A single-domain antibody against cd22 and uses thereof

Abstract

This invention belongs to the field of immunology and relates to a single-domain antibody against CD22 and its uses. The single-domain antibody is composed of heavy chains, including heavy chain CDR1 shown in any one of SEQ ID NO:30-SEQ ID NO:34, heavy chain CDR2 shown in any one of SEQ ID NO:35-SEQ ID NO:39, and heavy chain CDR3 shown in any one of SEQ ID NO:40-SEQ ID NO:43. Compared with the prior art, the beneficial effects of this invention are: this invention uses bioengineering technology to screen for single-domain antibodies specifically targeting CD22, exhibiting good antibody affinity.

Description

Technical Field

[0001] This invention relates to a single-domain antibody (hereinafter abbreviated as "CD22 single-domain antibody") capable of specifically binding to CD22, a pharmaceutical composition containing the single-domain antibody as an active ingredient, and its use in pharmaceutical therapy. Background Technology

[0002] B cell receptor CD22, also known as sialic acid-binding Ig-like lectin 2 (Siglec-2), B lymphocyte cell adhesion molecule (BL-CAM), and T cell surface antigen Leu-14, 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 primarily involved in the production of mature B cells in the peripheral regions of bone marrow, blood, and lymphoid tissues.

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

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

[0005] CD22 is restricted to expression 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, immunotherapeutic drugs targeting CD22 include monoclonal antibody drugs, antibody-drug conjugates (ADCs), and CAR-T therapy. Summary of the Invention

[0006] The purpose of this patent is to provide a single-domain antibody that can specifically bind to CD22 and its uses.

[0007] A first aspect of the present invention provides a single-domain antibody against CD22, said single-domain antibody being composed of a heavy chain, the heavy chain comprising SEQ ID NO.

[0008] Heavy chain CDR1 shown in any of SEQ ID NO:30-SEQ ID NO:34, and heavy chain shown in any of SEQ ID NO:35-SEQ ID NO:39

[0009] CDR2 and any one of the heavy chains CDR3 shown in SEQ ID NO:40-SEQ ID NO:43. Anti-CD22 single-domain antibodies are those 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 in SEQ ID NO:32, CDR2 shown in SEQ ID NO:37, and CDR3 shown in SEQ ID NO:41;

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

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

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

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

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

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

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

[0019] All of the above sequences can be replaced with sequences that have "at least 80% homology" or sequences that replace only one or a few amino acids; preferably "at least 85% homology", more preferably "at least 90% homology", even more preferably "at least 95% homology", and most preferably "at least 98% homology".

[0020] In one embodiment, in any one or more CDRs of the heavy chain CDR1, CDR2, and CDR3, one to five arbitrary amino acid residues may be substituted with their conserved amino acids. Specifically, in the heavy chain CDR1, one to five amino acid residues may be substituted with their conserved amino acids; in the heavy chain CDR2, one to five amino acid residues may be substituted with their conserved amino acids; and in the heavy chain CDR3, one to five amino acid residues may be substituted with their conserved amino acids.

[0021] As used herein, the term "sequence homology" refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is typically expressed as a percentage. Preferably, homology is determined over the overall length of the sequences being compared. Thus, two copies having completely identical sequences have 100% homology.

[0022] In some embodiments, the inventive objective can also be achieved by substituting only one or a few amino acids compared to the aforementioned sequence, for example, by including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions. These variations include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, most preferably up to 5) at the C-terminus and / or N-terminus. In practice, 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 “conserved” amino acid substitutions, in which case the substitution would preferably be a conserved amino acid substitution. A conserved amino acid can generally be described as an amino acid residue substituted by another amino acid residue having a similar chemical structure, and this substitution has little or no effect on the function, activity, or other biological properties of the polypeptide. The conserved amino acid substitutions are common in the art. For example, a conserved amino acid substitution is the substitution of one or a few amino acids in the following groups (a)-(d) by another or a few amino acids in the same group: (a) polar negatively charged residues and their non-charged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. The particularly preferred conserved 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 or Gln; Phe is replaced by Met, Leu, or Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe or 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. Furthermore, those skilled in the art will understand that the inventiveness of the single-domain antibody lies in the CDR1-3 regions, while the frame region sequences FR1-4 are not immutable, and the sequences of FR1-4 can adopt conserved sequence variants of the sequences disclosed in this invention.

