Modified immunoglobulin variable weight domains with reduced immunogenicity

Abstract

This disclosure generally relates to single-domain antibodies (sdAbs), such as VH, VHH, and scFv polypeptide fragments, which include C-terminal modifications that reduce or eliminate binding to endogenous pre-existing antibodies; isolated polypeptides containing such modifications; nucleotides encoding such polypeptides; and methods for using such antibody polypeptides. TIFF2026521433000046.tif124165

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 506,797, filed on 7 June 2023. The disclosure of U.S. Provisional Patent Application No. 63 / 506,797 is incorporated herein by reference in its entirety for all purposes.

[0002] field This disclosure generally relates to single-domain antibodies (sdAbs), such as VH, VHH, and scFv polypeptide fragments, which include C-terminal modifications that reduce or eliminate binding to endogenous pre-existing antibodies; isolated polypeptides containing such modifications; nucleotides encoding such polypeptides; and methods for using such antibody polypeptides. [Background technology]

[0003] background In single-domain antibody fragments derived from camelid animals, such as VHH and scFv fragments, the exposed C-terminal VTVSS (SEQ ID NO:1) amino acid sequence is recognized by existing circulating antibodies in the immune system, resulting in an immunogenic response that limits the efficacy of therapeutic VHH and scFv drug therapies. Previous attempts have been made to reduce the immune response of these existing antibodies by modifying the C-terminal amino acid sequence of single-domain antibodies with the exposed C-terminal VTVSS (SEQ ID NO:1) amino acid sequence. For example, Nieba et al. have disclosed mutations at positions 11, 14, 41, 84, 87, and / or 89 (amino acid position numbering according to Kabat) in the VH region. WO11 / 07586 (Patent Document 1) discloses mutations at positions 99, 101, and / or 148 in the VL domain, or at positions 12, 97, 98, 99, 103, and / or 144 in the VH domain (corresponding to amino acid positions 11, 83, 84, 85, 89, and 103 according to Kabat). Not all such efforts have been sufficiently effective in eliminating the immune response of existing antibodies. Therefore, there is a great need to develop sdAbs that eliminate or further reduce the immune response of these existing antibodies. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] WO11 / 07586 [Overview of the project]

[0005] overview In one aspect, this disclosure relates to modifications of a newly exposed C-terminal VTVSS (SEQ ID NO:1) amino acid sequence to eliminate or reduce recognition by existing antibodies and the immune response caused by existing antibodies.

[0006] In one aspect, such amino acid sequence modifications to the C-terminal VTVSS (SEQ ID NO:1) amino acid sequence alter the neoepitope resulting from the newly exposed C-terminal VTVSS (SEQ ID NO:1) amino acid sequence on a single-chain antibody, such as a single-chain antibody including sdAb, VHH, single-chain, scFv, sdAb, Fab, diabody, cFab, a multi-domain antibody including a fusion of IgG or HSA with another single-chain antibody, or any other antigen-binding domain or Fc fusion protein that normally exposes a non-exposed N-terminal or C-terminal sequence.

[0007] In one aspect, the present disclosure relates to an isolated single-domain antibody comprising a C-terminal modification, the C-terminal modification comprising a substitution, addition, or deletion of at least one amino acid residue such that modification to the single-domain antibody eliminates the interaction of the single-domain antibody with at least one existing antibody without interfering with the binding of the single-domain antibody to its target. In one aspect, the C-terminal amino acid sequence of the single-domain antibody is exposed such that the exposed C-terminal can be utilized for interaction with an existing antibody.

[0008] In one aspect, the present disclosure provides a polypeptide comprising a single-domain antibody (sdAb) comprising a modified C-terminal amino acid sequence at a position corresponding to the endogenous sdAb amino acid residues (T / L 108 )V 109 T 110 V 111 S 112 S 113 (numbered according to the Kabat numbering scheme relative to the human VH carboxy-terminal amino acid residues). According to the present disclosure, the modified amino acid sequence comprises the formula X 108 X 109 X 110 V 111 X 112 X 113 Y, X 108 is selected from the group consisting of L, T, and Q, X 109It is selected from the group consisting of V, G, N, and L. X 110 It is selected from the group consisting of T and Q, X 111 is V, X 112 is selected from the group consisting of S, C, T, A, and G, or is arbitrarily absent, and X 113 is selected from the group consisting of S, C, A, G, and T, or is optionally absent. however, X 109 When V, then X 112 and X 113 It is not possible for both to be S, and X 112 and X 113 It is not possible for both to be C, and Y contains a polypeptide of 1 to 5 amino acids, and such amino acids are independently selected from the group consisting of A, G, S, T, L, and V, or Y is optionally absent.

[0009] In some embodiments, sdAb is further modified to include amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A, numbered according to the Kabat numbering scheme.

[0010] In certain aspects of this disclosure, X 108 L is X 109 X is selected from the group consisting of V, G, and L. 110 is T, and X 112 X is selected from the group consisting of S, T, and C. 113 Y is selected from the group consisting of S, T, C, and A, and Y contains a polypeptide comprising 1 to 5 amino acids independently selected from the group consisting of A, G, S, T, L, and V, and Y is optionally absent. In other embodiments, amino acid sequence X 109 X 110 V 111 X 112 X 113 V 109 T110 V 111 S 112 A 113 Therefore, Y is AA.

[0011] In some embodiments, sdAb is an amino acid sequence X selected from the group consisting of GTVSS (SEQ ID NO:3), LTVSS (SEQ ID NO:27), NTVSS (SEQ ID NO:22), VTVCS (SEQ ID NO:8), VTVSC (SEQ ID NO:10), VTVTS (SEQ ID NO:23), and VTVTT (SEQ ID NO:24). 109 X 110 V 111 X 112 X 113 Includes.

[0012] In some embodiments, the polypeptides of this disclosure exhibit reduced binding to existing antibodies.

[0013] In some embodiments, single-domain antibodies are VHH.

[0014] In another context, this disclosure is based on the formula: VHH1-L n -VHH2 This invention relates to a polypeptide in which VHH1 is the first VHH, L is a polypeptide linker containing 1 to 50 amino acids, n is 0 or 1, VHH2 is the second VHH, and the second VHH may be the same as or different from VHH1.

[0015] In some embodiments, VHH1 and VHH2 independently bind to the extracellular domain of the cytokine receptor. In some embodiments, the cytokine receptor to which VHH1 and VHH2 bind is selected from the group consisting of IL2Rα, IL2Rβ, IL2Rγ, IL10Rα, IL10Rβ, IL12Rβ1, IL12Rβ2, IL18Rα, IL18Rβ, IL22R1, IL27Rα, gp130, IL23R, IL28Rα, IFNRγ1, IFNRγ2, and IL21R. In some embodiments, VHH1 and VHH2 selectively bind to a pair of cytokine receptors selected from the following pairs: IL10Rα / IL10Rβ, IL27Rα / gp130, IFNγR1 / IFNγR2, IL10Rβ / IL28Rα, IL2Rβ / IL2Rγ, IL18Rα / IL18Rβ, IL22R1 / IL10Rβ, IL10Rα / IL2Rγ, IL2Rβ / IL2Rγ, IL10R1 / IFNRγ, IFNRγ / IL28Rα, IL12Rβ1 / IL12Rβ2, IL12Rβ1 / IL23R, and IL10Rα / IL2Rγ.

[0016] The above formula VHH1-L n In -VHH2, the linker molecule L may be a GS linker. Various suitable GS linkers are described and illustrated herein.

[0017] In some embodiments, the polypeptide is pegylated. In some embodiments, the polypeptide is conjugated with an Fc domain.

[0018] In some embodiments, single-domain antibodies and their antigen-binding fragments can be conjugated with a variety of other chemical entities, including antibody-drug conjugates (ADCs).

[0019] The single-domain antibodies or antigen-binding fragments described herein may also be used for diagnostic purposes.

[0020] The modified polypeptides described herein exhibit reduced immunogenicity attributable to existing antibodies when administered to human subjects, compared to single-domain polypeptides having the endogenous heavy chain C-terminal amino acid sequence VTVSS (SEQ ID NO:1).

[0021] The Disclosure also includes methods for treating a mammalian subject suffering from a disease, disorder, or condition, comprising the step of administering a therapeutically effective amount of the polypeptide of the Disclosure to the mammalian subject. In some embodiments, the Disclosure includes the administration of the polypeptide of the Disclosure by administration of a drug comprising a nucleic acid sequence encoding the polypeptide of the Disclosure with a modified C-terminus. In some embodiments, the Disclosure provides a method for reducing the immune response to a single-domain antibody. In some embodiments, the Disclosure includes a method for reducing the immunogenicity of a single-domain antibody due to an existing antibody (innate immune response) or to a B-cell derived antibody (via adaptive immunity) by using a modified C-terminal amino acid sequence disclosed herein, a pharmaceutically acceptable formulation of the modified sdAb and polypeptide described herein, a nucleic acid encoding such sdAb and polypeptide, a vector comprising such nucleic acid, and a host cell comprising such vector.

[0022] In some embodiments, the formula VHH1-L includes a first cytokine receptor subunit and a second cytokine receptor subunit. n - A cytokine receptor-binding polypeptide of VHH2, wherein either VHH1 or VHH2 is a VHH that selectively binds to the extracellular domain of a first cytokine receptor subunit, and the other VHH1 or VHH2 is a VHH that selectively binds to the extracellular domain of a second cytokine receptor subunit, where L is a polypeptide linker, n=0 (absent) or (1) present, and VHH2 is a polypeptide whose amino acids are numbered according to the Kabat numbering scheme, formula X 108 V 109 T 110 V 111 S 112 S 113The amino acid sequence of Y is included, and X108 is selected from the group consisting of L, T, and Q, X 109 X is selected from the group consisting of V, G, N, and L. 110 X is selected from the group consisting of T and Q. 111 V is X 112 is selected from the group consisting of S, C, T, A, and G, or is optionally absent, X 113 (a)X 109 If is V, then X112 and X113 cannot both be S, and (b)X 112 and X 113 A cytokine receptor-binding polypeptide is provided, wherein neither of the two is C, and Y comprises a polypeptide comprising 1 to 5 amino acids independently selected from the group consisting of A, G, S, T, L, and V, or Y is optionally absent, and the polypeptide is optionally further modified to include amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A, numbered according to the Kabat numbering scheme.

[0023] In some embodiments, equation X of VHH2 108 V 109 T 110 V 111 S 112 S 113 The amino acid sequence of Y is selected from the group consisting of SEQ ID NO: 2 to 24 and optionally further includes amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A amino acid residues numbered according to the Kabat numbering scheme. In some embodiments, the cytokine receptor-binding polypeptide is the one with formula X in VHH2. 108 V 109 T 110 V 111 S 112 S 113It exhibits reduced immunogenicity compared to polypeptides lacking the Y amino acid sequence. In some embodiments, n=1, and L is a polypeptide linker with 1 to 50 amino acids. In some embodiments, n=1, and L is a polypeptide linker selected from the group consisting of SEQ ID NO: 25 to 48.

[0024] In some embodiments, the cytokine receptor-binding polypeptide is modified to have an extended half-life in vivo. In some embodiments, the cytokine receptor-binding polypeptide is pegylated.

[0025] Nucleic acid sequences encoding cytokine receptor-binding polypeptides described above or elsewhere in this specification are also provided.

[0026] In some embodiments, the cytokine receptor is the IL10 receptor, the first cytokine receptor subunit is IL10Ra, and the second cytokine receptor subunit is IL10Rb. In some embodiments, the cytokine receptor-binding polypeptide contains an amino acid sequence having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 50-72. In some embodiments, the cytokine receptor-binding polypeptide exhibits reduced immunogenicity compared to DR2485aa (SEQ ID NO: 49). In some embodiments, the cytokine receptor-binding protein contains an amino acid sequence selected from the group consisting of SEQ ID NO: 50-72. In some embodiments, the cytokine receptor-binding protein contains an amino acid sequence selected from the group consisting of SEQ ID NO: 50-72. In some embodiments, the cytokine receptor is the IL18 receptor, the first cytokine receptor subunit is IL18Ra, and the second cytokine receptor subunit is IL18Rb. In some embodiments, the cytokine receptor-binding polypeptide contains an amino acid sequence having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 111-134. In some embodiments, the cytokine receptor-binding polypeptide exhibits reduced immunogenicity compared to R3905aa (SEQ ID NO: 110). In some embodiments, the cytokine receptor-binding protein contains an amino acid sequence selected from the group consisting of SEQ ID NO: 111-134. In some embodiments, the cytokine receptor-binding protein contains an amino acid sequence selected from the group consisting of SEQ ID NO: 111-134.

[0027] In some embodiments, the cytokine receptor is the IL2 receptor, the first cytokine receptor subunit is IL2Rb (CD122), and the second cytokine receptor subunit is IL2Rg (CD132). In some embodiments, the cytokine receptor is the IL18 receptor, the first cytokine receptor subunit is IL18Ra, and the second cytokine receptor subunit is IL18Rb. In some embodiments, the cytokine receptor is the IL27 receptor, the first cytokine receptor subunit is IL27Ra, and the second cytokine receptor subunit is gp130. In some embodiments, the cytokine receptor is the IL22 receptor, the first cytokine receptor subunit is IL22Ra, and the second cytokine receptor subunit is IL12Rb. In some embodiments, the cytokine receptor is the IL4 receptor, the first cytokine receptor subunit is IL4Ra, and the second cytokine receptor subunit is IL2Rg (CD132). In some embodiments, the cytokine receptor is the IL7 receptor, the first cytokine receptor subunit is IL7Ra, and the second cytokine receptor subunit is IL2Rg(CD132). In some embodiments, the cytokine receptor is the IL9 receptor, the first cytokine receptor subunit is IL9Ra, and the second cytokine receptor subunit is IL2Rg(CD132). In some embodiments, the cytokine receptor is the IL12 receptor, the first cytokine receptor subunit is IL12Ra, and the second cytokine receptor subunit is IL12Rb.

[0028] Formula VHH1-L n -VHH2 is a divalent IL6R / HSA-binding polypeptide, where either VHH1 or VHH2 is a VHH that selectively binds to the extracellular domain of IL6Ra, and the other VHH1 or VHH2 is a VHH that selectively binds to human serum albumin, where L is a polypeptide linker, n=0 (absent) or (1) present, and VHH2 is a polypeptide whose amino acids are numbered according to the Kabat numbering scheme, formula X 108 V 109 T110 V 111 S 112 S 113 comprising the amino acid sequence of Y, X 108 is selected from the group consisting of L, T, and Q, X 109 is selected from the group consisting of V, G, N, and L, X 110 is selected from the group consisting of T and Q, X 111 is V, X 112 is selected from the group consisting of S, C, T, A, and G or is optionally absent, X 113 is selected from the group consisting of S, C, A, G, and T or is optionally absent, provided that (a) if X 109 is V, then X 112 and X 113 are not both S, and (b) X 112 and X 113 are not both C, and Y comprises a polypeptide comprising 1 to 5 amino acids independently selected from the group consisting of A, G, S, T, L, and V or Y is optionally absent, and the polypeptide is optionally further modified to comprise an amino acid substitution selected from the group consisting of L11S, L11Q, L11G, and P14A numbered according to the Kabat numbering scheme. Also provided is a bivalent IL6R / HSA binding polypeptide. In some embodiments, the formula X of VHH2 108 V 109 T 110 V 111 S 112 S 113 The amino acid sequence of Y is selected from the group consisting of SEQ ID NO: 2 - 24 and optionally further comprises an amino acid substitution selected from the group consisting of L11S, L11Q, L11G, and P14A in which the amino acid residues are numbered according to the Kabat numbering scheme. In some embodiments, the bivalent IL6R / HSA binding polypeptide has the formula X in VHH2 108 V 109 T 110 V 111 S 112 S 113It exhibits reduced immunogenicity compared to polypeptides lacking the Y amino acid sequence. In some embodiments, n=1, and L is a polypeptide linker with 1 to 50 amino acids. In some embodiments, n=1, and L is a polypeptide linker selected from the group consisting of SEQ ID NO: 25 to 48.

[0029] In some embodiments, the divalent IL6R / HSA-binding polypeptide is modified to extend its half-life in vivo. In some embodiments, the divalent IL6R / HSA-binding polypeptide is pegylated. In some embodiments, the divalent IL6R / HSA-binding polypeptide contains an amino acid sequence having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 85-109. In some embodiments, the divalent IL6R / HSA-binding polypeptide exhibits immunogenicity compared to DR2514aa (SEQ ID NO: 84). In some embodiments, the divalent IL6R / HSA-binding polypeptide contains an amino acid sequence selected from the group consisting of SEQ ID NO: 85-109.

[0030] Formula X, in which amino acids are numbered according to the Kabat numbering scheme. 108 V 109 T 110 V 111 S 112 S 113 Anti-HSA (human serum albumin) VHH, which contains the amino acid sequence Y and selectively binds to human serum albumin, and X 108 X is selected from the group consisting of L, T, and Q. 109 X is selected from the group consisting of V, G, N, and L. 110 X is selected from the group consisting of T and Q. 111 V is X 112 is selected from the group consisting of S, C, T, A, and G, or is optionally absent, X 113 (a)X109 If V, then X 112 and X 113 (b)X 112 and X 113 Anti-HSA (human serum albumin) VHH is also provided, wherein both are not C, and Y comprises a polypeptide comprising 1 to 5 amino acids independently selected from the group consisting of A, G, S, T, L, and V, or Y is optionally absent, and the polypeptide is optionally further modified to include amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A, numbered according to the Kabat numbering scheme. In some embodiments, amino acid sequence X 108 V 109 T 110 V 111 S 112 S 113 Y is selected from the group consisting of SEQ ID NO: 2 to 24, and optionally further includes amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A, whose amino acid residues are numbered according to the Kabat numbering scheme. In some embodiments, anti-HSA VHH is formula X 108 V 109 T 110 V 111 S 112 S 113It exhibits reduced immunogenicity compared to polypeptides lacking the Y amino acid sequence. In some embodiments, anti-HSA is modified to extend its half-life in vivo. In some embodiments, anti-HSA is pegylated. In some embodiments, anti-HSA contains an amino acid sequence having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 160-194. In some embodiments, anti-HSA VHH exhibits reduced immunogenicity compared to the polypeptide of SEQ ID NO: 159. In some embodiments, anti-HSA contains an amino acid sequence selected from the group consisting of SEQ ID NO: 160-194. Exemplary anti-HSA VHHs are shown in Table 9 as SEQ ID NO: 160-194 derived from reference sequence DR2830aa (SEQ ID NO: 159). [Brief explanation of the drawing]

[0031] Description of the drawing [Figure 1] Figure 1 is a scatter plot comparing the normalized percentage binding activity (and estimated immunogenicity) of various VHH constructs of this disclosure with wild-type sdAb C-terminal amino acid motif VTVSS (SEQ ID NO:1) and other amino acid VTVSS (SEQ ID NO:1) variants described in the scientific literature. [Figure 2] Figure 2 is a scatter plot comparing the normalized binding activity (and expected immunogenicity) of the VHH construct C-terminal amino acid modifications of this disclosure, in terms of bioluminescence measurements (relative luminescence, i.e., RLU), with wild-type sdAb C-terminal amino acid motif VTVSS (SEQ ID NO:1) and other amino acid VTVSS (SEQ ID NO:1) variants described in the scientific literature, as will be further fully described in Example 1 herein. [Figure 3] Figure 3 is a graph in violin-type scatter plot format showing the data from Table 12. [Figure 4] Figure 4 is a scatter plot comparing the normalized binding activity (and expected immunogenicity) of the αIL6R-αHSA VHH dimer containing the amino acid modification of this disclosure with that of a parent reference molecule, in terms of bioluminescence measurements (relative luminescence, i.e., RLU), as will be further fully described in Example 2 herein. [Figure 5] Figure 5 is a scatter plot comparing the normalized binding activity (and expected immunogenicity) of the αIL18Rα-αIL18Rβ VHH dimer containing the amino acid modification of this disclosure with that of a parent reference molecule, in terms of bioluminescence measurements (relative luminescence, i.e., RLU), as will be further fully described in Example 3 of this specification. [Figure 6] Figure 6 is a scatter plot comparing the normalized binding activity (and expected immunogenicity) of the -αHSA VHH dimer molecules containing the amino acid modification of this disclosure to the parent reference molecule, in terms of bioluminescence measurements (relative luminescence, i.e., RLU), as will be further fully described in Example 4 of this specification. [Modes for carrying out the invention]

[0032] Detailed explanation This disclosure provides engineered sdAbs comprising one or more amino acid substitutions, deletions, and / or additions that result in an improved reduction of immunogenicity derived from an existing endogenous human antibody compared to the parental sdAb from which the engineered sdAb was supplied. In some embodiments, the engineered sdAb also exhibits binding affinity, specificity, stability, or expression efficiency comparable to the parental sdAb from which the engineered sdAb was supplied.

[0033] In some embodiments, the sdAb of the present disclosure comprises one or more amino acid substitutions, deletions, and / or additions in the complementarity-determining region (CDR) (also known as the hypervariable region (HVR)) of the parent antibody from which the sdAb is supplied.

[0034] The modified immunoglobulin heavy chain single-domain antibodies (sdAbs) and fragments of this disclosure are VHH, V NAR , manipulated V H or V K VHH can be obtained from a variety of sources, including but not limited to domains. VHH can be prepared from heavy-chain-only antibodies and libraries of camelid animals. NAR This can be produced from antibodies containing only the heavy chain of cartilaginous fish and their libraries. H and V K Various methods have been employed to produce monomeric sdAbs from domains, including interface manipulation and selection of specific germline families. In some embodiments, the modified sdAbs of this disclosure are human or humanized.

[0035] Endogenous human anti-drug antibodies (ADAs) are frequently observed when the exposed carboxyl terminus is present in the sdAb. This disclosure provides mutations within the sdAb carboxyl-terminal region that block or reduce ADA recognition. In some embodiments, sdAbs derived from non-human sources (e.g., camelids) are modified by incorporating amino acid substitutions, particularly in surface-exposed amino acid residues or sequences. Such modifications include, for example, the exchange of an amino acid residue or sequence with an amino acid or sequence characteristic of antibodies derived from human sources. In such cases, the sdAb is referred to as “humanized,” as further detailed below.

[0036] In some embodiments, the sdAb of this disclosure may also include one or more amino acid substitutions or deletions in other regions of the sdAb. For example, in some embodiments, one or more amino acid substitutions or deletions may be added within framework region 1 (FW1). In other embodiments, the sdAb includes one or more amino acid substitutions or deletions within framework region 2 (FW2). In other embodiments, the sdAb includes one or more amino acid substitutions or deletions within framework region 3 (FW3). In other embodiments, the sdAb includes one or more amino acid substitutions or deletions within framework region 4 (FW4). In some embodiments, the sdAb is modified within a single region. In other embodiments, the sdAb includes one or more amino acid substitutions or deletions in one or more of FW1, FW2, FW3, and FW4. For example, the sdAb may be modified within FW1 and FW4.

[0037] As described in the definitions and references below, all amino acid numbering of immunoglobulin heavy chains used herein utilizes the Kabat numbering scheme.

