.

[0485] Immunoglobulin sequences (such as V HH The production of V antibodies is widely described in various publications, such as 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 the target antigen. The V antibodies obtained from the immunization are injected into the camelid. HH The library is directed against V that bind (or do not bind) to the target antigen HH Conduct further screening.

[0486] In the context of the present invention, immunoglobulin sequences from different sources can be used, including mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences. In the context of the present invention, fully human, humanized or chimeric sequences are also included. In the context of the present invention, camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences or camelized domain antibodies (e.g., camelized dAbs described by Ward et al. (Nature, 341:544, 1989)) are also included (see, for example, WO94 / 04678, and Davies and Riechmann, "'Camelising' 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).

[0487] "Humanized V HH "Contains naturally occurring V HH The amino acid sequence of the domain corresponds to the amino acid sequence of the naturally occurring V HH One or more amino acid residues in the amino acid sequence of the sequence (and in particular the framework sequence) are replaced by V residues of a conventional 4-chain antibody from a human (e.g., as shown above). H Humanization can be achieved by replacing one or more amino acid residues present at one or more corresponding positions in the V domain. This can be done in a manner known per se, as will be clear to the skilled person, for example based on the further description herein and prior art (e.g., WO 2008 / 020079). Likewise, it should be noted that such humanized V HH can be obtained in any suitable manner known per se and is therefore not strictly limited to having used a compound comprising naturally occurring V HH Preferably, if the building block of the present invention is V HH , then V HH It is a humanized V HH .

[0488] "Camel-derived V H "Contains naturally occurring V HThe amino acid sequence of the domains corresponds to the amino acid sequence of the 4-chain antibody but has been "camelized", i.e. by replacing the naturally occurring V domains from a conventional 4-chain antibody with the V domains from the 4-chain antibody. H One or more amino acid residues in the amino acid sequence of the domain are replaced by the V HH Camelization is achieved by replacing one or more amino acid residues present at one or more corresponding positions in the V domain. This can be done in a manner known per se, which will be clear to the skilled person, for example based on the further description of this application and the prior art (e.g., WO 2008 / 020079). As defined herein, such "camelization" substitutions are typically inserted into and / or present in V H -V L In one embodiment, the amino acid positions of the camelid V-linked proteins are used to generate or design camelized V-linked proteins. H The starting material or starting point of V H The sequence is from mammalian V H sequence, or human V H Sequence, such as V H 3 sequences. However, it should be noted that such camelized V H , and is therefore not strictly limited to having used a H The polypeptides obtained by using the polypeptide domain as the starting material.

[0489] The structure of the ISVD sequence can be considered to be composed of four framework regions ("FR"), which are respectively 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"), and the framework regions are interrupted by three complementarity determining regions ("CDR"), which are respectively referred to in the art and herein as "complementarity determining region 1" ("CDR1"), "complementarity determining region 2" ("CDR2"), and "complementarity determining region 3" ("CDR3").

[0490] In addition, as further described in paragraph q) on pages 58 and 59 of WO 2008 / 020079, according to the method provided by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, Maryland, publication number 91), V HThe amino acid residues of the ISVD are numbered according to the universal numbering of domains as described in Riechmann and Muyldermans, 2000 (J. Immunol. Methods, 240(1-2):185-195; see, for example, the reference to this publication. Figure 2 ) applied to V from Camelidae HH It should be noted that, as is known in the art, H domain and V HH As is well known in the art, the total number of amino acid residues in each CDR can 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 means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. H domain and V HH The total number of amino acid residues in a domain is typically between 110 and 120, often ranging between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0491] In this application, CDR sequences may also be described according to Kabat numbering with AbM CDR annotation as described in Kontermann and Dübel (eds., 2010, Antibody Engineering, Vol. 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51). According to this method, FR1 comprises the amino acid residues at positions 1-25, CDR1 comprises the amino acid residues at positions 26-35, FR2 comprises the amino acids at positions 36-49, CDR2 comprises the amino acid residues at positions 50-58, FR3 comprises the amino acid residues at positions 59-94, CDR3 comprises the amino acid residues at positions 95-102, and FR4 comprises the amino acid residues at positions 103-113.

[0492] The determination of the CDR regions can also be performed according to different methods. In the CDR determination according to Kabat, the FR1 of the ISVD comprises the amino acid residues at positions 1-30, the CDR1 of the ISVD comprises the amino acid residues at positions 31-35, the FR2 of the ISVD comprises the amino acids at positions 36-49, the CDR2 of the ISVD comprises the amino acid residues at positions 50-65, the FR3 of the ISVD comprises the amino acid residues at positions 66-94, the CDR3 of the ISVD comprises the amino acid residues at positions 95-102, and the FR4 of the ISVD comprises the amino acid residues at positions 103-113.

[0493] In such immunoglobulin sequences, the framework sequence may be any suitable framework sequence, and examples of suitable framework sequences will be clear to the skilled person, e.g. based on standard manuals and the further disclosure and prior art mentioned in this application.

[0494] The framework sequence is an immunoglobulin framework sequence or a suitable combination of framework sequences derived from immunoglobulin framework sequences, for example by humanization or camelization. For example, the framework sequence can be derived from a light chain variable domain (e.g., V L sequence) and / or heavy chain variable domain (e.g., V H Sequence or V HH In one aspect, the framework sequence is derived from V HH A framework sequence of a sequence, wherein the framework sequence can optionally be partially or fully humanized; or a conventional V H Sequence (as defined herein).

[0495] In particular, the framework sequence present in the ISVD sequence mentioned in the present invention may contain one or more signature residues (as defined in this application) such that the ISVD sequence is ISVD, such as V HH , including humanized V HH or camel-derived V H Some non-limiting examples of suitable combinations of such architectural sequences will become clear from the further disclosure of this application.

[0496] However, it should be noted that, in the context of the present invention, the origin of the ISVD sequence or the origin of the nucleotide sequence used to express it is not limited, and the manner in which the ISVD sequence or nucleotide sequence is generated or obtained or has been generated or obtained is also not limited. Thus, the ISVD sequence can be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In specific but non-limiting aspects, the ISVD sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including but not limited to a "humanized" (as defined herein) immunoglobulin sequence (such as a partially or fully humanized mouse or rabbit immunoglobulin sequence, and in particular a partially or fully humanized V HH sequences), "camelized" (as defined herein) immunoglobulin sequences, as well as immunoglobulin sequences obtained by techniques such as affinity maturation (e.g., starting from synthetic, random, or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to those skilled in the art, or any suitable combination of any of the foregoing.

[0497] Similarly, the nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence and may, for example, be a sequence isolated by PCR from a suitable naturally occurring template (e.g. DNA or RNA isolated from a cell), a nucleotide sequence that has been isolated from a library (and in particular an expression library), a nucleotide sequence that has been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), a nucleotide sequence that has been prepared by PCR using overlapping primers or a nucleotide sequence that has been prepared using DNA synthesis techniques known per se.

[0498] As mentioned above, the ISVD precursor is preferably V HH , including humanized V HH or camel-derived V H or a suitable fragment thereof, more preferably a humanized V HH or a suitable fragment thereof. The resulting protein-based building blocks should be soluble, have a globular 3D structure and not specifically bind to human proteins, preferably should not specifically bind to any non-protein molecules, and preferably should not specifically bind to V as described above. HH Preferably, as described above, the molecule (comprising at least one V-based HH (including humanized V HH or camel-derived V H) a building block derived from a protein and at least one cargo attached thereto via said at least one coupling site or connection point) does not specifically bind to any non-proteinaceous molecule and / or does not specifically bind to V HH (including humanized V HH or camel-derived V H ) precursor specifically binds to any non-human protein.

[0499] Furthermore, preferably, the at least one ISVD-based vector building block (i) does not specifically bind to any human cell and / or cell type, or binds to any of the human cells and / or cell types with a specific binding affinity greater than 5×10 -4 mol / L K D (K D value) (preferably as determined by a cell binding assay) binds to human cells and / or cell types, (ii) does not specifically bind to any microorganism (such as bacteria, fungi, protists, yeasts) and / or any virus, or binds to a specific concentration greater than 5×10 -4 K in mol / L (preferably as determined by cell binding assays and / or SPR as described herein) D (K D value) binds to microorganisms (e.g., bacteria, fungi, protists, yeasts) and / or viruses), and / or (iii) 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 with a binding capacity greater than 5x10 -4 K in mol / L (preferably as determined by cell binding assays and / or SPR as described herein) D (K D value) binds to biomolecules (including human and non-human biomolecules).