[0023] The term "anti-CD22 single-domain antibody" in this invention includes not only complete single-domain antibodies but also fragments, derivatives, and analogs of said anti-CD22 single-domain antibodies. As used herein, the terms "fragment," "derivative," and "analyte" have the same meaning and refer to polypeptides that substantially retain the same biological function or activity as the antibodies of this invention. The polypeptide fragments, derivatives, or analogs of this invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved 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 substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends 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 (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with an Fc tag). Based on 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 as follows:

[0025] The FR1 or a variant thereof shown in any one of SEQ ID NO:15-18, wherein the variant thereof contains a substitution of up to 5 amino acids in the FR1;

[0026] The FR2 or a variant thereof shown in any one of SEQ ID NO:19-21, wherein the variant thereof contains a substitution of up to 5 amino acids in the FR2;

[0027] The FR3 or a variant of FR3 shown in any one of SEQ ID NO:22-26, wherein the variant of FR3 contains substitutions of up to 5 amino acids in the FR3;

[0028] The FR4 or a variant thereof shown in any one of SEQ ID NO:27-29, wherein the variant of the FR4 contains substitutions of up to 5 amino acids.

[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 an amino acid sequence of a single-domain antibody capable of binding to CD22, wherein the amino acid sequences of the single-domain antibody are as shown in SEQ ID NO. 1-7; or, compared with any one of SEQ ID NO: 1-7, at least one amino acid residue in the FR1, FR2, FR3 or FR4 sequence of the single-domain antibody is replaced by a conserved 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%, and 100% sequence homology with an amino acid sequence selected from SEQ ID NO: 1-7, and is capable of specifically binding to the CD22 protein.

[0032] A third aspect of the invention is to provide an Fc fusion antibody or humanized antibody that provides any of the aforementioned single-domain antibodies against CD22.

[0033] A fourth aspect of the present invention is to provide a nucleotide molecule encoding the aforementioned anti-CD22 single-domain antibody, the aforementioned Fc fusion antibody, or the aforementioned humanized antibody, wherein the nucleotide sequences are as shown in SEQ ID NO: 8-14, or the amino acid sequences encoded by the nucleotide sequences are the same as those 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%, and 100% sequence homology with nucleotide sequences selected from SEQ ID NO: 8-14, and the anti-CD22 single-domain antibody encoded therein 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 a single-domain antibody, an Fc fusion antibody, or a humanized antibody against CD22, the nucleotide sequences of which are shown in SEQ ID NO: 8-14, or the amino acid sequences encoded by the nucleotide sequences are the same as those 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 or its Fc fusion antibody or humanized antibody encoding anti-CD22 is integrated into RJK-V4-hFC4 by genetic engineering). Other universal expression vectors may also be selected as needed.

[0037] A sixth aspect of the present invention is to provide a host cell capable of expressing the aforementioned anti-CD22 single-domain antibody, Fc fusion antibody, or humanized antibody, or an expression vector comprising the aforementioned. Preferably, the host cell is a bacterial cell, fungal cell, or mammalian cell.

[0038] In another preferred embodiment, the host cell includes prokaryotic 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, bacteriophages, or combinations thereof.

[0040] In another preferred embodiment, the prokaryotic cells are selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or combinations thereof.

[0041] In another preferred embodiment, the eukaryotic cells are selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosomalidomiae, Trichoderma, or combinations thereof.

[0042] In another preferred embodiment, the eukaryotic cells are selected from the group consisting of insect cells such as armyworms, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or combinations thereof.

