WO2020114358A1

WO2020114358A1 - Cd19 antibody and uses thereof

Info

Publication number: WO2020114358A1
Application number: PCT/CN2019/122486
Authority: WO
Inventors: 董军纪; 陈超; 于婷婷; 徐乐; 危喜玲; 李志广; 李想; 曾怡; 李少燕; 陈小锋; 李文佳
Original assignee: 广东东阳光药业有限公司
Priority date: 2018-12-03
Filing date: 2019-12-02
Publication date: 2020-06-11
Also published as: CN111253487B; CN111253487A

Abstract

Provided are a specifically binding CD19 antibody or an antigen binding fragment thereof, wherein the antibody binds to a CD19 protein having 3.5μg/mL or lower IC50, and the uses the antibody or the antigen binding fragment thereof in preparing a drug.

Description

CD19 antibody and its application

Technical field

The present application relates to the field of biomedicine, in particular to an antibody or antigen-binding fragment that specifically binds to CD19 protein.

Background technique

B lymphocyte antigen CD19, also known as CD19 molecule (differentiation cluster 19). In normal lymphoid tissues, CD19 is expressed in B cells of the germinal center and large dendritic cells in the follicular dendritic cells, mantle cells, and T cell regions between follicles. CD19 plays two main roles in human B cells, one is to act as an adaptor protein to recruit cytoplasmic signaling proteins to the membrane, and the other is to act within the CD19/CD21 complex to lower the threshold of the B cell receptor signaling pathway . CD19 protein is a target protein that exists stably on the surface of B lymphocytes. It exists in all stages of B cell maturation. Most hematoma-related tumors (B-ALL/CLL/B-NHL, etc.) highly express CD19. Hematopoietic stem cells and other normal cells are not expressed, so CD19 has become an important target for the treatment of hematoma-related tumors.

However, the currently developed CD19 antibodies have low specificity and limited tumor suppressive capacity, so it is urgent to develop new anti-CD19 drugs for new drug development.

Summary of the invention

The present application provides an antibody or antigen-binding fragment that specifically binds to CD19 and uses thereof. The present application also provides a fusion protein comprising the antibody or antigen-binding fragment thereof, a nucleic acid molecule encoding the antibody or antigen-binding protein thereof, a vector containing the nucleic acid molecule, a cell containing the vector or the nucleic acid molecule, etc. . The anti-CD19 antibody provided in this application has one or more of the following properties: 1) can specifically bind to the CD19 protein; 2) has different affinity characteristics for different epitopes of the CD19 protein; 3) has a good targeted killing effect ; 4) has higher stability; 5) has higher purity; 6) can be used to treat cancer.

In one aspect, the present application provides an antibody, antigen-binding fragment or variant thereof that binds to CD19 protein with an IC ₅₀ value of 3.5 μg/ml or lower.

In certain embodiments, the antibody is selected from any one of the group consisting of monoclonal antibodies, single chain antibodies, chimeric antibodies, murine antibodies, and humanized antibodies.

In certain embodiments, the antigen-binding fragment is selected from the group consisting of: Fab, Fab', F(ab)2, F(ab')2, Fv, and scFv.

In certain embodiments, the variant is selected from the group consisting of: 1) a protein or polypeptide substituted, deleted, or added with one or more amino acids in the antibody or the antigen-binding fragment thereof; and 2) with The antibody or the antigen-binding fragment of the protein or polypeptide has more than 90% sequence homology.

On the other hand, the present application provides an antibody, antigen-binding fragment or variant thereof, which competes with a reference antibody for binding to the CD19 protein, wherein the reference antibody comprises light Chain variable region and heavy chain variable region, the light chain variable region of the reference antibody comprises LCDR1-3, and the amino acid sequence of the LCDR1 is selected from the amino acid sequence shown in any one of the following group: SEQ ID NO : 4 and SEQ ID NO: 18; the amino acid sequence of the LCDR2 is selected from the amino acid sequences shown in any of the following groups: SEQ ID NO: 5 and SEQ ID NO: 19; and the LCDR3 amino acid sequence is selected from The amino acid sequence shown in any of the following group: SEQ ID NO: 6 and SEQ ID NO: 20; and the heavy chain variable region of the reference antibody comprises HCDR1-3, and the amino acid sequence of the HCDR1 is selected from The amino acid sequence shown in any one of the groups: SEQ ID NO: 7, SEQ ID NO: 21 and SEQ ID NO: 32; the amino acid sequence of the HCDR2 is selected from the amino acid sequence shown in any one of the following groups: SEQ ID NO: 8, SEQ ID NO: 22 and SEQ ID NO: 33; and the amino acid sequence of the HCDR3 is selected from the amino acid sequences shown in any of the following groups: SEQ ID NO: 9, SEQ ID NO: 23 and SEQ ID NO: 34.

In certain embodiments, the amino acid sequence of the light chain variable region of the reference antibody is selected from the amino acid sequences shown in any of the following groups: SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO :46, and the amino acid sequence of the heavy chain variable region of the reference antibody is selected from the amino acid sequences shown in any of the following groups: SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52.

In certain embodiments, the antibody 3.5μg / ml or less of IC ₅₀ values combined with the CD19 protein.

On the other hand, the present application provides an antibody, antigen-binding fragment or variant thereof, the antibody, antigen-binding fragment or variant thereof comprises LCDR1, and the LCDR1 comprises an amino acid selected from any one of the following group Sequence: SEQ ID NO: 4 and SEQ ID NO: 18. In certain embodiments, it comprises LCDR2, and the LCDR2 comprises an amino acid sequence selected from any one of the group consisting of SEQ ID NO: 5 and SEQ ID NO: 19. In certain embodiments, it includes LCDR3, and the LCDR3 includes an amino acid sequence selected from any one of the group consisting of SEQ ID NO: 6 and SEQ ID NO: 20. In certain embodiments, it comprises a light chain variable region VL, and the light chain variable region VL comprises an amino acid sequence selected from any one of the group consisting of SEQ ID NO: 42, SEQ ID NO: 44 And SEQ ID NO: 46.

In certain embodiments, it comprises HCDR1, and the HCDR1 comprises an amino acid sequence selected from any one of the group consisting of SEQ ID NO: 7, SEQ ID NO: 21 and SEQ ID NO: 32. In certain embodiments, it comprises HCDR2, and the HCDR2 comprises an amino acid sequence selected from any one of the group consisting of SEQ ID NO: 8, SEQ ID NO: 22, and SEQ ID NO: 33. In certain embodiments, it comprises HCDR3, and the HCDR3 comprises an amino acid sequence selected from any one of the group consisting of SEQ ID NO: 9 and SEQ ID NO: 23 and SEQ ID NO: 34. In certain embodiments, it comprises a heavy chain variable region VH, and the heavy chain variable region VH comprises an amino acid sequence selected from any one of the group consisting of SEQ ID NO: 48, SEQ ID NO: 50 And SEQ ID NO: 52.

In some embodiments, the antibody or antigen-binding fragment thereof is scFv. In some embodiments, the scFv comprises an amino acid sequence selected from any one of the group consisting of SEQ ID NO: 56, SEQ ID NO: 58 and SEQ ID NO: 60.

In certain embodiments, the CD19 protein is mouse CD19 protein.

In another aspect, the present application provides a fusion protein comprising the antibody, antigen-binding fragment or variant thereof described herein. In certain embodiments, the fusion protein is a chimeric antigen receptor.

In certain embodiments, the chimeric antigen receptor comprises an extracellular hinge region, a transmembrane domain, a costimulatory domain, and a CD3ζ intracellular signal transduction domain.

In certain embodiments, the extracellular hinge region is selected from CD8 hinge or IgG4 hinge.

In certain embodiments, the transmembrane domain is selected from the α, β, or ζ chain of the T cell receptor, CD28, CD3e, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64 , CD80, CD86, CD134, CD137 and CD154. In certain embodiments, the transmembrane domain is selected from CD8-1 or CD8-2.

In certain embodiments, the costimulatory domain is selected from CD27, CD28, 4-1BB, OX40, or ICOS.

In certain embodiments, in the chimeric antigen receptor, the extracellular hinge region is selected from a CD8 hinge or an IgG4 hinge; the transmembrane domain is selected from CD8-1 or CD8-2; the co-stimulation The domain is selected from 4-1BB or OX40.

In certain embodiments, in the chimeric antigen receptor, the N-terminus of the CD8-1 transmembrane is connected to the C-terminus of the CD8 hinge, and the C-terminus of the CD8-1 transmembrane is linked to the 4 The N terminal of -1BB is connected, the C terminal of 4-1BB is connected to the N terminal of OX40, and the C terminal of OX40 is connected to the N terminal of CD3ζ.

In certain embodiments, in the chimeric antigen receptor, the N-terminus of the CD8-2 transmembrane is connected to the C-terminus of the IgG4 hinge, and the C-terminus of the CD8-2 transmembrane is linked to the 4 The N terminal of -1BB is connected, the C terminal of 4-1BB is connected to the N terminal of OX40, and the C terminal of OX40 is connected to the N terminal of CD3ζ.

In another aspect, the present application provides isolated one or more nucleic acid molecules that encode the antibodies, antigen-binding fragments or variants described herein, and/or that encode the fusion proteins described herein.

In another aspect, the present application provides one or more vectors, which comprise one or more nucleic acid molecules described in the present application.

In another aspect, the present application provides one or more cells comprising one or more nucleic acid molecules described herein or one or more vectors described herein.

On the other hand, the present application provides a method for preparing the antibody, antigen-binding fragment or variant thereof, and/or the fusion protein described in the present application. The antigen-binding fragments or variants thereof, and/or the cells described herein are cultured under the conditions for the expression of the fusion protein described herein. In certain embodiments, the methods described herein include harvesting the antibody, antigen-binding fragment or variant thereof, and/or the fusion protein.

On the other hand, the present application provides a pharmaceutical composition comprising the antibody described herein, its antigen-binding fragment or variant, the nucleic acid molecule described herein, the vector described herein, and the application described herein Cells and/or fusion proteins described herein, and optionally pharmaceutically acceptable adjuvants.

On the other hand, the present application provides the antibody, antigen-binding fragment or variant thereof, and/or the use of the fusion protein in the preparation of a medicament for preventing or treating a disease or disorder.

In certain embodiments, the disease or disorder is selected from any one of the group consisting of tumors and autoimmune diseases.

In certain embodiments, the tumor is selected from any one of the group consisting of B-cell subtype non-Hodgkin's malignant lymphoma (NHL), Burkitt's lymphoma, multiple myeloma, pre-B Acute lymphoblastic leukemia and other malignant lung tumors derived from early B-cell precursors, common acute lymphocytic leukemia, T chronic lymphocytic leukemia, hairy cell leukemia, non-acute lymphoblastic leukemia, Fahrenheit macroglobulin blood Disease, prolymphocytic leukemia, plasmacytoma, osteosclerotic myeloma, plasmacytic leukemia, monoclonal gammopathy (MGUS), smoldering multiple myeloma (SMM), chronic multiple myeloma (IMM) ), Hodgkin's malignant lymphoma, gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, prostate cancer, cervical cancer, adrenal tumor and bladder Tumor.

In certain embodiments, the autoimmune disease is selected from any one of the group consisting of rheumatoid arthritis, multiple sclerosis, and CD19 ⁺ leukemia.