[0038] Wild-type human V H Carboxyterminant residue Leu / Thr 108 Val 109 Thr 110 Val 111 Ser 112 Ser 113 Exemplary modifications using FW4 of sdAb, aligned with or corresponding to the above, are described herein. Amino acid numbers 108-113 are numbered according to the Kabat numbering scheme.

[0039] definition Unless otherwise defined, scientific and technical terms used in this disclosure shall have meanings generally understood by those skilled in the art. Furthermore, unless specifically required by context, singular terms shall include plurals and plural terms shall include singulars. Generally, the terminology and techniques used herein in relation to cell culture and tissue culture, molecular biology, and protein and oligo or polynucleotide chemistry, and hybridization are well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation and lipofection). Enzyme reactions and purification techniques are performed as commonly accomplished in the art or according to the manufacturer's specifications as described herein. The aforementioned techniques and procedures are generally performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this disclosure. The terminology and experimental procedures and techniques used herein in relation to analytical chemistry, synthetic organic chemistry, and medicinal chemistry and pharmaceutical chemistry are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, prescription, delivery, and patient treatment.

[0040] All patents and publications referenced herein are incorporated herein by reference to the same extent that each individual patent and publication is introduced in detail and individually.

[0041] The following terms used in this disclosure shall be understood to have the meanings set forth below unless otherwise specified.

[0042] As used herein, the terms “specifically binding,” “immunoreactive,” or “made against” mean that an antibody reacts with one or more antigenic determinants of a desired antigen and does not react with other polypeptides, or has very low affinity (K d >10 -6 This means binding via ). Antibodies include polyclonal, monoclonal, chimeric, dAb (domain antibody), single-chain, Fab, Fab' and F(ab')2 fragments, Fv, scFv, Fab expression libraries, and single-domain antibody (sdAb) fragments, e.g., VHH, V NAR , manipulated V H or V K This includes, but is not limited to, the following:

[0043] The term "antigen-binding site" or "binding region" refers to the portion of an immunoglobulin molecule that is involved in or influences antigen binding. The antigen-binding site is formed by amino acid residues in the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three distinctly different regions within the V regions of the heavy and light chains, called "hypervariable regions," are located between adjacent regions that are more conserved than the "hypervariable regions," known as "framework regions," or "FR." The "framework regions," or "FR," are naturally occurring amino acid sequences between the hypervariable regions of immunoglobulins and adjacent to them. In antibody molecules, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form the antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the antigen it binds to, and it binds to it via non-covalent chemical interactions.

[0044] The common numbering used in the Kabat numbering system for standard antibodies is described in Kabat et al., Sequence of proteins of immunological interest. US Public Health Services, NIH Bethesda, Md., Publication No. 91. As described in Riechmann and Muyldermans. 2000. J Immunol Methods 240(1-2):185-195, the standard Kabat numbering scheme is applied to VHH domains derived from camelids. According to this numbering scheme, the amino acid residues were numbered as follows (amino acid residue numbers in parentheses): FR1 (1-30), CDR1 (31-35), FR2 (36-49), CDR2 (50-65), FR3 (66-94), CDR3 (95-102), and FR4 (103-113). It is known in the art that the total number of amino acid residues in the CDR of VH and VHH polypeptides derived from single-domain antibodies may vary and may not exactly match the total number of amino acid residues indicated by the standard Kabat numbering scheme (i.e., one or more positions according to Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number considered by Kabat numbering). Therefore, the numbering according to the standard Kabat numbering scheme may or may not correspond to the actual numbering of amino acid residues in the actual sequence used by Kabat for sdAb. However, generally speaking, according to the standard Kabat numbering system and regardless of the number of amino acid residues in the CDR, position 1 according to Kabat numbering corresponds to the start of FR1 and vice versa; position 36 according to Kabat numbering corresponds to the start of FR2 and vice versa; position 66 according to Kabat numbering corresponds to the start of FR3 and vice versa; and position 103 according to Kabat numbering corresponds to the start of FR4 and vice versa.

[0045] As used herein, the term “epitope” includes any protein domain involved in specific binding to or by immunoglobulins, their fragments, or T cell receptors. Epitope domains typically consist of a chemically active surface group of a molecule, such as an amino acid or sugar side chain. Typically, epitope domains have specific three-dimensional structural features as well as specific charge features. An antibody is said to “specifically bind” to an antigen when its dissociation constant is ≤1 μM; for example, ≤100 nM, or alternatively ≤10 nM, or alternatively ≤1 nM.

[0046] As used herein, the terms “immunological binding” and “immunological binding properties” refer to a type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen to which the immunoglobulin exhibits specificity. The strength or affinity of an immunological binding interaction is expressed by the interaction dissociation constant (K). d It can be expressed as K. d The smaller the value, the greater the affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method measures the rate of formation and dissociation of the antigen-binding site / antigen complex, which depends on the concentration of the complex partner, the affinity of the interaction, and geometric parameters that affect the rate in both directions. Thus, the "on-rate constant" (k on ) and "off rate constant" (k off Both can be determined by calculating the concentrations and actual rates of association and dissociation, as described in Nature 361:186-87. 1993. For example, as outlined in Davies et al. 1990. Annual Rev Biochem. 59:439-473, k off / k on The ratio allows for the cancellation of all parameters unrelated to affinity, and the dissociation constant K dEquilibrium binding constant (K) is equal to the value obtained by assays such as radioactive ligand binding assays, surface plasmon resonance (SPR), flow cytometry binding assays, or similar assays known to those skilled in the art. d The antibodies of this disclosure are said to "specifically bind" to the antigen when the concentration is ≤1 μM, or alternatively ≤100 nM, or alternatively ≤10 nM, or alternatively ≤100 pM to approximately 1 pM.

[0047] The term "substantial identity" as applied to polypeptides means that two peptide sequences share at least 80 percent sequence identity, or alternatively at least 90 percent sequence identity, or alternatively at least 95 percent sequence identity, or alternatively at least 99 percent sequence identity, when optimally aligned using the standard default gap weights of the GAP software program.

[0048] In some embodiments, minor variations in the amino acid sequence of an antibody or immunoglobulin molecule are considered to be encompassed by modifications involving “conservative amino acid substitutions” with alternative amino acids having similar properties. Conservative amino acid substitutions typically involve the substitution of one amino acid residue with another amino acid residue having a side chain with similar chemical properties, which is equivalent to the exchange of an amino acid residue without changing or significantly changing the antibody’s activity or binding properties. Conservative amino acid substitutions are intended to be within the scope of this disclosure. Conservative amino acid substitutions are performed within families of amino acids having side chains with similar chemical properties. Natural amino acids can be broadly divided into the following families of amino acids with similar chemical properties: (1) acidic amino acids, including aspartic acid and glutamic acid; (2) basic amino acids, including lysine, arginine, and histidine; (3) nonpolar amino acids, including alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids, including glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other families of amino acids with similar chemical properties include: (i) serine and threonine, which are in the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are in the amide-containing family; (iii) alanine, valine, leucine, and isoleucine, which are in the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are in the aromatic family.For example, it is reasonable to expect that isolated exchanges of leucine and isoleucine or valine, aspartic acid and glutamic acid, threonine and serine, or similar exchanges of amino acids with structurally related amino acids, will not significantly affect the binding or properties of the resulting molecule, especially if the exchange involves amino acids located within framework sites not directly involved in binding. Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative. Fragments or analogues of antibody or immunoglobulin molecules can be readily prepared by those skilled in the art. In some embodiments, the amino and carboxyl terms of the fragment or analogue arise near the boundaries of the functional domain. Structural and functional domains can be identified by comparing nucleotide and amino acid sequence data with public sequence databases or sequence databases protected by intellectual property rights. In some embodiments, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that arise in other proteins of known structure and function. Methods for identifying protein sequences that fold into known three-dimensional structures are publicly known, for example, as described in Bowie et al. 1991. Science 253:164. Thus, the aforementioned examples demonstrate that those skilled in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with this disclosure. In some embodiments, amino acid substitutions (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for protein complex formation, (4) alter binding affinity, or (4) confer or modify other physicochemical or functional properties of such analogues. Analogues may include various mutaines of sequences other than the native peptide sequence. For example, one or more amino acid substitutions (e.g., conserved amino acid substitutions) may be added to the native sequence, for example, to a polypeptide portion outside the domain that forms intermolecular contacts. Conserved amino acid substitutions should not significantly or substantially alter the structural features of the parent sequence.For example, exchange amino acids should not tend to disrupt helices that arise in the parent sequence, nor should they tend to disrupt other types of secondary structures in the parent sequence. Examples of polypeptide secondary and tertiary structures recognized in the art are described, for example, by Creighton et al. 1984. Proteins, Structures and Molecular Principles; Branden et al. 1991. Introduction to Protein Structure (Garland Publishing, New York, NY); and Thornton et al. 1991. Nature 354:105.

[0049] As used herein, the term “polypeptide fragment” means a polypeptide having one or more amino-terminant or carboxyl-terminant deletions, but whose remaining amino acid sequence is identical or similar to the corresponding positions in the put forward, for example, the natural sequence put forward from a full-length cDNA sequence. The fragments typically have a length of at least 5, 6, 8, or 10 amino acids; in some embodiments, at least 14 amino acids; in some embodiments, at least 20 amino acids; in some embodiments, at least 50 amino acids; and in some embodiments, at least 70 amino acids.

[0050] In this disclosure and claims, terms such as “comprises,” “comprising,” “containing,” “having,” “includes,” and “including” are technical terms having the “open-ended” interpretation as defined by U.S. patent law, allowing for the addition of elements not explicitly listed, which still form a construct within the claims. The terms “essentially from” or “essentially” also have the meaning as defined by U.S. patent law, which are also “open-ended,” allowing for additional elements not explicitly listed, where the basic or novel features of the explicitly listed elements are not substantially altered by the presence of the additional listed elements, excluding the prior art aspects.

[0051] The term “fragment” means a portion of a polypeptide or nucleic acid molecule. In some embodiments, this portion contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total length of the reference nucleic acid molecule or polypeptide. The fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0052] The term "single-domain antibody" or "sdAb" refers to an antibody that has a single monomeric variable antibody domain, which may contain one variable domain of a heavy-chain antibody (VH) or a common IgG molecule. sdAbs can selectively bind to specific antigens. The term single-domain antibody (sdAb) also refers to VHH polypeptides, which are the variable domains of heavy-chain-only polypeptide molecules. Single-domain antibodies can be obtained by immunizing dromedary camels, camels, llamas, alpacas, or sharks with a desired antigen and then isolating the mRNA encoding the variable regions (VNAR and VHH) of the heavy-chain antibody. Alternatively, sdAbs can be produced from common mouse, rabbit, or human IgG, which has four strands. Humans can also produce sdAbs by randomly creating stop codons in the light chain. The term "single-domain antibody" or "sdAb" also generally refers to a single-chain variable fragment (scFv), which is a fusion protein of the variable region of a variable heavy chain fragment (VH) and the variable region of a variable light chain fragment (VL) of an immunoglobulin molecule, linked by a short linker peptide of approximately 10 to 25 amino acids. The linker is usually rich in glycine if mobile, and serine or threonine amino acids if soluble, and may link the N-terminus of VH to the C-terminus of VL (i.e., having the amino acid sequence structure VL-linker-VH, where the VH region has a C-terminal VTVSS (SEQ ID NO:1) motif), or vice versa (i.e., having the amino acid sequence structure VH-linker-VL, where the VL region has a C-terminal sequence that may be the same as, or similar to, the C-terminal VTVSS (SEQ ID NO:1) motif of VH). The scFv protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker. Regarding modifications to the VTVSS (SEQ ID NO:1) motif disclosed herein.

[0053] As used herein, the term "VHH" refers to a single monomeric variable domain (i.e., a "heavy-chain only" variable domain) derived from a single heavy-chain polypeptide. A heavy-chain antibody is a functional antibody that has two heavy chains bound together and lacks a light chain. Such antibodies may be found in or produced from camelid mammals (e.g., camels, llamas) that naturally lack a light chain. VHHs may be obtained by immunizing camelids (including camels, llamas, and alpacas) to obtain complete camelid antibodies that can serve as a source of VHHs (see, for example, Hamers-Casterman, et al. (1993) Nature 363:446-448), or by screening libraries constructed within a VHH framework (e.g., phage libraries). VHH antibodies were initially described as the antigen-binding immunoglobulin (variable) domain of “heavy-chain antibodies” (i.e., “antibodies lacking a light chain”; Hamers-Casterman et al. 1993. Nature 363:446-448). The term “VHH domain” is used to distinguish these variable domains from the heavy-chain variable domains present in conventional four-chain antibodies (referred to herein as “VH domains” or “VH”) and the light-chain variable domains present in conventional four-chain antibodies (referred to herein as “VL domains” or “VL”).VHH is detailed in Muyldermans. 2001. Reviews in Molec Biotechnol. 74:277-302, and the following patent applications are mentioned as general background technology: International Patent Publication Nos. WO9404678, WO9504079, and WO9634103 of Vrije Universiteit Brussel; WO9425591, WO9937681, WO0040968, WO0043507, WO0065057, WO0140310, WO0144301, EP1134231 and WO0248193 of Unilever; Vlaams Instituut voor Biotechnologie (VI B) WO9749805, WO0121817, WO03035694, WO03054016 and WO03055527; WO03050531 from Algonomics NV and Ablynx NV; WO0190190 from the National Research Council of Canada; WO03025020 (EP1433793) from the Institute of Antibodies; and Ablynx This is further detailed in the international patent publication numbers WO2004041867, WO2004041862, WO2004041865, WO2004041863, WO2004062551, WO2005044858, WO200640153, WO2006079372, WO2006122786, WO06122787, WO2006122825, WO2008101985, WO2008142164, and WO2015173325 by NV, as well as in further published patent applications by Ablynx NV. Further prior art described in these applications, in particular the list of references described on pages 41-43 of international application WO2006040153, is also referred to herein. These lists and references are incorporated herein by reference. Methods for obtaining VHH domains that bind to specific antigens or epitopes have been previously described, for example, in WO2006 / 040153 and WO2006 / 122786.As will be explained in more detail, VHH domains derived from camelids may be “humanized” or “human-like” by manipulation, for example, by exchanging one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues that occur at corresponding positions in the VH domain from a conventional four-chain antibody derived from humans. The humanized VHH domain may contain one or more full-human framework region sequences, and in a more specific embodiment, may optionally contain human framework region sequences or portions thereof derived from DP-29, DP-47, DP-51, combined with a JH sequence such as JH5. VHH CDRs can be grafted into several types of binding proteins (e.g., antibodies), and these CDRs retain binding. Once VHH CDRs are grafted onto a framework, they can be manipulated to have desired binding behavior. For example, VHH may be genetically linked to an Fc-domain, other nanobodies, peptide tags, or toxins, or chemically conjugated to drugs, radionuclides, photosensitizers, and nanoparticles at specific sites. See Bannas et al. 2017. Front Immunol. 8:1603. In certain embodiments of the above method, the binding protein is selected from single-chain variable fragments (scFv); recombinant camelid heavy chain-only antibodies (VHH); shark heavy chain-only antibodies (VNAR); microproteins; dalpin; anticarin; adnectin; aptamers; Sac7d derivatives (affitin, e.g., nanophytin; see Journal of Molecular Biology 383(5):1058-68, these contents are incorporated herein by reference), Fv; Fab; Fab'; and F(ab')2. In embodiments, the binding protein is a heterodimer. For example, the binding protein has greater efficacy than each of its individual monomers. In an alternative embodiment, the heteromultimer neutralizing binding protein is a multimer, and the multimer components are linked by non-covalent or covalent bonds.VHH is an antibody-derived therapeutic protein that incorporates the unique structural and functional properties of natural heavy-chain antibodies. VHH technology is based on fully functional antibodies derived from camelid animals that lack a light chain. These heavy-chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Cloned and isolated VHH domains are stable polypeptides with the antigen-binding ability of the original heavy-chain antibodies. See U.S. Patent No. 5,840,526 by Castoran et al., issued November 24, 1998; and U.S. Patent No. 6,015,695 by Castoran et al., issued January 18, 2000. Each of these is incorporated herein by reference in whole. VHH is marketed under the NANOBODIES® trademark by Ablynx Inc. (Ghent, Belgium). Appropriate methods for generating or isolating antibody fragments having the required binding specificity and affinity are described herein, including, for example, methods for selecting recombinant antibodies from a library by PCR (see Ladner Patent No. 5,455,030 issued October 3, 1995, and Devy et al. Patent No. 7,745,587 issued January 29, 2010, each of which is incorporated herein by reference in its entirety).

[0054] In some embodiments herein, VHH is a bispecific VHH consisting of two separate VHH molecules that are the same or different, or that bind to the same or different epitopes. 2 A binding molecule may also be used. The equilibrium dissociation constant between VHH and the receptor (e.g., the first or second receptor of a native or unnative receptor pair) is approximately 10 when confirmed, for example, by scatchard analysis (Munsen et al. 1980. Analyt Biochem. 107:220-239). 6 M or more, alternatively about 10 8 M+, alternatively about 10 10 M or more, alternatively about 10 11 M+, alternatively about 10 10 M or more, alternatively about 10 12If M is greater than, then VHH as described in this specification. 2 The binding molecule may bind to the receptor in some embodiments. Standardized protocols for producing single-domain antibodies from camelids are well known in the scientific literature. See, for example, Vincke et al. 2012. Methods in Molecular Biology. Chapter 8. Walker, J. (Humana Press, Totowa NJ). Specific binding can be evaluated using techniques known in the art, including but not limited to competitive ELISA, BIACORE® assay, and / or KINEXA® assay. In some embodiments, the VHH described herein can be humanized to include a human framework region. Examples of human germlines that can be used to produce humanized VHH include, but are not limited to, VH3-23 (e.g., UniProt ID: P01764), VH3-74 (e.g., UniProt ID: A0A0B4J1X5), VH3-66 (e.g., UniProt ID: A0A0C4DH42), VH3-30 (e.g., UniProt ID: P01768), VH3-11 (e.g., UniProt ID: P01762), and VH3-9 (e.g., UniProt ID: P01782). 2 : The term "VHH" used in this specification 2 " and "Bispecific VHH 2 The terms "VHH dimer" and "VHH-binding domain" are used synonymously to refer to a subtype of the single-domain antibody-binding molecule of the Disclosure wherein both the first sdAb and the second sdAb are VHH, the first VHH binds to a first receptor or its domain or subunit, and the second VHH binds to a second receptor or its domain or subunit. The first VHH-binding domain may be the same as or different from the second VHH-binding domain. 2These may be linked by covalent bonds via a linker, or they may be directly linked by covalent bonds at the C-terminal amino acid of the first VHH (VHH1) and the N-terminal amino acid of the second VHH (VHH2).

[0055] Alternatively, the dimeric variable domain may be split into monomers from a common immunoglobulin G (IgG) derived from human or mouse. Single-domain antibodies also include sdAbs derived from light chains that specifically bind to a target epitope.

[0056] As used herein, “range” as provided herein includes all values ​​within that range, and includes any specified value within that range. For example, the range 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0057] Unless explicitly stated or evident from the context, the terms “a,” “an,” and “the” as used herein mean either singular or plural. Unless explicitly stated or evident from the context, the term “or” as used herein includes both referents.

[0058] Unless otherwise explicitly stated or evident from the context, the term “approximately” as used herein means within the standard tolerance in the art, for example, within two standard deviations of the mean. “Approximately” can be understood as being within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise evident from the context, all numerical values ​​provided herein are modified by the term “approximately.”

[0059] Any term not expressly defined herein has its usual meaning in the art, and its meaning is as defined in, for example, standard handbooks such as Sambrook et al. 1989. Molecular Cloning: A Laboratory Manual 2nd. Ed., Vols. 1-3 (Cold Spring Harbor Laboratory Press); Ausubel et al. 1987. Current Protocols in Molecular Biology (Green Publishing and Wiley Interscience, New York); Lewin. 1985. Genes II (John Wiley & Sons, New York, NY); Old et al. 1981. Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2nd edition (University of California Press, Berkeley, Calif.); Roitt et al. 2001. Immunology, 6th Ed. (Mosby / Elsevier, Edinburgh); Roitt et al. 2001. Roitt's Essential Immunology, 10th This is known to those skilled in the art as described in Ed. (Blackwell Publishing, UK); and Janeway et al. 2005. Immunobiology, 6th Ed. (Garland Science Publishing / Churchill Livingstone, New York), as well as in the general background art cited herein.

[0060] Amino acid numbering of single-domain antibodies The amino acid residues of VHH described herein are numbered according to a general numbering scheme for VH domains developed by Kabat et al., as applied to VHH domains derived from camelids in Riechmann et al. 2000. J Immunol Methods 23:240 and (1-2):185-195, or as otherwise referred to herein. Generally, according to this numbering system, VHH FR1 contains amino acid residues at positions 1-30, VHH CDR1 contains amino acid residues at positions 31-35B (where 35B is an amino acid insertion), VHH FR2 contains amino acid residues at positions 36-49, VHH CDR2 contains amino acid residues at positions 50-65, VHH FR3 contains amino acid residues at positions 66-92, VHH CDR3 contains amino acid residues at positions 93-102, and VHH FR4 contains amino acid residues at positions 103-113. According to the Kabat numbering scheme, the C-terminal amino acid sequence (L / T)VTVSS is L / T 108 V 109 T 110 V 111 S 112 S 113 They are numbered as follows. The number of amino acid residues in each CDR of a VH or VHH antibody may vary, may not exactly match the total number of amino acid residues indicated by the Kabat numbering scheme, one or more positions according to the Kabat numbering scheme may not be occupied in the sequence, or the sequence may contain more amino acid residues than predicted by Kabat numbering.

[0061] As described herein, the summary of the disclosure, brief description of the disclosure, detailed description of the disclosure, drawings, sequence listings (and corresponding SEQ ID NOs), examples, and experimental data are provided solely for the purpose of illustrating specific aspects of the disclosure and should not be understood or construed as limiting the scope of the disclosure or the appended claims unless expressly indicated herein.

[0062] As briefly explained above, in full-size conventional four-chain monoclonal antibodies, and in "heavy-chain only" antibodies, such as those found in camelids, the remaining part of the antibody, for example, the CH1 region in conventional monoclonal antibodies and the hinge region in camelid heavy-chain antibodies, can be conjugated to the C-terminus of the sdAb, and it is known to those skilled in the art that such full-size antibodies can be protected from such protein interference.

[0063] Single domain antibodies (sdAbs) can be used, for example, Hamers-Casterman et al. 1993. Naturally Occurring Antibodies Devoid of Light Chains. Nature 363: 446-448; Nguyen et al., 2000. Camel Heavy-chain Antibodies: Diverse Germline V(H)H and Specific Mechanisms Enlarge the Antigen-binding Repertoire. EMBO, 19: 921-30; Achour et al. 2008. Tetrameric and Homodimeric Camelid IgGs Originate from the Same IgH Locus. J Immunol. 181: 2001-2009; Harmsen et al. 2000. Llama Heavy-chain V Regions Consist of at Least Four Distinct Subfamilies Revealing Novel Sequence Features. Mol Immunol. These are antibody fragments consisting of a single monomeric variable antibody domain that selectively binds to a specific antigen, as described in 37:579-590; Ghahroudi et al. 1997. Selection and Identification of Single Domain Antibody Fragments from Camel Heavy-chain Antibodies. FEBS Lett. 414:521-526; and Vincke et al. General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold. J Biol Chem. 284(5):3273-84. The contents of each of these are incorporated herein by reference in their entirety.

