WO2023216218A1

WO2023216218A1 - Antibody specifically binding to cd22, and preparation method therefor and use thereof

Info

Publication number: WO2023216218A1
Application number: PCT/CN2022/092634
Authority: WO
Inventors: 杜靓; 万婷婷; 徐溜溜; 张红艳; 郭晓月; 金理娜; 陈亚莉; 汤荣; 金夷
Original assignee: 上海吉倍生物技术有限公司
Priority date: 2022-05-13
Filing date: 2022-05-13
Publication date: 2023-11-16

Abstract

The present application belongs to the technical field of biological immunity, and specifically relates to a monoclonal antibody capable of specifically binding to CD22 and an antigen-binding fragment thereof. The present application further relates to a method for preparing the antibody and the antigen-binding fragment thereof and the use of the antibody and the antigen-binding fragment thereof.

Description

CD22 specific binding antibodies and preparation methods and uses thereof

Technical field

This application belongs to the field of biological immunity technology, and specifically relates to monoclonal antibodies that can specifically bind to human CD22 and their antigen-binding fragments. The present application also relates to preparation methods and uses of the antibodies and antigen-binding fragments thereof.

Background technique

Hematoma is a type of cancer that affects blood cells and includes three main types: leukemia, lymphoma, and myeloma. Of these, leukemia is found in the blood and bone marrow, lymphoma primarily affects the lymphatic system, and myeloma is a blood disorder that specifically targets plasma cells. Each year, these types of cancer account for approximately 10% of all new cancer diagnoses. In the United States, someone is diagnosed with blood cancer every 3 minutes. Leukemia is the most common cancer in children, accounting for 28% of all childhood cancers. To date, more than 1.2 million people in the United States have blood cancer or are in remission. Currently, chemotherapy is the main treatment strategy for leukemia, and in recent years patients have begun using more intensive, long-term chemotherapy regimens, which has resulted in greater responses to treatment. However, these treatments can also cause serious side effects, such as severe damage to the body's white blood cells, and patients may need to take other medications to help prevent or alleviate these side effects. At the same time, long-term chemotherapy also faces the limitation of chemoresistance, which cannot be properly solved so far. Compared with traditional chemotherapy, targeted immunotherapy targets biomarkers on the surface of tumor cells and selects corresponding drugs that can specifically kill tumor cells without drug resistance. Approximately 60-80% of B-cell malignancies express CD22, making it a potential target for targeted immunotherapy.

CD22 is a member of the sialic acid-binding immunoglobulin-like lectin (Siglecs) family and one of the inhibitory co-receptors on the surface of B cells. It is involved in regulating B cell migration, B lymphocyte survival, signal transduction, and B cell Homing and cell adhesion, and are closely related to B cell development, differentiation and function. CD22 is a 140KDa single-pass transmembrane glycoprotein on the surface of B cells. Its extracellular domain (ECD) consists of 7 immunoglobulin (Ig) domains (d1-d7) and contains 12 N-linked glycosylation. site. CD22 is restrictedly expressed on the surface of mature B cells and most malignant B lymphoma cells, such as refractory and relapsed B acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma ( NHL) and hairy cell leukemia, but is not expressed in normal tissues, including hematopoietic stem cells and hematopoietic precursors of B lymphocytes, and can be transferred to the plasma membrane as B cells mature until plasma cells differentiate, but plasma cells do not express this molecule . Currently, the types of immunotherapy drugs that target CD22 include monoclonal antibody drugs, antibody conjugates (ADC), and CAR-T therapy. Compared with other B lymphocyte lineage surface antigens, CD22 is a better internalized antigen. Anti-CD22 antibodies can be rapidly internalized after binding to CD22. To take advantage of this unique property, antibodies are often used as targeting moieties to selectively Deliver radionuclides, toxins or ribonucleases into CD22 tumor cells.

CD22 is expressed in more than 90% of patients with refractory and relapsed B-acute lymphoblastic leukemia (ALL), and current complete response rates for adult refractory and relapsed acute B-cell leukemia are 60-90% . However, many patients with complete remission will relapse, and only about 30-50% will have disease-free survival of 3 years or more. Pfizer launched Besponsa (Inotuzumab Ozogamicin) in December 2021, an anti-CD22 antibody conjugated with calicheamicin. The complete response rate of this treatment group for refractory and relapsed acute B-cell leukemia was significantly higher than that of standard chemotherapy. group, 80.7% and 29.4% respectively; among patients with complete response, the Inotuzumab Ozogamicin group had a higher percentage below the threshold of minimal residual disease (0.01% bone marrow blasts) (78.4% vs. 28.1%, P<0.001 ); the response period was longer in the Inotuzumab Ozogamicin group (median, 4.6 months vs. 3.1 months); in a survival analysis including 326 patients, the progression-free survival period in the Inotuzumab Ozogamicin group was significantly longer (median , 5.0 months vs. 1.8 months); the median overall survival was 7.7 months and 6.7 months.

Based on this, there is a need to develop better antibodies that specifically bind CD22 for the treatment of CD22-expressing tumors.

Contents of the invention

In this application, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used in this article are routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand this application, definitions and explanations of relevant terms are provided below.

Definition of Terms

As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). Antibody light chains can be classified into kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the interaction of immunoglobulins with host tissues or factors, including various cells of the immune system (e.g., effector cells) and classical complement. Binding of the first component of the system (C1q). The VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs). Each _VH and _VL consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain/light chain pair respectively form the antigen-binding site. The assignment of amino acids to each region or domain can follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901- 917; Definition of Chothia et al. (1989) Nature 342:878-883.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable regions of the heavy chain and light chain each contain three CDRs, named CDR1, CDR2 and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883) or IMGT numbering system (Lefranc et al. , Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

In the present invention, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies of the invention, or antigen-binding fragments thereof, are preferably determined by the Kabat, Chothia, or IMGT numbering systems.

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

The term "antibody" is not limited to any particular method of producing the antibody. This includes, for example, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibodies may be of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody Specific binding to an antigen, which is also called an "antigen-binding moiety." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), which is incorporated herein by reference in its entirety for all purposes. It can be obtained by recombinant DNA technology Or antigen-binding fragments of the antibody are generated by enzymatic or chemical cleavage of the intact antibody. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab') ₂ , Fd, Fv, complementarity determining region (CDR) fragments, scFv, diabody, single domain antibody, chimeric antibody, linear antibody, nanobody (technology from Domantis), probody and such polypeptides, which contain sufficient antigen to confer specificity to the polypeptide At least a portion of an antibody with binding capacity. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

As used herein, the term "full-length antibody" means an antibody consisting of two "full-length heavy chains" and two "full-length light chains." Among them, "full-length heavy chain" refers to a polypeptide chain that consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), and a heavy chain in the direction from the N end to the C end. It consists of a constant region CH2 domain and a heavy chain constant region CH3 domain; and, when the full-length antibody is of IgE isotype, optionally also includes a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains. The full-length antibody of the present invention can be from a single species, such as human; it can also be a chimeric antibody or a humanized antibody. The full-length antibody of the present invention contains two antigen-binding sites formed by VH and VL pairs respectively, and these two antigen-binding sites specifically recognize/bind the same antigen.