[0500] As mentioned above, the ISVD precursor is preferably a VHH, a humanized VHH or a camelized VH (e.g. ISVD) or a suitable fragment thereof, more preferably a humanized The resulting protein-based building blocks should be soluble, have a globular 3D structure and not specifically bind to human proteins, preferably should not specifically bind to any non-protein molecules, and preferably should not specifically bind to VHHs, humanized VHHs or camelized VHs as described above (e.g. Preferably, as described above, the molecule (comprising at least one VHH-based, humanized VHH-based or camelized VH-based (e.g. The ISVD)-derived protein building block and at least one cargo attached thereto via said at least one coupling site or connection point) does not specifically bind to any non-proteinaceous molecule and / or does not specifically bind to VHH, humanized VHH or camelized VH (e.g. Any non-human protein to which the ISVD) precursor specifically binds. For a general description of ISVD, reference is made to this specification and the prior art cited in this application. However, in this respect, it should be noted that this specification and the prior art primarily describe the so-called “V H Category 3 ISVD, that is, with V H The human germline sequences of the three classes (such as DP-47, DP-51 or DP-29) have high sequence homology However, it should be noted that the present invention in its broadest sense can generally be used with any type of ISVD, and also uses, for example, the so-called "V H Category 4 ISVD, that is, with V H The human germline sequences of type 4 (such as DP-78) have high sequence homology ISVD, for example as described in WO 2007 / 118670.

[0501] In one embodiment, at least one protein-based carrier building block comprised in a molecule of the invention is derived from a family of proteins belonging to the so-called "V H Category 3 ISVD, that is, with V H The human germline sequences of the three classes (such as DP-47, DP-51 or DP-29) have high sequence homology The ISVD, as long as the protein-based building blocks are soluble, has a globular 3D structure and does not specifically bind to human proteins, preferably does not specifically bind to any non-protein human molecule, and preferably also does not specifically bind to any non-human protein to which the ISVD precursor specifically binds as described above.

[0502] generally, ISVD (especially V HH Sequences, including (partially) humanized V HH Sequence and camelization V H A sequence) may be characterized by the presence of one or more "Hallmark residues" (as described herein) in one or more framework sequences (also as further described herein). Typically, An ISVD can be defined as an immunoglobulin sequence with the following (general) structure:

[0503] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0504] wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and wherein one or more of the Hallmark residues are as further defined herein.

[0505] In particular, An ISVD may be an immunoglobulin sequence with the following (general) structure:

[0506] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0507] wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and wherein the framework sequence is as further defined herein.

[0508] More specifically, An ISVD may be an immunoglobulin sequence with the following (general) structure:

[0509] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0510] wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and wherein:

[0511] One or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to Kabat numbering are selected from the Hallmark residues mentioned in Table 3 below.

[0512] Table 3: Hallmark residues in ISVD (according to Kabat numbering)

[0513]

[0514]

[0515] therefore, An ISVD can be defined as an amino acid sequence with the following (general) structure:

[0516] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0517] wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and wherein the amino acid residue(s) 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.

[0518] For example, when the protein-based building block of the invention is based on an ISVD, it may be derived from an antiviral ISVD, such as from an antiviral V HH or ISVD. For example, the building blocks of the present invention may be derived from a functional ISVD (i.e., an ISVD that specifically binds to human proteins and / or non-human proteins (such as viral proteins and / or bacterial proteins) and / or non-protein molecules (such as human non-protein molecules)), said building block having been engineered / modified such that it no longer specifically binds to any human protein, preferably such that it has been engineered / modified such that it also no longer specifically binds to any non-human protein (such as bacterial and / or viral protein) to which it was originally bound, as described in detail above, and / or preferably such that it also no longer specifically binds to any non-protein molecule to which it was originally bound (if any). In a further preferred embodiment, the ISVD-based building blocks of the present invention are derived from an ISVD, such as from a heavy chain ISVD, preferably from ISVD, the building blocks have been further engineered / modified to include mutations that prevent / eliminate binding of pre-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 of pre-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 preferably be Val, Thr or Leu, preferably Leu; and / or the amino acid at position 110 (according to Kabat) may preferably be 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 building blocks based on ISVD may contain a C-terminal extension of 1-5 amino acids selected from any naturally occurring amino acids.

[0519] The resulting ISVD-based building blocks can be derived from variants of anti-hRSV ISVD, such as the variants of anti-hRSV ISVD depicted in Table A-2 starting on page 69 of WO 2018 / 099968. In a preferred embodiment, the resulting ISVD-based building blocks are derived from variants of ISVD RSV001A04 (SEQ ID NO.: 179 in this specification, also referred to as RSV001A04), and are described in detail in SEQ ID NO.: 5 (referred to as NC41) in Table A-1 on page 388 of WO 2010 / 139808. In this specific embodiment, the protein-based carrier building blocks derived from RSV001A04 do not specifically bind to any human protein. In this embodiment, the "building block precursor" (or "ISVD precursor") is RSV001A04, SEQ ID NO.: 179:

[0520] EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSS.

[0521] Once an ISVD is selected as a starting point (as an "ISVD precursor," see above), residues that are preferably located in solvent-accessible positions should be identified to generate at least one coupling site, as described in detail above in this specification. Additionally or alternatively, one or more coupling sites may already be present in the ISVD precursor as reactive groups in the side chains of amino acids that are preferably located in solvent-accessible positions or as free N-terminal primary amines and / or free C-terminal carboxylic acids.

[0522] For example, preferably one or more of the identified residues at solvent accessible positions in the amino acid sequence of the ISVD precursor are replaced with cysteine, lysine, tyrosine and / or an unnatural amino acid.

[0523] In one embodiment, at least one protein-based building block comprised in a molecule of the invention is derived from an ISVD, such as one belonging to the so-called "V H 3" ISVDs, wherein the resulting building blocks comprise at least one cysteine, at least one lysine, at least one unnatural amino acid and / or at least one tyrosine, preferably located at one or more solvent accessible positions. In a further embodiment, at least one protein-based building block comprised in a molecule of the invention is derived from an ISVD, such as one belonging to the so-called "V H3” ISVDs, wherein the resulting building blocks comprise at least one engineered cysteine, at least one engineered lysine, at least one unnatural amino acid, and / or at least one engineered tyrosine, preferably located at one or more solvent accessible positions.

[0524] Preferably, when the building blocks of the molecules of the invention are derived from ISVD (preferably from V HH (including humanized V HH or camel-derived V H )or ISVD, as described above), it comprises a leucine at position 108 (according to Kabat numbering). In other embodiments, when the building blocks of the molecules of the invention are derived from an ISVD as described above, it comprises a valine at position 11, a leucine at position 89 and / or a leucine at position 108 (according to Kabat numbering).