[0064] Single-domain antibodies typically have a molecular weight of about 150–160 kDa, which is considerably smaller than typical antibodies with two protein heavy chains and two light chains, ranging from about 12–15 kDa. Single-domain antibodies also have a molecular weight of about 50 kDa, which is smaller than Fab fragments with one light chain and half a heavy chain, and a molecular weight of about kDa, which is smaller than single-chain variable fragments (scFv) with two variable domains, one derived from the light chain and the other from the heavy chain.

[0065] Single-domain antibodies possess a complementary determining region that is part of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, naturally occurring light-chain-deficient antibodies, single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single-domain scaffolds other than those derived from antibodies. Single-domain antibodies can be obtained from any species, including, but not limited to, mice, humans, camels, llamas, goats, rabbits, and cattle. In some embodiments, single-domain antibodies are naturally occurring single-domain antibodies known as light-chain-deficient heavy-chain antibodies (VH). Variable domains derived from naturally occurring light-chain-deficient heavy-chain antibodies are referred to herein as VHH to distinguish them from conventional VH of four-chain immunoglobulins. Such VHH molecules can be obtained from antibodies produced in camelid species such as camels, llamas, dromedaries, alpacas, and guanacos. Non-camelid species may also naturally produce light-chain-deficient heavy-chain antibodies.

[0066] Single-domain antibodies can be obtained by immunizing dromedary camels, camels, llamas, alpacas, or sharks with the desired antigen and then isolating the mRNA encoding the heavy-chain antibody. Genetic libraries of single-domain antibodies containing millions of clones are generated by reverse transcription and polymerase chain reaction. Screening techniques such as phage display and yeast surface display are commonly used to identify clones that bind to the antigen of interest, as described, for example, by Ghahroudi et al. (cited above) and Desmyter et al. 1997. Selection and Identification of Single Domain Antibody Fragments from Camel Heavy-chain Antibodies. FEBS Letters 414(3):521-526. Another method for constructing sdAbs uses a gene library derived from animals that have never been pre-immunized, known as a "naive library." Such naive libraries typically contain only antibodies with low affinity to the desired antigen, and therefore require an additional step of affinity maturation by random mutagenesis, as described by Saerens et al. 2008. Single-domain Antibodies as Building Blocks for Novel Therapeutics. Current Opinions in Pharmacology 8(5):600-608. The most potent clones are identified, and their DNA sequences are optimized, for example, to improve their stability against enzymes. Another common approach is to humanize the sdAb to inhibit the immunological response of human organisms to the antibody. Humanization is usually not a problem because there is a high degree of homology between camelid VHH and human VH fragments, as described by Saerens et al. 2008.Humanized sdAbs are produced by translating optimized single-domain antibody DNA in Escherichia coli (E. coli), S. cerevisiae, or other suitable organisms. sdAb fragments can also be obtained from conventional antibodies. In some embodiments, the sdAbs of this disclosure can be produced from common four-strand mouse IgG or human IgG, as described, for example, Holt et al. 2003. Domain Antibodies: Proteins for Therapy. Trends in Biotechnology 21(11):484-490. This process typically utilizes a gene library derived from an immunized or naive donor and antigen display methods to identify the most specific antigen. However, this approach results in common IgG consisting of two domains (VH and VL) that constitute the binding region. Since these two IgG domains are lipophilic, they dimerize or aggregate, thus requiring an additional monomerization step by exchanging lipophilic amino acids for hydrophilic amino acids. This often results in a loss of affinity for the antigen, as described, for example, in Borrebaeck et al. 2002. Antibody Evolution Beyond Nature. Nature Biotechnology 20(12):1189-90. If affinity can be maintained, single-domain antibodies can be produced in Escherichia coli, S. cerevisiae, or other organisms. Modification within the human or humanized single-domain antibody fragments described herein is useful regardless of the production method, and any sdAb fragment can be used.

[0067] This disclosure describes modifications of sdAbs to reduce or eliminate interference by ADA. In one aspect, such modifications involve exchanging, eliminating, or adding specific amino acid residues to the C-terminus of a variable domain of an sdAb, such as a VH or VHH antibody or antibody fragment. Various conventional methods have been used to produce monomeric sdAbs from heterodimeric VH and VK domains, including interface manipulation and selection of specific germline families. In some aspects, the modified sdAbs of this disclosure are human or humanized VH or VHH antibodies or fragments thereof. Endogenous ADA, particularly anti-human single-domain antibody (sdAb) antibodies, are primarily observed in sdAbs with exposed C-terminal amino acids.

[0068] This disclosure provides mutations within the C-terminal region of camelid, human, or humanized sdAb that reduce or eliminate immunogenicity induced by ADA. In some embodiments, the camelid, human, or humanized sdAb C-terminal mutations include amino acid substitutions of endogenous or wild-type amino acids. In some embodiments, the human or humanized sdAb C-terminal mutations include amino acid additions to existing amino acids. In some embodiments, the modifications within the C-terminal region of camelid, human, or humanized sdAb include amino acid deletions to existing amino acids. In some embodiments, the mutations within the C-terminal region of human or humanized sdAb include one or more amino acid substitutions, additions, or deletions to existing amino acids. Such sdAb modifications of this disclosure inhibit ADA recognition and immunogenic response without substantially reducing the binding affinity, specificity, expression, or stability of this protein.

[0069] Examples of such C-terminal substitutions, deletions, and additions to sdAbs are described herein. The mutations described herein may also be applied to constructs that are polyvalent, multispecific (e.g., bispecific), or multiparatopic (e.g., biparatopic) antibody constructs, in some embodiments, having a C-terminal variable domain, where two or more sdAbs are directly linked or linked via one or more suitable linkers, and having a solvent-exposed variable domain, for example, an sdAb having a C-terminal heavy-chain variable domain and scFv. Such constructs may consist entirely of VH domains (e.g., VHH domains, humanized VHH domains, or camelized VH domains), for example, directly linked or linked via one or more suitable linkers. For example, other examples of such constructs can be fabricated as described by Conrath et al. 2001. JBC 276:10(9), 7346, and Muyldermans. 2001. Reviews in Mol Biotechnol. 74:27.

[0070] As described herein, an sdAb is generally defined as an amino acid sequence that contains an immunoglobulin fold or, under appropriate physiological conditions, can form an immunoglobulin fold to form an immunoglobulin variable domain (e.g., a VH, VL, or VHH domain). Such an sdAb can form or produce an immunoglobulin variable domain containing a functional antigen-binding site without requiring interaction with another immunoglobulin variable domain (e.g., a VH-VL interaction). Examples of immunoglobulin single variable domains currently known in the art include, for example, single-chain Fv(scFv) domains, single-domain antibodies (sdAbs), variable heavy chain (VH) antibodies, and variable heavy chain-only (VHH) antibodies (i.e., single variable domains located in single heavy chain antibodies), which are generally obtained from, but not exclusively from, camelid heavy chain variable domains.

[0071] The embodiments of the disclosure described herein are intended to apply to, and are suitable for application to, single-domain antibodies (sdAbs) that include, are based on, or are derived from heavy-chain variable domains, such as VH domains (including human VH domains), VHH domains including humanized and sequence-optimized VHH domains, or camelized VH domains. Such sdAbs may be synthetic, for example, obtained starting from a synthetic library, obtained based on a specific framework region, semi-synthetic, for example, obtained by humanization, camelization, sequence optimization, affinity maturation or CDR grafting, or obtained, for example, by starting from a natural VH or VHH domain or a completely natural VH or VHH domain. The aspects of this disclosure are described and illustrated herein primarily in relation to sdAbs that are based on, derived from, or similar to VH or VHH domains and that retain at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0072] This disclosure provides a single-domain antibody (sbAb) or its antigen-binding fragment, comprising at least one mutation that reduces or blocks the recognition of the sdAb by an antibody that specifically recognizes a single-domain antibody. In some embodiments, the mutation is at least one mutation in the framework region, e.g., the C-terminal region of framework region 4 of the sdAb. Surprisingly, it has been found that, according to this disclosure, adding, replacing, or removing specific amino acid residues from the C-terminal region of the sdAb can substantially, essentially eliminate the immunogenicity caused by ADA.

[0073] In some embodiments, the mutation is an amino acid substitution in the carboxy-terminal region of the framework 4 (FW4) region. In other embodiments, the mutation is or includes a carboxy-terminal extension in the FW4 region. In some embodiments, the mutation is a combination of substitution and / or extension at the C-terminus of the FW4 region. In some embodiments, the carboxy-terminal amino acid modification includes a C-terminal amino acid sequence modification comprising an amino acid sequence selected from the group consisting of amino acid sequences with SEQ ID NO: 2 to 24.

[0074] sdAb, VHH, sdAb-based biological agents, or VHH-based biological agents may also be functionalized to extend the half-life of the construct by including a moiety or binding unit that extends the half-life of the construct. Examples of such functionalizations, moieties, or binding units will be apparent to those skilled in the art and may include, for example, those described herein, and may include, for example, pegylation, fusion with serum albumin, or fusion with serum proteins, such as peptides or binding units that can bind to serum albumin. Such serum-albumin-binding peptides or binding domains may be any suitable serum-albumin-binding peptides or binding domains that can extend the half-life of the construct (compared to the same construct without the serum-albumin-binding peptide or binding domain), and may be serum-albumin-binding peptides such as those described in WO2008 / 068280 and WO2009 / 127691, or serum-albumin-bound sdAb, e.g., serum-albumin-bound VHH, e.g., Alb-1 or a humanized version of Alb-1, e.g., Alb-8, as disclosed in WO06 / 122787.

[0075] In vitro assays may be used to evaluate the immunogenicity of the modified sdAb of this disclosure. For example, such assays may include ELISA, MSD, Biacore®, or other similar techniques, e.g., those described in Examples 1 and 2 below. It is thought that blood or serum or other biological fluids, e.g., from a particular individual or population, as referred herein, may, under certain circumstances, contain certain pre-existing or endogenous proteins that may nonspecifically bind to sdAb and interfere with the signal in certain assays used to analyze blood or serum samples obtained from such individuals. This disclosure also addresses the issue of nonspecific protein interference when assaying samples obtained from subjects that have been previously administered sdAb, and when assaying samples obtained from subjects that have been administered sdAb.

[0076] The modified sdAbs of this disclosure can also be used to reduce or avoid protein interference or signaling resulting from nonspecific binding in immunoassays performed on biological samples, such as blood or serum samples, obtained from subjects to whom sdAb-based biological drugs have been administered. Such samples are referred to herein as “test samples” or “assay samples.” Examples of immunoassays that can be used to detect the presence of ADA, or to characterize ADA, or to confirm ADA-induced immunogenicity before administering sdAb-based drugs include the immunoassays described in the Guideline on the Clinical Investigation of the Pharmacokinetics of Therapeutic Proteins (document dated 27 January 2007, CHMP / EWP / 89249 / 2004) issued by the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMEA). Modified sdAbs can be used to predict, eliminate, or reduce such protein interference in “anti-drug antibody” or “ADA” assays performed on test samples of biological fluids taken from subjects who are intended to be administered with or have previously been administered with an sdAb (e.g., VHH, or, as further defined herein, an sdAb-based or VHH-based biological therapeutic agent).Accordingly, in one aspect, the compounds and methods of the present disclosure can be used to predict, avoid, or reduce aspecific signals typically associated with such protein interference in biological test samples obtained from subjects who may be administered or have previously been administered one or more such sdAb or VHH, or, as further defined herein, sdAb-based or VHH-based biological therapeutic agents, such samples are suitable or intended for use in immunological assays such as the ADA assay. Such biological samples may be whole blood, serum or plasma, ocular fluid, bronchoalveolar fluid / BALF, cerebrospinal fluid, or any other suitable biological fluid, or samples suitable for use in immunoassays, particularly the ADA assay.

[0077] This disclosure describes modifications that can be made to variable domains, as previously disclosed in the art, to reduce, eliminate, substantially eliminate, or essentially eliminate such protein interference. In one non-limiting aspect, such modifications may include the step of eliminating, substituting, or adding one or more amino acid residues to the C-terminal region of a variable domain in accordance with this disclosure. Surprisingly, it has been found that substituting, eliminating, or adding one amino acid residue to the C-terminus of a particular different variable domain or construct based thereon can reduce or eliminate the ADA-induced immunogenicity of an antibody-based biotherapy product. In some embodiments, the modification involves the step of substituting one amino acid residue (residence). In other embodiments, the modification involves the step of substituting one amino acid residue in combination with the step of adding one or more amino acid residues to the C-terminus of sdAb. In combination with the above substitutions and additions to the C-terminus of sdAb, other mutations within the C-terminal region or within the framework region are also included in the scope of this disclosure. For example, as described in WO08 / 020079, US20160207981A1, and US20200355221, it is well known that mutations can be made in C-terminal amino acid residues (including those at positions explicitly mentioned in Nieba et al. and WO11 / 07586) in order to humanize the variable domain (including, but not limited to, the VHH domain) or to “camelize” the VH domain.

[0078] The modifications disclosed herein can be applied to any variable domain that is not linked to or otherwise bound to a constant domain (or to another group or peptide moiety that functions to "shield," cover, or "fill" the C-terminal region of a variable domain), and more generally, to any variable domain having a solvent-exposed C-terminal region. As stated above, the methods, assays, and modifications may be applied to heavy chain variable domains (VH domains), and according to a particular aspect of this disclosure, they may be applied to VHH domains. The modifications described herein can also be applied to other constructs having a solvent-exposed variable domain at the C-terminus, e.g., single-chain Fv(scFv) having a heavy chain variable domain at the C-terminus.

[0079] The modifications, methods, and assays described herein may also be applied to protein constructs containing one or more variable domains, and to such constructs in which the variable domains form the C-terminal portion of the construct, or in which the C-terminal region of the variable domains is exposed to the solvent. The methods, assays, and modifications described herein are applicable to constructs in which the VH domain (and VHH domain) forms the C-terminal portion of the construct or is otherwise exposed to the solvent.

[0080] The modifications described herein may also be applied to sdAbs containing, based on, or derived from heavy chain variable domains, e.g., VH domains (including human VH domains) and VHH domains (including humanized and sequence-optimized VHH domains), or camelized VH domains. These may be synthetic VH or VHH domains (e.g., sdAbs obtained starting from synthetic libraries or based on certain framework regions), semi-synthetic VH or VHH domains (e.g., sdAbs obtained starting from natural VH or VHH domains and then humanized, camelized, sequence-optimized, or obtained by affinity maturation or CDR grafting), or entirely natural VH or VHH domains. Accordingly, this disclosure will be further illustrated herein, in non-limiting examples, with respect to sdAbs based on VH or VHH domains and sdAbs obtained therefrom.

[0081] In some embodiments, the sdAb of this disclosure is modified within a single region. In other embodiments, the sdAb is modified in multiple regions. For example, the sdAb may be modified within the VTVSS (SEQ ID NO: 1) classical wild-type sequence, as well as at other locations, e.g., amino acid positions 8, 9, 10, 11, 12, or 13 of FW1, or by adding further amino acids. Modifications to the endogenous wild-type sdAb (e.g., VHH or scFv) C-terminal amino acid motif LVTVSS (SEQ ID NO: 20) or TVTVSS (SEQ ID NO: 21) (collectively referred to herein as "(T / L)VTVSS") located in FW4 as described herein may be combined with modifications at amino acid positions 11 and 14 of FW1. In some embodiments, the modifications may include L11S, L11Q, L11G, or P14A. The sdAb modifications described herein eliminate or reduce ADA-based immunogenicity caused by existing endogenous antibodies without substantially reducing the protein's binding affinity, specificity, expressibility, or stability.

[0082] Accordingly, in one aspect, the present disclosure relates to a camelized VH, VH, or sdAb, or a drug based on an sdAb, or a drug based on VHH, or a VHH or VH domain, which has another sdAb at its C-terminus, for example, a VH domain, or an sdAb derived from a VH domain, or an sdAb based on or obtained from the amino acid sequence of VHH or a VH domain, wherein the sdAb or VHH has an amino acid sequence at its C-terminus in which the classical wild-type C-terminal sequence (T / L)VTVSS C-terminal sequence is modified.

[0083] In one aspect, this disclosure relates to endogenous sdAb amino acid residues (T / L) that have been numbered according to the Kabat numbering scheme for human VH carboxy-terminal amino acid residues. 108 )V 109 T 110 V 111 S 112 S 113 Regarding single-domain antibodies (sdAb) containing a modified amino acid sequence at a corresponding position, the modified amino acid sequence is as shown in Table 1 below, with respect to formula X 108 X 109 X 110 V 111 X 112 X 113 Y (where "V" represents the amino acid valine) and X 108 X 109 X 110 V 111 X 112 X 113 One or more amino acid substitutions targeting [position]; and / or one or more C-terminal amino acid additions at position Y.

[0084] (Table 1) Overview of C-terminal amino acid substitutions, additions, and exclusions TIFF2026521433000002.tif107146

[0085] Polypeptide modifications disclosed herein may optionally further include one or more amino acid substitutions at positions 11, 12, 13, and 14, for example, substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A. As used above, the single-letter amino acid abbreviations T, L, Q, G, N, S, C, and A represent the amino acids threonine, lysine, glutamine, glycine, asparagine, serine, cysteine, and alanine, respectively.

[0086] In one aspect, this disclosure relates to endogenous sdAb amino acid residues (T / L) that have been numbered according to the Kabat numbering scheme for human VH carboxy-terminal amino acid residues. 108 )V 109 T 110 V 111 S 112 S 113 A single-domain antibody (sdAb) containing a modified amino acid sequence at a position corresponding to formula X 108 X 109 X 110 V 111 X 112 X 113 Including Y, X 108 However, selected from the group consisting of L, T, and Q, X 109 However, selected from the group consisting of V, G, N, and L, X 110 However, selected from the group consisting of T and Q, X 111 V is, X 112 However, it is selected from the group consisting of S, C, T, A, and G, or it is arbitrarily absent, and X 113 However, it is selected from the group consisting of S, C, A, G, and T, or it is optionally absent. however, (a)X 109 When V, then X 112 and X 113 It is not possible for both to be S, and (b)X 112and X 113 It is not possible for both to be C, and This relates to a single-domain antibody (sdAb) in which Y comprises a polypeptide of 1 to 5 amino acids, such amino acids being independently selected from the group consisting of A, G, S, T, L, and V, or Y is optionally absent.

[0087] sdAb has been optionally further modified to include amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A, numbered according to the Kabat numbering scheme.

[0088] In some cases, X 108 L is X 109 X is selected from the group consisting of V, G, and L. 110 is T, and X 112 X is selected from the group consisting of S, T, and C. 113 X is selected from the group consisting of S, T, C, and A. 114 The amino acid sequence AA comprises a polypeptide containing 1 to 5 amino acids independently selected from the group consisting of A, G, S, T, L, and V, where Y is either absent or a dipeptide. 109 X 110 V 111 X 112 X 113 sdAb having includes, but is not limited to, one of the following variants: GTVSS (SEQ ID NO: 3), LTVSS (SEQ ID NO: 27), VTVCS (SEQ ID NO: 8), VTVSC (SEQ ID NO: 10), NTVSS (SEQ ID NO: 22), VTVTS (SEQ ID NO: 23), and VTVTT (SEQ ID NO: 24). In certain embodiments, Y is AA. In other embodiments, amino acid sequence X 109 X 110 V 111 X 112 X 113 is VTVSA (SEQ ID NO: 6), and Y is AA. In some embodiments, amino acid sequence X 109 X110 V 111 X 112 X 113 This is selected from the group consisting of GTVSS (SEQ ID NO:3), LTVSS (SEQ ID NO:27), VTVCS (SEQ ID NO:8), VTVSC (SEQ ID NO:10), NTVSS (SEQ ID NO:22), VTVTS (SEQ ID NO:23), and VTVTT (SEQ ID NO:24). In some embodiments, Y is AA. In some embodiments, amino acid sequence X 109 X 110 V 111 X 112 X 113 is VTVSA (SEQ ID NO: 6), and Y is AA. In some embodiments, sdAb is VHH. In some embodiments, sdAb is formula VHH1-L n -VHH2 polypeptide, where VHH1 is VHH, L is a polypeptide linker containing 1 to 50 amino acids, n is 0 or 1, and VHH2 is VHH as described in claim 6.

[0089] In some embodiments, the C-terminus of the single-domain antibody of this disclosure has an amino acid sequence selected from the group consisting of one of the amino acid sequence ID numbers 2 to 24 in Table 2 below.

[0090] (Table 2) TIFF2026521433000003.tif172128

[0091] The C-terminal amino acid sequences listed in Table 2 above are located at the C-terminus of framework region 4 (FR4) of sdAb, e.g., VHH or scFv, which have FR and CDR domain structures FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0092] In some embodiments, endogenous sdAb amino acid residues (T / L) are numbered according to the Kabat numbering scheme for human VH carboxy-terminal amino acid residues. 108 )V 109 T 110 V111 S 112 S 113 The C-terminal amino acid sequences corresponding to the positions listed in Table 2, with SEQ ID NO: 2-24. The modified amino acid sequence is given by formula X. 108 X 109 X 110 V 111 X 112 X 113 Including Y, the position of sdAb in the C-terminal domain is as shown in Table 3 below.

[0093] (Table 3) TIFF2026521433000004.tif126146

[0094] In some embodiments, the C-terminus of the single-domain antibody of this disclosure is VTVSSAA (SEQ ID NO: 2). In some embodiments, the C-terminus of the single-domain antibody of this disclosure is GTVSS (SEQ ID NO: 3). In some embodiments, the C-terminus of the single-domain antibody of this disclosure is LTVSS (SEQ ID NO: 4). In some embodiments, the C-terminus of the single-domain antibody of this disclosure is LTVSSAA (SEQ ID NO: 5). In some embodiments, the C-terminus of the single-domain antibody of this disclosure is VTVSA (SEQ ID NO: 6). In some embodiments, the C-terminus of the single-domain antibody of this disclosure is VTVSAAA (SEQ ID NO: 7). In some embodiments, the C-terminus of the single-domain antibody of this disclosure is VTVCS (SEQ ID NO: 8). In some embodiments, the C-terminus of the single-domain antibody of this disclosure is VTVCSA (SEQ ID NO: 9). In some embodiments, the C-terminus of the single-domain antibody of this disclosure is VTVSCSA (SEQ ID NO: 10). In some embodiments, the C-terminus of the single-domain antibody of this disclosure is VTVSC (SEQ ID NO: 11). In some embodiments, the C-terminus of the single-domain antibody of this disclosure is VTVSCAA (SEQ ID NO: 12). In some embodiments, the C-terminus of the single-domain antibody of this disclosure is LLTVSS (SEQ ID NO: 13). In some embodiments, the C-terminus of the single-domain antibody of this disclosure is LLTVSSA (SEQ ID NO: 14). In some embodiments, the C-terminus of the single-domain antibody of this disclosure is LLTVSSAA (SEQ ID NO: 15). In some embodiments, the C-terminus of the single-domain antibody of this disclosure is LLTVSS (SEQ ID NO: 13) further including the L11S substitution. In some embodiments, the C-terminus of the single-domain antibody of this disclosure is LLTVSSA (SEQ ID NO: 14) further including the L11S substitution. In some embodiments, the C-terminus of the single-domain antibody of this disclosure is LLTVSSAA (SEQ ID NO: 15) further containing an L11S substitution. In some embodiments, the C-terminus of the single-domain antibody of this disclosure is LVTVTT (SEQ ID NO: 16).In some embodiments, the C-terminus of the single-domain antibody of this disclosure is LVTVTTAA (SEQ ID NO: 17). In some embodiments, the C-terminus of the single-domain antibody of this disclosure further includes an L11S substitution (SEQ ID NO: 16). In some embodiments, the C-terminus of the single-domain antibody of this disclosure further includes an L11S substitution (SEQ ID NO: 17). In some embodiments, the C-terminus of the single-domain antibody of this disclosure is TLTVSS (SEQ ID NO: 18). In some embodiments, the C-terminus of the single-domain antibody of this disclosure is TLTVSSA (SEQ ID NO: 19). In some embodiments, the C-terminus of the single-domain antibody of this disclosure is TLTVSSAA (SEQ ID NO: 20). In some embodiments, the C-terminus of the single-domain antibody of this disclosure is TVTVSS (SEQ ID NO: 21). In some embodiments, the C-terminus of the single-domain antibody of this disclosure is NTVSS (SEQ ID NO: 22). In some embodiments, the C-terminus of the single-domain antibody of this disclosure is VTVTS (SEQ ID NO: 23). In some embodiments, the C-terminus of the single-domain antibody of this disclosure is VTVTT (SEQ ID NO: 24).