As used herein, the term "Fd" means an antibody fragment consisting of VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544 546 ( 1989)); the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F(ab') ₂ fragment" means an antibody fragment consisting of two fragments connected by a disulfide bridge on the hinge region An antibody fragment of a Fab fragment; the term "Fab'fragment" means the fragment obtained by reducing the disulfide bond connecting the two heavy chain fragments in the F(ab') ₂ fragment, consisting of a complete light chain and the Fd of the heavy chain. Fragment (consisting of VH and CH1 domains).

As used herein, the term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen-binding site. It is generally believed that six CDRs confer the antigen-binding specificity of an antibody. However, even a variable region (such as an Fd fragment, which contains only three CDRs specific for the antigen) can recognize and bind the antigen, although its affinity may be lower than that of the intact binding site.

As used herein, the term "Fc" means a region formed by disulfide bonding of the second and third constant regions of the first heavy chain of an antibody to the second and third constant regions of the second heavy chain. Antibody fragments. The Fc fragment of an antibody has many different functions but does not participate in antigen binding.

As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains connected by a linker (see, e.g., Bird et al., Science 242:423 -426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: _NH2 -VL-linker-VH-COOH or _NH2 -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) ₄ can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present invention are provided by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between VH and VL of scFv. In certain embodiments of the invention, scFv can form di-scFv, which refers to two or more individual scFvs connected in series to form an antibody. In certain embodiments of the invention, scFv can form (scFv) ₂ , which refers to two or more individual scFvs joining in parallel to form an antibody.

As used herein, the term "diabody" means one whose VH and VL domains are expressed on a single polypeptide chain but using a linker that is too short to allow pairing between the two domains of the same chain, This forces the domain to pair with the complementary domain of the other chain and creates two antigen binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2:1121-1123 (1994)).

As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art, which refers to an antibody composed of a single monomeric variable domain (e.g., a single heavy chain variable An antibody fragment consisting of a region) that retains the ability to specifically bind to the same antigen that the full-length antibody binds. Single domain antibodies are also called nanobodies.

Each of the above antibody fragments retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen.

Antigen-binding fragments of an antibody (e.g., the above-described antibody fragments) can be obtained from a given antibody (e.g., the antibodies provided by the invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation methods) ), and the antigen-binding fragments of the antibody are screened for specificity in the same manner as for intact antibodies.

As used herein, when the term "antibody" is mentioned, it includes not only intact antibodies but also antigen-binding fragments of the antibodies, unless the context clearly indicates otherwise.

As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the light chain or/and heavy chain is derived from an antibody (which may originate from a specific species or belong to a specific species). a specific antibody class or subclass), and the other part of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but regardless of However, it still retains the binding activity to the target antigen (U.S.P 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)). In certain embodiments, the term "chimeric antibody" may include antibodies in which the heavy and light chain variable regions of the antibody are derived from a first antibody and the heavy and light chain constant regions of the antibody are derived from a second antibody.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the first amino acid sequence or nucleic acid sequence to best match the second amino acid or nucleic acid sequence). Good comparison). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. Molecules are identical when a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (ie, percent identity = number of identical overlapping positions/total number of positions x 100%). In certain embodiments, both sequences are the same length.

Determination of percent identity between two sequences can also be accomplished using mathematical algorithms. One non-limiting example of a mathematical algorithm for comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Improved in .Acad.Sci.U.S.A.90:5873-5877. Such algorithms were integrated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.

As used herein, the term "variant", in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing substitutions, deletions, or additions of amino acid residues. In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (ie, by covalently linking any type of molecule to the polypeptide or peptide). For example, and without limitation, polypeptides may be modified, e.g., by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, Attached to cellular ligands or other proteins, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Furthermore, a variant has a similar, identical or improved function to the polypeptide or peptide from which it is derived.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K _D ) of the interaction. In the present invention, the term " _KD " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.

The specific binding properties between two molecules can be determined using methods known in the art. One approach involves measuring the rate at which antigen binding site/antigen complexes form and dissociate. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361 :186-187). The ratio kdis/kon is equal to the dissociation constant _KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). K _D , kon and kdis values can be measured by any valid method. In certain embodiments, dissociation constants can be measured in Biacore using surface plasmon resonance (SPR). Alternatively, bioluminescence interferometry or Kinexa can be used to measure dissociation constants.

As used herein, a detectable label of the invention may be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides fluorescein (e.g., ^3H , ^125I , ^35S , ^14C , or ^32P ), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC) , phycoerythrin (PE), Texas red, rhodamine, quantum dots or cyanine dye derivatives (such as Cy7, Alexa750)), luminescent substances (such as chemiluminescent substances, such as acridinium ester compounds, lumi Noradrenaline and its derivatives, ruthenium derivatives such as ruthenium terpyridine), magnetic beads (e.g.,

), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and avidin modified to bind the above labels (e.g., streptavidin ) of biotin.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Polyomavacuolating viruses (such as SV40). A vector can contain a variety of expression control elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain an origin of replication site.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

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

The twenty conventional amino acids involved in this article have been prepared following conventional usage. See, e.g., Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" means a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, They are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers Agents, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffer. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Agents that maintain osmotic pressure include, but are not limited to, sugar, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of active ingredients in medicines, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose) , lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient includes sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions). In certain exemplary embodiments, such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), dextrose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.

As used herein, the term "prevention" refers to a method performed to prevent or delay the occurrence of a disease or condition or symptom in a subject. As used herein, the term "treatment" refers to a method performed to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of the extent of the disease, stabilization (i.e., no worsening) of the state of the disease, delaying or slowing the progression of the disease, ameliorating or alleviating the disease. status, and relief of symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" may also refer to prolonging survival compared to expected survival if not receiving treatment.

As used herein, the term "effective amount" refers to an amount sufficient to obtain, at least in part, the desired effect. For example, a disease-preventing effective amount refers to an amount sufficient to prevent, prevent, or delay the occurrence of the disease; a disease-treating effective amount refers to an amount sufficient to cure or at least partially prevent the disease and its complications in patients who already suffer from the disease. quantity. Determining such effective amounts is well within the capabilities of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall status of the patient's own immune system, the patient's general condition such as age, weight and gender, the manner in which the drug is administered, and other treatments administered concurrently etc.

This application uses a CD22 high-expressing cell line for immunization. In the preliminary screening stage, the binding of antibodies and CD22 at the cellular level is examined, and this is used as a basis for screening to screen out antibodies that specifically bind CD22. In the subsequent screening verification, the anti-CD22 antibody Inotuzumab (the antibody sequence is derived from Inotuzumab Ozogamicin, an antibody-conjugated drug, purchased from Pfizer, KR20170049617) was used as the benchmark antibody. In vitro activity testing showed that the antibody of the present invention was more effective than the benchmark antibody. It shows outstanding advantages in combination, internalization and killing. Therefore, this antibody has great application potential in the treatment of CD22-expressing tumors.

