CN114230673A

CN114230673A - Fusion protein and application thereof

Info

Publication number: CN114230673A
Application number: CN202111414759.0A
Authority: CN
Inventors: 李光超; 罗敏; 丁雯; 王学俊
Original assignee: Guangzhou Bio Gene Technology Co Ltd
Current assignee: Guangzhou Bio Gene Technology Co Ltd
Priority date: 2021-11-25
Filing date: 2021-11-25
Publication date: 2022-03-25

Abstract

The invention discloses a fusion protein, which comprises a chimeric antigen receptor targeting CD19, a linker element and a CXCR5 protein which are sequentially connected from an N end to a C end; the chimeric antigen receptor comprises a CD19 antibody, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain connected in series from the N-terminus to the C-terminus. The invention also discloses an isolated nucleic acid, and the fusion protein is obtained by expression of the isolated nucleic acid. The invention also discloses a vector containing the nucleic acid. The invention also discloses a T cell. The invention also discloses a T cell containing the T cell. The invention also discloses application of the T cell in preparing a medicament for treating B cell lymphoma.

Description

Fusion protein and application thereof

Technical Field

The invention relates to the field of biological medicine, and particularly relates to a fusion protein and application thereof.

Background

CD19 is a transmembrane glycoprotein playing an important regulatory role in the activation and proliferation process of B lymphocytes, and is widely present on the surface of over 95% of B cell malignancies, mainly including acute B-cell leukemia, chronic lymphocytic leukemia/small lymphocytic lymphoma, and B cell non-hodgkin lymphoma (diffuse large B-cell lymphoma DLBCL, primary mediastinal B-cell lymphoma PMBCL, and follicular lymphoma FL), while its expression in normal tissues is limited to the B lymphocyte lineage.

CD 19-targeted CAR-T cell therapy opens a new model for the treatment of refractory and relapsed B-cell malignancies by virtue of excellent therapeutic effects, and published data show that the complete remission rate of anti-CD19 CAR-T cell therapy refractory and relapsed B-ALL can be as high as 70% -90%.

In recent years, the anti-CD19 CAR-T therapy range has gradually expanded from acute and chronic B-cell leukemia to the treatment of less aggressive or indolent B-cell lymphoma diseases, such as B-NHL, marginal zone B lymphoma MZBL, and the like. However, similar therapeutic effects are not successfully replicated in these patients, such as the complete remission rate of anti-CD19 CAR-T for treating B-NHL is only 43% -59%, the effect of anti-CD19 CAR-T drugs in treating B-cell lymphoma is not expected, and the development of more effective CAR-T therapeutic products against B-cell lymphoma is urgently needed.

B-cell lymphomas are a type of lymphoid malignancies with high heterogeneity in tissue morphology, immunophenotype, genetic biology, clinical therapy, and prognosis. Unlike hematologic leukemias, B-cell lymphomas often form tumors in situ or at metastases and form a tumor microenvironment around them similar to solid tumors, inhibiting and blocking migration of CAR-T cells to the tumor site. Therefore, successful transfer of CAR-T cells to the site of lymphoma to allow better drug efficacy is a problem that needs to be addressed first when CAR-T is treating B-cell lymphoma.

Disclosure of Invention

Based on the above, there is a need for a fusion protein and an application thereof, which can transfer CAR-T cells to B cell lymphoma sites with high efficiency, so that the CAR-T cells can exert better drug efficacy.

The first purpose of the invention is to provide a fusion protein, which comprises a chimeric antigen receptor targeting CD19, a linker element and a CXCR5 protein which are sequentially connected from N end to C end;

the chimeric antigen receptor comprises a CD19 antibody, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain connected in series from the N-terminus to the C-terminus.

The second purpose of the invention is to provide an isolated nucleic acid, and the fusion protein is obtained by expression of the isolated nucleic acid.

The third object of the present invention is to provide a vector containing the nucleic acid.

The fourth object of the present invention is to provide a cell comprising the nucleic acid or the vector, or a cell membrane having the chimeric antigen receptor and the CXCR5 protein of the fusion protein expressed on the surface thereof.

The fifth object of the present invention is to provide a pharmaceutical composition comprising said cells.

The sixth purpose of the invention is to provide the application of the cell in preparing a medicine for treating B cell lymphoma.

The CD19 antibody specific targeting B cells and CXCL13 factor secretion characteristics of CXCL 5 specific tendency tumors are combined, the anti-CD19 CAR-T cells of the high expression chemokine receptor CXCR5 are obtained, the modified T cells can be guided to directionally migrate and enrich to and kill tumor cells towards B cell lymphoma areas of the chemokine CXCL13 high expression, and therefore the problem that the B cell lymphoma treatment effect of the existing anti-CD19 CAR-T treatment method is poor is hopefully solved, the tumors are thoroughly killed, and the CR rate is improved.