[0095] In some embodiments, the disclosure provides a VHH in which the C-terminus of the VHH comprises an amino acid sequence selected from the group consisting of amino acid SEQ ID NO: 2 to 24, and the single-domain antibody optionally further comprises one or more amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A, numbered according to the Kabat numbering scheme.

[0096] In some embodiments, sdAb is given by the formula VHH1-L n -VHH2 polypeptide, where VHH1 and VHH2 each independently bind to different molecules. In one embodiment, VHH1 binds to a molecule expressed on the surface of a first cell, and VHH2 binds to a molecule expressed on the surface of a second cell. In some embodiments, the first and second cells are different cell types. In one embodiment, the polypeptide of formula VHH1-L n- One of the VHH1 or VHH2 polypeptides of VHH2 binds to antigens preferentially expressed on the surface of cancer cells, and the other of VHH1 or VHH2 binds to antigens preferentially expressed on the surface of immune cells, such as activated immune cells and other pro-inflammatory cells. In some embodiments, the antigens expressed on the surface of cancer cells are selected from a group consisting of tumor antigens for which antibody-binding molecules have been identified, and their clinical therapeutic targets are CD19 (e.g., hematological malignancies, e.g., ALLs, CLLs, B-cell lymphomas), CD20 (e.g., refractory or relapsed CD20 +CD22 (B-cell lymphoma), BCMA (e.g., multiple myeloma, Carpenter, et al. (2013) Clin Cancer Res; 19(8); 2048-60), CD30 (e.g., CD30+ lymphoma including Hodgkin lymphoma; Grover, (2019) BMC Cancer 19, 203), CD70 (e.g., acute myeloid leukemia (AML; Sauer, et al (2019) Blood 134 (Supplement_1): 1932), Lewis Y (e.g., AML; Ritchie, et al (2013) Molecular Therapy 21(11):2122-9), GD2 (e.g., glioma; Mount, (2018) Nat Med 24, 572-579), GD3 (e.g., metastatic melanoma and neuroectodermal tumors; Agnes, et al. (2010) DOI: 10.1158 / 1078-0432.CCR-10-0043), mesothelin (e.g., mesothelioma, lung, pancreatic, breast, ovarian, and other solid tumors; Beatty, et al. Al., (2014) Cancer Immunol Research 2(2)), ROR-1 (e.g., chronic lymphocytic leukemia; Aghebati-Maleki, et al (2017) Biomedicine and Pharmacology 88: 814-822), CD44 (e.g., AML and multiple myeloma; Casuccia, et al (2013) Blood 122 (20): 3461-3472), CD171 (e.g., neuroblastoma; Kunkele, et al (2017) Clin Cancer Research 23(2):466-477); EGP2, EphA2 (e.g., gliablastoma; Yi, et al (2018) Molecular Therapy: Methods & Clinical Development 9:70-80), ErbB2, ErbB3 / 4, FAP, FAR The compound includes IL11Ra, PSCA (prostate cancer), PSMA (prostate cancer), NCAM, HER2, NY-ESO-1, MUC1, CD123, FLT3, B7-H3, CD33, IL1RAP, CLL1 (CLEC12A)PSA, CEA, VEGF, VEGF-R2, c-Met, glycolipid F77, FAP, EGFRvIII, MAGE A3, 5T4, WT1, KG2D ligand, folate receptor (FRa), and Wnt1 antigen, CD123. Furthermore, ABD may exhibit specificity for multiple tumor antigens (e.g., CD19 and CD20 as described in Zah, et al (2016) Cancer Immunol Res; 4(6); 498-508; CD19 and CD22 as described in Tu, et al (2019) Frontiers in Oncology 9:1350).

[0097] In one embodiment, formula VHH1-L nEither VHH1 or VHH2 of the VHH2 polypeptide binds to antigens preferentially expressed on the surface of immune cells, including but not limited to activated immune cells or pro-inflammatory immune cells, including, but not limited to, IL1R1 receptor, IL-1 receptor accessory protein, IL6 receptor subunit (IL6R), HLA-DR, HLA-DRα-chain, HLA-DRβ-chain, TNFR1, TNFR2, CD4, CD8, F4 / 80, CCR2, CD169, CX3CR1, CD206, CD163, and Lyve1.

[0098] In some embodiments, sdAb is given by the formula VHH1-L n -VHH2 polypeptide, where VHH1 and VHH2 each independently bind to the extracellular domain of the cytokine receptor. In some embodiments, sdAb is a polypeptide of the formula VHH1-L n -VHH2 polypeptide, the cytokine receptor is selected from the group consisting of IL2Rα, IL2Rβ, IL2Rγ, IL10Rα, IL10Rβ, IL12Rβ1, IL12Rβ2, IL18Rα, IL18Rβ, IL22R1, IL27Rα, gp130, IL23R, IL28Rα, IFNRγ1, IFNRγ2, and IL21R. In some embodiments, sdAb is a polypeptide of formula VHH1-L n -VHH2 polypeptides, VHH1 and VHH2 selectively bind to a pair of cytokine receptors selected from the following set: IL10Rα / IL10Rβ, IL27Rα / gp130, IFNγR1 / IFNγR2, IL10Rβ / IL28Rα, IL2Rβ / IL2Rγ, IL18Rα / IL18Rβ, IL22R1 / IL10Rβ, IL10Rα / IL2Rγ, IL2Rβ / IL2Rγ, IL10R1 / IFNRγ, IFNRγ / IL28Rα, IL12Rβ1 / IL12Rβ2, IL12Rβ1 / IL23R, IL12Rβ2 / gp130, and IL10Rα / IL2Rγ.

[0099] In some embodiments, sdAb is a VHH that selectively binds to the extracellular domain of a cytokine receptor subunit. In some embodiments, the cytokine receptor is selected from the group consisting of IL2Ra, IL2Rb, IL2Rg, IL6Ra, IL10Ra, IL10Rb, IL12Rb1, IL12Rb2, IL18Ra, IL18Rb, IL22R1, IL27Ra, gp130, IL23R, IL28Rα, IFNRγ1, IFNRγ2, and IL21R.

[0100] In some embodiments, sdAb is a polypeptide that binds to human serum albumin.

[0101] In some embodiments, sdAb is given by the formula VHH1-L n -VHH2 polypeptide, where L is a linker containing amino acids G and S. In some embodiments, sdAb is a pegylated polypeptide. In some embodiments, sdAb is a polypeptide conjugated with an Fc domain.

[0102] In some embodiments, the polypeptide, when administered to human subjects, exhibits reduced immunogenicity to existing endogenous antibodies compared to the polypeptide of the corresponding native human sequence VTVSS (SEQ ID NO:1).

[0103] In another context, this disclosure relates to formula VHH1-L n Regarding the polypeptide of -VHH2, VHH1 is VHH, L is a polypeptide linker containing 1 to 50 amino acids, n is 0 or 1, and VHH2 is sdAb VHH.

[0104] In some embodiments, VHH1 and VHH2 independently bind to the extracellular domain of a cytokine receptor. For example, VHH1 and VHH2 may independently bind to one or more cytokine receptors such as IL2Rα, IL2Rβ, IL2Rγ, IL10Rα, IL10Rβ, IL12Rβ1, IL12Rβ2, IL18Rα, IL18Rβ, IL22R1, IL27Rα, gp130, IL23R, IL28Rα, IFNRγ1, and IFNRγ2. VHH1 and VHH2 may also selectively bind to pairs of cytokine receptors such as IL10Rα / IL10Rβ;IL27Rα / gp130;IFNγR1 / IFNγR2, IL10Rβ / IL28Rα, IL2Rβ / IL2Rγ, IL18Rα / IL18Rβ, IL22R1 / IL10Rβ, IL10Rα / IL2Rγ, IL2Rβ / IL2Rγ, IL10R1 / IFNRγ, IFNRγ / IL28Rα, IL12Rβ1 / IL12Rβ2, IL12Rβ1 / IL23R, and IL10Rα / IL2Rγ. With respect to the above pairs of cytokine receptors, the forward slash symbol " / " indicates that the cytokine pair can be linked in any order. For example, the cytokine pair "IL10Rα / IL10Rβ" may be directed as "IL10Rα-IL10Rβ", or alternatively as "IL10Rβ-IL10Rα".

[0105] In some embodiments, this disclosure relates to formula VHH1-L n - A cytokine receptor-binding polypeptide of VHH2, wherein the cytokine comprises a first cytokine receptor subunit and a second cytokine receptor subunit, where either VHH1 or VHH2 is a VHH that selectively binds to the extracellular domain of the first cytokine receptor subunit, and the other VHH1 or VHH2 is a VHH that selectively binds to the extracellular domain of the second cytokine receptor subunit, where L is a polypeptide linker, n=0 (absent) or (1) present, and VHH2 is a polypeptide whose amino acids are numbered according to the Kabat numbering scheme, formula X 108 V 109 T 110 V 111 S112 S 113 Contains the amino acid sequence Y, X 108 X is selected from the group consisting of L, T, and Q. 109 X is selected from the group consisting of V, G, N, and L. 110 X is selected from the group consisting of T and Q. 111 V is X 112 is selected from the group consisting of S, C, T, A, and G, or is optionally absent, X 113 (a)X 109 If V, then X 112 and X 113 (b)X 112 and X 113 The present invention provides cytokine receptor-binding polypeptides in which both are not C, and Y comprises a polypeptide comprising 1 to 5 amino acids independently selected from the group consisting of A, G, S, T, L, and V, or Y is optionally absent, and the polypeptide is further modified to optionally include amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A, numbered according to the Kabat numbering scheme. In some embodiments, the formula VHH1-L n -A cytokine receptor-binding polypeptide of VHH2, where formula X of VHH2 108 V 109 T 110 V 111 S 112 S 113 The amino acid sequence of Y is selected from the group consisting of SEQ ID NO: 2 to 24, and optionally further includes amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A, in which amino acid residues are numbered according to the Kabat numbering scheme. In some embodiments, the cytokine receptor-binding polypeptide is the one with formula X in VHH2. 108 V 109 T 110 V 111 S 112 S 113It exhibits reduced immunogenicity compared to polypeptides lacking the Y amino acid sequence. In some embodiments of the cytokine receptor-binding polypeptide, n=1 and L is a polypeptide linker of 1 to 50 amino acids. In some embodiments, the cytokine receptor-binding polypeptide is n=1 and L is a polypeptide linker selected from the group consisting of SEQ ID NO: 25 to 48. In some embodiments, the cytokine receptor-binding polypeptide is further modified to extend its half-life in vivo. In some embodiments, the cytokine receptor-binding polypeptide is pegylated. In some embodiments, this disclosure provides nucleic acid sequences encoding cytokine receptor-binding polypeptides. In some embodiments, the cytokine receptor is the IL10 receptor, the first cytokine receptor subunit is IL10Ra, and the second cytokine receptor subunit is IL10Rb. In some embodiments, the cytokine receptor is the IL2 receptor, the first cytokine receptor subunit is IL2Rb(CD122), and the second cytokine receptor subunit is IL2Rg(CD132). In some embodiments, the cytokine receptor is the IL18 receptor, the first cytokine receptor subunit is IL18Ra, and the second cytokine receptor subunit is IL18Rb. In some embodiments, the cytokine receptor is the IL27 receptor, the first cytokine receptor subunit is IL27Ra, and the second cytokine receptor subunit is gp130. In some embodiments, the cytokine receptor is the IL22 receptor, the first cytokine receptor subunit is IL22Ra, and the second cytokine receptor subunit is IL12Rb. In some embodiments, the cytokine receptor is the IL4 receptor, the first cytokine receptor subunit is IL4Ra, and the second cytokine receptor subunit is IL2Rg(CD132). In some embodiments, the cytokine receptor is the IL7 receptor, the first cytokine receptor subunit is IL7Ra, and the second cytokine receptor subunit is IL2Rg(CD132).In some embodiments, the cytokine receptor is the IL9 receptor, the first cytokine receptor subunit is IL9Ra, and the second cytokine receptor subunit is IL2Rg(CD132). In some embodiments, the cytokine receptor is the IL12 receptor, the first cytokine receptor subunit is IL12Ra, and the second cytokine receptor subunit is IL12Rb.

[0106] IL10 receptor binding molecule In some embodiments, this disclosure relates to formula VHH1-L n -VHH2 is an IL10 receptor-binding polypeptide, where either VHH1 or VHH2 is a VHH that selectively binds to the extracellular domain of IL10Ra, and the other VHH1 or VHH2 is a VHH that selectively binds to the extracellular domain of IL10Rb, where L is a polypeptide linker, n=0 (absent) or (1) present, and VHH2 is a polypeptide whose amino acids are numbered according to the Kabat numbering scheme, formula X 108 V 109 T 110 V 111 S 112 S 113 Contains the amino acid sequence Y, X 108 X is selected from the group consisting of L, T, and Q. 109 X is selected from the group consisting of V, G, N, and L. 110 X is selected from the group consisting of T and Q. 111 V is X 112 is selected from the group consisting of S, C, T, A, and G, or is optionally absent, X 113 (a)X 109 If V, then X 112 and X 113 (b)X 112 and X 113The present invention provides an IL10 receptor-binding polypeptide in which both are not C, and Y comprises a polypeptide comprising 1 to 5 amino acids independently selected from the group consisting of A, G, S, T, L, and V, or Y is optionally absent, and the polypeptide is further modified to optionally include amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A, numbered according to the Kabat numbering scheme. In some embodiments, the formula VHH1-L n - A VHH2 IL10 receptor-binding polypeptide, where VHH2 is given by formula X 108 V 109 T 110 V 111 S 112 S 113 The amino acid sequence of Y is selected from the group consisting of SEQ ID NO: 2 to 24, and optionally further includes amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A, in which amino acid residues are numbered according to the Kabat numbering scheme. In some embodiments, the IL10 receptor-binding polypeptide is the one with formula X in VHH2. 108 V 109 T 110 V 111 S 112 S 113It exhibits reduced immunogenicity compared to polypeptides lacking the Y amino acid sequence. In some embodiments of the IL10 receptor-binding polypeptide, n=1 and L is a polypeptide linker of 1 to 50 amino acids. In some embodiments of the IL10 receptor-binding polypeptide, n=1 and L is a polypeptide linker selected from the group consisting of SEQ ID NO: 25 to 48. In some embodiments, the IL10 receptor-binding polypeptide is further modified to extend its half-life in vivo. In some embodiments, the IL10 receptor-binding polypeptide is pegylated. In some embodiments, this disclosure provides nucleic acid sequences encoding the IL10 receptor-binding polypeptide. In some embodiments, the IL10 receptor-binding polypeptide contains an amino acid sequence having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 50-72, and the IL10 receptor-binding polypeptide exhibits reduced immunogenicity compared to DR2485 (SEQ ID NO: 49). In some embodiments, the IL10 receptor-binding polypeptide contains an amino acid sequence selected from the group consisting of SEQ ID NO: 50-72. Exemplary IL10 receptor-binding molecules described herein are shown in Table 4 as SEQ ID NO: 50-72 derived from the reference sequence DR2485aa (SEQ ID NO: 49).

[0107] (Table 4) Amino acid sequence of IL-10Ra-IL-10Rb dimer TIFF2026521433000005.tif160143TIFF2026521433000006.tif197143TIFF20265214330 00007.tif197143TIFF2026521433000008.tif197143TIFF2026521433000009.tif197143

[0108] In some embodiments, the Disclosure provides nucleic acid molecules comprising nucleic acid sequences encoding the polypeptides of Table 4 above, as shown in Table 5 below.

[0109] (Table 5) Nucleic acid sequences encoding the IL-10Ra-IL-10Rb dimer TIFF2026521433000010.tif184152TIFF2026521433000011.tif184152TIFF2026521433000012.tif18415 2TIFF2026521433000013.tif184152TIFF2026521433000014.tif184152TIFF2026521433000015.tif97152

[0110] Divalent IL6R / HSA binding molecule In some embodiments, this disclosure relates to formula VHH1-L n - A divalent IL6R / HSA-binding polypeptide of VHH, wherein either VHH1 or VHH2 is a VHH that selectively binds to the extracellular domain of IL6Ra, and the other VHH1 or VHH2 is a VHH that selectively binds to human serum albumin, where L is a polypeptide linker, n=0 (absent) or (1) present, and VHH2 is a polypeptide whose amino acids are numbered according to the Kabat numbering scheme, formula X 108 V 109 T 110 V 111 S 112 S 113 Contains the amino acid sequence Y, X 108 X is selected from the group consisting of L, T, and Q. 109 X is selected from the group consisting of V, G, N, and L. 110 X is selected from the group consisting of T and Q. 111 V is X 112 is selected from the group consisting of S, C, T, A, and G, or is optionally absent, X 113 (a)X 109 If V, then X 112 and X 113 (b)X 112 and X 113The present invention provides a divalent IL6R / HSA-binding polypeptide in which both are not C, and Y comprises a polypeptide comprising 1 to 5 amino acids independently selected from the group consisting of A, G, S, T, L, and V, or Y is optionally absent, and the polypeptide is further modified to optionally include amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A, numbered according to the Kabat numbering scheme. In some embodiments, the formula VHH1-L n -A divalent IL6R / HSA-binding polypeptide of VHH2, where formula X of VHH2 108 V 109 T 110 V 111 S 112 S 113 The amino acid sequence of Y is selected from the group consisting of SEQ ID NO: 2 to 24, and optionally further includes amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A, in which amino acid residues are numbered according to the Kabat numbering scheme. In some embodiments, the divalent IL6R / HSA-binding polypeptide is the one with formula X in VHH2. 108 V 109 T 110 V 111 S 112 S 113It exhibits reduced immunogenicity compared to polypeptides without the Y amino acid sequence. In some embodiments, the divalent IL6R / HSA-binding polypeptide is n=1, and L is a polypeptide linker of 1 to 50 amino acids. In some embodiments, the divalent IL6R / HSA-binding polypeptide is n=1, and L is a polypeptide linker selected from the group consisting of SEQ ID NO: 25 to 48. In some embodiments, the divalent IL6R / HSA-binding polypeptide is further modified to extend its half-life in vivo. In some embodiments, the divalent IL6R / HSA-binding polypeptide is pegylated. In some embodiments, the disclosure provides nucleic acid sequences encoding the divalent IL6R / HSA-binding polypeptide. In some embodiments, the divalent IL6R / HSA-binding polypeptide comprises an amino acid sequence having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 85 to 109. In some embodiments, the divalent IL6R / HSA-binding polypeptide contains an amino acid sequence selected from the group consisting of SEQ ID NO: 85-109.

[0111] The exemplary divalent IL6R / HSA-binding polypeptide molecules described herein are shown as SEQ ID NO: 85-109 in Table 6, derived from the reference sequence DR2514aa (SEQ ID NO: 84).

[0112] (Table 6) Amino acid sequence of αIL-6R-αHSA VHH dimer TIFF2026521433000016.tif242148TIFF2026521433000017.tif214148TIFF2026521433000018.tif214148TIFF2026521433000019.tif129148

[0113] IL18 receptor binding molecule In some embodiments, this disclosure relates to formula VHH1-L n-VHH2 is an IL18 receptor-binding polypeptide, where either VHH1 or VHH2 is a VHH that selectively binds to the extracellular domain of IL18Ra, and the other VHH1 or VHH2 is a VHH that selectively binds to the extracellular domain of IL18Rb, where L is a polypeptide linker, n=0 (absent) or (1) present, and VHH2 is a polypeptide whose amino acids are numbered according to the Kabat numbering scheme, formula X 108 V 109 T 110 V 111 S 112 S 113 Contains the amino acid sequence Y, X 108 X is selected from the group consisting of L, T, and Q. 109 X is selected from the group consisting of V, G, N, and L. 110 X is selected from the group consisting of T and Q. 111 V is X 112 is selected from the group consisting of S, C, T, A, and G, or is optionally absent, X 113 (a)X 109 If V, then X 112 and X 113 (b)X 112 and X 113 The present invention provides an IL18 receptor-binding polypeptide in which both are not C, and Y comprises a polypeptide comprising 1 to 5 amino acids independently selected from the group consisting of A, G, S, T, L, and V, or Y is optionally absent, and the polypeptide is further modified to optionally include amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A, numbered according to the Kabat numbering scheme. In some embodiments, the formula VHH1-L n - A VHH2 IL18 receptor-binding polypeptide, where VHH2 is given by formula X 108 V 109 T 110 V 111 S 112 S 113The amino acid sequence of Y is selected from the group consisting of SEQ ID NO: 2 to 24, and optionally further includes amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A, in which amino acid residues are numbered according to the Kabat numbering scheme. In some embodiments, the IL18 receptor-binding polypeptide is the one with formula X in VHH2. 108 V 109 T 110 V 111 S 112 S 113 It exhibits reduced immunogenicity compared to polypeptides lacking the Y amino acid sequence. In some embodiments of the IL18 receptor-binding polypeptide, n=1 and L is a polypeptide linker of 1 to 50 amino acids. In some embodiments of the IL18 receptor-binding polypeptide, n=1 and L is a polypeptide linker selected from the group consisting of SEQ ID NO: 25 to 48. In some embodiments, the IL18 receptor-binding polypeptide is further modified to extend its half-life in vivo. In some embodiments, the IL18 receptor-binding polypeptide is pegylated. In some embodiments, this disclosure provides nucleic acid sequences encoding the IL18 receptor-binding polypeptide. In some embodiments, the IL18 receptor-binding polypeptide comprises an amino acid sequence having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 111 to 134. In some embodiments, the IL18 receptor-binding polypeptide contains an amino acid sequence selected from the group consisting of SEQ ID NO: 111-134.

[0114] The exemplary IL18 receptor-binding molecules described herein are shown in Table 7 as SEQ ID NO: 111-134, derived from the reference sequence DR3905aa (SEQ ID NO: 110).