Therefore, in a first aspect, the present application provides an antibody or an antigen-binding fragment thereof that specifically binds to CD22 protein, the antibody or an antigen-binding fragment thereof comprising:

(a) Heavy chain variable region (VH) containing the following three complementarity determining regions (CDRs):

(i) VH CDR1, which consists of the following sequence: SEQ ID NO: 3, or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions) or add) sequence,

(ii) VH CDR2, which consists of the following sequence: SEQ ID NO: 4, or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions) or addition), and (iii) VH CDR3, which consists of the following sequence: SEQ ID NO: 5 or 13, or has one or several amino acid substitutions, deletions or additions (e.g. 1, 2 Sequence with substitution, deletion or addition of one or three amino acids);

and / or,

(b) A light chain variable region (VL) containing the following three complementarity determining regions (CDRs):

(iv) VL CDR1, which consists of the following sequence: SEQ ID NO: 6, or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions) or add) sequence,

(v) VL CDR2, which consists of the following sequence: SEQ ID NO: 7, or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions) or addition) sequence, and (vi) VL CDR3, which consists of the following sequence: SEQ ID NO: 8, or has one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions).

In certain embodiments, the substitutions of any one of (i)-(vi) are conservative substitutions.

In certain embodiments, the CDRs described in any of (i)-(vi) are defined according to the Kabat, IMGT, or Chothia numbering systems.

In certain embodiments, the CDRs of any of (i)-(vi) are defined according to the Kabat numbering system.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

(1) The following 3 heavy chain CDRs: VH CDR1 as shown in SEQ ID NO:3, VH CDR2 as shown in SEQ ID NO:4, VH CDR3 as shown in SEQ ID NO:5; and/or, The following 3 light chain CDRs: VL CDR1 as shown in SEQ ID NO:6, VL CDR2 as shown in SEQ ID NO:7, VL CDR3 as shown in SEQ ID NO:8; or

(2) The following 3 heavy chain CDRs: VH CDR1 as shown in SEQ ID NO:3, VH CDR2 as shown in SEQ ID NO:4, VH CDR3 as shown in SEQ ID NO:13; and/or, The following three light chain CDRs: VL CDR1 as shown in SEQ ID NO:6, VL CDR2 as shown in SEQ ID NO:7, and VL CDR3 as shown in SEQ ID NO:8.

In certain embodiments, the antibody or antigen-binding fragment thereof as described above, wherein the antibody or antigen-binding fragment thereof comprises:

(a) Heavy chain variable region (VH) comprising an amino acid sequence selected from the following:

(i) The sequence shown in SEQ ID NO: 1 or 11;

(ii) Compared with the sequence shown in SEQ ID NO: 1 or 11, there is a substitution, deletion or addition of one or several amino acids (such as a substitution, deletion of 1, 2, 3, 4 or 5 amino acids) or add) sequence; or

(iii) Be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% identical to the sequence shown in SEQ ID NO: 1 or 11 Sequences that have %, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

and / or

(b) A light chain variable region (VL) comprising an amino acid sequence selected from:

(iv) The sequence shown in SEQ ID NO: 2 or 12;

(v) Compared with the sequence shown in SEQ ID NO: 2 or 12, it has one or several amino acid substitutions, deletions or additions (such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions) or add) sequence; or

(vi) Be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% identical to the sequence shown in SEQ ID NO: 2 or 12 %, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

In certain embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

(1) VH having the sequence shown in SEQ ID NO:1 and VL having the sequence shown in SEQ ID NO:2, or

(2) VH having the sequence shown in SEQ ID NO: 11 and VL having the sequence shown in SEQ ID NO: 12.

In certain embodiments, the antibody or antigen-binding fragment thereof as described above, wherein the antibody or antigen-binding fragment thereof comprises a constant region derived from a human immunoglobulin or a variant thereof.

(a) The heavy chain constant region (CH) of a human immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence from which it is derived (e.g. , substitution, deletion or addition of up to 20, up to 15, up to 10 or up to 5 amino acids or any combination thereof; for example, substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids. addition or any combination thereof); and/or

(b) A light chain constant region (CL) of a human immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence from which it is derived (e.g. , substitution, deletion or addition of up to 20, up to 15, up to 10 or up to 5 amino acids or any combination thereof; for example, substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids. addition or any combination thereof).

In some embodiments, the variant of the heavy chain constant region (CH) may have one or more conservative substitutions of amino acids compared to the sequence from which it is derived. In such embodiments, the variant of the heavy chain constant region (CH) may have the same or substantially the same effector function as the wild-type sequence from which it is derived.

In certain embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgGl, IgG2, IgG3 or IgG4 heavy chain constant region.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain constant region (CH) set forth in SEQ ID NO:9.

In certain embodiments, the light chain constant region is a kappa light chain constant region or a lambda light chain constant region.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises the light chain constant region (CL) set forth in SEQ ID NO: 10.

In certain embodiments, the antibody or antigen-binding fragment thereof as described above, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab') ₂ , Fv, disulfide-linked Fv, BsFv, dsFv, (dsFv) ₂ , dsFv-dsFv', scFv, scFv dimer, camelized single chain domain antibody, diabody, ds diabody, Nanobody, Single domain antibody (sdAb), bivalent domain antibody; and/or, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a multispecific antibody.

The antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering and recombinant technology. For example, DNA molecules encoding the heavy chain and light chain genes of the antibody of the present invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into host cells. Then, the transfected host cells are cultured under specific conditions and express the antibody of the invention.

The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)) . Alternatively, these antigen-binding fragments can also be produced directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000) )). For example, Fab’ fragments can be obtained directly from host cells; Fab’ fragments can be chemically coupled to form F(ab’)2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)). In addition, Fv, Fab or F(ab’)2 fragments can also be directly isolated from the recombinant host cell culture medium. Those of ordinary skill in the art are well aware of other techniques for preparing such antigen-binding fragments.

In certain embodiments, the antibody or antigen-binding fragment thereof is as described above, wherein the antibody or antigen-binding fragment thereof is labeled. In certain embodiments, the antibody or antigen-binding fragment thereof carries a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or Biotin.

In another aspect, the present application provides an isolated nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described above, or a heavy chain variable region and/or a light chain variable region thereof.

In certain embodiments, the nucleic acid molecule comprises a sequence set forth in SEQ ID NO: 14 or 16. In certain embodiments, the nucleic acid molecule comprises a sequence set forth in SEQ ID NO: 15 or 17.

In certain embodiments, the heavy chain variable region has the sequence set forth in SEQ ID NO: 14 or 16.

In certain embodiments, the light chain variable region has the sequence set forth in SEQ ID NO: 15 or 17.

In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention and a first nucleotide sequence encoding said antibody or antigen-binding fragment thereof A second nucleotide sequence of a light chain or light chain variable region, wherein said first nucleotide sequence and said second nucleotide sequence are present on the same or different separate nucleic acid molecules. When the first nucleotide sequence and the second nucleotide sequence are present on different isolated nucleic acid molecules, the isolated nucleic acid molecule of the present invention includes a third nucleotide sequence containing the first nucleotide sequence. a nucleic acid molecule and a second nucleic acid molecule containing said second nucleotide sequence.

