WO2024000259A1

WO2024000259A1 - Antibody specifically binding to cd22, preparation method therefor and use thereof on bispecific cart

Info

Publication number: WO2024000259A1
Application number: PCT/CN2022/102313
Authority: WO
Inventors: 杜靓; 牟男; 万婷婷; 于跃; 徐溜溜; 张红艳; 金夷
Original assignee: 上海吉倍生物技术有限公司
Priority date: 2022-06-29
Filing date: 2022-06-29
Publication date: 2024-01-04

Abstract

The present application relates to the technical field of biological immunity, and in particular, to a monoclonal antibody capable of specifically binding to CD22 and an antigen-binding fragment thereof. The present application also relates to a bispecific chimeric antigen receptor, a preparation method therefor and a use thereof.

Description

CD22-specific binding antibodies, preparation methods thereof and their use in bispecific CART

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 bispecific chimeric antigen receptors and preparation methods and uses thereof.

Background technique

Immune cell therapy uses the tumor patient's autologous immune cells, such as T cells, which are amplified and cultured in vitro and genetically modified and then infused back into the cancer patient to eliminate the tumor. To date, successful immune cell therapies include tumor infiltrating T cell therapy (TIL, Tumor Infiltrating T cell therapy), T cell receptor therapy (TCR-T, T Cell Receptor T cell therapy) and chimeric antigen receptor T cell therapy (TCR-T, T Cell Receptor T cell therapy). CART, Chimeric Antigen Receptor T cell therapy). CART is a new type of anti-cancer therapy. Cancer patients' T cells are cultured and expanded in vitro and genetically engineered using viral vectors to generate CART cells. CART cells can recognize and activate tumor-specific antigens (such as CD19), and specifically lyse tumor cells to kill them. For tumor purposes, it is characterized by no HLA (Human Leukocyte Antigen) restriction, short treatment cycle, fast curative effect, high complete response rate, and even complete cure.

The most successful case of CAR-T cell therapy is a series of clinical trials targeting CD19 for B-cell tumors. CD19 is a cell surface antigen specifically expressed on various differentiation stages of B lymphocytes. Most malignant tumors derived from the B lineage, including B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia and non-Hodgkin lymphoma cells, express CD19. , so it is considered an ideal target for CAR-T treatment of B-cell tumors. Clinical trial results show that the cure rate of CD19 CAR-T for acute B-lymphoblastic leukemia (B-ALL) has reached 90%. Facing the threat of CAR-T cell therapy, some tumor cells will regulate the CD19 expressed by cancer cells. Level up and escape the attack. In a clinical trial called PLAT-02 conducted at Seattle Children's Hospital, up to 93% of patients with relapsed or refractory ALL achieved good response after receiving CD19 CAR-T cell therapy, but in the end 50% The patients all relapsed, and researchers found that in some relapsed leukemia patients, there was no expression of CD19 protein, but expression of CD22 protein. In order to reduce the recurrence rate by up to 50%, researchers have been working to improve this experimental treatment.

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 closely related to the development, differentiation and function of B cells. CD22 is restrictedly expressed on the surface of mature B cells and most malignant B lymphoma cells, making it one of the popular targets for the treatment of autoimmune diseases and B cell malignancies. Currently, the types of immunotherapy drugs that target CD22 include monoclonal antibody drugs, antibody conjugates (ADCs) and CAR-T therapy.

At the end of 2017, Crystal Mackall's team at Stanford University School of Medicine and the National Cancer Institute (NCI) publicly reported the results of a phase I clinical trial of CD22 CAR-T in patients with relapsed B-ALL in the authoritative academic journal "Nature Medicine". In a phase 1 clinical trial, 15 patients whose disease had relapsed or failed to respond to CD19-targeting CAR-T therapy were treated with CD22-targeting CAR-T therapy, 10 of whom had previously received CD19-targeting therapy. But resistance develops. It was found that 1 of 6 patients who received the lowest dose achieved complete remission; 11 of 15 patients who received the higher dose achieved remission, with a response rate of 73% and a median response time of 6 months. , three of them still maintained complete remission at 6, 9 and 21 months after receiving treatment. Furthermore, this therapy was well tolerated by patients. The Journal of Clinical Oncology published the results of a Phase I clinical trial of CD22 CAR-T cell therapy in 2020. The trial enrolled a total of 64 patients, and 58 patients aged 4 to 31 years with CD22+ B-cell malignancies (56 of whom were ALL) received varying doses following conditioning therapy with fludarabine and cyclophosphamide. CD22 CAR-T cell therapy (dose 1: 3×10 ⁵ /kg; dose 2: 1×10 ⁶ /kg; dose 3: 3×10 ⁶ /kg). Among these patients, 88% and 33% had previously received CD19-targeted therapy and CD22-targeted therapy, respectively. The study results showed that 70% of patients achieved complete response (CR) and the median overall survival was 13.4 months. Among patients with complete responses, 87.5% had a negative minimal residual disease response, and the median recurrence-free survival was 6 months. Prior CD19-targeted therapy or hematopoietic stem cell transplantation did not affect response rate, but prior CD22-targeted therapy was associated with poorer response. At relapse, most patients are CD22-/dim. In terms of safety, 86% and 33% of patients experienced cytokine release syndrome (CRS) and neurological toxicity, respectively, most of which were grade 1-2 (90% and 95%). However, two grade 5 adverse events occurred in the dose 2 group, one of which was related to CRS. In addition, it is noteworthy that hemophagocytic lymphohistiocytosis (HLH)/macrophage activation syndrome (MAS)-like events occurred only in patients who developed CRS (19 cases in total, 38%), and the onset was delayed . Among them, 14 cases required treatment, and 8 cases received treatment with the interleukin-1 receptor antagonist anakinra, which did not impair CAR-T cell activity.