[0115] (Table 7) Amino acid sequence of IL-18Ra-IL-18Rb dimer TIFF2026521433000020.tif209152TIFF2026521433000021.tif209152TIFF2026521433000022.tif209152TIFF2026521433000023.tif124152

[0116] The polypeptides described in the above table can be encoded by DNA sequences as shown in Table 8 below.

[0117] (Table 8) Nucleic acid sequences of IL-18Ra-IL-18Rb VHH dimers TIFF2026521433000024.tif179149TIFF2026521433000025.tif179149TIFF2026521433000026.tif179149TIFF2026521433000027.tif179149TIFF2026521433000028.tif179149TIFF2026521433000029.tif179149TIFF2026521433000030.tif179149TIFF2026521433000031.tif179149TIFF2026521433000032.tif179149TIFF2026521433000033.tif179149TIFF2026521433000034.tif179149TIFF2026521433000035.tif179149 [[ID=I2]]

[0118] Anti-HSA VHH In some embodiments, the disclosure provides an anti-HSA VHH that selectively binds to human serum albumin, wherein the HSA VHH has the formula X in which the amino acids are numbered according to the Kabat numbering scheme 108 V 109 T 110 V 111 S 112 S 113 Y, and X 108 is selected from the group consisting of L, T, and Q, and X 109 is selected from the group consisting of V, G, N, and L, and X 110 is selected from the group consisting of T and Q, and X111 V is X 112 is selected from the group consisting of S, C, T, A, and G, or is optionally absent, X 113 (a)X 109 If V, then X 112 and X 113 (b)X 112 and X 113 The present invention provides an anti-HSA VHH in which both are not C, and Y comprises a polypeptide comprising 1 to 5 amino acids independently selected from the group consisting of A, G, S, T, L, and V, or Y is optionally absent, and the polypeptide is optionally further modified to include amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A, numbered according to the Kabat numbering scheme. In some embodiments of the HSA VHH, formula X 108 V 109 T 110 V 111 S 112 S 113 The amino acid sequence of Y is selected from the group consisting of SEQ ID NO: 2 to 24 and optionally further includes amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A, in which amino acid residues are numbered according to the Kabat numbering scheme. In some embodiments, anti-HSA VHH is formula X 108 V 109 T 110 V 111 S 112 S 113It exhibits reduced immunogenicity compared to a polypeptide lacking the amino acid sequence of Y. In some embodiments, the HSA VHH is modified to extend its half-life in vivo. In some embodiments, the anti-HSA VHH is pegylated. In some embodiments, the present disclosure provides a nucleic acid sequence encoding the anti-HSA VHH. In some embodiments, the anti-HSA VHH has at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 160-194 and exhibits reduced immunogenicity compared to the polypeptide of SEQ ID NO: 159. In some embodiments, the anti-HSA VHH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 160-194. Exemplary anti-HSA VHHs are shown in Table 9 as SEQ ID NOs: 160-194 derived from the reference sequence DR2830aa (SEQ ID NO: 159).

[0119] (Table 9) Amino acid sequences of anti-HSA VHH monomers TIFF2026521433000,036.tif230,143TIFF2026521433000,037.tif214,143TIFF2026521433000,038.tif79,143

[0120] In some embodiments, an anti-HSA VHH molecule containing amino acid substitutions exhibiting reduced immunogenicity can be conjugated (covalently linked) with a second molecule (e.g., a therapeutic protein or antibody) to extend the in vivo half-life of a therapeutic protein. In one embodiment, the disclosure provides a conjugate comprising a therapeutic protein and an anti-HSA sdAb polypeptide having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 160-194. In one embodiment, the present disclosure provides a conjugate comprising a therapeutic protein and an anti-HSA sdAb polypeptide having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 160-194, and exhibiting reduced immunogenicity compared to a conjugate containing a polypeptide with SEQ ID NO: 159.

[0121] In some embodiments, the disclosure relates to compositions in which the polypeptide is pegylated. In other embodiments, the disclosure relates to compositions in which the polypeptide is conjugated with an Fc domain.

[0122] In other embodiments, the polypeptide exhibits reduced immunogenicity when administered to human subjects compared to the polypeptide of the corresponding native human sequence VTVSS.

[0123] In some embodiments, the natural C-terminal amino acid sequence VTVSS of the sdAb or sdAb-containing construct may also be replaced with a non-natural C-terminal amino acid sequence, for example, when the C-terminus is an sdAb or VHH derived from VH.

[0124] Linker In some embodiments, the covalent bonds of each sdAb dimer may further include linkers. For example, VHH1 and VHH2 may be linked together by a linker represented by "L".

[0125] The linker is a molecule selected from the group including, but not limited to, peptide linkers and chemical linkers. In some embodiments, the linker connects the C-terminus of a first sdAb to the N-terminus of a second sdAb. In some embodiments, the linker connects the C-terminus of a second sdAb to the N-terminus of a first sdAb.

[0126] In some embodiments, the linker is a peptide linker. The peptide linker may contain 1 to 50 amino acids (e.g., 2 to 50, 5 to 50, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 2 to 45, 2 to 40, 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 5 amino acids). In some embodiments, linker "L" is a GS linker. Glycine and glycine-serine polymers are relatively structurally indeterminate and may act as neutral linkers between components. An example of a glycine polymer is (G) n , glycine-alanine polymer, alanine-serine polymer, glycine-serine polymer (for example, (G m S o ) n (GSGGS) n , (G m S o G m ) n , (G m S o G m S o G m ) n (GSGGS m ) n (GSGS m G) n , and (GGGS m ) nThis includes, as well as combinations thereof, and other mobile linkers, where m, n, and o are each independently selected from an integer of at least 1 to 20 (e.g., 1 to 18, 2 to 16, 3 to 14, 4 to 12, 5 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Exemplary mobile peptide linkers useful in the preparation of sdAb-binding molecules and polypeptides of this disclosure may include, but are not limited to, the specific linkers shown in Table 10.

[0127] (Table 10) Exemplary polypeptide linkers TIFF2026521433000039.tif179146

[0128] In some embodiments, a chemical linker may covalently link a first domain and a second domain. The term “linker” refers to a link between two elements, such as polypeptide domains. The linker may be covalent or a peptide linker. The linkage may be a chemical bond, e.g., an amide bond, a disulfide bond, or any bond created from a chemical reaction or chemical conjugate. The linker may also be a peptide linker containing amino acids or polypeptides that link two protein domains, providing space or mobility between the two protein domains. Examples of chemical linkers include arylacetylenes, ethylene glycol oligomers containing 2-10 monomer units, diamines, dibasic acids, amino acids, or combinations thereof, in the novel polypeptides described above. L is a “GSA” linker selected from the group consisting of linkers containing amino acid residues G, S, and A, as shown in the table above.

[0129] As further described herein, any modified protein or polypeptide may be a construct containing, for example, two or more sdAbs (e.g., two or more VHHs, or scFvs with VH domains linked together) linked via one or more suitable linkers. Thus, for example, such a construct may be a divalent, trivalent, tetravalent, or pentavalent construct (e.g., a divalent, trivalent, tetravalent, or pentavalent VHH construct), or a divalent, trivalent, tetravalent, or pentavalent construct (e.g., a divalent, trivalent, tetravalent, or pentavalent VHH construct), which may be a bispecific, triplicate, or biparatopic construct (e.g., a monospecific, bispecific, or biparatopic construct that can also bind to serum albumin or another serum protein, or, in the case of half-life extension, polyethylene glycol (PEG)). It is known in the art that a conjugate of a molecule with an antibody containing an sdAb or antibody fragment that binds to human serum albumin can extend the half-life of the molecule in vivo.

[0130] This disclosure provides an anti-HSA VHH molecule containing amino acid substitutions exhibiting reduced immunogenicity, which can be conjugated (covalently linked) with a second molecule (e.g., a therapeutic protein or antibody) to extend the in vivo half-life of a therapeutic protein. In particular, this disclosure provides a conjugate comprising a therapeutic protein and an anti-HSA sdAb monomer, wherein the anti-HSA sdAb monomer polypeptide has at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 160-194.

[0131] As described herein, it is also assumed that this disclosure may apply to other proteins or polypeptides having a VH-domain at the C-terminus (and antibody fragments, e.g., Fab fragments or other proteins or polypeptides based on antibody fragments, e.g., scFv). Thus, in another aspect, this disclosure applies to V at the C-terminus H The domain relates to a protein or polypeptide (e.g., scFv) having the amino acid sequence described above.

[0132] Immunoconjugate The antibodies disclosed herein, for example, single-domain antibodies and their antigen-binding fragments, can also be conjugated with various other chemical entities, such as drug small molecules, enzymes, liposomes, polymers, such as polyethylene glycol (PEG), radionuclides, and antibody Fc domains. Such antibodies and fragments are useful for therapeutic, diagnostic, kit, or other purposes, and include, for example, antibodies coupled with dyes, radioisotopes, enzymes, or metals, such as colloidal gold (see, for example, Le Doussal et al. 1991. J Immunol. 146:169-175; Gibellini et al. 1998. J Immunol. 160:3891-3898; Hsing and Bishop. 1999. J Immunol. 162:2804-2811; Everts et al. 2002. J Immunol. 168:883-889). Any method known in the art for conjugating the single-domain antibody and its antigen-binding fragment of the present invention with various parts can be used, including the methods described by Hunter, et al. 1962. Nature. 144:945; David et al. 1974. Biochemistry 13:1014; Pain et al. 1981. J Immunol Meth. 40:219; and Nygren J. 1982. Histochem and Cytochem. 30:407. Methods for conjugating antibodies and fragments are conventional and well known in the art.

[0133] Such conjugated portions may be conjugated with the single-domain antibody or fragment as a fusion protein at any suitable position on the single-domain antibody or fragment, for example, at either the N-terminus or the C-terminus, or they may be conjugated with the side chain of an antibody residue at, for example, the sulfhydryl or SH group of a cysteine ​​residue, for example, at a cysteine ​​amino acid residue at amino acid position 112 or 113.

[0134] In some embodiments, the single-domain antibodies and antigen-binding fragment compounds disclosed herein may be conjugated with polymers. Such polymers include, but are not limited to, hydrophilic polymers such as polyethylene glycol (PEG), e.g., PEG having molecular weights of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30 kDa, or 40 kDa, dextran, and monomethoxypolyethylene glycol (mPEG). Conjugation with PEG or mPEG may extend the biological (e.g., serum) half-life of the compound. To pegylate an antibody or fragment, the antibody or fragment is typically reacted with a reactive form of polyethylene glycol (PEG), e.g., a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups are attached to the antibody or antibody fragment. In some embodiments, pegylation is carried out via an acylation or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono(C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody or fragment being pegylated is an unglycosylated antibody or fragment. Methods for pegyrating proteins are well known in the art and can be applied to the antibodies of this disclosure. See, for example, EP 0 154 316 and EP 0 401 384, Lee et al. 1999. Bioconj Chem. 10:973-981 (PEG-conjugated single-chain antibodies), and Wen et al. 2001. Bioconj Chem. 12:545-553 (Antibodies conjugated with PEG attached to the radioactive metal chelating agent diethylenetriaminepentaacetic acid (DTPA)).

[0135] In other embodiments, single-domain antibodies or antigen-binding fragments described herein can be conjugated with therapeutic agents having immunostimulatory (agonist) or immunoinhibitory (antagonist) activity to form antibody-drug conjugate (ADC) compounds. Various forms of ADCs and methods for producing ADCs are well known in the art. Suitable therapeutic agents include, for example, cytotoxic agents (e.g., chemotherapeutic agents), toxins (e.g., enzymatically active toxins or fragments thereof derived from bacteria, fungi, plants, or animals), and / or radioisotopes (i.e., radioconjugates). Further suitable agents include, for example, antimetabolites, alkylating agents, DNA subgroove binding agents, DNA intercalators, DNA crosslinkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. In some embodiments, immunosuppressive cells, such as regulatory T cells, can be depleted, for example, from the tumor microenvironment by using an ADC having the single-domain antibody or its antigen-binding fragment described herein (for example, conjugated with a cytotoxic agent) that binds to immunosuppressive cells.

[0136] In other embodiments, the single-domain antibodies or antigen-binding fragments disclosed herein may also include fluorophores, such as rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanates, phycoerythrin, phycocyanin, allophycocyanin, o-phthaladehyde, fluoresamine, 152Eu, dansyl, umbelliferone, luciferin, luminal labeling, isoluminal labeling, aromatic acridinium ester labeling, imidazole labeling, acridimium salt labeling, oxalate ester labeling, aequorin labeling, 2,3-dihydrophthalazinedione, biotin / avidin, spin labeling, and fluorescent or chemiluminescent labeling including stable free radicals may be conjugated. Single-domain antibodies or antigen-binding antibody fragments disclosed herein may also be radiolabeled, for example, 99 Tc, 90 Y, 111 In, 32 P, 14 C, 125 I, 3 H, 131 I, 11 C, 15 O, 13 N, 18 F, 35 S, 51 Cr, 57 To, 226 Ra, 60 Co, 59 Fe, 57 Se, 152 EU, 67 CU, 217 Carbon, 211 At, 212 Pb, 47 Sc, 109 Pd, 234 Th, and 40 K, 157 Gd, 55 Mn, 52 Tr, and 56 It may be conjugated with Fe. In some embodiments, the radionuclide used in the preparation of a radioconjugated single-domain antibody or fragment thereof is 212 Bi, 131 I, 131 In, 90 Y, and 186 It is Re.

[0137] ADCs may be formed using enzymatically active toxins and their fragments, such as diphtheria A chain, unbound active fragments of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modesine A chain, α-sarcin, Aleurites fordii protein, diansin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, geronin, mitogellin, restrictocin, phenomycin, neomycin, and trichothecene. Further examples of cytotoxic or cytotoxic agents include, for example, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracine dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and its analogues or homologs.Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechloretamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine).

[0138] In ADCs, the antibody and therapeutic agent are preferably conjugated via a cleavable linker such as a peptidyl, disulfide, or hydrazone linker. More preferably, the linker is a peptidyl linker, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, Glu, or other peptidyl linkers known to those skilled in the art. ADCs can be prepared as described in U.S. Patent Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publication Nos. WO02 / 096910; WO07 / 038658; WO07 / 051081; WO07 / 059404; WO08 / 083312; and WO08 / 103693; U.S. Patent Application Publication Nos. 20060024317; 20060004081; and 20060247295. These disclosures are incorporated herein by reference.

[0139] Immunoconjugates can also be used to modify certain biological responses, and the drug moiety should not be construed as limited to classical chemotherapeutic agents. For example, the drug moiety may be a protein or polypeptide having a desired biological activity (e.g., lymphokine, tumor necrosis factor, IFNγ, growth factor).

[0140] Immunoconjugates can be prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutareldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., tolyene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies (see, e.g., PCT Publication No. WO94 / 11026).

[0141] Techniques for conjugating such therapeutic portions with antibodies are well-known. For example, Arnon et al. 1985. Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy. In Monoclonal Antibodies And Cancer Therapy. Reisfeld et al. eds. Alan R. Liss, Inc. pp. 243-56; Hellstrom et al. 1987. Antibodies For Drug Delivery. In Controlled Drug Delivery (2nd ed.) Robinson et al. eds., Marcel Dekker, Inc. pp. 623-53; Thorpe. Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review. In Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. eds., pp. 475-506; 1985. Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy. In Monoclonal See Antibodies For Cancer Detection And Therapy, Baldwin et al. eds. Academic Press. pp. 303-16; and Thorpe et al. 1982. The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates. Immunol Rev. 62:119-58.

[0142] Pharmaceutical preparations Furthermore, this disclosure relates to a pharmaceutical composition comprising an sdAb (preferably a therapeutic sdAb), or at least one sdAb, for example, a protein or polypeptide containing at least one therapeutic sdAb, at least one suitable carrier, diluent, or excipient suitable for pharmaceutical use, and optionally one or more further active substances, wherein the sdAb, protein, or polypeptide is an sdAb, protein, or polypeptide further described herein (i.e., an sdAb, protein, or polypeptide that conforms to one or more aspects described herein, in particular an sdAb, protein, or polypeptide that conforms to one or more aspects described on the previous page, and more specifically an sdAb, protein, or polypeptide having a C-terminal / C-terminal sequence that conforms to one or more aspects described herein). Such compositions, carriers, diluents, or excipients may be, for example, the compositions, carriers, diluents, or excipients described in WO08 / 020079 for pharmaceutical compositions comprising a protein or polypeptide containing VHH or at least one sdAb, where the sdAb may be VHH.

[0143] Furthermore, this disclosure relates to an sdAb, or a protein or polypeptide comprising at least one sdAb, for use in the therapy of diseases in humans (e.g., patients requiring such treatment), wherein the VHH, protein, or polypeptide is a VHH, protein, or polypeptide further described herein, for example, in some embodiments, a VHH, protein, or polypeptide that conforms to one or more of the aspects described herein, in other embodiments, a VHH, protein, or polypeptide having a C-terminal / C-terminal sequence that conforms to one or more of the aspects described herein.

[0144] Furthermore, this disclosure relates to the use of an sdAb, or a protein or polypeptide comprising at least one sdAb, in the preparation of a pharmaceutical composition, wherein the sdAb, protein, or polypeptide is an sdAb, protein, or polypeptide further described herein, for example, an sdAb, protein, or polypeptide that conforms to one or more aspects described herein, in some aspects conforming to one or more aspects described on the previous page, or an sdAb, protein, or polypeptide having a C-terminal / C-terminal sequence that conforms to one or more aspects described herein.

[0145] Furthermore, this disclosure relates to a treatment method, comprising the step of administering to a human subject (e.g., a patient requiring such treatment) an sdAb, or a protein or polypeptide containing at least one sdAb, in the preparation of a pharmaceutical composition, wherein the sdAb, protein or polypeptide is an sdAb, protein or polypeptide further described herein, i.e., an sdAb, protein or polypeptide conforming to one or more aspects described herein, in some embodiments, an sdAb, protein or polypeptide conforming to one or more aspects described on the preceding page, or in some embodiments, an sdAb, protein or polypeptide having a C-terminal sequence conforming to one or more aspects described herein, or to a pharmaceutical composition (as described above) containing at least one such sdAb, protein or polypeptide.

[0146] In another aspect, this disclosure relates to the therapeutic use of the sdAbs, proteins, and polypeptides described herein as such therapeutic use (or the clinical development of such sdAbs, proteins, and polypeptides for such therapeutic use), including the preliminary use of an ADA assay to determine whether the sdAbs, proteins, or polypeptides exhibit immunogenicity and may produce ADA when administered to human subjects. In this regard, it is also clear that concerns about possible immunogenism must be addressed when the therapeutic agent is used over a longer period (weeks, months, or years), or when it has a half-life of at least 3 days, e.g., at least 1 week, and up to 10 days, or longer (preferably represented by t1 / 2-β) in human subjects.

[0147] Accordingly, according to a particular aspect of this disclosure, the sdAbs, proteins, polypeptides, or pharmaceutical compositions described herein are intended for the treatment of chronic diseases in humans, and / or such sdAbs, proteins, polypeptides described herein are intended to be present in the circulation of a subject (e.g., at a pharmacologically active level) for a period of at least one week, preferably at least two weeks, for example, at least one month or more (i.e., at a therapeutically active dose), and / or such sdAbs, proteins, polypeptides described herein are intended to be present in a human subject for at least three days, for example, The sdAb, protein, polypeptide having a half-life of at least one week and up to 10 days or longer (e.g., represented by t1 / 2-β), and / or such sdAb protein, polypeptide, or pharmaceutical composition described herein is intended to be administered to humans in two or more doses over a period of at least three days, e.g., at least one week, e.g., at least two weeks, or at least one month, or even longer (e.g., at least three months, at least six months, or at least one year), or in some embodiments, to be administered over a long period.

[0148] Furthermore, this disclosure provides a method for (substantially) reducing or essentially preventing the tendency of sdAb, VHH, or sdAb-based drugs, or VHH-based drugs, to cause immunogenic protein interference, comprising at least (1) optionally, a step of determining the tendency of sdAb, VHH, sdAb-based drugs, or VHH-based drugs to cause protein interference using a method comprising at least steps (i) and (ii) as referred herein, and (2) sdAb or VHH, or sdAb-based drugs or VHH-based drugs The present invention relates to a method comprising the step of modifying an sdAb, VHH, an sdAb-based drug, or a VHH-based drug by introducing one or more amino acid substitutions, additions, or deletions in the C-terminal sdAb or VHH (if any) of the drug, and in some embodiments, by introducing one or more amino acid substitutions or additions in the C-terminal region of the sdAb or VHH, or in the C-terminal region of the sdAb-based drug or a VHH-based drug (if any).

[0149] Humanization In some embodiments, sdAb containing the C-terminal modification described herein can be humanized to include a human framework region. To demonstrate the usefulness of the humanized versions of the VHH component and VHH molecule of sdAb, the humanized versions are prepared and evaluated for binding to target receptors using binding assays such as surface plasmon resonance spectroscopy (Biacore®).

[0150] When humanizing target-binding VHHs, one consideration for humanized VHH design is the amino acid distribution at each position of the non-human framework, suggesting amino acid residues that, when modified, could introduce significant alterations to the protein's secondary and tertiary structures. Examples of human germlines that can be used to create humanized VHHs include, but are not limited to, VH3-23 (e.g., UniProt ID: P01764), VH3-74 (e.g., UniProt ID: A0A0B4J1X5), VH3-66 (e.g., UniProt ID: A0A0C4DH42), VH3-30 (e.g., UniProt ID: P01768), VH3-11 (e.g., UniProt ID: P01762), and VH3-9 (e.g., UniProt ID: P01782). Certain framework residues in the classic camel VHH sequence VH3-66 (Uniprot A0A0C4DH42) include positions V37, G44, L45, and W47, and vernier zone residues R94 and W103 may also be residues that are not easily modified. To identify the amino acids in the highly conserved camel VHH framework region, the VHH sdAb sequence is obtained, and the amino acid distribution at each position is evaluated using an in-house developed R script. The amino acid distribution chart allows for the identification of rare residues at each position.

[0151] ADA test The protein sequences disclosed herein may be tested in direct anti-single-domain antibody (ADA) detection assays to confirm the presence of pre-existing ADA in serum derived from human donors. Generally, pre-existing antibodies in human serum can be identified by ELISA, in which sdAbs are immobilized on plates and blocked with BSA. In some embodiments, human serum (neat or diluted with PBS + 0.01% Tween-20) is then incubated with the plate-bound sdAbs. In some embodiments, unbound serum IgG is washed away, and any remaining antibodies against the sdAbs are detected using anti-human Igκ and anti-human IgλHRP conjugated antibodies.

[0152] Indirect ADA assays are useful for analyzing multiple variants of a particular sdAb to measure sdAb-specific serum antibody reactivity. In some embodiments, indirect ADA involves immobilization of the sdAb and blocking with BSA. Human serum (neat, or diluted with PBS + 0.01% Tween-20) is then pre-incubated with the soluble sdAb (the same sdAb, or a variant of the parent sdAb with the modification described herein) and then added to the plate-bound sdAb. If the soluble sdAb variant can block ADA recognition of the immobilized sdAb, it indicates that the soluble sdAb was recognized by ADA and is therefore an indirect measure of the pre-existing ADA response. Conversely, if the soluble sdAb cannot prevent ADA recognition of the immobilized sdAb, it indicates that the soluble sdAb variant is non-immunogenic or was not recognized by the pre-existing ADA in another manner. Generally, 8 to 16 assays are performed in parallel using serum from separate donors previously confirmed to contain ADA.