[0525] In one embodiment, at least one protein-based carrier building block present in a molecule of the invention comprises or alternatively consists of: SEQ ID NO.: 186:

[0526] X1VX2LX3EX4X5GX6X7X8X9X 10 X 11 GX 12 X 13 X 14 IX 15 CX 16 AX 17 X 18 X 19 X 20 LX 21 X 22 X 23 VLGWFRX 24 AX25X 26 X 27 X 28 X 29 X 30 FVAAINX 31 X 32 X 33 X 34 X 35 X 36 X 37 X 38 PX 39 X 40 VX 41 X 42 X 43 FX 44 IX 45 X 46 X47 X 48 X 49 X 50 X 51 TGX 52 LX 53 MX 54 X 55 LX 56 X 57 X 58 DX 59 AX 60 YX 61 CGAGX 62 PX 63 X 64 X 65 X 66 AYX 67 X 68 X 69 X 70 SYX 71 X 72 X 73 GX 74 X 75 TX 76 VX 77 VX 78 X 79 X 80 X 81 X 82 ,

[0527] in

[0528] 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;

[0529] X2 (position 3 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, such as cysteine;

[0530] X3 (position 5 according to Kabat numbering) can be Val or any amino acid having a reactive group in its side chain, such as cysteine;

[0531] X4 (position 7 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0532] X5 (position 8 according to Kabat numbering) can be Gly or any amino acid with a reactive group in its side chain, such as cysteine;

[0533] X6 (position 10 according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as cysteine;

[0534] 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, such as cysteine;

[0535] X8 (position 12 according to Kabat numbering) can be Val or any amino acid having a reactive group in its side chain, such as cysteine;

[0536] X9 (position 13 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, such as cysteine;

[0537] X 10 (position 14 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, such as cysteine;

[0538] X 11 (position 15 according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as cysteine;

[0539] X 12 (position 17 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0540] X 13 (position 18 according to Kabat numbering) can be Leu or any amino acid having a reactive group in its side chain, such as cysteine;

[0541] X 14 (position 19 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0542] X 15 : (position 21 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0543] X 16 : (position 23 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, such as cysteine;

[0544] X 17 : (position 25 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0545] X 18 : (position 26 according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as cysteine;

[0546] X 19 : (position 27 according to Kabat numbering) can be Gly or any amino acid with a reactive group in its side chain, such as cysteine;

[0547] X 20 : (position 28 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0548] X 21 : (position 30 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0549] X 22 : (position 31 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine;

[0550] X 23 : (position 32 according to Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, such as cysteine;

[0551] X 24 : (position 39 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, such as cysteine;

[0552] X 25 : (position 41 according to Kabat numbering) can be Pro or any amino acid with a reactive group in its side chain, such as cysteine;

[0553] X 26 : (position 42 according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as cysteine;

[0554] X 27 : (position 43 according to Kabat numbering) can be Lys or any amino acid having a reactive group in its side chain, such as cysteine;

[0555] X 28 : (position 44 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, such as cysteine;

[0556] X29 : (position 45 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, such as cysteine;

[0557] X 30 : (position 46 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, such as cysteine;

[0558] X 31 : (position 52a according to Kabat numbering) can be Trp or any amino acid having a reactive group in its side chain, such as cysteine;

[0559] X 32 : (position 53 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, such as cysteine;

[0560] X 33 : (position 54 according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as cysteine;

[0561] X 34 : (position 55 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as cysteine;

[0562] X 35 : (position 56 according to Kabat numbering) can be He or any amino acid having a reactive group in its side chain, such as cysteine;

[0563] X 36 : (position 57 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, such as cysteine;

[0564] X 37 : (position 58 according to Kabat numbering) can be He or any amino acid having a reactive group in its side chain, such as cysteine;

[0565] X 38 : (position 59 according to Kabat numbering) can be Gly or any amino acid with a reactive group in its side chain, such as cysteine;

[0566] X 39 : (position 61 according to Kabat numbering) can be Pro or any amino acid with a reactive group in its side chain, such as cysteine;

[0567] X 40: (position 62 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine;

[0568] X 41 : (position 64 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, such as cysteine;

[0569] X 42 : (position 65 according to Kabat numbering) can be Gly or any amino acid with a reactive group in its side chain, such as cysteine;

[0570] X 43 : (position 66 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, such as cysteine;

[0571] X 44 : (position 68 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, such as cysteine;

[0572] X 45 : (position 70 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0573] X 46 : (position 71 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, such as cysteine;

[0574] X 47 : (position 72 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as cysteine;

[0575] X 48 : (position 73 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine;

[0576] X 49 : (position 74 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, such as cysteine;

[0577] X 50 : (position 75 according to Kabat numbering) can be Lys or any amino acid with a reactive group in its side chain, such as cysteine;

[0578] X 51: (position 76 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine;

[0579] X 52 : (position 79 according to Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, such as cysteine;

[0580] X 53 : (position 81 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, such as cysteine;

[0581] X 54 : (position 82a according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine;

[0582] X 55 : (position 82b according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0583] X 56 : (position 83 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, such as cysteine;

[0584] X 57 : (position 84 according to Kabat numbering) can be Pro or any amino acid with a reactive group in its side chain, such as cysteine;

[0585] X 58 : (position 85 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as cysteine;

[0586] X 59 : (position 87 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, such as cysteine;

[0587] 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 Ile; preferably Leu, Val, Ser or Glu, more preferably Leu or Val, or any other amino acid having a reactive group in its side chain, such as cysteine;

[0588] X 61: (position 91 according to Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, such as cysteine;

[0589] X 62 : (position 96 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, such as cysteine;

[0590] X 63 : (position 98 according to Kabat numbering) can be Leu or any amino acid with a reactive group in its side chain, such as cysteine;

[0591] X 64 : (position 99 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine;

[0592] X 65 : (position 100 according to Kabat numbering) can be Pro or any amino acid with a reactive group in its side chain, such as cysteine;

[0593] X 66 : (position 100a according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as cysteine;

[0594] X 67 : (position 100d according to Kabat numbering) can be He or any amino acid having a reactive group in its side chain, such as cysteine;

[0595] X 68 : (position 100e according to Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, such as cysteine;

[0596] X 69 : (position 100f according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as cysteine;

[0597] 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;

[0598] X 71 : (position 101 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as cysteine;

[0599] X 72: (position 102 according to Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, such as cysteine;

[0600] X 73 : (position 103 according to Kabat numbering) can be Trp or any amino acid having a reactive group in its side chain, such as cysteine;

[0601] X 74 : (position 105 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, such as cysteine;

[0602] X 75 : (position 106 according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as cysteine;

[0603] 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, such as cysteine;

[0604] X 77 : (position 110 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, such as cysteine;

[0605] X 78 : (position 112 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0606] X 79 : (position 113 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0607] X 80 : absent or Gly;

[0608] X 81 : absent or Gly;

[0609] X 82 : does not exist or is Cys,

[0610] or a sequence having 80% or greater identity to SEQ ID NO.: 186, preferably a sequence having 85% or greater, 90% or greater, 95% or greater, 97% or greater or 99% or greater sequence identity to SEQ ID NO.: 186, 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, 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, such as 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 does not specifically bind to any non-human protein to which it was originally bound (such as bacterial and / or viral proteins, as described in detail above), and / or preferably does not specifically bind to any non-proteinaceous molecule to which it was originally bound (if any, all as described in detail above). Preferably, as described above, the molecule (comprising at least one such building block based on a protein derived from an ISVD and at least one cargo attached thereto via said at least one coupling site or connection point) does not specifically bind to any non-proteinaceous molecule and / or does not specifically bind to any non-human protein to which the ISVD precursor specifically binds.

[0611] Preferably, the protein-based vector building block of the present invention 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.

[0612] In a further preferred embodiment, additionally or alternatively, the protein-based vector building block of the present invention comprises or alternatively consists of SEQ ID NO.: 186 as defined above, wherein SEQ ID NO.: 186 has been further engineered / modified to include mutations that prevent / eliminate binding of pre-existing antibodies / factors. Examples of such mutations are described, for example, in WO 2012 / 175741 and WO 2015 / 173325. For example, in order to prevent / eliminate binding of pre-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 acids.

[0613] Thus, the present invention 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 amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 according to 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 as defined above, wherein SEQ ID NO.: 186 has been further engineered / modified to include mutations that prevent / eliminate binding of pre-existing antibodies / factors. Examples of such mutations are described, for example, in WO 2012 / 175741 and WO 2015 / 173325. For example, in order to prevent / eliminate binding of pre-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 acids.