On the other hand, the present application provides a method for inhibiting the biological activity of CD19 protein, which comprises administering the antibody, antigen-binding fragment or variant thereof, the nucleic acid molecule, the carrier, the carrier Cells and/or the fusion protein.

On the other hand, the application provides the antibody, antigen-binding fragment or variant thereof, the nucleic acid molecule, the carrier, the cell and/or the fusion protein described in the application Application of reagents for detecting tumors.

Those skilled in the art can easily gain insight into other aspects and advantages of the present application from the detailed description below. Only the exemplary embodiments of the present application are shown and described in the detailed description below. As those skilled in the art will recognize, the content of this application enables those skilled in the art to make changes to the disclosed specific embodiments without departing from the spirit and scope of the invention involved in this application. Accordingly, the drawings and descriptions in the specification of the present application are merely exemplary, not limiting.

BRIEF DESCRIPTION

The specific features of the invention to which this application relates are shown in the appended claims. The characteristics and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and drawings described in detail below. The brief description of the drawings is as follows:

Figure 1 shows the ELISA activity detection results of the CD19 antibody described in this application.

Figure 2 shows the results of the CD19 antibody flow cytometry assay described in this application.

Figure 3 shows the detection results of the CD19 antibody Western blot method described in this application.

Fig. 4 shows the results of the flow cytometry detection of the CD19 antibody described in this application at different concentrations.

FIG. 5 shows a schematic diagram of the CAR structure constructed in the embodiment of the present application.

Figure 6 shows the test results of anti-CD19CAR-T positive rate.

detailed description

The following describes the implementation of the invention of the present application by specific specific examples. Those skilled in the art can easily understand other advantages and effects of the invention of the present application by the content disclosed in this specification. The present application provides an antibody that can specifically bind to CD19 protein, which can specifically bind to CD19 protein and possess different affinity characteristics for different epitopes of CD19. The CD19 antibody has good targeted killing effect, higher stability, and higher purity. The CD19 antibody can be used to treat cancer.

The present invention is further described below: In the present invention, unless otherwise stated, the scientific and technical terms used in this application have the meanings generally understood by those skilled in the art. In addition, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and laboratory procedures used in this application are all widely used terms and routine procedures in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

In this application, the term "antibody" generally refers to a polypeptide molecule that can specifically recognize and/or neutralize a specific antigen. For example, an antibody may include an immunoglobulin composed of at least two heavy (H) chains and two light (L) chains connected to each other by a disulfide bond, and include any molecule that includes an antigen binding portion thereof. The term "antibody" includes monoclonal antibodies, antibody fragments, or antibody derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies (eg, dAbs), single chain antibodies (eg, scFv), And antibody fragments that bind to the antigen (eg, Fab, Fab' and (Fab) 2 fragments). In the present application, the term "monoclonal antibody" (monoclonal antibody) (mAb) generally refers to an antibody produced by only one type of immune cell, and can also be produced by the fusion of the immune cell that makes this antibody and cancer cells Hybridoma cells are produced. In the present application, the term "single chain antibody" (single chain antibody) (sFv) generally refers to an antibody composed of an antibody heavy chain variable region and a light chain variable region connected by a linker. ScFv can better retain its affinity for antigen, and has the characteristics of small molecular weight, strong penetration and weak antigenity. In this application, the term "humanized antibody" generally refers to an antibody re-expressed by murine monoclonal antibody modified by gene cloning and DNA recombination technology, most of its amino acid sequence is replaced by human sequence, and the parent mouse monoclonal is basically retained The affinity and specificity of the antibody, which reduces its heterogeneity, is beneficial to the human body. In the present application, the term "chimeric antibody" generally refers to an antibody composed of the V region gene of a murine antibody and the C region gene of a human antibody, because it reduces the components of the murine origin, thereby reducing the murine origin Adverse reactions caused by sexual antibodies and help improve efficacy. In the present application, the term "murine antibody" generally refers to the fusion of B cells derived from immunized mice with myeloma cells, and then selecting a mouse hybrid fusion cell that can both wirelessly propagate and secrete antibodies, and Screening, antibody preparation and purification. The term "antibody" also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells as well as any antibody fragments and derivatives thereof that bind to antigens described herein. Each heavy chain may be composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain may be composed of a light chain variable region (VL) and a light chain constant region. VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FR). Each VH and VL may be composed of three CDRs and four FR regions, and they may be arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component of the classical complement system (Clq).

In the present application, the term "antibody binding fragment" generally refers to one or more fragments of an antibody that perform the function of specifically binding an antigen. The antigen-binding function of an antibody can be achieved by the full-length fragment of the antibody. The antigen-binding function of an antibody can also be achieved by the following fragments: (1) Fab fragment, that is, a monovalent fragment composed of VL, VH, CL, and CH domains; (2) F(ab') 2 fragment, including A bivalent fragment of two Fab fragments connected by a disulfide bond at the hinge region; (3) Fd fragments composed of VH and CH domains; (4) Fv fragments composed of VL and VH domains of one arm of an antibody; (5) A dAb fragment composed of VH domains (Ward et al., (1989) Nature 341: 544-546); (6) the isolated complementarity determining region (CDR) and (7) two optionally connected by a linker A combination of three or more separate CDRs. In addition, it can also include a monovalent single-chain molecule Fv (scFv) formed by pairing VL and VH (see Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. 85 : 5879-5883). The "antigen binding portion" may also include an immunoglobulin fusion protein, the fusion protein comprising a binding domain selected from: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) CH2 constant region of the immunoglobulin heavy chain fused to the hinge region; and (3) CH3 constant region of the immunoglobulin heavy chain fused to the CH2 constant region. An antibody specifically binding to CD19 protein provided by the present application, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, scFv, Fab', F(ab)2, F(ab')2 and dAb .

In the present application, the term "CD19 protein" generally refers to a transmembrane glycoprotein unique to the B cell line of about 95 kDa that is mainly expressed in early B cells. CD19 protein is expressed in normal and malignant B lymphocytes and is regarded as the most reliable surface marker during B cell development. In this application, the CD19 protein may be a human CD19 protein. For example, the human CD19 protein expressed in the present application may include the amino acid sequence shown in NCBI accession number AAB60697. In certain embodiments, the CD19 protein may be a CD19 recombinant protein, and the recombinant protein may be the extracellular region of the CD19 protein. For example, the CD19 recombinant protein may be ab234966 of abcam, a protein with amino acid sequence numbers from 20 to 291.

In the present application, the term "fusion protein" (fusion protein) is also referred to as chimeric protein (chimeric protein), and usually refers to the expression product of two genes obtained by DNA recombination technology after recombination. Fusion protein technology is a purposeful gene fusion and protein expression method for obtaining a large number of standard fusion proteins. Using fusion protein technology, it is possible to construct and express novel target proteins with multiple functions. Translation of the fusion gene produces a single or multiple polypeptides with the functional properties of each original protein of the derivat. The recombinant fusion protein artificially produced by recombinant DNA technology can be used for biological research or treatment. The chimera or chimera generally represents a fusion protein made of polypeptides with different functions or physicochemical characteristics. The fusion protein may include a chimeric antigen receptor formed by fusing the antigen-binding region scFv, CD3ζ chain or intracellular part of the antibody, which may include tumor-associated antigen-binding region, extracellular hinge region, transmembrane region and other domains. The tumor-associated antigen-binding region can be derived from the antigen-binding region of an antibody, such as scFv.

In the present application, the term "chimeric antigen receptor" (CAR) is a fusion protein of the variable region of a single chain antibody and a T cell signaling molecule. It allows T cells to recognize specific antigens in a non-MHC-restricted manner and exert a killing effect. In the present application, the term "single-chain antibody" may be an antibody composed of the heavy chain variable region and the light chain variable region connected by a linker peptide. CAR is the core component of chimeric antigen receptor T cells (CAR-T), which may include CD19 binding domain, transmembrane domain, costimulatory domain and intracellular signal transduction domain.

In the present application, the term "transmembrane domain" (transmembrane domain) generally refers to a domain in CAR that passes through a cell membrane, which is connected to an intracellular signal transduction domain and plays a role in transmitting signals.

In the present application, the term "costimulatory domain" generally refers to an intracellular domain that can provide an immunocostimulatory molecule that is a cell surface molecule required for an effective response of lymphocytes to an antigen.

In this application, the term “intracellular signaling domain” generally refers to a component of CAR signaling located in a cell, which includes a signaling domain and a domain that specifically binds to the receptor component, for example: It can be selected from CD3ζ intracellular domain, CD28 intracellular domain, 4-1BB intracellular domain and OX40 intracellular domain.

The term "variant" related to antibodies means in the present application that it has been substituted by at least 1, such as 1-30, or 1-20 or 1-10, such as 1 or 2 or 3 or 4 or 5 amino acids, Antibodies with deletion and/or insertion that have a target antibody region of amino acid changes (eg, heavy chain variable region or light chain variable region or heavy chain CDR region or light chain CDR region), where the variant substantially retains the antibody before the change Molecular biological properties. In one aspect, this application encompasses any antibody variants described in this application. In certain embodiments, the antibody variant retains at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding capacity) of the pre-altered antibody. In some embodiments, the change does not cause the antibody variant to lose its binding to the antigen, but optionally can impart properties such as increased antigen affinity and different effector functions. It can be understood that the heavy chain variable region or light chain variable region, or each CDR region of an antibody may be changed individually or in combination. In certain embodiments, the amino acid changes in one or more or all three heavy chain CDRs are no more than 1, 2, 3, 4, 5, 6, 6, 7, 8, 9 Or 10. In some embodiments, the amino acid change may be an amino acid substitution, for example, it may be a conservative substitution. In certain embodiments, the antibody variant has at least 80%, 85%, 90%, or 95% or 99% or higher amino acid identity over the antibody sequence region of interest.

In this application, the term "isolated" generally refers to an antibody that has been separated from components in its natural environment. In certain embodiments, the antibody is purified to greater than 95% or 99% purity by, for example, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or Reverse phase HPLC). For a review of methods for evaluating antibody purity, see Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

In the present application, the term "nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, isolated from its natural environment or artificially synthesized, or analogs thereof. The nucleic acid molecules described in this application may be isolated. For example, it can be produced or synthesized by: (i) amplified in vitro, such as polymerase chain reaction (PCR) amplification, (ii) cloned and recombinantly produced, (iii) purified , Such as by enzyme digestion and gel electrophoresis fractionation, or (iv) synthetic, for example by chemical synthesis. In certain embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology. In this application, nucleic acids encoding the antibodies or antigen-binding fragments thereof can be prepared by a variety of methods known in the art. These methods include, but are not limited to, the use of restriction fragment manipulation or the use of synthetic oligonucleotides Overlapping extension PCR, specific operations can be found in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al. Current Protocols Molecular Biology, Greene Publishing and Wiley-Interscience, New York, 1993.