[0153] For example, in the clinical development of biological drug molecules, drug molecules are tested to evaluate their immunogenicity, particularly their ability to induce so-called "anti-drug antibodies," or "ADAs." This is confirmed using so-called "anti-drug antibody" or "ADA assays," such as immunoassays (see, for example, the reviews by Shankar et al., 2008. Journal of Pharmaceutical and Biomedical Analysis. 48:1267-1281; and Mire-Sluis et al. 2004. J Immunol Meth. 289:1-16; Peng et al. 2011. J Pharm. and Biomed. Analysis. 54:629-635; and Loyet et al. 2009. J Immunol Meth. 345:17-28). Such ADA assays and methods for performing them are commonplace in the field of pharmacology and are routinely used in the clinical development of biological drug products (and are required by various regulatory authorities worldwide).

[0154] For example, various ADA assay formats are known, such as the "ELISA-bridging format," "ELISA direct format," "indirect format," radioimmunoprecipitation assay (RIP), "surface plasmon resonance," and "electrochemiluminescence-bridging format," as described by Mire-Sluis et al. and Peng et al. Other formats for performing ADA immunoassays are well known to those skilled in the art.

[0155] A variety of commercially available technology platforms suitable for setting up and performing ADA assays can be used. These include, but are not limited to, the MSD platform (Meso Scale Diagnostics), Gyrolab (Gyros), and the octet platform (Fortebio).

[0156] Recently, techniques for predicting, detecting, and reducing nonspecific protein interference were disclosed in assays involving immunoglobulin single variable domains, as disclosed in US20200325221A1. In such methods, various modifications of immunogenic sdAbs were evaluated using ADA assays. Enbrel (TNFR2ECD-Fc) and BSA were used as non-ADA recognition controls. In some methods, purified serum IgG was coated as a positive control of the secondary antibody.

[0157] Indirect ADA assays are sometimes used to demonstrate the effects of multiple modifications to humanized VHH compared to humanized VHH having a native carboxy-terminal sequence, VTVSS (SEQ ID NO:1).

[0158] Diagnostic use The single-domain antibodies described herein may also be used for diagnostic purposes. Such sdAbs can be conjugated with appropriate detectable agents to form immunoconjugates. For diagnostic purposes, appropriate agents include radioisotopes for whole-body imaging, and detectable labels including radioisotopes, enzymes, fluorescent labels, and other appropriate antibody tags for sample testing.

[0159] Detectable labels may be any of the various types currently used in the field of in vitro diagnostics, including particle labels, isotopes, chromophores, fluorescent markers, luminescence markers, metallic labels (e.g., CyTOF, in the case of imaging mass cytometry), phosphorescent markers, as well as enzymatic labels that convert a particular substrate into a detectable marker, and polynucleotide tags that become apparent after amplification, such as by polymerase chain reaction. Suitable enzymatic labels include horseradish peroxidase and alkaline phosphatase. For example, the label may be an alkaline phosphatase detected by measuring the presence or formation of chemiluminescence after conversion of a 1,2-dioxetane substrate, such as adamantylmethoxyphosphoryloxyphenyldioxetane (AMPPD), disodium 3-(4-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.13,7}decane}-4-yl)phenylphosphate (CSPD), as well as CDP and CDP-Star®, or other luminescence substrates well known to those skilled in the art, such as chelates of suitable lanthanides including terbium(III) and europium(III). The means of detection are confirmed by the selected label. The appearance of the label or its reaction product can be observed with the naked eye, or with instruments such as a spectrophotometer, luminometer, or fluorometer, in accordance with standard practice, if the label is a particle and accumulates at an appropriate level.

[0160] Preferably, the conjugation method yields non-immunogenic or reduced immunogenic linkages, such as peptide-(i.e., amide-), sulfide-, (sterically hindered), disulfide-, hydrazone-, and ether linkages. These linkages are nearly non-immunogenic and exhibit reasonable stability in serum (see, e.g., Senter, PD, 2009. Curr Opin Chem Biol. 13:235-244; and PCT publication numbers WO2009 / 059278 and WO95 / 17886).

[0161] Depending on the biochemical properties of the aforementioned moiety and antibody, various conjugate strategies can be used. If the moiety is natural or a recombinant of 50-500 amino acids, there are standard procedures in textbooks on the chemistry for synthesizing protein conjugates that can be easily modeled by those skilled in the art (see, for example, Hackenberger, CPR, and Schwarzer, D. 2008. Angew Chem Int Ed Engl. 47:10030-10074). In one embodiment, a reaction is used between the maleinimido moiety and a cysteine ​​residue within the antibody or moiety. This is a particularly suitable coupling chemistry when the Fab or Fab'-fragment of the antibody is used.

[0162] Generally, site-directed reactions and covalent couplings are based on converting native amino acids into amino acids that have orthogonal reactivity with respect to the reactivity of other functional groups present. For example, certain cysteines within rare contexts can be enzymatically converted in aldehydes (see Frese MA and Dierks T. 2009. Chem Bio Chem. 10:425-427). Desired amino acid modifications can also be obtained by utilizing the specific enzymatic reactivity between a particular enzyme and the native amino acids in the sequence (see, for example, Taki M et al. 2004. Prot Eng Des Sel. 17:119-126; Gautier A. et al. 2008. Chem Biol. 15:128-136; and Protease-catalyzed formation of CN bonds is used by Bordusa, F. 2008. Highlights in Bioorganic Chemistry pp389-403). Site-directed reactions and covalent coupling can also be achieved through selective reactions between terminal amino acids and appropriate modifying reagents. Site-directed covalent coupling can be achieved using the reactivity of N-terminal cysteine ​​with benzonitrile (see Ren H. et al. 2009. Angew Chem Int Ed Engl. 48:9658-9662).

[0163] The aforementioned portion may also be a synthetic peptide or peptide mime. When polypeptides are chemically synthesized, amino acids with orthogonal chemical reactivity can be incorporated during such synthesis (see, for example, de Graaf AJ et al. 2009. Bioconjug Chem. 20:1281-1295). A wide variety of orthogonal functional groups are at issue and can be introduced into synthetic peptides, so the conjugation of such peptides with linkers is standard chemistry.

[0164] In other embodiments, the present invention relates to a pharmaceutical composition comprising a single-domain variable antibody (sdAb) having a modified amino acid sequence corresponding to the C-terminal endogenous sdAb amino acid residue described herein, at least one suitable carrier, diluent, or excipient (i.e., suitable for pharmaceutical use), and optionally one or more further active substances. Such a composition, carrier, diluent, or excipient may be, for example, as described in WO08 / 020079 for a pharmaceutical composition comprising an sdAb protein or polypeptide containing at least one VH or VHH polypeptide.

[0165] In other embodiments, the present invention relates to a pharmaceutical composition comprising a single-domain variable antibody (sdAb) having a modified amino acid sequence corresponding to the C-terminal endogenous sdAb amino acid residue described herein, at least one suitable carrier, diluent, or excipient (i.e., suitable for pharmaceutical use), and optionally one or more further active substances for use in the therapy of a disease.

[0166] In other embodiments, the present invention relates to a pharmaceutical composition comprising a treatment method, which includes administering a pharmaceutical composition comprising a single-domain variable antibody (sdAb) described herein, or at least one such sdAb, protein, or polypeptide, to a human subject (for example, a patient requiring such treatment).

[0167] In relation to the above, it is clear that the therapeutic use of the sdAbs, proteins, and polypeptides described herein is a very important aspect of the present invention. This is because such therapeutic use (or the clinical development of such sdAbs, proteins, and polypeptides for such therapeutic use) may involve the use of ADA assays to determine whether the sdAb, protein, or polypeptide is immunogenic, or whether its immunogenicity against endogenous pre-existing antibodies has been reduced or eliminated (i.e., whether it can produce ADA when administered to human subjects). In this regard, it is also clear that particular concerns must be addressed regarding potential immunogenicity when the therapeutic agent is used over a longer period (weeks, months, or years) and / or when it has a half-life of at least 3 days, e.g., at least 1 week, and up to 10 days, or longer (preferably represented by t1 / 2-β), in human subjects.

[0168] Accordingly, according to a particular aspect of the present invention, the sdAb, protein, polypeptide, or pharmaceutical composition described herein is intended to treat a chronic disease in humans, and / or such sdAb, protein, polypeptide described herein is intended to be present in the circulation of a subject (i.e., at a pharmacologically active level) for a period of at least one week, preferably at least two weeks, for example, at least one month (i.e., at a therapeutically active dose), and / or such sdAb, protein, polypeptide described herein is intended to be present in a human subject for at least three days, for example. For example, an sdAb, protein, polypeptide having a half-life of at least one week and up to 10 days or longer (e.g., represented by t1 / 2-β), and / or such sdAb proteins, polypeptides, or pharmaceutical compositions described herein are administered to humans in two or more doses over a period of at least three days, e.g., at least one week, e.g., at least two weeks, or at least one month, or even longer (i.e., at least three months, at least six months, or at least one year), or are intended for long-term administration.

[0169] Recombinant production In another aspect, the sdAb molecules of this disclosure are produced by recombinant DNA technology. In a typical implementation of polypeptide recombinant production, a nucleic acid sequence encoding the desired polypeptide is incorporated into an expression vector suitable for the host cell in which expression is to be performed. This nucleic acid sequence is functionally ligated to one or more expression regulatory sequences encoded by the vector and functions in the target host cell. The recombinant protein may be recovered by disrupting the host cell, or it may be recovered from the cell culture medium if a secretion leader sequence (signal peptide) is incorporated into the polypeptide.

[0170] In some embodiments, sdAb molecules are produced by a recombinant method using a nucleic acid sequence encoding an sdAb molecule (or a fusion protein containing an sdAb molecule). The nucleic acid sequence encoding the desired sdAb molecule can be synthesized by chemical means using an oligonucleotide synthesizer.

[0171] Nucleic acid molecules are not limited to sequences that encode polypeptides. They may also include some or all of the non-coding sequences upstream or downstream of the coding sequence. Those skilled in molecular biology are familiar with routine procedures for isolating nucleic acid molecules. For example, nucleic acid molecules can be produced by treating genomic DNA with restriction endonucleases or by performing polymerase chain reactions (PCR). If the nucleic acid molecule is ribonucleic acid (RNA), the molecule can be produced, for example, by in vitro transcription.

[0172] Nucleic acid molecules encoding sdAb molecules (and their fusions) may differ from those occurring in nature, but may contain natural sequences encoding the same polypeptide due to genetic code degeneracy. These nucleic acid molecules may consist of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA, e.g., those produced by phosphoramidite-based synthesis), or combinations or modifications of nucleotides within these types of nucleic acids. Furthermore, nucleic acid molecules may be double-stranded or single-stranded (i.e., either sense strand or antisense strand).

[0173] Nucleic acid sequences encoding sdAb molecules may be obtained from various commercial suppliers that provide custom-ordered nucleic acid sequences. Amino acid sequence variants of the sdAb molecules of this disclosure are prepared by introducing appropriate nucleotide changes into the coding sequence based on the genetic code well known in the art. Such variants are insertions, substitutions, and / or specified deletions of residues, as mentioned herein. Any combination of insertions, substitutions, and / or specified deletions is added to arrive at the final construct, provided that the final construct has the desired biological activity as defined herein.

[0174] Methods for constructing the DNA sequence encoding the sdAb molecule and expressing that sequence in a properly transformed host include, but are not limited to, the use of PCR-assisted mutagenesis. Mutations consisting of deletions or additions of amino acid residues to the sdAb molecule can also be introduced using standard recombination methods. If deletions or additions occur, the nucleic acid molecule encoding the sdAb molecule is optionally digested with an appropriate restriction endonuclease. The resulting fragment may be expressed directly or further manipulated, for example, by ligation to a second fragment. Ligation may be easier if the two ends of the nucleic acid molecule contain complementary nucleotides that overlap, but blunt-end fragments can also be ligated. Various mutant sequences can also be constructed using nucleic acids generated by PCR.

[0175] The sdAb molecules of this disclosure may be produced not only directly by recombination, but also as fusion polypeptides with heterologous polypeptides, such as a signal sequence, or other polypeptides having specific cleavage sites at the N-terminus or C-terminus of a mature sdAb molecule. Generally, the signal sequence may be a component of the vector or part of a coding sequence inserted into the vector. The selected heterologous signal sequence is preferably recognized and processed by the host cell (i.e., cleaved by a signal peptidase). The incorporation of the signal sequence depends on whether it is desirable for the recombinant cell from which the sdAb molecule is produced to secrete the sdAb molecule. If the selected cell is a prokaryote, it is generally preferable that the DNA sequence does not code for the signal sequence. When the recombinant host cell is a yeast cell such as Saccharomyces cerevisiae, an α-conjugation factor secretion signaling sequence may be used to cause sdAb molecules to be secreted extracellularly into the culture medium, as described in Singh, U.S. Patent No. 7,198,919B1, published April 3, 2007.

[0176] If the sdAb molecule to be expressed is expressed as a chimeric protein (e.g., a fusion protein containing the sdAb molecule and a heterologous polypeptide sequence), the chimeric protein may be encoded by a hybrid nucleic acid molecule containing a first sequence encoding all or part of the sdAb molecule and a second sequence encoding all or part of the heterologous polypeptide. For example, the sdAb molecule described herein may be fused with a hexahistidine / octahistidine tag to facilitate the purification of the protein expressed by bacteria, or with a hemagglutinin tag to facilitate the purification of the protein expressed in eukaryotic cells. The first and second should not be understood as limitations on the orientation of the elements of the fusion protein, as the heterologous polypeptide can be ligated to either the N-terminus and / or C-terminus of the sdAb molecule. For example, the N-terminus may be ligated to a targeting domain, and the C-terminus may be ligated to a hexahistidine tag purification handle.

[0177] A back-translated gene can be constructed using the complete amino acid sequence of the polypeptide (or fusion / chimera) to be expressed. DNA oligomers containing the nucleotide sequence encoding the sdAb molecule can be synthesized. For example, several small oligonucleotides encoding a portion of the desired polypeptide can be synthesized and then ligated together. Individual oligonucleotides typically contain a 5' or 3' overhang for complementary assembly.

[0178] In some embodiments, the nucleic acid sequence encoding the sdAb molecule may be “codon-optimized” to facilitate expression in a particular host cell type. Techniques for codon optimization in a wide variety of expression systems, including mammalian host cells, yeast host cells, and bacterial host cells, are well known in the art, and online tools for providing codon-optimized sequences for expression in various host cell types are publicly available. For example, see Hawash, et al., (2017) 9:46-53, and Mauro and Chappell in Recombinant Protein Expression in Mammalian Cells: Methods and ProtocolsSee David Hacker (Human Press New York) for further information. Additionally, there are various web-based online software packages freely available to assist in the preparation of codon-optimized nucleic acid sequences.

[0179] Once assembled (by synthesis, site-directed mutagenesis, or other means), the nucleic acid sequence encoding the sdAb molecule is inserted into the expression vector. Various expression vectors are available for use in different host cells and are typically selected based on the host cell for expression. An expression vector typically includes, but is not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Vectors include viral vectors, plasmid vectors, and embedded vectors. Plasmids are an example of a non-viral vector.

[0180] To facilitate the efficient expression of recombinant polypeptides, the nucleic acid sequence encoding the polypeptide sequence to be expressed is functionally ligated to transcriptional and translational regulatory sequences that function in the selected expression host.

[0181] Expression vectors typically contain a selection gene, also known as a selection marker. This gene encodes a protein necessary for the survival or proliferation of transformed host cells grown in a selective culture medium. Host cells not transformed with a vector containing the selection gene will not survive in the culture medium. Typical selection genes encode (a) proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) proteins that compensate for nutritional deficiencies; or (c) proteins that supply essential nutrients unavailable from the complex medium.

[0182] The expression vectors for sdAb molecules described herein contain a regulatory sequence that is recognized by a host organism and functionally linked to a nucleic acid sequence encoding the sdAb molecule. The terms “regulatory sequence,” “regulatory sequence,” or “expression regulatory sequence” are used herein synonymously to refer to promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals). For example, see Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, CA, USA). Regulatory sequences include those that induce constitutive expression of nucleotide sequences in many types of host cells, and those that induce expression of nucleotide sequences only in certain host cells (e.g., tissue-specific regulatory sequences). It is understood by those skilled in the art that the design of expression vectors may depend on factors such as the selection of host cells to be transformed and the desired level of protein expression. In selecting an expression regulatory sequence, various factors understood by those skilled in the art must be considered. These include, for example, the relative strength of the sequence, its controllability, and, in particular, its compatibility with the actual DNA sequence encoding the sdAb molecule, with respect to its potential secondary structure.

[0183] In some embodiments, the regulatory sequence is a promoter, and the promoter is selected, for example, based on the cell type to which expression is desired. A promoter is an uncoding sequence located upstream (5') of the start codon of a structural gene (typically within about 100–1000 bp) that controls the transcription and translation of a specific functionally linked nucleic acid sequence. Such promoters are typically divided into two classes: inductive promoters and constitutive promoters. Inductive promoters are promoters that initiate high levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of nutrients or temperature changes. Many promoters recognized by various potential host cells are well known.

[0184] The T7 promoter can be used in bacteria, the polyhedrin promoter in insect cells, and the cytomegalovirus promoter or metallothionein promoter in mammalian cells. Similarly, in higher eukaryotes, tissue-specific and cell-type-specific promoters are widely available. These promoters are so named because of their ability to induce the expression of nucleic acid molecules in their respective tissues or cell types within the body. Those skilled in the art are well aware of the many promoters and other regulatory elements that can be used to induce nucleic acid expression.

[0185] Transcription from a vector in mammalian host cells may be controlled by promoters obtained from the genomes of viruses, e.g., polyomavirus, fowlpox virus, adenovirus (e.g., human adenovirus serotype 5), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus (e.g., mouse stem cell virus), hepatitis B virus, most preferably Simian virus 40 (SV40), heterozoan promoters, e.g., actin promoter, PGK (phosphoglycerate kinase), or immunoglobulin promoter, or heat shock promoter, if such promoters are compatible with the host cell line. Conveniently, the early and late promoters of the SV40 virus can be obtained as SV40 restriction fragments that also contain the SV40 virus origin of replication.

[0186] Transcription in higher eukaryotes is often increased by inserting enhancer sequences into vectors. Enhancers are typically cis-acting DNA elements of about 10–300 bp that act on promoters to increase transcription. Enhancers have been found to be relatively directional and position-independent, located 5' and 3' relative to the transcription unit, within introns, and even within the coding sequence itself. Many enhancer sequences derived from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin) are now known. However, enhancers derived from eukaryotic viruses are commonly used. Examples include the SV40 enhancer located late at the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer located late at the origin of replication, and the adenovirus enhancer. Enhancers may be spliced ​​and placed at the 5' or 3' side of the coding sequence in the expression vector, preferably located 5' from the promoter. Expression vectors used in eukaryotic host cells also contain sequences necessary for transcription termination and mRNA stabilization. Such sequences can generally be obtained from the 5' untranslated region, and sometimes the 3' untranslated region, of eukaryotic or viral DNA or cDNA. Standard techniques are used to construct appropriate vectors containing one or more of the components listed above.

[0187] In addition to sequences that facilitate the transcription of the inserted nucleic acid molecule, the vector may also contain an origin of replication and other genes encoding selection markers. For example, the neomycin resistance (neoR) gene confers G418 resistance to cells expressing the neomycin resistance (neoR) gene, thus enabling phenotypic selection of transfected cells. Further examples of marker or reporter genes include β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding β-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT). Those skilled in the art can easily determine whether a particular regulatory element or selection marker is suitable for use in a particular experimental context.

[0188] The correct assembly of the expression vector can be confirmed by nucleotide sequencing, restriction enzyme mapping, and expression of a biologically active polypeptide in a suitable host.

[0189] Furthermore, this disclosure provides prokaryotic or eukaryotic cells containing and expressing a nucleic acid molecule encoding the sdAb molecule. The cells of this disclosure are transfected cells, i.e., cells into which a nucleic acid molecule, such as a nucleic acid molecule encoding a mutant IL-2 polypeptide, has been introduced by recombinant DNA. Progeny of such cells are also considered to be within the scope of this disclosure.

[0190] Host cells are typically selected according to their compatibility with the selected expression vector, the toxicity and secretory properties of the product encoded by the DNA sequence of the present invention, their ability to correctly fold polypeptides, fermentation or culture requirements, and the ease of purifying the product encoded by the DNA sequence. Suitable host cells for cloning or expressing DNA in a vector as used herein are prokaryotes, yeasts, or higher eukaryotic cells.

[0191] In some embodiments, recombinant sdAb molecules can also be produced in eukaryotes such as yeast or human cells. Suitable eukaryotic host cells include insect cells (examples of baculovirus vectors usable for protein expression in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39)); yeast cells (examples of vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, This includes pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187:195)); or mammalian cells (mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187:195)).

[0192] Examples of useful mammalian host cell lines include mouse L cells (LM[TK-], ATCC#CRL-2648), monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651); human fetal kidney cells (HEK293 cells or HEK293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR(CHO); mouse Sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); and human lung cells (W138, ATCC CCL 34). 75); human liver cells (Hep G2, HB 8065); mouse mammary gland tumors (MMT 060562, ATCC CCL51); TRI cells; MRC5 cells; FS4 cells; and human hepatome strain (HepG2). In mammalian cells, the regulatory function of expression vectors is often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and Simianvirus 40.

[0193] The sdAb molecule may be produced in a prokaryotic host such as the bacterium Escherichia coli, or in a eukaryotic host such as insect cells (e.g., Sf21 cells) or mammalian cells (e.g., COS cells, NIH3T3 cells, or HeLa cells). These cells are available from many suppliers, including the American Type Culture Collection (Manassas, Va.). When selecting an expression system, the only issue is that the components are compatible with each other. Those skilled in the art can make such a decision. Furthermore, if guidance is needed when selecting an expression system, those skilled in the art can consult Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, New York, NY, 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987).

[0194] In some embodiments, the resulting sdAb molecules may or may not be glycosylated, depending on the host organism used to produce mutein. If bacteria are selected as the host cell, the produced sdAb molecules will not be glycosylated. On the other hand, eukaryotic cells typically glycosylate sdAb molecules.

[0195] In some embodiments, the amino acid sequence of sdAb incorporated into the sdAb molecule (particularly the CDR sequence) may contain a glycosylation motif, in particular an N-linked glycosylation motif of the sequence Asn-X-Ser(NXS) or Asn-X-Thr(NXT), where X is any amino acid other than proline. In such cases, it is desirable to eliminate the N-linked glycosylation motif by modifying the sequence of such an N-linked glycosylation motif to inhibit glycosylation. In some embodiments, the N-linked glycosylation motif is disrupted by incorporating a conserved amino acid substitution of the Asn(N) residue of the N-linked glycosylation motif.

[0196] For further expression systems for prokaryotic and eukaryotic cells, see Chapters 16 and 17 of Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY). See also Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.).