2019 Blood magazine Sequential CD19-22 CART therapy induces sustained remission in children with r/r B-ALL. This study used CD19- and CD22-CART cell sequential infusion to treat 20 children with r/r B-ALL, achieving one-year disease-free survival (LFS) and overall survival (OS) of 79.5% and 92.3, respectively. %'s excellent efficacy is not only much higher than that of single-target CD19- or CD22-CART, but also performs better than other sequential or "cocktail therapies". This research provides a solution to the problem of high relapse after CART cell therapy, and is also an international breakthrough in CART cell therapy for leukemia.

Therefore, there is a need to provide new bispecific CARTs targeting CD19 and CD22 to expand the indications and applicable populations of the existing hematoma pipeline products, improve drug efficacy, and reduce the recurrence rate after treatment.

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 "chimeric antigen receptor," abbreviated as CAR, refers to a cell surface receptor that can recognize a specific antigen (e.g., a tumor antigen) and contains an extracellular domain capable of recognizing the specific antigen (e.g., recognizing and binding Antigen-binding fragments of antibodies to specific antigens) and intracellular domains capable of transmitting extracellular signals to the interior of the cell (also known as signaling domains, such as the zeta chain of CD3 or the intracellular portion of FcεRIγ). T cells that carry and express such chimeric antigen receptors are called CAR-T cells, which are able to recognize and bind to specific antigens and cells expressing the specific antigens (such as tumor cells) through the extracellular domain, and through intracellular The signal transduction effect of the domain activates the immune response, releases a large number of multiple effectors, and efficiently kills cells expressing the specific antigen (such as tumor cells), thereby exerting a therapeutic effect (such as treating tumors).

As used herein, the terms "bispecific chimeric antigen receptor" or "dual-target chimeric antigen receptor" have the same meaning and refer to a cell surface that can recognize two specific antigens (e.g., tumor antigens) Receptors, which typically comprise a first antigen-binding domain (e.g., an antigen-binding fragment) for a first antigen, a second antigen-binding domain (e.g., an antigen-binding fragment) for a second antigen, and a hinge region (spacer) ), transmembrane domain, costimulatory domain (cytoplasmic region), etc.

As used herein, the term "spacer" or "hinge region" refers to a polypeptide linking region that provides a flexible structure that can connect the antigen-binding region to a transmembrane domain. Generally, they are flexible enough to allow the antigen-binding region to be oriented in different directions and bind to the antigen. The hinge region may be naturally occurring or non-naturally occurring, including but not limited to altered hinge regions as described in U.S. Patent No. 5,677,425. In certain embodiments, the hinge region may comprise an entire hinge region derived from an antibody of a different class or subclass than that of the CH1 domain antibody.

As used herein, the term "transmembrane domain" is a hydrophobic alpha helix that spans a membrane. Different transmembrane domains can lead to different receptor stabilities. The transmembrane domain is inserted between the spacer domain and the costimulatory domain (cytoplasmic domain). In some embodiments, the transmembrane domain is inserted between the spacer and one or more costimulatory regions. In some embodiments, the linker is between a transmembrane domain and one or more costimulatory regions.

As used herein, the term "cytoplasmic domain" or "costimulatory domain" refers to the region through which, following antigen recognition, receptors assemble and signals are transmitted to the cell. In some embodiments, the costimulatory domain is part of the cytoplasmic domain.

As used herein, the term "signaling domain" refers to a region that provides a distinct and detectable signal (e.g., by a cell of one or more species) in response to activation by binding of an antigen to an antigen-binding domain. Increased production of various cytokines; changes in target gene transcription; changes in protein activity; changes in cell behavior, such as cell death; cell proliferation; cell differentiation; cell survival; regulation of cell signaling responses, etc.). In certain embodiments, the signaling domain acts together with a costimulatory domain to transmit a signal.