[0614] In one embodiment, at least one protein-based carrier building block present in a molecule of the invention comprises or alternatively consists of: SEQ ID NO.: 206:

[0615] X 1a VQLVEX1GGGZ1VX2AGGX3LX4IX5CX6AX7X 7b GX 7c LSX8YVLGWFR QAPGX9X 10 REFVAAINWRGX 11 ITIGPPX 12 VEX 13 RFX 14 IX 15 RX 16 NX 17 X 18 NTG YLQMNX 19 LAPX 19b DTAZ2YYCGAGTPLNPX 20AYIYX 21 WSYDYWGX 22 GTZ3VTVX 23 SX 24 X 25 X 26

[0616] in

[0617] X 1a (position 1 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, such as cysteine;

[0618] X1 (position 7 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0619] 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;

[0620] X2 (position 13 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, such as cysteine;

[0621] X3 (position 17 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0622] X4 (position 19 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0623] X5: (position 21 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0624] X6: (position 23 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, such as cysteine;

[0625] X7: (position 25 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0626] X 7b : (position 26 according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as cysteine;

[0627] X 7c : (position 28 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0628] X8: (position 31 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine;

[0629] X9: (position 43 according to Kabat numbering) can be Lys or any amino acid having a reactive group in its side chain, such as cysteine;

[0630] X 10 : (position 44 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, such as cysteine;

[0631] X 11 : (position 55 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as cysteine;

[0632] X 12 : (position 62 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine;

[0633] X 13 : (position 65 according to Kabat numbering) can be Gly or any amino acid with a reactive group in its side chain, such as cysteine;

[0634] X 14 : (position 68 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, such as cysteine;

[0635] X 15 : (position 70 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0636] X 16 : (position 72 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as cysteine;

[0637] X 17 : (position 74 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, such as cysteine;

[0638] X 18: (position 75 according to Kabat numbering) can be Lys or any amino acid with a reactive group in its side chain, such as cysteine;

[0639] X 19 : (position 82b according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0640] X 19b : (position 85 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as cysteine;

[0641] 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;

[0642] X 20 : (position 100a according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as cysteine;

[0643] X 21 : (position 100f according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as cysteine;

[0644] X 22 : (position 105 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, such as cysteine;

[0645] 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;

[0646] X 23 : (position 112 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0647] X 24 : absent or Gly;

[0648] X 25 : absent or Gly;

[0649] X 26 : does not exist or is Cys,

[0650] or a sequence having 80% or greater identity to SEQ ID NO.: 206, preferably a sequence having 85% or greater, 90% or greater, 95% or greater, 97% or greater or 99% or greater sequence identity to SEQ ID NO.: 206, 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, 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, such as 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 does not specifically bind to any non-human protein to which it was originally bound (such as bacterial and / or viral proteins, as described in detail above), and / or preferably does not specifically bind to any non-proteinaceous molecule to which it was originally bound (if any, all as described in detail above). Preferably, as described above, the molecule (comprising at least one such building block based on a protein derived from an ISVD and at least one cargo attached thereto via said at least one coupling site or connection point) does not specifically bind to any non-proteinaceous molecule and / or does not specifically bind to any non-human protein to which the ISVD precursor specifically binds.

[0651] Preferably, the protein-based vector building block of the present invention 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.

[0652] In a further preferred embodiment, additionally or alternatively, the protein-based vector building block of the present invention comprises or alternatively consists of SEQ ID NO.: 206 as defined above, wherein SEQ ID NO.: 206 has been further engineered / modified to include mutations that prevent / eliminate binding of pre-existing antibodies / factors. Examples of such mutations are described, for example, in WO 2012 / 175741 and WO 2015 / 173325. For example, in order to prevent / eliminate binding of pre-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 acids.

[0653] Thus, the present invention 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 amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to 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 as defined above, wherein SEQ ID NO.: 206 has been further engineered / modified to include mutations that prevent / eliminate binding of pre-existing antibodies / factors. Examples of such mutations are described, for example, in WO 2012 / 175741 and WO 2015 / 173325. For example, in order to prevent / eliminate binding of pre-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 acids.

[0654] In one embodiment, at least one protein-based carrier building block present in a molecule of the invention comprises or alternatively consists of: SEQ ID NO.: 185:

[0655] EVQLVEX1GGGZ1VX2AGGX3LX4IX5CX6AX7GGSLSX8YVLGWFRQAP GX9X 10 REFVAAINWRGX 11 ITIGPPX 12 VEX 13 RFX 14 IX 15 RX 16 NX 17 X 18 NTGYLQ MNX 19 LAPDDTAZ2YYCGAGTPLNPX 20 AYIYX 21 WSYDYWGX 22 GTZ3VTVX23 SX 24 X 25 X 26

[0656] in

[0657] X1 (position 7 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0658] 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;

[0659] X2 (position 13 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, such as cysteine;

[0660] X3 (position 17 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0661] X4 (position 19 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0662] X5: (position 21 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0663] X6: (position 23 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, such as cysteine;

[0664] X7: (position 25 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0665] X8: (position 31 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine;

[0666] X9: (position 43 according to Kabat numbering) can be Lys or any amino acid having a reactive group in its side chain, such as cysteine;

[0667] X 10 : (position 44 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, such as cysteine;

[0668] X 11 : (position 55 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as cysteine;

[0669] X 12 : (position 62 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine;

[0670] X 13 : (position 65 according to Kabat numbering) can be Gly or any amino acid with a reactive group in its side chain, such as cysteine;

[0671] X 14 : (position 68 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, such as cysteine;

[0672] X 15 : (position 70 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0673] X 16 : (position 72 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as cysteine;

[0674] X 17 : (position 74 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, such as cysteine;

[0675] X 18 : (position 75 according to Kabat numbering) can be Lys or any amino acid with a reactive group in its side chain, such as cysteine;

[0676] X 19 : (position 82b according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0677] 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;

[0678] X 20: (position 100a according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as cysteine;

[0679] X 21 : (position 100f according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as cysteine;

[0680] X 22 : (position 105 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, such as cysteine;

[0681] 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;

[0682] X 23 : (position 112 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine;

[0683] X 24 : absent or Gly;

[0684] X 25 : absent or Gly;

[0685] X 26 : does not exist or is Cys,

[0686] or a sequence having 80% or greater identity to SEQ ID NO.: 185, preferably a sequence having 85% or greater, 90% or greater, 95% or greater, 97% or greater or 99% or greater sequence identity to SEQ ID NO.: 185, 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, 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, such as 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 does not specifically bind to any non-human protein to which it was originally bound (such as bacterial and / or viral proteins, as described in detail above), and / or preferably does not specifically bind to any non-proteinaceous molecule to which it was originally bound (if any, all as described in detail above). Preferably, as described above, the molecule (comprising at least one such building block based on a protein derived from an ISVD and at least one cargo attached thereto via said at least one coupling site or connection point) does not specifically bind to any non-proteinaceous molecule and / or does not specifically bind to any non-human protein to which the ISVD precursor specifically binds.

[0687] Preferably, the protein-based vector building block of the present invention 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.

[0688] In a further preferred embodiment, additionally or alternatively, the protein-based vector building block of the present invention comprises or alternatively consists of SEQ ID NO.: 185 as defined above, wherein SEQ ID NO.: 185 has been further engineered / modified to include mutations that prevent / eliminate binding of pre-existing antibodies / factors. Examples of such mutations are described, for example, in WO 2012 / 175741 and WO 2015 / 173325. For example, in order to prevent / eliminate binding of pre-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 acids.

[0689] Thus, the present invention 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 amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 according to 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 as defined above, wherein SEQ ID NO.: 185 has been further engineered / modified to include mutations that prevent / eliminate binding of pre-existing antibodies / factors. Examples of such mutations are described, for example, in WO 2012 / 175741 and WO 2015 / 173325. For example, in order to prevent / eliminate binding of pre-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 acids.

[0690] In one embodiment, the protein-based carrier building block comprises at least one amino acid having a reactive group in its side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine, in at least one of the following solvent accessible positions; such as three amino acids having reactive groups in their side chains, such as three cysteines, or three lysines, or three tyrosines, or three unnatural amino acids, preferably three cysteines, in the following solvent accessible positions in SEQ ID NO.: 179, numbered according to Kabat:

[0691] *43, 100f, 105; or

[0692] *43, 75, 100a; or

[0693] *21, 68, 100f; or

[0694] *7, 44, 55; or

[0695] *13, 72, 100a;

[0696] *13, 31, 100f; or

[0697] *C-terminal Cys(-GGC),

[0698] Even more preferably, at least one of the following solvent accessible positions in SEQ ID NO.: 179 (according to Kabat numbering):

[0699] *43, 100f, 105; or

[0700] *43, 75, 100a.