In the present application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host to transfer the inserted nucleic acid molecule into and/or between host cells. The vector may include a vector mainly used for inserting DNA or RNA into a cell, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA. The carrier also includes carriers having multiple functions described above. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, by culturing a suitable host cell containing the vector, the vector can produce the desired expression product. In the present application, one or more of the nucleic acid molecules may be included in the vector. In addition, the vector may also contain other genes, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. In addition, the vector may also contain expression control elements that allow the coding region to be correctly expressed in a suitable host. Such control elements are well known to those skilled in the art. For example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In some embodiments, the expression control sequence is an adjustable element. The specific structure of the expression control sequence may vary according to the function of the species or cell type, but usually contains 5'untranscribed sequences and 5'and 3'untranslated sequences involved in transcription and translation initiation, such as TATA box, Cap sequence, CAAT sequence, etc. For example, the 5' non-transcriptional expression control sequence may comprise a promoter region, and the promoter region may comprise a promoter sequence for transcriptional control of functionally linked nucleic acids.

In the present application, the term "cell" generally refers to an individual cell or cell that can express the antibody, antigen-binding fragment or variant thereof described herein, and can or already contains a plasmid or vector including the nucleic acid molecule described herein Line or cell culture. The cells may be prokaryotic cells (such as E. coli) or eukaryotic cells (such as yeast cells, COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells or bone marrow Tumor cells). In some embodiments, the cell may be an immune cell. For example, plasma cells, cytotoxic T cells, NK cells, APSC pluripotent cells, mast cells, Ramos cells, NALM-6 cells.

In this application, the term "pharmaceutically acceptable adjuvant" generally refers to a pharmaceutically acceptable formulation carrier, solution, or additive that enhances the properties of the formulation. Such additives are well known to those skilled in the art.

In this application, the term "cancer" generally refers to or describes the physiological condition of a mammal, which is typically characterized by unregulated cell proliferation or survival. In this application, hyperproliferative diseases called cancer include, but are not limited to, solid tumors, such as occur in breast, respiratory tract, brain, reproductive organs, digestive tract, urethra, eyes, liver, skin, head and neck, thyroid, parathyroid Cancer of the glands, and their distant metastasis. Such diseases also include lymphoma, sarcoma and leukemia. Examples of breast cancer include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, breast ductal carcinoma in situ, and breast lobular carcinoma in situ. Examples of cancers of the respiratory tract include, but are not limited to, small cell lung cancer and non-small cell lung cancer, as well as bronchial adenoma and pleural lung blastoma. Examples of brain cancer include, but are not limited to, brain stem and hypothalamic keratinoma, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, and neuroectodermal and pineal tumors. Male genital tumors include but are not limited to prostate and testicular cancer. Female genital tumors include but are not limited to endometrial cancer, cervical cancer, ovarian cancer, vaginal cancer and vulvar cancer, as well as uterine tumors. Gastrointestinal tumors include but are not limited to anal, colon, colorectal, esophageal, gallbladder, stomach, pancreas, rectum, small intestine, and salivary gland cancers. Urethral tumors include but are not limited to bladder, penis, kidney, renal pelvis, ureter, and urethral cancer. Eye cancers include but are not limited to intraocular melanoma and retinoblastoma. Examples of liver cancer include, but are not limited to, hepatocellular carcinoma (hepatocellular carcinoma with or without fibrous lamellar variation), cholangiocarcinoma (intrahepatic cholangiocarcinoma), and mixed hepatocellular cholangiocarcinoma. Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma type skin cancer. Head and neck cancers include but are not limited to laryngeal/ hypopharyngeal/nasopharyngeal/oropharyngeal cancers, and lips and oral cavity cancers. Lymphomas include, but are not limited to, AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and central nervous system lymphoma. Sarcomas include, but are not limited to, soft tissue sarcoma, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma. Leukemias include but are not limited to acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and hairy cell leukemia.

In the present application, the term "autoimmune disease" generally refers to an autoimmune disease that refers to a disease caused by an immune response of the body to self-antigens that causes damage to its own tissues. The immune system defends the body from invasion (recognition) by foreign or dangerous substances, including microorganisms, parasites, malignant tumor cells, and transplanted organs and tissues. Under normal circumstances, the immune system only reacts to foreign or dangerous substances, but does not react to antigens in its own tissues. However, sometimes there is abnormal immune function, which treats its own tissues as foreign, and produces antibodies (called autoantibodies) or immune cells to attack its own cells or tissues. This response is called an autoimmune response. It causes inflammation and tissue damage. This reaction may cause autoimmune diseases, but some people produce very little autoantibodies without autoimmune diseases. Common autoimmune diseases include rheumatoid arthritis, multiple sclerosis, and CD19 ⁺ leukemia. Additional diseases considered to be autoimmune include Addison's disease, polymyositis,

Syndrome, progressive systemic sclerosis, multiple cases of glomerulonephritis (kidney inflammation) and some cases of infertility.

The term "and/or" should be understood to mean any one of the alternatives or two of the alternatives.

In this application, the term "about" generally refers to a range of 0.5%-10% above or below the specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.

In this application, the term "comprising" generally refers to the meaning of including, encompassing, containing, or including. In some cases, it also means "for" and "consisting of".

Antibody or antigen-binding fragment

The present application provides an antibody, antigen-binding fragment or variant thereof, which may be 3.5 μg/mL or less (eg, 3.5 μg/ml or less, 3.4 μg/mL or less, 3.3 μg/mL or more Low, 3.2μg/mL or lower, 3.15μg/mL or lower, 3.1μg/ml or lower, 3.0μg/mL or lower, 2.9μg/mL or lower, 2.8μg/mL or lower, 2.5μg / mL or less, or 2.1μg / mL or less) and IC ₅₀ values with CD19 binding proteins. For example, the IC ₅₀ value is determined by an ELISA method, and the IC ₅₀ value may be 3.12 μg/mL or lower.

In the present application, the antibody, antigen-binding fragment or variant thereof may be 5 μg/mL or lower (eg, 5 μg/mL or lower, 4.5 μg/mL or lower, 4 μg/mL or lower, ₅₀ value of 3.5μg / mL or less, 3μg / mL or less, 2.5μg / mL or less, 2μg / mL or less, 1.5μg / mL or less, 1μg / mL or less) and an IC CD19 protein combined. For example, the IC ₅₀ value is determined by flow cytometry, and the IC ₅₀ value may be 5 μg/mL or less.

In the present application, the antibody may be selected from any of the following group: monoclonal antibody, single chain antibody, chimeric antibody, murine antibody, and humanized antibody. In this application, the antigen-binding fragment may be selected from the group consisting of: Fab, Fab', F(ab)2, F(ab')2, Fv, and scFv.

In this application, the variant may be selected from the following group: 1) One or more (eg, 1-2, 1-3) are substituted, deleted, or added to the antibody or the antigen-binding fragment thereof , 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10 or more) amino acid protein or polypeptide; and 2) More than 90% (eg, at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97) of the antibody or the antigen-binding fragment thereof %, about 98%, about 99% or more) of sequence homology protein or polypeptide. In the present application, the variant has substantially the same function as the antibody and its antigen-binding fragment (for example, it can specifically bind to the CD19 protein).

In this application, the antibody, antigen-binding fragment or variant thereof can compete with a reference antibody for binding to the CD19 protein. In the present application, the reference antibody may comprise a light chain variable region and a heavy chain variable region, the light chain variable region of the reference antibody may comprise LCDR1-3, and the amino acid sequence of the LCDR1 may be selected from The amino acid sequence shown in any one of the following group or a variant thereof: SEQ ID NO: 4 and SEQ ID NO: 18; the amino acid sequence of the LCDR2 may be selected from the amino acid sequence shown in any one of the following group or Variants: SEQ ID NO: 5 and SEQ ID NO: 19; and the amino acid sequence of the LCDR3 can be selected from the amino acid sequences shown in any of the following groups or variants thereof: SEQ ID NO: 6 and SEQ ID NO :20; and the heavy chain variable region of the reference antibody may include HCDR1-3, and the amino acid sequence of the HCDR1 may be selected from the amino acid sequence shown in any one of the following group or a variant thereof: SEQ ID NO: 7. SEQ ID NO: 21 and SEQ ID NO: 32; the amino acid sequence of the HCDR2 may be selected from the amino acid sequence shown in any one of the following groups or a variant thereof: SEQ ID NO: 8, SEQ ID NO: 22 And SEQ ID NO: 33; and the amino acid sequence of the HCDR3 can be selected from the amino acid sequence shown in any one of the following group or a variant thereof: SEQ ID NO: 9, SEQ ID NO: 23 and SEQ ID NO: 34 .

In the present application, the sequence of the light chain variable region of the reference antibody may be selected from the amino acid sequence shown in any one of the following groups or a variant thereof: SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO: 46; the sequence of the heavy chain variable region of the reference antibody may be selected from the amino acid sequence shown in any one of the following group or a variant thereof: SEQ ID NO: 48, SEQ ID NO: 50 and SEQ ID NO: 52.

The antibodies, antigen-binding fragments or variants thereof described herein may comprise antibody light chains or fragments thereof.

In this application, the light chain may include a light chain variable region, and the light chain variable region may include LCDR1, and the amino acid sequence of the LCDR1 may be selected from the amino acid sequence shown in any one of the following group or Variants: SEQ ID NO: 4 and SEQ ID NO: 18. The light chain variable region may include LCDR2, and the amino acid sequence of the LCDR2 may be selected from the amino acid sequence shown in any one of the following group or a variant thereof: SEQ ID NO: 5 and SEQ ID NO: 19. The light chain variable region may include LCDR3, and the amino acid sequence of the LCDR3 may be selected from the amino acid sequence shown in any one of the following group or a variant thereof: SEQ ID NO: 6 and SEQ ID NO: 20.

In the present application, the light chain variable region of the antibody, antigen-binding fragment or variant thereof may comprise LFR1, and the amino acid sequence of the LFR1 may be selected from the amino acid sequence shown in any one of the following group or a variant thereof : SEQ ID NO: 10, SEQ ID NO: 24 and SEQ ID NO: 35. The light chain variable region may include LFR2, and the amino acid sequence of the LFR2 may be selected from the amino acid sequence shown in any one of the following group or a variant thereof: SEQ ID NO: 11 and SEQ ID NO: 25. The light chain variable region may include LFR3, and the amino acid sequence of the LFR3 may be selected from the amino acid sequence shown in any one of the following group or a variant thereof: SEQ ID NO: 12 and SEQ ID NO: 26. The light chain variable region may include LFR4, and the amino acid sequence of the FR4 may be selected from the amino acid sequence shown in any one of the following group or a variant thereof: SEQ ID NO: 13, SEQ ID NO: 27, and SEQ ID NO:36.

In the present application, the sequence of the light chain variable region may be selected from the amino acid sequence shown in any one of the following group or a variant thereof: SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO: 46 .

The antibodies, antigen-binding fragments or variants thereof described herein may comprise antibody heavy chains or fragments thereof.

In the present application, the light chain may include a heavy chain variable region, and the heavy chain variable region may include HCDR1, and the amino acid sequence of the HCDR1 may be selected from the amino acid sequences shown in any one of the following groups or Variants: SEQ ID NO: 7, SEQ ID NO: 21 and SEQ ID NO: 32. The variable region of the heavy chain may comprise HCDR2, and the amino acid sequence of the HCDR2 may be selected from the amino acid sequence shown in any one of the following group or a variant thereof: SEQ ID NO: 8, SEQ ID NO: 22 and SEQ ID NO:33. The variable region of the heavy chain may include HCDR3, and the amino acid sequence of the HCDR3 may be selected from the amino acid sequence shown in any one of the following group or a variant thereof: SEQ ID NO: 9, SEQ ID NO: 23, and SEQ ID NO:34.