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. (1987and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342:878-883 definition.

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. 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 is composed 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-controlling 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. Furthermore, a CD19 and CD22 bispecific CART was constructed based on the development of CD22 antibodies. CD19 and CD22 bispecific CART can improve the killing efficiency, amplification efficiency and persistence of CD19 low-expressing tumor cells. It can expand the indications and suitable groups of existing hematoma pipeline products, improve drug efficacy, and reduce the recurrence rate after treatment. Of great significance.

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 has one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) 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).

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:

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.

(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 10;

(ii) Compared with the sequence shown in SEQ ID NO: 1 or 10, 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 10 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 11;

(v) Compared with the sequence shown in SEQ ID NO: 2 or 11, 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 11 %, 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: 10 and VL having the sequence shown in SEQ ID NO: 11.

In certain embodiments, 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 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: 12.

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: 13.

In certain embodiments, 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 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: 16 or 18. In certain embodiments, the nucleic acid molecule comprises a sequence set forth in SEQ ID NO: 17 or 19.

In certain embodiments, the heavy chain variable region has the sequence set forth in SEQ ID NO: 16 or 18.

In certain embodiments, the light chain variable region has the sequence set forth in SEQ ID NO: 17 or 19.

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 or antigen-binding fragment thereof) with a second binding specificity for a second target (eg, CD19).

In another aspect, the present application provides a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD22 protein, the antigen-binding domain comprising an antibody or an antigen-binding fragment thereof as described above.

In certain embodiments, the chimeric antigen receptor further comprises one or more domains selected from:

(a) Hinge area;

(b) Transmembrane domain;

(c) costimulatory domain; or

(d) Signaling domain.

In certain embodiments, the chimeric antigen receptor includes, from N-terminus to C-terminus, in order: VH of the antigen-binding domain, VL of the antigen-binding domain, hinge region, transmembrane domain, and co-stimulatory domain, and signaling domains.

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

In certain embodiments, the bispecific chimeric antigen receptor further comprises a second antigen binding domain that specifically binds a second target (e.g., CD19); wherein the first antigen binding domain optionally is connected to the N-terminus and/or C-terminus (eg N-terminus) of the second antigen binding domain through a linker.

In certain embodiments, the second antigen-binding domain is an antibody or antigen-binding fragment thereof that specifically binds CD19.

In certain embodiments, the bispecific chimeric antigen receptor further comprises one or more domains selected from:

(a) Hinge area;

(b) Transmembrane domain;

(c) costimulatory domain; or

(d) Signaling domain.

In certain embodiments, the bispecific chimeric antigen receptor comprises, from the N-terminus to the C-terminus, in order: VL of the second antigen-binding domain, VH of the first antigen-binding domain, and the first antigen-binding domain. VL, second antigen-binding domain of VH, hinge region, transmembrane domain, costimulatory domain, and signaling domain.

In certain embodiments, the VL of the second antigen binding domain and the VH of the first antigen binding domain are connected by a linker. In certain embodiments, the VH of the first antigen binding domain and the VL of the first antigen binding domain are connected by a linker. In certain embodiments, the VL of the first antigen binding domain and the VH of the second antigen binding domain are connected by a linker.

In certain embodiments, the VH of the second antigen binding domain is set forth in SEQ ID NO: 14. In certain embodiments, the VL of the second antigen binding domain is set forth in SEQ ID NO: 15.

In some embodiments, the sequence of the linker can refer to the sequence disclosed in the prior art, for example, Mare W, Bell B A, Sheau-Line F, et al. An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability[J].Protein Eng, 1993(8):989-995.

In certain embodiments, the linker has the sequence set forth in SEQ ID NO: 24 or 25.

In certain embodiments, the dual specific chimeric antigen receptor contains the sequence set forth in SEQ ID NO: 9 or 26.

In certain embodiments, the hinge region is a hinge region selected from the following proteins: CD8, CD28, 4-1BB, or any combination thereof. In certain embodiments, the hinge region contains or consists of the amino acid sequence set forth in SEQ ID NO:20.

In certain embodiments, the transmembrane domain is a transmembrane domain selected from the following proteins: CD8, CD28, CD3ζ, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, 4-1BB, CD154, or any combination thereof. In certain embodiments the transmembrane domain contains or consists of the amino acid sequence set forth in SEQ ID NO: 21.

In certain embodiments, the costimulatory domain contains or consists of the amino acid sequence set forth in SEQ ID NO: 22.

