Anti-CD22 single-domain antibody and application thereof

Abstract

The invention belongs to the field of immunology, and relates to an anti-CD22 single-domain antibody and application thereof. The single-domain antibody is composed of heavy chains, and the heavy chains comprise a heavy chain CDR1 as shown in any one of SEQ ID NO: 30 to SEQ ID NO: 34, a heavy chain CDR2 as shown in any one of SEQ ID NO: 35 to SEQ ID NO: 39 and a heavy chain CDR3 as shown in any one of SEQ ID NO: 40 to SEQ ID NO: 43. Compared with the prior art, the invention has the beneficial effects that the specific single-domain antibody aiming at CD22 is screened by using a biological genetic engineering technology, and the affinity of the antibody is better.

Description

Technical Field

[0001] The present invention relates to a single-domain antibody capable of specifically binding to CD22 (hereinafter, its abbreviation is "CD22 single-domain antibody"), a pharmaceutical composition containing the single-domain antibody as an active ingredient, and its pharmaceutical therapeutic use. Background Art

[0002] The B cell receptor CD22 is also known as sialic acid-binding Ig-like lectin 2 (Siglec-2), B lymphocyte cell adhesion molecule (BL-CAM), T cell surface antigen Leu-14, and belongs to the immunoglobulin superfamily and the SIGLEC (sialic acid-binding Ig-like lectin) family. The phenotype of CD22-deficient mice indicates that CD22 is mainly involved in the generation of mature B cells in bone marrow tissue, blood, and the marginal zone of lymphoid tissue.

[0003] CD22 recruits the tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1) to the immunoreceptor tyrosine-based inhibitory motif (ITIM) and inhibits B cell receptor (BCR)-induced normal B cell Ca 2+ signaling. CD22 specifically interacts with ligands carrying α2-6-linked sialic acid.

[0004] As an inhibitory coreceptor of the B cell receptor (BCR), CD22 plays a key role in establishing the signaling threshold for B cell activation. Like other coreceptors, the ability of CD22 to regulate B cell signaling critically depends on its proximity to the BCR, which in turn is governed by the binding of its extracellular domain to α2,6-linked sialic acid ligands.

[0005] CD22 is restrictedly expressed on the surface of mature B cells and most malignant B lymphoma cells, making it one of the popular targets for the treatment of autoimmune diseases and B cell malignancies. Currently, the types of immunotherapeutic drugs targeting CD22 include monoclonal antibody drugs, antibody-drug conjugates (ADCs), and CAR-T therapies. Summary of the Invention

[0006] The object of the present invention is to provide a single-domain antibody capable of specifically binding to CD22 and its uses.

[0007] The first aspect of the present invention provides a single-domain antibody against CD22, which is composed of a heavy chain, and the heavy chain includes a heavy chain CDR1 shown in any one of SEQ ID

[0008] NO:30 - SEQ ID NO:34, and a heavy chain shown in any one of SEQ ID NO:35 - SEQ ID NO:39

[0009] The heavy chain CDR3 shown by any one of CDR2 and SEQ ID NO:40 - SEQ ID NO:43. The single - domain antibody against CD22 is the single - domain antibody targeting CD22.

[0010] Preferably, the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are one of the following (1)-(7):

[0011] (1) CDR1 shown by SEQ ID NO:32, CDR2 shown by SEQ ID NO:37, CDR3 shown by SEQ ID NO:41;

[0012] (2) CDR1 shown by SEQ ID NO:31, CDR2 shown by SEQ ID NO:37, CDR3 shown by SEQ ID NO:41;

[0013] (3) CDR1 shown by SEQ ID NO:33, CDR2 shown by SEQ ID NO:38, CDR3 shown by SEQ ID NO:42;

[0014] (4) CDR1 shown by SEQ ID NO:32, CDR2 shown by SEQ ID NO:37, CDR3 shown by SEQ ID NO:41;

[0015] (5) CDR1 shown by SEQ ID NO:34, CDR2 shown by SEQ ID NO:39, CDR3 shown by SEQ ID NO:43;

[0016] (6) CDR1 shown by SEQ ID NO:33, CDR2 shown by SEQ ID NO:35, CDR3 shown by SEQ ID NO:43;

[0017] (7) CDR1 shown by SEQ ID NO:30, CDR2 shown by SEQ ID NO:36, CDR3 shown by SEQ ID NO:40.

[0018] The above 7 CDR combinations (1)-(7) correspond to SEQ ID NO.1 - 7 in sequence.

[0019] All the above - mentioned sequences can be replaced by sequences with "at least 80% homology" to this sequence or sequences with only one or a few amino acid substitutions; preferably "at least 85% homology", more preferably "at least 90% homology", more preferably "at least 95% homology", and most preferably "at least 98% homology".

[0020] In one embodiment, in any one or more of the CDRs of the heavy chain CDR1, CDR2, and CDR3, one to five arbitrary amino acid residues can be replaced with their conservative amino acids respectively. Specifically, in the heavy chain CDR1, 1 to 5 amino acid residues can be replaced by their conservative amino acids; in the heavy chain CDR2, 1 to 5 amino acid residues can be replaced by their conservative amino acids; in the heavy chain CDR3, 1 to 5 amino acid residues can be replaced by their conservative amino acids.

[0021] As used herein, the term "sequence homology" refers to the degree to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is usually expressed as a percentage. Preferably, homology is determined over the entire length of the sequences being compared. Thus, two copies with exactly the same sequence have 100% homology.