[0701] Thus, in one embodiment, the protein-based building blocks of the invention comprise or alternatively consist of one of the following sequences:

[0702] *SEQ ID NO.: 185, wherein

[0703] X1: (position 7 according to Kabat numbering) is Ser;

[0704] Z1: (position 11 according to Kabat numbering) is Leu or Val;

[0705] X2: (position 13 according to Kabat numbering) is Gln;

[0706] X3: (position 17 according to Kabat numbering) is Ser;

[0707] X4: (position 19 according to Kabat numbering) is Ser;

[0708] X5: (position 21 according to Kabat numbering) is Ser;

[0709] X6: (position 23 according to Kabat numbering) is Ala;

[0710] X7: (position 25 according to Kabat numbering) is Ser;

[0711] X8: (position 31 according to Kabat numbering) is Asn;

[0712] X9: (position 43 according to Kabat numbering) can be any amino acid having a reactive group in its side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine;

[0713] X 10 : (position 44 according to Kabat numbering) is Glu;

[0714] X 11 : (position 55 according to Kabat numbering) is Asp;

[0715] X 12 : (position 62 according to Kabat numbering) is Asn;

[0716] X 13 : (position 65 according to Kabat numbering) is Gly;

[0717] X 14 : (position 68 according to Kabat numbering) is Thr;

[0718] X 15 : (position 70 according to Kabat numbering) is Ser;

[0719] X 16 : (position 72 according to Kabat numbering) is Asp;

[0720] X 17 : (position 74 according to Kabat numbering) is Ala;

[0721] X 18 : (position 75 according to Kabat numbering) is Lys;

[0722] X 19 : (position 82b according to Kabat numbering) is Ser;

[0723] Z2: (position 89 according to Kabat numbering) is Val or Leu;

[0724] X 20 : (position 100a according to Kabat numbering) is Gly;

[0725] X 21 : (position 100f according to Kabat numbering) can be any amino acid having a reactive group in its side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine;

[0726] X 22 : (position 105 according to Kabat numbering) can be any amino acid having a reactive group in its side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine;

[0727] Z3: (position 108 according to Kabat numbering) is Gln or Leu;

[0728] X 23 : (position 112 according to Kabat numbering) is Ser;

[0729] X 24 : does not exist;

[0730] X 25 : does not exist;

[0731] X 26 : does not exist, or

[0732] *SEQ ID NO.: 185, wherein

[0733] X1: (position 7 according to Kabat numbering) is Ser;

[0734] Z1: (position 11 according to Kabat numbering) is Leu or Val;

[0735] X2: (position 13 according to Kabat numbering) is Gln;

[0736] X3: (position 17 according to Kabat numbering) is Ser;

[0737] X4: (position 19 according to Kabat numbering) is Ser;

[0738] X5: (position 21 according to Kabat numbering) is Ser;

[0739] X6: (position 23 according to Kabat numbering) is Ala;

[0740] X7: (position 25 according to Kabat numbering) is Ser;

[0741] X8: (position 31 according to Kabat numbering) is Asn;

[0742] X9: (position 43 according to Kabat numbering) can be any amino acid having a reactive group in its side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine;

[0743] X 10 : (position 44 according to Kabat numbering) is Glu;

[0744] X 11 : (position 55 according to Kabat numbering) is Asp;

[0745] X 12 : (position 62 according to Kabat numbering) is Asn;

[0746] X 13 : (position 65 according to Kabat numbering) is Gly;

[0747] X 14 : (position 68 according to Kabat numbering) is Thr;

[0748] X 15 : (position 70 according to Kabat numbering) is Ser;

[0749] X 16 : (position 72 according to Kabat numbering) is Asp;

[0750] X 17 : (position 74 according to Kabat numbering) is Ala;

[0751] X 18 : (position 75 according to Kabat numbering) can be any amino acid having a reactive group in its side chain, such as cysteine, or lysine, or tyrosine, or an unnatural amino acid, preferably cysteine;

[0752] X 19 : (position 82b according to Kabat numbering) is Ser;

[0753] Z2: (position 89 according to Kabat numbering) is Val or Leu;

[0754] X 20 : (position 100a according to Kabat numbering) can be any amino acid havi...

Claims

1. A molecule comprising at least one protein-based building block, wherein the at least one protein-based building block: a) contains at least two coupling sites or attachment points; b) having a molecular weight of about 2.5 to about 70 kDa; c) having a spherical three-dimensional (3D) structure; d) having a solubility of 10 mg / mL or greater, as measured in an aqueous solution at room temperature, wherein the aqueous solution is citrate buffer or PBS at a pH of 7.0 or 7.4; e) does not specifically bind to any human protein, or binds to any protein at a rate greater than 5 x 10 -4 mol / L K D The value is bound to one or more human proteins as determined by surface plasmon resonance, such as described in Ober et al., 2001, Intern. Immunology 13: 1551-1559; and f) does not comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NOs.: 1 to 34 shown in Tables A-1 and A-2 of WO 2016 / 055656 and / or SEQ ID NOs.: 1 to 12 shown in Table A-1 of WO 2010 / 139808.

2. A molecule according to claim 1, wherein one, preferably at least two, of said at least two coupling sites or attachment points are present at solvent accessible positions in said protein-based building block.

3. The molecule according to any one of claims 1 to 2, wherein at least one of the attachment points or coupling sites, preferably at least two attachment points or coupling sites, more preferably all attachment points or coupling sites are engineered attachment points or coupling sites.

4. The molecule according to any one of claims 1 to 3, wherein the at least two attachment points or coupling sites are reactive groups present in the side chain of any amino acid in the protein-based carrier building block, preferably reactive groups 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 an unnatural amino acid.

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

6. The molecule according to any one of claims 1 to 5, wherein the at least one protein-based building block does not specifically bind to any non-protein molecules such as DNA, RNA, lipids or polysaccharides, or binds to any protein-based building block at a rate greater than 5 x 10 -4 mol / L K D The value binds to one or more non-protein molecules.

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

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

9. The molecule of claim 8, wherein the ISVD-based building block is derived from V H , humanized V H 、People V H 、V HH , humanized V HH or camel-derived V H (derived from heavy chain ISVD), preferably from a H Category 3" ISVD.

10. The molecule according to any one of claims 8 to 9, wherein the building block derived from ISVD is derived from RSV001A04 (SEQ ID NO.: 179).