In certain embodiments, the signaling domain is an intracellular domain selected from the group consisting of: CD3ζ, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB, PD1, DAPLO, CDS, ICAM-1, LFA-1(CDLLA/CD18), ICOS(CD278), NKG2D, GITR, TLR2, or any combination thereof. In certain embodiments, the signaling domain is the intracellular domain of CD3ζ. In certain embodiments, the signaling domain contains or consists of the amino acid sequence set forth in SEQ ID NO: 23.

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

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 is a lentiviral, adenoviral, or retroviral vector.

In another aspect, the present application provides a host cell comprising a nucleic acid molecule as described above or a vector as described above.

In certain embodiments, the host cells are engineered immune cells.

In certain embodiments, the engineered immune cells secretively express a bispecific chimeric antigen receptor as described above.

In certain embodiments, the engineered immune cells are selected from T cells, NK cells, γδ T cells, NKT cells, or any combination thereof.

In certain embodiments, the host cells are obtained from (eg, isolated from) peripheral blood mononuclear cells (PBMC).

On the other hand, the present application provides a method for preparing a host cell as described above, which includes: (1) providing immune cells; (2) converting the isolated nucleic acid molecule as described above or the vector as described above Introduce the immune cells.

In certain embodiments, the immune cells are selected from T cells, NK cells, γδ T cells, NKT cells, or any combination thereof.

In certain embodiments, in step (1), the immune cells are pretreated, and the pretreatment includes sorting, activation and/or proliferation of immune cells.

In certain embodiments, the nucleic acid molecule or vector is introduced into the host cell in step (2) by viral infection or by non-viral vector transfection.

In some embodiments, step (2) is followed by a step of amplifying the immune cells obtained in step (2).

On the other hand, the present application provides a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described above, or an isolated nucleic acid molecule as described above, or a carrier as described above, or a vector as described above. The host cell as described above, or the multi-characteristic molecule as described above, or the bispecific chimeric antigen receptor as described above, or the isolated nucleic acid molecule as described above, or the vector as described above, or Host cells as described previously.

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 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 an immunoconjugate comprising an antibody or an 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 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.

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 isolated nucleic acid molecules as described above, or vectors as described above, or host cells as described above, or multi-characteristic molecules as described above, or bispecific embedded molecules as described above The combined antigen receptor, or the isolated nucleic acid molecule as described above, or the vector as described above, or the host cell is contacted as described above.

In another aspect, the present application provides a method for inhibiting the growth of tumor cells expressing CD22 and CD19 and/or killing the tumor cells, which comprises combining the tumor cells with an effective amount of the bispecific as described above. The chimeric antigen receptor, or the isolated nucleic acid molecule as described above, or the vector as described above, or the host cell as described above, or the pharmaceutical composition as described above is contacted.

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, or the isolated nucleic acid molecule as mentioned above, or the vector as mentioned above, or the host cell as mentioned above, or the multi-characteristic molecule as mentioned above, or the dual-molecule as mentioned above Specific chimeric antigen receptor, or an isolated nucleic acid molecule as described above, or a vector as described above, or a host cell as described above, or a pharmaceutical composition as described above.

In certain embodiments, the tumor expresses CD22. In certain embodiments, the tumor expresses CD22 and CD19.

In certain embodiments, the tumor involves CD22-expressing tumor cells. In certain embodiments, the tumor involves tumor cells expressing CD22 and CD19.

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.

On the other hand, the application provides the antibody or antigen-binding fragment thereof as described above, or the isolated nucleic acid molecule as described above, or the vector as described above, or the host cell as described above, or The multi-characteristic molecule as mentioned above, or the bispecific chimeric antigen receptor as mentioned above, or the isolated nucleic acid molecule as mentioned above, or the vector as mentioned above, or the host as mentioned above Use of cells, or a pharmaceutical composition as described above, in the preparation of a medicament for preventing and/or treating tumors in a subject.

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 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 an antibody or an 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

Note: scFv-BBz-1 sequence: the underlined parts are 7E11VL, 15E11VH, 15E11VL, 7E11VH, hinge region and costimulatory domain; the italicized parts are signal peptide, transmembrane region and CD3ζ region;

scFv-BBz-2 sequence: The underlined parts are 7E11VL, 15E11-2VH, 15E11-2VL, 7E11VH, hinge region and co-stimulatory domain; the italicized parts are signal peptide, transmembrane region and CD3ζ region.

beneficial effects

The monoclonal antibodies of the present application (eg, antibody 15E11, antibody 15E11-2) can bind to CD22 protein or cells expressing CD22 protein with high specificity. Furthermore, a CD19 and CD22 bispecific CART was constructed based on the anti-CD22 antibody. Compared with the benchmark bispecific CART, the bispecific CART of the present application can improve the killing efficiency, amplification efficiency and persistence of CD19 low-expressing tumor cells, and can also inhibit tumor growth in mice. Therefore, the monoclonal antibodies and bispecific CART of the present application have high clinical application value and high application potential in targeted therapy of tumors.