In the present application, the heavy chain variable region of the antibody, antigen-binding fragment or variant thereof may include HFR1, and the amino acid sequence of the HFR1 may be selected from the amino acid sequences shown in any one of the following groups or variants thereof : SEQ ID NO: 14, SEQ ID NO: 28 and SEQ ID NO: 37. The variable region of the heavy chain may include HFR2, and the amino acid sequence of the HFR2 may be selected from the amino acid sequence shown in any one of the following group or a variant thereof: SEQ ID NO: 15, SEQ ID NO: 29, and SEQ ID NO:38. The heavy chain variable region may include HFR3, and the amino acid sequence of the HFR3 may be selected from the amino acid sequence shown in any one of the following group or a variant thereof: SEQ ID NO: 16, SEQ ID NO: 30, and SEQ ID NO:39. The variable region of the heavy chain may include HFR4, and the amino acid sequence of the HFR4 may be selected from the amino acid sequence shown in any one of the following group or a variant thereof: SEQ ID NO: 17, SEQ ID NO: 31, and SEQ ID NO:40.

In the present application, the sequence of the variable region of the heavy chain may be selected from the amino acid sequence shown in any one of the following groups or a variant thereof: SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 .

In certain embodiments, the amino acid sequence of LCDR1 in the antibody or antigen-binding fragment thereof described herein may include SEQ ID NO: 4 or its variant; the amino acid sequence of LCDR2 may include SEQ ID NO: 5 or its variant ; The amino acid sequence of LCDR3 may include SEQ ID NO: 6 or its variant; and the amino acid sequence of HCDR1 may include SEQ ID NO: 7 or its variant; the amino acid sequence of HCDR2 may include SEQ ID NO: 8 or its variant; The amino acid sequence of HCDR3 may include SEQ ID NO: 9 or a variant thereof. For example, the antibody or antigen-binding fragment thereof may include antibody 25C5-2 or an antibody having the same LCDR1-3 and HCDR1-3 as it. The amino acid sequence of LFR1 in the antibody or antigen-binding fragment thereof described in this application may include SEQ ID NO: 10 or its variant; the amino acid sequence of LFR2 may include SEQ ID NO: 11 or its variant; the amino acid sequence of LFR3 may include SEQ ID NO: 12 or its variant; the amino acid sequence of LFR4 may include SEQ ID NO: 13 or its variant; and the amino acid sequence of HFR1 may include SEQ ID NO: 14 or its variant; the amino acid sequence of HFR2 may include SEQ ID NO: 15 or a variant thereof; the amino acid sequence of HFR3 may include SEQ ID NO: 16 or a variant thereof; the amino acid sequence of HFR4 may include SEQ ID NO: 17 or a variant thereof. For example, the antibody or antigen-binding fragment thereof may include antibody 25C5-2 or antibodies having the same LFR1-4 and HFR1-4. In certain embodiments, the light chain of the antibody or antigen-binding fragment thereof described herein may include a light chain variable region, and the amino acid sequence of the light chain variable region may include SEQ ID NO: 42 or a variant thereof ; And wherein the heavy chain may comprise a heavy chain variable region, the amino acid sequence of the heavy chain variable region may include SEQ ID NO: 48 or a variant thereof. For example, the antibody or antigen-binding fragment thereof may include antibody 25C5-2 or an antibody having the same light chain variable region and heavy chain variable region as the same.

For example, the antibody described in this application may be 25C5-2. Among them, the amino acid sequence of LCDR1-3 of antibody 25C5-2 is shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the amino acid sequence of LFR1-4 is SEQ ID NO: 10, SEQ respectively ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13; VL amino acid sequence is shown in SEQ ID NO: 42; HCDR1-3 amino acid sequences are shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9; the amino acid sequences of HFR1-4 are shown in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively; the amino acid sequence of VH is shown in SEQ ID NO: 48.

In certain embodiments, the antibody 25C5-2 may be scFv, and the scFv is VL-VH (that is, VL is connected to the N-terminus of VH through the linker peptide), wherein the amino acid sequence of the linker peptide may be as SEQ ID NO: 54 shows. The amino acid sequence of the scFv may be as shown in SEQ ID NO: 56.

For example, the antibody described in this application may be 10H10-1. Among them, the amino acid sequence of LCDR1-3 of antibody 10H10-1 is shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 respectively; the amino acid sequence of LFR1-4 is SEQ ID NO: 24 and SEQ respectively ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27 are shown; the amino acid sequence of VL is shown in SEQ ID NO: 44; the amino acid sequences of HCDR1-3 are shown in SEQ ID NO: 21 and SEQ ID NO: 22 and SEQ ID NO: 23; the amino acid sequences of HFR1-4 are shown in SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31; the amino acid sequence of VH is shown in SEQ ID NO: 50.

In some embodiments, the antibody 10H10-1 may be scFv, and the scFv is VL-VH (that is, VL is connected to the N-terminus of VH through the linker peptide), wherein the amino acid sequence of the linker peptide may be as SEQ ID NO: 54 shows. The amino acid sequence of the scFv may be as shown in SEQ ID NO: 58.

For example, the antibody described in this application may be 16F10. Among them, the amino acid sequences of LCDR1-3 of antibody 16F10 are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the amino acid sequences of LFR1-4 are SEQ ID NO: 35, SEQ ID NO :11, SEQ ID NO:12 and SEQ ID NO:36; VL amino acid sequence is shown as SEQ ID NO:46; HCDR1-3 amino acid sequences are shown as SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO: 34; the amino acid sequences of HFR1-4 are shown in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40 respectively; the amino acid sequence of VH is shown in SEQ ID NO :52 shown.

In some embodiments, the antibody 16F10 may be scFv, and the scFv is VL-VH (that is, VL is connected to the N-terminus of VH through the linker peptide), wherein the amino acid sequence of the linker peptide may be as SEQ ID NO:54 shown. The amino acid sequence of the scFv may be as shown in SEQ ID NO: 60.

Fusion protein

In another aspect, the present application provides a fusion protein comprising the antibody, antigen-binding fragment or variant thereof. In the present application, the fusion protein may be a chimeric antigen receptor (CAR), which may include the antibody and T cell signaling molecule described in the present application. For example, the CAR may include domains such as a CD19 binding domain, a transmembrane domain, a hinge region, a costimulatory domain, and an intracellular signal transduction domain.

In the present application, the antibody may be a single chain antibody. In certain embodiments, the antibody may comprise the amino acid sequence shown in SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60 or a functional variant thereof.

For example, the single chain antibody may include 25C5-2, the amino acid sequence of which is shown in SEQ ID NO: 56. The amino acid sequence of LCDR1-3 of single-chain antibody 25C5-2 is shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the amino acid sequence of VL is shown in SEQ ID NO: 42; HCDR1- The amino acid sequence of 3 is shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; the amino acid sequence of VH is shown in SEQ ID NO: 48.

For example, the single-chain antibody may include 10H10-1, and its amino acid sequence is shown in SEQ ID NO: 58. The amino acid sequence of LCDR1-3 of single-chain antibody 10H10-1 is shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 respectively; the amino acid sequence of VL is shown in SEQ ID NO: 44; HCDR1- The amino acid sequence of 3 is shown in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23 respectively; the amino acid sequence of VH is shown in SEQ ID NO: 50.

For another example, the single-chain antibody may include 16F10, and its amino acid sequence is shown in SEQ ID NO: 60. The amino acid sequences of LCDR1-3 of single-chain antibody 16F10 are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the amino acid sequence of VL is shown in SEQ ID NO: 46; HCDR1-3 The amino acid sequence is shown in SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34 respectively; the amino acid sequence of VH is shown in SEQ ID NO: 52.

In the present application, the CD19 binding domain may comprise an antibody, antigen-binding fragment or variant that specifically binds CD19. The antibodies, antigen-binding fragments or variants thereof described herein may comprise antibody light chains or fragments thereof. The light chain may include a light chain variable region, and the light chain variable region may include LCDR1, LCDR2, and LCDR3. The amino acid sequence of the LCDR1 may be selected from the amino acid sequence shown in any one of the following group or a variant thereof: SEQ ID NO: 4 and SEQ ID NO: 18. The amino acid sequence of the LCDR2 may be selected from the amino acid sequence shown in any one of the following group or a variant thereof: SEQ ID NO: 5 and SEQ ID NO: 19. The amino acid sequence of the LCDR3 may be selected from the amino acid sequence shown in any one of the following group or a variant thereof: SEQ ID NO: 6 and SEQ ID NO: 20. In the present application, the sequence of the light chain variable region may be selected from the amino acid sequence shown in any one of the following group or a variant thereof: SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO: 46 . In the present application, the light chain may include a heavy chain variable region, and the heavy chain variable region may include HCDR1, and the amino acid sequence of the HCDR1 may be selected from the amino acid sequences shown in any one of the following groups or Variants: SEQ ID NO: 7, SEQ ID NO: 21 and SEQ ID NO: 32. The variable region of the heavy chain may comprise HCDR2, and the amino acid sequence of the HCDR2 may be selected from the amino acid sequence shown in any one of the following group or a variant thereof: SEQ ID NO: 8, SEQ ID NO: 22 and SEQ ID NO:33. The variable region of the heavy chain may include HCDR3, and the amino acid sequence of the HCDR3 may be selected from the amino acid sequence shown in any one of the following group or a variant thereof: SEQ ID NO: 9, SEQ ID NO: 23, and SEQ ID NO:34. In the present application, the sequence of the variable region of the heavy chain may be selected from the amino acid sequence shown in any one of the following groups or a variant thereof: SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 .

In the present application, the CAR may include a hinge region, which may connect the antibody and the transmembrane domain. For example, the hinge region may be selected from CD8, whose amino acid sequence is shown in SEQ ID NO: 62; or it may be selected from IgG4, whose amino acid sequence is shown in SEQ ID NO: 64.

In the present application, the CAR may include a transmembrane domain. For example, the transmembrane domain may be selected from the α, β, or ζ chain of the T cell receptor, CD28, CD3e, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86 , CD134, CD137 and CD154 and other polypeptides. In the present application, the transmembrane domain may be CD8-1, which may include the amino acid sequence shown in SEQ ID NO: 66 or a functional variant thereof; it may also be CD8-2, which may contain SEQ ID NO : The amino acid sequence shown in 68 or a functional variant thereof.

In this application, the CAR may include a costimulatory domain. For example, the costimulatory domain may be selected from polypeptides such as CD28, 4-1BB, OX40, and ICOS. In the present application, the costimulatory domain may be 4-1BB, which may contain the amino acid sequence shown in SEQ ID NO: 70 or a functional variant thereof; it may also be OX40, which may contain SEQ ID NO: 72. Shown amino acid sequence or functional variants.

In the present application, the CAR may include a label detection signal, and the label detection signal may be a fluorescent protein. For example, GFP, RFP or YFP. The label detection signal may be located at the C-terminal of the CAR.

In the present application, the CAR may include a Kozak sequence, the nucleotide sequence of which is shown in SEQ ID NO:1. The Kozak sequence may be located at the N-terminus of the CAR.