11. The molecule according to any one of claims 8 to 10, wherein the building block derived from ISVD comprises or alternatively consists of: SEQ ID NO.: 186: X1VX2LX3EX4X5GX6X7X8X9X 10 X 11 GX 12 X 13 X 14 IX 15 CX 16 SURE 17 X 18 X 19 X 20 LX 21 X 22 X 23 VLGWFRX 24 SURE 25 X 26 X27X 28 X 29 X 30 FVAAINX 31 X 32 X 33 X 34 X 35 X 36 X 37 X 38 PX 39 X 40 VX 41 X 42 X 43 FX 44 IX 45 X 46 X 47 X 48 X 49 X 50 X 51 TGX 52 LX 53 MX 54 X 55 LX 56 X 57 X 58 DX 59 SURE 60 YX 61 CGAGX 62 PX 63 X 64 X 65 X 66 AYX 67 X 68 X 69 X 70 SYX 71 X 72 X 73 GX 74 X 75 TX 76 VX 77 VX78X 79 X 80 X 81 X 82 in 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, such as cysteine; X3 (position 5 according to Kabat numbering) can be Val or any amino acid having a reactive group in its side chain, such as cysteine; X4 (position 7 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine; X5 (position 8 according to Kabat numbering) can be Gly or any amino acid with a reactive group in its side chain, such as cysteine; X6 (position 10 according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as 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, 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, such as cysteine; X9 (position 13 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, such as cysteine; X 10 (position 14 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, such as cysteine; X 11 (position 15 according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as cysteine; X 12 (position 17 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine; X 13 (position 18 according to Kabat numbering) can be Leu or any amino acid having a reactive group in its side chain, such as cysteine; X 14 (position 19 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine; X 15 : (position 21 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine; X 16 : (position 23 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, such as cysteine; X 17 : (position 25 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine; X 18 : (position 26 according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as cysteine; X 19 : (position 27 according to Kabat numbering) can be Gly or any amino acid with a reactive group in its side chain, such as cysteine; X 20 : (position 28 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine; X 21 : (position 30 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine; X 22 : (position 31 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine; X 23 : (position 32 according to Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, such as cysteine; X 24 : (position 39 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, such as cysteine; X 25 : (position 41 according to Kabat numbering) can be Pro or any amino acid with a reactive group in its side chain, such as cysteine; X 26 : (position 42 according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as cysteine; X 27 : (position 43 according to Kabat numbering) can be Lys or any amino acid having a reactive group in its side chain, such as cysteine; X 28 : (position 44 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, such as cysteine; X 29 : (position 45 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, such as cysteine; X 30 : (position 46 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, such as cysteine; X 31 : (position 52a according to Kabat numbering) can be Trp or any amino acid having a reactive group in its side chain, such as cysteine; X 32 : (position 53 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, such as cysteine; X 33 : (position 54 according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as cysteine; X 34 : (position 55 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as cysteine; X 35 : (position 56 according to Kabat numbering) can be He or any amino acid having a reactive group in its side chain, such as cysteine; X 36 : (position 57 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, such as cysteine; X 37 : (position 58 according to Kabat numbering) can be He or any amino acid having a reactive group in its side chain, such as cysteine; X 38 : (position 59 according to Kabat numbering) can be Gly or any amino acid with a reactive group in its side chain, such as cysteine; X 39 : (position 61 according to Kabat numbering) can be Pro or any amino acid with a reactive group in its side chain, such as cysteine; X 40 : (position 62 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine; X 41 : (position 64 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, such as cysteine; X 42 : (position 65 according to Kabat numbering) can be Gly or any amino acid with a reactive group in its side chain, such as cysteine; X 43 : (position 66 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, such as cysteine; X 44 : (position 68 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, such as cysteine; X 45 : (position 70 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine; X 46 : (position 71 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, such as cysteine; X 47 : (position 72 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as cysteine; X 48 : (position 73 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine; X 49 : (position 74 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, such as cysteine; X 50 : (position 75 according to Kabat numbering) can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X 51 : (position 76 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine; X 52 : (position 79 according to Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, such as cysteine; X 53 : (position 81 according to Kabat numbering) can be Gln or any amino acid having a reactive group in its side chain, such as cysteine; X 54 : (position 82a according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine; X 55 : (position 82b according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine; X 56 : (position 83 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, such as cysteine; X 57 : (position 84 according to Kabat numbering) can be Pro or any amino acid with a reactive group in its side chain, such as cysteine; X 58 : (position 85 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as cysteine; X 59 : (position 87 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, such as 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 Ile; preferably Leu, Val, Ser or Glu, more preferably Leu or Val, or any other amino acid having a reactive group in its side chain, such as cysteine; X 61 : (position 91 according to Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, such as cysteine; X 62 : (position 96 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, such as cysteine; X 63 : (position 98 according to Kabat numbering) can be Leu or any amino acid with a reactive group in its side chain, such as cysteine; X 64 : (position 99 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine; X 65 : (position 100 according to Kabat numbering) can be Pro or any amino acid with a reactive group in its side chain, such as cysteine; X 66 : (position 100a according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as cysteine; X 67 : (position 100d according to Kabat numbering) can be He or any amino acid having a reactive group in its side chain, such as cysteine; X 68 : (position 100e according to Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, such as cysteine; X 69 : (position 100f according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as 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, such as cysteine; X 72 : (position 102 according to Kabat numbering) can be Tyr or any amino acid having a reactive group in its side chain, such as cysteine; X 73 : (position 103 according to Kabat numbering) can be Trp or any amino acid having a reactive group in its side chain, such as cysteine; X 74 : (position 105 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, such as cysteine; X 75 : (position 106 according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as 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, such as cysteine; X 77 : (position 110 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, such as cysteine; X 78 : (position 112 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine; X 79 : (position 113 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine; X 80 : absent or Gly; X 81 : absent or Gly; X 82 : does not exist or is Cys, or a sequence having 80% or greater identity to SEQ ID NO.: 186, preferably a sequence having 85% or greater, 90% or greater, 95% or greater, 97% or greater, or 99% or greater sequence identity to SEQ ID NO.: 186, 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, 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, such as 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 molecule according to any one of claims 8 to 11, wherein the building block derived from ISVD comprises or alternatively consists of: SEQ ID NO.: 206: X 1a VQLVEX1GGGZ1VX2AGGX3LX4IX5CX6AX7X 7b GX 7c LSX8YVLGWFRQAPGX9X 10 REFVAAINWRGX 11 ITIGPPX 12 VEX 13 RFX 14 IX 15 RX 16 NX 17 X 18 NTGYLQMNX 19 LAPX 19b DTAZ2YYCGAGTPLNPX 20 AYIYX 21 WSYDYWGX 22 GTZ3VTVX 23 SX 24 X 25 X 26 in X 1a (position 1 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, such as cysteine; X1 (position 7 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as 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, such as cysteine; X3 (position 17 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine; X4 (position 19 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine; X5: (position 21 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine; X6: (position 23 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, such as cysteine; X7: (position 25 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine; X 7b : (position 26 according to Kabat numbering) can be Gly or any amino acid having a reactive group in its side chain, such as cysteine; X 7c : (position 28 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine; X8: (position 31 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine; X9: (position 43 according to Kabat numbering) can be Lys or any amino acid having a reactive group in its side chain, such as cysteine; X 10 : (position 44 according to Kabat numbering) can be Glu or any amino acid having a reactive group in its side chain, such as cysteine; X 11 : (position 55 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as cysteine; X 12 : (position 62 according to Kabat numbering) can be Asn or any amino acid having a reactive group in its side chain, such as cysteine; X 13 : (position 65 according to Kabat numbering) can be Gly or any amino acid with a reactive group in its side chain, such as cysteine; X 14 : (position 68 according to Kabat numbering) can be Thr or any amino acid having a reactive group in its side chain, such as cysteine; X 15 : (position 70 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine; X 16 : (position 72 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as cysteine; X 17 : (position 74 according to Kabat numbering) can be Ala or any amino acid having a reactive group in its side chain, such as cysteine; X 18 : (position 75 according to Kabat numbering) can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X 19 : (position 82b according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine; X 19b : (position 85 according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as 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 Gly or any amino acid having a reactive group in its side chain, such as cysteine; X 21 : (position 100f according to Kabat numbering) can be Asp or any amino acid having a reactive group in its side chain, such as cysteine; X 22 : (position 105 according to Kabat numbering) can be Arg or any amino acid having a reactive group in its side chain, such as 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 according to Kabat numbering) can be Ser or any amino acid having a reactive group in its side chain, such as cysteine; X 24 : absent or Gly; X 25 : absent or Gly; X 26 : does not exist or is Cys, or a sequence having 80% or greater identity to SEQ ID NO.: 206, preferably a sequence having 85% or greater, 90% or greater, 95% or greater, 97% or greater, or 99% or greater sequence identity to SEQ ID NO.: 206, 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, 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, such as 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 building block is derived from the polypeptide defined by SEQ ID NO.:

187.