[0022] In some embodiments, sequences that differ from the foregoing sequences by only one or a few amino acid substitutions, for example, sequences containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, can also achieve the object of the invention. These variant forms include (but are not limited to): deletion, insertion, and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. In fact, when determining the degree of sequence homology between two amino acid sequences or when determining the combination of CDR1, CDR2, and CDR3 in a single-domain antibody, those skilled in the art can consider so-called "conservative" amino acid substitutions, in which case the substitution will preferably be a conservative amino acid substitution. The conservative amino acids are usually described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, and the substitution has little or no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are common in the art. For example, conservative amino acid substitutions are substitutions of one or a few amino acids within one of the following groups (a)-(d) by another or a few amino acids within the same group: (a) polar negatively charged residues and their uncharged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. Particularly preferred conservative amino acid substitutions are as follows: Asp is replaced by Glu; Asn is replaced by Gln or His; Glu is replaced by Asp; Gln is replaced by Asn; His is replaced by Asn or Gln; Arg is replaced by Lys; Lys is replaced by Arg, Gln; Phe is replaced by Met, Leu, Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe, Trp; Ala is replaced by Gly or Ser; Ser is replaced by Thr; Thr is replaced by Ser; Gly is replaced by Ala or Pro; Met is replaced by Leu, Tyr, or Ile; Leu is replaced by Ile or Val; Ile is replaced by Leu or Val; Val is replaced by Ile or Leu; Cys is replaced by Ser. Additionally, those skilled in the art know that the creativity of a single-domain antibody lies in the CDR1-3 regions, and the sequences of the framework regions FR1-4 are not immutable. The sequences of FR1-4 can adopt conservative sequence variants of the sequences disclosed in the present invention.

[0023] As used herein, the term "anti-CD22 single-domain antibody" of the present invention not only includes the complete single-domain antibody, but also includes fragments, derivatives and analogs of the anti-CD22 single-domain antibody. As used herein, the terms "fragment", "derivative" and "analog" have the same meaning and all refer to polypeptides that substantially maintain the same biological function or activity as the antibody of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substituent group in one or more amino acid residues, or (iii) polypeptides formed by fusing a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or a secretion sequence or a sequence used to purify this polypeptide or a proprotein sequence, or a fusion protein formed with an Fc tag). According to the teachings herein, these fragments, derivatives and analogs are within the scope well known to those skilled in the art.

[0024] In a preferred embodiment, the antibody sequence further includes a framework region FR; the framework region FR includes the amino acid sequences of FR1, FR2, FR3 and FR4; the amino acid sequences of the framework region FR are respectively:

[0025] FR1 or a variant of FR1 shown by any one of SEQ ID NO: 15-18, and the variant of FR1 contains at most 5 amino acid substitutions in the FR1;

[0026] FR2 or a variant of FR2 shown by any one of SEQ ID NO: 19-21, and the variant of FR2 contains at most 5 amino acid substitutions in the FR2;

[0027] FR3 or a variant of FR3 shown by any one of SEQ ID NO: 22-26, and the variant of FR3 contains at most 5 amino acid substitutions in the FR3;

[0028] FR4 or a variant of FR4 shown by any one of SEQ ID NO: 27-29, and the variant of FR4 contains at most 5 amino acid substitutions in the FR4.

[0029] In a preferred embodiment, the single-domain antibody specifically binds to the extracellular domain of the CD22 antigen.

[0030] A second aspect of the present invention is to provide the amino acid sequences of single-domain antibodies capable of binding to CD22, wherein the amino acid sequences of the single-domain antibodies are respectively shown in SEQ ID NO. 1-7; or compared with any one of SEQ ID NO: 1-7, at least 1 amino acid residue in the FR1, FR2, FR3 or FR4 sequence is replaced by a conservative amino acid and still can specifically bind to CD22; or the single-domain antibody has at least 80% sequence homology with the amino acid sequences of SEQ ID NO. 1-7 and is capable of specifically binding to the CD22 protein.

[0031] In one embodiment, the anti-CD22 single-domain antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology with the amino acid sequences selected from SEQ ID NO: 1-7 and is capable of specifically binding to the CD22 protein.

[0032] A third aspect of the present invention is to provide an Fc fusion antibody or a humanized antibody of the anti-CD22 single-domain antibody as described above.

[0033] A fourth aspect of the present invention is to provide nucleotide molecules encoding the anti-CD22 single-domain antibody or the Fc fusion antibody or the humanized antibody as described above, wherein the nucleotide sequences are respectively shown in SEQ ID NO: 8-14, or the amino acid sequences encoded by the nucleotide sequences are the same as the amino acid sequences encoded by any one of SEQ ID NO: 8-14, or have at least 95% sequence homology with any one of SEQ ID NO: 8-14.

[0034] In one embodiment, the nucleic acid molecule encoding the anti-CD22 single-domain antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology with the nucleotide sequences selected from SEQ ID NO: 8-14, and the anti-CD22 single-domain antibody encoded by it is capable of specifically binding to the CD22 protein.

[0035] A fifth aspect of the present invention is to provide an expression vector comprising a nucleotide molecule encoding an anti-CD22 single-domain antibody or an Fc fusion antibody or a humanized antibody, wherein the nucleotide sequences are respectively shown in SEQ ID NO: 8-14, or the amino acid sequences encoded by the nucleotide sequences are the same as the amino acid sequences encoded by any one of SEQ ID NO: 8-14, but the nucleotide sequences are different.

[0036] In a preferred embodiment, the expression vector used is RJK-V4-hFC4 (a nucleotide molecule encoding a single-domain antibody against CD22, or its Fc fusion antibody or humanized antibody is integrated into RJK-V4-hFC4 by genetic engineering means), and other common expression vectors can also be selected as needed.

[0037] The sixth aspect of the present invention is to provide a host cell capable of expressing the aforementioned single-domain antibody against CD22, Fc fusion antibody or humanized antibody, or a host cell comprising the aforementioned expression vector. Preferred host cells are bacterial cells, fungal cells or mammalian cells.

[0038] In another preferred embodiment, the host cell includes prokaryotic cells or eukaryotic cells, including bacteria and fungi.

[0039] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, phages, or combinations thereof.

[0040] In another preferred embodiment, the prokaryotic cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, Lactobacillus, Streptomyces, Proteus mirabilis, or combinations thereof.

[0041] In another preferred embodiment, the eukaryotic cell is selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, or combinations thereof.

[0042] In another preferred embodiment, the eukaryotic cell is selected from the group consisting of insect cells such as Spodoptera frugiperda, plant cells such as Nicotiana tabacum, BHK cells, CHO cells, COS cells, myeloma cells, or combinations thereof.

[0043] In another preferred embodiment, the host cell is suspension ExpiCHO-S cells.

[0044] In another preferred embodiment, the host cell is suspension 293F cells.