In this application, the CAR may include a leader sequence whose nucleotide sequence is shown in SEQ ID NO: 2 (the nucleotide sequence does not include the Kozak sequence). The leader sequence may be located at the N-terminus of the CAR.

In certain embodiments, the CAR of the present application may include a Kozak sequence, a leader sequence, a CD19 binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain in sequence from the N-terminus. In certain factual ways, the CAR described in this application may include the Kozak sequence, the leader sequence, the CD19 binding domain, the transmembrane domain, the costimulatory domain, the intracellular signaling domain, and the label detection signal in sequence from the N-terminus .

For example, the CAR described in this application may be CAR25C5-2, the amino acid sequence of LCDR1-3 is shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the amino acid sequence of VL is SEQ ID NO: 42; the amino acid sequences of HCDR1-3 are shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; the amino acid sequence of VH is shown in SEQ ID NO: 48; VH and VL The sequence of the connecting peptide between them is shown in SEQ ID NO: 54; the nucleotide sequence of its Kozak sequence is shown in SEQ ID NO: 1; the amino acid sequence of its leader sequence is shown in SEQ ID NO: 3; its hinge The amino acid sequence of the region is shown in SEQ ID NO: 62; the amino acid sequence of its transmembrane domain is shown in SEQ ID NO: 66; the amino acid sequence of its costimulatory domain is shown in SEQ ID NO: 70; its CD3ζ cell The amino acid sequence of the inner signaling domain is shown in SEQ ID NO: 74.

For another example, the CAR described in this application may be CAR10H10-1, and the amino acid sequence of LCDR1-3 is shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively; the amino acid sequence of VL is SEQ ID NO: 44; the amino acid sequences of HCDR1-3 are shown in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23 respectively; the amino acid sequence of VH is shown in SEQ ID NO: 50; VH and VL The amino acid sequence of the connecting peptide is shown in SEQ ID NO: 54; the nucleotide sequence of its Kozak sequence is shown in SEQ ID NO: 1; the amino acid sequence of its leader sequence is shown in SEQ ID NO: 3; The amino acid sequence of the hinge region is shown in SEQ ID NO: 62; the amino acid sequence of its transmembrane domain is shown in SEQ ID NO: 66; the amino acid sequence of its costimulatory domain is shown in SEQ ID NO: 70; its CD3ζ The amino acid sequence of the intracellular signaling domain is shown in SEQ ID NO: 74.

For another example, the CAR described in this application may be CAR16F10, and the amino acid sequences of LCDR1-3 are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the amino acid sequence of VL is SEQ ID NO: 46; the amino acid sequence of HCDR1-3 is shown in SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34 respectively; the amino acid sequence of VH is shown in SEQ ID NO: 52; between VH and VL The amino acid sequence of the connecting peptide is shown in SEQ ID NO: 54; the nucleotide sequence of its Kozak sequence is shown in SEQ ID NO: 1; the amino acid sequence of its leader sequence is shown in SEQ ID NO: 3; its hinge region The amino acid sequence is shown in SEQ ID NO: 62; the amino acid sequence of its transmembrane domain is shown in SEQ ID NO: 66; the amino acid sequence of its costimulatory domain is shown in SEQ ID NO: 70; its CD3ζ cell The amino acid sequence of the inner signaling domain is shown in SEQ ID NO: 74.

Nucleic acid molecule, carrier, cell and preparation method

In another aspect, the present application provides isolated one or more nucleic acid molecules, which can encode the antibodies, antigen-binding fragments or variants described herein, and/or, encode the fusion protein. The isolated nucleic acid molecule encoding the antibody of the present application may comprise the nucleic acid sequence shown in SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59 or a functional variant thereof. The nucleic acid molecules described in this application may be isolated. For example, it can be produced or synthesized by: (i) amplified in vitro, such as polymerase chain reaction (PCR) amplification, (ii) cloned and recombinantly produced, (iii) purified , Such as by enzyme digestion and gel electrophoresis fractionation, or (iv) synthetic, for example by chemical synthesis. In certain embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.

On the other hand, the present application provides one or more vectors, which may contain one or more nucleic acid molecules described in the present application. In this application, the vector may be PXC17.4 or pCDNA3.4. For example, the PXC17.4 or pCDNA3.4 vector may include the nucleic acid sequence shown in SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59 or a functional variant thereof. In addition, the vector may also contain other genes, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. In addition, the vector may also contain expression control elements that allow the coding region to be correctly expressed in a suitable host. Such control elements are well known to those skilled in the art. For example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In some embodiments, the expression control sequence is an adjustable element. The specific structure of the expression control sequence may vary according to the function of the species or cell type, but usually contains 5'untranscribed sequences and 5'and 3'untranslated sequences involved in transcription and translation initiation, such as TATA box, Cap sequence, CAAT sequence, etc. For example, the 5' non-transcriptional expression control sequence may comprise a promoter region, and the promoter region may comprise a promoter sequence for transcriptional control of functionally linked nucleic acids. One or more nucleic acid molecules described herein can be operably linked to the expression control element. The vector may include, for example, a plasmid, cosmid, virus, bacteriophage, or other vectors commonly used in, for example, genetic engineering.

In another aspect, the present application provides one or more cells, which may comprise one or more nucleic acid molecules described herein or one or more vectors described herein. In the present application, the cells may be selected from PBMC, CD4, CD8, NK and other cells. In some embodiments, each or each cell may contain one or one vector described herein. In some embodiments, each or each cell may contain multiple (eg, 2 or more) or multiple (eg, 2 or more) vectors described herein. In the present application, the vector can be introduced into immune effector cells. The vector described in the present application can be introduced into the cells by methods known in the art. In this application, the vector described in this application can be introduced into the cells by methods known in the art, such as electroporation, lipofectamine transfection (lipofectamine 2000, Invitrogen), and the like. For example, electrical transduction can be performed by Bio-Rad's electrical transduction tool. For another example, transduction can be performed by Gibco's transfection kit.

On the other hand, the present application provides a method for preparing the antibody, antigen-binding fragment or variant thereof, and/or the fusion protein described in the present application, the method may include An antigen-binding fragment or variant thereof, and/or the fusion protein described herein under the conditions for expressing the cells described herein may further comprise harvesting the antibody, antigen-binding fragment or variant thereof, and/or , The fusion protein.

Pharmaceutical composition, pharmaceutical use

On the other hand, the present application provides a pharmaceutical composition, which may comprise the antibody described herein, the antigen-binding fragment or variant thereof, the nucleic acid molecule described herein, the carrier described herein, or the The cells and/or fusion proteins described herein, and optionally a pharmaceutically acceptable adjuvant. The pharmaceutically acceptable adjuvants may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes and/or nonionic surfaces Active agent, etc. In the present application, the pharmaceutical composition may be formulated for oral administration, intravenous administration (eg, intravenous injection, IV), intramuscular administration (eg, intramuscular injection, IM), the original in the tumor site Site administration, inhalation, rectal administration, vaginal administration, transdermal administration (eg, subcutaneous injection, IC) or administration via subcutaneous depot.

On the other hand, the present application provides the antibody, antigen-binding fragment or variant thereof, and/or the use of the fusion protein in the preparation of a medicament for preventing or treating a disease or disorder, wherein the medicament is used for Treatment of tumors and autoimmune diseases.

On the other hand, the present application provides the antibody, antigen-binding fragment or variant thereof, and/or the fusion protein, which treats tumors and autoimmune diseases.

In another aspect, the present application provides a method for treating tumors and autoimmune diseases, comprising administering the antibody, antigen-binding fragment or variant thereof, and/or the fusion protein to a patient.

In certain embodiments, the tumor is selected from any one of the group consisting of B-cell subtype non-Hodgkin's malignant lymphoma (NHL), Burkitt's lymphoma, multiple myeloma, pre-B Acute lymphoblastic leukemia and other malignant lung tumors derived from early B-cell precursors, common acute lymphocytic leukemia, T chronic lymphocytic leukemia, hairy cell leukemia, non-acute lymphoblastic leukemia, Fahrenheit macroglobulin blood Disease, prolymphocytic leukemia, plasmacytoma, osteosclerotic myeloma, plasmacytic leukemia, monoclonal gammopathy (MGUS), smoldering multiple myeloma (SMM), chronic multiple myeloma (IMM) ), Hodgkin's malignant lymphoma, gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, prostate cancer, cervical cancer, adrenal tumor and bladder Tumor. In certain embodiments, the autoimmune disease is selected from any one of the group consisting of rheumatoid arthritis, multiple sclerosis, and CD19 ⁺ leukemia.

On the other hand, the present application provides a method for inhibiting the biological activity of CD19 protein, which comprises administration of the antibody described herein, antigen-binding fragment or variant thereof, the nucleic acid molecule described herein, the The carrier, the cell described in this application and/or the fusion protein described in this application.

On the other hand, the present application provides the antibody, antigen-binding fragment or variant thereof, the nucleic acid molecule described herein, the vector described herein, the cell described herein, and/or the The application of the fusion protein described above in the preparation of reagents for diagnosis or detection of tumors. In some embodiments, the reagent for diagnosing or detecting tumors can specifically recognize CD19 protein and/or cancer cells bearing CD19 protein. In some embodiments, the reagents for diagnosing or detecting tumors can be diagnosed in vitro by methods such as enzyme-linked immunoassay, chemiluminescence, and immunoturbidimetry. For example, the detection can be performed by enzyme-linked immunosorbent assay, using the tumor marker CD19 protein as a stationary phase, and competing with the sample to be tested for the antibody described in this application. In some embodiments, the reagent for diagnosing or detecting tumors can react with a substrate of horseradish peroxidase to develop a color for diagnosis of tumors.

Without intending to be bound by any theory, the following embodiments are only for explaining the working manner of the device, method and system of the present application, and are not intended to limit the scope of the invention of the present application.

Examples

Example 1 Preparation of CD19 antibody

1.1 Immunization of experimental animals

For the production of CD19 antibodies, six weeks old Babl/c female mice (Hunan Slake Biotechnology Co., Ltd.) were immunized with purified recombinant CD19 protein (ACRO, CD9-H52H2) and CD19 ⁺ Ramos cells (ATCC). The mice used were SPF Grade (specfic pathogen free). Mice can be immunized by subcutaneous immunization with recombinant protein and intraperitoneal injection of Ramos cells (purchased from ATCC). The mice are six weeks old after the first antigen infusion. For the first immunization, the recombinant CD19 protein and Freund's complete adjuvant (purchased from Sigma) were used to immunize them at a ratio of v:v = 1:1. After subcutaneous immunization of the extremities, 50 μg each was used. One week later, a second immunization was performed, using recombinant CD19 protein and Freund's incomplete adjuvant (purchased from Sigma) at a ratio of v:v = 1:1, and then subcutaneously immunized extremities, each 50 μg. A week later, a third immunization was performed, and Ramos cells (purchased from ATCC) were used for intraperitoneal immunization. Ramos cells were collected, washed twice with serum-free RPMI-1640 medium (purchased from Sigma), and the cell density was diluted to 10 ⁷ cells/mL. Each mouse was then injected subcutaneously with 1 mL of Ramos cell solution for immunization. A week later, the fourth immunization was performed, the immunization was performed with the Ramos cell solution, and 1 mL of Ramos cell solution was injected subcutaneously into each mouse for immunization. One week after the fourth immunization, the eyeball blood was taken and the serum antibody titer was detected by ELISA. If the antibody dilution is not less than 10 ⁵ , the immunization is completed; if the antibody dilution is greater than 10 ⁵ , the above-mentioned recombinant CD19 protein and Freund’s Complete adjuvant (purchased from Sigma) was mixed thoroughly in the ratio of v:v=1:1, and then subcutaneous immunization of the extremities was performed, each 50 μg, and then tested again until the immunization was completed.