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

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

[0045] A 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 NO. 1-7, or a single-domain antibody having at least 80% homology with the single-domain antibody in SEQ ID NO. 1-7, or a fragment, derivative, or analogue of the aforementioned anti-CD22 single-domain antibody, or a multi-epitope antibody, multi-specific antibody, or multivalent antibody comprising the aforementioned single-domain antibody or its fragments, derivatives, or analogues; for example, the multi-epitope antibody may consist of more than one sequence in SEQ ID NO. 1-7; the multivalent antibody may consist of a sequence in SEQ ID NO. 1-7 repeated a certain number of times; the multi-specific antibody includes, but is not limited to, bispecific antibodies and trispecific antibodies; furthermore, 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 chimeric antigen receptors (CARs) in CAR-T therapy. These recombinant proteins specifically bind to the target cell surface antigen CD22, achieving precise anchoring of the target cells. They can also be used as components of antibody-drug conjugates (ADCs) to carry small molecule drugs to work together to target cells.

[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 sequence of the single-domain antibody is as shown in SEQ ID NO. 1-7 or has at least 80% homology with SEQ ID NO. 1-7. The second antigen-binding portion can also be as shown in SEQ ID NO. 1-7 or has at least 80% homology with SEQ ID NO. 1-7 (the sequences of the first antigen-binding portion and the second antigen-binding portion are different). The second antigen-binding portion can also be another type of antibody, such as an antibody against CD19, CD20, CD38, TLR9, CD3, CD28, or PDL1 (which can be a monoclonal antibody, a polyclonal antibody, a single-domain antibody, or any other form of antibody). The first antigen-binding portion and the second antigen-binding portion are fused together, for example, through the Fc segment of human IgG.

[0047] Trispecific antibodies consist of a first antigen-binding moiety, a second antigen-binding moiety, and a third antigen-binding moiety. These three moieties are fused together and are different antibody sequences, targeting up to three targets, such as CD20, CD22, and CD38.

[0048] An eighth aspect of the invention is to provide a pharmaceutical composition comprising the aforementioned CD22-binding single-domain antibody and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally determined based on the isoelectric point of the antibody (the pH of the aqueous carrier medium must deviate from the isoelectric point of the antibody by approximately 2).

[0049] The pharmaceutical compositions of this invention can be directly bound to CD22 protein molecules, and therefore can be used to treat diseases (including but not limited to autoimmune diseases and hematologic malignancies). Furthermore, they can be used in combination with other therapeutic agents or treatment modalities. Other therapeutic agents can be any other existing drugs for treating the relevant disease; other treatment modalities include chemotherapy, radiotherapy, etc.

[0050] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 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 solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions.

[0051] A ninth aspect of the present invention is to provide a medicament for treating a disease, comprising the aforementioned single-domain antibody for binding the CD22 protein as an active ingredient. In a preferred embodiment, the disease includes, but is not limited to, autoimmune diseases and hematologic malignancies.

[0052] A 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 cells described in the sixth aspect of the invention under conditions suitable for generating single-domain antibodies, thereby obtaining a culture containing the single-domain antibody against CD22; and

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

[0055] (c) Optionally, purify and / or modify the 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 agent in the preparation of a medicament for treating a disease.

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

[0058] In a preferred embodiment, the disease includes autoimmune diseases and hematologic malignancies.

[0059] In a preferred embodiment, the diseases include systemic lupus erythematosus, rheumatoid arthritis, Sjögren'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 lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, and CD22-positive B-cell acute lymphoblastic leukemia.

[0060] Beneficial effects

[0061] Compared with the prior art, the beneficial effects of the present invention are:

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

[0063] (2) The single-domain antibody obtained by the present invention has a flexible expression system. 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) The single-domain antibody obtained by the present invention has simple multi-combination form modification. It can be obtained by simple tandem through genetic engineering to obtain multivalent and multispecific antibodies. Moreover, its immune heterogeneity is very low and it will not produce a strong immune response without humanization modification.