[0045] The seventh aspect of the present invention is to provide a recombinant protein comprising the aforementioned anti-CD22 single-domain antibody. The recombinant protein may be the single-domain antibody shown in SEQ ID NOs. 1-7 above, or a single-domain antibody having at least 80% homology with SEQ ID NOs. 1-7, or a fragment, derivative, or analogue of the aforementioned anti-CD22 single-domain antibody, or a multi-epitope antibody, multi-specific antibody, and multivalent antibody comprising the aforementioned single-domain antibody or its fragment, derivative, or analogue; for example, the multi-epitope antibody may be composed of more than one sequence among SEQ ID NOs. 1-7; the multivalent antibody may be composed of one of the sequences among SEQ ID NOs. 1-7 arranged repeatedly several times; the multi-specific antibody includes, but is not limited to, bispecific antibodies and trispecific antibodies; in addition, the recombinant protein may also be an Fc fusion antibody or a humanized antibody. All of the above recombinant proteins can be used alone or as a component of a chimeric antigen receptor (CAR) in CAR-T therapy. The recombinant protein specifically binds to the target cell surface antigen CD22 to achieve precise anchoring of the target cell; it can also be used as a component of an antibody-drug conjugate (ADC) to carry small molecule drugs to jointly act on the target cell.

[0046] The bispecific antibody includes a first antigen-binding portion and a second antigen-binding portion. The first antigen-binding portion is a single-domain antibody, and the amino acid sequences of the single-domain antibody are respectively as shown in SEQ ID NOs. 1-7 or have at least 80% homology with SEQ ID NOs. 1-7. The second antigen-binding portion may also be as shown in SEQ ID NOs. 1-7 or have at least 80% homology with SEQ ID NOs. 1-7 (the sequences of the first antigen-binding portion and the second antigen-binding portion are different), and the second antigen-binding portion may also be another antibody, such as an antibody against CD19, CD20, CD38, TLR9, CD3, CD28, PDL1 (which may be a monoclonal antibody, polyclonal antibody, single-domain antibody, or any other form of antibody); the first antigen-binding portion and the second antigen-binding portion are fused to each other, for example, through the Fc segment of human IgG to achieve the mutual fusion of the first antigen-binding portion and the second antigen-binding portion.

[0047] The trispecific antibody includes a first antigen-binding portion, a second antigen-binding portion, and a third antigen-binding portion. The first antigen-binding portion, the second antigen-binding portion, and the third antigen-binding portion are fused to each other and are different antibody sequences, targeting at most 3 targets, such as CD20, CD22, and CD38.

[0048] The eighth aspect of the present invention is to provide a pharmaceutical composition comprising the aforementioned single-domain antibody that binds to CD22 and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, where the pH is usually determined according to the isoelectric point of the antibody (the pH of the aqueous carrier medium needs to deviate from the isoelectric point of the antibody and is approximately 2 different from the isoelectric point of the antibody).

[0049] The pharmaceutical composition of the present invention can be directly used to bind to the CD22 protein molecule, and thus can be used to treat diseases (including but not limited to autoimmune diseases and hematological tumors). In addition, other therapeutic agents or treatment modalities can be used in combination. Other therapeutic agents can be any existing drugs for treating related diseases; other treatment modalities, such as chemotherapy, radiotherapy, etc.

[0050] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001 - 99 wt%, preferably 0.01 - 90 wt%, more preferably 0.1 - 80 wt%) of the aforementioned single-domain antibody and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and their combinations. The pharmaceutical preparation should match the administration method. The pharmaceutical composition of the present invention can be made into an injectable form, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injectables and solutions should be manufactured under aseptic conditions.

[0051] The ninth aspect of the present invention is to provide a medicament for treating diseases, which comprises the aforementioned single-domain antibody for binding to the CD22 protein as an active ingredient. In a preferred embodiment, the diseases include but are not limited to autoimmune diseases and hematological tumors.

[0052] The tenth aspect of the present invention provides a method for generating a single-domain antibody against CD22, comprising the steps of:

[0053] (a) Culturing the host cell described in the sixth aspect of the present invention under conditions suitable for generating a single-domain antibody, thereby obtaining a culture containing the anti-CD22 single-domain antibody; and

[0054] (b) Separating or recovering the anti-CD22 single-domain antibody from the culture; and

[0055] (c) Optionally, purifying and / or modifying the anti-CD22 single-domain antibody obtained in step (b).

[0056] The eleventh aspect of the present invention is to provide the use of the aforementioned anti-CD22 single-domain antibody, the aforementioned Fc fusion antibody or humanized antibody, recombinant protein, the aforementioned pharmaceutical composition or medicament in the preparation of a drug for treating diseases.

[0057] In a preferred embodiment, the disease can be any disorder associated with abnormal expression of CD22.

[0058] In a preferred embodiment, the diseases include autoimmune diseases and hematological tumors.

[0059] In a preferred embodiment, the diseases include systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, hairy cell leukemia, precursor B-cell acute lymphoblastic leukemia, precursor B-cell lymphoblastic leukemia lymphoma, acute lymphoblastic leukemia, relapsed acute lymphoblastic leukemia, chronic myeloid leukemia, B-cell lymphoma, B-cell chronic lymphocytic leukemia, non-Hodgkin lymphoma, multiple myeloma, and CD22-positive B-cell acute lymphoblastic leukemia.

[0060] Advantages

[0061] Compared with the prior art, the advantages of the present invention are as follows:

[0062] (1) The single-domain antibody of the present invention specifically targets the CD22 protein with the correct spatial structure.

[0063] (2) For the single-domain antibody obtained in the present invention, the expression system can be flexibly selected. It can be expressed in a prokaryotic system or in a eukaryotic system of yeast cells or mammalian cells. Moreover, its expression cost in the prokaryotic expression system is low, which can reduce the later production cost.

[0064] (3) For the single-domain antibody obtained in the present invention, the modification of the multi-combination form of the antibody is simple. Multivalent and multispecific antibodies can be obtained by simply concatenating through genetic engineering. Moreover, its immune heterogeneity is very low, and a strong immune response will not be generated without humanization modification.

[0065] (4) For the single-domain antibody obtained in the present invention, its affinity range is wider. Before affinity maturation, its affinity range can range from the nM level to the pM level, providing multiple choices for antibodies for different later uses. Brief Description of the Drawings

[0066] In order to more clearly illustrate the technical solutions of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.

