A protein binding to serum albumin and its use

Abstract

The present application belongs to the field of biopharmaceutical technology. More specifically, the present application relates to a humanized single-domain antibody that binds to serum albumin, as well as a multispecific antibody, fusion protein, and conjugate comprising the single-domain antibody. The humanized single-domain antibody of the present application is capable of efficiently binding to human serum albumin, thereby extending the in vivo half-life of the molecule to which it is attached. The humanized single-domain antibody of the present application, as well as the multispecific antibody, fusion protein, and conjugate comprising the single-domain antibody, can be used to prepare a medicament for the prevention and / or treatment or adjuvant treatment of tumors, infections, autoimmune diseases, metabolic-related diseases, or developmental-related diseases.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology. More specifically, the present application relates to a single-domain antibody that binds to serum albumin and a multispecific antibody, fusion protein and conjugate comprising the single-domain antibody. The single-domain antibody can prolong the in vivo half-life of an active agent connected thereto. Technical Background

[0002] To achieve therapeutic effects, most protein drugs often require multiple, high-dose injections, increasing the treatment burden and reducing patient compliance. Consequently, various approaches to prolonging the effectiveness of proteins have been developed. These include mutational modification of proteins to improve stability and prevent their recognition and degradation by proteases. However, mutations can easily lead to immunogenicity, reducing the drug's in vivo function. Another approach is PEG conjugation, which increases the protein's hydration radius to prevent rapid clearance by the kidneys while shielding some protease sites and slowing degradation. However, high-molecular-weight PEG can easily lead to loss of protein activity and poses the risk of renal accumulation leading to glomerular hollowing.

[0003] This field urgently needs better protein long-acting technology, which should be applied to clinical drug development to meet medication needs and improve patient compliance. Summary of the Invention

[0004] To address the current challenges of long-acting proteins, camel-derived antibodies that specifically bind to human serum albumin have attracted the attention of researchers. These antibodies can extend the half-life of drugs by binding to human serum albumin, and their small molecular weight can avoid the adverse effects of fusion proteins such as HSA and Fc on activity, processing, and efficacy. However, camel-derived antibodies are highly immunogenic when introduced into the human body, and are prone to producing anti-drug antibodies.

[0005] Therefore, in order to reduce the production of anti-drug antibodies and improve the safety of drugs, camel-derived antibodies need to be humanized. However, due to the matching problem between the CDR of the donor antibody (e.g., camel-derived antibody) and the FR of the receptor antibody (e.g., human antibody), the expected properties of humanized antibodies (e.g., antigen specificity, affinity, reactivity, etc.) are generally lower than those of non-human donor antibodies (e.g., camel-derived antibodies). In this regard, although researchers in the field have made some progress in the humanization of antibodies, how to fully humanize a certain donor antibody so that the humanized antibody produced has both the highest possible degree of humanization and the expected properties of the donor antibody can be retained as much as possible, the prior art does not provide guidance. Technicians need to explore, explore and transform specific donor antibodies, and it is possible to obtain humanized antibodies that have both a high degree of humanization and retain the expected properties of specific donor antibodies through a lot of creative work.

[0006] In this application, the inventors have devoted a lot of creative work to conduct in-depth research and transformation of camel-derived antibodies, thereby developing humanized single-domain antibodies.

[0007] The humanized single-domain antibody of the present application is extremely advantageous. It not only retains (or even improves) the functions and properties of the parent camel-derived single-domain antibody, such as binding to human serum albumin with very high affinity and specificity, and has the potential to be used to extend the in vivo half-life of the active agent connected thereto, but also has a very high degree of humanization and can be safely administered to human subjects without eliciting or less eliciting an immunogenic response. In addition, the humanized single-domain antibody of the present application has the possibility of being produced in a microbial expression system at a relatively low cost, and has great potential as a chronic disease treatment drug required for long-term high doses. Therefore, the antibody of the present application has significant clinical value.

[0008] Single domain antibodies

[0009] In a first aspect, the present application provides a humanized single-domain antibody or an antigen-binding fragment thereof, wherein the single-domain antibody is basically composed of four framework regions (FRs) and three complementarity determining regions (CDRs), and the four framework regions are FR1-FR4.

[0010] In certain embodiments, the FRs comprise, according to the definition of the IMGT numbering system, FR1 having a sequence of SEQ ID NO: 10 or 17, or a sequence having one or several amino acid substitutions, deletions, additions, or any combination thereof (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 10 or 17; FR2 having a sequence of SEQ ID NO: 11 or 18, or a sequence having one or several amino acid substitutions, deletions, additions, or any combination thereof (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 11 or 18; FR3 having a sequence of SEQ ID NO: 12 or 19, or a sequence having one or several amino acid substitutions, deletions, additions, or any combination thereof (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 12 or 19; and FR4 having a sequence of SEQ ID NO: 13 or 19. NO:13 or an FR4 having a sequence with one or more amino acid substitutions, deletions, additions or any combination thereof (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto; and, the FR1-FR4 further comprises one or more selected from the following: L at position 5, A at position 24, L at position 50, S at position 83, R at position 95, L at position 123.

[0011] In certain embodiments, the FRs comprise, according to the definition of the IMGT numbering system, FR1 having a sequence of SEQ ID NO: 10 or 17, or a sequence having one or several amino acid substitutions, deletions, additions, or any combination thereof (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 10 or 17; FR2 having a sequence of SEQ ID NO: 11 or 18, or a sequence having one or several amino acid substitutions, deletions, additions, or any combination thereof (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 11 or 18; FR3 having a sequence of SEQ ID NO: 12 or 19, or a sequence having one or several amino acid substitutions, deletions, additions, or any combination thereof (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 12 or 19; and FR4 having a sequence of SEQ ID NO: 13 or 19. NO:13 or an FR4 having one or more amino acid substitutions, deletions, additions or any combination thereof (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto; and, the FR1-FR4 further comprises: L at position 5, A at position 24, L at position 50, S at position 83, R at position 95, and L at position 123.

[0012] In certain embodiments, the FRs further comprise one or more selected from the group consisting of L or S at position 54, A or P at position 96, A or T at position 105, and E or I at position 106, as defined by the IMGT numbering system.

[0013] In certain embodiments, the FRs further comprise an S at position 54, a P at position 96, a T at position 105, and an I at position 106, as defined by the IMGT numbering system.

[0014] In certain embodiments, the FRs are defined according to the Kabat numbering system and comprise: FR1 having a sequence of SEQ ID NO: 14 or 20, or a sequence having one or several amino acid substitutions, deletions, additions, or any combination thereof (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 14 or 20; FR2 having a sequence of SEQ ID NO: 15 or 21, or a sequence having one or several amino acid substitutions, deletions, additions, or any combination thereof (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 15 or 21; FR3 having a sequence of SEQ ID NO: 16 or 22, or a sequence having one or several amino acid substitutions, deletions, additions, or any combination thereof (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 16 or 22; and FR4 having a sequence of SEQ ID NO: 17 or 18. NO:13 or an FR4 having one or more amino acid substitutions, deletions, additions or any combination thereof (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto; and, the FR1-FR4 further comprises one or more selected from the following: L at position 5, A at position 23, L at position 45, S at position 74, R at position 83, L at position 108.

[0015] In certain embodiments, the FRs are defined according to the Kabat numbering system and comprise: an FR1 having a sequence of SEQ ID NO: 14 or 20, or a sequence having one or several amino acid substitutions, deletions, additions, or any combination thereof (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 14 or 20; an FR2 having a sequence of SEQ ID NO: 15 or 21, or a sequence having one or several amino acid substitutions, deletions, additions, or any combination thereof (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 15 or 21; an FR3 having a sequence of SEQ ID NO: 16 or 22, or a sequence having one or several amino acid substitutions, deletions, additions, or any combination thereof (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 16 or 22; and an FR4 having a sequence of SEQ ID NO: 17 or 18. NO:13 or an FR4 having one or more amino acid substitutions, deletions, additions or any combination thereof (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto; and, the FR1-FR4 further comprises: L at position 5, A at position 23, L at position 45, S at position 74, R at position 83, and L at position 108.

[0016] In certain embodiments, the FRs further comprise one or more selected from the group consisting of L or S at position 49, A or P at position 84, A or T at position 93, and E or I at position 94, as defined by the Kabat numbering system.

[0017] In certain embodiments, the FRs comprise one or more selected from the group consisting of: S at position 49, P at position 84, T at position 93, and I at position 94, as defined by the Kabat numbering system.

[0018] In certain embodiments, the single domain antibody or antigen binding fragment thereof comprises: an E or a Q at position 1, as defined by the IMGT or Kabat numbering system. In certain embodiments, the single domain antibody or antigen binding fragment thereof comprises: an E at position 1.

[0019] In a second aspect, the present application provides a single-domain antibody or antigen-binding fragment thereof that specifically binds to serum albumin, wherein the single-domain antibody is substantially composed of four framework regions (FRs) and three complementarity determining regions (CDRs), wherein the four framework regions are FR1-FR4, and the three complementarity determining regions are CDR1-CDR3, respectively. The antigen-binding fragment comprises at least a portion of the single-domain antibody, which portion is sufficient to confer upon the fragment the ability to specifically bind to serum albumin. The single-domain antibody or antigen-binding fragment thereof specifically binds to human serum albumin.

[0020] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises, as defined by the IMGT numbering system:

[0021] The following three complementarity determining regions (CDRs): CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 6 or SEQ ID NO: 37; and,

[0022] the following four framework regions (FRs): FR1 having a sequence of SEQ ID NO: 10 or a sequence having one or several amino acid substitutions, deletions, additions or any combination thereof (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto, FR2 having a sequence of SEQ ID NO: 11 or a sequence having one or several amino acid substitutions, deletions, additions or any combination thereof (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto, FR3 having a sequence of SEQ ID NO: 12 or a sequence having one or several amino acid substitutions, deletions, additions or any combination thereof (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto, FR4 having a sequence of SEQ ID NO: 13 or a sequence having one or several amino acid substitutions, deletions, additions or any combination thereof (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto;

[0023] Furthermore, the single-domain antibody or antigen-binding fragment thereof comprises one or more selected from the following: L at position 5, A at position 24, L at position 50, S at position 83, R at position 95, and L at position 123.

[0024] In certain embodiments, FR1 is SEQ ID NO: 10 or a sequence having one or several amino acid substitutions (e.g., 1, 2, or 3 amino acid substitutions) as compared thereto, FR2 is SEQ ID NO: 11 or a sequence having one or several amino acid substitutions (e.g., 1, 2, or 3 amino acid substitutions) as compared thereto, FR3 is SEQ ID NO: 12 or a sequence having one or several amino acid substitutions (e.g., 1, 2, or 3 amino acid substitutions) as compared thereto, and FR4 is SEQ ID NO: 13 or a sequence having one or several amino acid substitutions (e.g., 1, 2, or 3 amino acid substitutions) as compared thereto;

[0025] Furthermore, the single-domain antibody or antigen-binding fragment thereof comprises: L at position 5, A at position 24, L at position 50, S at position 83, R at position 95, and L at position 123.

[0026] In certain embodiments, the substitutions described in any of the above embodiments are conservative substitutions.

[0027] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises:

[0028] The following three complementarity determining regions (CDRs): CDR1 of SEQ ID NO: 7, CDR2 of SEQ ID NO: 8, and CDR3 of SEQ ID NO: 9; and,

[0029] the following four framework regions (FRs): FR1 having a sequence of SEQ ID NO: 14 or a sequence having one or several amino acid substitutions, deletions, additions or any combination thereof (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, FR2 having a sequence of SEQ ID NO: 15 or a sequence having one or several amino acid substitutions, deletions, additions or any combination thereof (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, FR3 having a sequence of SEQ ID NO: 16 or a sequence having one or several amino acid substitutions, deletions, additions or any combination thereof (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, FR4 having a sequence of SEQ ID NO: 13 or a sequence having one or several amino acid substitutions, deletions, additions or any combination thereof (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;

[0030] Furthermore, the single-domain antibody or antigen-binding fragment thereof comprises one or more selected from the following: L at position 5, A at position 23, L at position 45, S at position 74, R at position 83, and L at position 108.

[0031] In certain embodiments, FR1 is SEQ ID NO: 14 or a sequence having one or several amino acid substitutions (e.g., 1, 2, or 3 amino acid substitutions) as compared thereto, FR2 is SEQ ID NO: 15 or a sequence having one or several amino acid substitutions (e.g., 1, 2, or 3 amino acid substitutions) as compared thereto, FR3 is SEQ ID NO: 16 or a sequence having one or several amino acid substitutions (e.g., 1, 2, or 3 amino acid substitutions) as compared thereto, and FR4 is SEQ ID NO: 13 or a sequence having one or several amino acid substitutions (e.g., 1, 2, or 3 amino acid substitutions) as compared thereto;

[0032] Furthermore, the single-domain antibody or antigen-binding fragment thereof comprises: L at position 5, A at position 23, L at position 45, S at position 74, R at position 83, and L at position 108.

[0033] In certain embodiments, the substitutions described in any of the above embodiments are conservative substitutions.

[0034] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprising the above-mentioned substitutions, deletions or additions retains the reduced immunogenicity against humans and / or the activity of specifically binding to human serum albumin of the single domain antibody or antigen-binding fragment thereof from which it is derived.

[0035] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises one or more selected from the group consisting of: L or S at position 54, A or P at position 96, A or T at position 105, E or I at position 106, as defined by the IMGT numbering system. In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises: L or S at position 54, A or P at position 96, A or T at position 105, E or I at position 106.

[0036] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises one or more selected from the group consisting of: S at position 54, P at position 96, T at position 105, and I at position 106, as defined by the IMGT numbering system. In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises: S at position 54, P at position 96, T at position 105, and I at position 106.

[0037] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises one or more selected from the group consisting of L or S at position 49, A or P at position 84, A or T at position 93, and E or I at position 94, as defined by the Kabat numbering system. In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises L or S at position 49, A or P at position 84, A or T at position 93, and E or I at position 94.

[0038] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises one or more selected from the group consisting of: S at position 49, P at position 84, T at position 93, and I at position 94, as defined by the Kabat numbering system. In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises: S at position 49, P at position 84, T at position 93, and I at position 94.

[0039] In certain embodiments, the single domain antibody or antigen binding fragment thereof comprises: an E or a Q at position 1, as defined by the IMGT or Kabat numbering system. In certain embodiments, the single domain antibody or antigen binding fragment thereof comprises: an E at position 1.

[0040] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises: E at position 1, L at position 5, A at position 24, L at position 50, S at position 54, S at position 83, R at position 95, P at position 96, T at position 105, I at position 106, L at position 123, as defined by the IMGT numbering system.

[0041] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises: E at position 1, L at position 5, A at position 23, L at position 45, S at position 49, S at position 74, R at position 83, P at position 84, T at position 93, I at position 94, and L at position 108, as defined by the Kabat numbering system.

[0042] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises: Q at position 1, L at position 5, A at position 24, L at position 50, S at position 54, S at position 83, R at position 95, P at position 96, T at position 105, I at position 106, and L at position 123, as defined by the IMGT numbering system.

[0043] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises: Q at position 1, L at position 5, A at position 23, L at position 45, S at position 49, S at position 74, R at position 83, P at position 84, T at position 93, I at position 94, and L at position 108 as defined by the Kabat numbering system.

[0044] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises: Q at position 1, L at position 5, A at position 24, L at position 50, L at position 54, S at position 83, R at position 95, A at position 96, A at position 105, E at position 106, L at position 123, as defined by the IMGT numbering system.

[0045] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises: Q at position 1, L at position 5, A at position 23, L at position 45, L at position 49, S at position 74, R at position 83, A at position 84, A at position 93, E at position 94, and L at position 108, as defined by the Kabat numbering system.

[0046] In certain embodiments, FR1 is selected from SEQ ID NO: 10 or 17, FR2 is selected from SEQ ID NO: 11 or 18, FR3 is selected from SEQ ID NO: 12 or 19, and FR4 is selected from SEQ ID NO: 13, as defined by the IMGT numbering system.

[0047] In certain embodiments, FR1 is selected from SEQ ID NO: 14 or 20, FR2 is selected from SEQ ID NO: 15 or 21, FR3 is selected from SEQ ID NO: 16 or 22, and FR4 is selected from SEQ ID NO: 13, as defined by the Kabat numbering system.

[0048] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises, as defined by the IMGT numbering system, a CDR1 having a sequence of SEQ ID NO: 4, a CDR2 having a sequence of SEQ ID NO: 5, a CDR3 having a sequence of SEQ ID NO: 6; and a FR1 having a sequence of SEQ ID NO: 10, a FR2 having a sequence of SEQ ID NO: 11, a FR3 having a sequence of SEQ ID NO: 12, and a FR4 having a sequence of SEQ ID NO: 13.

[0049] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises, as defined by the Kabat numbering system, a CDR1 of SEQ ID NO: 7, a CDR2 of SEQ ID NO: 8, and a CDR3 of SEQ ID NO: 9; and a FR1 of SEQ ID NO: 14, a FR2 of SEQ ID NO: 15, a FR3 of SEQ ID NO: 16, and a FR4 of SEQ ID NO: 13.

[0050] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises the sequence set forth in SEQ ID NO: 3 or a variant sequence thereof, wherein the variant sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 3. Preferably, the substitutions are conservative substitutions.

[0051] In certain embodiments, the variant sequence comprises one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in at least one CDR and / or at least one FR compared to SEQ ID NO: 3. Preferably, the substitutions are conservative substitutions.