Description of drawings

Figure 1 shows the application of the mouse antibody 15E11 and CD22-expressing cells Daudi (Figure 1A), NALM6 (Figure 1B), CHOS-CD22 (Figure 1C), HEK293-CD22 (Figure 1D), CHOS (Figure 1E) and HEK293 (Figure 1F).

Figure 2 shows a schematic structural diagram of the GV401 vector.

Figure 3 shows the results of IL2 release by cytokines after target cells were treated with bispecific CART 7E11-15E11 and control (MockT, bispecific CART 7E11-m971) of the present application.

Figure 4 shows the results of cytokine IFNγ release after the dual-specific CART 7E11-15E11 and control (MockT, dual-specific CART 7E11-m971) of the present application treat target cells.

Figure 5 shows the binding of humanized antibody 15E11-2 to tumor cells that naturally express CD22 (Daudi).

Figure 6 shows the results of IL2 release by cytokines after target cells were treated with bispecific CART 7E11-15E11-2 and control (MockT, bispecific CART 7E11-m971, CART 7E11) of the present application.

Figure 7 shows the results of cytokine IFNγ release after the dual-specific CART 7E11-15E11-2 of the present application and the control (MockT, dual-specific CART 7E11-m971, CART 7E11) treated target cells.

Figure 8 shows the results of tumor growth in mice after the bispecific CART 7E11-15E11-2 of the present application and the control (MockT, bispecific CART 7E11-m971, CART 7E11) treated mice.

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 enzymes was in accordance with the conditions recommended by the product manufacturer. If specific conditions are not specified in the examples, the conditions should be carried out in accordance with conventional conditions or 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 15E11. The binding conditions of 15E11 to cells Daudi, NALM6, CHOS-hCD22, HEK293-hCD22, CHOS and HEK293 are shown in Figure 1 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 the 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 15E11 murine 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: In vitro activity detection of chimeric anti-CD22 and anti-CD19 bispecific CART

Construction of chimeric anti-CD22 and anti-CD19 bispecific CART: First, the anti-CD19 antibody 7E11 (Shanghai Gibe Biotechnology Co., Ltd., CN107793480A) light chain variable region (SEQ ID NO: 15), anti-CD22 antibody 15E11 heavy chain variable region region (SEQ ID NO:1), as well as the anti-CD22 antibody 15E11 light chain variable region (SEQ ID NO:2), and the anti-CD19 antibody 7E11 heavy chain variable region (SEQ ID NO:14) are connected through a linker; then connected to On the sequence of BBz (including hinge region, transmembrane region, 4-1BB costimulatory domain and CD3ζ), the signal peptide (its amino acid sequence is shown in SEQ ID NO: 27) is then connected. The constructed scFv-BBz-1 (its amino acid sequence is shown in SEQ ID NO:9, where the underlined parts of SEQ ID NO:9 are 7E11VL, 15E11VH, 15E11VL, 7E11VH, hinge region and costimulatory domain; The italicized parts are the signal peptide, transmembrane region and CD3ζ), which were inserted into the GV401 vector through the BamHI and EcoRI restriction sites (as shown in Figure 2). The constructed dual-specific CART was named 7E11-15E11.

The control anti-CD22 antibody was m971 (US2020283522), which is currently in the first phase of clinical trials. The construction method of the control chimeric anti-CD22 and anti-CD19 bispecific CART 7E11-m971 is the same as above, except that it contains different antibody sequences. The construction method of control CART 7E11 is the same as above, except that it contains different antibody sequences.

Lentivirus packaging: The GV401 vector containing the scFv mutant BBz and the two plasmids pCMV-VSVGenv and pCMV-Gag/pol are transiently transfected into 293T cells using the calcium phosphate precipitation method at a mass ratio of 3:2:2, 48-72 Collect the supernatant after 1 hour, which contains VSVG-packaged lentiviral particles.

Activation and infection of T cells: Peripheral blood mononuclear cells (PBMC) isolated from healthy people were cultured in X-vivo15 medium containing 200IU/ml hIL2 and 10% FBS; anti-CD3 (OKT3 clone) and CD28 antibodies ( 15E8 clone) was activated for 24 hours, and lentivirus containing 7E11-15E11 and 7E11-m971 was added. CD19 and CD22 were expressed on T cells 72 hours after infection, and the infection efficiency was detected. The specific results are shown in Table 3.