[0067] Figure 1 It is the enrichment situation of the library for screening the anti-CD22 antibody in Example 3;

[0068] Figure 2It is the graph for measuring the antibody-antigen binding dose-effect curve in Example 12 (6C10, 9B2);

[0069] Figure 3 It is the graph for measuring the antibody-antigen binding dose-effect curve in Example 12 (14C2, 14G2);

[0070] Figure 4 It is the graph for measuring the antibody-antigen binding dose-effect curve in Example 12 (21A3);

[0071] Figure 5 It is the graph for measuring the antibody-antigen binding dose-effect curve in Example 12 (22H10, 10F9, Tab1 and hIgG). Detailed implementation mode

[0072] The present invention will be further described in detail below in conjunction with examples, so that those skilled in the art can implement it according to the description in the specification.

[0073] As used herein, "single-domain antibody" (sdAb, also called nanobody or VHH by the developer Ablynx) is well known to those skilled in the art. A single-domain antibody is an antibody whose complementarity-determining region is part of a single-domain polypeptide. Therefore, a single-domain antibody contains a single complementarity-determining region (a single CDR1, a single CDR2, and a single CDR3). Examples of single-domain antibodies are antibodies that only have a heavy chain (this antibody does not naturally contain a light chain), single-domain antibodies derived from conventional antibodies, and engineered antibodies.

[0074] Single-domain antibodies can be derived from any species, including mice, humans, camels, llamas, goats, rabbits, and cows. For example, naturally occurring VHH molecules can be derived from antibodies provided by camelid species (such as camels, dromedaries, llamas, and guanacos). Like intact antibodies, single-domain antibodies can selectively bind to specific antigens. A single-domain antibody can only contain the variable domain of the immunoglobulin chain, which has CDR1, CDR2, and CDR3, as well as framework regions.

[0075] As used herein, the term "sequence homology" refers to the degree to which two (nucleotide or amino acid) sequences have the same residues at the same positions in an alignment, and is usually expressed as a percentage. Preferably, homology is determined over the entire length of the sequences being compared. Therefore, two copies with exactly the same sequence have 100% homology.

[0076] As used herein, the term "Fc fusion antibody" refers to a novel protein produced by fusing the Fc segment of a target antibody with a functional protein molecule with biological activity using genetic engineering techniques.

[0077] The term "humanized antibody" refers to an antibody obtained by fusing the heavy chain variable region of a target antibody (such as an animal antibody) with the constant region of a human antibody, or by transplanting the complementarity-determining regions (CDR1-3 sequences) of the target antibody into the variable region of a human antibody, or by mutating the amino acids of the target antibody according to the characteristics of the human antibody framework regions (FR1-4). Humanized antibodies can be obtained by synthetic methods or site-directed mutagenesis methods.

[0078] In the present invention, sequences with high homology to the sequences of CDR1-3 disclosed in the present invention can also yield single-domain antibodies against the CD22 protein. In some embodiments, sequences having "at least 80% homology", or "at least 85% homology", "at least 90% homology", "at least 95% homology", "at least 98% homology" with the sequences in SEQ ID NO.1-7 can all achieve the object of the invention.

[0079] In some embodiments, sequences that differ from the sequences of SEQ ID NOs: 1-7 by only one or a few amino acid substitutions, for example, sequences containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, can also achieve the object of the invention. In fact, when determining the degree of sequence homology between two amino acid sequences or when determining the combination of CDR1, CDR2, and CDR3 in a single-domain antibody, those skilled in the art may consider so-called "conservative" amino acid substitutions. In the case of substitutions, the substitutions will preferably be conservative amino acid substitutions. The conservative amino acids are generally described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, and the substitution has little or no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are common in the art. For example, conservative amino acid substitutions are substitutions of one or a few amino acids within one of the following groups (a)-(d) by another or a few amino acids within the same group: (a) polar negatively charged residues and their uncharged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. Particularly preferred conservative amino acid substitutions are as follows: Asp is replaced by Glu; Asn is replaced by Gln or His; Glu is replaced by Asp; Gln is replaced by Asn; His is replaced by Asn or Gln; Arg is replaced by Lys; Lys is replaced by Arg, Gln; Phe is replaced by Met, Leu, Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe, Trp; Ala is replaced by Gly or Ser; Ser is replaced by Thr; Thr is replaced by Ser; Gly is replaced by Ala or Pro; Met is replaced by Leu, Tyr, or Ile; Leu is replaced by Ile or Val; Ile is replaced by Leu or Val; Val is replaced by Ile or Leu; Cys is replaced by Ser. In addition, those skilled in the art know that the creativity of a single-domain antibody lies in the CDR1-3 regions, and the sequences of the framework regions FR1-4 are not immutable. The sequences of FR1-4 can adopt conservative sequence variants of the sequences disclosed in the present invention.

[0080] The preferred host cells of the present invention are bacterial cells, fungal cells, or mammalian cells.

[0081] This patent prepares the target protein and its truncated form through genetic engineering techniques. Then, the obtained antigen protein is used to immunize the Inner Mongolia Alxa Bactrian camel. After multiple immunizations, peripheral blood lymphocytes or spleen cells of the camel are obtained. Through genetic engineering methods, the coding sequences of the variable regions of camel antibodies are recombined into a phage display vector. Through phage display technology, specific antibodies against the antigen protein are screened out, and their ability to bind to the antigen and their application in the treatment of autoimmune diseases and other aspects are further detected.

[0082] Now, the above technical solution will be disassembled and detailed, and described in the form of specific examples:

[0083] Example 1: Preparation of human CD22 recombinant extracellular domain protein:

[0084] The human recombinant extracellular domain protein used in this patent is obtained by the company's own expression and purification. The specific design scheme of the expression vector for human recombinant CD22 protein is as follows:

[0085] (1) Retrieve the coding sequence of CD22 in NCBI, and its accession number is NM_001771.3. The accession number of the amino acid sequence encoded by this sequence is NP_001762.2.

[0086] (2) Use gene synthesis to clone the nucleotide sequence encoding the extracellular region of CD22 (amino acids 20 - 687) into the vector pcDNA3.4. Perform Sanger sequencing on the constructed vector, compare it with the original sequence. After confirmation, extract a large amount of the recombinant plasmid, remove endotoxin, and transfect suspension 293F cells for the expression and purification of the target protein. The purity reaches over 90%, meeting the requirements for animal immunization.