[0052] In certain embodiments, the variant sequence retains reduced immunogenicity against humans and / or specific binding activity to human serum albumin compared to the camelid antibody.

[0053] In certain embodiments, the variant comprises one or more selected from the group consisting of: L at position 5, A at position 24, L at position 50, S at position 83, R at position 95, L at position 123, as defined by the IMGT numbering system. In certain embodiments, the variant comprises: L at position 5, A at position 24, L at position 50, S at position 83, R at position 95, L at position 123.

[0054] In certain embodiments, the variant comprises one or more selected from the group consisting of: L at position 5, A at position 23, L at position 45, S at position 74, R at position 83, L at position 108, as defined by the Kabat numbering system. In certain embodiments, the variant comprises: L at position 5, A at position 23, L at position 45, S at position 74, R at position 83, L at position 108.

[0055] In certain embodiments, the variant further comprises one or more selected from the group consisting of: S at position 54, P at position 96, T at position 105, I at position 106, as defined by the IMGT numbering system. In certain embodiments, the variant further comprises: S at position 54, P at position 96, T at position 105, I at position 106.

[0056] In certain embodiments, the variant is selected from one or more of the following, as defined by the Kabat numbering system: S at position 49, P at position 84, T at position 93, I at position 94. In certain embodiments, the variant comprises: S at position 49, P at position 84, T at position 93, I at position 94.

[0057] In certain embodiments, the variant further comprises: an E at position 1 as defined by the IMGT or Kabat numbering system.

[0058] In certain exemplary embodiments, the single-domain antibody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO:3.

[0059] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises, as defined by the IMGT numbering system, a CDR1 of SEQ ID NO: 4, a CDR2 of SEQ ID NO: 5, a CDR3 of SEQ ID NO: 37, and a FR1 of SEQ ID NO: 17, a FR2 of SEQ ID NO: 18, a FR3 of SEQ ID NO: 19, and a FR4 of SEQ ID NO: 13.

[0060] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises, as defined by the Kabat numbering system, a CDR1 of SEQ ID NO: 7, a CDR2 of SEQ ID NO: 8, a CDR3 of SEQ ID NO: 9; and a FR1 of SEQ ID NO: 20, a FR2 of SEQ ID NO: 21, a FR3 of SEQ ID NO: 22, and a FR4 of SEQ ID NO: 13.

[0061] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises: a sequence as set forth in SEQ ID NO: 1 or a variant sequence thereof, wherein the variant sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 1. Preferably, the substitutions are conservative substitutions.

[0062] In certain embodiments, the variant sequence comprises one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in at least one CDR and / or at least one FR compared to SEQ ID NO: 1. Preferably, the substitutions are conservative substitutions.

[0063] In certain embodiments, the variant sequence retains reduced immunogenicity against humans and / or specific binding activity to human serum albumin compared to the camelid antibody.

[0064] In certain embodiments, the variant comprises one or more selected from the group consisting of: L at position 5, A at position 24, L at position 50, S at position 83, R at position 95, L at position 123, as defined by the IMGT numbering system. In certain embodiments, the variant comprises: L at position 5, A at position 24, L at position 50, S at position 83, R at position 95, L at position 123.

[0065] In certain embodiments, the variant comprises one or more selected from the group consisting of: L at position 5, A at position 23, L at position 45, S at position 74, R at position 83, L at position 108, as defined by the Kabat numbering system. In certain embodiments, the variant comprises: L at position 5, A at position 23, L at position 45, S at position 74, R at position 83, L at position 108.

[0066] In certain embodiments, the variant comprises one or more selected from the group consisting of: L at position 54, A at position 96, A at position 105, E at position 106, as defined by the IMGT numbering system. In certain embodiments, the variant further comprises: L at position 54, A at position 96, A at position 105, E at position 106.

[0067] In certain embodiments, the variant comprises one or more selected from the group consisting of: L at position 49, A at position 84, A at position 93, E at position 94, as defined by the Kabat numbering system. In certain embodiments, the variant comprises: L at position 49, A at position 84, A at position 93, E at position 94, as defined by the Kabat numbering system.

[0068] In certain embodiments, the variant further comprises: a Q at position 1 as defined by the IMGT or Kabat numbering systems.

[0069] In certain exemplary embodiments, the single-domain antibody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 1.

[0070] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises, as defined by the IMGT numbering system, a CDR1 of SEQ ID NO: 4, a CDR2 of SEQ ID NO: 5, a CDR3 of SEQ ID NO: 6; and a FR1 of SEQ ID NO: 17, a FR2 of SEQ ID NO: 11, a FR3 of SEQ ID NO: 12, and a FR4 of SEQ ID NO: 13.

[0071] In certain embodiments, according to the Kabat numbering system, the CDR1 of SEQ ID NO: 7, the CDR2 of SEQ ID NO: 8, and the CDR3 of SEQ ID NO: 9; and the FR1 of SEQ ID NO: 20, the FR2 of SEQ ID NO: 15, the FR3 of SEQ ID NO: 16, and the FR4 of SEQ ID NO: 13.

[0072] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises the sequence of SEQ ID NO: 2 or a variant thereof, wherein the variant sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 2. Preferably, the substitutions are conservative substitutions.

[0073] In certain embodiments, the variant sequence has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions) in at least one CDR and / or at least one FR compared to SEQ ID NO: 2. Preferably, the substitutions are conservative substitutions.

[0074] In certain embodiments, the variant sequence retains reduced immunogenicity against humans and / or specific binding activity to human serum albumin compared to the camelid antibody.

[0075] In certain embodiments, the variant comprises one or more selected from the group consisting of: L at position 5, A at position 24, L at position 50, S at position 83, R at position 95, L at position 123, as defined by the IMGT numbering system. In certain embodiments, the variant comprises: L at position 5, A at position 24, L at position 50, S at position 83, R at position 95, L at position 123.

[0076] In certain embodiments, the variant comprises one or more selected from the group consisting of: L at position 5, A at position 23, L at position 45, S at position 74, R at position 83, L at position 108, as defined by the Kabat numbering system. In certain embodiments, the variant comprises: L at position 5, A at position 23, L at position 45, S at position 74, R at position 83, L at position 108.

[0077] In certain embodiments, the variant further comprises one or more selected from the group consisting of: S at position 54, P at position 96, T at position 105, I at position 106, as defined by the IMGT numbering system. In certain embodiments, the variant further comprises: S at position 54, P at position 96, T at position 105, I at position 106.

[0078] In certain embodiments, the variant comprises one or more selected from the group consisting of: S at position 49, P at position 84, T at position 93, I at position 94, as defined by the Kabat numbering system. In certain embodiments, the variant comprises: S at position 49, P at position 84, T at position 93, I at position 94.

[0079] In certain embodiments, the single variant further comprises: a Q at position 1 as defined by the IMGT or Kabat numbering system.

[0080] In certain exemplary embodiments, the single-domain antibody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 2.

[0081] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof is a humanized VHH. A humanized VHH refers to a single-domain antibody obtained by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VHH domain with one or more amino acid residues present at corresponding positions in a VH domain from a human antibody.

[0082] In certain embodiments, the degree of humanization of the segment encoded by the V gene of the single-domain antibody or its antigen-binding fragment is not less than 79%, for example, 80%, 85%, 90%, 93%, 95%, 97%, or 100%.

[0083] In certain embodiments, the degree of humanization of the segment encoded by the J gene of the single-domain antibody or antigen-binding fragment thereof is not less than 89%, for example, 90%, 93%, 95%, 97%, 98%, or 100%.

[0084] In certain embodiments, the single domain antibody or antigen binding fragment thereof binds to human serum albumin with an EC50 of less than about 40 nM, such as less than about 30 nM, 20 nM, 10 nM, 8 nM, 5 nM, 3 nM, 1 nM or less. In certain embodiments, the EC50 is measured by ELISA technology.

[0085] The single-domain antibodies of the present application are not limited to a specific biological source or specific preparation method. For example, the single-domain antibodies of the present application can be obtained by: (1) "humanizing" a naturally occurring VHH domain or by expressing a nucleic acid encoding such a humanized VHH domain; (2) using synthetic or semi-synthetic techniques to prepare a protein, polypeptide or other amino acid sequence; (3) using nucleic acid synthesis techniques to prepare a nucleic acid encoding a single-domain antibody and then expressing the nucleic acid obtained; and / or (4) by any combination of the foregoing.

[0086] Multispecific antibodies

[0087] In a third aspect, the present application provides a multispecific antibody comprising the single-domain antibody or antigen-binding fragment thereof described in the second aspect of the present application, and another antibody or antigen-binding fragment thereof, or antibody analog.

[0088] In certain embodiments, the additional antibody, or antigen-binding fragment thereof, or antibody analog has a different binding specificity than the aforementioned single domain antibody or antigen-binding fragment thereof. In certain embodiments, the additional antibody, or antigen-binding fragment thereof, or antibody analog does not bind to serum albumin.

[0089] In certain embodiments, the single domain antibody or antigen binding fragment thereof is capable of extending the in vivo half-life of the additional antibody or antigen binding fragment thereof, or antibody analog to which it is linked, and the extension of the in vivo half-life is relative to the in vivo half-life of the additional antibody or antigen binding fragment thereof, or antibody analog in the absence of the single domain antibody or antigen binding fragment thereof.

[0090] In certain embodiments, the multispecific antibody comprises one single-domain antibody or antigen-binding fragment thereof, and one or more (e.g., two) additional antibodies or antigen-binding fragments thereof, or antibody analogs; wherein the multiple additional antibodies or antigen-binding fragments thereof, or antibody analogs may have different binding specificities from each other.

[0091] In certain embodiments, the multispecific antibody comprises a single domain antibody or antigen-binding fragment thereof and an additional antibody or antigen-binding fragment thereof, or antibody analog.

[0092] In certain embodiments, the multispecific antibody comprises one single domain antibody or antigen-binding fragment thereof, and two additional antibodies or antigen-binding fragments thereof, or antibody analogs.

[0093] In certain embodiments, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0094] In certain embodiments, the additional antibody or antigen-binding fragment thereof, or antibody analog has molecule or antigen binding specificity to at least one of a tumor-specific antigen (TSA), a tumor-associated antigen (TAA), a T cell engager molecule, a tumor immunity-related molecule, or an autoimmune regulation-related molecule.

[0095] In certain embodiments, the tumor-specific antigen (TSA) or tumor-associated antigen (TAA) includes but is not limited to: claudin, P53, KRAS, NRAS, alpha-fetoprotein, carcinoembryonic antigen, ganglioside, mucin.

[0096] In certain embodiments, the additional antibody or antigen-binding fragment thereof, or antibody analog has the activity of binding to one or more molecules associated with tumor immunity, wherein the one or more molecules associated with tumor immunity include: PD-1, PD-L1, CTLA4, LAG3.

[0097] In certain embodiments, the additional antibody or antigen-binding fragment thereof, or antibody analog has binding specificity for one or more T cell engager molecules including but not limited to CD19, CD20.

[0098] In certain preferred embodiments, the additional antibody or antigen-binding fragment thereof, or antibody analog has antigen-binding specificity for one or more autoimmune regulatory targets including but not limited to IL6, IL6R, TNFα.

[0099] In certain preferred embodiments, the additional antibody or antigen-binding fragment thereof, or antibody analog has antigen-binding specificity for at least one of PD-1, PD-L1, CD19, CD20, CTLA4, IL6, IL6R or TNFα.

[0100] In certain embodiments, the multispecific antibody is a bispecific antibody, wherein the bispecificity is the binding specificity of "HSA and PD-L1", "HSA and PD-1", "HSA and CTLA4", "HSA and CD19", "HSA and CD20", "HSA and IL6", "HSA and IL6R" or "HSA and TNFα".

[0101] In certain embodiments, the multispecific antibody is a trispecific antibody. In certain embodiments, the trispecificity is binding to "HSA, PD-1 and PD-L1", "HSA, PD-1 and CTLA4", "HSA, PD-L1 and CTLA4", "HSA, CD19 and CD20", "HSA, IL6R and TNFα", or "HSA, IL6 and TNFα".

[0102] In certain embodiments, the multispecific antibody is a tetraspecific antibody. In certain embodiments, the tetraspecificity of the multispecific antibody includes binding specificity for "HSA, PD-1 and PD-L1", "HSA, PD-1 and CTLA4", "HSA, PD-L1 and CTLA4", "HSA, CD19 and CD20", "HSA, IL6R and TNFα", or "HSA, IL6 and TNFα".

[0103] In certain embodiments, the multispecific antibody has the following structure from its N-terminus to its C-terminus: ALB or BLA, wherein: A is the single-domain antibody or antigen-binding fragment thereof of the present application, L is a linker or deletion, and B is one or more additional antibodies or antigen-binding fragments thereof, or antibody analogs; wherein the multiple additional antibodies or antigen-binding fragments thereof, or antibody analogs have the activity of binding to the same antigen or different antigens.

[0104] In certain embodiments, the multispecific antibody has a structure selected from the following from its N-terminus to the C-terminus: B1-L1-A-L2-B2, A-L1-B1-L2-B2, B1-L1-B2-L2-A, wherein: A is the single-domain antibody or antigen-binding fragment thereof of the present application, L1 and L2 are each independently a linker or a deletion, and B1 and B2 are each independently another antibody or antigen-binding fragment thereof, or an antibody analog; wherein B1 and B2 have the activity of binding to the same antigen or different antigens.

[0105] In certain embodiments, L, L1, and L2 are each independently (G4S) n , wherein n is selected from an integer of 0-10, and L, L1 and L2 are the same or different from each other. In certain embodiments, n is selected from an integer of 0-6, such as 0, 1, 2, 3, 4, 5 or 6. In certain embodiments, L, L1 and L2 are each independently selected from the sequence shown in SEQ ID NO: 33 or 34.

[0106] In certain embodiments, the additional antibody or antigen-binding fragment thereof, or antibody analog is selected from a full-length antibody, a scFv, a single domain antibody, or an antibody analog.

[0107] In certain embodiments, the multispecific antibody is a bispecific antibody, which comprises a single-domain antibody or antigen-binding fragment thereof of the present application, and one or more additional antibodies or antigen-binding fragments thereof, or antibody analogs, wherein the one or more additional antibodies or antigen-binding fragments thereof, or antibody analogs do not bind to serum albumin, and the multiple antibodies or antigen-binding fragments thereof, or antibody analogs have the same antigen-binding activity.

[0108] In certain embodiments, the multispecific antibody is a bispecific antibody having the following structure: N-single domain antibody or antigen-binding fragment thereof of the present application-(Linker)-one of the other antibodies or antigen-binding fragments thereof, or antibody analogs.

[0109] In certain embodiments, the multispecific antibody has the following structure: N-single-domain antibody or antigen-binding fragment thereof of the present application-(Linker)-plurality of the additional antibodies or antigen-binding fragments thereof, or antibody analogs. The additional antibodies or antigen-binding fragments thereof, or antibody analogs are optionally connected by a linker.

[0110] In certain embodiments, the multispecific antibody has the following structure: N-plurality of additional antibodies, antigen-binding fragments thereof, or antibody analogs-(Linker)-single-domain antibody or antigen-binding fragment thereof of the present application. The plurality of additional antibodies, antigen-binding fragments thereof, or antibody analogs are optionally connected by a linker.

[0111] In certain embodiments, the multispecific antibody is a bispecific antibody having the following structure: N-another antibody or antigen-binding fragment thereof, or antibody analog-(Linker)-single domain antibody or antigen-binding fragment thereof of the present application.

[0112] In certain embodiments, the multispecific antibody is a trispecific antibody having the following structure: N-another antibody or its antigen-binding fragment, or antibody analog-(Linker)-the single domain antibody or its antigen-binding fragment of the present application-(Linker)-another antibody or its antigen-binding fragment, or antibody analog.

[0113] In the embodiments described above, the single-domain antibody or antigen-binding fragment thereof of the present application is linked to the N-terminus or C-terminus of the heavy chain or light chain of one or more additional antibodies or antigen-binding fragments thereof.

[0114] In certain embodiments, the multispecific antibody is a trispecific antibody, comprising the single domain antibody or antigen-binding fragment thereof of the present application and another antibody or antigen-binding fragment thereof, wherein the other antibody or antigen-binding fragment thereof provides the trispecific antibody with two additional antigen-binding activities. The construction of the trispecific antibody can be flexible, and the other antibodies or antigen-binding fragments thereof can all be located at the N-terminus or C-terminus of the single domain antibody or antigen-binding fragment thereof of the present application, or each can be located at the N-terminus or C-terminus of the single domain antibody or antigen-binding fragment thereof. The presence, length, and number of linkers can also be adjusted according to the purpose of constructing the trispecific antibody.

[0115] In the embodiment described above, the multispecific antibody has (G4S) n A linker wherein n is selected from an integer from 0 to 10. In certain embodiments, n is selected from an integer from 0 to 6, such as 0, 1, 2, 3, 4, 5 or 6.

[0116] In certain embodiments, the multispecific antibody described above has a linker shown in SEQ ID NO: 33 or 34.

[0117] Fusion protein

[0118] In a fourth aspect, the present application provides a fusion protein comprising the single-domain antibody or antigen-binding fragment thereof according to the second aspect, and another biologically active polypeptide.

[0119] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof is capable of extending the in vivo half-life of another biologically active polypeptide to which it is linked, and the extension of the in vivo half-life is relative to the in vivo half-life of the other biologically active polypeptide in the absence of the single-domain antibody or antigen-binding fragment thereof.