[0065] (4) The single-domain antibody obtained by the present invention has a wider affinity range. Before affinity maturation, its affinity range can be from nM to pM, providing multiple options for antibodies for different purposes in the later stage. Attached Figure Description

[0066] To more clearly illustrate the technical solution of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0067] Figure 1 The enrichment status of the library for screening CD22-targeting antibodies in Example 3;

[0068] Figure 2The antibody-antigen binding dose-response curve (6C10, 9B2) is shown in Example 12.

[0069] Figure 3 The antibody-antigen binding dose-response curve (14C2, 14G2) is shown in Example 12.

[0070] Figure 4 This is the antibody-antigen binding dose-response curve determination diagram (21A3) from Example 12;

[0071] Figure 5 The above is a dose-response curve of antibody-antigen binding in Example 12 (22H10, 10F9, Tab1 and hIgG). Detailed Implementation

[0072] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0073] As used herein, “sdAb” (also referred to as nanobodies 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 include antibodies containing only heavy chains (which naturally do not contain light chains), 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 cattle. For example, naturally occurring VHH molecules can be derived from antibodies provided by camelid species such as camels, dromedaries, llamas, and guanacos. Like complete antibodies, single-domain antibodies can selectively bind to specific antigens. Single-domain antibodies may contain only variable domains of the immunoglobulin chain, which have CDR1, CDR2, and CDR3, as well as a frame region.

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

[0076] As used in this article, the term "Fc fusion antibody" refers to a novel protein produced by fusing the Fc fragment of a target antibody with a biologically active functional protein molecule 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 region (CDR1-3 sequence) of a target antibody into the variable region of a human antibody, or by mutating the target antibody according to the characteristics of the human antibody backbone region (FR1-4). Humanized antibodies can be produced using synthetic methods or site-directed mutagenesis methods.

[0078] In this invention, sequences with high homology to the CDR1-3 sequences disclosed herein 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," or "at least 98% homology" with the sequences in SEQ ID NO. 1-7 can achieve the purpose of the invention.

[0079] In some embodiments, the inventive objective can also be achieved by replacing only one or a few amino acids compared to the sequences in SEQ ID NO: 1-7, for example, by including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions. In practice, 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 “conserved” amino acid substitutions. In the case of substitution, the substitution will preferably be a conserved amino acid substitution, which can generally be described as an amino acid residue replaced by another amino acid residue having a similar chemical structure, and this substitution has little or no effect on the function, activity, or other biological properties of the polypeptide. The conserved amino acid substitutions are common in the art. For example, a conserved amino acid substitution is the substitution of one or a few amino acids in the following groups (a)-(d) by another or a few amino acids in the same group: (a) polar negatively charged residues and their non-charged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. The particularly preferred conserved 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 or Gln; Phe is replaced by Met, Leu, or Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe or 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. Furthermore, those skilled in the art will understand that the inventiveness of the single-domain antibody lies in the CDR1-3 regions, while the frame region sequences FR1-4 are not immutable, and the sequences of FR1-4 can adopt conserved sequence variants of the sequences disclosed in this invention.

[0080] The preferred host cell of the present invention is a bacterial cell, a fungal cell, or a mammalian cell.

[0081] This patent involves preparing a target protein and a truncated form of the target protein using genetic engineering technology. The obtained antigen protein is then used to immunize Bactrian camels in Alashan, Inner Mongolia. After multiple immunizations, peripheral blood lymphocytes or spleen cells of the camels are obtained. Through genetic engineering, the variable region coding sequence of the camel-derived antibody is recombined into a phage display vector. Specific antibodies against the antigen protein are screened using phage display technology, and their ability to bind to the antigen and their application in the treatment of autoimmune diseases are further tested.

[0082] The above technical solution will now be broken down and explained in detail, and described with specific embodiments:

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

[0084] The human recombinant extracellular domain protein used in this patent was expressed and purified by the company itself. The specific design scheme of the expression vector for the human recombinant CD22 protein is as follows:

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

[0086] (2) The nucleotide sequence encoding the extracellular terminus of CD22 (amino acids 20-687) was cloned into the vector pcDNA3.4 using gene synthesis. The constructed vector was subjected to Sanger sequencing, and after comparison with the original sequence and confirmation that it was correct, the recombinant plasmid was extracted in large quantities, endotoxin was removed, and it was transfected into suspension 293F cells for expression and purification of the target protein. The purity reached more than 90%, which met the requirements for animal immunization.