In another aspect, the application provides a vector comprising a nucleic acid molecule as described above. In certain embodiments, the vector is a cloning vector or an expression vector.

In certain embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention and a light chain encoding said antibody or antigen-binding fragment thereof. or a second nucleotide sequence of the light chain variable region, wherein said first nucleotide sequence and said second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence exist on different vectors, the vector of the present invention includes a first vector containing the first nucleotide sequence and a vector containing the A second vector for a second nucleotide sequence.

In another aspect, the application also provides a host cell comprising a nucleic acid molecule or vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., E. coli cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., small mouse cells, human cells, etc.).

In another aspect, the application provides a method for preparing an antibody or an antigen-binding fragment thereof as described above, which includes culturing a host cell as described above under conditions that allow expression of the antibody or an antigen-binding fragment thereof, and The antibody or antigen-binding fragment thereof is recovered from the cultured host cell culture.

In another aspect, the application provides multispecific molecules comprising an antibody or antigen-binding fragment thereof as described above.

In certain embodiments, the multispecific molecule specifically binds CD22 and additionally specifically binds one or more other targets.

In certain embodiments, the multispecific molecule further comprises at least one molecule (eg, a second antibody) having a second binding specificity for a second target.

In another aspect, the application provides immunoconjugates comprising an antibody or antigen-binding fragment thereof as described above and a therapeutic agent linked to the antibody or antigen-binding fragment thereof.

In certain embodiments, the therapeutic agent is selected from cytotoxic agents.

In certain embodiments, the therapeutic agent is selected from the group consisting of alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and random combination.

In certain embodiments, the immunoconjugate is an antibody-drug conjugate (ADC).

In another aspect, the application provides a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described above, or a multispecific molecule as described above, or an immunoconjugate as described above, and a pharmaceutically acceptable carriers and/or excipients.

In certain embodiments, pharmaceutical compositions further comprise additional pharmaceutically active agents.

In certain embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an alkylating agent, a mitosis inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors or oncolytic viruses.

In certain embodiments, the antibody or antigen-binding fragment or multispecific molecule or immunoconjugate thereof and the additional pharmaceutically active agent are provided as separate components or as components of the same composition.

In certain exemplary embodiments, the pharmaceutically acceptable carrier and/or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), dextrose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.

In another aspect, the present application provides a kit containing the antibody or antigen-binding fragment thereof as described above.

In certain embodiments, the antibody or antigen-binding fragment thereof carries a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or Biotin.

In certain embodiments, the kit further includes a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof as described above.

In certain embodiments, the second antibody further includes a detectable label, such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), or biotin.

In another aspect, the present application provides a chimeric antigen receptor comprising an antigen-binding domain of an antibody as described above, or an antigen-binding fragment thereof.

In certain embodiments, the antigen-binding domain comprises the heavy chain variable region and the light chain variable region of an antibody or antigen-binding fragment thereof as described above.

In certain embodiments, the antigen binding domain is a scFv.

In certain embodiments, the antigen-binding receptor comprises an antigen-binding fragment of an antibody as described above.

In certain embodiments, the antigen-binding receptor is expressed by immune effector cells (eg, T cells).

In another aspect, the application provides an isolated nucleic acid molecule encoding a chimeric antigen receptor as described above.

In another aspect, the application provides a host cell comprising a vector as described above.

In another aspect, the present application also provides a method for reducing the expression level of CD22 on the cell surface, which includes combining the cells with the antibody or antigen-binding fragment thereof, or the multispecific molecule , or the immunoconjugate, or the pharmaceutical composition, or the chimeric antigen receptor, or the host cell contact, such that the expression level of CD22 on the cell surface is reduced; wherein, The cells express CD22 on their surface.

In certain embodiments, the cells are CD22-expressing tumor cells.

On the other hand, the present application provides a method for inhibiting the growth of CD22-expressing tumor cells and/or killing the tumor cells, which includes combining the tumor cells with an effective amount of the aforementioned antibody or its antigen. Fragments, or multispecific molecules as described above, or immunoconjugates as described above, or pharmaceutical compositions as described above, or chimeric antigen receptor contacts as described above.

In another aspect, the present application provides a method for preventing and/or treating tumors in a subject, the method comprising administering to a subject in need thereof an effective amount of an antibody as described above or The antigen-binding fragment thereof is a multispecific molecule as described above, an immunoconjugate as described above, a pharmaceutical composition as described above, or a chimeric antigen receptor as described above.

In certain embodiments, the tumor expresses CD22.

In certain embodiments, the tumor involves CD22-expressing tumor cells. In certain embodiments, the CD22 is expressed on the surface of the tumor cells.

In certain embodiments, the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, Melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cells Cancer and other hematological malignancies, such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-cell-rich lymphoma, EBV-positive and -negative PTLD and EBV-related Diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma and HHV8-related primary effusion lymphoma, Hodgkin lymphoma, central nervous system Systemic (CNS) tumors, such as primary CNS lymphoma, spinal tumors, and brainstem gliomas.

In certain embodiments, the subject is a mammal, such as a human.

In certain embodiments, the method further includes administering an additional drug with anti-tumor activity, such as an alkylating agent, a mitosis inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors or oncolytic viruses.

In certain embodiments, the methods further include administering additional anti-tumor therapy, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care.

On the other hand, the present application provides an antibody or an antigen-binding fragment thereof as described above, or a multispecific molecule as described above, or an immunoconjugate as described above, or a drug as described above. The composition, or the use of the chimeric antigen receptor as described above, in the preparation of a medicament for preventing and/or treating tumors in a subject.

In certain embodiments, the drug also contains an additional pharmaceutically active agent.

In certain embodiments, the tumor expresses CD22.

In certain embodiments, the subject is a mammal, such as a human.

On the other hand, the present application provides a method for detecting the presence or amount of CD22 in a sample, which includes the following steps:

(1) Contact the sample with the antibody or antigen-binding fragment thereof as described above;

(2) Detect the formation of a complex between the antibody or its antigen-binding fragment and CD22 or detect the amount of the complex.

In certain embodiments, the antibody or antigen-binding fragment thereof is detectably labeled.

In certain embodiments, the CD22 is human CD22.

In certain embodiments, the methods are used for therapeutic purposes, diagnostic purposes, or non-therapeutic non-diagnostic purposes.

In certain embodiments, the method is an immunological assay, such as a western blot, enzyme immunoassay (eg, ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay.

In another aspect, the present application provides a method for detecting whether a tumor can be treated by anti-tumor therapy targeting CD22, comprising the following steps:

(1) Contact the sample containing the tumor cells with the antibody or antigen-binding fragment thereof as described above;

(2) Detect the formation of a complex between the antibody or its antigen-binding fragment and CD22.

In certain embodiments, the CD22 is human CD22.

In another aspect, the present application provides the use of the antibody or antigen-binding fragment thereof as described above in the preparation of a kit for detecting whether a tumor can be treated by anti-tumor therapy targeting CD22.