[0120] In certain embodiments, the additional biologically active polypeptide is a polypeptide or protein having therapeutic activity, binding activity, or enzymatic activity.

[0121] In certain embodiments, the additional biologically active polypeptide is an additional antibody or antigen-binding fragment thereof, or an antibody analog.

[0122] In certain embodiments, the additional biologically active polypeptide is linked directly or via a linker to the N-terminus and / or C-terminus of the single domain antibody or antigen-binding fragment thereof.

[0123] In certain embodiments, the linker is (G4S)n, where n is an integer greater than or equal to 0. In certain embodiments, n is an integer selected from 0-6, such as 0, 1, 2, 3, 4, 5 or 6. In certain embodiments, the linker is as shown in SEQ ID NO: 33 or 34.

[0124] In certain embodiments, the fusion protein has the following structure from its N-terminus to its C-terminus: ALB or BLA, wherein: A is the single-domain antibody or antigen-binding fragment thereof of the present application, L is a linker or deletion, and B is one or more additional biologically active polypeptides.

[0125] In certain embodiments, the fusion protein has a structure selected from the following from its N-terminus to the C-terminus: B1-L1-A-L2-B2, A-L1-B1-L2-B2, B1-L1-B2-L2-A, wherein: A is the single-domain antibody or antigen-binding fragment thereof of the present application, L1 and L2 are each independently a linker or a deletion, and B1 and B2 are each independently another biologically active polypeptide.

[0126] In certain embodiments, L, L1, and L2 are each independently (G4S) n , wherein n is selected from an integer of 0-10, and L, L1 and L2 are the same or different from each other. In certain embodiments, n is selected from an integer of 0-6, such as 0, 1, 2, 3, 4, 5 or 6. In certain embodiments, L, L1 and L2 are each independently selected from the sequence shown in SEQ ID NO: 33 or 34.

[0127] In certain embodiments, the fusion protein comprises one or more (e.g., two) of the single-domain antibodies or antigen-binding fragments thereof and one or more (e.g., two) of the additional biologically active polypeptides; wherein the multiple single-domain antibodies or antigen-binding fragments thereof are the same or different, and the multiple additional biologically active polypeptides are the same or different.

[0128] In certain embodiments, the fusion protein comprises one of the single-domain antibodies or antigen-binding fragments thereof and one of the additional biologically active polypeptides.

[0129] In certain embodiments, the fusion protein comprises one of the single-domain antibodies or antigen-binding fragments thereof and two of the additional biologically active polypeptides.

[0130] In certain embodiments, the two additional biologically active polypeptides are each linked directly or via a linker to the N-terminus and / or C-terminus of the single domain antibody or antigen-binding fragment thereof, respectively.

[0131] In certain embodiments, the two additional biologically active polypeptides are linked to each other directly or via a linker, and the two additional biologically active polypeptides linked to each other are further linked to the N-terminus or C-terminus of the single domain antibody or antigen-binding fragment thereof directly or via a linker.

[0132] In certain embodiments, the two additional biologically active polypeptides are each linked directly or through a linker to the N-terminus and C-terminus, or the C-terminus and N-terminus, of the single-domain antibody or antigen-binding fragment thereof.

[0133] In certain embodiments, the biologically active polypeptide is selected from colony stimulating factor (CSF), G protein-coupled receptor, IL-15, IL15RD, IL15RD and IL-15 fusion protein, human growth hormone (GH), cytokine binding protein, protein A, allergy inhibitory factor, necroptosis glycoprotein, immunotoxin, lymphotoxin, tumor suppressor, transforming growth factor, α1-antitrypsin, albumin, α-lactalbumin, apolipoprotein-E, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, bone stimulating protein, calcitonin, atrial natriuretic peptide, chondroitin, elcatonin, connective tissue activating factor, tissue factor pathway inhibitor, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing factor, autotoxin, lactoferrin, myostatin, a member of the heat shock protein family (such as heat shock protein 70, GP96), or any combination thereof.

[0134] In certain embodiments, the biologically active polypeptide is selected from G-CSF, GH, IL-15, IL15RD and IL-15 fusion protein, or any combination thereof.

[0135] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 3 or a variant sequence thereof, wherein the variant sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO: 3, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 3, and the variant sequence retains the reduced immunogenicity to humans and / or specific binding activity to human serum albumin of the sequence from which it is derived; and, according to the IMGT numbering system, the single domain antibody or antigen-binding fragment thereof comprises: E at position 1, L at position 5, A at position 24, L at position 50, at position 108;

[0136] Furthermore, the bioactive polypeptide is selected from G-CSF, GH, IL-15, IL15RD, IL15RD and IL-15 fusion protein, or any combination thereof.

[0137] In certain embodiments, the additional biologically active polypeptide is selected from the group consisting of the sequence set forth in SEQ ID NO: 23 or a variant thereof having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO: 23. The variant sequence retains the biological function of the biologically active polypeptide sequence from which it is derived, e.g., stimulation of neutrophil production.

[0138] In certain embodiments, the additional biologically active polypeptide is selected from the following sequences: the sequence set forth in SEQ ID NO: 27 or a variant sequence thereof, wherein the variant sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO: 27. The variant sequence retains the biological function of the biologically active polypeptide sequence from which it is derived, for example, promoting human growth.

[0139] In certain embodiments, the additional biologically active polypeptide is selected from the following sequences: a sequence as set forth in any one of SEQ ID NOs: 29-31, or a variant sequence thereof, wherein the variant sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the aforementioned sequences. The variant sequence retains the biological function of the biologically active polypeptide sequence from which it is derived, for example, stimulating NK cells and enhancing immunity.

[0140] In certain embodiments, the fusion protein has a sequence selected from the following: a sequence shown in any one of SEQ ID NOs: 24, 25, 26, or a variant sequence thereof, wherein the variant sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence shown in any one of SEQ ID NOs: 24, 25, 26. The variant sequence retains the biological function of the sequence from which it is derived, for example, reduced immunogenicity to humans compared to camel-derived antibodies, activity that specifically binds to human serum albumin, and activity that stimulates neutrophil production.

[0141] In certain embodiments, the fusion protein has a sequence selected from the group consisting of: a sequence shown in SEQ ID NO: 28 or 38, or a variant sequence thereof, wherein the variant sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28 or 38. The variant sequence retains the biological function of the sequence from which it is derived, for example, reduced immunogenicity against humans compared to camelid antibodies, activity that specifically binds to human serum albumin, and activity that promotes human growth.

[0142] In certain embodiments, the fusion protein has a sequence selected from the following: the sequence shown in SEQ ID NO: 32 or a variant sequence thereof, wherein the variant sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO: 32. The variant sequence retains the biological function of the sequence from which it is derived, for example, reduced immunogenicity against humans compared to camel-derived antibodies, activity that specifically binds to human serum albumin, and activity that stimulates NK cells and enhances immunity.

[0143] In certain embodiments, the fusion protein comprises the single-domain antibody or antigen-binding fragment thereof according to the second aspect and G-CSF (e.g., G-CSF as shown in SEQ ID NO: 23), and the fusion protein has an EC of less than about 40 nM, for example, less than about 30 nM, 20 nM, 15 nM, 10 nM, 8 nM, 5 nM, 3 nM, 1 nM or less. 50 Binds to human serum albumin; preferably, the EC 50 Measured by ELISA technology.

[0144] In certain embodiments, the fusion protein comprises the single domain antibody or antigen-binding fragment thereof according to the second aspect and GH (e.g., the GH shown in SEQ ID NO: 27), and the fusion protein has an EC of less than about 40 nM, such as less than about 30 nM, 20 nM, 15 nM, 10 nM, 8 nM, 5 nM, 3 nM, 1 nM or less. 50 Binds to human serum albumin; preferably, the EC 50 Measured by ELISA technology.

[0145] In certain embodiments, the fusion protein comprises the single-domain antibody or antigen-binding fragment thereof according to the second aspect and one selected from IL-15, IL15RD, or a fusion protein of IL15RD and IL-15 (e.g., a fusion protein of IL15RD and IL-15 as shown in SEQ ID NO: 31), wherein the fusion protein has an EC of less than about 40 nM, for example, less than about 30 nM, 20 nM, 15 nM, 10 nM, 8 nM, 5 nM, 3 nM, 1 nM or less. 50 Binds to human serum albumin; preferably, the EC 50 Measured by ELISA technology.

[0146] In certain embodiments, the fusion protein comprises the single-domain antibody or antigen-binding fragment thereof described in the second aspect, another antibody or antigen-binding fragment thereof, or antibody analog, and a biologically active polypeptide. The other antibody or antigen-binding fragment thereof, or antibody analog has a different binding specificity than the single-domain antibody or antigen-binding fragment thereof, exemplary antibodies or antigen-binding fragments thereof, or antibody analogs are as described in the multispecific antibodies section of this specification, and exemplary biologically active polypeptides are as described above.

[0147] Conjugate

[0148] In a fifth aspect, the present application provides a conjugate comprising the single-domain antibody or antigen-binding fragment thereof according to the second aspect and a chemotherapy drug, a radiopharmaceutical, or a developer connected to the single-domain antibody or antigen-binding fragment thereof.

[0149] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof is capable of extending the in vivo half-life of a chemotherapeutic drug, radiopharmaceutical, or imaging agent to which it is linked, and the extension of the in vivo half-life is relative to the in vivo half-life of the chemotherapeutic drug, radiopharmaceutical, or imaging agent in the absence of the single-domain antibody or antigen-binding fragment thereof.

[0150] In certain embodiments, the chemotherapy drug is selected from one or more of the following: 13-cis-Retinoic Acid, 2-CdA, 2-Chlorodeoxyadenosine, 5-Azacitidine, 5-Fluorouracil, 5-FU, 6-Mercaptopurine, 6-MP, 6-TG, 6-Thioguanine, A, Abraxane, Actinomycin-D, ALIMTA, Alitretinoin, All-transretinoic Acid, Acid, Altretamine, Amethopterin, Amifostine, Aminoglutethimide, Anagrelide, Anastrozole, Arabinosylcytosine, Ara-C, Arsenic Trioxide, Asparaginase, ATRA, Azacitidine, BCG, BCNU, Bexarotene, Bicalutamide, BiCNU, Bleomycin, Bortezomib, Busulfan, C225, Calcium folinate Leucovorin, Camptothecin-11, Capecitabine, CaracTM, Carboplatin, Carmustine, Carmustine Wafer, Conside CC-5013, CCNU, CDDP, CeeNU, Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine, Cortisone, CPT-11, Cyclophosphamide, Cytarabine, Cytarabine Liposomal, Dacarbazine, Dacogen, Dactinomycin, Darbepoetin Alfa, Dasatinib,Daunomycin, Daunorubicin, Daunorubicin Hydrochloride, Daunorubicin Liposomal, Decadron, Decitabine, DepoCyt™, Dexamethasone, Dexamethasone acetate, Dexamethasone Sodium Phosphate, Dexamethasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel, Doxorubicin, Doxorubicin Liposomal liposomal, Droxia™, DTIC, Eligard™, Ellence™, Eloxatin™, Epirubicin, Epoetin alfa, Erbitux™, Erlotinib, Erwinia L-asparaginase, Estramustine, Ethyol, Etoposide, Etoposide phosphate Phosphate, Exemestane, Filgrastim, Floxuridine, Fludarabine, Fluorouracil, Fluorouracil (Cream), Fluoxymesterone, Flutamide, Folinic Acid Acid), Fulvestrant, Gefitinib, Gemcitabine, Ozogamicin, GleevecTM, ​​Wafer, Goserelin, Hexadrol, Hexamethylmelamine, HMM, Hydrocortisone acetate, Hydrocortisone, Hydrocortisone sodium phosphate, Hydrocortisone sodium succinate,Hydrocortone Phosphate, Hydroxyurea, Idarubicin, Ifosfamide, Imatinib mesylate, Imidazole Carboxamide, Irinotecan, Isotretinoin, Lapatinib, L-Asparaginase, LCR, Lenalidomide, Letrozole, Leucovorin, Chlorambucil, Leukine™, Leuprolide, Leurocristine, Leustatin™, Liposomal Ara-C, Liquid Lomustine ine), L-PAM, L-Sarcolysin, LupronM, Maxidex, Mechlorethamine, Mechlorethamine Hydrochloride, Megestrol, Megestrol Acetate, Melphalan, Mercaptopurine, Mesna, MesnexTM, Methotrexate, Methotrexate Sodium Sodium, Methylprednisolone, Mitomycin, Mitomycin-C, Mitoxantrone, MTC, MTX, Mustine, Mylocel™, Nelarabine, Neulasta™, Nilutamide, Nitrogen Mustard, Octreotide, Octreotide acetate acetate), OnxalTM, Oprevelkin, Oxaliplatin, Paclitaxel, Protein-bound Paclitaxel, Pamidronate, Panitumumab, Pegaspargase, PEG-L-asparaginase, Pemetrexed, Pentostatin,Phenylalanine Mustard, Prednisolone, Prednisone, Procarbazine, Prolifeprospan 20 with Carmustine Implant, Raloxifene, Rubidomycin Hydrochloride, Sandostatin, Sargramostim, Sorafenib, SPRYCEL™, STI-571, Streptozocin, SU11248, Sunitinib, Tamoxifen, Taxol, Temozolomide, Teniposide, TESPA, Thalidomide, Thioguanine, Thioguanine Thiophosphoamide, Thiotepa, Topotecan, Toremifene, Tretinoin, Trexall™, TSPA, VCR, Vectibix™, Viadur™, Vinblastine, Vinblastine Sulfate, Vincasar, Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VM-26, Vorinostat, VP-16, Zevalin™, Zoledronic acid, or Zolinza.

[0151] In certain embodiments, the radiopharmaceutical is selected from one or more of the following: carbon-11, carbon-14, chromium-51, cobalt-57, cobalt-58, erbium-169, fluorine-18, gallium-67, gold-198, indium-111, indium-113m, iodine-123, iodine-125, iodine-131, iron-59, krypton-81m, nitrogen-13, oxygen-15, phosphorus-32, rhenium-186, rubidium-82, samarium-153, selenium-75, strontium-89, technetium-99m, thallium-201, tritium, xenon-127, xenon-133, or yttrium-90.

[0152] In certain embodiments, the imaging agent is selected from one or more of the following: gadolinium, magnetite, manganese, technetium, I125, I131, P32, Tl201, iopamidol, or PET-FDG.

[0153] Preparation of antibodies and fusion proteins

[0154] The single domain antibody, multispecific antibody or fusion protein of the present application can be prepared by various methods known in the art, for example, by genetic engineering recombinant technology. For example, a DNA molecule encoding the single domain antibody, multispecific antibody or fusion protein of the present application is obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into a host cell. The transfected host cell is then cultured under specific conditions and expresses the single domain antibody, multispecific antibody or fusion protein of the present application.

[0155] The antigen-binding fragments of the present application can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). In addition, these antigen-binding fragments can also be directly produced by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, Fab' fragments can be directly obtained from host cells; Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). In addition, Fv, Fab, or F(ab')2 fragments can also be directly isolated from the culture medium of recombinant host cells. The preparation of the single domain antibody or antigen binding fragment thereof of the present application can also refer to Muyldermans, Reviews in Molecular Biotechnology 74, 277-302 (2001), J. Sambrook & D. Russell, Molecular Cloning: A Laboratory Manual (3 rd

[00145] (Cold Spring Harbor Laboratory Press, 2000), or its Chinese translation published by Science Press of China. Other techniques for preparing these antigen-binding fragments are well known to those skilled in the art.

[0156] In a sixth aspect, the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the single-domain antibody or antigen-binding fragment thereof described in the second aspect, the multispecific antibody described in the third aspect, or the fusion protein described in the fourth aspect. Based on codon degeneracy known in the art, in certain embodiments, the nucleotide sequence can be replaced based on codon degeneracy. In certain embodiments, the nucleotide sequence is codon-optimized.

[0157] In certain embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding the single-domain antibody or antigen-binding fragment thereof of the second aspect, a nucleotide sequence encoding the multispecific antibody of the third aspect, or a nucleotide sequence encoding the fusion protein of the fourth aspect. In certain embodiments, the nucleotide sequence is selected from: (a). The nucleotide sequence as shown in SEQ ID NO: 35 or a degenerate sequence thereof, or (b). A sequence substantially identical to the nucleotide sequence of (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity, or a sequence having one or more nucleotide substitutions compared to the nucleotide sequence of (a)), or (c). A sequence that differs from the nucleotide sequence of (a) by no more than 3, 6, 15, 30, or 45 nucleotides.

[0158] In certain embodiments, the isolated nucleic acid molecule comprises (a) a sequence as set forth in SEQ ID NO: 36 or a degenerate sequence thereof, or (b) a sequence substantially identical to the nucleotide sequence of (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity, or a sequence having one or more nucleotide substitutions, compared to the nucleotide sequence of (a)), or (c) a sequence that differs from the nucleotide sequence of (a) by no more than 3, 6, 15, 30, or 45 nucleotides.

[0159] In a seventh aspect, the present application provides a vector comprising the isolated nucleic acid molecule described in the sixth aspect. In certain embodiments, the vector of the present application is, for example, a plasmid, a cosmid, a phage, a lentivirus, etc. In certain embodiments, the vector is capable of expressing the single-domain antibody or antigen-binding fragment thereof, multispecific antibody, or fusion protein of the present application in a subject (e.g., a mammal, such as a human).