1.2 Fusion and selection of hybridoma cells

To screen Babl/c immunized mice that produce antibodies that specifically bind to CD19, the sera of the immunized mice were tested by ELISA as described by Fishwild et al. (1996). In short, the mice whose serum antibody dilution reached more than 10 ⁵ after immunization with recombinant CD19 protein in Example 1.2 were taken, and their spleen was removed after being sacrificed by neck removal. The spleen was crushed and separated using a 70 μm sieve (corning) to obtain a single For B cells, the B cells and SP2/0 cells were mixed at a ratio of 1:10, and the B cells and SP2/0 cells were fused into hybridoma cells by means of PEG (purchased from Sigma). The fused cells were dispensed into 96-well cell culture plates in an amount of 200 μL per well, for a total of 30 96-well cell culture plates. The 96-well cell culture plate was cultured in a 5% CO ₂ cell incubator (Thermo 150i) at 37°C, and then a fusion cell line secreting highly active antibodies was screened by ELISA as described by Fishwild et al. (1996).

1ng/μL of purified recombinant CD19 dissolved in PBS was coated on an ELISA detection plate (purchased from Xiamen Yijiamei), 100μL per well. After incubating at 37°C for 2 hours, it was then blocked with 5% chicken serum dissolved in PBS/Tween (0.05%), 280 μL per well, and then incubated at 37°C for 2 hours before use. 100 μL of supernatant from hybridoma fusion cells was added to each well, and then incubated at 37° C. ambient temperature for 30 minutes. The plate was washed 5 times with PBS/Tween, and 100 μL of goat-anti-human IgG polyclonal antibody conjugated with horseradish peroxidase (HRP) was added to each well and incubated at 37°C for 30 minutes. After washing 5 times with PBS/Tween, 100 μL of TMB color developing solution (Shandong Zibo Yunqiao Biotechnology Co., Ltd.) was added to each well. After incubation at 37° C. for 5 minutes, 50 μL of 2M sulfuric acid was added to stop the reaction, and passed through a microplate reader ( Wavelength 450nm) Read the OD value, take the hybridoma fusion cell line with an OD value higher than 1.0, and select the monoclonal cell line that can specifically bind to CD19 by limiting dilution method (see Zhang Yue, Qu Huihua, Wu Tingting, Etc. Preparation and identification of monoclonal antibodies to glycyrrhizic acid[J]. Journal of Pharmaceutical Analysis, 2013, 33(5): 770-774.).

1.3 Anti-CD19 different epitope and affinity antibody screening

Three anti-CD19 murine antibodies with different affinities and binding epitopes were screened by hybridoma fusion technology and named 25C5-2, 10H10-1 and 16F10 respectively. Sequencing by Sanger high-throughput sequencing technology (see Tsiatis A, C, Norris-Kirby A, Rich R, G, and others. .The Journal of Molecular Diagnostics,2010,12(4):425-432.) Obtained the nucleotide sequences of the heavy chain and light chain variable regions of three antibodies, of which the VL amino acid sequence of 25C5-2 is as SEQ ID NO: 42, the VH amino acid sequence is shown in SEQ ID NO: 48; the 10H10-1 VL amino acid sequence is shown in SEQ ID NO: 44, the VH amino acid sequence is shown in SEQ ID NO: 50; 16F10 is the VL amino acid The sequence is shown in SEQ ID NO: 46, and the VH amino acid sequence is shown in SEQ ID NO: 52.

1.4 Synthetic CD19 antibody in scFv form

The VL-VH connecting peptide between the VL and VH is artificially synthesized, and its nucleotide sequence is shown in SEQ ID NO:53. In addition, the nucleotide sequences of VL and VH of the three strains of antibodies that can specifically bind to CD19 obtained by sequencing in the previous step are artificially synthesized, and then connected first to construct the CD19 antibody in scFv format.

Specifically, to obtain the 25C5-2 antibody, first synthesize its VL, whose nucleotide sequence is shown in SEQ ID NO: 41; then synthesize the VL-VH linking peptide, whose nucleotide sequence is shown in SEQ ID NO: 53 Then, its VH is synthesized, and its nucleotide sequence is shown in SEQ ID NO: 47. The nucleotide sequences of VL, VL-VH linking peptide and VL are connected end to end to obtain the complete 25C5-2scFv sequence. The nucleotide sequence is shown in SEQ ID NO: 55.

Similarly, to obtain the 10H10-1 antibody, first synthesize its VL whose nucleotide sequence is shown in SEQ ID NO: 43; then synthesize the VL-VH connecting peptide whose nucleotide sequence is shown in SEQ ID NO: 53 ; Then synthesize its VH, and its nucleotide sequence is shown as SEQ ID NO:49. The nucleotide sequences of VL, VL-VH connecting peptide and VH are connected end to end to obtain the complete 10H10-1scFv sequence. The nucleotide sequence is shown in SEQ ID NO: 57.

Similarly, to obtain the 16F10 antibody, first synthesize its VL, whose nucleotide sequence is shown in SEQ ID NO: 45; then synthesize the VL-VH connecting peptide, whose nucleotide sequence is shown in SEQ ID NO: 53; then Synthesize its VH, and its nucleotide sequence is shown in SEQ ID NO: 51. The nucleotide sequences of VL, VL-VH connecting peptide and VL are connected end to end to obtain the complete 16F10scFv sequence. The nucleotide sequence is shown in SEQ ID NO:59.

1.5 Vector construction and antibody expression

The scFv gene synthesized in the previous step was amplified by E. coli to extract the plasmid, digested with BamHI and SaiL, and then ligated into the expression vector PXC17.4, etc., electroporation was performed using Bio-Rad's electrical transduction tool, and the expression vector was introduced into CHO The cells are expressed, and then the expression of the antibody is detected.

Example 2 CD19 antibody activity test (ELISA)

The CD19 antibody prepared in Example 1 was detected by ELISA. Take 100 μg of CD19 protein (purchased from acro), add 300 μL of ultrapure water (purified water meter purchased from PALL) to fully dissolve, and obtain a CD19 protein dilution with a concentration of 0.33 μg/μL. A 96-well test plate was coated with 100 ng per well.

Dilute CD19 protein to 1μg/μL with PBS buffer solution and take 40mL; and take 120μL of CD19 protein dissolved in ultrapure water above to 40mL of PBS buffer solution, mix the two thoroughly and follow the amount of 100μL per well Add to 96-well test plate and incubate at 37°C for 2 hours. After the incubation was completed, the remaining protein solution in the 96-well plate was removed, and 280 L (1% BSA + PBS) blocking solution was added to each well. After incubation at 37C for 2 hours, the solution was stored at 4C for later use.

The antibodies 16F10, 25C5-2, and 10H10-1 prepared in Example 1 were used for detection, and FMC-63 (Merck, selection MAB1794) was used as a positive control (all anti-CD19 positive controls used in all examples of this application were It is FMC-63. For the scfv nucleotide sequence, please refer to CN201180067173.X SEQ ID NO.14, and the amino acid sequence is SEQ ID NO.20), without adding primary antibody or secondary antibody as a negative control.

Dilute the antibody to be tested with PBS to a concentration of 0.1 mg/mL for later use. Add 100 μL of PBS to the detection plate according to the method of multiple dilution. Add 100 μL of the diluted 0.1 mg/mL antibody solution to the first detection well, mix thoroughly, and then take 100 μL to the next gradient in a similar manner. Perform 11 gradient dilutions similarly, repeating each sample once. The diluted test plate was incubated at 37°C for 30 minutes.

After the incubation, the plate was washed 5 times, 280 μL each time. After the plate was washed, the plate was gently patted several times to remove the residual washing solution. Then, add the prepared goat anti-human secondary antibody dilution solution (the goat anti-human secondary antibody was purchased from abcam and diluted with PBS at a ratio of 1:10000) according to the amount of 100 μL per well. After incubating at 37°C for 30 minutes, the plate was washed . After washing the plate, add TMB color developing solution according to 100 μL of each well, incubate at 37°C for 5 minutes and add 2M sulfuric acid stop solution according to the amount of 50 μL per well to stop the reaction. Set the MD-M2E (Molecular Instrument Company of America) microplate reader to read the OD value at 450nm, and use the software MD-M2E for data processing. The results are shown in Figure 1. The results show that 10H10-1 has the strongest affinity for CD19 protein, and it still has strong binding capacity even at low concentrations. The detection results of 16F10 and 25C5-2 are similar.

Example 3 CD19 antibody activity test

3.1 Cell processing

Collect NALM-6 cells in a 50mL centrifuge tube, centrifuge at 400g for 5 minutes to collect the cells, take 20mL of PBS pre-cooled to 4°C to resuspend the cells, centrifuge to collect the cells, wash the cells with PBS three times according to this method, use 13mL After resuspending the cells in PBS and counting, the cell concentration was 3.92×10 ⁶ cells/mL, and the cell survival rate was 98%. Dispense 600μL of each cell into a flow detection tube for a total of 25 cells.

3.2 CD19 antibody dilution

The CD19 antibody was diluted with PBS according to the following gradient. The specific operation is as follows: take the antibodies 16F10, 10H10-1 and 25C5-2 prepared in Example 1 and dilute them to 50 μg/mL, 10 μg/mL, 5 μg/mL, 2.5 μg/mL and 1.25 μg/mL respectively. 200 μL was added to the cell pellet collected by centrifugation to resuspend the cells. After reacting at 4° C. for 1 hour, the cells were collected by centrifugation and washed three times with PBS. After the reaction was completed, centrifuge at 4°C for 5 min at a speed of 400g, collect the cells, add 1mL of PBS pre-cooled to 4°C and centrifuge and wash twice according to the above centrifugation conditions, then collect the cell pellet and discard the supernatant.

3.3 Secondary antibody incubation

After FITC-labeled goat anti-mouse antibody (Abcam Anti-Mouse IgG H&L (FITC) ab6785) was diluted at a ratio of 1:300 (PBS dilution), 500 μL of FITC-labeled goat anti-mouse was added to each detection tube Resuspend the cells in the antibody dilution, and add 500μL LITC-labeled goat anti-mouse antibody dilution to the FITC-labeled goat anti-mouse antibody control group. Add 500 μL of PBS to the negative control group. After incubating at 4°C for 1 hour, wash the cells three times with PBS as described above Finally, resuspend and wash the centrifuged cells with 200 μL PBS for flow cytometry.

3.4 Flow detection

First, resuspend the cells obtained in the previous step in PBS buffer, add the CD19 antibodies 16F10, 10H10-1, 25C5-2 obtained in Example 1 without adding primary and secondary antibodies as negative controls.