14. A molecule according to claim 13, wherein at least one protein-based building block comprises or alternatively consists of: SEQ ID NO.: 188: X1X2GX3X4LLX5AAX6X7X8X9X 10 X 11 X 12 VX 13 X 14 LMX 15 X 16 X 17 SURE 18 VX 19 SURE 20 X 21 X 22 X 23 GX 24 TPLHLAX 25 X26X 27 X 28 X 29 X 30 IVX 31 VLLX 32 X 33 X 34 SURE 35 VX 36 SURE 37 DX 38 X 39 GATPLHLAX 40 X 41 X 42 X 43 X 44 X 45 IVX 46 VLLX 47 X 48 X49AX 50 VX 51 SURE 52 DX 53 X 54 GATPLHX 55 AAX 56 X 57 X 58 X 59 X 60 X 61 IVX 62 X 63 LX 64 X 65 X 66 X 67 SURE 68 X 69 X 70 SURE 71 DX 72 X 73 X74X 75 TAX 76 X 77 ISX 78 X 79 X 80 X 81 X 82 X 83 X 84 LAX 85 X 86 LX 87 X 88 X 89 X 90 , in X1 can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X2 can be Leu or any amino acid with a reactive group in its side chain, such as cysteine; X3 can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X4 can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X5 can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X6 can be Arg or any amino acid with a reactive group in its side chain, such as cysteine; X7 can be Ala or any amino acid with a reactive group in its side chain, such as cysteine; X8 can be Gly or any amino acid with a reactive group in its side chain, such as cysteine; X9 can be Gln or any amino acid with a reactive group in its side chain, such as cysteine; X 10 It can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X 11 It can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X 12 It can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X 13 It can be Arg or any amino acid with a reactive group in its side chain, such as cysteine; X 14 It can be Ile or any amino acid with a reactive group in its side chain, such as cysteine; X 15 It can be Ala or any amino acid with a reactive group in its side chain, such as cysteine; X 16 It can be Asn or any amino acid with a reactive group in its side chain, such as cysteine; X 17 It can be Gly or any amino acid with a reactive group in its side chain, such as cysteine; X 18 It can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X 19 It can be Asn or any amino acid with a reactive group in its side chain, such as cysteine; X 20 It can be His or any amino acid with a reactive group in its side chain, such as cysteine; X 21 Can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X 22 Can be Thr or any amino acid with a reactive group in its side chain, such as cysteine; X 23 Can be Phe or any amino acid with a reactive group in its side chain, such as cysteine; X 24 Can be Phe or any amino acid with a reactive group in its side chain, such as cysteine; X 25 Can be Leu or any amino acid with a reactive group in its side chain, such as cysteine; X 26 Can be Tyr or any amino acid with a reactive group in its side chain, such as cysteine; X 27 Can be Gly or any amino acid with a reactive group in its side chain, such as cysteine; X 28 It can be His or any amino acid with a reactive group in its side chain, such as cysteine X 29 Can be Leu or any amino acid with a reactive group in its side chain, such as cysteine X 30 It can be Glu or any amino acid with a reactive group in its side chain, such as cysteine X 31 It can be Glu or any amino acid with a reactive group in its side chain, such as cysteine X 32 It can be Lys or any amino acid with a reactive group in its side chain, such as cysteine X 33 Can be Asn or any amino acid with a reactive group in its side chain, such as cysteine X 34 It can be Gly or any amino acid with a reactive group in its side chain, such as cysteine X 35 It can be Asp or any amino acid with a reactive group in its side chain, such as cysteine X 36 Can be Asn or any amino acid with a reactive group in its side chain, such as cysteine X 37 It can be Asp or any amino acid with a reactive group in its side chain, such as cysteine X 38 It can be Ser or any amino acid with a reactive group in its side chain, such as cysteine X 39 Can be Tyr or any amino acid with a reactive group in its side chain, such as cysteine; X 40 Can be Met or any amino acid having a reactive group in its side chain, such as cysteine; X 41 Can be Arg or any amino acid with a reactive group in its side chain, such as cysteine; X 42 Can be Gly or any amino acid with a reactive group in its side chain, such as cysteine; X 43 Can be His or any amino acid with a reactive group in its side chain, such as cysteine; X 44 Can be Leu or any amino acid with a reactive group in its side chain, such as cysteine; X 45 Can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X 46 Can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X 47 Can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X 48 Can be Tyr or any amino acid with a reactive group in its side chain, such as cysteine; X 49 Can be Gly or any amino acid with a reactive group in its side chain, such as cysteine; X 50 Can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X 51 Can be Asn or any amino acid with a reactive group in its side chain, such as cysteine; X 52 Can be Ala or any amino acid with a reactive group in its side chain, such as cysteine; X 53 Can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X 54 Can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X 55 Can be Leu or any amino acid with a reactive group in its side chain, such as cysteine; X 56 Can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X 57 Can be Ala or any amino acid with a reactive group in its side chain, such as cysteine; X 58 Can be Gly or any amino acid with a reactive group in its side chain, such as cysteine; X 59 Can be His or any amino acid with a reactive group in its side chain, such as cysteine; X 60 Can be Leu or any amino acid with a reactive group in its side chain, such as cysteine; X 61 Can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X 62 Can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X 63 Can be Val or any amino acid with a reactive group in its side chain, such as cysteine; X 64 Can be Leu or any amino acid with a reactive group in its side chain, such as cysteine; X 65 Can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X 66 Can be Asn or any amino acid with a reactive group in its side chain, such as cysteine; X 67 Can be Gly or any amino acid with a reactive group in its side chain, such as cysteine; X 68 Can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X 69 Can be Val or any amino acid with a reactive group in its side chain, such as cysteine; X 70 Can be Asn or any amino acid with a reactive group in its side chain, such as cysteine; X 71 Can be Gln or any amino acid with a reactive group in its side chain, such as cysteine; X 72 Can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X 73 Can be Phe or any amino acid with a reactive group in its side chain, such as cysteine; X 74 Can be Gly or any amino acid with a reactive group in its side chain, such as cysteine; X 75 Can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X 76 Can be Phe or any amino acid with a reactive group in its side chain, such as cysteine; X 77 Can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X 78 Can be He or any amino acid having a reactive group in its side chain, such as cysteine; X 79 Can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X 80 Can be Asn or any amino acid with a reactive group in its side chain, such as cysteine; X 81 Can be Gly or any amino acid with a reactive group in its side chain, such as cysteine; X 82 Can be Asn or any amino acid with a reactive group in its side chain, such as cysteine; X 83 It can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X 84 It can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X 85 It can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X 86 It can be Ile or any amino acid with a reactive group in its side chain, such as cysteine; X 87 It can be Gln or any amino acid with a reactive group in its side chain, such as cysteine; X 88 It can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X 89 Can be absent or Leu; X 90 Can be absent or Cys or a sequence having 80% or greater identity to SEQ ID NO.: 188, preferably a sequence having 85% or greater, 90% or greater, 95% or greater, 97% or greater, or 99% or greater 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, 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, such as 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 molecule according to any one of claims 13 to 14, wherein the at least one protein-based building block comprises or alternatively consists of: SEQ ID NO.: 189: DLGKX1LLEAARAGQDDEVRILMANGADVNAHDTFGFTPLHLAALYGHLX2IVEVLLKNGAX3VNAX4DSYGATPLHLAAMRGHLX5IVX6VLLKYGAX7VX8AX9DEX 10 GATPLHLAAKAGHLX 11 IVEVLLKNGAX 12 VNAQDKFGKTAFDISIX 13 NGNEX 14 LAEILQX 15 X 16 X 17 , in X1 can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X2 can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X3 can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X4 can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X5 can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X6 can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X7 can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X8 can be Asn or any amino acid with a reactive group in its side chain, such as cysteine; X9 can be Ala or any amino acid with a reactive group in its side chain, such as cysteine; X 10 It can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X 11 It can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X 12 It can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X 13 It can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X 14 It can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X 15 It can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X 16 Can be absent or Leu; X 17 Can be absent or Cys, or a sequence having 80% or greater identity to SEQ ID NO.: 189, preferably a sequence having 85% or greater, 90% or greater, 95% or greater, 97% or greater, or 99% or greater sequence identity to SEQ ID NO.: 189, 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, 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, such as 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 according to claim 8, wherein the building block derived from CSK1 is derived from the polypeptide defined by SEQ ID NO.:

190.