Table 3: T cell infection efficiency

	感染效率CD19(％)Infection efficiency CD19 (%)	感染效率CD22(％)Infection efficiency CD22 (%)
7E11-15E117E11-15E11	1.471.47	26.826.8
7E11-m9717E11-m971	4.24.2	12.412.4
MockTMockT	2.132.13	1.031.03

Cytokine release experiment: Resuspend the target cells (K562, K562-CD19, K562-CD22, K562-CD19-CD22) in 1640 culture medium containing 2% FBS to 1×10 ⁵ /ml, and take a U-shaped bottom 96 well plate, add 100ul; after diluting the T cell clone to 1×10 ⁵ /ml, add 100ul to each well, incubate overnight, remove 50ul of the supernatant, use the FACS method to measure IL2 and IFNγ cytokines, and calculate the cells by the ratio to the background release The fold change of factor release and the specific results of cytokine IL2 and IFNγ release are shown in Figure 3 and Figure 4.

As can be seen from Figure 3 and Figure 4, after the dual-specific CART 7E11-15E11 of the present application treats target cells (K562-CD19, K562-CD22, K562-CD19-CD22), the cytokines (IL2 and IFNγ) released by the cells are significantly increased. higher than the control bispecific CART 7E11-m971.

Example 4: 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 mouse antibody 15E11 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 15E11, which was named 15E11-2. The heavy chain variable region and light chain variable region of 15E11-2 are as shown in SEQ ID NO: 10 and As shown in 11, the heavy chain constant region of the antibody is SEQ ID NO:12, and the light chain constant region is SEQ ID NO:13.

Example 5: Evaluation of antigen-binding activity of humanized anti-CD22 antibodies

Place 200,000 CD22 naturally expressing tumor cells (Daudi) 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°C 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. A flow cytometer (BD Company, model Canto II) was used to measure and read the cells in the experimental plate. During the measurement, 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 concentration, and the ordinate uses the average fluorescence intensity value. According to the curve Fit the EC50 of the anti-CD22 antibody.

The binding conditions of humanized antibody 15E11-2 and CD22 naturally expressing tumor cells (Daudi) are shown in Figure 5. Table 4 lists the EC50 and maximum binding value (Top MFI) of humanized antibodies binding to tumor cells. The results show that humanized antibody 15E11-2 has good binding activity to CD22 on the membrane surface.

Table 4: Binding of antibodies to CD22-positive tumor cells

Example 6: In vitro activity detection of humanized anti-CD22 and anti-CD19 bispecific CART

Humanized anti-CD22 and anti-CD19 bispecific CART constructs: anti-CD19 antibody 7E11 light chain variable region (SEQ ID NO:15), anti-CD22 antibody 15E11-2 heavy chain variable region (SEQ ID NO:10), and Anti-CD22 antibody 15E11-2 light chain variable region (SEQ ID NO:11), anti-CD19 antibody 7E11 heavy chain variable region (SEQ ID NO:14) are connected through a linker, then connected to the BBz sequence, and then connected to the signal peptide (The amino acid sequence is shown in SEQ ID NO: 27). The constructed scFv-BBz-2 (amino acid sequence is shown in SEQ ID NO:26, where the underlined parts of SEQ ID NO:26 are 7E11VL, 15E11-2VH, 15E11-2VL, 7E11VH, hinge region and co-stimulation Structural domain; the italicized parts are signal peptide, transmembrane region and CD3ζ) were inserted into the GV401 vector (same as Figure 2) through the BamHI and EcoRI restriction sites. The constructed dual-specific CART was named 7E11-15E11-2, and its structure is similar to The 7E11-15E11 constructed in Implementation 3 is exactly the same. The only difference is that the sequences of 15E11VH and 15E11VL are replaced by the sequences of 15E11-2VH and 15E11-2VL respectively. The dual-specific CART7E11-m971 and CART 7E11 are used as controls.

Activation and infection of T cells: Peripheral blood mononuclear cells (PBMC) isolated from healthy people were cultured in X-vivo15 medium containing 200IU/ml hIL2 and 10% FBS; anti-CD3 (OKT3 clone) and CD28 antibodies ( 15E8 clone) was activated for 24 hours, and the lentivirus containing 7E11-15E11-2 and 7E11-m971 was added. The infection efficiency was detected by T cell expression of CD19 and CD22 72 hours after infection. The specific results are shown in Table 5.

Table 5: T cell infection efficiency

	感染效率CD19/CD22(％)Infection efficiency CD19/CD22 (%)
7E11-15E11-27E11-15E11-2	43.443.4
7E11-m9717E11-m971	75.375.3
7E117E11	81.581.5
MockTMockT	0.0180.018

Cytokine release experiment: Resuspend the target cells (K562, K562-CD19, K562-CD22, K562-CD19-CD22, NALM6-LUC) to 1×10 ⁵ /ml in 1640 medium containing 2% FBS, and take U Type-bottom 96-well plate, add 100ul; dilute the T cell clone to 1×10 ⁵ /ml, add 100ul to each well, incubate overnight, remove 50ul of supernatant, use FACS method to measure IL2 and IFNγ cytokines, and compare with background release Ratio, fold change of cytokine release was calculated, and the specific results of cytokine IL2 and IFNγ release are shown in Figures 6 and 7.