[0160] In an eighth aspect of the present application, a host cell is provided, which comprises the nucleic acid molecule of the sixth aspect and / or the vector of the seventh aspect. The host cell can be a eukaryotic cell (e.g., mammalian cell, insect cell, yeast cell) or a prokaryotic cell (e.g., Escherichia coli). Suitable eukaryotic cells include but are not limited to NSO cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include but are not limited to Sf9 cells. In certain embodiments, the host cell is Escherichia coli.

[0161] The present application also provides a method for preparing the single-domain antibody or antigen-binding fragment thereof described in the second aspect, the multispecific antibody described in the third aspect, or the fusion protein described in the fourth aspect, which comprises culturing the host cell described in the eighth aspect under conditions that allow expression of the protein (e.g., the single-domain antibody or antigen-binding fragment thereof, multispecific antibody, or fusion protein), and recovering the single-domain antibody or antigen-binding fragment thereof, multispecific antibody, or fusion protein from the cultured host cell culture.

[0162] In certain embodiments, the host cell is E. coli.

[0163] In certain embodiments, the recovery includes collecting the cells and purifying them, including the step of subjecting the renatured product of the protein inclusion bodies to chromatography. In certain embodiments, the cells are collected by centrifugation and the inclusion bodies are obtained by crushing the cells. In certain embodiments, the inclusion bodies are further washed with a washing solution to remove host proteins or impurities. In certain embodiments, the inclusion bodies are denatured and renatured, and then eluted on a chromatography column to obtain the single-domain antibody or its antigen-binding fragment, multi-specific antibody, or fusion protein. In certain embodiments, the washing solution is 20mM Tris + 1mM EDTA + 0.1% TritonX-001 + 1.5M urea (pH 8.0) and / or 20mM Tris-HCl + 1mM EDTA (pH 8.5).

[0164] Uses and pharmaceutical compositions

[0165] In a ninth aspect, the present application provides a pharmaceutical composition comprising the single-domain antibody or antigen-binding fragment thereof described in the second aspect, the multispecific antibody described in the third aspect, the fusion protein described in the fourth aspect, the conjugate described in the fifth aspect, the isolated nucleic acid molecule described in the sixth aspect, the vector described in the seventh aspect, or the host cell described in the eighth aspect; and a pharmaceutically acceptable carrier and / or excipient. The pharmaceutical composition exhibits a prolonged in vivo half-life compared to a corresponding pharmaceutical composition lacking the single-domain antibody or antigen-binding fragment thereof.

[0166] In certain embodiments, the pharmaceutical composition of the present application comprises the single-domain antibody or antigen-binding fragment thereof of the present application, and an active agent linked to the single-domain antibody or antigen-binding fragment thereof.

[0167] In certain embodiments, the active agent is another antibody or its antigen-binding fragment, or antibody analog. In certain embodiments, the other antibody or its antigen-binding fragment, or antibody analog is as defined in the third aspect of the present application. In certain embodiments, the pharmaceutical composition comprises a multispecific antibody of the present application.

[0168] In certain embodiments, the active agent is another biologically active polypeptide. In certain embodiments, the other biologically active polypeptide is as defined in the fourth aspect of the present application. In certain embodiments, the pharmaceutical composition comprises a fusion protein of the present application.

[0169] In certain embodiments, the active agent is a chemotherapeutic drug, a radiopharmaceutical, or an imaging agent. In certain embodiments, the chemotherapeutic drug, radiopharmaceutical, or imaging agent is as defined in the fifth aspect of the present application. In certain embodiments, the pharmaceutical composition comprises a conjugate of the present application.

[0170] In certain embodiments, the pharmaceutical composition of the present application comprises the vector or host cell of the present application, and a pharmaceutically acceptable carrier and / or excipient. In such embodiments, the host cell comprises an isolated nucleic acid molecule or vector as described above.

[0171] In certain embodiments, the pharmaceutical composition may further comprise additional pharmaceutically active agents, such as drugs having anti-tumor activity; preferably, the additional pharmaceutically active agents include anti-PD-1 / PD-L1 antibodies, anti-CTLA-4 antibodies, anti-TIM-3 antibodies, anti-LAG-3 antibodies or anti-TIGIT antibodies.

[0172] In certain embodiments, in the pharmaceutical composition, the single-domain antibody or antigen-binding fragment thereof, multispecific antibody, fusion protein or conjugate of the present application and the additional pharmaceutically active agent are provided as separate components or as components of the same composition. Thus, the antibody or antigen-binding fragment thereof of the present application and the additional pharmaceutically active agent can be administered simultaneously, separately or sequentially.

[0173] In the tenth aspect of the present application, a kit is provided, comprising the antibody or antigen-binding fragment thereof described in the second aspect, the multispecific antibody described in the third aspect, the fusion protein described in the fourth aspect, the conjugate described in the fifth aspect, the isolated nucleic acid molecule described in the sixth aspect, the vector described in the seventh aspect, the host cell described in the eighth aspect, or the pharmaceutical composition described in the ninth aspect; and optionally instructions for use.

[0174] In another aspect, the present application provides use of the single-domain antibody or antigen-binding fragment thereof according to the second aspect for prolonging the in vivo half-life of an active agent linked thereto.

[0175] In another aspect, the present application provides a method for extending the in vivo half-life of an active agent, comprising linking the active agent to the single domain antibody or antigen-binding fragment thereof according to the second aspect.

[0176] In certain embodiments, the extension of in vivo half-life is relative to the in vivo half-life of the active agent in the absence of the single domain antibody or antigen-binding fragment thereof.

[0177] In certain embodiments, the active agent is another antibody or antigen-binding fragment thereof, or antibody analog. In certain embodiments, the other antibody or antigen-binding fragment thereof, or antibody analog is as defined in the third aspect of the present application.

[0178] In certain embodiments, the active agent is another biologically active polypeptide. In certain embodiments, the other biologically active polypeptide is as defined in the fourth aspect of the present application.

[0179] In certain embodiments, the active agent is a chemotherapeutic drug, a radiopharmaceutical, or an imaging agent. In certain embodiments, the chemotherapeutic drug, a radiopharmaceutical, or an imaging agent is as defined in the fifth aspect of the present application.

[0180] In certain embodiments, the linkage is fusion or chemical coupling.

[0181] On the other hand, the present application provides the use of the single-domain antibody or antigen-binding fragment thereof of the second aspect, the multispecific antibody of the third aspect, the fusion protein of the fourth aspect, the conjugate of the fifth aspect, the isolated nucleic acid molecule of the sixth aspect, the vector of the seventh aspect, or the host cell of the eighth aspect in the preparation of a drug, wherein the drug exhibits an extended in vivo half-life relative to a corresponding drug lacking the single-domain antibody or antigen-binding fragment thereof.

[0182] In certain embodiments, the drug is a protein drug.

[0183] In certain embodiments, the present application provides use of the single-domain antibody or antigen-binding fragment thereof according to the second aspect in the preparation of a drug, wherein the drug exhibits a prolonged in vivo half-life relative to a corresponding drug lacking the single-domain antibody or antigen-binding fragment thereof.

[0184] In certain embodiments, the present application provides use of the multispecific antibody described in the third aspect in the preparation of a drug, wherein the multispecific antibody comprises the single-domain antibody or antigen-binding fragment thereof of the present application, and another antibody or antigen-binding fragment thereof, or antibody analog; wherein the drug exhibits a prolonged in vivo half-life relative to a corresponding drug lacking the single-domain antibody or antigen-binding fragment thereof.

[0185] In certain embodiments, the present application provides use of the fusion protein described in the fourth aspect in the preparation of a drug, wherein the fusion protein comprises the single-domain antibody or its antigen-binding fragment of the present application, and another biologically active polypeptide; wherein the drug shows a prolonged in vivo half-life relative to a corresponding drug lacking the single-domain antibody or its antigen-binding fragment.

[0186] In certain embodiments, the present application provides use of the conjugate described in the fifth aspect in the preparation of a drug, wherein the conjugate comprises the single-domain antibody or antigen-binding fragment thereof of the present application, and a chemotherapy drug, a radioactive drug or an imaging agent; wherein the drug exhibits an in vivo half-life that is prolonged compared to a corresponding drug lacking the single-domain antibody or antigen-binding fragment thereof.

[0187] In certain embodiments, the present application provides use of the isolated nucleic acid molecule described in the sixth aspect in preparing a drug.

[0188] In certain embodiments, the present application provides use of the carrier described in the seventh aspect in preparing a drug.

[0189] In certain embodiments, the present application provides use of the host cell described in the eighth aspect in preparing a drug.

[0190] On the other hand, the present application provides the use of the single-domain antibody or its antigen-binding fragment described in the second aspect, the multispecific antibody described in the third aspect, the fusion protein described in the fourth aspect, the conjugate described in the fifth aspect, the isolated nucleic acid molecule described in the sixth aspect, the vector described in the seventh aspect, or the host cell described in the eighth aspect in the preparation of a drug for the prevention and / or treatment or adjuvant treatment of tumors, infections, autoimmune diseases, metabolism-related diseases, or development-related diseases.

[0191] In certain embodiments, the present application provides the use of the multispecific antibody described in the third aspect in the preparation of a medicament for the prevention and / or treatment or adjuvant treatment of tumors, infections, autoimmune diseases, metabolism-related diseases, or development-related diseases, wherein the multispecific antibody comprises the single-domain antibody or its antigen-binding fragment, and another antibody or its antigen-binding fragment, or antibody analog; wherein the other antibody or its antigen-binding fragment, or antibody analog has binding specificity for one or more molecules or antigens in tumor antigens (e.g., tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs)) and / or immune checkpoint proteins.

[0192] In certain embodiments, the present application provides the use of the fusion protein described in the fourth aspect in the preparation of a medicament for the prevention and / or treatment or adjuvant treatment of tumors, infections, autoimmune diseases, metabolism-related diseases, or development-related diseases, wherein the fusion protein comprises the single-domain antibody or its antigen-binding fragment, and another biologically active polypeptide; wherein the other biologically active polypeptide is a polypeptide or protein with anti-tumor activity.

[0193] In certain embodiments, the present application provides a fusion protein for the preparation of a drug for preventing or treating neutropenia or leukopenia. The fusion protein comprises any single-domain antibody or antigen-binding fragment thereof described in any second aspect of the present application and G-CSF (e.g., G-CSF shown in SEQ ID NO.23). Preferably, the fusion protein comprises any sequence in SEQ ID NOs:24-26. The fusion protein has the following uses: (1) promoting the proliferation and / or differentiation of hematopoietic stem cells, (2) increasing the number of neutrophils or leukocytes in the blood, (3) activating the function of neutrophils in the blood, (4) alleviating or eliminating bone marrow hematopoietic suppression, (5) preventing or treating infection, (6) preventing and / or treating neutropenia and / or leukopenia caused by tumor treatment, or (7) any combination of (1)-(6).

[0194] In certain embodiments, the present application provides the use of the multispecific antibody of the third aspect, the fusion protein of the fourth aspect, the conjugate of the fifth aspect, or the pharmaceutical composition of the ninth aspect in the preparation of a drug, wherein the drug is used to: (1) promote the proliferation and / or differentiation of hematopoietic stem cells, (2) increase the number of neutrophils or leukocytes in the blood, (3) activate the function of neutrophils in the blood, (4) reduce or eliminate bone marrow hematopoietic suppression, (5) prevent or treat infection, (6) prevent and / or treat neutropenia and / or leukopenia caused by tumor treatment, or (7) any combination of (1)-(6). In certain embodiments, the multispecific antibody, fusion protein, conjugate, and / or pharmaceutical composition includes G-CSF.

[0195] In certain embodiments, the neutropenia or leukopenia is associated with tumor treatment, such as tumor radiotherapy or chemotherapy. In certain embodiments, the neutropenia or leukopenia is associated with infection, such as human immunodeficiency viruses (HIV) infection.

[0196] In certain embodiments, the aforementioned fusion protein can be used in bone marrow transplantation. The aforementioned fusion protein plays one or more of the above functions in bone marrow transplantation, shortening the time it takes for a patient to acquire immunity.

[0197] In certain embodiments, the present application provides a use of a fusion protein in the preparation of a medicament for promoting physical development and / or promoting height growth. The fusion protein comprises any single-domain antibody or antigen-binding fragment thereof described in the second aspect of the present application and a growth hormone polypeptide (e.g., human growth hormone as shown in SEQ ID NO.27). Preferably, the fusion protein comprises the sequence shown in SEQ ID NO:28 or 38. The fusion protein has the following uses: (1) promoting protein synthesis, affecting fat and mineral metabolism, (2) regulating metabolism, promoting physical development, promoting height growth, (3) promoting the growth of bones, muscles, internal organs and the whole body of mammals, or (4) any combination of (1)-(3).

[0198] In certain embodiments, the present application provides a use of the multispecific antibody of the third aspect, the fusion protein of the fourth aspect, the conjugate of the fifth aspect, or the pharmaceutical composition of the ninth aspect in the preparation of a drug, wherein the drug is used to: (1) promote protein synthesis, affect fat and mineral metabolism, (2) regulate metabolism, promote body development, promote height growth, (3) promote the growth of bones, muscles, internal organs, and the whole body of a mammal, or (4) any combination of (1)-(3). In certain embodiments, the multispecific antibody, fusion protein, conjugate, and / or pharmaceutical composition includes growth hormone.

[0199] In certain embodiments, the mammal is a human; preferably, the mammal is a child.

[0200] In certain embodiments, the mammal is a non-human mammal. In such embodiments, the fusion protein can be used as a veterinary drug, for example, administered to meat-producing livestock such as pigs, cattle, and sheep; for example, for producing lean pigs.

[0201] In certain embodiments, the present application provides a use of a fusion protein in the preparation of a drug, wherein the drug is used to select one or more of the following: (1) stimulate the activation and proliferation of T lymphocytes, natural killer (NK) cells and / or NKT cells, and promote the survival of T lymphocytes, natural killer (NK) cells and / or NKT cells; (2) induce B lymphocyte proliferation and differentiation, enhance immunoglobulin secretion, and maintain memory cells and B cell differentiation; (3) induce the activity of lymphokine-activated killer cells (LAK); (4) synergistically stimulate NK cells to secrete cytokines (such as IFN-γ) with IL-12; (5) stimulate an immune response (such as an innate immune response and / or an adaptive immune response, such as a humoral immune response and / or a cellular immune response) in a subject; for example, stimulate an immune response against a tumor; and / or, (6) prevent and / or treat a tumor, such as non-small cell lung cancer, in a subject. The fusion protein comprises any single-domain antibody or antigen-binding fragment thereof according to the second aspect of the present application and a polypeptide having IL-15 activity, such as IL-15 (e.g., SEQ ID NO: 30), IL15RD (e.g., SEQ ID NO: 29), a fusion protein of IL15RD and IL-15 (e.g., SEQ ID NO: 31), or any combination thereof. In certain embodiments, the fusion protein has the sequence set forth in SEQ ID NO: 32.

[0202] In certain embodiments, the above fusion protein is co-administered with another pharmaceutically active agent (e.g., a drug with anti-tumor activity). In certain embodiments, the above fusion protein is co-administered with an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor is selected from anti-PD-1 / PD-L1 antibodies, anti-CTLA-4 antibodies, anti-TIM-3 antibodies, anti-LAG-3 antibodies, anti-TIGIT antibodies, or any combination thereof.

[0203] In certain embodiments, the above-mentioned fusion protein is administered in combination with an anti-PD-1 / PD-L1 antibody. In certain embodiments, the anti-PD-1 / PD-L1 antibody comprises a heavy chain variable region and / or a light chain variable region of any one of the following groups: (1) a heavy chain variable region and / or a light chain variable region of Nivolumab or a variant thereof, (2) a heavy chain variable region and / or a light chain variable region of Pembrolizumab or a variant thereof, (3) a heavy chain variable region and / or a light chain variable region of Atezolizumab or a variant thereof, (4) a heavy chain variable region and / or a light chain variable region of Durvalumab or a variant thereof, (5) a heavy chain variable region and / or a light chain variable region of Avelumab or a variant thereof, (6) a heavy chain variable region and / or a light chain variable region of αPD-1 or a variant thereof, wherein the αPD-1 has a heavy chain shown in SEQ ID NO: 39 and a heavy chain variable region shown in SEQ ID NO: 40. The light chain shown in NO:40, (7) the heavy chain variable region and / or light chain variable region of 5C10H2L2 or 5C10H2L2-IgG1mt, 5C10H2L2 and 5C10H2L2-IgG1mt refer to Chinese patent application CN201780002182.8. Wherein, the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions. In certain embodiments, the anti-PD-1 / PD-L1 antibody is selected from: Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab, Avelumab, αPD-1, 5C10H2L2 or 5C10H2L2-IgG1mt.

[0204] In certain embodiments, the present application provides the use of the multispecific antibody of the third aspect, the fusion protein of the fourth aspect, the conjugate of the fifth aspect, or the pharmaceutical composition of the ninth aspect in the preparation of a drug, wherein the drug is used to select one or more of the following: (1) stimulate the activation and proliferation of T lymphocytes, natural killer (NK) cells and / or NKT cells, and promote the survival of T lymphocytes, natural killer (NK) cells and / or NKT cells; (2) induce B lymphocyte proliferation and differentiation, enhance immunoglobulin secretion, and maintain memory cells and B cell differentiation; (3) induce the activity of lymphokine-activated killer cells (LAK); (4) synergistically stimulate NK cells to secrete cytokines (such as IFN-γ) with IL-12; (5) stimulate an immune response (e.g., an innate immune response and / or an adaptive immune response, such as a humoral immune response and / or a cellular immune response) in a subject; for example, stimulate an immune response against a tumor; and / or (6) prevent and / or treat a tumor, such as non-small cell lung cancer, in a subject. In certain embodiments, the multispecific antibody, fusion protein, conjugate and / or pharmaceutical composition comprises an additional biologically active polypeptide selected from IL-15, IL15RD, a fusion protein of IL15RD and IL-15, and any combination thereof.