[0197] The expression constructs disclosed herein can be introduced into host cells to produce the sdAb molecules disclosed herein. An expression vector containing a nucleic acid sequence encoding the sdAb molecule is introduced into prokaryotic or eukaryotic host cells by conventional transformation or transfection methods. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY) and other standard molecular biology laboratory manuals. To facilitate transfection of target cells, target cells may be directly exposed with a non-viral vector under conditions that facilitate the uptake of the non-viral vector. Examples of conditions that facilitate the uptake of foreign nucleic acids by mammalian cells are well known in the art and include, but are not limited to, chemical means (e.g., Lipofectamine®, Thermo-Fisher Scientific), high salt levels, and magnetic fields (electroporation).

[0198] Cells may be cultured in conventional nutrient media, which may be modified as appropriate for promoter induction, transformant selection, or amplification of genes encoding desired sequences. Mammalian host cells can be cultured in a variety of media. Suitable commercially available media for culturing host cells include Ham's F10 (Sigma), Minimum Essential Medium ((MEM), Sigma), RPMI1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma). To any of these media, hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphates), buffers (e.g., HEPES), nucleosides (e.g., adenosine and thymidine), antibiotics, trace elements, and glucose or equivalent energy sources may be added as needed. Any other necessary supplements may also be included in appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature and pH, have been previously used with host cells selected for expression and are apparent to those skilled in the art.

[0199] If a secretion leader sequence is used, the recombination-produced sdAb molecular polypeptide can be recovered from the culture medium as a secretion polypeptide. Alternatively, the sdAb molecular polypeptide can also be recovered from host cell lysates. During purification, protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) may be used during the recovery from cell lysates to inhibit proteolysis, and antibiotics may be included to prevent the growth of exogenous contaminants.

[0200] Various purification processes, such as affinity chromatography, are known and used in the art. Affinity chromatography typically utilizes highly specific binding sites present in biological macromolecules to separate molecules capable of binding to specific ligands. The ligand is covalently attached to an insoluble porous support medium in such a way that the ligand is explicitly presented on the protein sample, thereby separating and purifying a second species from the mixture using the innate specific binding of one molecular species. Antibodies are typically used in affinity chromatography. Size selection processes may also be used to separate proteins according to their size, for example, gel filtration chromatography (also known as size exclusion chromatography or molecular sieve chromatography). In gel filtration, a protein solution is passed through a column packed with a semipermeable porous resin. The semipermeable resin has a range of pore sizes that determines the size of the proteins that can be separated by the column.

[0201] Recombinant sdAb molecules from transformed hosts can be purified according to any suitable method. Recombinant sdAb molecules may be isolated from inclusion bodies produced in E. coli using cation exchange, gel filtration, and / or reverse-phase liquid chromatography, or from conditioned mediums derived from mammalian or yeast cultures producing a particular mutaine. Substantially purified forms of recombinant sdAb molecules can be purified from expression systems using routine biochemical procedures and used, for example, as described herein as therapeutic agents.

[0202] In some embodiments in which the sdAb molecule is expressed with a purification tag as discussed above, this purification handle may be used to isolate the sdAb molecule from cell lysates or cell culture media. When the purification tag is a chelated peptide, methods for isolating such molecules using immobilized metal affinity chromatography are well known in the art. See, for example, Smith et al., U.S. Patent No. 4,569,794.

[0203] The biological activity of the recovered sdAb molecules can be assayed for activity by any suitable method known in the art, and may be evaluated in a substantially purified form, or as part of a cell lysate or cell culture medium when a secretion leader sequence is used for expression.

[0204] Pharmaceutical preparations In another context, the sdAb molecules described herein (and / or nucleic acids encoding the sdAb molecules, or recombinant cells incorporating nucleic acid sequences and modified to express the sdAb molecules) can be incorporated into compositions comprising a pharmaceutical composition. Such compositions typically comprise the polypeptide or nucleic acid molecule and a pharmaceutically acceptable carrier. The pharmaceutical composition is formulated to be compatible with its intended route of administration, and the sdAb molecules are compatible with therapeutic use in which they are administered to subjects requiring treatment or prevention.

[0205] The carrier may include a sterile diluent, such as water for injection, saline solution, non-volatile oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvent. The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate fluidity can be maintained by using a surfactant, such as sodium dodecyl sulfate, by using a coating such as lecithin, or, in the case of a dispersion, by maintaining the required particle size. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS).

[0206] The term buffer solution includes buffering agents such as acetates, citrates, or phosphates, and agents for adjusting the tonicity, such as sodium chloride or dextrose. The pH can be adjusted using an acid or base, such as sodium dihydrogen phosphate and / or sodium hydrogen phosphate, hydrochloric acid, or sodium hydroxide (for example, to a pH of 7.2–7.8, e.g., 7.5).

[0207] Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components derived from those listed above. For sterile powders for sterile injection preparation, preferred preparation methods are vacuum drying and freeze-drying of the powder of the active ingredient and any further desired components from its previously sterile filtered solution.

[0208] Pharmaceutical preparations for parenteral administration to subjects must be sterile and fluid to facilitate syringability. They must be stable under manufacturing and storage conditions and stored in a manner that prevents contamination. Microbial activity can be inhibited by various antimicrobial and antifungal agents, e.g., drugs such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; and chelating agents such as ethylenediaminetetraacetic acid, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. Sterile solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent, along with one or a combination of the components listed above as needed, and then filtration by sterile filtration.

[0209] In many cases, it is preferable to include an isotonic agent in the composition, such as sugar, polyhydric alcohol, such as mannitol, sorbitol, or sodium chloride.

[0210] Administration Some aspects of the therapeutic methods of the present disclosure involve administering a pharmaceutical formulation containing an sdAb molecule (and / or a nucleic acid encoding an sdAb molecule, or a recombinantly modified host cell expressing an sdAb molecule) to a subject requiring treatment. The pharmaceutical formulations containing the sdAb molecule of the present disclosure may be administered to a subject requiring treatment or prevention by various routes of administration, including parenteral administration, oral routes, local routes, or inhalation routes.

[0211] In some embodiments, the methods of the present disclosure involve parenteral administration of a pharmaceutical formulation containing an sdAb molecule (and / or a nucleic acid encoding an sdAb molecule, or a recombinantly modified host cell expressing an sdAb molecule) to a subject requiring treatment. Examples of parenteral administration routes include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), transmucosal, and rectal administration. Parenteral formulations, including solutions or suspensions used for parenteral applications, may include a vehicle, a carrier, and a buffer. Pharmaceutical formulations for parenteral administration include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for immediate preparation of sterile injections or dispersions. Parenteral preparations may be contained in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. In one embodiment, the formulation is provided in a pre-filled syringe.

[0212] In some embodiments, the methods of the present disclosure involve the oral administration of a pharmaceutical formulation containing an sdAb molecule (and / or a nucleic acid encoding an sdAb molecule, or a recombinantly modified host cell expressing an sdAb molecule) to a subject requiring treatment. If an oral composition is used, it generally includes an inert diluent or an edible carrier. For oral therapeutic administration, the active compound can be incorporated with an excipient and used in the form of tablets, lozenges, or capsules, such as gelatin capsules. The oral composition may also be prepared using a fluid carrier for use as an oral rinse. A pharmaceutically suitable binder and / or adjuvant material may be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients: binders, e.g., crystalline cellulose, tragacanth gum, or gelatin; excipients, e.g., starch or lactose; disintegrants, e.g., alginic acid, Primogel®, or corn starch; lubricants, e.g., magnesium stearate or Sterotes®; fluidizers, e.g., colloidal silicon dioxide; sweeteners, e.g., sucrose or saccharin; or flavorings, e.g., peppermint, methyl salicylate, or orange flavor, or any compound of similar properties.

[0213] In some embodiments, the methods of the present disclosure involve the inhalation administration of a pharmaceutical formulation containing an sdAb molecule (and / or a nucleic acid encoding the sdAb molecule, or a recombinantly modified host cell expressing the sdAb molecule) to a subject requiring treatment. In the case of inhalation administration, the sdAb molecule, or the nucleic acid encoding them, is delivered in the form of an aerosol spray from a suitable spray, such as a pressurized container or dispenser containing a gas, such as carbon dioxide, or from a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798.

[0214] In some embodiments, the methods of the present disclosure involve mucosal or transdermal administration of a pharmaceutical formulation containing an sdAb molecule (and / or a nucleic acid encoding an sdAb molecule, or a recombinantly modified host cell expressing an sdAb molecule) to a target requiring treatment. In the case of mucosal or transdermal administration, a permeation agent suitable for the barrier to be penetrated is used in the formulation. Such permeation agents are generally known in the art and, for example, in the case of mucosal administration, include surfactants, bile salts, and fusidic acid derivatives. Mucosal administration can be achieved using nasal sprays or, in the case of rectal delivery, suppositories (e.g., conventional suppository bases, e.g., suppositories or retained enemas using cocoa butter and other glycerides). In the case of transdermal administration, the active compound is formulated in the form of an ointment, plaster, gel, or cream, as is generally known in the art, and may incorporate a permeation enhancer such as ethanol or lanolin.

[0215] In some embodiments of the methods of this disclosure, the sdAb molecule is administered to a subject requiring treatment in the form of a formulation for long-term release of the sdAb molecular agent. Examples of long-term release formulations of injectable compositions can be achieved by including an absorption-delaying agent, such as aluminum monostearate and gelatin, in the composition. In one embodiment, the sdAb molecule or nucleic acid is prepared together with a sustained-release formulation comprising a carrier, such as a graft and a microencapsulation delivery system, that protects the sdAb molecule from rapid elimination from the body. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such formulations can be prepared using standard techniques. These materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (containing liposomes targeted to infected cells using monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared, for example, by methods known to those skilled in the art, as described in U.S. Patent No. 4,522,811.

[0216] In some aspects of the methods of this disclosure, delivery of the sdAb molecule to a subject requiring treatment is achieved by administration of a nucleic acid encoding the sdAb molecule. Methods for administering the nucleic acid encoding the sdAb molecule to a subject include, but are not limited to, the methods described by McCaffrey et al. (Nature (2002) 418:6893), Xia et al. (Nature Biotechnol. (2002) 20:1006-1010), or Putnam (Am. J. Health Syst. Pharm. (1996) 53: 151-160 erratum at Am. J. Health Syst. Pharm. (1996) 53:325), and are achieved by transfection or infection using methods known in the art. In some embodiments, the sdAb molecule is administered to a subject by administering a pharmaceutically acceptable formulation of a recombinant expression vector containing a nucleic acid sequence encoding the sdAb molecule, functionally linked to one or more expression regulatory sequences operable in mammalian subjects. In some embodiments, expression regulatory sequences operable in a limited range of cell types (or single cell types) may be selected to facilitate the selective expression of the sdAb molecule in a specific target cell type. In one embodiment, the recombinant expression vector is a viral vector. In some embodiments, the recombinant vector is a recombinant viral vector. In some embodiments, the recombinant viral vector is a recombinant adeno-associated virus (rAAV) or recombinant adenovirus (rAd), in particular a replication-deficient adenovirus derived from human adenovirus serotype 3 and / or 5. In some embodiments, the replication-deficient adenovirus has one or more modifications to the E1 region that interfere with the virus's ability to initiate the cell cycle and / or apoptotic pathway in human cells. The replication-deficient adenovirus vector may optionally include a deletion in the E3 domain. In some embodiments, the adenovirus is a replication-competent adenovirus. In some embodiments, the adenovirus is a replication-competent recombinant virus that has been engineered to selectively replicate in a target cell type.

[0217] In some embodiments, particularly when administering sdAb molecules to a target, especially to treat intestinal diseases or bacterial infections in the target, the nucleic acid encoding the sdAb molecule may be delivered to the target by administration of a recombinantly modified bacteriophage vector encoding the sdAb molecule. As used herein, the terms “prokaryotic virus,” “bacteriophage,” and “phage” are used synonymously to describe any variety of bacterial viruses that infect bacteria and replicate within them. Bacteriophages selectively infect prokaryotic cells, and therefore, the expression of the sdAb molecule is restricted to prokaryotic cells in the target, while expression in mammalian cells is avoided. A wide variety of bacteriophages capable of selectively infecting a broad range of bacterial cells have been identified and extensively characterized in the scientific literature. In some embodiments, phages are modified to remove adjacent motifs (PAMs). Removing the Cas9 sequence from the phage genome reduces the ability of target prokaryotic cells' Cas9 endonucleases to neutralize invading phages encoding SdAb molecules.

[0218] In some embodiments of the methods described herein, delivery of the sdAb molecule to a subject requiring treatment is achieved by administering recombinant host cells modified to express the sdAb molecule administered in the therapeutic and prophylactic uses described herein. In some embodiments, the recombinant host cells are mammalian cells, such as human cells.

[0219] In some embodiments, the nucleic acid sequence encoding the sdAb molecule (or a vector containing it) may be maintained extrachromosomally in a host cell modified by recombination for administration. In other embodiments, the nucleic acid sequence encoding the sdAb molecule may be incorporated into the genome of the host cell to be administered using at least one endonuclease to facilitate the insertion of the nucleic acid sequence into the cell's genomic sequence. As used herein, the term “endonuclease” refers to a wild-type or variant enzyme capable of catalyzing the cleavage of nucleic acid bonds within a DNA or RNA molecule, preferably a DNA molecule. An endonuclease is called a “rare-cutting” endonuclease when it has a polynucleotide recognition site that is more than about 12 base pairs (bp), more preferably 14–55 bp in length. Rare-cutting endonucleases may be used to inactivate a gene at a locus, or to incorporate a transgene by homologous recombination (HR), i.e., by inducing a DNA double-strand break (DSB) at the locus, and by gene repair mechanisms to insert exogenous DNA into this locus. Examples of rare-cutting endonucleases include homing endonucleases (Grizot, et al (2009) Nucleic Acids Research 37(16):5405-5419), chimeric zinc finger nucleases (ZFNs) resulting from the fusion of manipulated zinc finger domains (Porteus M and Carroll D., Gene targeting using zinc finger nucleases (2005) Nature Biotechnology 23(3):967-973), TALEN nucleases, Cas9 endonucleases derived from the CRISPR system, or modified restriction endonucleases for extended sequence specificity (Eisenschmidt, et al. 2005; 33(22): 7039-7047).

[0220] In some embodiments, particularly when administering sdAb molecules to the intestinal tract, sdAb may be delivered to the target by recombinantly modified prokaryotic cells (e.g., Lactobacillus lacti). The use of engineered prokaryotic cells to deliver recombinant proteins to the intestinal tract is known in the art. See, for example, Lin et al. 2017. Microb Cell Fact 16:148. In some embodiments, engineered bacterial cells expressing sdAb may be administered orally, typically dissolved in an aqueous suspension, or rectally (e.g., by enema).

[0221] How to use Furthermore, the present disclosure provides a method for treating a subject suffering from a disease disorder or condition by administering a therapeutically effective amount of the present disclosure of sdAb molecules (or nucleic acids encoding sdAb, including recombinant viruses encoding sdAb).

[0222] The following examples illustrate the use of the VTVSS (SEQ ID NO:1) variant of this disclosure, exemplified by the use of the IL10Ra-IL10Rb VHH construct as a vehicle for modifying the endogenous VTVSS (SEQ ID NO:1) motif. [Examples]

[0223] Example 1 - Existing MSD method for detecting human anti-VHH anti-drug antibodies (ADA): To evaluate the effect of the amino acid substitutions of this disclosure on reducing immunogenicity, the IL10Ra / IL10Rb VHH dimers with SEQ ID NO: 49-59 were used as test materials in the following experiments. The polypeptide molecules listed in Table 11 below were used in the following experiments. The reference sequence DR2485aa (SEQ ID NO: 49) retains the characteristic wild-type VTVSS (SEQ ID NO: 1) C-terminal motif, while the derivative molecules incorporate various amino acid substitutions that reduce immunogenicity, as can be demonstrated for the amino acid sequences shown above.

[0224] (Table 11) IL10Ra / IL10Rb VHH test articles and the corresponding nucleic acids encoding them TIFF2026521433000040.tif111147

[0225] The IL10Ra / IL10Rb VHH dimers described above were produced by recombination using the corresponding DNA sequences shown in the horizontal rows of Table 11 above. Briefly, the nucleic acids encoding the IL10Ra / IL10Rb VHH test items listed in Table XXx further incorporated a 5' sequence encoding a mouse IgH signal peptide (Uniprot Reference: Q99LA6) upstream of the IL10Ra / IL10Rb VHH dimer polypeptide encoding sequence. The signal peptide and the nucleic acid sequences encoding the IL10Ra / IL10Rb VHH dimer were inserted downstream of the CMV promoter in the pExSYN2.0 plasmid, a derivative of the pcDNA3.4 (ThermoFisher) expression vector, using EcoR1 and Not1 restriction endonucleases. The resulting expression vector was used to transfect expi293 cells, which were then cultured in Expi293 expression medium (ThermoFisher Scientific) for 96 hours. This protein was purified by affinity chromatography using MabSelect® VH3 resin (Cytiva).

[0226] The VHH dimer produced according to the above procedure was diluted to 5 ug / mL with phosphate-buffered saline (PBS, Gibco, catalog number 10010-031) and transferred to the wells of a standard 96-well multi-array MSD plate (Meso Scale Discovery, catalog number L15XA-3). The plate was placed on a plate shaker and shaken briefly at room temperature, then transferred to 4C and incubated overnight to coat the plate. Some wells were left empty as a negative control. After incubation, all plate wells were washed three times with 1x TRIS-buffered saline (TBS [20x], Thermo Scientific Pierce, catalog number P128258). The wells were then blocked with a PBS solution containing 5% w / v bovine serum albumin (BSA, BoldBio, catalog number A-421-50) and incubated at room temperature for 2 hours with shaking. The plate was washed again as described above.

[0227] During incubation, human serum samples (BioIVT, catalog number HUMANSRM-0101139) derived from each individual donor were separately diluted to 5% serum in 1% w / v PBS BSA solution. The pooled human serum samples (Gemini Bio, catalog number 507533011) were similarly diluted. After washing, the diluted human serum was transferred to wells and incubated at room temperature for 1 hour with shaking. The plates were washed again as described above. A solution containing Biotin-SP AffiniPure Goat Anti-Human IgG, Fcγ (Jackson ImmunoResearch, catalog number 128-065-098), diluted 1:20,000 in 1% w / v PBS BSA solution, was added to the wells treated with the diluted human serum. A 1% w / v PBS BSA solution containing 1:4,000 MonoRab Rabbit anti-Camelid VHH-biotin (GenScript, catalog number A01995-200) and 1:20,000 Biotin-SP AffiniPure Goat Anti-Alpaca IgG, VHH domain (Jackson ImmunoResearch, catalog number 125-065-232) was added to wells coated with VHH but not treated with human serum, which served as a positive control. The plates were then incubated at room temperature for 1 hour with shaking.

[0228] After incubation, the plate was washed again as described above. A 1% w / v PBS BSA solution containing 0.125 ug / mL Streptavidin-Sulfotag (MesoScale Discovery, catalog number R32AD-1) was added to all wells of the plate and incubated at room temperature for 1 hour with shaking. The wells were then washed again as described above. 150 uL of MSD Gold Read Buffer B (MesoScale Discovery, catalog number R60AM-4) was added to each well of the plate. The plate was then read using a MESO QuickPlex SQ 120MM instrument.

[0229] Table 12 shows the assay results used to measure existing human anti-VHH ADA. Mutations exhibit varying efficacy in the remission of binding of existing human ADA to camelid VHH compared to parental VHH. These results are also shown graphically in Figures 1, 2, and 3.

[0230] (Table 12) Binding activity of IL10Ra-IL10Rb VHH dimer TIFF2026521433000041.tif111146

[0231] In summary, all mutations tested reduced ADA binding compared to the C-terminal sequence of DR2485, which is endogenous with the parental wild-type VTVSS (SEQ ID NO:1). Confirmed by Friedman's multiple comparison test and Dunn's correction for multiple comparison, all mutations except DR2486 (VTVSSAA), DR2493 (VTVSA), and DR2498 (VTVSCAA) showed a significant reduction in ADA binding. Importantly, DR2493 (VTVSA) significantly increased ADA binding in some donors. DR2485 showed a relatively weak ADA signal without additional mutations.

[0232] Example 2: Evaluation of sequence modification in the αIL-6R-αHSA VHH dimer A significant portion of human subjects exhibited existing anti-drug antibodies (ADAs) produced against the exposed C-terminus of several sdAbs. Rossotti, et al. (2022), Immunogenicity and humanization of single-domain antibodies. FEBS J, 289: 4304-4327. Volbarilizumab (also known as ALX-0061 in the literature) is a bispecific fusion protein in which affinity-matured humanized llama-derived anti-hIL6R (αIL6R)VHH and anti-HSA (αHSA)VHH are linked by a GGGGSGGGS (SEQ ID NO:48) linker, and is produced in Pichia pastoris. Van Roy, et al. (2105) Arthritis Res Ther. 17(1):135. Bovalilizumab has been reported to be quite immunogenic in primates (Van Roy, et al., see above), and a significant proportion of human subjects treated in a Phase II trial developed treatment-emergent ADA (Rossotti, et al., see above; Ackaert, et al. (2021) Front Immunol. 12:632687). Therefore, volvalilizumab was selected as the base molecule to evaluate the sequence modifications described herein that reduce immunogenicity.

[0233] A bovalilizumab analog containing amino acid sequences derived from the anti-IL6R (αIL6R)VHH and anti-HSA (αHSA)VHH domains of bovalilizumab, linked via the GGGS (SEQ ID NO: 47) linker, was prepared to form DR2514aa (SEQ ID NO: 84). In bovalilizumab, the carboxyl terminus of the aIL16R VHH domain of bovalilizumab is linked to the amino terminus of the αHSA VHH domain of bovalilizumab via the GGGGSGGGS (SEQ ID NO: 48) linker. In contrast, in DR2514aa, the carboxyl terminus of the aIL16R VHH domain of DR2514aa is linked to the amino terminus of the αHSA VHH domain of DR2514aa via the GGGS (SEQ ID NO: 47) linker.

[0234] A series of αIL-6R-αHSA VHH dimer molecules containing the immunogenicity-reducing modifications described herein were designed based on DR2514aa, and their amino acid sequences are shown in Table 6. The αIL-6R-αHSA VHH dimer polypeptides shown in Table 6 were prepared by constructing synthetic nucleic acid sequences encoding a polypeptide further containing a 5' sequence encoding a mouse IgH signal peptide (Uniprot Reference: Q99LA6) upstream of the αIL-6R-αHSA VHH dimer polypeptide encoding sequence. The nucleic acid sequences encoding the signal peptide and the mature αIL-6R-αHSA VHH dimer were inserted downstream of the CMV promoter in the pExSYN2.0 plasmid, a derivative of the pcDNA3.4 (ThermoFisher) expression vector, using EcoR1 and Not1 restriction endonucleases. Expi293 cells were transfected with the resulting expression vector and cultured in Expi293 expression medium (ThermoFisher Scientific) for 96 hours. This protein was purified by affinity chromatography using MabSelect® VH3 resin (Cytiva). Polypeptides with Sequence ID NO: 85-93 were not efficiently expressed using the aforementioned system and were therefore not included in the following experiments; thus, polypeptides corresponding to SEQ ID NO: 84 and 94-109 were used in the following experiments.