In certain embodiments, the CD22 is human CD22.

The following examples are intended to better illustrate the present application and should not be construed as limiting the scope of the present application. All of the specific compositions, materials, and methods described below, in whole or in part, are within the scope of this application. These specific compositions, materials, and methods are not intended to limit the application but are merely illustrative of specific embodiments within the scope of the application. Equivalent compositions, materials and methods may be developed by those skilled in the art without inventive steps and without departing from the scope of the present application. It will be understood that various modifications to the methods of the present application may be made and still be included within the scope of the present application. The inventors intend that such modifications be included within the scope of the present application.

sequence information

Information on the sequences involved in this application is shown in Table 1 below.

Table 1. Sequence information

beneficial effects

The monoclonal antibodies of the present application (eg, antibody 13G9, antibody hu13G9) can bind to CD22 protein or cells expressing CD22 protein with high specificity. Compared with existing antibodies, the antibody hu13G9 of the present application has higher antigen-binding activity, stronger internalization ability on tumor cells, and can effectively induce the killing of CD22-expressing tumor cells. Therefore, the monoclonal antibody of the present application has high clinical application value and high application potential in targeted therapy of tumors.

Description of the drawings

Figure 1 shows the application of mouse antibody 13G9 and CD22-expressing cells Daudi (Figure 1A), NALM6 (Figure 1B), CHOS-hCD22 (Figure 1C), HEK293-hCD22 (Figure 1D), CHOS (Figure 1E) and HEK293 (Figure 1F).

Figure 2 shows the binding activity of antibodies (the application's antibody hu13G9 and the standard antibody Inotuzumab) to Raji cells.

Figure 3 shows the binding activity of antibodies (the application's antibody hu13G9 and the reference antibody Inotuzumab) to HT cells.

Figure 4 shows the binding activity of antibodies (the application's antibody hu13G9 and the standard antibody Inotuzumab) to K562-CD22 cells.

Figure 5 shows the internalization ability of antibodies (the application's antibody hu13G9 and the reference antibody Inotuzumab) on NALM6 cells.

Figure 6 shows the internalization ability of antibodies (the application's antibody hu13G9 and the reference antibody Inotuzumab) on Raji cells.

Figure 7 shows the internalization ability of antibodies (the application's antibody hu13G9 and the reference antibody Inotuzumab) on HT cells.

Figure 8 shows the internalization ability of antibodies (the application's antibody hu13G9 and the reference antibody Inotuzumab) on K562-CD19L-CD22H cells.

Figure 9 shows the internalization and killing ability of antibodies (the application's antibody hu13G9 and the standard antibody Inotuzumab) on NALM6 cells.

Figure 10 shows the internalization and killing ability of antibodies (the application's antibody hu13G9 and the standard antibody Inotuzumab) on Raji cells.

Detailed ways

The present application will now be described with reference to the following examples which are intended to illustrate, rather than limit, the application.

Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this application basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and The method was carried out as described in F. M. Ausubel et al., Compiled Experimental Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases was in accordance with the conditions recommended by the product manufacturer. If the specific conditions are not specified in the examples, the conditions should be carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially. Those skilled in the art will appreciate that the embodiments describe the present application by way of example and are not intended to limit the scope of protection claimed by the present application.

Example 1: Generation of mouse anti-human CD22 antibodies

Human CD22 (hCD22) was overexpressed on HEK293 cells (ATCC) and CHOS cells (Invitrogen) through lentiviral infection (MOI=3-10, 5μg/ml polybrene). The lentivirus was provided by Shanghai Gene Medical Technology Co., Ltd. After the cells were infected for 72 hours, corresponding antibiotics were added and cultured for 2-4 weeks, amplified and cryopreserved to obtain two overexpression cell lines, HEK293-hCD22 and CHOS-hCD22. , for subsequent experiments.

In order to obtain anti-human CD22 antibodies, the constructed CHOS-hCD22 cells overexpressing human CD22 were used to immunize Balb/c mice (Beijing Vital River Experimental Animal Technology Co., Ltd., strain code 216); the primary immunization adjuvant was completely free His adjuvant CFA (InvivoGen Company, product number vac-cfa-60), and subsequent immune adjuvants use IFA (InvivoGen Company, product number vac-ifa-60); the immune route is subcutaneous multiple points. After multiple immunizations, the spleen cells of the immunized mice were fused with mouse myeloma cells SP2/0 using the polyethylene glycol method to obtain B cell fusions that can express antibodies and proliferate indefinitely in vitro, and were selectively cultured in HAT Cultured in the substrate. The fused hybridoma cells were plated in a 96-well cell culture plate, and by detecting the ability of the antibodies in the supernatant to bind CD22 at the cellular level, the target positive clones were screened and 2-3 rounds of subcloning were performed.

High-throughput screening of mouse anti-binding cell levels: Separate plating was performed in the screen by using human CD22-expressing cells (HEK293-hCD22). Dilute 6,000 cells into 100 μL of complete culture medium, use a flat-bottomed 96-well plate, and allow the cells to adhere or sink to the bottom of the well overnight. Remove the supernatant the next day. Add 100 μL of hybridoma supernatant to be screened to the cell plate and incubate at room temperature for 1 hour. After removing the supernatant, add 100 μL of secondary antibody (DyLight488 goat anti-mouse IgG (Abcam catalog number ab97015)) to each well at a concentration of 3 μg/mL, and incubate at room temperature for 0.5 hours. After the staining is completed, remove the supernatant, add 100 μL of DPBS to each well, and incubate. machine for readings. A full field cytoscan analyzer (Nexcelom, model

Image Cytometer) to read the test plate. During the measurement, the second antibody is selected to correspond to the fluorescence channel and the bright field channel to perform high-speed scanning and imaging of the cells in the well. The imaging obtained by the fluorescence channel counts the antibody-bound cells according to the fluorescently labeled cell morphology and fluorescence intensity setting parameters. The imaging obtained by the brightfield channel counts adherent cells according to the cell morphology setting parameters, and then the two sets of data are compared. In addition, the percentage of cells showing fluorescence that binds to the antibody to the total number of cells is obtained. Based on this ratio, the binding effect of the antibody in the fusion tumor supernatant to CD22-expressing cells was determined.

Flow cytometric evaluation of mouse anti-CD22 binding: 200,000 CD22-expressing cells Daudi (Chinese Academy of Sciences), NALM6 (ATCC), CHOS-hCD22, HEK293-hCD22, CHOS (Invitrogen), HEK293 (ATCC) were placed in FACS buffer (PBS+ 2% FBS)/well, add the mouse antibody to be tested and incubate at 4 degrees Celsius for 1 hour. Centrifuge to remove the supernatant, wash twice with FACS buffer, add secondary antibody (DyLight488 goat anti-mouse IgG, Abcam catalog number ab97015) and incubate at 4 degrees Celsius for 0.5 hours. After the staining is completed, centrifuge to remove the supernatant, wash twice with FACS buffer, resuspend the cells in FACS buffer, and then put the cells on the machine for reading. The experimental cells were measured and read using a flow cytometer (BD Company, model CantoII). During the measurement, first circle the cell position based on FSC and SSC, and then select the second antibody corresponding to the fluorescent channel and SSC to analyze the cells.