The flow detection voltage is set according to the following steps: After a sufficient amount of sheath liquid is filled in the flow sheath liquid barrel, the flow cytometer (BD verse) is preheated for 20 minutes, the flow detection software is opened, and the FSC/ adhesion to the SSC, FSC and SSC detection Videos in FIG appropriate detection gate, the amount of the detection cell 10 set the detection threshold of ^4, FSC-H and then draw a second detector detecting FIG FIGS-a FSC, defined The detection cells on the second detection chart come from P1 gate, and set the appropriate P2 gate on the second detection chart, set the third detection chart COUNT and FITC-A, define the detection data of the third picture from P2 gate After the related test chart is drawn, set the detection voltage with the control NALM-6 cells so that the NALM-6 cells are displayed at the center of the first test chart. After the FITC-A detection signal is between 10 ^{1 and} 10 ² , start Test NALM-6 cells, collect data and set up a control group, save the test voltage and related parameters, and then start the test sample test, and record the related data. The results of 16F10, 10H10-1, 25C5-2 antibody concentration of 5 μg/mL and FMC63 concentration of 0.025 μg/mL are plotted. The results are shown in FIG. 2.

The results showed that 16F10, 25C5-2, and 10H10-1 all had similar binding ability to CD19 expressed on the surface of tumor cells NALM-6, of which 16F10 had the strongest binding ability, followed by 10H10-1 and 25C5-2.

Example 4 CD19 antibody Western blot detection

Take a concentration of 1 mg/mL CD19 extracellular domain protein (purchased from abcam, ab234966, amino acid sequence number 20 to 291) 10 μL, and 10mL 2× loading buffer (which contains 20mM DTT), and mix well, Boil in a water bath at 95°C for 5 minutes. Then perform the SDS-PAGE electrophoresis experiment (containing 12% SDS) according to the conventional operations in the prior art, and perform the electrophoresis test on the CD19 antibodies 10H10-1 and 25C5-1 obtained in Example 1, without adding primary antibody and secondary antibody Antibody was used as a negative control. Marker used in electrophoresis was purchased from full-type gold, catalog number: DM201, lot: K51027, and 5 μL of each sample was added. During electrophoresis. Use concentrated gel (5% concentrated gel, 2mL, purified water 1.4mL, 30% Acr-Bis 0.33mL, 1M pH8.8 Tris 0.25mL, 12% SDS (sodium dodecyl sulfonate) 0.02mL, 10% Ammonium persulfate 0.02ml, TEMED (tetramethyldiethylamine) 0.002mL) was concentrated, the voltage was 100V during concentration; use separation gel (10% concentrated gel, 5mL, purified water 1.3mL, 30% Acr-Bis 1.7mL, 1M pH8.8Tris 1.9mL, 12% SDS (sodium dodecyl sulfonate) 0.05mL, 10% ammonium persulfate 0.05mL, TEMED (tetramethyldiethylamine) 0.002mL) for separation The voltage is 140V. Stop electrophoresis when bromophenol blue reaches the bottom of the gel.

The film transfer was performed at a constant voltage of 100V, and the film transfer time was 45 minutes. The polyvinylidene fluoride (PVDF) membrane was placed in a 5% skimmed milk powder solution (PBST solution configuration) for 1 hour, washed 3 times with PBST solution, and washed slowly on a side swing shaker for about 5 minutes each time. After blocking, the CD19 antibodies 10H10-1, 25C5-1 and the positive control FMC-63 obtained in Example 1 were diluted with PBST to a concentration of 20 μg/mL and reacted with 5% skim milk-blocked PVDF membrane for 1 hour at room temperature. Then wash with PBST three times, each time on the side shaker and shake slowly for about 5 minutes. After the PBST washing is completed, a HRP-labeled goat anti-mouse secondary antibody diluted with 1:5000PBST is added, and after 45 minutes of reaction at room temperature, the reaction is washed three times with PBST after the reaction is completed, each washing for about 5 minutes. ECL luminescent solution (purchased from BIO-RAD) was added to take photos to detect the protein, and the exposure time was 1 second during the collection.

The results are shown in Figure 3. The results show that 10H10-1 can react with the denatured CD19 recombinant protein, and the positive controls FMC63 and 25C5-2 do not react with the denatured CD19 recombinant protein.

Example 5 CD19 scfv antibody flow cytometry

5.1 Cell processing

Take the same NALM-6 cells as in Example 3, collect NALM-6 cells into a 15ml centrifuge tube, centrifuge at 400g for 5 minutes to collect the cells, discard the supernatant, and use the same volume as the discarded supernatant The cells collected by centrifugation were resuspended in PBS buffer solution, and after 3 repetitions, the cells were resuspended with 10 mL of PBS buffer solution. Take 20μl of the cell suspension after washing with PBS and add it to a sterile 1.5ml centrifuge tube. Then add equal volume of trypan blue and mix gently. Take 20μL and add it to the Count Sart cell counting plate. Place the counting plate in the Count Sart In the cell counter, click the cell count to read the data. After counting, the cell concentration was 5.37×10 ⁵ /mL. The washed cell suspension was then divided into flow detection tubes, 1 mL per tube. Reserve a tube as a negative control.

5.2 CD19 scfv antibody dilution

Dilute the CD19 antibodies 10H10-1 and 25C5-2 to be tested to a concentration of 200 μg/mL in PBS, and then dilute them at the ratios of 1:20, 1:80, 1:320, 1:1280, and 1:5120 respectively. To 10μg/mL, 2.5μg/mL, 0.625μg/mL, 0.312μg/mL, 0.156μg/mL, and then added to 1mL NALM-6 cell suspension, mixed thoroughly, and reacted at 4℃ for 2 hours. The cells were collected by centrifugation at 4°C for 5 minutes at a speed of 400g, and 1mL of PBS buffer solution pre-cooled to 4°C was added. Wash twice by centrifugation according to the above centrifugation conditions, then collect the cell pellet and discard the supernatant, add PE-labeled goat anti-human antibody diluted 1:1000 in advance to the cell pellet, and react at 4°C for 1 h. After the reaction was completed, the cells were centrifuged at 4°C for 5 min at 400g to collect the cells, added with 1mL of PBS buffer solution pre-cooled to 4°C, centrifuged and washed twice according to the above centrifugation conditions, the cell pellet was collected, the supernatant was discarded, and finally 200μL of PBS was used The buffer solution was resuspended to wash the centrifuged cells for flow cytometry.

5.3 Flow detection

First, resuspend the cells obtained in the previous step in PBS buffer, add the scFv of the CD19 antibodies 10H10-1 and 25C5-2 obtained in Example 1, without adding primary and secondary antibodies as negative controls.

The flow detection voltage is set according to the following steps: After a sufficient amount of sheath liquid is filled in the flow sheath liquid barrel, the flow cytometer (BD verse) is preheated for 20 minutes, the flow detection software is opened, and the FSC/ adhesion to the SSC, FSC and SSC detection Videos in FIG appropriate detection gate, the amount of the detection cell 10 set the detection threshold of ^4, FSC-H and then draw a second detector detecting FIG FIGS-a FSC, defined The detection cells of the second detection chart come from P1 gate, and set the appropriate P2 gate on the second detection chart, set the third detection chart COUNT and FITC-A, define the detection data of the third picture from P2 gate, After the relevant detection chart is drawn, set the detection voltage with the control NALM-6 cells so that the NALM-6 cells are displayed in the center of the first detection chart. After the FITC-A detection signal is between 10-10 ² , start to detect NALM -6 cells, collect data to set up a control group, save the test voltage and related parameters, and then start the test sample test, and record the relevant data.

The results are shown in Figure 4. 25C5-2 and 10H10-1 have obvious ability to bind CD19 positive cells. Among them, 10H10-1 has the strongest binding ability, followed by 25C5-2.

Example 6 Anti-CD19CAR-T construction and positive rate detection

6.1 Synthesis of anti-CD19CAR structure

The obtained anti-CD19scfv and different functional regions form a CAR structure and then the CAR structure is obtained by gene synthesis (the schematic diagram of the CAR structure is shown in Figure 5)

Among them, the nucleotide sequence of the leader is shown in SEQ ID NO. 2; the nucleotide sequence of the hinge region (Hinge) is shown in SEQ ID NO. 61, and the nucleotide sequence of the transmembrane structure (TM) is shown in SEQ ID NO.65, 4-1BB nucleotide sequence is shown in SEQ ID NO.69, OX40 nucleotide sequence is shown in SEQ ID NO.71, CD3zeta nucleotide sequence is shown in SEQ ID NO.73 As shown. The anti-CD19scfv obtained through screening in this application was constructed with 10H10-1 (SEQ ID NO.57) and 25C5-2 (SEQ ID NO.55) as examples, and the positive drug CAR-T (anti-CD19scfv (FMC-63 (Merck))

Synthetic genes are amplified by the large intestine, and plasmids are extracted. They are digested with BamHI and SaiL and then ligated into expression vectors. The plasmids are amplified by stabl3 to prepare plasmids for constructing CAR-T cells

6.2 T cell isolation activation

Separate and collect PBMC cells in whole blood by Ficoll reagent method, separate CD4+ and CD8+ T cells by magnetic bead negative selection method, add activated magnetic beads CD3 to the separated T cells at a ratio of 1:1 (number ratio) /CD28, T cells are activated for 24 hours for electro-transfer test.

6.3 Construction of CAR-T cells

The constructed anti-CD19CAR-T expression vector was prepared according to the amount of 3.3μg corresponding to 4×10 ⁶ T cells. The electrotransformation conditions were followed to collect T cells after electroporation according to the program set together, and the CAR-T cell positive rate and CAR- were detected in 24 hours. T cells kill vitality.

6.4 Positive rate of CAR-T cells by flow cytometry

Cultures were harvested electroporation T cells after 24 hours, washed with PBS 3 times in accordance with an amount of 10 ⁶ cells one aliquoted into different flow detection cuvette, and 10 ⁶ cells were added to 2μLFITC labeled CD19 protein 4 ℃ tending 2 After 3 hours, wash with PBS three times, resuspend the cells with 300 μL of PBS, and then check the positive rate of CAR-T cells by flow cytometry. The results are shown in Figure 6.

Example 7 Anti-CD19CAR-T tumor killing activity detection

7.1 Target cell labeling

Collect NALM-6 in the logarithmic growth phase and wash the cells once with 0.01 MPBS. The cells were resuspended in a complete medium to a density of 1×10 ⁶ cell/mL suspension. Take 3 mL of the cell suspension, add 5 μL of BATDA and mix well, and incubate at 37° C. under 5% CO ₂ for 5 min.

The labeled NALM-6 cells were washed 4 times with washing solution (1×PBS+20 mM Hepes). After the final wash, resuspend the cells in complete medium and adjust the cell density to 8×10 ⁴ cell/mL.

2. Co-incubation of target cells

After the above 1) target cells are resuspended, an appropriate amount of cell suspension is immediately centrifuged, and the supernatant is taken as a background test well.

Take 100 μL (8000cell/well) of the cell suspension of 1) and inoculate in 96well V-bottom plate. 100μL complete medium solution containing 1% TritonX-100 was added to the maximum release well, 100μL complete medium solution was added to the spontaneous release well, and 100μL effector cells were added to the test well (efficiency target ratio 1:1).

After incubating for 1-4h, centrifuge at 500g at room temperature for 5min.