Example 7: In vivo efficacy verification of humanized anti-CD22 and anti-CD19 bispecific CART

To verify the efficacy of CART in vivo, the tumor-bearing cells used were NALM6-LUC, the positive control group was bispecific CART 7E11-m971 (m971US2020283522) and CART 7E11, and the negative control was MockT. Each mouse was given 1 × 10 ⁶ cells, and 1E7CART cells were given on day 6.

Preparation of NALM6-LUC cells: The ffluc (firefly Luciferase) gene is inserted into the Ubc promoter-ffluc-IRES-PuroR reading frame through standard molecular biology methods, and the reading frame is inserted into the GV260 vector (Jikai Gene) and packaged into lentivirus , by infecting NALM6 cells and testing Luciferase expression after screening with 1ug/ml puromycin.

NDG mouse late-stage tumor-bearing model: On the first day, 1×10 ⁶ Raji-ffluc cells were resuspended in 200ul PBS solution; 4-6 weeks old NDG mice were administered via the tail vein. On

day

6, 150 mg/ml luciferin was administered intraperitoneally to the mice, and the fluorescence intensity was measured (PerkinElmer IVIS Spectrum) 10 minutes later.

Administration: After tumor expansion was confirmed on the 6th day, 100ul PBS, 5×10 ⁶ 7E11-15E11-2, 7E11-m971, 7E11 and MockT CART cells were administered to the mice respectively via the tail vein (6 in each group).

On day 29, the quantitative results of mouse in vivo imaging showed that bispecific CART 7E11-15E11-2 was more effective than bispecific CART 7E11-m971 and CART 7E11 in the NALM6-LUC model. The tumor growth curve is shown in Figure 8. As can be seen from Figure 8, the bispecific CART of the present application has the best inhibitory effect on tumor growth.

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 has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions) or add) 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:

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.
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 10;

(ii) Compared with the sequence shown in SEQ ID NO: 1 or 10, 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 10 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 11;

(v) Compared with the sequence shown in SEQ ID NO: 2 or 11, 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 11 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: 10 and VL having the sequence shown in SEQ ID NO: 11.
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: 12;

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: 13.
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 (eg, CD19).
A chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD22 protein, the antigen-binding domain comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5;

Preferably, the chimeric antigen receptor further comprises one or more domains selected from the group consisting of:

(a) Hinge area;

(b) Transmembrane domain;

(c) costimulatory domain; or

(d) Signaling domain.
A bispecific chimeric antigen receptor comprising the first antigen-binding domain of the antibody or antigen-binding fragment thereof according to any one of claims 1-5;

Preferably, the bispecific chimeric antigen receptor further comprises a second antigen binding domain that specifically binds a second target (eg, CD19); wherein the first antigen binding domain is optionally linked by a linker To the N-terminus and/or C-terminus (e.g., N-terminus) of the second binding domain of the antigen;

Preferably, the second antigen-binding domain is an antibody that specifically binds CD19 or an antigen-binding fragment thereof;

Preferably, the bispecific chimeric antigen receptor further comprises one or more domains selected from the following:

(a) Hinge area;

(b) Transmembrane domain;

(c) costimulatory domain; or

(d) Signaling domain;

Preferably, the bispecific chimeric antigen receptor includes in sequence from N-terminus to C-terminus: the VL of the second antigen-binding domain, the VH of the first antigen-binding domain, the VL of the first antigen-binding domain, and the VL of the first antigen-binding domain. VH of two antigen-binding domains, hinge region, transmembrane domain, costimulatory domain, and signaling domain;

Preferably, the VL of the second antigen-binding domain and the VH of the first antigen-binding domain are connected through a linker; preferably, the VH of the first antigen-binding domain and the VL of the first antigen-binding domain are connected through a linker. Connection; Preferably, the VL of the first antigen-binding domain and the VH of the second antigen-binding domain are connected through a linker;

Preferably, the VH of the second antigen-binding domain is as shown in SEQ ID NO:14; Preferably, the VL of the second antigen-binding domain is as shown in SEQ ID NO:15;

Preferably, the linker has the sequence shown in SEQ ID NO: 24 or 25;

Preferably, the dual-specific chimeric antigen receptor contains the sequence shown in SEQ ID NO: 9 or 26.
The bispecific chimeric antigen receptor of claim 12, wherein the bispecific chimeric antigen receptor has one or more characteristics selected from the following:

(1) The hinge region contains or consists of the amino acid sequence shown in SEQ ID NO:20;

(2) The transmembrane domain contains or consists of the amino acid sequence shown in SEQ ID NO: 21;

(3) The costimulatory domain contains or consists of the amino acid sequence shown in SEQ ID NO: 22; or

(4) The signaling domain is the intracellular domain of CD3ζ; preferably, the signaling domain contains or consists of the amino acid sequence shown in SEQ ID NO: 23.
An isolated nucleic acid molecule encoding the chimeric antigen receptor of claim 11 or the bispecific chimeric antigen receptor of claim 12 or 13.
A vector comprising the nucleic acid molecule of claim 14;

Preferably, the vector is a cloning vector or an expression vector; preferably, the vector is a lentivirus, adenovirus or retrovirus vector.
A host cell comprising the nucleic acid molecule of claim 14 or the vector of claim 15;

Preferably, the host cells are engineered immune cells;

Preferably, the engineered immune cells secrete and express the chimeric antigen receptor described in claim 11 or the bispecific chimeric antigen receptor described in claim 12 or 13;

Preferably, the engineered immune cells are selected from T cells, NK cells, γδ T cells, NKT cells, or any combination thereof;

Preferably, the host cells are obtained from (eg, isolated from) peripheral blood mononuclear cells (PBMC).
A method for preparing the host cell of claim 16, comprising: (1) providing immune cells; (2) introducing the isolated nucleic acid molecule of claim 14 or the vector of claim 15 into the immune cells;

Preferably, the immune cells are selected from T cells, NK cells, γδ T cells, NKT cells, or any combination thereof;

Preferably, in step (1), the immune cells are pretreated, and the pretreatment includes sorting, activation and/or proliferation of immune cells;

Preferably, in step (2), the nucleic acid molecule or vector is introduced into the host cell by viral infection or by non-viral vector transfection;

Preferably, the step of amplifying the immune cells obtained in step (2) is further included after step (2).
A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 5, or the isolated nucleic acid molecule of claim 6, or the vector of claim 7, or the host cell of claim 8, or the host cell of claim 8 The multi-characteristic molecule of claim 10, or the chimeric antigen receptor of claim 11, or the bispecific chimeric antigen receptor of claim 12 or 13, or the isolated nucleic acid molecule of claim 14, or the vector of claim 15, Or the host cell of claim 16;

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.
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 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 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 to 5, or the claim The isolated nucleic acid molecule of claim 6, or the vector of claim 7, or the host cell of claim 8, or the multi-characteristic molecule of claim 10, or the chimeric antigen receptor of claim 11, or the dual compound of claim 12 or 13 Specific chimeric antigen receptor, or the isolated nucleic acid molecule of claim 14, or the vector of claim 15, or the host cell contact of claim 16.
A method for inhibiting the growth of tumor cells expressing CD22 and CD19 and/or killing the tumor cells, which comprises combining the tumor cells with an effective amount of the chimeric antigen receptor of claim 11, or the chimeric antigen receptor of claim 12 or 13 The bispecific chimeric antigen receptor, or the isolated nucleic acid molecule of claim 14, or the vector of claim 15, or the host cell of claim 16, or the pharmaceutical composition of claim 18.
A method for preventing and/or treating tumors in a subject, the method comprising administering an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 5 to a subject in need thereof, Or the isolated nucleic acid molecule of claim 6, or the vector of claim 7, or the host cell of claim 8, or the multi-characteristic molecule of claim 10, or the chimeric antigen receptor of claim 11, or claim 12 or The dual-specific chimeric antigen receptor of claim 13, or the isolated nucleic acid molecule of claim 14, or the vector of claim 15, or the host cell of claim 16, or the pharmaceutical composition of claim 18;

Preferably, the tumor expresses CD22; preferably, the tumor expresses CD22 and CD19;

Preferably, the tumor involves tumor cells expressing CD22; Preferably, the tumor involves tumor cells expressing CD22 and CD19;

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.
The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, or the isolated nucleic acid molecule of claim 6, or the vector of claim 7, or the host cell of claim 8, or the multi-characteristic molecule of claim 10, Or the chimeric antigen receptor of claim 11, or the bispecific chimeric antigen receptor of claim 12 or 13, or the isolated nucleic acid molecule of claim 14, or the vector of claim 15, or the host of claim 16 Cells, or the use of the pharmaceutical composition of claim 18 in the preparation of a medicament for preventing and/or treating tumors in a subject;

Preferably, the tumor expresses CD22; preferably, the tumor expresses CD22 and CD19;

Preferably, the tumor involves tumor cells expressing CD22; Preferably, the tumor involves tumor cells expressing CD22 and CD19;

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.
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.