As a result, a mouse antibody with good binding activity was obtained, which was named 13G9. The binding conditions of 13G9 to cells Daudi, NALM6, CHOS-hCD22, HEK293-hCD22, CHOS and HEK293 are shown in Figure 1A to Figure 1F respectively. .

Example 2: Variable region sequence determination of mouse anti-human CD22 antibody

Collect hybridoma cells by centrifugation. Add 1ml TRIzol and 0.2ml chloroform for every 5-10×10 ⁶ cells. Shake vigorously for 15 seconds and leave at room temperature for 3 minutes. Centrifuge to remove the water phase and add 0.5ml isopropanol. Leave at room temperature for 10 minutes and collect the precipitate. , washed with ethanol and dried to obtain RNA. Add template RNA and primers to the pre-cooled centrifuge tube, make sure the primers and template are correctly paired, then perform the reverse transcription process, and then perform PCR amplification. Add 2.5 μl of dNTP/ddNTP mixture to each microcentrifuge tube, and incubate the mixture at 37°C for 5 minutes and set aside. In an empty microcentrifuge tube, add 1 pmol of PCR amplified double-stranded DNA, 10 pmol of sequencing primer, 2 μl of 5× sequencing buffer, add double-distilled water to a total volume of 10 μl, heat at 96°C for 8 minutes, and cool on ice for 1 minutes, centrifuge at 10,000 g for 10 seconds at 4°C. Add 2 μl of pre-cooled labeling mixture (0.75 μmol/L each of dCTP, dGTP, and dTTP), 5 μCi of α-32P-dATP, 1 μl of 0.1 mol/L DDT, and 2 U of sequencing enzyme. Add water to 15 μl. Mix well and place on ice for 2 minutes. , marking newly synthesized DNA strands. Add 3.5 μl of labeling reaction mixture to the prepared microcentrifuge tube and incubate at 37°C for 5 minutes. Add 4 μl of stop solution to each tube. The samples were thermally denatured in a water bath at 80°C for 5 minutes, and 2 μl of each lane was added to the sequencing gel. These fragments were separated by electrophoresis and sequence information was collected.

The VH and VL sequences of the 13G9 mouse antibody are shown in Table 1. Further, the method described by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), pp. 647-669 page), the CDR sequence of the mouse monoclonal antibody was determined. The sequence information of the antibody is shown in Tables 1 and 2.

Table 2: Sequence information of mouse-derived antibodies

Example 3: Humanization of mouse anti-human CD22 antibody

In order to improve the sequence homology between the candidate antibody and the human antibody and reduce the immunogenicity of the antibody to humans, the murine antibodies provided in the above examples were humanized designed and prepared, and the murine antibodies were humanized using methods known in the art. CDR regions inserted into human framework sequences (see Winter, U.S. Patent Nos. 5,225,539; Queen et al., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370; and Lo, Benny, K.C., editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004).

Specifically, the heavy chain and light chain CDR regions of the mouse antibody 13G9 were moved to the FR framework of the corresponding humanized template, and a series of back mutations were performed on the amino acid residues in the FR region of the humanized template. , so that the humanized antibody retains the antigen-binding ability of the mouse antibody as much as possible. According to the above method, the inventors prepared a humanized antibody of murine antibody 13G9, which was named hu13G9. The heavy chain variable region and light chain variable region of hu13G9 are as shown in SEQ ID NO: 11 and 12 respectively. The sequence of the antibody heavy chain CDR3N-S mutation is SEQ ID NO:13, the heavy chain constant region of the antibody is SEQ ID NO:9, and the light chain constant region is SEQ ID NO:10.

Example 4: Evaluation of antigen-binding activity of humanized anti-CD22 antibodies

Place 200,000 CD22-expressing cells Raji (Chinese Academy of Sciences), HT (ATCC), K562-CD22 (self-built, the construction method is the same as CHOS-hCD22 and HEK293-hCD22) in FACS buffer for later use, using a round-bottom low-adsorption 96-well plate. Antibody samples were serially diluted using FACS buffer. Add diluted antibodies to the cell plate, add FACS buffer to the corresponding negative control wells, and incubate at 4 degrees Celsius for 1 hour. After centrifuging to remove the supernatant, wash twice with FACS buffer, add secondary antibody (DyLight488 goat anti-human IgG, Abcam catalog number ab97003) to each well and incubate at 4 degrees Celsius for another 0.5 hours. After the staining is completed, centrifuge to remove the supernatant, wash twice with FACS buffer, add FACS buffer to each well to resuspend the cells, and then use the machine for reading. Use a flow cytometer (BD Company, model Canto II) to measure and read the cells in the experimental plate. When measuring, first circle the cell position based on FSC and SSC, then select the second antibody corresponding to the fluorescence channel and SSC to analyze the cells. Use GraphPad for data analysis. The abscissa uses the logarithm of the antibody molar concentration, and the ordinate uses the average fluorescence intensity value. According to The EC50 of the anti-CD22 antibody was obtained by curve fitting.

The binding of hu13G9 and the benchmark antibody Inotuzumab to three types of CD22-expressing cells (Raji, HT, and K562-CD22) are shown in Figures 2 to 4 respectively. Table 3 lists the EC50 and maximum binding value (Top MFI) of the two antibodies on tumor cells. The results show that hu13G9 has better binding activity to membrane surface CD22 than Inotuzumab.

Table 3 Binding of antibodies to CD22 positive tumor cells

Example 5: Anti-CD22 antibody-mediated internalization of CD22

Anti-CD22 antibody-mediated CD22 internalization was tested by flow cytometry. Determine the relationship between internalization caused by the antibody and time; incubate the antibody to be tested with pHAB Reactive Dyes (Promega, G9845) at room temperature for 1 hour, remove the uncoupled dye by ultrafiltration, and remove the CD22-expressing cells at 37°C. Tumor cells NALM6 (ATCC), Raji (Chinese Academy of Sciences), HT (ATCC), K562-CD19L-CD22H (self-constructed, the construction method is the same as CHOS-hCD22 and HEK293-hCD22) were incubated with labeled antibodies for different times, and then analyzed by flow cytometry Cytometric analysis of the proportion of CD22 internalization mediated by anti-CD22 antibodies.

The results are shown in Figures 5 to 8. hu13G9 mediated the internalization of CD22 on the surface of tumor cells to varying degrees, and its internalization ability on these four types of tumor cells was stronger than that of the benchmark antibody Inotuzumab.