3. Determination of cell killing activity

Take 20 μL of supernatant to a flat-bottom 96-well plate and add 200 μL of Europium Solution.

Shake at room temperature in the dark for 15 minutes.

The fluorescence value was measured by HTRF method (excitation light 340 nm, emission light 615 nm).

4. Data processing

Calculate the cell killing rate (%) of the tested cells as follows:

Cell killing rate (%) = (Test well reading-Spontaneous release well reading) / (Maximum release well reading-Spontaneous release well reading) × 100

5. As shown in the results in Table 1, CAR-T constructed with 25C5 as an example has stronger killing activity than the positive control.

Table 1 CD19-CAR-T to NALM-6 kill rate (%, (Mean ± SEM, n = 3)

The foregoing detailed description is provided by way of explanation and example, and is not intended to limit the scope of the appended claims. Various changes to the embodiments listed in the present application will be obvious to those of ordinary skill in the art, and remain within the scope of the appended claims and equivalents thereof.

Claims

The antibody, its antigen-binding fragment or variant, binds to the CD19 protein with an IC50 value of 3.5 μg/mL or lower.
The antibody, antigen-binding fragment or variant thereof according to claim 1, which is selected from any one of the group consisting of a monoclonal antibody, a single chain antibody, a chimeric antibody, a murine antibody and a humanized antibody.
The antibody, antigen-binding fragment or variant thereof according to any one of claims 1-2, the antigen-binding fragment is selected from the group consisting of: Fab, Fab', F(ab)2, F(ab')2, Fv And scFv.
The antibody, antigen-binding fragment or variant thereof according to any one of claims 1 to 3, said variant being selected from the group consisting of:

1) A protein or polypeptide in which one or more amino acids have been substituted, deleted, or added to the antibody or the antigen-binding fragment thereof; and

2) A protein or polypeptide having more than 90% sequence homology with the antibody or the antigen-binding fragment thereof.
An antibody, an antigen-binding fragment or variant thereof, which competes with a reference antibody for binding to the CD19 protein, wherein the reference antibody includes a light chain variable region and a heavy chain variable region, and the light chain of the reference antibody may The variable region contains LCDR1-3, and the amino acid sequence of LCDR1 is selected from the amino acid sequences shown in any of the following groups: SEQ ID NO: 4 and SEQ ID NO: 18; the amino acid sequence of LCDR2 is selected from the group The amino acid sequence shown in any one: SEQ ID NO: 5 and SEQ ID NO: 19; and the amino acid sequence of the LCDR3 is selected from the amino acid sequence shown in any one of the following group: SEQ ID NO: 6 and SEQ ID NO:20; and,

The heavy chain variable region of the reference antibody comprises HCDR1-3, and the amino acid sequence of the HCDR1 is selected from the amino acid sequences shown in any of the following groups: SEQ ID NO: 7, SEQ ID NO: 21 and SEQ ID NO: 32; the amino acid sequence of the HCDR2 is selected from the amino acid sequences shown in any of the following groups: SEQ ID NO: 8, SEQ ID NO: 22 and SEQ ID NO: 33; and the amino acid sequence selection of the HCDR3 The amino acid sequence shown in any one of the following group: SEQ ID NO: 9, SEQ ID NO: 23 and SEQ ID NO: 34.
The antibody, antigen-binding fragment or variant thereof according to claim 5, wherein the amino acid sequence of the light chain variable region of the reference antibody is selected from the amino acid sequence shown in any one of the following group: SEQ ID NO: 42. SEQ ID NO: 44 and SEQ ID NO: 46, and the amino acid sequence of the heavy chain variable region of the reference antibody is selected from the amino acid sequences shown in any of the following groups: SEQ ID NO: 48, SEQ ID NO: 50 and SEQ ID NO: 52.
An antibody, an antigen-binding fragment or variant thereof, includes LCDR1, and the LCDR1 includes an amino acid sequence selected from any one of the following group: SEQ ID NO: 4 and SEQ ID NO: 18.
The antibody, antigen-binding fragment or variant thereof according to claim 7, which comprises LCDR2, and the LCDR2 comprises an amino acid sequence selected from any one of the group consisting of SEQ ID NO: 5 and SEQ ID NO: 19.
The antibody, antigen-binding fragment or variant thereof according to claim 7 or 8, which comprises LCDR3, and the LCDR3 comprises an amino acid sequence selected from any one of the group consisting of SEQ ID NO: 6 and SEQ ID NO:20.
The antibody, antigen-binding fragment or variant thereof according to any one of claims 7-9, which comprises a light chain variable region VL, and the light chain variable region VL comprises any one selected from the group consisting of The shown amino acid sequence: SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO: 46.
The antibody, antigen-binding fragment or variant thereof according to any one of claims 7-10, which comprises HCDR1, and the HCDR1 comprises an amino acid sequence selected from any one of the following group: SEQ ID NO: 7 , SEQ ID NO: 21, SEQ ID NO: 32.
The antibody, antigen-binding fragment or variant thereof according to any one of claims 7 to 11, which comprises HCDR2, and the HCDR2 comprises an amino acid sequence selected from any one of the following group: SEQ ID NO: 8 , SEQ ID NO: 22 and SEQ ID NO: 33.
The antibody, antigen-binding fragment or variant thereof according to any one of claims 7-12, which comprises HCDR3, and the HCDR3 comprises an amino acid sequence selected from any one of the group consisting of SEQ ID NO: 9 , SEQ ID NO: 23 and SEQ ID NO: 34.
The antibody, antigen-binding fragment or variant thereof according to any one of claims 7-13, which comprises a heavy chain variable region VH, and the heavy chain variable region VH comprises any one selected from the group consisting of The amino acid sequence shown: SEQ ID NO: 48, SEQ ID NO: 50 and SEQ ID NO: 52.
The antibody, antigen-binding fragment or variant thereof according to any one of claims 1-14, wherein the antibody or antigen-binding fragment thereof is scFv.
The antibody, antigen-binding fragment or variant thereof according to claim 15, wherein the scFv comprises an amino acid sequence selected from any one of the group consisting of SEQ ID NO: 56, SEQ ID NO: 58 and SEQ ID NO :60.
The antibody, antigen-binding fragment or variant thereof according to any one of claims 1-16, wherein the CD19 protein is human CD19 protein.
A fusion protein comprising the antibody according to any one of claims 1-17, an antigen-binding fragment or a variant thereof.
The fusion protein according to claim 18, wherein the fusion protein is a chimeric antigen receptor.
The fusion protein of claim 19, wherein the chimeric antigen receptor comprises an extracellular hinge region, a transmembrane domain, a costimulatory domain, and a CD3ζ intracellular signal transduction domain.
The fusion protein of claim 20, wherein the extracellular hinge region is selected from a CD8 hinge or an IgG4 hinge.
The fusion protein according to claim 20 or 21, wherein the transmembrane domain is selected from α, β or ζ chain of T cell receptor, CD28, CD3e, CD45, CD4, CD5, CD8, CD9, CD16, CD22 , CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
The fusion protein of claim 22, wherein the transmembrane domain is selected from CD8-1 or CD8-2.
The fusion protein according to any one of claims 20-23, wherein the costimulatory domain is selected from CD27, CD28, 4-1BB, OX40 or ICOS.
The fusion protein according to any one of claims 20-24, wherein the chimeric antigen receptor, the extracellular hinge region is selected from the CD8 hinge or IgG4 hinge; the transmembrane domain is selected from CD8-1 Or CD8-2; the costimulatory domain is selected from 4-1BB or OX40.
The fusion protein of claim 25, wherein the chimeric antigen receptor, the N-terminus of the CD8-1 transmembrane is connected to the C-terminus of the CD8 hinge, and the C-terminus of the CD8-1 transmembrane It is connected to the N terminal of the 4-1BB, the C terminal of the 4-1BB is connected to the N terminal of the OX40, and the C terminal of the OX40 is connected to the N terminal of the CD3ζ.
The fusion protein of claim 25, wherein the chimeric antigen receptor, the N-terminus of the CD8-2 transmembrane is connected to the C-terminus of the IgG4 hinge, and the C-terminus of the CD8-2 transmembrane It is connected to the N terminal of the 4-1BB, the C terminal of the 4-1BB is connected to the N terminal of the OX40, and the C terminal of the OX40 is connected to the N terminal of the CD3ζ.
An isolated nucleic acid molecule or molecules encoding the antibody, antigen-binding fragment or variant of any one of claims 1-17, and/or encoding the antibody of any one of claims 18-27 The fusion protein mentioned.
One or more vectors comprising one or more nucleic acid molecules according to claim 28.
One or more cells comprising one or more nucleic acid molecules according to claim 28 or one or more vectors according to claim 29.
A method for preparing the antibody according to any one of claims 1-17, an antigen-binding fragment or variant thereof, and/or the fusion protein according to any one of claims 18-28, the method comprising The antibody according to any one of claims 1-17, an antigen-binding fragment or variant thereof, and/or, under the conditions for expression of the fusion protein according to any one of claims 18-27, the culture according to claim 30 Cell.
The method of claim 31, comprising harvesting the antibody, antigen-binding fragment or variant thereof, and/or the fusion protein.
A pharmaceutical composition comprising the antibody according to any one of claims 1-17, an antigen-binding fragment or variant thereof, the fusion protein according to any one of claims 18-27, and the nucleic acid according to claim 28 Molecule, the carrier of claim 29, and/or, the cell of claim 30, and optionally a pharmaceutically acceptable adjuvant.
The antibody according to any one of claims 1-17, an antigen-binding fragment or variant thereof, and/or the fusion protein according to any one of claims 18-27 in the preparation of a medicament for preventing or treating a disease or disorder Use in.
The use according to claim 34, the disease or disorder is selected from any one of the group consisting of tumors and autoimmune diseases.
The use according to claim 35, the tumor is selected from any one of the group consisting of B-cell subtype non-Hodgkin's malignant lymphoma (NHL), Burkitt's lymphoma, multiple myeloma, Pre-B acute lymphoblastic leukemia and other malignant lung tumors derived from early B-cell precursors, common acute lymphocytic leukemia, T chronic lymphocytic leukemia, hairy cell leukemia, non-acute lymphoblastic leukemia, Fahrenheit Proteinemia, prolymphocytic leukemia, plasmacytoma, osteosclerotic myeloma, plasmacytic leukemia, monoclonal gammopathy (MGUS), smoldering multiple myeloma (SMM), chronic multiple myeloma (IMM), Hodgkin's malignant lymphoma, gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, prostate cancer, cervical cancer, adrenal tumors And bladder tumors.
The use according to claim 36, the autoimmune disease is selected from any one of the group consisting of rheumatoid arthritis, multiple sclerosis and CD19+ leukemia.
A method for inhibiting the biological activity of CD19 protein, which comprises administering the antibody according to any one of claims 1-17, an antigen-binding fragment or variant thereof, the fusion protein according to any one of claims 18-27, the right The nucleic acid molecule of claim 28, the vector of claim 29, and/or the cell of claim 30.
The antibody according to any one of claims 1-17, an antigen-binding fragment or variant thereof, the fusion protein according to any one of claims 18-27, the nucleic acid molecule according to claim 28, or The use of said carrier, and/or the cell of claim 30, in the preparation of reagents for diagnosis or detection of tumors.