[0205] In certain embodiments, the present application provides the use of the conjugate described in the fifth aspect in the preparation of a drug for the prevention and / or treatment or adjuvant treatment of tumors, infections, autoimmune diseases, metabolism-related diseases, or development-related diseases, wherein the conjugate comprises the single-domain antibody or its antigen-binding fragment, and a chemotherapy drug, a radiopharmaceutical, or an imaging agent.

[0206] On the other hand, the present application provides a method for preventing and / or treating and / or adjuvant treating tumors, infections, autoimmune diseases, metabolism-related diseases, or development-related diseases, wherein the method comprises administering to a subject in need thereof an effective dose of the single-domain antibody or antigen-binding fragment thereof described in the second aspect, the multispecific antibody described in the third aspect, the fusion protein described in the fourth aspect, the conjugate described in the fifth aspect, the isolated nucleic acid molecule described in the sixth aspect, the vector described in the seventh aspect, the host cell described in the eighth aspect, or the pharmaceutical composition described in the ninth aspect.

[0207] In certain embodiments, the present application provides a method for preventing and / or treating neutropenia or leukopenia, comprising administering to an individual in need thereof an effective dose of the fusion protein of the fourth aspect, wherein the fusion protein comprises G-CSF. Preferably, the fusion protein comprises any one of SEQ ID NOs: 24-26. Preferably, the individual is a human. Preferably, the neutropenia or leukopenia is related to tumor treatment.

[0208] In certain embodiments, the present application provides a method for promoting physical development and / or height growth, comprising administering to an individual in need thereof an effective dose of the fusion protein of the fourth aspect, wherein the fusion protein comprises a growth hormone polypeptide. Preferably, the fusion protein comprises the sequence set forth in SEQ ID NO: 28 or 38. Preferably, the individual is a human. Preferably, the individual is a child.

[0209] In certain embodiments, the present application provides a method for stimulating the activation and proliferation of T lymphocytes, natural killer (NK) cells and / or NKT cells in a subject, and promoting the survival of T lymphocytes, natural killer (NK) cells and / or NKT cells; (2) inducing B lymphocyte proliferation and differentiation, enhancing immunoglobulin secretion, and maintaining memory cell and B cell differentiation in a subject; (3) inducing the activity of lymphokine-activated killer cells (LAK) in a subject; (4) synergistically stimulating NK cells to secrete cytokines (such as IFN-γ) in a subject with IL-12; (5) stimulating an immune response (e.g., an innate immune response and / or an adaptive immune response, such as a humoral immune response and / or a cellular immune response) in a subject; for example, stimulating an immune response against a tumor; and / or (6) preventing and / or treating a tumor, such as non-small cell lung cancer, in a subject. The method comprises administering to a subject in need thereof an effective dose of the fusion protein of the fourth aspect, wherein the fusion protein comprises a polypeptide having IL-15 activity, such as IL-15 (e.g., SEQ ID NO: 30), IL15RD (e.g., SEQ ID NO: 29), a fusion protein of IL15RD and IL-15 (e.g., SEQ ID NO: 31), or any combination thereof. Preferably, the fusion protein comprises the sequence set forth in SEQ ID NO: 32. In certain embodiments, the subject is a human. Preferably, the subject suffers from a tumor.

[0210] In certain embodiments, the fusion protein comprising a polypeptide having IL-15 activity is co-administered with another pharmaceutically active agent (e.g., a drug with anti-tumor activity), for example, simultaneously, separately, or sequentially. In certain embodiments, the fusion protein is co-administered with an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor is selected from anti-PD-1 / PD-L1 antibodies, anti-CTLA-4 antibodies, anti-TIM-3 antibodies, anti-LAG-3 antibodies, anti-TIGIT antibodies, or any combination thereof.

[0211] In certain embodiments, the fusion protein comprising a polypeptide having IL-15 activity is administered in combination with an anti-PD-1 / PD-L1 antibody. In certain embodiments, the anti-PD-1 / PD-L1 antibody comprises a heavy chain variable region and / or a light chain variable region of any one of the following groups: (1) a heavy chain variable region and / or a light chain variable region of nivolumab or a variant thereof, (2) a heavy chain variable region and / or a light chain variable region of pembrolizumab or a variant thereof, (3) a heavy chain variable region and / or a light chain variable region of atezolizumab or a variant thereof, (4) a heavy chain variable region and / or a light chain variable region of durvalumab or a variant thereof, (5) a heavy chain variable region and / or a light chain variable region of avelumab or a variant thereof, (6) a heavy chain variable region and / or a light chain variable region of αPD-1 or a variant thereof, wherein αPD-1 has a heavy chain shown in SEQ ID NO:39 and a light chain shown in SEQ ID NO:40, and (7) a heavy chain variable region and / or a light chain variable region of 5C10H2L2 or 5C10H2L2-IgG1mt. wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0212] The single domain antibody or its antigen-binding fragment, multispecific antibody, fusion protein, conjugate or pharmaceutical composition of the present application can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The single domain antibody or its antigen-binding fragment, multispecific antibody, fusion protein, conjugate or pharmaceutical composition of the present application should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such an injection can be a sterile injection solution. For example, a sterile injection solution can be prepared by the following method: a single domain antibody or its antigen binding fragment, multispecific antibody, fusion protein, conjugate or pharmaceutical composition of the present application is incorporated into a suitable solvent at a necessary dose, and optionally, other desired ingredients (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof) are incorporated at the same time, followed by filtration sterilization. In addition, the sterile injection solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use.

[0213] The single domain antibody of the present application or its antigen-binding fragment, multi-specific antibody, fusion protein, conjugate or pharmaceutical composition can be applied by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, inguinal, intravesical, topical (such as, powder, ointment or drops), or nasal route. However, for many therapeutic uses, preferred route of administration / mode is parenteral administration (such as intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Technicians should understand that route of administration and / or mode will change according to the intended purpose. In a preferred embodiment, the single domain antibody of the present application or its antigen-binding fragment, multi-specific antibody, fusion protein, conjugate or pharmaceutical composition is given by intravenous infusion or injection.

[0214] The pharmaceutical composition of the present application may include a "therapeutically effective amount" or a "prophylactically effective amount" of the single domain antibody or its antigen-binding fragment, multispecific antibody, fusion protein or conjugate of the present application. A "prophylactically effective amount" refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of the single domain antibody or its antigen-binding fragment, multispecific antibody, fusion protein, conjugate or pharmaceutical composition of the present application may vary according to the following factors: the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0215] The single-domain antibody of the present application can efficiently bind to human serum albumin, thereby achieving a significant effect of extending the half-life, and because of its small molecular weight, it can avoid the adverse effects of fusion proteins such as HSA and Fc on activity, process and efficacy. At the same time, the single-domain antibody of the present application has an extremely high degree of humanization and can be safely administered to human subjects without triggering an immunogenic response. In addition, the single-domain antibody or fusion protein of the present application can be produced in a microbial expression system at a relatively low cost, and has great potential as a drug for the treatment of chronic diseases that require long-term, high-dose treatments. In addition, the expression amount and quality of the humanized single-domain antibody of the present application are significantly improved compared to camel-derived antibodies, which reduces aggregate formation and is conducive to improving protein stability. Therefore, the antibody or antigen-binding fragment of the present application has significant clinical value.

[0216] Definition of Abbreviations and Terms

[0217] CDR Complementarity determining region in immunoglobulin variable region

[0218] FR Antibody framework region: amino acid residues in the variable region of an antibody other than CDR residues

[0219] VH antibody heavy chain variable region

[0220] VL Antibody light chain variable region

[0221] VHH variable region domain of heavy chain antibody

[0222] IgG immunoglobulin G

[0223] Kabat The immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, eg, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0224] Chothia The immunoglobulin numbering system proposed by Chothia et al. is a classical rule identifying CDR region boundaries based on the position of structural loop regions (see, eg, Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

[0225] IMGT is based on the international ImMunoGeneTics information system initiated by Lefranc et al. For the Immunofluorescence Immunofluorescence (IMGT) numbering system, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.

[0226] K D Equilibrium dissociation constant

[0227] K a Binding rate constant

[0228] K d Dissociation rate constant

[0229] In this application, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, laboratory procedures such as cell culture, biochemistry, nucleic acid chemistry, and immunology used herein are conventional procedures widely used in the corresponding fields. To facilitate a better understanding of this application, definitions and explanations of relevant terms are provided below.

[0230] As used herein, the term "human serum albumin (HSA)" has a meaning well known to those skilled in the art. It is the most common blood protein produced naturally in the human circulatory system and has the longest half-life in the human body, with a half-life of up to 19 days. HSA and recombinant human albumin (rHSA) both have a similarly long circulatory half-life in humans. Studies have shown that therapeutic proteins genetically fused to human albumin can acquire the circulatory half-life characteristic of albumin.

[0231] As used herein, the term "granulocyte colony-stimulating factor (G-CSF)" has a meaning well known to those skilled in the art. It is a long-chain polypeptide glycoprotein derived from monocytes and fibroblasts that can induce the proliferation and differentiation of hematopoietic stem cells and increase the number of neutrophils in the blood. It also stimulates the release of mature neutrophils from the bone marrow and activates neutrophil function. It has three main functions in the body: 1. It acts on bone marrow stem cells and neutrophil precursor cells to drive the differentiation, proliferation, and maturation of neutrophils; 2. It activates mature neutrophils to participate in immune responses; and 3. It cooperates with other cytokines, such as stem cell factor IL-3, to promote hematopoietic function.

[0232] As used herein, the term "in vivo half-life" has a meaning well known to those skilled in the art and can generally be defined as the time required for the serum concentration of a molecule to decrease by 50% in vivo (e.g., due to degradation of the ligand and / or clearance or sequestration of the ligand by natural mechanisms). Methods for determining in vivo half-life are well known to those skilled in the art and can be determined, for example, by pharmacokinetic analysis, as described in Kenneth, A et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al., Pharmacokinetic analysis: A Practical Approach (1996). Reference is also made to "Pharmacokinetics", M Gibaldi & D Perron, Marcel Dekker, 2nd revised edition (1982).

[0233] The term "antibody" herein is used in the broadest sense. "Antibodies" include complete monoclonal antibodies, single domain antibodies, heavy chain antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), and antibody fragments (e.g., antigen-binding fragments) with the desired biological activity. Basic four-chain antibodies generally refer to four heteromeric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain and light chain has regularly spaced intrachain disulfide bridges. Each heavy chain and light chain has a variable domain (VH and VL) at its respective N-terminus. The variable domains of each heavy chain / light chain pair form an antigen binding site, respectively. The term "antibody" is not limited to any particular method for producing antibodies. For information on the types, structures, biological activities, and preparation methods of antibodies, see Fundamental Immunology (William E. Paul, 7th Edition, LWW, 2012); Antibody Engineering Springer Lab Manual (Roland Kontermann, Stefan Dübel, ISBN 978-3-662-04605-0 (eBook)); Schiel JE, Davis DL, Borisov OV, State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody C Haracteriazation Volume 1. Monoclonal Antibody Therapeutics: Structure, Function, and Regulatory Space (American Chemical Society, 2014). Antigen-binding fragments are antibody fragments that retain the ability to bind to specific antigens, non-limiting examples of which include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies, domain antibodies, or antigen-binding fragments of single-domain antibodies, which contain at least a portion of an antibody sufficient to confer specific antigen-binding ability.

[0234] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003).

[0235] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0236] As used herein, the term "camel-derived antibodies" refers to antibodies produced by Camelidae animals (including camels, alpacas, and llamas) against an antigen following immunization or antigen invasion. It is known to those skilled in the art that among the antibodies produced by Camelidae animals, there are "heavy-chain antibodies" (HCAbs) that lack light chains. These antibodies contain only a single heavy chain variable domain (VHH) and two conventional CH2 and CH3 regions. The separately cloned and expressed VHH region exhibits excellent structural stability and antigen-binding activity. VHH is the smallest unit currently known to bind to a target antigen.

[0237] As used herein, the terms "single domain antibody (sdAb)", "domain antibody" and "nanobody" are used interchangeably and refer to antibody fragments consisting of a single variable domain (e.g., heavy chain variable region) in an antibody. Typically, a single domain antibody, domain antibody or nanobody consists of four framework regions and three complementarity determining regions, wherein the four framework regions are FR1-FR4 and the three complementarity determining regions are CDR1-CDR3. In certain embodiments, the single domain antibody of the present application may have a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In certain embodiments, the single domain antibody is a humanized VHH. These antibodies do not require a light chain variable region and can bind to the antigen with high affinity and specificity. Compared with antibodies composed of heavy and light chains, nanobodies have high solubility and high stability to heat, pH, proteases and other deforming agents, and only single chain expression is required for large-scale production.

[0238] As used herein, the term "multispecific antibody" refers to an antibody with multiple different antigen-binding specificities, including, for example, bispecific antibodies, trispecific antibodies, and tetraspecific antibodies. A "bispecific antibody" refers to an antibody with two different antigen-binding specificities, which is a conjugate formed by a first antibody (or fragment thereof) and a second antibody (or fragment thereof) or antibody analog via a coupling arm, wherein the coupling method includes but is not limited to chemical reaction, genetic fusion, and enzymatic catalysis. A trispecific antibody is an antibody with three different antigen-binding specificities, and a tetraspecific antibody is an antibody with four different antigen-binding specificities.

[0239] As used herein, "antibody mimetic" refers to an antibody that specifically binds to an antigen like an antibody, but does not have an antibody structure. They are typically artificial peptides or proteins with a molar mass of approximately 3 to 20 kDa. For example, ankyrin repeat proteins (DARPins) and fynomers. Designed ankyrin repeat proteins (DARPins) can be linked to IgG antibodies, scFv-Fc antibody fragments, or a combination thereof, such as CN104341529A. Anti-IL-17a fynomers are bound to anti-IL-6R antibodies, such as WO2015141862A1.

[0240] As used herein, "gene" refers to a nucleic acid that encodes a protein, or a nucleic acid that regulates protein expression. The heavy chain variable region of an antibody, or immunoglobulin, is encoded by a variable segment (V gene), a diversity segment (D gene), and a joining segment (J gene). V(D)J gene rearrangement is one of the mechanisms that generate antibody diversity.

[0241] As used herein, the term "germline antibody gene" refers to an immunoglobulin sequence encoded by non-lymphocytes that has not undergone the genetic rearrangement and maturation process that leads to the expression of specific immunoglobulins. One advantage provided by the various embodiments of the present application stems from the recognition that germline antibody genes retain more of the important amino acid sequence structure characteristic of individual animal species than mature antibody genes. Therefore, when therapeutically applied to that species, they are less likely to be recognized as foreign substances by that species.

[0242] As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., FR) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc. The donor antibody can be a camel-derived antibody with the expected properties (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and / or ability to enhance immune response).

[0243] As used herein, the term "humanization degree" is an indicator used to evaluate the amino acid residue identity between an antibody and a human. The degree of humanization of a humanized antibody can be estimated, for example, by using the IMGT website DomainGapAlign to predict the homology between the sequence tested and the same human domain.

[0244] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (K) of the interaction. D ) or half-maximal effect concentration (EC 50 )express.

[0245] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate at which the antigen binding site / antigen complex forms and dissociates. The "association rate constant" (K a or K on ) and the “dissociation rate constant” (K d or K off ) Both can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio kd / kon is equal to the dissociation constant K D(See Davies et al., Annual Rev Biochem, 1990; 59: 439-473). K can be measured by any effective method. D , K on and K off In certain embodiments, the dissociation constant can be measured using bioluminescence interferometry (e.g., the ForteBio Octet method). Alternatively, the dissociation constant can be measured using surface plasmon resonance (e.g., Biacore) or Kinexa.

[0246] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0247] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0248] As used herein, the term "identity" refers to the match between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Typically, two sequences are compared when they are aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0249] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0250] The compilation of the twenty conventional amino acids involved herein follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this application, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably, consisting of two or more amino acids. And in this application, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala, arginine can be represented by R or Arg, aspartic acid can be represented by D or Asp, cysteine ​​can be represented by C or Cys, glutamine can be represented by Q or Gln, glutamic acid can be represented by E or Glu, histidine can be represented by H or His, isoleucine can be represented by I or Ile, glycine can be represented by G or Gly, asparagine can be represented by N or Asn, leucine can be represented by L or Leu, lysine can be represented by K or Lys, methionine can be represented by M or Met, phenylalanine can be represented by F or Phe, coline can be represented by P or Pro, serine can be represented by S or Ser, threonine can be represented by T or Thr, tryptophan can be represented by W or Trp, tyrosine can be represented by Y or Tyr, and valine can be represented by V or Val.

[0251] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein), or degradation products thereof (such as lactalbumin hydrolysate), etc.

[0252] As used herein, the term "prevention" refers to the method implemented in order to prevent or delay the occurrence of disease or illness or symptom in subject's body. As used herein, the term "treatment" refers to the method implemented in order to obtain beneficial or required clinical outcome. For the purpose of the application, beneficial or required clinical outcome includes but is not limited to, alleviating symptoms, narrowing the scope of the disease, stabilizing the state of (that is, no longer worsening) disease, delaying or slowing down the development of the disease, improving or alleviating the state of the disease, alleviating symptoms (no matter in part or in whole), alleviating or improving prognosis, reducing or suppressing the recurrence of disease, preventing or alleviating the generation of complication etc., whether detectable or undetectable. In addition, "treatment" can also refer to, compared with the expected survival (if not receiving treatment), prolonging survival.