Example 6: Antibody-induced internalized killing

Target cells used CD22 naturally expressing tumor cells NALM6 (ATCC) and Raji (Chinese Academy of Sciences), and the experiment was performed in a 96-well flat-bottomed cell plate (Corning 3903). Serially diluted antibodies were added to Fab-Zap (Advanced Targeting Systems, IT-51) and incubated at 37°C for 15 minutes. Add 3,000 tumor cells to each well and react at 37°C for 72 hours. At the end of the reaction, CellTiter-GloTM reagent (Promega, G755A) is added for fluorescence development. The cell plate is measured with a Tecan Spark10 microplate reader for luminescence readings. Data analysis uses GraphPad. The abscissa uses the logarithm of the antibody molar concentration. The ordinate uses the cell killing percentage. The cell killing percentage = 100-100×sample luminescence reading/(maximum luminescence reading - minimum luminescence reading), where: max. The luminescence reading value is the well with Fab-Zap and cells added, and the minimum luminescence reading value is the well without cells. The EC50 of internalization killing induced by anti-CD22 antibody is fitted according to the curve.

The anti-CD22 antibody hu13G9 and the benchmark antibody Inotuzumab induce internalization and killing effects on two types of tumor cells (NALM6, Raji) that naturally express human CD22, as shown in Figure 9 and Figure 10 respectively. Table 4 lists the EC50 and maximum value (Top value) of internalized killing of antibodies in different tumor cells. The results show that 13G9VH1(N-G)/VL1 can effectively induce the killing of CD22-expressing tumor cells and is stronger than the benchmark antibody Inotuzumab.

Table 4 Antibodies’ internalized killing effect on CD22-positive tumor cells

Example 7: Anti-CD22 antibody binding affinity analysis

The CD22 antigen selected in this example is human Siglec-2/CD22 Protein (KACTUS, SIG-HM122-100ug). Anti-CD22 antibody binding to human CD22 protein was measured using probeLife's Gator label-free analyzer device. The biosensor uses a protein A probe, loaded with a target antibody whose concentration is confirmed by A280, and then loaded with CD22 protein. The kinetic association and dissociation rates were determined at 5-6 concentrations for CD22 protein at pH 7.4, and the KD was determined.

The binding affinity results of anti-CD22 antibodies are shown in Table 5, which lists the binding and dissociation constants of anti-CD22 antibodies and the corresponding KD values. The results showed that the binding affinity constant of humanized antibody hu13G9 to human CD22 protein was stronger than that of the standard antibody Inotuzumab.

Table 5 Anti-CD22 antibody binding affinity constants

	Koff(1/s)Koff(1/s)	Kon(1/Ms)Kon(1/Ms)	KD(M)KD(M)
hu13G9hu13G9	6.38E-046.38E-04	1.13E+051.13E+05	5.65E-095.65E-09
InotuzumabInotuzumab	2.38E-032.38E-03	6.73E+046.73E+04	3.54E-083.54E-08

Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all teachings that have been published, and these changes are within the protection scope of the present invention. . The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

An antibody or an antigen-binding fragment thereof that specifically binds to CD22 protein, the antibody or an antigen-binding fragment thereof comprising:

(a) Heavy chain variable region (VH) containing the following three complementarity determining regions (CDRs):

(i) VH CDR1, which consists of the following sequence: SEQ ID NO: 3, or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions) or add) sequence,

(ii) VH CDR2, which consists of the following sequence: SEQ ID NO: 4, or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions) or add) sequence, and

(iii) VH CDR3, which consists of the following sequence: SEQ ID NO: 5 or 13, or has one or several amino acid substitutions, deletions or additions thereto (e.g. 1, 2 or 3 amino acid substitutions) , missing or added) sequence;

and / or,

(b) A light chain variable region (VL) containing the following three complementarity determining regions (CDRs):

(iv) VL CDR1, which consists of the following sequence: SEQ ID NO: 6, or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions) or add) sequence,

(v) VL CDR2, which consists of the following sequence: SEQ ID NO: 7, or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions) or addition) sequence, and (vi) VL CDR3, which consists of the following sequence: SEQ ID NO: 8, or has one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) sequence;

Preferably, the substitution described in any one of (i)-(vi) is a conservative substitution;

Preferably, the CDR described in any one of (i)-(vi) is defined according to Kabat, IMGT or Chothia numbering system;

Preferably, the CDR described in any one of (i)-(vi) is defined according to the Kabat numbering system;

Preferably, the antibody or antigen-binding fragment thereof comprises:

(1) The following 3 heavy chain CDRs: VH CDR1 as shown in SEQ ID NO:3, VH CDR2 as shown in SEQ ID NO:4, VH CDR3 as shown in SEQ ID NO:5; and/or, The following 3 light chain CDRs: VL CDR1 as shown in SEQ ID NO:6, VL CDR2 as shown in SEQ ID NO:7, VL CDR3 as shown in SEQ ID NO:8; or

(2) The following 3 heavy chain CDRs: VH CDR1 as shown in SEQ ID NO:3, VH CDR2 as shown in SEQ ID NO:4, VH CDR3 as shown in SEQ ID NO:13; and/or, The following three light chain CDRs: VL CDR1 as shown in SEQ ID NO:6, VL CDR2 as shown in SEQ ID NO:7, and VL CDR3 as shown in SEQ ID NO:8.
The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises:

(a) Heavy chain variable region (VH) comprising an amino acid sequence selected from the following:

(i) The sequence shown in SEQ ID NO: 1 or 11;

(ii) Compared with the sequence shown in SEQ ID NO: 1 or 11, there is a substitution, deletion or addition of one or several amino acids (such as a substitution, deletion of 1, 2, 3, 4 or 5 amino acids) or add) sequence; or

(iii) Be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% identical to the sequence shown in SEQ ID NO: 1 or 11 Sequences that have %, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

and / or

(b) A light chain variable region (VL) comprising an amino acid sequence selected from:

(iv) The sequence shown in SEQ ID NO: 2 or 12;

(v) Compared with the sequence shown in SEQ ID NO: 2 or 12, it has one or several amino acid substitutions, deletions or additions (such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions) or add) sequence; or

(vi) Be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% identical to the sequence shown in SEQ ID NO: 2 or 12 Sequences that have %, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

Preferably, the substitutions described in (ii) or (v) are conservative substitutions;

Preferably, the antibody or antigen-binding fragment thereof comprises:

(1) VH having the sequence shown in SEQ ID NO:1 and VL having the sequence shown in SEQ ID NO:2, or

(2) VH having the sequence shown in SEQ ID NO: 11 and VL having the sequence shown in SEQ ID NO: 12.
The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a constant region derived from human immunoglobulin or a variant thereof;

Preferably, the antibody or antigen-binding fragment thereof comprises:

(a) The heavy chain constant region (CH) of a human immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence from which it is derived (e.g. , substitution, deletion or addition of up to 20, up to 15, up to 10 or up to 5 amino acids or any combination thereof; for example, substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids. addition or any combination thereof); and/or

(b) A light chain constant region (CL) of a human immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence from which it is derived (e.g. , substitution, deletion or addition of up to 20, up to 15, up to 10 or up to 5 amino acids or any combination thereof; for example, substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids. addition or any combination thereof);

Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region;

Preferably, the antibody or antigen-binding fragment thereof comprises the heavy chain constant region (CH) shown in SEQ ID NO: 9;

Preferably, the light chain constant region is a kappa light chain constant region or a lambda light chain constant region;