[0087] Example 2: Construction of a single-domain antibody library against CD22 protein:

[0088] Mix 1 mg of the human recombinant CD22 protein purified in Example 1 with an equal volume of Freund's complete adjuvant, and immunize an Inner Mongolia Alxa Bactrian camel once a week for a total of 7 consecutive immunizations. Except for the first immunization, the remaining six immunizations are carried out by mixing 1 mg of CD22 protein with an equal volume of Freund's incomplete adjuvant for animal immunization. This immunization process is to intensively stimulate the camel to produce antibodies against the CD22 protein.

[0089] After the animal immunization is completed, draw 150 mL of peripheral blood lymphocytes from the camel and extract the RNA of the cells. Use the extracted total RNA to synthesize cDNA, and amplify VHH (heavy chain variable region of antibody) with cDNA as the template through nested PCR reaction.

[0090] Then, the pMECS vector and the VHH fragment were digested with restriction endonucleases respectively, and then the digested fragments and the vector were ligated. The ligated fragments were electrotransformed into competent cells TG1 to construct a phage display library of CD22 protein and the library capacity was determined. The library capacity was approximately 1×10 9 , and at the same time, the correct insertion rate of the library in the target fragment was detected by colony PCR identification.

[0091] The results showed that after PCR amplification of 30 colonies randomly selected from the library, 28 clones could amplify bands of the predicted size, and 2 clones amplified incorrect bands. Therefore, the correct insertion rate was 28÷30×100%≈93%.

[0092] Example 3: Screening of single-domain antibodies against CD22 protein:

[0093] Take 200 μL of the recombinant TG1 cells in Example 2 and culture them in 2×TY medium. During this period, add 40 μL of helper phage VCSM13 to infect TG1 cells and culture overnight to amplify the phage. The next day, precipitate the phage with PEG / NaCl and centrifuge to collect the amplified phage.

[0094] Dilute 500 μg of CD22 protein in 100 mM NaHCO 3 with a pH of 8.3 and conjugate it on an ELISA plate, and place it at 4°C overnight. At the same time, set up a negative control well (medium control); the next day, add 200 μL of 3% skim milk and block it at room temperature for 2 h; after the blocking is completed, add 100 μl of the amplified phage library (about 2×10 11 phage particles), and let it act at room temperature for 1 h; after acting for 1 hour, wash it 15 times with PBS + 0.05% Tween-20 to wash away the unbound phage.

[0095] Dissociate the phage specifically binding to CD22 protein with trypsin at a final concentration of 25 mg / mL and infect Escherichia coli TG1 cells in the logarithmic growth phase. Culture at 37°C for 1 h, produce and collect the phage for the next round of screening, and repeat the same screening process for 1 round to gradually obtain enrichment.

[0096] When the enrichment multiple reaches more than 10 times, the enrichment effect is as Figure 1 shown.

[0097] Figure 1Among them, P / N = the number of monoclonal bacteria grown after the phages eluted from the positive wells in biopanning infect TG1 bacteria / the number of monoclonal bacteria grown after the phages eluted from the positive wells in biopanning infect TG1 bacteria. This parameter will gradually increase after enrichment occurs; I / E = the total amount of phages added to the positive wells in each round of biopanning / the total amount of phages eluted from the positive wells in each round of biopanning. This parameter will gradually approach 1 after enrichment occurs.

[0098] Example 4: Screening for specific positive clones against CD22 using the enzyme-linked immunosorbent assay (ELISA) of phages:

[0099] According to the screening method in Example 3 above, the single-domain antibody against CD22 protein was screened for 4 rounds. The phage enrichment factor of the anti-CD22 protein reached more than 10. After the screening ended, 384 single colonies were selected from the obtained positive clones and inoculated into 96-deep well plates containing 2×TY medium with 100 μg / mL ampicillin, and a blank control was set. After culturing at 37°C until the logarithmic phase, IPTG with a final concentration of 1 mM was added, and the culture was continued overnight at 28°C.

[0100] The crude antibody was obtained by osmotic lysis method; the CD22 recombinant protein was respectively diluted to 100 mM in NaHCO 3 and 100 μg of the protein was coated overnight at 4°C in an enzyme-linked immunosorbent assay plate (ELISA plate). 100 μL of the obtained crude antibody solution was transferred to the ELISA plate with the antigen added and incubated at room temperature for 1 h; the unbound antibody was washed away with PBST, 100 μl of Mouse Anti-HA tag Antibody (HRP) (mouse anti-HA horseradish peroxidase-labeled antibody, ThermoFisher) diluted 1:2000 was added, and the incubation was continued at room temperature for 1 h; the unbound antibody was washed away with PBST, the horseradish peroxidase chromogenic solution was added, and after reacting at 37°C for 15 min, the stop solution was added, and the absorbance was read at a wavelength of 450 nm on an enzyme-linked immunosorbent assay reader.

[0101] When the OD value of the sample well is more than 5 times that of the control well, it is determined as a positive clone well; the bacteria in the positive clone well are transferred and shaken in LB medium containing 100 μg / mL ampicillin for plasmid extraction and sequencing.

[0102] The gene sequences of each clone were analyzed using the sequence alignment software VectorNTI. Clones with identical CDR1, CDR2, and CDR3 sequences were regarded as the same clone, while clones with different sequences were regarded as different clones. Finally, single-domain antibodies specifically targeting the CD22 protein were obtained (SEQ ID NO.1 - 7 and clones with sequences not shown, including 1B8, 2A11, 4B9, 4E10, 12A3, 12E8, 12H9, 15H3, 17C4, 21B10, 21D3, 22C10, 22D4, 22H3, 23A12, 23F4).

[0103] The amino acid sequence of its antibody has the structure of FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4, constituting the entire VHH. The obtained single-domain antibody recombinant plasmid can be expressed in a prokaryotic system, and finally, single-domain antibody protein is obtained.

[0104] The CDR and FR sequences of the 7 single-domain antibodies are shown in Tables 1 - 7, and the amino acid sequences and nucleotide sequences of the 7 single-domain antibodies are shown in Table 8.