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

[0088] One mg of the purified human recombinant CD22 protein obtained in Example 1 was mixed with an equal volume of Freund's complete adjuvant and used to immunize a Bactrian camel from Alashan, Inner Mongolia. The camel was immunized once a week for a total of 7 weeks. Except for the first immunization, the remaining six immunizations were performed by mixing one mg of CD22 protein with an equal volume of Freund's incomplete adjuvant. This immunization process was intended to concentrate the stimulation of the camel to produce antibodies against CD22 protein.

[0089] After animal immunization, 150 mL of peripheral blood lymphocytes were collected from camels, and RNA was extracted from the cells. cDNA was synthesized using the extracted total RNA, and VHH (antibody heavy chain variable region) was amplified using nested PCR with the cDNA as a template.

[0090] Then, the pMECS vector and VHH fragment were digested with restriction endonucleases, and the digested fragments were ligated to the vector. The ligated fragments were electroporated into competent TG1 cells to construct a phage display library of the CD22 protein, and the library size was determined to be approximately 1 × 10⁻⁶. 9 Meanwhile, the correct insertion rate of the target fragment in the library was detected by colony PCR.

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

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

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

[0094] 500 μg of CD22 protein diluted in 100 mM pH 8.3 NaHCO3 was coupled onto an ELISA plate and incubated overnight at 4°C. A negative control well (culture medium control) was also included. The next day, 200 μL of 3% skim milk was added, and the plate was blocked at room temperature for 2 hours. After blocking, 100 μL of the amplified phage library (approximately 2 × 10⁻⁶) was added. 11 (1 phage particle), incubate at room temperature for 1 hour; after 1 hour, wash 15 times with PBS + 0.05% Tween-20 to remove unbound phage.

[0095] Phages that specifically bind to CD22 protein were dissociated using trypsin at a final concentration of 25 mg / mL and then used to infect E. coli TG1 cells in the logarithmic growth phase. The cells were cultured at 37°C for 1 h to produce and collect phages for the next round of screening. The same screening process was repeated once to gradually enrich the cells.

[0096] When the enrichment factor reaches 10 times or more, the enrichment effect is as follows: Figure 1 As shown.

[0097] Figure 1In this context, P / N = the number of monoclonal bacteria grown from phages eluted from positive wells in the biopanning process after infecting TG1 bacteria / the number of monoclonal bacteria grown from phages eluted from negative wells after infecting TG1 bacteria. This parameter gradually increases after enrichment occurs. I / E = the total number of phages added to positive wells in each round of the biopanning process / the total number of phages eluted from positive wells in each round of the biopanning process. This parameter gradually approaches 1 after enrichment occurs.

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

[0099] Four rounds of screening were conducted on single-domain antibodies against the anti-CD22 protein according to the screening method in Example 3 above. The phage enrichment factor against the anti-CD22 protein reached more than 10. After the screening, 384 single colonies were selected from the positive clones and inoculated into 96-well plates of 2×TY medium containing 100 μg / mL ampicillin. A blank control was set up. After incubation at 37°C to the logarithmic phase, IPTG was added to a final concentration of 1 mM and incubated overnight at 28°C.

[0100] Crude antibody was obtained using the osmotic burst method. CD22 recombinant protein was released into 100 mM NaHCO3 (pH 8.3), and 100 μg of protein was coated overnight at 4°C in an ELISA plate. 100 μL of the obtained crude antibody extract was transferred to an ELISA plate containing the antigen and incubated at room temperature for 1 h. Unbound antibody was washed away with PBST, and 100 μL of Mouse Anti-HA tag Antibody (HRP) (Mouse anti-HA horseradish peroxidase labeled antibody, ThermoFisher) diluted 1:2000 was added. The plate was incubated at room temperature for 1 h. Unbound antibody was washed away with PBST, and horseradish peroxidase chromogenic solution was added. The reaction was carried out at 37°C for 15 min, and then stop solution was added. The absorbance was read at 450 nm using an ELISA reader.