[0253] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human.

[0254] As used herein, the term "about" means plus or minus 10% of the numerical value recited herein.

[0255] As used herein, the term "multiple" means ≥2.

[0256] Chinese does not have grammatical rules for singular or plural. Whether it is singular or plural depends on the context or common sense in the field. If it refers to the plural, it is correct to add "one or more" before the noun when translating.

[0257] The embodiments of the present application will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only intended to illustrate the present application and are not intended to limit the scope of the present application. Based on the following detailed description of the drawings and preferred embodiments, the various objects and advantages of the present application will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0258] Figure 1 G-CSF-PEG conjugation reaction detected by reducing SDS-PAGE. 1: Unconjugated G-CSF; 2: 0.5 h conjugation; 3: 4.5 h conjugation; 4: 20 h conjugation; 5: 24 h conjugation; M: Protein molecular weight marker. The conjugation reaction yielded a single conjugated product, while some exhibited unconjugated and multiple PEG-conjugated products.

[0259] Figure 2 : SDS-PAGE analysis of PEG-conjugated G-CSF purification. 1: First elution peak of the cation of the conjugated product, non-reduced; 2: First elution peak of the cation of the conjugated product, reduced; 3: Second elution peak of the cation of the conjugated product, non-reduced; 4: Second elution peak of the cation of the conjugated product, reduced; M: Protein molecular weight standard.

[0260] Figure 3 : Expression and detection of camelid and humanized antibodies. M: protein molecular weight marker; 1: HZ1; 2: HZ2; 3: HZ21; 4: MSA21.

[0261] Figure 4: SDS-PAGE analysis of fusion protein expression. 1: Uninduced; 2: Precipitate after HZ21NG expression; 3: Supernatant after HZ21NG expression; 4: Whole cells after HZ21NG expression; 5: Precipitate after GNHZ21 expression; 6: Supernatant after GNHZ21 expression; 7: Whole cells after GNHZ21 expression; 8: Supernatant after GHZ21G expression; 9: Precipitate after GHZ21G expression; 10: Whole cells after GHZ21G expression; 11: Uninduced; M: Protein molecular weight marker. The fusion proteins HZ21NG, GNHZ21, and GHZ21G were expressed at levels of 70%, 65%, and 30% of the total protein, respectively, and were all expressed as inclusion bodies.

[0262] Figure 5 Comparison of purification yields of G-CSF and fusion protein HZ21NG. The yield of HZ21NG is significantly better than that of PEG coupling.

[0263] Figure 6 Fusion protein stimulated NFS-60 cell proliferation activity assay. HZ21NG showed consistent NFS-60 cell proliferation activity with that of Rebactam and was more potent than that of Synrebactam.

[0264] Figure 7 :Detection of neutrophil proliferation activity stimulated by fusion protein in rats.

[0265] Figure 8 :Detection of fusion protein activity in cyclophosphamide-treated model rats. Compared with the model group, the fusion protein can significantly improve the symptoms of neutropenia.

[0266] Figure 9 : Pharmacokinetics of fusion protein HZ21NG in rats.

[0267] Figure 10 :Pharmacokinetics of fusion protein HZ21NG in cynomolgus monkeys.

[0268] Figure 11 : Expression and purification of humanized antibody and human growth hormone fusion proteins GHNHZ21 and HZ21NGH by SDS-PAGE. M: Protein molecular weight standard; 1: Uninduced; 2: Supernatant after expression and fragmentation; 3: Elution of target protein GHNHZ21; 4: Elution of target protein HZ21NGH.

[0269] Figure 12 :In vitro cell activity detection of growth hormone fusion protein.

[0270] Figure 13: SDS-PAGE analysis of the expression of humanized antibody and interleukin fusion protein HZ21RD15. 1: Uninduced; 2: Precipitate after expression and disruption; 3: Supernatant after expression and disruption; 4: Whole cells after expression and disruption; 5: Supernatant of inclusion body wash; 6: Purified inclusion body; M: Protein molecular weight standard.

[0271] Figure 14 : Fusion protein HZ21RD15 combined with αPD-1 enhances tumor killing.

[0272] Figure 15 :The fusion protein HZ21RD15 increases CD8 + 、CD56 + Immune cell ratio.

[0273] Sequence information

[0274] The information of the sequences involved in this application is described in the table below.

[0275]

[0276] DETAILED DESCRIPTION

[0277] The embodiments of the present application will be described in detail below with reference to the examples. Those skilled in the art will appreciate that the following examples are merely illustrative of the present application and should not be construed as limiting the scope of the present application. Specific techniques or conditions not specified in the examples are described in accordance with the techniques or conditions described in the literature in this area (e.g., with reference to " Molecular Cloning Experiment Guide ", 3rd edition, Science Press, by Huang Peitang, etc., with reference to J. Sambrook, etc.) or according to product specifications. Reagents or instruments not specified by manufacturer are all conventional products that can be purchased on the market.

[0278] Reference Sample Preparation Example 1: G-CSF Expression and Purification

[0279] The G-CSF gene was optimized and synthesized by GenScript Biotech Co., Ltd. according to the codons of E. coli, constructed into the expression vector pET28a, and transformed into E. coli BL21 (DE3) competent cells after sequencing verification. A single clone was picked and transferred to LB medium containing kanamycin, cultured with shaking at 37°C overnight, and inoculated into LB medium containing kanamycin at a ratio of 1:100, and cultured with shaking at 37°C until the OD 600The expression level of the protein was approximately 0.8. 0.5 mM IPTG was added for induction for 4 hours. The cells were collected for purification, and the expression level was 45% of the total protein by SDS-PAGE electrophoresis. The induced cells were collected by centrifugation, resuspended and washed once with 20 mM PB (pH 7.4), and then collected by centrifugation. The cells were resuspended in 20 mM PB (pH 7.4), lysed by ultrasonication, and centrifuged at 10,000 g for 15 minutes to collect inclusion bodies. The crude inclusion bodies were resuspended in Wash Buffer I (20 mM Tris + 1 mM EDTA + 0.1% Triton X-001 + 1.5 M urea (pH 8.0)) and Wash Buffer II (20 mM Tris-HCl + 1 mM EDTA (pH 8.5)) and stirred at room temperature for 1 hour to solubilize some soluble host proteins and remove impurities such as nucleic acids. The purified inclusion bodies were collected by centrifugation. Take 1g of refined inclusion bodies and resuspend them in denaturation buffer. Take the denatured supernatant and slowly dilute it into the renaturation solution. After renaturation, use dilute hydrochloric acid to adjust the pH. After centrifugation, load the supernatant onto a cationic chromatography column and elute to obtain 80mg of the target protein. The purity was 100% as determined by SDS-PAGE electrophoresis.

[0280] Reference Sample Preparation Example 2: G-CSF Conjugation with PEG and Purification

[0281] According to the PEG coupling method known in the prior art, 80 mg of purified G-CSF stock solution was adjusted to a concentration of 1 mg / mL, PEG and a reducing agent, sodium cyanoborohydride solution, were added, stirred thoroughly, and reacted at 2-8°C. The coupling products were collected at 0.5 h, 4.5 h, 20 h, and 24 h, and detected by SDS-PAGE electrophoresis. The results were as follows: Figure 1 , some proteins were coupled to PEG after 0.5h reaction, the reaction reached stability after 20h, and there was no significant change in coupling efficiency after 24h. According to grayscale analysis, about 68% of the protein was coupled to one PEG, about 4% of the protein was coupled to multiple PEGs, and 27% of the protein was not coupled. The product was adjusted to pH with dilute hydrochloric acid, loaded onto a cationic chromatography column, and eluted with a NaCl gradient buffer to finally obtain 38mg (based on protein quantification) of PEG-GCSF protein. SDS-PAGE electrophoresis detection, the results are as follows Figure 2 The first elution peak was all uncoupled G-CSF, and the second elution peak was PEG-GCSF. The protein purity was greater than 98% as determined by SDS-PAGE electrophoresis.

[0282] Example 1: Nanobody Humanization

[0283] Camel-derived antibodies have strong immunogenicity when introduced into the human body, and are prone to producing anti-drug antibodies. To reduce the production of anti-drug antibodies and improve drug safety, camel-derived antibodies need to be humanized. The camel-derived antibody MSA21 (see SEQ ID NO. 1 in WO2004041865) was humanized using a CDR-grafted antibody humanization method. Briefly, humanization involves the following steps:

[0284] 1. Based on sequence homology and HLA-DR affinity, human embryonic framework sequences with high homology and low affinity are selected for CDR transplantation, and the amino acids with low frequency of occurrence in the corresponding positions of human embryonic genes in the camel-derived sequence are replaced with the corresponding human amino acids.

[0285] 2. Using computer simulation technology, we examined its spatial binding mode, calculated electrostatic force, van der Waals force, hydrophilicity and entropy value, retained camel-derived amino acids at key sites, and performed a series of reverse mutations to enable the humanized antibody to retain the antigen-binding ability of the camel-derived antibody as much as possible.

[0286] The humanized sequences HZ1 (SEQ ID NO: 1), HZ2 (SEQ ID NO: 2), and HZ21 (SEQ ID NO: 3) were designed through the above steps. According to the Kabat numbering system, HZ1, HZ2, and HZ21 sequences have leucine (Leu) at position 5, alanine (Ala) at position 23, leucine (Leu) at position 45, serine (Ser) at position 74, arginine (Arg) at position 83, and leucine (Leu) at position 108; and HZ1 sequence has leucine (Leu) at position 49, alanine (Ala) at position 84, alanine (Ala) at position 93, and glutamic acid (Glu) at position 94. According to the IMGT numbering system, the HZ1, HZ2, and HZ21 sequences have leucine (Leu) at position 5, alanine (Ala) at position 24, leucine (Leu) at position 50, serine (Ser) at position 83, arginine (Arg) at position 95, and leucine (Leu) at position 123; the HZ1 sequence has leucine (Leu) at position 54, alanine (Ala) at position 96, alanine (Ala) at position 105, and glutamic acid (Glu) at position 106.

[0287] By analyzing the isoelectric points of the above-mentioned humanized antibodies, the isoelectric point of HZ2 is 8.73, which will affect the stability of the antibody or its fusion protein or its conjugate; the inventors unexpectedly found that the isoelectric point of HZ21 is 8.08, and the structure is more stable, thereby ensuring the stability of the protein and preventing it from being quickly cleared.

[0288] Therefore, further analysis of HZ21 showed that the degree of humanization of its V gene coding segment was increased from 84.4% to 89.6% compared with the camel source, and the degree of humanization of the J gene coding segment was increased from 90% to 100%. The antibody has a higher degree of humanization, avoiding the immunogenicity that may be caused by camel-derived amino acids, and at the same time obtaining a humanized antibody with good stability.

[0289] The AbYsis website (http: / / www.abysis.org / abysis / ) was used to annotate the antibody CDR regions of the amino acid sequences of HZ1, HZ2, and HZ21. The results are shown in Table 1:

[0290] Table 1: Humanized single domain antibodies and their CDR sequences

[0291]

[0292] Example 2: Expression of humanized single domain antibodies and verification of binding activity

[0293] 2.1 Detection of camelid and humanized antibody expression

[0294] The gene sequences of the camel-derived antibody MSA21 and the humanized antibodies HZ1, HZ2, and HZ21 were commissioned to GenScript Biotech Co., Ltd. for gene optimization and synthesis according to the Escherichia coli codon. A signal peptide was added to the 5'-end of the gene, and 6×His was added to the 3'-end. The genes were constructed into the pET28a vector and transformed into Escherichia coli BL21 (DE3) cells for expression. Single clones were picked and cultured overnight in LB medium. The cells were inoculated at a ratio of 1:100 into autoinduction medium (12 g of tryptone, 24 g of yeast extract, 10 mL of glycerol, 50 mM Na2HPO4, 50 mM KH2PO4, 25 mM (NH4)4SO4). After sterilization and cooling, 1 ml of 0.22 μm-filtered 50% glucose, 40 ml of 0.22 μm-filtered 10% lactose, and 2 ml of 0.22 μm-filtered 1 M MgSO4 were added. The cells were cultured at 37°C for 24 h, and the supernatant was collected by centrifugation at 8000 g for 20 min. The supernatant of the induced culture medium was purified using Ni+ chromatography medium, and the MSA21, HZ1, HZ2, and HZ21-his proteins were eluted and ultrafiltrated into PBS pH 7.4. The expression levels were quantitatively calculated and the purity was detected by SDS-PAGE electrophoresis. The results are shown in Table 2 and Figure 3 The results showed that the humanization of Example 1 and the mutation modification based on electrostatic force and hydrophilicity improved the expression amount and quality of the single-domain antibody, reduced aggregate formation, and was beneficial to improving protein stability.

[0295] Table 2: Expression detection of camel-derived and humanized antibodies

[0296] Protein name Expression level (mg / L) Monomer purity% Polymer content% MSA21 32 92 8 HZ1 28 99 1 HZ2 35 98 2 HZ21 50 98 2

[0297] 2.2 Binding affinity detection of HZ21-his protein with albumins from different species

[0298] The binding affinity of HZ21-his protein to albumins from different species was detected by ELISA. Human albumin (HSA), rat albumin (RSA), and monkey albumin (CSA) were diluted to 10 μg / mL with coating solution and added to the enzyme-labeled wells at 100 μL / well for coating overnight at 4°C; the coating solution was discarded, the plates were washed once with PBST, 250 μL of PBS + 5% skim milk was added to each well, and the plates were blocked at 37°C for 2 h; after patting dry, 100 μL of HZ21-his protein diluted in PBS + 5% skim milk was added to each well and incubated at 37°C for 2 h; the plates were washed three times with PBST, and HRP-labeled rabbit anti-His tag antibody diluted 1:10,000 in PBS + 5% skim milk was added at 100 μL / well and incubated at 37°C for 1 h; the plates were washed six times with PBST, TMB color development solution was added at 100 μL / well, and the reaction was allowed to react at room temperature for 5 min. The reaction was terminated by adding stop solution at 50 μL / well, and the optical density was measured at 450 nm. The data were exported to Graphpad, and the EC50 of the fusion protein HZ21-his binding to HSA, rat RSA, and monkey albumin CSA was calculated. The EC50 of HZ21-his binding to HSA, rat RSA, and monkey albumin CSA were 10.10nM, 121.30nM, and 4.67nM, respectively. This result indicates that the humanized Single domain The antibody has nM-level binding ability to albumin and can be further used in fusion protein construction.

[0299] Example 3: Construction, expression and purification of a fusion protein of a humanized single domain antibody and G-CSF

[0300] The structure of the fusion protein of humanized single-domain antibody and G-CSF is shown in Table 3. The humanized antibody HZ21 gene was spliced ​​to the 5' end or 3' end of G-CSF by overlapping PCR, or the G-CSF gene was spliced ​​at both ends of the HZ21 gene. To ensure the functional activity of the protein, the middle of each gene was connected by GGGGSGGGGS. The resulting gene was constructed into the pET28a vector, and the encoded proteins were named HZ21NG, GNHZ21 and GHZ21G respectively. The constructed vector was transformed into Escherichia coli BL21 (DE3), and a single clone was picked to LB medium containing kanamycin, cultured at 37°C with shaking overnight, inoculated into LB medium containing kanamycin at a ratio of 1:100, and cultured at 37°C with shaking until OD 600 About 0.8, add 0.5mM IPTG to induce for 4h, collect the cells for purification. After ultrasonic disruption of the cells, take the whole cells, centrifuge and take the supernatant and precipitate for SDS-PAGE electrophoresis detection. The results are as follows Figure 4The expression levels of fusion proteins were high, with HZ21NG, GNHZ21, and GHZ21G accounting for 70%, 65%, and 30% of the total protein, respectively, and all expressed as inclusion bodies (in the precipitate after crushing).

[0301] The cells were disrupted and washed according to the method of Reference Sample Preparation Example 1, and 1 g of HZ21NG was purified and inclusion bodies were purified according to the method of Reference Sample Preparation Example 1. The eluted product was diluted with 20 mM sodium acetate buffer and loaded onto an anion exchange column. The flowthrough was collected and subjected to SDS-PAGE electrophoresis, size exclusion-high performance liquid chromatography (SEC-HPLC) and endotoxin detection (limulus amebocyte lysate detection method). The results are shown in Table 4. The amount of purified protein is as follows Figure 5 As shown, the purity and yield of HZ21NG fusion protein were high (1 g inclusion body gave 100 mg HZ21NG), and the yield was significantly higher than that of PEG-GCSF (1 g inclusion body gave 38 mg PEG-GCSF protein).