[0105] Table 1 CDR1 sequences of 7 antibodies

[0106]

[0107]

[0108] Table 2 CDR2 sequences of 7 antibodies

[0109]

[0110] Table 3 CDR3 sequences of 7 antibodies

[0111]

[0112] Table 4 FR1 sequences of 7 antibodies

[0113]

[0114] Table 5 FR2 sequences of 7 antibodies

[0115]

[0116] Table 6 FR3 sequences of 7 antibodies

[0117]

[0118] Table 7 FR4 sequences of 7 antibodies

[0119]

[0120]

[0121]

[0122]

[0123] Table 8 Amino acid sequences and nucleic acid sequences of 7 single-domain antibodies

[0124] Example 5: Purification and expression of specific single-domain antibodies against CD22 protein in host bacterium Escherichia coli

[0125] The plasmids (pMECS-VHH) of different clones obtained by sequencing analysis in Example 4 were electrotransformed into Escherichia coli HB2151, and then spread on an LB + amp + glucose culture plate containing ampicillin and glucose, and cultured overnight at 37°C; single colonies were selected and inoculated into 5 mL of LB culture medium containing ampicillin, and cultured overnight on a shaker at 37°C.

[0126] 1 mL of the overnight culture was inoculated into 330 mL of TB culture medium, and cultured on a shaker at 37°C. When the OD600nm value reached 0.6 - 0.9, 1 M IPTG was added, and the culture was continued overnight on a shaker at 28°C; the bacteria were centrifuged, and the Escherichia coli was collected. An antibody crude extract was obtained by osmotic lysis.

[0127] Antibodies were purified by nickel column affinity chromatography. The purified partial single-domain antibodies included 6C10, 9B2, 10F9, 14C2, 14G2, 21A3, and 22H10.

[0128] Example 6: Construction of eukaryotic expression vector of Fc fusion antibody of single-domain antibody against CD22

[0129] (1) Subclone the target sequence obtained in Example 4 into a eukaryotic expression vector: The antibody screened in Example 4 was subjected to Sanger sequencing to obtain its nucleotide sequence;

[0130] (2) Synthesize the above nucleotide sequences (such as SEQ ID NO. 8 - 14) into the vector RJK-V4-hFC4 designed and modified by the company by sequence synthesis to obtain a recombinant eukaryotic expression vector. The modification method of this vector is as described in Example 10;

[0131] (3) Transform the recombinant eukaryotic expression vector constructed in step (2) into Escherichia coli DH5α, culture it, and extract the plasmid to remove endotoxin;

[0132] (4) Sequence and identify the plasmid after extraction;

[0133] (5) Prepare the confirmed recombinant vector for subsequent eukaryotic cell transfection and expression. After expressing the Fc protein of VHH by the method of Example 7 or 8, purify the above antibody by the method of Example 9.

[0134] Example 7: Expression of a single-domain antibody against CD22 protein in suspension ExpiCHO-S cells

[0135] (1) Three days before transfection, passage and expand ExpiCHO-S cells at 2.5×10 5 / mL, transfer the calculated required cell volume to a 500 mL shake flask containing fresh pre-warmed 120 mL (final volume) of ExpiCHO TM expression medium; adjust the cell concentration to approximately 4×10 TM -6×10 6 viable cells / mL; 6

[0136] (2) One day before transfection, dilute the ExpiCHO-S TM cells to a concentration of 3.5×10 6 viable cells / mL and incubate the cells overnight;

[0137] (3) On the day of transfection, measure the cell density and percentage of viable cells. The cell density before transfection should reach approximately 7×10 6 -10×10 6 viable cells / mL;

[0138] (4) Dilute the cells to 6×10 TM viable cells / mL with fresh pre-warmed ExpiCHO 6 expression medium. Transfer the calculated required cell volume to a 500 mL shake flask containing fresh pre-warmed 100 mL (final volume) of ExpiCHO TM expression medium;

[0139] (5) Gently invert and mix the ExpiFectamine TM CHO reagent, dilute the ExpiFectamine TM CHO reagent with 3.7 mL of OptiPRO TM medium, swirl or mix;

[0140] (6) Dilute the plasmid DNA with 4 mL of chilled OptiPRO TM medium and swirl to mix;

[0141] (7) Incubate the ExpiFectamine CHO / plasmid DNA (the plasmid DNA is the eukaryotic expression vector of the Fc-fusion antibody of the anti-CD22 single-domain antibody prepared in Example 6) complex at room temperature for 1 - 5 minutes, and then gently add it to the prepared cell suspension. Gently swirl the flask during the addition process.

[0142] (8) Incubate the cells in a shaking incubator at 37 °C in 8% CO 2 2, humidified air.

[0143] (9) Add 600 μL of ExpiFectamine TM CHO Enhancer and 24 mL of ExpiCHO feed 1 day (18 - 22 hours) after transfection.

[0144] (10) Collect the supernatant approximately 8 days after transfection (when the cell viability is less than 70%).

[0145] Example 8: Expression of the anti-CD22 protein single-domain antibody in suspension 293F cells

[0146] Recombinant single-domain antibody expression experimental procedure (taking a 500 mL flask as an example):

[0147] (1) Three days before transfection, passage and expand the 293F cells at 2.5×10 5 / mL, and transfer the calculated required cell volume to a 500 mL flask containing 120 mL (final volume) of pre-warmed OPM-293CD05 Medium. Adjust the cell concentration to approximately 2×10 6 -3×10 6 viable cells / mL.

[0148] (2) On the day of transfection, measure the cell density and the percentage of viable cells. The cell density should reach approximately 2×10 6 -3×10 6 viable cells / mL before transfection.

[0149] (3) Dilute the cells to 1×10 6 viable cells / mL with pre-warmed OPM-293CD05 Medium. Calculate the required cell volume and transfer it to a 500 mL flask containing 100 mL (final volume) of pre-warmed medium.

[0150] (4) Dilute the PEI (1 mg / mL) reagent with 4 mL of Opti-MEM medium, and mix well by swirling or pipetting up and down; dilute the plasmid DNA (the plasmid DNA is the eukaryotic expression vector of the Fc fusion antibody of the anti-CD22 single-domain antibody prepared in Example 6) with 4 mL of Opti-MEM medium, mix well by swirling, and filter through a 0.22-μm filter tip. Incubate at room temperature for 5 min.