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

[0102] Gene sequences of each clone were analyzed using the sequence alignment software VectorNTI. Clones with identical CDR1, CDR2, and CDR3 sequences were considered the same clone, while clones with different sequences were considered different clones. Ultimately, 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 the antibody is 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 to ultimately obtain the single-domain antibody protein.

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

[0119]

[0120]

[0121]

[0122]

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

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

[0125] The plasmids (pMECS-VHH) of different clones obtained from sequencing analysis in Example 4 were electroporated into Escherichia coli HB2151 and plated on LB+amp+glucose culture plates containing ampicillin and glucose, and incubated overnight at 37°C. Single colonies were selected and inoculated into 5 mL of LB culture medium containing ampicillin and incubated overnight on a shaker at 37°C.

[0126] Inoculate 1 mL of overnight cultured bacteria into 330 mL of TB culture medium and incubate at 37°C in a shaker. When the OD600nm value reaches 0.6-0.9, add 1 M IPTG and incubate overnight at 28°C in a shaker. Centrifuge to collect E. coli and obtain crude antibody extract using the osmotic rupture method.

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

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

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

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

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

[0132] (4) The extracted plasmids were then sequenced and identified.

[0133] (5) After confirming the recombinant vector, prepare it 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 single-domain antibody against CD22 protein in suspension ExpiCHO-S cells

[0135] (1) Three days before transfection, use 2.5×10 5 / mL cell passage and expansion culture ExpiCHO-S TM Cells, the calculated desired cell volume, were transferred to 120 mL (final volume) of fresh, preheated ExpiCHO. TM In a 500 mL shake flask of expression medium, the cell concentration was increased to approximately 4 × 10⁻⁶ cells / mL. 6 -6×10 6 live cells / mL;

[0136] (2) One day before transfection, ExpiCHO-S TM Cells were diluted to a concentration of 3.5 × 10⁻⁶. 6 Live cells / mL, incubate cells overnight;

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

[0138] (4) Use fresh ExpiCHO preheated to 37°C TM The expression medium was used to dilute the cells to 6 × 10⁶. 6 viable cells / mL. The calculated desired cell volume was transferred to 100 mL (final volume) of fresh, preheated ExpiCHO. TM In a 500 mL shake flask of expression medium;

[0139] (5) Gently invert to mix ExpiFectamine TM CHO reagent, using 3.7 mL OptiPRO TM Culture medium for diluting ExpiFectamine TM CHO reagent, vortex or mix well;

[0140] (6) Use 4 mL of refrigerated OptiPRO TM Dilute the plasmid DNA in the culture medium and vortex 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 while gently shaking the flask during the addition process.

[0142] (8) The cells were cultured with shaking in humidified air at 37°C and 8% CO2.

[0143] (9) Add 600ul of ExpiFectamine on the first day after transfection (18-22 hours later). TM CHO Enhancer and 24mLExpiCHO feed.

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

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

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

[0147] (1) Three days before transfection, use 2.5×10 5 After passage and expansion of 293F cells at / mL, the calculated desired cell volume was transferred to a 500mL shake flask containing 120mL (final volume) of fresh, preheated OPM-293CD05 Medium to achieve a cell concentration of approximately 2×10⁻⁶. 6 -3×10 6 Live cells / mL.

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

[0149] (3) Dilute the cells to 1×10⁻⁵ using preheated OPM-293CD05 Medium. 6 1 live cells / mL. Calculate the required cell volume and transfer it to a 500 mL shake flask containing 100 mL (final volume) of fresh, preheated culture medium.

[0150] (4) Dilute PEI (1 mg / mL) reagent with 4 mL Opti-MEM medium, and mix by swirling or pipetting; dilute 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 Opt-MEM medium, mix by swirling, and filter with a 0.22 μm filter. 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 while gently shaking the flask during the addition process.