Preferably, the antibody or antigen-binding fragment thereof comprises the light chain constant region (CL) shown in SEQ ID NO: 10.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv, BsFv, dsFv , (dsFv) 2 , dsFv-dsFv', scFv, scFv dimer, camelized single chain domain antibody, diabody, ds diabody, nanobody, single Domain antibody (sdAb), bivalent domain antibody; and/or, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a multispecific antibody.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof carries a label; preferably, the antibody or antigen-binding fragment thereof carries a detectable label, Examples include enzymes (eg horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (eg chemiluminescent substances) or biotin.
An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or its heavy chain variable region and/or light chain variable region.
A vector comprising the nucleic acid molecule of claim 6; preferably, the vector is a cloning vector or an expression vector.
A host cell comprising the nucleic acid molecule of claim 6 or the vector of claim 7.
A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, comprising culturing the host cell according to claim 8 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and The antibody or antigen-binding fragment thereof is recovered from the cultured host cell culture.
A multispecific molecule comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-5;

Preferably, the multispecific molecule specifically binds CD22 and additionally specifically binds one or more other targets;

Preferably, the multispecific molecule further comprises at least one molecule (eg, a second antibody) having a second binding specificity for a second target.
An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 and a therapeutic agent connected to the antibody or antigen-binding fragment thereof;

Preferably, the therapeutic agent is selected from cytotoxic agents;

Preferably, the therapeutic agent is selected from the group consisting of alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and any combination thereof;

Preferably, the immunoconjugate is an antibody-drug conjugate (ADC).
A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the multispecific molecule according to claim 10 or the immunoconjugate according to claim 11, and a pharmaceutical Acceptable carriers and/or excipients;

Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent;

Preferably, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radioactive Nuclides, radiosensitizers, anti-angiogenic agents, cytokines, molecular targeted drugs, immune checkpoint inhibitors or oncolytic viruses;

Preferably, said antibody or antigen-binding fragment thereof or multispecific molecule or immunoconjugate and said additional pharmaceutically active agent are provided as separate components or as components of the same composition.
A kit containing the antibody or antigen-binding fragment thereof according to any one of claims 1-5;

Preferably, the antibody or antigen-binding fragment thereof carries a detectable label, such as an enzyme (such as horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance) or biotin;

Preferably, the kit further includes a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof according to any one of claims 1-5;

Preferably, the second antibody further includes a detectable label, such as an enzyme (eg horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg a chemiluminescent substance) or biotin.
A chimeric antigen receptor comprising the antigen-binding domain of the antibody or antigen-binding fragment thereof according to any one of claims 1-5;

Preferably, the antigen-binding domain comprises the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof according to any one of claims 1-5;

Preferably, the antigen-binding domain is scFv;

Preferably, the antigen-binding receptor comprises an antigen-binding fragment of the antibody of any one of claims 1-5;

Preferably, the antigen-binding receptor is expressed by immune effector cells, such as T cells.
A method for inhibiting the growth of CD22-expressing tumor cells and/or killing the tumor cells, which includes combining the tumor cells with an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1-5, or The multispecific molecule of claim 10, or the immunoconjugate of claim 11, or the pharmaceutical composition of claim 12, or the chimeric antigen receptor contact of claim 14.
A method for preventing and/or treating tumors in a subject, the method comprising administering an effective amount of the antibody or antigen binding thereof according to any one of claims 1 to 5 to a subject in need thereof. Fragment, or the multispecific molecule of claim 10, or the immunoconjugate of claim 11, or the pharmaceutical composition of claim 12, or the chimeric antigen receptor of claim 14;

Preferably, the tumor expresses CD22;

Preferably, the tumor involves tumor cells expressing CD22; preferably, the CD22 is expressed on the surface of the tumor cells;

Preferably, the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head Neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cell carcinoma and other malignancies Hematologic disorders, such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-cell-rich lymphoma, EBV-positive and -negative PTLD, and EBV-related diffuse large B-cell lymphoma cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma and HHV8-related primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) Tumors, such as primary CNS lymphoma, spinal tumors, and brainstem glioma;

Preferably, the subject is a mammal, such as a human;

Preferably, the method further includes the administration of additional drugs with anti-tumor activity, such as alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radioactive Nuclides, radiosensitizers, anti-angiogenic agents, cytokines, molecular targeted drugs, immune checkpoint inhibitors or oncolytic viruses;

Preferably, the method further comprises administering additional anti-tumor therapy, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormonal therapy, gene therapy or palliative therapy.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the multispecific molecule according to claim 10, or the immunoconjugate according to claim 11, or the drug according to claim 12 The composition, or the use of the chimeric antigen receptor according to claim 14 in the preparation of a medicament for preventing and/treating tumors in a subject;

Preferably, the medicament also contains an additional pharmaceutically active agent;

Preferably, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radioactive Nuclides, radiosensitizers, anti-angiogenic agents, cytokines, molecular targeted drugs, immune checkpoint inhibitors or oncolytic viruses;

Preferably, the tumor expresses CD22;

Preferably, the tumor involves tumor cells expressing CD22; preferably, the CD22 is expressed on the surface of the tumor cells;

Preferably, the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head Neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cell carcinoma and other malignancies Hematologic disorders, such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-cell-rich lymphoma, EBV-positive and -negative PTLD, and EBV-related diffuse large B-cell lymphoma cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma and HHV8-related primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) Tumors such as primary CNS lymphoma, spinal tumors, and brainstem gliomas.

Preferably, the subject is a mammal, such as a human.
A method for detecting the presence or amount of CD22 in a sample, comprising the following steps:

(1) contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1-5;

(2) detecting the formation of a complex between the antibody or its antigen-binding fragment and CD22 or detecting the amount of the complex;

Preferably, the antibody or antigen-binding fragment thereof is detectably labeled;

Preferably, said CD22 is human CD22.
A method for detecting whether a tumor can be treated by anti-tumor therapy targeting CD22, comprising the following steps:

(1) Contact the sample containing the tumor cells with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5;

(2) detecting the formation of a complex between the antibody or its antigen-binding fragment and CD22;

Preferably, the antibody or antigen-binding fragment thereof is detectably labeled;

Preferably, said CD22 is human CD22;

Preferably, the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head Neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cell carcinoma and other malignancies Hematologic disorders, such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-cell-rich lymphoma, EBV-positive and -negative PTLD, and EBV-related diffuse large B-cell lymphoma cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma and HHV8-related primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) Tumors such as primary CNS lymphoma, spinal tumors, and brainstem gliomas.
The use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 in the preparation of a kit for detecting whether tumors can be treated by anti-tumor therapy targeting CD22;

Preferably, the antibody or antigen-binding fragment thereof is detectably labeled;

Preferably, said CD22 is human CD22;

Preferably, the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head Neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cell carcinoma and other malignancies Hematologic disorders, such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-cell-rich lymphoma, EBV-positive and -negative PTLD, and EBV-related diffuse large B-cell lymphoma cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma and HHV8-related primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) Tumors such as primary CNS lymphoma, spinal tumors, and brainstem gliomas.