[0151] (5) Add the diluted PEI reagent to the diluted DNA, and mix by inverting. Incubate the PEI / plasmid DNA complex at room temperature for 15 - 20 minutes, then gently add it to the prepared cell suspension, and gently swirl the flask during the addition.

[0152] (6) Incubate the cells at 37 °C, 5% CO 2 2, and shake culture at 120 rpm.

[0153] (7) Add 5 mL of OPM-CHO PFF05 feed at 24 h and 72 h after transfection.

[0154] (8) Collect the supernatant at about 7 days after transfection (when the cell viability is lower than 70%).

[0155] Example 9: Purification of the anti-CD22 protein single-domain antibody

[0156] (1) Filter the protein expression supernatant obtained in Example 7 or 8 through a 0.45-μm disposable filter tip to remove insoluble impurities;

[0157] (2) Purify the above filtrate by affinity chromatography using a protein purifier. Utilize the ability of human Fc to bind to Protein A, and use agarose beads conjugated with Protein A for purification;

[0158] (3) Flow the filtrate through the Protein A pre-packed column at a flow rate of 1 mL / min. In this step, the target protein in the filtrate will bind to the packing material;

[0159] (4) Wash the impurity proteins bound to the column with low-salt and high-salt buffer solutions;

[0160] (5) Elute the target protein bound to the column with a low-pH buffer solution;

[0161] (6) Immediately add the eluate to a Tris-HCl solution with a pH of 9.0 for neutralization;

[0162] (7) After dialysis of the above neutralized protein solution, perform SDS-PAGE analysis. After determining that the protein purity is above 95% and the concentration is above 0.5 mg / mL, store it at low temperature for later use.

[0163] Example 10: Construction of Eukaryotic Expression Vector RJK-V4-hFC4 of Single-Domain Antibody

[0164] The target vector RJK-V4-hFC4, which is common for the mentioned nanobodies, is modified on the basis of the invitrogen commercial vector pCDNA3.4 (vector data link: https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / pcdna3_4_topo_ta_cloning_kit_man.pdf) by fusing the Fc segment in the heavy chain coding sequence of human IgG4. That is, this vector contains the hinge region (Hinge), CH2, and CH3 regions of the IgG4 heavy chain. The specific modification scheme is as follows:

[0165] (1) Select the restriction enzyme cleavage sites XbaI and AgeI on pcDNA3.4;

[0166] (2) Introduce a multiple cloning site (MCS, Multiple Cloning Site) and a 6×His tag at the 5' end and 3' end of the Fc fragment coding sequence by overlapping PCR, respectively;

[0167] (3) Amplify the above fragments by PCR using a pair of primers with XbaI and AgeI restriction enzyme cleavage sites, respectively;

[0168] (4) Digest pcDNA3.4 and the recombinant DNA fragment in (3) with the restriction enzymes XbaI and AgeI, respectively;

[0169] (5) Ligate the digested vector and the inserted fragment under the action of T4 ligase, then transform the ligation product into Escherichia coli, amplify, verify by sequencing, and obtain the recombinant plasmid.

[0170] Example 11: Expression and Purification of Tool Antibody (Tab1) Targeting Human CD22

[0171] In this article, Tab1 is Inotuzumab Ozogamicin; the antibody sequence of this drug found by searching was entrusted to General Biosystems (Anhui) Co., Ltd. for codon optimization in the mammalian cell expression system and cloned into the RJK-V4-hFC4 vector. After resistance screening, plasmid-positive bacteria were selected for amplification, and the plasmid was extracted using a plasmid midiprep kit (Macherey Nagel, Cat#740412.50). According to 100 μg of plasmid (40 μg heavy chain + 60 μg light chain) added per 100 mL of cells, transient expression was carried out in 293F cells (culture medium: FreeStyle 293 Expression medium, Thermo, Cat#12338026 + F-68, Thermo, Cat#24040032) using PEI; 5% by volume of 10% Peptone (Sigma, Cat#P0521-100G) was added 6 - 24 h after transfection, and 2 cultured at 130 rpm for about 7 - 8 days; when the cell viability dropped to 50%, the expression supernatant was collected and purified using a Protein A (GE, Cat#17-5438-02) gravity column; after dialysis with PBS, the concentration was measured using Nanodrop, the purity was identified by SEC, and the binding ability was verified by indirect ELISA;

[0172] Tab1 (Tab1 fused and expressed with hFC4) obtained by this method had a concentration of not less than 2 mg / ml and a purity of greater than 95%.

[0173] Example 12: Determination of the antigen-binding dose-response curve of the antibody

[0174] This example was carried out using the standard enzyme-linked immunosorbent assay (ELISA) operation procedure.

[0175] (1) Coat 50 μL of 1 μg / mL human CD22 protein overnight at 4°C.

[0176] (2) Wash the plate; add 200 μL of 5% milk and block at 37°C for 2 h.

[0177] (3) Dilute VHH-hFc to 2 μg / mL, and then serially dilute the antibody 5-fold for a total of 8 concentration gradients. Here, VHH-hFc refers to the Fc fusion antibody of the single-domain antibody against CD22 protein prepared in Example 8 (expressed in 293F cells) purified by Example 9. In addition, hIgG (actually hIgG4) and Tab1 controls were also set; Tab1 was prepared in Example 11;

[0178] (4) Wash the plate; add 50 μL of the single-domain antibody diluted in step (3), with two replicates, and incubate at 37°C for 1 h.

[0179] (5) Wash the plate; add 50 μL of HRP-Goat anti hIgG secondary antibody and incubate at 37 °C for 30 min.

[0180] (6) Wash the plate (wash several times); add 50 μL of TMB that has been pre-restored to room temperature and react at room temperature in the dark for 15 min.

[0181] (7) Add 50 μL of termination solution (1N HCl) and save the reading on the microplate reader.

[0182] (8) Plot the curve and calculate the EC50, as Figures 2 - 5 shown, where hIgG refers to the isotype control, an immunoglobulin molecule that does not bind to any target, and is obtained by commercial purchase. Among them, Figures 2 - 5 are the results of the samples (including 6C10, 9B2, 10F9, 14C2, 14G2, 21A3, 22H10 and antibody strains 1B8, 2A11, 4B9, 4E10, 12A3, 12E8, 12H9, 15H3, 17C4, 21B10, 21D3, 22C10, 22D4, 22H3, 23A12, 23F4 whose sequences are not shown).