[0152] (6) Culture the cells at 37°C, 5% CO2, and 120 rpm with shaking.

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

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

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

[0156] (1) The protein expression supernatant obtained in Example 7 or 8 was filtered with a 0.45 μm disposable filter to remove insoluble impurities;

[0157] (2) The above filtrate was purified by affinity chromatography using a protein purifier. The agarose packing material coupled with Protein A was used to purify the filtrate by utilizing the ability of human Fc to bind to Protein A.

[0158] (3) Pass the filtrate through a pre-packed Protein A 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 buffers;

[0160] (5) Use a low pH buffer to systemically bind the target protein on the column;

[0161] (6) Quickly add the eluent to a Tris-HCl solution with a pH of 9.0 to neutralize it;

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

[0163] Example 10: Construction of the single-domain antibody eukaryotic expression vector RJK-V4-hFC4

[0164] The commonly used target vector for nanobodies, RJK-V4-hFC4, is a modified version of Invitrogen's commercial vector pCDNA3.4 (vector data link: https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / pcdna3_4_topo_ta_cloning_kit_man.pdf), which incorporates the Fc region of the human IgG4 heavy chain coding sequence. Specifically, this vector contains the CH2 and CH3 hinge regions of the IgG4 heavy chain. The specific modification scheme is as follows:

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

[0166] (2) Multiple cloning sites (MCS) and 6×His tags were introduced at the 5' and 3' ends of the Fc fragment coding sequence, respectively, by overlapping PCR.

[0167] (3) The above fragment was amplified by PCR using a pair of primers with XbaI and AgeI restriction sites respectively;

[0168] (4) The recombinant DNA fragments in pcDNA3.4 and (3) were digested with restriction endonucleases XbaI and AgeI, respectively;

[0169] (5) The digested vector and the insert fragment were ligated with T4 ligase, and then the ligation product was transformed into E. coli, amplified, sequenced and verified to obtain the recombinant plasmid.

[0170] Example 11: Expression and purification of a tool antibody (Tab1) targeting human CD22

[0171] In this study, Tab1 refers to Inotuzumab Ozogamicin. The antibody sequence of this drug was commissioned to General Biosystems (Anhui) Co., Ltd. for codon optimization in a mammalian cell expression system and cloned into the RJK-V4-hFC4 vector. After antibiotic screening, plasmid-positive bacteria were selected for amplification, and plasmids were extracted using a plasmid extraction kit (Macherey Nagel, Cat#740412.50). 100 μg of plasmid (40 μg heavy chain + 60 μg light chain) was added per 100 mL of cells, and PEI was used to transiently transfect 293F cells (medium: FreeStyle 293Expression medium, Thermo, Cat#12338026+F-68, Thermo, Cat#24040032). 6–24 h after transfection, 5% volume of 10% Peptone (Sigma, Cat#P0521-100G) was added, and the cells were cultured in 8% CO2 at 130 rpm for approximately 7–8 days. When cell viability decreased to 50%, the expression supernatant was collected and purified using a gravity column with Protein A (GE, Cat#17-5438-02). After dialysis with PBS, the concentration was determined using Nanodrop, purity was identified by SEC, and binding capacity was verified by indirect ELISA.

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

[0173] Example 12: Determination of Antibody-Antigen Binding Dose-Reaction Curve

[0174] This embodiment uses the standard enzyme-linked immunosorbent assay (ELISA) procedure.

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

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

[0177] (3) VHH-hFc was diluted to 2 μg / mL, and then the antibody was serially diluted 5-fold to 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 in Example 9. In addition, hIgG (actually hIgG4) and Tab1 controls were also set up; Tab1 was prepared in Example 11;

[0178] (4) Wash the plate; add 50 μL of the single-domain antibody obtained by dilution in step (3), double 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 brought to room temperature beforehand, and react at room temperature in the dark for 15 min.