[0302] Table 3: Construction methods of fusion proteins

[0303] Protein name Protein structure (N-terminus → C-terminus) Connector L SEQ ID NO: HZ21NG HZ21-L-GCSF <![CDATA[(GGGGS)2]]> 24 GNHZ21 GCSF-L-HZ21 <![CDATA[(GGGGS)2]]> 25 GHZ21G GCSF-L-HZ21-L-GCSF <![CDATA[(GGGGS)2]]> 26

[0304] Table 4: Detection of HZ21NG after purification

[0305] protein SDS-PAGE SEC-HPLC endotoxin Protein HZ21NG 98％ 99.7％ 0.55EU / mg 100mg

[0306] Example 4: In vitro binding activity detection of fusion protein HZ21NG

[0307] The binding affinity of HZ21NG to different proteins was tested by ELISA. Human, rat, and monkey albumin were diluted to 10 μg / mL in coating buffer and added to the enzyme-labeled wells at 100 μL / well for overnight coating at 4°C. The coating buffer was discarded, the plates were washed once with PBST, and 250 μL of PBS + 5% skim milk was added to each well and blocked at 37°C for 2 h. After patting dry, 100 μL of HZ21NG serially diluted with PBS + 5% skim milk was added to each well and incubated at 37°C for 2 h. The plates were washed three times with PBST, and 100 μL / well of rabbit anti-human G-CSF antibody diluted 1:1000 in PBS + 5% skim milk was added and incubated at 37°C for 1 h. The plates were then washed three times with PBST. HRP goat anti-rabbit antibody diluted 1:2000 in PBS + 5% skim milk was added (100 μL / well) and incubated at 37°C for 1 hour. The cells were washed six times with PBST and developed with TMB colorimetric solution (100 μL / well) for 5 minutes at room temperature. The reaction was terminated with stop solution (50 μL / well) and the optical density was measured at 450 nm. Data were exported to GraphPad to calculate the EC50 of binding of the fusion protein HZ21NG to human (HSA), rat (RSA), and monkey albumin (CSA). The EC50s for HZ21NG binding to human (HSA), rat (RSA), and monkey albumin (CSA) were 2.10 nM, 174.90 nM, and 1.58 nM, respectively. These results demonstrate that the humanized antibody fused to the bioactive protein retains comparable albumin binding capacity to the humanized antibody itself, demonstrating that the bioactive protein-humanized nanobody fusion protein can bind to albumin.

[0308] Example 5: In vitro cell activity detection of fusion protein HZ21NG

[0309] NFS-60 cell proliferation activity assay. NFS-60 cells were cultured in 1640+10% FBS+10 ng / mL G-CSF, centrifuged at 100 g for 5 min, and washed with 1640+10% FBS medium without G-CSF to remove residual G-CSF. The cells were counted and the 1×10 4 100 μL / well of each cell was added to a 96-well plate; 100 μL / well of the fusion protein HZ21NG, Rebactam (purchased from Qilu Pharmaceutical, short-acting G-CSF), and Xinrebactam (purchased from Qilu Pharmaceutical, PEG-GCSF) were diluted in 1640+10% FBS+0.2% HSA medium without G-CSF, and 100 μL / well of each cell was added to the cells. The cells were cultured at 37°C for 48 hours, and 20 μL / well of CCK8 was added. The cells were cultured at 37°C for 4 hours, and the OD450nm reading was performed. The data were imported into Graphpad to calculate the protein-stimulated proliferation activity. The results are shown in Figure 2. Figure 6The fusion protein HZ21NG stimulated the proliferation activity of NFS-60 (EC50=1.65pM), which was equivalent to that of Rebactam (EC50=1.52pM), with no significant difference in activity. It was higher than the activity of long-acting PEG-GCSF Xinrebactam (EC50=3.92pM), indicating that the fusion protein retained the biological activity of GCSF and had better in vitro activity than the long-acting PEG-GCSF Xinrebactam.

[0310] The above results indicate that the fusion protein HZ21NG retains the ability of the single-domain antibody to bind to albumin without affecting the biological function of G-CSF, suggesting that the fusion protein may have the function of long-term stimulation of neutrophils.

[0311] Example 6: Effect of fusion protein HZ21NG on rat neutrophil proliferation

[0312] The functional differences between the fusion protein and Rebactam and New Rebactam in stimulating the proliferation of neutrophils in normal rats were tested. SD rats were randomly divided into 4 groups according to body weight, with 3 rats in each group, and administered subcutaneously at the back of the neck (day 0). The Rebactam and New Rebactam groups (200 μg / kg according to protein quantification) and HZ21NG were given an equimolar dose (370 μg / kg according to protein quantification), and the vehicle group (20 mM acetic acid, pH 4.0) were all given a single dose of 10 mL / kg. After administration, 150 μL of orbital blood was collected from the rats every day, and the neutrophil content was detected by hematology analyzer. The results are as follows Figure 7 Under the same G-CSF molar dose conditions, HZ21NG reached its highest level on the first day after administration, with total neutrophil counts increasing 6.6-fold compared to the vehicle group, while Synreb increased by 5.6-fold, returning to vehicle group levels on the fourth day. Neutrophil counts peaked on the first day after Synreb administration, increasing 3.8-fold compared to the vehicle group, but then quickly lost their stimulatory function, returning to vehicle group levels on the second day. These results suggest that the fusion protein HZ21NG has the ability to long-lastingly stimulate neutrophil proliferation in vivo, and is slightly superior to the long-acting Synreb.

[0313] Example 7: Effect of fusion protein HZ21NG on neutrophil proliferation in chemotherapy model rats

[0314] Cyclophosphamide was used to model rats, and the functional differences in neutrophil proliferation stimulated by fusion protein and xinruibai were tested. SD rats were randomly divided into 3 groups according to body weight, with 5 rats in each group, and all rats were intravenously injected with 50 mg / kg of cyclophosphamide (day 0). 24 hours after modeling, subcutaneous administration was performed on the back of the neck. The model group was given an equal volume of HZ21NG buffer (20mM acetic acid, pH4.0), the xinruibai group (100μg / kg according to protein quantification), and the HZ21NG group were given an equimolar dose of fusion protein (169μg / kg according to protein quantification), all with a single dose of 10mL / kg. After administration, 150μL of orbital blood was collected from the rats every day, and the neutrophil content was detected by hematology analyzer. The results are as follows Figure 8 The neutrophil count of the rats in the model group decreased rapidly, below 1×10 9 / L for 3 days, while after the application of HZ21NG, it was less than 1×10 9 / L, indicating that HZ21NG can significantly improve the symptoms of neutrophil reduction in rats; after administration of Xinruibai, the number of neutrophils in rats was less than 1×10 9 / L, but the minimum total neutrophil count in the HZ21NG group (0.94±1.32) was higher than that in the model group (0.32±0.25) and the Xinruibai group (0.32±0.40). Under the same G-CSF molar dose conditions, HZ21NG and PEG-coupled long-acting Xinruibai showed similar trends, but the fusion protein stimulated greater neutrophil proliferation, with a higher minimum value than Xinruibai and a faster recovery to normal levels. These results indicate that HZ21NG has superior activity compared to Xinruibai.

[0315] Example 8: Pharmacokinetics of the fusion protein HZ21NG in rats

[0316] SD rats were randomly divided into two dose groups (1 mg / kg and 3 mg / kg) based on body weight, with 10 rats (half male and half female) in each group. 2 mL / kg of the drug was administered subcutaneously at the back of the neck (at hour 0). Blood samples were collected before dosing and at 0.5, 2, 6, 24, 48, 72, 96, 120, and 168 hours after dosing. Blood samples were transferred to labeled sampling tubes containing separation gel and coagulant and centrifuged at 1500g for 10 minutes at room temperature. Serum was centrifuged and transferred to new labeled centrifuge tubes for PK analysis. The ELISA plate was pre-coated with Human G-CSF Antibody (R&D, MAB214) as a capture reagent. After blocking and washing, the standard curve sample, blank sample, quality control sample and the sample to be tested (all samples were pre-treated with diluent at a ratio of 1:30 before addition) were added to the ELISA plate for incubation. After washing away the free HZ21NG, Human G-CSF Biotinylated Antibody (R&D, BAF214) was added. After incubation, the free antibody was removed. After washing, SA-HRP secondary antibody was added, and finally the substrate TMB was added for color development. After termination, the OD value was read at a dual wavelength of 450 / 630 nm. The HZ21NG concentration in serum was calculated according to the standard curve, and the toxicokinetic parameters were calculated using WinNonlin software. The results are shown in Figure 2. Figure 9 As shown in Table 5, the half-life of HZ21NG in rats was 4.5 h in the 1 mg / kg dose group and 5.3 h in the 3 mg / kg dose group. After G-CSF was fused with a humanized anti-albumin single-domain antibody, the fusion protein HZ21NG had a significantly prolonged half-life in vivo (the reported half-life of G-CSF in rats is 1.8 h, see GNCox et al. Experimental Hematology 32 (2004): 441–449).

[0317] Table 5: PK parameters of fusion protein HZ21NG in rats

[0318]

[0319] Example 9: Pharmacokinetics of the fusion protein HZ21NG in cynomolgus monkeys

[0320] Three-year-old cynomolgus monkeys were randomly divided into two dose groups (0.2 mg / kg and 0.6 mg / kg), with 10 monkeys in each group (half male and half female). 0.5 mL / kg was administered subcutaneously at the back of the neck (hour 0). Blood samples were collected before administration and at 0.5, 2, 6, 24, 48, 72, 96, 120, and 168 hours after administration. The HZ21NG concentration was detected and calculated using the same method as in Example 8. The results are shown in Figure 8. Figure 10As shown in Table 6, HZ21NG showed a significantly prolonged half-life in cynomolgus monkeys, reaching 16-22 h (while the reported half-life of G-CSF in cynomolgus monkeys is 3.5 h, see JAMES F. ELIASON et al. STEM CELLS. 18 (2000): 40-45).

[0321] Table 6: PK parameters of fusion protein HZ21NG in cynomolgus monkeys

[0322] dose <![CDATA[t 1 / 2 ]]> <![CDATA[T max ]]> <![CDATA[C max ]]> <![CDATA[AUC last ]]> (mg / kg) (hr) (hr) (ug / mL) (hr*ug / mL) 0.2 22.33 13.2 0.7493 24.714 0.6 16.731 26.4 2.737 135.19

[0323] In vivo PK studies in rats and crab-eating macaques have shown that the fusion of G-CSF with a humanized anti-albumin single-domain antibody can significantly prolong the in vivo half-life of G-CSF, thereby achieving the goal of long-term functional protein.

[0324] Example 10: Construction and expression of humanized single domain antibody and growth hormone fusion protein

[0325] The structure of the humanized single-domain antibody and growth hormone (GH) fusion protein is shown in Table 7. The humanized antibody HZ21 gene was spliced ​​to the 5'-end or 3'-end of the human growth hormone GH gene by overlapping PCR. The middle of the gene was connected by (GGGGS)2. A signal peptide sequence was added to the 5'-end of the full-length gene. The resulting gene was constructed into the pET28a vector. The protein fused to the 5'-end was named HZ21NGH, and the protein fused to the 3'-end was named GHNHZ21. The constructed plasmid was transformed into Escherichia coli BL21 (DE3), and a single clone was picked to induce expression. After ultrasonic disruption, the supernatant was collected by centrifugation and purified using Protein A to obtain the target protein. The results are shown in Figure 7. Figure 11 The fusion proteins HZ21NGH and GHNHZ21 were expressed soluble and purified to obtain a 35 kDa protein, which was consistent with the molecular weight of the target protein.

[0326] Table 7: Construction of fusion proteins

[0327] Protein name Protein structure (N-terminus → C-terminus) Connector L SEQ ID NO: GHNHZ21 GH-L-HZ21 <![CDATA[(GGGGS)2]]> 28 HZ21NGH HZ21-L-GH <![CDATA[(GGGGS)2]]> 38

[0328] Example 11: In vitro cell activity detection of growth hormone fusion protein

[0329] The GH gene was constructed into pET28a vector by adding signal peptide sequence to 5' and 6×his tag sequence to 3'. The vector was transformed into BL21 (DE3) and single clone was selected to induce expression. After induction, the expression supernatant was collected by centrifugation and purified by Ni +The protein was purified by column chromatography, eluted by ultrafiltration into PBS (pH 7.4), and sterilized by filtration for activity testing. The protein was named GH-His. The full-length human growth hormone receptor (GHR, NP_000154.1) gene was constructed and inserted into the lentiviral expression vector pLVX-IRES-puro. The virus was packaged and used to infect BaF3 cells. After puromycin selection and stabilization, the protein was used for activity evaluation. The cells were named BaF3-GHR.

[0330] Stable BaF3-GHR cells were cultured in 1640+10% FBS+1 ng / mL mIL-3+0.5 μg / mL puromycin. Before use, the cells were washed three times with 1640 medium to remove mIL-3. After culturing in 1640+10% FBS for 6 h, the cells were centrifuged and the cell density was adjusted to 4×10 cells / mL in 1640+10% FBS. 5 / mL, take 100μL of cells and add them to a 96-well plate; 1640+10% FBS gradient dilution of GH-his, HZ21NGH, GHNHZ21 protein, 100μL / well added to the cell plate, continue to culture at 37℃, 5% CO2 for 48h. Add CCK8 at 20μL / well, continue to culture at 37℃ for 4h, and measure OD450 readings with a microplate reader. The results are as follows Figure 12 As shown in Table 8, GH-his did not stimulate the proliferation of blank BaF3 cells (BaF3+GH-his), while GH-his and fusion proteins could stimulate the proliferation of BaF3-GHR cells. The activity of humanized single-domain antibodies fused to the N-terminus or C-terminus of growth hormone had no significant difference from that of growth hormone, and the activity of HZ21NGH was slightly better than that of GHNHZ21.

[0331] Table 8: Cell proliferation activity of growth hormone and fusion protein

[0332] sample GH-His HZ21NGH GHNHZ21 EC50 (nM) 0.051 0.036 0.091

[0333] Example 12: Construction and expression of humanized antibody and interleukin-15 fusion protein

[0334] The structure of the humanized single-domain antibody and interleukin-15 fusion protein is shown in Table 9. Interleukin-15 (IL15) has three receptors, IL15Rα and IL-2Rβ and IL-2Rγ, which are shared receptors with interleukin-2. The sushi domain (IL15RD) of IL15Rα stimulates the enhancement of NK cell activity after binding to IL15. The full-length genes of humanized antibodies, IL15RD and IL15 were constructed by overlapping PCR. Each gene was connected by a linker (GGGGS) 4. The resulting gene was constructed into the pET28a vector and the encoded protein was named HZ21RD15. The constructed plasmid was transformed into Escherichia coli BL21 (DE3) and detected by electrophoresis after induction. As a result, the target protein was obtained and expressed as inclusion bodies. After ultrasonic disruption of the bacteria, the inclusion bodies were collected by centrifugation and washed with washing buffer to obtain refined inclusion bodies. The results are shown in Figure 2. Figure 13 After induction, the expression level of the fusion protein accounted for 30% of the total protein. After washing, the purified inclusion bodies with a purity of 80% were obtained and used for subsequent purification.

[0335] Table 9: Construction of fusion proteins

[0336] Protein name Protein structure (N-terminus → C-terminus) Connector L SEQ ID NO: HZ21RD15 HZ21-L-IL15RD-L-IL15 <![CDATA[(GGGGS)4]]> 32

[0337] Example 13: In vivo efficacy study of fusion protein HZ21RD15

[0338] Female B-NDG mice aged 6-8 weeks were subcutaneously inoculated with 5 × 10 6 HCC827 tumor cells (human non-small cell lung cancer cells); 3-4 days before administration, huPBMCs were revived, resuspended in PBS, and injected intraperitoneally into each mouse at 3×10 6 (0.2ml) huPBMCs. The average tumor volume of immune reconstitution mice reached 70mm 3 Up to 120mm 3 At the same time, mice with tumors that were too small or too large were eliminated, and the remaining mice were randomly divided into 3 groups, with 5 mice in each group. The grouping and dosage of each group were as follows: hIgG1 group 2 mg / kg, αPD-1 antibody (its heavy chain is shown in SEQ ID NO: 39, and its light chain is shown in SEQ ID NO: 40) group 1.5 mg / kg, combined group αPD-1 antibody dose 1.5 mg / kg, HZ21RD15 dose 50 μg / kg, intravenous administration twice a week.

[0339] Tumor diameter was measured with a vernier caliper twice a week, and human CD45 in the tumor was detected by flow cytometry 21 days after administration. + CD3 + CD8 + T cells and human CD45 + CD3 - CD56+ T cell ratio.

[0340] The results are as follows Figure 14 、 15 Compared with the hIgG1 group, the αPD-1 group and the combined group showed the effect of inhibiting tumors. After αPD-1 was combined with HZ21RD15, the anti-tumor activity of αPD-1 was enhanced. At the same time, the human immune cells in the tumor, as well as CD45 + CD3 + CD8 + T cells, CD45 + CD3 - CD56 + The significant increase in the proportion of T cells demonstrates that the fusion protein HZ21RD15 has the biological activity of stimulating immune cell proliferation and / or reducing tumor infiltration, and also has the function of enhancing the anti-tumor activity of immune checkpoint inhibitor antibodies. Therefore, combining the fusion protein HZ21RD15 with immune checkpoint inhibitors has the advantage of enhancing immune response and anti-tumor activity.

[0341] In summary, the present application has obtained a single-domain antibody that binds to albumin with a high degree of humanization by humanizing camel-derived antibodies; the fusion protein constructed based on the single-domain antibody and another active protein has albumin binding activity, and has in vitro activity consistent with the active protein, and in vivo biological function. It is worth noting that the fusion protein greatly prolongs the in vivo half-life of the active protein, and is not easy to induce immunogenic reactions and has high safety. The fusion protein based on the single-domain antibody has the advantages of small molecular weight and prokaryotic expression. Compared with the existing PEG-coupled long-acting drugs on the market, it does not require coupling, has a simple production process, a high yield, and has higher activity than PEG-coupled long-acting drugs, which is significantly better than the current long-acting method. In addition, the expression amount and quality of the humanized antibody obtained in the present application are significantly improved compared to the camel-derived antibody, and the aggregate formation is reduced, which is conducive to improving protein stability.