[0183] From Figures 2 - 5 it can be seen that compared with the hIgG and Tab1 controls, 6C10, 9B2, 10F9, 14C2, 14G2, 21A3, 22H10 all show excellent binding performance to the CD22 antigen, with better affinity and specificity.

[0184] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form. Any technical solutions obtained by using equivalent replacements or equivalent transformations fall within the protection scope of the present invention.

Claims

1. A single-domain antibody against CD22, characterized in that: the single-domain antibody is composed of a heavy chain, and the heavy chain includes a heavy chain CDR1 shown by any one of SEQ ID NO: 30 - SEQ ID NO: 34, a heavy chain CDR2 shown by any one of SEQ ID NO: 35 - SEQ ID NO: 39, and a heavy chain CDR3 shown by any one of SEQ ID NO: 40 - SEQ ID NO:

43.

2. The single-domain antibody against CD22 according to claim 1, characterized in that: the amino acid sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 are one of the following (1) - (7): (1) CDR1 shown by SEQ ID NO: 32, CDR2 shown by SEQ ID NO: 37, CDR3 shown by SEQ ID NO: 41; (2) CDR1 shown by SEQ ID NO: 31, CDR2 shown by SEQ ID NO: 37, CDR3 shown by SEQ ID NO: 41; (3) CDR1 shown by SEQ ID NO: 33, CDR2 shown by SEQ ID NO: 38, CDR3 shown by SEQ ID NO: 42; (4) CDR1 shown by SEQ ID NO: 32, CDR2 shown by SEQ ID NO: 37, CDR3 shown by SEQ ID NO: 41; (5) CDR1 shown by SEQ ID NO: 34, CDR2 shown by SEQ ID NO: 39, CDR3 shown by SEQ ID NO: 43; (6) CDR1 shown by SEQ ID NO: 33, CDR2 shown by SEQ ID NO: 35, CDR3 shown by SEQ ID NO: 43; (7) CDR1 shown by SEQ ID NO: 30, CDR2 shown by SEQ ID NO: 36, CDR3 shown by SEQ ID NO:

40.

3. The single-domain antibody against CD22 according to claim 1, characterized in that: the single-domain antibody further includes a framework region FR; the framework region FR includes the amino acid sequences of FR1, FR2, FR3, and FR4; the amino acid sequences of the framework region FR are respectively: FR1 shown by any one of SEQ ID NO: 15 - 18 or a variant of FR1, and the variant of FR1 contains at most 5 amino acid substitutions in FR1; FR2 shown by any one of SEQ ID NO: 19 - 21 or a variant of FR2, and the variant of FR2 contains at most 5 amino acid substitutions in FR2; FR3 shown by any one of SEQ ID NO: 22 - 26 or a variant of FR3, and the variant of FR3 contains at most 5 amino acid substitutions in FR3; An FR4 or a variant of FR4 shown by any one of SEQ ID NO: 27-29, wherein the variant of FR4 contains substitutions of up to 5 amino acids in the FR4.

4. The anti-CD22 single-domain antibody according to claim 1, wherein: the single-domain antibody specifically binds to the extracellular domain of the CD22 antigen.

5. An anti-CD22 single-domain antibody, wherein: the amino acid sequence of the single-domain antibody is respectively shown by any one of SEQ ID NO: 1-7; or compared with any one of SEQ ID NO: 1-7, at least 1 amino acid residue in the FR1, FR2, FR3 or FR4 sequence is replaced by a conservative amino acid.

6. An Fc fusion antibody or a humanized antibody of the anti-CD22 single-domain antibody according to any one of claims 1 to 5.

7. A recombinant protein, wherein, the recombinant protein comprises the anti-CD22 single-domain antibody according to any one of claims 1 to 5, or is a fragment, derivative or analogue of the anti-CD22 single-domain antibody according to any one of claims 1 to 5, or is a bispecific antibody, multi-epitope antibody, multispecific antibody and multivalent antibody comprising the aforementioned single-domain antibody, its fragment, derivative or analogue; or alternatively, the recombinant protein comprises the Fc fusion antibody or humanized antibody according to claim 6.

8. A nucleotide molecule encoding the anti-CD22 single-domain antibody according to any one of claims 1 to 5, wherein: its nucleotide sequence is respectively shown by any one of SEQ ID NO: 8-14, or the amino acid sequence encoded by the nucleotide sequence is the same as the amino acid sequence encoded by any one of SEQ ID NO: 8-14.

9. An expression vector, wherein: it contains a nucleotide molecule encoding the anti-CD22 single-domain antibody according to any one of claims 1 to 5 or the Fc fusion antibody or humanized antibody according to claim 6 or the nucleotide molecule according to claim 8.

10. A host cell, wherein: it can express the anti-CD22 single-domain antibody according to any one of claims 1 to 5 or the Fc fusion antibody or humanized antibody according to claim 6, or it contains the expression vector according to claim 9.

11. A pharmaceutical composition, wherein: the pharmaceutical composition contains the anti-CD22 single-domain antibody selected from any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

12. An agent for treating a disease, wherein: it contains the anti-CD22 single-domain antibody according to any one of claims 1 to 5 as an active ingredient.

13. Use of the anti-CD22 single-domain antibody according to any one of claims 1 to 5, the Fc fusion antibody or humanized antibody according to claim 6, the recombinant protein according to claim 7, the pharmaceutical composition according to claim 11 or the agent according to claim 12 in the preparation of a drug for treating a disease.

14. The use according to claim 13, wherein: The diseases include autoimmune diseases and hematological tumors.

15. The use according to claim 13, wherein: the diseases include systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, hairy cell leukemia, precursor B-cell acute lymphoblastic leukemia, precursor B-cell lymphoblastic lymphoma, acute lymphoblastic leukemia, relapsed acute lymphoblastic leukemia, chronic myeloid leukemia, B-cell lymphoma, B-cell chronic lymphocytic leukemia, non-Hodgkin lymphoma, multiple myeloma, and CD22-positive B-cell acute lymphoblastic leukemia.

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