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

[0182] (8) Plot the curve and calculate EC50, as follows: Figures 2-5 As shown, hIgG refers to the isotype control, an immunoglobulin molecule that does not bind to any target and is commercially available. Figures 2-5 The results for the sample (containing 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).

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

[0184] The foregoing has shown and described 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 way, and all technical solutions obtained by equivalent substitution or equivalent transformation 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 variable region, which includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3. The amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are as follows (1) or (2): (1) CDR1 shown in SEQ ID NO:32, CDR2 shown in SEQ ID NO:37, and CDR3 shown in SEQ ID NO:41; (2) CDR1 shown in SEQ ID NO:31, CDR2 shown in SEQ ID NO:37, and CDR3 shown in SEQ ID NO:

41.

2. The anti-CD22 single-domain antibody according to claim 1, characterized in that: The single-domain antibody further includes a frame region FR; the frame region FR includes the amino acid sequences of FR1, FR2, FR3, and FR4; the amino acid sequences of the frame region FR are as follows: The FR1 or a variant thereof shown in SEQ ID NO:15 or 17, wherein the variant thereof contains a substitution of up to 5 amino acids in the FR1; The FR2 or a variant thereof shown in SEQ ID NO:20, wherein the variant thereof contains substitutions of up to 5 amino acids; The FR3 or a variant thereof shown in SEQ ID NO: 25, wherein the variant thereof contains substitutions of up to 5 amino acids; The FR4 or a variant thereof shown in SEQ ID NO:28 or 29, wherein the variant thereof contains substitutions of up to 5 amino acids.

3. The anti-CD22 single-domain antibody according to claim 1, characterized in that: The single-domain antibody specifically binds to the extracellular domain of the CD22 antigen.

4. A single-domain antibody against CD22, characterized in that: The amino acid sequences of the single-domain antibody are shown in SEQ ID NO: 1, 2 or 4, respectively; or, compared with SEQ ID NO: 1, 2 or 4, at least one amino acid residue in the FR1, FR2, FR3 or FR4 sequence of the single-domain antibody is replaced by a conserved amino acid.

5. The Fc fusion antibody or humanized antibody of the single-domain anti-CD22 antibody according to any one of claims 1 to 4.

6. A nucleotide molecule encoding a single-domain antibody against CD22 according to any one of claims 1 to 4, characterized in that: The nucleotide sequences are shown in SEQ ID NO: 8, 9 or 11, respectively, or the amino acid sequences encoded by the nucleotide molecules are the same as those encoded by SEQ ID NO: 8, 9 or 11.

7. An expression vector, characterized in that: It comprises a nucleotide molecule encoding a single-domain antibody against CD22 as described in any one of claims 1 to 4, or an Fc fusion antibody or humanized antibody as described in claim 5, or a nucleotide molecule as described in claim 6.

8. A host cell, characterized in that: It can express the single-domain antibody against CD22 as described in any one of claims 1 to 4, or the Fc fusion antibody or humanized antibody as described in claim 5, or it may contain the expression vector as described in claim 7.

9. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises a single-domain antibody against CD22 selected from any one of claims 1 to 4, and a pharmaceutically acceptable carrier.

10. A medicine for treating diseases, characterized in that: It comprises an anti-CD22 single-domain antibody as the active ingredient of any one of claims 1 to 4.

11. The use of the anti-CD22 single-domain antibody according to any one of claims 1 to 4, the Fc fusion antibody or humanized antibody according to claim 5, the pharmaceutical composition according to claim 9, or the agent according to claim 10 in the preparation of a medicament for treating a disease, characterized in that: The diseases mentioned are systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, hairy cell leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, B-cell chronic lymphocytic leukemia, non-Hodgkin's lymphoma, or multiple myeloma.

12. The use according to claim 11, characterized in that, The acute lymphoblastic leukemia is relapsed acute lymphoblastic leukemia, precursor B-cell acute lymphoblastic leukemia, or CD22-positive B-cell acute lymphoblastic leukemia; the non-Hodgkin lymphoma is B-cell lymphoma or precursor B-cell lymphoblastic leukemia lymphoma.

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