[0342] Although the specific embodiments of the present application have been described in detail, it will be understood by those skilled in the art that various modifications and replacements may be made to those details based on all disclosed teachings, and these changes are all within the scope of protection of the present application. The full scope of the present application is given by the appended claims and any equivalents thereof. SEQUENCE LISTING <110> Sichuan Kelun Botai Biopharmaceutical Co., Ltd. <120> A protein binding to serum albumin and its use <130> IDC200393 <150> CN201911099739.1 <151> 2019-11-12 <160> 40 <170> PatentIn version 3.5 <210> 1 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Humanized HZ1 VHH <400> 1 [[ID=TW=20]]Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Phe 20 25 30 Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Leu Gly Ile Ser Ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Glu Gly Gly Ser Leu Asn Pro Gly Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 2 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Humanized HZ2 VHH <400> 2 Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Phe 20 25 30 Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Asn Pro Gly Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 3 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Humanized HZ21 VHH <400> 3 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Phe 20 25 30 Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Asn Pro Gly Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 4 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> IMGT HZ21 / hz1 / hz2 CDR1 <400> 4 Gly Phe Thr Phe Ser Arg Phe Gly 1 5 <210> 5 <211> 8 <212> PRT <213> Artificial sequence <220> <223> IMGT HZ21 / hz1 / hz2 CDR2 <400> 5 Ile Ser Ser Leu Gly Asp Ser Thr 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial sequence <220> <223> IMGT HZ21 / hz2 VHH CDR3 <400> 6 Thr Ile Gly Gly Ser Leu Asn Pro 1 5 <210> 7 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Kabat HZ21 / HZ2 / HZ1 VHH CDR1 <400> 7 Arg Phe Gly Met Thr 1 5 <210> 8 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Kabat HZ21 / HZ2 / HZ1 VHH CDR2 <400> 8 Gly Ile Ser Ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 9 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Kabat HZ21 / HZ2 / HZ1 VHH CDR3 <400> 9 Gly Gly Ser Leu Asn Pro 1 5 <210> 10 <211> 25 <212> PRT <213> Artificial sequence <220> <223> IMGT HZ21 VHH FR1 <400> 10 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 11 <211> 17 <212> PRT <213> Artificial sequence <220> <223> IMGT HZ21 / HZ2 VHH FR2 <400> 11 Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 15 Gly <210> 12 <211> 38 <212> PRT <213> Artificial sequence <220> <223> IMGT HZ21 / HZ2 VHH FR3 <400> 12 Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 13 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> IMGT / Kabat HZ21 / HZ2 / HZ1 VHH FR4 <400> 13 Gly Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 14 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Kabat HZ21 VHH FR1 <400> 14[[ID=三十九]] [[ID=四十]]Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 <210> 15 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Kabat HZ21 / HZ2 VHH FR2 <400> 15 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 <210> 16 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Kabat HZ21 / HZ2 VHH FR3 <400> 16 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile 20 25 30 <210> 17 <211> 25 <212> PRT <213> Artificial sequence <220> <223> IMGT HZ1 / HZ2 VHH FR1 <400> 17 Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 18 <211> 17 <212> PRT <213> Artificial sequence <220> <223> IMGT HZ1 VHH FR2 <400> 18 Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Leu 1 5 10 15 Gly <210> 19 <211> 38 <212> PRT <213> Artificial sequence <220> <223> IMGT HZ1 VHH FR3 <400> 19 Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 20 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Kabat HZ1 / HZ2 VHH FR1 <400> 20 Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 <210> 21 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Kabat HZ1 VHH FR2 <400> 21 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Leu 1 5 10 <210> 22 <211> 32 <212> PRT <213> artificial sequence <220> <223> Kabat HZ1 VHH FR3 <400> 22 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Glu 20 25 30 <210> 23 <211> 175 <212> PRT <213> Homo sapiens <400> 23 Met Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu 1 5 10 15 Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu 20 25 30 Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 35 40 45 Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser 50 55 60 Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His 65 70 75 80 Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile 85 90 95 Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala 100 105 110 Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala 115 120 125 Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala 130 135 140 Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser 145 150 155 160 Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 165 170 175 <210> 24 <211> 300 <212> PRT <213> artificial sequence <220> <223> HZ21NG <400> 24 Met Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg 20 25 30 Phe Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Gly Ile Ser Ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Thr Ile Gly Gly Ser Leu Asn Pro Gly Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Pro 115 120 125 Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu 130 135 140 Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys 145 150 155 160 Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu 165 170 175 Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser 180 185 190 Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu 195 200 205 Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu 210 215 220 Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala 225 230 235 240 Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu 245 250 255 Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg 260 265 270 Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu 275 280 285 Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 290 295 300 <210> 25 <211> 300 <212> PRT <213> artificial sequence <220> <223> GNHZ21 <400> 25 Met Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu 1 5 10 15 Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu 20 25 30 Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 35 40 45 Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser 50 55 60 Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His 65 70 75 80 Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile 85 90 95 Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala 100 105 110 Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala 115 120 125 Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala 130 135 140 Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser 145 150 155 160 Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Gly 165 170 175 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser 180 185 190 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 195 200 205 Ala Ser Gly Phe Thr Phe Ser Arg Phe Gly Met Thr Trp Val Arg Gln 210 215 220 Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Ile Ser Ser Leu Gly 225 230 235 240 Asp Ser Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 245 250 255 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 260 265 270 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Asn 275 280 285 Pro Gly Gly Gln Gly Thr Leu Val Thr Val Ser Ser 290 295 300 <210> 26 <211> 484 <212> PRT <213> Artificial Sequence <220> <223> GHZ21G <400> 26 Met Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu 1 5 10 15 Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu 20 25 30 Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 35 40 45 Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser 50 55 60 Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His 65 70 75 80 Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile 85 90 95 Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala 100 105 110 Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala 115 120 125 Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala 130 135 140 Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser 145 150 155 160 Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Gly 165 170 175 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser 180 185 190 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 195 200 205 Ala Ser Gly Phe Thr Phe Ser Arg Phe Gly Met Thr Trp Val Arg Gln 210 215 220 Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Ile Ser Ser Leu Gly 225 230 235 240 Asp Ser Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 245 250 255 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 260 265 270 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Asn 275 280 285 Pro Gly Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 290 295 300 Ser Gly Gly Gly Gly Ser Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro 305 310 315 320 Gln Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly 325 330 335 Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys 340 345 350 His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp 355 360 365 Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys 370 375 380 Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln 385 390 395 400 Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu 405 410 415 Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu 420 425 430 Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro 435 440 445 Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala 450 455 460 Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His 465 470 475 480 Leo Ala Gln Pro <210> 27 <211> 191 <212> PRT <213> Homo sapiens <400> 27 Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg 1 5 10 15 Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu 20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro 35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg 50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu 65 70 75 80 Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val 85 90 95 Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp 100 105 110 Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser 130 135 140 Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160 Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe 165 170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe 180 185 190 <210> 28 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> GHNHZ21 <400> 28 Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg 1 5 10 15 Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu 20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro 35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg<00013​​​​​​Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val 85 90 95 Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp 100 105 110 Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser 130 135 140 Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160 Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe 165 170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Gly 180 185 190 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser 195 200 205 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 210 215 220 Ala Ser Gly Phe Thr Phe Ser Arg Phe Gly Met Thr Trp Val Arg Gln 225 230 235 240 Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Ile Ser Ser Leu Gly 245 250 255 Asp Ser Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 260 265 270 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 275 280 285 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Asn 290 295 300 Pro Gly Gly Gln Gly Thr Leu Val Thr Val Ser Ser 305 310 315 <210> 29 <211> 65 <212> PRT <213> Homo sapiens(智人) <400> 29 Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val 1 5 10 15 Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30 Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45 Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60 Arg 65 <210> 30 <211> 114 <212> PRT <213> Homo sapiens (Homo sapiens) <400> 30 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 31 <211> 199 <212> PRT <213> Artificial sequence <220> <223> IL15RD-IL15 <400> 31 Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val 1 5 10 15 Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30 Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45 Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60 Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 65 70 75 80 Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys 85 90 95 Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr 100 105 110 Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe 115 120 125 Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile 130 135 140 His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser 145 150 155 160 Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu 165 170 175 Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val 180 185 190 Gln Met Phe Ile Asn Thr Ser 195 <210> 32 <211> 335 <212> PRT <213> Artificial Sequence <220> <223> HZ21RD15 <400> 32 Met Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg 20 25 30 Phe Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Gly Ile Ser Ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Thr Ile Gly Gly Ser Leu Asn Pro Gly Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Ile Thr Cys Pro Pro Pro Met Ser 130 135 140 Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg 145 150 155 160 Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser 165 170 175 Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp 180 185 190 Thr Thr Pro Ser Leu Lys Cys Ile Arg Gly Gly Gly Gly Ser Gly Gly 195 200 205 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val 210 215 220 Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met 225 230 235 240 His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys 245 250 255 Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser 260 265 270 Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile 275 280 285 Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser 290 295 第300 Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe 305 310 315 320 Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser 325 330 335 <210> 33 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Linker 1 <400> 33 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 34 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Linker 2 <400> 34 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 It should be noted that there may be some inaccuracies in the translation due to the unclear meaning of some parts in the original text (such as "第300" which might be a wrong expression). It is recommended to double-check with the original source for a more accurate understanding.Gly Gly Gly Ser 20 <210> 35 <211> 345 <212> DNA <213> Artificial sequence <220> <223> Humanized HZ21 VHH nucleic acid <400> 35 gaagttcagc tgctggaaag cggtggcggt ctggttcaac cgggcggtag cctgcgtctg 60 agctgcgcgg cgagcggttt cacctttagc cgtttcggta tgacctgggt gcgtcaagcg 120 ccgggcaagg gtctggagtg ggttagcggc atcagcagcc tgggtgacag caccctgtac 180 gcggatagcg tgaagggccg ttttaccatt agccgtgaca acagcaaaaa caccctgtat 240 ctgcagatga acagcctgcg tccggaagat accgcggttt actattgcac catcggcggt 300 agcctgaacc cgggcggtca aggtaccctg gtgaccgttt ctagc 345 <210> 36 <211> 900 <212> DNA <213> Artificial sequence <220> <223> HZ21NG nucleic acid <400> 36 atggaagttc agctgctgga aagcggtggc ggtctggttc aaccgggcgg tagcctgcgt 60 ctgagctgcg cggcgagcgg tttcaccttt agccgtttcg gtatgacctg ggtgcgtcaa 120 gcgccgggca agggtctgga gtgggttagc ggcatcagca gcctgggtga cagcaccctg 180 tacgcggata gcgtgaaggg ccgttttacc attagccgtg acaacagcaa aaacaccctg 240 tatctgcaga tgaacagcct gcgtccggaa gataccgcgg tttactattg caccatcggc 300 ggtagcctga acccgggcgg tcaaggtacc ctggtgaccg tttctagcgg tggtggcggt 360 tctggtggcg gtggtagcac accattaggt ccggcgagca gcctgccgca gagcttcctg 420 ctgaagtgcc tggagcaagt gcgtaaaatc caaggtgacg gcgcggcgct gcaagaaaag 480 ctgtgcgcga cctacaaact gtgccacccg gaggaactgg ttctgctggg tcacagcctg 540 ggcattccgt gggcgccgct gagcagctgc ccgagccagg cgctgcaact ggcgggttgc 600 ctgagccagc tgcacagcgg tctgttcctg tatcagggcc tgctgcaagc gctggagggt 660 atcagcccgg aactgggtcc gaccctggat accctgcaac tggacgtggc ggattttgcg 720 accaccattt ggcagcaaat ggaggaactg ggtatggcgc cggcgctgca gccgacccaa 780 ggtgcgatgc cggcgttcgc gagcgcgttt cagcgtcgtg cgggtggcgt gctggttgcg 840 agccacctgc aaagcttcct ggaagtgagc taccgtgttc tgcgtcacct ggcgcagccg 900 <210> 37 <211> 8 <212> PRT <213> Synthetic Sequence <220> <223> HZ1 IMGT CDR3 <400> 37 Ala Glu Gly Gly Ser Leu Asn Pro 1 5 <210> 38 <211> 316 [[ID=Z4]]<212> PRT <213> Synthetic Sequence <220> <223> HZ21NGH <400> 38 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 1o 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Phe 20 25 30 Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 It should be noted that there seems to be a typo in the original text where "1o" appears in line 36 of the English translation. It should probably be "10".Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Asn Pro Gly Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Phe Pro Thr 115 120 125 Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg 130 135 140 Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr 145 150 155 160 Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser 165 170 175 Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr 180 185 190 Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile 195 200 205 Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn 210 215 220 Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys 225 230 235 240 Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly 245 250 255 Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp 260 265 270 Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu 275 280 285 Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile 290 295 300 Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe 305 310 315 <210> 39 <211> 446 <212> PRT <213> Artificial sequence <220> <223> PD-1 antibody heavy chain <400> 39 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Asn Trp Val Arg Glu Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Asn Pro Lys Tyr Gly Asp Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Ile Arg Leu Phe Asp Ser Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 40 <211> 214 <212> PRT <213> artificial sequence <220> <223> PD-1 antibody <400> 40 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Pro Ser Gly Asn Val His Asn Tyr 20 25 30 Phe Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys His His Tyr Tyr Thr Ala Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210

Claims

1. A single domain antibody that specifically binds to serum albumin, wherein: The single domain antibody comprises: (1) According to the IMGT numbering system, the CDR1 of SEQ ID NO: 4, the CDR2 of SEQ ID NO: 5, and the CDR3 of SEQ ID NO: 6; and the FR1 of SEQ ID NO: 10, the FR2 of SEQ ID NO: 11, the FR3 of SEQ ID NO: 12, and the FR4 of SEQ ID NO: 13; or (2) According to the Kabat numbering system, the CDR1 of SEQ ID NO: 7, the CDR2 of SEQ ID NO: 8, and the CDR3 of SEQ ID NO: 9; and the FR1 of SEQ ID NO: 14, the FR2 of SEQ ID NO: 15, the FR3 of SEQ ID NO: 16, and the FR4 of SEQ ID NO:

13.

2. The single domain antibody according to claim 1, wherein The single-domain antibody comprises the sequence shown in SEQ ID NO:

3.

3. The single domain antibody according to claim 1, wherein The single domain antibody is a humanized VHH, and: (i) the degree of humanization of the segment encoded by the V gene of the single-domain antibody is not less than 79%; or (ii) The degree of humanization of the segment encoded by the J gene of the single-domain antibody is not less than 89%.

4. The single domain antibody according to claim 1, wherein The single domain antibody has an EC of less than 20 nM 50 Binds to human serum albumin.

5. The single domain antibody according to claim 4, wherein The EC 50 Measured by ELISA technology.

6. A fusion protein comprising the single-domain antibody according to any one of claims 1 to 5 and another biologically active polypeptide, wherein the sequence of the fusion protein is: SEQ ID NO: 24, 25, 26, 28, 32 or 38.

7. An isolated nucleic acid molecule encoding the single domain antibody according to any one of claims 1 to 5 or the fusion protein according to claim 6.

8. The isolated nucleic acid molecule of claim 7, comprising the nucleotide sequence set forth in SEQ ID NO: 35 or SEQ ID NO:

36.

9. A vector comprising the isolated nucleic acid molecule of claim 7 or 8.

10. The vector according to claim 9, wherein The vector is a cloning vector.

11. The vector according to claim 9, wherein The vector is an expression vector.

12. A host cell comprising the isolated nucleic acid molecule of claim 7 or 8 and / or the vector of any one of claims 9 to 11.

13. A method for preparing the single-domain antibody according to any one of claims 1 to 5 or the fusion protein according to claim 6, comprising culturing the host cell according to claim 12 under conditions that allow protein expression, and recovering the single-domain antibody or fusion protein from the culture obtained by culturing the host cell.

14. The method of claim 13, wherein The recovery includes collecting bacterial bodies and purifying them, including the step of performing chromatography on the renatured products of protein inclusion bodies.

15. A pharmaceutical composition comprising the single domain antibody according to any one of claims 1 to 5, the fusion protein according to claim 6, the isolated nucleic acid molecule according to claim 7 or 8, the vector according to any one of claims 9 to 11 or the host cell according to claim 12, and a pharmaceutically acceptable carrier and / or excipient.

16. The pharmaceutical composition according to claim 15, wherein The pharmaceutical composition comprises a fusion protein comprising the single-domain antibody according to any one of claims 1 to 5, IL15RD and IL15, and the sequence of the fusion protein is shown in SEQ ID NO: 32; The pharmaceutical composition further comprises another pharmaceutically active agent, which is an anti-PD-1 antibody, the heavy chain of which is shown in SEQ ID NO: 39, and the light chain of which is shown in SEQ ID NO:

40.

17. A kit comprising the single domain antibody of any one of claims 1 to 5, the fusion protein of claim 6, the isolated nucleic acid molecule of claim 7 or 8, the vector of any one of claims 9 to 11, the host cell of claim 12, or the pharmaceutical composition of claim 15 or 16.

18. Use of a fusion protein in preparing a drug, wherein: The sequence of the fusion protein is shown in SEQ ID NO: 24, The drug is used to: Prevention and / or treatment of cyclophosphamide-induced neutropenia.

19. Use of a fusion protein and an anti-PD-1 antibody in the preparation of a drug, wherein: The sequence of the fusion protein is shown in SEQ ID NO: 32, the heavy chain of the anti-PD-1 antibody is shown in SEQ ID NO: 39, and the light chain is shown in SEQ ID NO: 40; The drug is used to: Treating non-small cell lung cancer.

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