17. The molecule of claim 16, wherein said at least one protein-based building block comprises or alternatively consists of: SEQ ID NO.: 191: X1X2X3X4IX5X6SX7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 VX 19 LPX 20 X 21 X 22 AX 23 X 24 VX 25 X 23b X 24b X 25b X 26 MX 27 X 28 X 29 X 30 WX 31 X 32 LX 33 VX 34 QX 35 X 36 X 37 WX 38 HX 39 X 40 X 41 X 42 X 43 X 44 X 45 X 46 X 47 ILLFX 48 X 49 X 50 X 51 X 52 X 53 X54X 55 X 56 X 57 , in X1 can be Ser or any amino acid with a reactive group in its side chain, such as cysteine; X2 can be His or any amino acid with a reactive group in its side chain, such as cysteine; X3 can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X4 can be Gln or any amino acid with a reactive group in its side chain, such as cysteine; X5 can be Tyr or any amino acid with a reactive group in its side chain, such as cysteine; X6 can be Tyr or any amino acid with a reactive group in its side chain, such as cysteine; X7 can be Asp or any amino acid having a reactive group in its side chain, such as cysteine; X8 can be Lys or any amino acid having a reactive group in its side chain, such as 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 with a reactive group in its side chain, such as cysteine; X 11 Can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X 12 Can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X 13 Can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X 14 Can be Phe or any amino acid with a reactive group in its side chain, such as cysteine; X 15 Can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X 16 Can be Tyr or any amino acid with a reactive group in its side chain, such as cysteine; X 17 Can be Arg or any amino acid with a reactive group in its side chain, such as cysteine; X 18 Can be His or any amino acid with a reactive group in its side chain, such as cysteine; X 19 Can be Met or any amino acid having a reactive group in its side chain, such as cysteine; X 20 Can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X 21 Can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X 22 Can be He or any amino acid having a reactive group in its side chain, such as cysteine; X 23 Can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X 24 Can be Leu or any amino acid with a reactive group in its side chain, such as cysteine; X 25 Can be Pro or any amino acid with a reactive group in its side chain, such as cysteine; X 23b Can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X 24b Can be Thr or any amino acid with a reactive group in its side chain, such as cysteine; X 25b Can be His or any amino acid with a reactive group in its side chain, such as cysteine; X 26 Can be Leu or any amino acid with a reactive group in its side chain, such as cysteine; X 27 Can be Ser or any amino acid with a reactive group in its side chain, such as cysteine; X 28 Can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X 29 Can be Ser or any amino acid with a reactive group in its side chain, such as cysteine; X 30 Can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X 31 Can be Arg or any amino acid with a reactive group in its side chain, such as cysteine; X 32 Can be Asn or any amino acid with a reactive group in its side chain, such as cysteine; X 33 Can be Gly or any amino acid with a reactive group in its side chain, such as cysteine; X 34 Can be Gln or any amino acid with a reactive group in its side chain, such as cysteine; X 35 It can be Ser or any amino acid with a reactive group in its side chain, such as cysteine; X 36 It can be Gln or any amino acid with a reactive group in its side chain, such as cysteine; X 37 It can be Gly or any amino acid with a reactive group in its side chain, such as cysteine; X 38 It can be Val or any amino acid with a reactive group in its side chain, such as cysteine; X 39 It can be Tyr or any amino acid with a reactive group in its side chain, such as cysteine; X 40 It can be Met or any amino acid with a reactive group in its side chain, such as cysteine; X 41 It can be Ile or any amino acid with a reactive group in its side chain, such as cysteine; X 42 It can be His or any amino acid with a reactive group in its side chain, such as cysteine; X 43 It can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X 44 It can be Pro or any amino acid with a reactive group in its side chain, such as cysteine; X 45 It can be Glu or any amino acid with a reactive group in its side chain, such as cysteine; X 46 It can be Pro or any amino acid with a reactive group in its side chain, such as cysteine; X 47 It can be His or any amino acid with a reactive group in its side chain, such as cysteine; X 48 It can be Arg or any amino acid with a reactive group in its side chain, such as cysteine; X 49 It can be Arg or any amino acid with a reactive group in its side chain, such as cysteine; X 50 It can be Pro or any amino acid with a reactive group in its side chain, such as cysteine; X 51 It can be Leu or any amino acid with a reactive group in its side chain, such as cysteine; X 52 It can be Pro or any amino acid with a reactive group in its side chain, such as cysteine; X 53 It can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X 54 It can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X 55 It can be Pro or any amino acid with a reactive group in its side chain, such as cysteine; X 56 It can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X 57 It can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; or a sequence having 80% or greater identity to SEQ ID NO.: 191, preferably a sequence having 85% or greater, 90% or greater, 95% or greater, 97% or greater, or 99% or greater sequence identity to SEQ ID NO.: 191, 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, 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, such as 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 molecule according to any one of claims 16 to 17, wherein the at least one protein-based building block comprises or alternatively consists of: SEQ ID NO.: 205: SHKQIYYSX1X2X3X4X5EEFEYRHVX6LPKDIAKLVPX7THLMSESEWRNLGVQQSX8GWVHYX9IHEPEPHILLFRRPLPKKPKX 10 , in X1 can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X2 can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X3 can be Tyr or any amino acid with a reactive group in its side chain, such as cysteine; X4 can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X5 can be Asp or any amino acid with a reactive group in its side chain, such as cysteine; X6 can be Met or any amino acid with a reactive group in its side chain, such as cysteine; X7 can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; X8 can be Gln or any amino acid with a reactive group in its side chain, such as cysteine; X9 can be Met or any amino acid with a reactive group in its side chain, such as cysteine; X 10 It can be Lys or any amino acid with a reactive group in its side chain, such as cysteine; or a sequence having 80% or greater identity to SEQ ID NO.: 205, preferably a sequence having 85% or greater, 90% or greater, 95% or greater, 97% or greater, or 99% or greater sequence identity to SEQ ID NO.: 205, 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, 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, such as 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. The molecule according to any one of claims 1 to 18, wherein the at least one protein-based building block comprises or consists of a polypeptide selected from the group consisting of SEQ ID NOs.: 80-105, 175, 199, 208 and / or 222-224.

20. The molecule according to any one of claims 1 to 19, wherein the molecule comprises at least one protein-based building block and at least one further moiety or cargo, preferably wherein the at least one further moiety or cargo is selected from a) half-life extension (HLE) component, and / or b) a targeting moiety, preferably a moiety that targets EGFR, such as a GE11 peptide; and / or c) therapeutic moieties or precursors therefrom; d) imaging part; e) toxicity part; f) siRNA; g) vitamins, preferably folic acid; h) Toll-like receptor agonists; i) a polysaccharide, preferably bimannose 6-phosphate (bisM6P) or mannose 6-phosphate (M6P); and / or j) lipids, preferably short-chain fatty acids.

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

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

23. The molecule according to claim 22, wherein the albumin binding polypeptide is an albumin binding ISVD, wherein preferably the albumin binding ISVD comprises or alternatively consists of a polypeptide as defined in any one of SEQ ID NOs.: 50-64 or 106, preferably a polypeptide as defined in SEQ ID NOs.: 63 or 106.

24. The molecule according to any one of claims 1 to 23, wherein the molecule comprises or alternatively consists of a polypeptide defined by any one of SEQ ID NOs.: 107-127, 170-174, 176 or 200.

25. A nucleic acid encoding the molecule of any one of claims 1 to 24, a portion of a molecule of any one of claims 1 to 24 and / or the protein-based building block of any one of claims 1-19. A vector comprising the nucleic acid of claim 25 .

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

28. A method of producing a molecule according to any one of claims 1 to 24, wherein the method comprises: a) expressing a nucleic acid sequence encoding at least one protein-based vector building block and / or the molecule or a portion of the molecule according to any one of claims 1 to 24 in a suitable host cell or host organism or in another suitable expression system; b) optionally isolating and / or purifying said at least one protein-based vector building block and / or said molecule or part of said molecule expressed in a); c) optionally coupling one or more (additional) cargoes to one or more attachment points or coupling sites of said protein-based carrier building blocks.

29. A method for producing a molecule according to any one of claims 1 to 24, wherein the method comprises: a) chemically synthesizing at least one protein-based carrier building block and / or said molecule or a portion 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 the at least one protein-based carrier building block and / or the molecule or a portion of the molecule synthesized in a); c) optionally coupling one or more (additional) cargoes to one or more attachment points or coupling sites of said protein-based carrier building blocks.

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 tumor 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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