[0235] Blood samples from 80 human donors were screened substantially in accordance with the method described in Example 1 to determine the presence of pre-existing antibodies against DR2514aa. To evaluate the immunogenicity-reducing effect of the sequence modifications of this disclosure, the donors who showed the highest levels of pre-existing antibodies against DR2514aa were used in the following experiments. Polypeptides with SEQ ID NO: 84 and 94-109 were incubated with human serum samples from each donor, and assays evaluating the binding of the human serum samples to ADA were performed substantially in accordance with Example 1 above. Table 13 below shows the results of the assays used to measure pre-existing human anti-VHH ADA. Normalized binding activity (and expected immunogenicity) was measured in terms of bioluminescence values ​​(relative luminescence, i.e., RLU). This data is shown in the form of a graph in Figure 4 of the attached drawings, with relative luminescence units (RLU) plotted on the vertical axis.

[0236] (Table 13) Evaluation of mean RLU of C-terminal modification constructs based on DR2514 in human blood donor samples TIFF2026521433000042.tif136143

[0237] Table 14 shows the results of a statistical analysis of standardized binding activity compared to the parent molecule DR2514.

[0238] (Table 14) Statistical analysis of immunogenicity of test materials in human blood donors compared to DR2514aa TIFF2026521433000043.tif132145

[0239] As illustrated by the data in Tables 13 and 14 and Figure 4, the majority (10 / 17) of the evaluated molecules containing the amino acid substitutions of this disclosure showed reduced ADA binding compared to the parent molecule DR2514. Multiple test articles showed a significant reduction in ADA binding when confirmed by Friedman multiple comparison tests and Dunn correction for multiple comparisons. From the data described above, it is demonstrated that molecules containing the immunogenicity-reducing amino acid sequence modifications of this disclosure uniformly reduced existing human ADA binding compared to the parent molecule DR2514.

[0240] Example 3 - Evaluation of immunogenicity of IL18R / IL18Ra VHH dimer sequence To further evaluate the effect of the modifications of this disclosure with respect to immunogenicity, a series of molecules based on the parent IL18 surrogate cytokine agonist (DR3905), including modifications to reduce immunogenicity, were prepared. These molecules contained a VHH molecule that binds to the IL18Ra receptor subunit and a second VHH that binds to the IL18Rb receptor subunit, linked via a 10-amino acid linker of sequence GGGGSGGGGS (SEQ ID NO: 27). The amino acid sequences of the IL18 VHH dimer parent molecule (DR3905) (SEQ ID NO: 110) and derivative molecules containing amino acid substitutions to reduce immunogenicity (SEQ ID NO: 111-134) are shown in Table 7 above. The test article polypeptides in Table 8 were prepared by constructing synthetic nucleic acid sequences encoding the above polypeptides, as shown in Table 9 below. The names of the nucleic acid sequences in Table 8 (e.g., DR3905-dna) reflect the sequences encoding the corresponding polypeptides in Table 7 (e.g., DR3905aa).

[0241] The nucleic acid sequences encoding the mature IL18 VHH dimer, as described in Table 9, were further modified by adding the 5' sequence encoding the mouse IgH signal peptide (Uniprot Reference: Q99LA6). The nucleic acid sequences encoding the signal peptide and the mature IL18 VHH dimer were inserted downstream of the CMV promoter in the pExSYN2.0 plasmid, a derivative of the pcDNA3.4 (ThermoFisher) expression vector, using EcoR1 and Not1 restriction endonucleases. The resulting expression vector was used to transfect expi293 cells, which were cultured in Expi293 expression medium (ThermoFisher Scientific) for 96 hours. The resulting protein was purified by affinity chromatography using MabSelect® VH3 resin (Cytiva). Polypeptides corresponding to SEQ ID NO: 110~134 were used in the following experiments.

[0242] Polypeptides with SEQ ID NO 110-134 were incubated with human serum samples derived from individual donors, and assays evaluating the binding of the human serum samples to ADA were performed substantially in accordance with Example 2 above. Table 15 shows the results of the assays used to measure existing human anti-VHH ADA. Normalized binding activity (and expected immunogenicity) was measured in terms of bioluminescence values ​​(relative luminescence, i.e., RLU). This data is shown in the form of a graph in Figure 5 of the attached drawings, with relative luminescence units (RLU) plotted on the vertical axis.

[0243] (Table 15) Evaluation of the binding activity of IL18 VHH dimer TIFF2026521433000044.tif193149

[0244] Table 16 shows the results of a statistical analysis of standardized binding activity compared to the parent molecule DR3905aa.

[0245] (Table 16) Statistical analysis of immunogenicity of test materials in human blood donors compared to DR3905aa TIFF2026521433000045.tif188155

[0246] As shown in Tables 15 and 16 and Figure 5 above, the majority (22 / 24) of the molecules containing immunogenicity-reducing amino acid substitutions disclosed herein showed reduced ADA binding compared to the parent DR3905aa molecule. Several showed significant reductions in ADA binding when confirmed by Friedman multiple comparison tests and Dunn corrections for multiple comparisons. D3905 showed a relatively weak ADA signal without additional mutations.

[0247] Example 4. Evaluation of the immunogenicity of anti-HSA VHH monomers As previously described in Example 2 above, the exposed carboxyl terminus of the VHH molecule is associated with pre-existing immunogenicity. A series of anti-HSA VHH monomers were prepared based on the anti-HSA VHH of DR2514aa. The parent VHH anti-HSA VHH monomer molecule, DR2830aa (SEQ ID NO: 159), and its derivatives (SEQ ID NO: 160-194) incorporating the amino acid sequence modifications of this disclosure are shown in Table 9 above.

[0248] The αHSA VHH monomer polypeptides represented by SEQ ID NO: 169-194 in Table 9 above were prepared by constructing a synthetic nucleic acid sequence encoding the polypeptide, further containing a 5' sequence encoding a mouse IgH signal peptide (Uniprot Reference: Q99LA6) upstream of the αHSA VHH monomer polypeptide encoding sequence. The nucleic acid sequences encoding the signal peptide and the αHSA VHH monomer were inserted downstream of the CMV promoter of the pExSYN2.0 plasmid, a derivative of the pcDNA3.4 (ThermoFisher) expression vector, using EcoR1 and Not1 restriction endonucleases. Expi293 cells were transfected with the resulting expression vector and cultured in Expi293 expression medium (ThermoFisher Scientific) for 96 hours. This protein was purified by affinity chromatography using MabSelect® VH3 resin (Cytiva). Polypeptides with Sequence ID NO: 178-189 were not efficiently expressed in the aforementioned system and were therefore not included in the following experiments; thus, polypeptides corresponding to SEQ ID NO: 159, 169-177, and 190-194 were used in the following experiments. Immunogenicity was evaluated substantially in accordance with the disclosure of Example 2. The data are shown in Figure 6 of the attached drawings. As illustrated by the data shown in Figure 6, the immunogenicity of anti-HSA monomer VHH was significantly reduced, including the modifications of this disclosure.

[0249] Other embodiments Although this disclosure has been described in the above description of this disclosure, the above description is intended to illustrate the scope of this disclosure and not to limit it. The scope of this disclosure is defined by the attached claims. Other aspects, advantages, and modifications are within the scope of the following:

Claims

1. A polypeptide comprising a single domain antibody (sdAb), wherein the sdAb has a modified C-terminal amino acid sequence corresponding to the C-terminal endogenous sdAb amino acid residues (T / L 108 )V 109 T 110 V 111 S 112 S 113 numbered according to the Kabat numbering scheme, and the modified amino acid sequence contains the formula X 108 X 109 X 110 V 111 X 112 X 113 Y, X 108 However, selected from the group consisting of L, T, and Q, X 109 However, selected from the group consisting of V, G, N, and L, X 110 However, selected from the group consisting of T and Q, X 111 V is, X 112 However, it is selected from the group consisting of S, C, T, A, and G, or it is optionally nonexistent, and X 113 However, it is selected from the group consisting of S, C, A, G, and T, or it is optionally absent. however, (a)X 109 When V, X 112 and X 113 It is not possible for both to be S, and (b)X 112 and X 113 It is not possible for both to be C, and Y contains a polypeptide comprising 1 to 5 amino acids independently selected from the group consisting of A, G, S, T, L, and V, or Y may be optionally absent, and The sdAb is optionally further modified to include amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A, numbered according to the Kabat numbering scheme. The aforementioned polypeptide.

2. X 108 L is, X 109 However, selected from the group consisting of V, G, and L, X 110 T is, X 112 However, selected from the group consisting of S, T, and C, X 113 However, selected from the group consisting of S, T, C, and A, The polypeptide according to claim 1, wherein Y is absent or is an amino acid sequence AA which is a dipeptide.

3. Amino acid sequence X 109 X 110 V 111 X 112 X 113 The polypeptide according to claim 2, selected from the group consisting of GTVSS (SEQ ID NO: 3), LTVSS (SEQ ID NO: 27), VTVCS (SEQ ID NO: 8), VTVSC (SEQ ID NO: 10), NTVSS (SEQ ID NO: 22), VTVTS (SEQ ID NO: 23), and VTVTT (SEQ ID NO: 24).

4. A polypeptide according to any one of claims 1 to 3, wherein Y is AA.

5. Amino acid sequence X 109 X 110 V 111 X 112 X 113 The polypeptide according to claim 2, wherein is VTVSA (SEQ ID NO: 6) and Y is AA.

6. The polypeptide according to any one of claims 1 to 5, wherein the sdAb is a fusion protein.

7. The polypeptide according to claim 6, wherein the fusion protein comprises a first VHH linked to a second VHH.

8. The polypeptide according to claim 7, wherein the first VHH has a C-terminus attached to the N-terminus of the second VHH.

9. Formula VHH1-L n - A polypeptide of VHH2, wherein VHH1 is VHH, L is a polypeptide linker containing 1 to 50 amino acids, n is 0 or 1, and VHH2 is VHH according to any one of claims 1 to 5.

10. The polypeptide according to claim 9, wherein VHH1 and VHH2 each independently bind to the extracellular domain of a cytokine receptor.

11. The polypeptide according to claim 9, wherein the cytokine receptor is selected from the group consisting of IL2Rα, IL2Rβ, IL2Rγ, IL10Rα, IL10Rβ, IL12Rβ1, IL12Rβ2, IL18Rα, IL18Rβ, IL22R1, IL27Rα, gp130, IL23R, IL28Rα, IFNRγ1, IFNRγ2, and IL21R.

12. The aforementioned VHH1 and VHH2 are the following set: The polypeptide according to claim 11, which selectively binds to a pair of cytokine receptors selected from IL10Rα / IL10Rβ, IL27Rα / gp130, IFNγR1 / IFNγR2, IL10Rβ / IL28Rα, IL2Rβ / IL2Rγ, IL18Rα / IL18Rβ, IL22R1 / IL10Rβ, IL10Rα / IL2Rγ, IL2Rβ / IL2Rγ, IL10R1 / IFNRγ, IFNRγ / IL28Rα, IL12Rβ1 / IL12Rβ2, IL12Rβ1 / IL23R, IL12Rβ2 / gp130, and IL10Rα / IL2Rγ.

13. The polypeptide according to any one of claims 9 to 12, wherein L is a linker containing amino acids G and S.

14. A polypeptide according to any one of claims 1 to 13, which is pegylated.

15. A polypeptide according to any one of claims 1 to 13, conjugated with an Fc domain.

16. The polypeptide according to any one of claims 1 to 15, which, when administered to a human subject, exhibits reduced immunogenicity against the human subject's existing antibodies (SEQ ID NO: 24) compared to an sdAb polypeptide containing the corresponding native human sequence VTVSS (SEQ ID NO: 1).

15. A pharmaceutical composition comprising the polypeptide described in any one of claims 1 to 14.

16. A nucleic acid encoding the polypeptide according to any one of claims 1 to 14.

17. A vector comprising the nucleic acid described in claim 16.

18. A host cell comprising the vector according to claim 17.

19. Formula VHH1-L n - A cytokine receptor-binding polypeptide of VHH2 comprising a first cytokine receptor subunit and a second cytokine receptor subunit, Either VHH1 or VHH2 is a VHH that selectively binds to the extracellular domain of a first cytokine receptor subunit, and the other VHH1 or VHH2 is a VHH that selectively binds to the extracellular domain of a second cytokine receptor subunit. L is a polypeptide linker, n = 0 (non-existence) or (1) exists, and The VHH2 is a compound of formula X, in which the amino acids are numbered according to the Kabat numbering scheme. 108 V 109 T 110 V 111 S 112 S 113 Contains the Y amino acid sequence, X 108 However, selected from the group consisting of L, T, and Q, X 109 However, selected from the group consisting of V, G, N, and L, X 110 However, selected from the group consisting of T and Q, X 111 V is, X 112 However, it is selected from the group consisting of S, C, T, A, and G, or it is optionally absent. X 113 However, it is selected from the group consisting of S, C, A, G, and T, or it is optionally absent. however, (a)X 109 If V, then X 112 and X 113 It is not possible for both to be S, and (b)X 112 and X 113 It is not possible for both to be C, and Y contains a polypeptide comprising 1 to 5 amino acids independently selected from the group consisting of A, G, S, T, L, and V, or Y may be optionally absent, and The polypeptide is optionally further modified to include amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A, numbered according to the Kabat numbering scheme. The cytokine receptor-binding polypeptide.

20. Equation X of VHH2 108 V 109 T 110 V 111 S 112 S 113 The cytokine receptor-binding polypeptide according to claim 19, wherein the amino acid sequence of Y is selected from the group consisting of SEQ ID NO: 2 to 24 and optionally further comprises amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A, and the amino acid residues are numbered according to the Kabat numbering scheme.

21. Equation X in VHH2 108 V 109 T 110 V 111 S 112 S 113 The cytokine receptor-binding polypeptide according to claim 19 or 20, exhibiting reduced immunogenicity compared to polypeptides lacking the Y amino acid sequence.

22. The cytokine receptor-binding polypeptide according to any one of claims 19 to 21, wherein n=1 and L is a polypeptide linker of 1 to 50 amino acids.

22. The cytokine receptor-binding polypeptide according to any one of claims 19 to 22, wherein n=1 and L is a polypeptide linker selected from the group consisting of SEQ ID NO: 25 to 48.

23. A cytokine receptor-binding polypeptide according to any one of claims 19 to 22, which is modified to extend its half-life in vivo.

24. A cytokine receptor-binding polypeptide according to any one of claims 19 to 23, which is pegylated.

25. A nucleic acid sequence encoding a cytokine receptor-binding polypeptide according to any one of claims 19 to 23.

26. The cytokine receptor-binding polypeptide according to any one of claims 19 to 23, wherein the cytokine receptor is an IL10 receptor, the first cytokine receptor subunit is IL10Ra, and the second cytokine receptor subunit is IL10Rb.

27. The cytokine receptor-binding polypeptide according to claim 26, comprising an amino acid sequence having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity with respect to an amino acid sequence selected from the group consisting of SEQ ID NO: 50 to 72.

28. The cytokine receptor-binding polypeptide according to claim 26, exhibiting reduced immunogenicity compared to DR2485aa (SEQ ID NO:49).

29. The cytokine receptor-binding polypeptide according to claim 26, wherein the cytokine receptor-binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 50 to 72.

30. The cytokine receptor-binding polypeptide according to claim 26, wherein the cytokine receptor-binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 50 to 72.

31. The cytokine receptor-binding polypeptide according to any one of claims 19 to 23, wherein the cytokine receptor is an IL18 receptor, the first cytokine receptor subunit is IL18Ra, and the second cytokine receptor subunit is IL18Rb.

32. The cytokine receptor-binding polypeptide according to claim 26, comprising an amino acid sequence having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity with respect to an amino acid sequence selected from the group consisting of SEQ ID NO: 111 to 134.

33. The cytokine receptor-binding polypeptide according to claim 26, exhibiting reduced immunogenicity compared to R3905aa (SEQ ID NO: 110).

34. The cytokine receptor-binding polypeptide according to claim 26, wherein the cytokine receptor-binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 111 to 134.

35. The cytokine receptor-binding polypeptide according to claim 26, wherein the cytokine receptor-binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 111 to 134.

36. The cytokine receptor-binding polypeptide according to any one of claims 19 to 23, wherein the cytokine receptor is an IL2 receptor, the first cytokine receptor subunit is IL2Rb(CD122), and the second cytokine receptor subunit is IL2Rg(CD132).

37. The cytokine receptor-binding polypeptide according to any one of claims 19 to 23, wherein the cytokine receptor is an IL18 receptor, the first cytokine receptor subunit is IL18Ra, and the second cytokine receptor subunit is IL18Rb.

38. The cytokine receptor-binding polypeptide according to any one of claims 19 to 23, wherein the cytokine receptor is an IL27 receptor, the first cytokine receptor subunit is IL27Ra, and the second cytokine receptor subunit is gp130.

39. The cytokine receptor-binding polypeptide according to any one of claims 19 to 23, wherein the cytokine receptor is an IL22 receptor, the first cytokine receptor subunit is IL22Ra, and the second cytokine receptor subunit is IL12Rb.

40. The cytokine receptor-binding polypeptide according to any one of claims 19 to 23, wherein the cytokine receptor is an IL4 receptor, the first cytokine receptor subunit is IL4Ra, and the second cytokine receptor subunit is IL2Rg(CD132).

41. The cytokine receptor-binding polypeptide according to any one of claims 19 to 23, wherein the cytokine receptor is an IL7 receptor, the first cytokine receptor subunit is IL7Ra, and the second cytokine receptor subunit is IL2Rg(CD132).

42. The cytokine receptor-binding polypeptide according to any one of claims 19 to 23, wherein the cytokine receptor is an IL9 receptor, the first cytokine receptor subunit is IL9Ra, and the second cytokine receptor subunit is IL2Rg(CD132).

43. The cytokine receptor-binding polypeptide according to any one of claims 19 to 23, wherein the cytokine receptor is an IL12 receptor, the first cytokine receptor subunit is IL12Ra, and the second cytokine receptor subunit is IL12Rb.

44. Formula VHH1-L n -VHH2 is a divalent IL6R / HSA-binding polypeptide, Either VHH1 or VHH2 is a VHH that selectively binds to the extracellular domain of IL6Ra, and The other of VHH1 or VHH2 is a VHH that selectively binds to human serum albumin. L is a polypeptide linker, n = 0 (non-existence) or (1) exists, and The VHH2 is a compound of formula X, in which the amino acids are numbered according to the Kabat numbering scheme. 108 V 109 T 110 V 111 S 112 S 113 Contains the Y amino acid sequence, X 108 However, selected from the group consisting of L, T, and Q, X 109 However, selected from the group consisting of V, G, N, and L, X 110 However, selected from the group consisting of T and Q, X 111 V is, X 112 However, it is selected from the group consisting of S, C, T, A, and G, or it is optionally absent. X 113 However, it is selected from the group consisting of S, C, A, G, and T, or it is optionally absent. however, (a)X 109 If V, then X 112 and X 113 It is not possible for both to be S, and (b)X 112 and X 113 It is not possible for both to be C, and Y contains a polypeptide comprising 1 to 5 amino acids independently selected from the group consisting of A, G, S, T, L, and V, or Y may be optionally absent, and The polypeptide is optionally further modified to include amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A, numbered according to the Kabat numbering scheme. The aforementioned divalent IL6R / HSA-conjugated polypeptide.

45. Formula X of VHH2 108 V 109 T 110 V 111 S 112 S 113 The amino acid sequence of Y is selected from the group consisting of SEQ ID NO: 2-24 and optionally further comprises an amino acid substitution selected from the group consisting of L11S, L11Q, L11G, and P14A, and the amino acid residues are numbered according to the Kabat numbering scheme. The bivalent IL6R / HSA binding polypeptide according to claim 44.

46. Formula X in VHH2 108 V 109 T 110 V 111 S 112 S 113 The bivalent IL6R / HSA-binding polypeptide according to claim 44 or 45, which exhibits reduced immunogenicity compared to a polypeptide lacking the amino acid sequence of Y.

46. The divalent IL6R / HSA-conjugated polypeptide according to any one of claims 44 to 46, wherein n=1 and L is a polypeptide linker of 1 to 50 amino acids.

47. The divalent IL6R / HSA-conjugated polypeptide according to any one of claims 44 to 46, wherein n=1 and L is a polypeptide linker selected from the group consisting of SEQ ID NO: 25 to 48.

48. A divalent IL6R / HSA-conjugated polypeptide according to any one of claims 44 to 47, wherein the polypeptide is modified to have an extended half-life in vivo.

49. A pegylated divalent IL6R / HSA-conjugated polypeptide according to any one of claims 44 to 48.

50. A divalent IL6R / HSA-binding polypeptide according to any one of claims 44 to 48, comprising an amino acid sequence having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity with respect to an amino acid sequence selected from the group consisting of SEQ ID NO: 85 to 109.

51. The divalent IL6R / HSA-binding polypeptide according to claim 50, exhibiting immunogenicity compared to DR2514aa (SEQ ID NO: 84).

52. The divalent IL6R / HSA-binding polypeptide according to claim 50, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 85 to 109.

53. Formula X, in which amino acids are numbered according to the Kabat numbering scheme. 108 V 109 T 110 V 111 S 112 S 113 Anti-HSA VHH, which contains the Y amino acid sequence and selectively binds to human serum albumin, X 108 However, selected from the group consisting of L, T, and Q, X 109 However, selected from the group consisting of V, G, N, and L, X 110 However, selected from the group consisting of T and Q, X 111 V is, X 112 However, it is selected from the group consisting of S, C, T, A, and G, or it is optionally absent. X 113 However, it is selected from the group consisting of S, C, A, G, and T, or it is optionally absent. however, (a)X 109 If V, then X 112 and X 113 It is not possible for both to be S, and (b)X 112 and X 113 It is not possible for both to be C, and Y contains a polypeptide comprising 1 to 5 amino acids independently selected from the group consisting of A, G, S, T, L, and V, or Y may be optionally absent, and The polypeptide is optionally further modified to include amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A, numbered according to the Kabat numbering scheme. The aforementioned anti-HSA VHH.

54. The aforementioned amino acid, `X` 108 V 109 T 110 V 111 S 112 S 113 The anti-HSA VHH according to claim 53, wherein Y is selected from the group consisting of SEQ ID NO: 2 to 24, and optionally further comprises amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A, wherein the amino acid residues are numbered according to the Kabat numbering scheme.

55. formula 108 V 109 T 110 V 111 S 112 S 113 The anti-HSA VHH according to claim 53 or 54, exhibiting reduced immunogenicity compared to polypeptides lacking the Y amino acid sequence.

56. The anti-HSA VHH according to any one of claims 53 to 55, wherein the anti-HSA is modified to have an extended half-life in vivo.

57. The anti-HSA VHH according to any one of claims 53 to 56, wherein the anti-HSA is pegged.

58. The anti-HSA VHH according to any one of claims 53 to 57, wherein the anti-HSA comprises an amino acid sequence having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 160 to 194.

59. The anti-HSA VHH according to claim 58, exhibiting reduced immunogenicity compared to the polypeptide SEQ ID NO:

159.

60. The anti-HSA VHH according to claim 58, wherein the anti-HSA comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 160 to 194.

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