Drawings

FIG. 1 is a graph showing the results of fusion proteins according to an embodiment of the present invention;

FIG. 2 is a schematic diagram of an anti-CD 19-CAR plasmid according to an embodiment of the present invention;

FIG. 3 is a schematic representation of an anti-CD 19-CXCR5 CAR plasmid according to one embodiment of the present invention;

FIG. 4 is a schematic representation of an anti-CD 19-CD28T & I-CXCR5 CAR plasmid according to one embodiment of the present invention;

FIG. 5 shows the binding of anti CD19-scFv and CD19 antigen by ELISA detection according to example 1 of the present invention, wherein A and B are a line graph and a bar graph, respectively;

FIG. 6 is a graph showing the interaction results of non-target binding of the membrane protein array validation antibody according to example 2 of the present invention;

FIG. 7 is a graph showing the results that the anti-CD19 scFv of example 3 of the present invention specifically recognizes a CD19 positive cell line;

FIG. 8A is a schematic diagram of a cell migration experiment in example 4 of the present invention;

FIG. 8B is a graph of the migration of CAR Ts cells according to example 4 of the invention as a function of time at various concentrations of recombinant human CXCL13 chemotaxis;

FIG. 9A is a graph of the IFN- γ secretion profile of normal human cells killed by anti-CD 19-CXCR5-CAR T (BG-T19C) according to example 5 of the present invention;

FIG. 9B is a graph of IL-2 secretion from normal human cells killed by anti-CD 19-CXCR5-CAR T (BG-T19C) according to example 5 of the present invention;

FIG. 10A is a diagram showing that ELISA of example 6 of the present invention detects the ability of anti-CD 19-CXCR5-CAR T cells to kill tumor target cells K562-CD19 in vitro;

FIG. 10B is an ELISA assay of anti-CD 19-CXCR5-CAR T cells killing tumor target cells Raji in vitro force diagram of example 6 of the present invention;

FIG. 10C is a diagram of the ELISA assay of anti-CD 19-CXCR5-CAR T cells killing tumor target cells in vitro, CD19 negative cells, K562 cells, according to example 6 of the present invention;

FIG. 11 is a schematic diagram of the experimental design of an animal according to example 7 of the present invention;

FIG. 12 is a graph showing tumor growth in vivo after CAR-T treatment in tumor-bearing mice as in example 7 of the invention.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

As used herein, the terms "comprising," "including," and "comprising" are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.

The invention mimics the natural chemokine-receptor system in the human body, so that the CAR-T cells are provided with tumor-derived chemokine receptors, and the CAR-T cells are driven to directionally aggregate to lymphoma regions.

CXCR5, Burkitt's lymphoma receptor 1 (BLR-1), is a member of the chemokine CXC receptor family, distributed primarily in peripheral mature B cells. CXCR5 is an important molecule for homing B lymphocytes to the lymph node follicles and for promoting lymph follicle staging and lymph node development. CXCL13, also known as B-cell chemokine 1 (BLC 1), is a member of the CXC family of chemokines and is distributed mainly in the stomach, liver and lymph nodes as a ligand for CXCR 5. CXCL13 is normally secreted by stromal cells in the B cell region of secondary lymphoid tissues and can also be expressed on follicular dendritic cell membranes. The CXCL13/CXCR5 plays a role in playing a role in biological effect, participates in the process that the immature B lymphocytes in the humoral circulation home to lymph nodes, promotes the development and construction of lymph nodes and secondary lymph tissues, promotes the contact and differentiation of the B cells in the secondary lymph nodes and antigens into functional B cells, and further coordinates humoral immunity.

Meanwhile, CXCL13 and receptor CXCR5 are also highly expressed in tumor tissues such as liver cancer, prostate cancer, stomach cancer, pancreatic cancer, breast cancer, lymphoma, and the like. The Tanyu et al have detected the expression of CXCR5 and CXCL13 in 5 cases of primary central nervous system lymphoma tissues and 10 cases of normal brain tissues, and the results show that the positive expression rates of CXCR5 and CXCL13 in tumor specimens are both 100% (5/5), and the positive expression rates in normal brain tissues are both 0 (0/10). Widney et al and Hussain et al in experimental studies on non-Hodgkin lymphoma have shown that the expression of chemokine CXCL13 in serum is significantly related to non-Hodgkin lymphoma, and that chemokine receptors and the ligand CXCL13 thereof are both expressed in cell lines of the non-Hodgkin lymphoma, CXCR5/CXCL13 have potential biological significance in the non-Hodgkin lymphoma, and CXCL13 can be used as a potential biological marker for diagnosing the non-Hodgkin lymphoma. In the case of clinical treatment of diffuse large B-cell lymphoma, the studies of tanghaman's macro show that, compared with CXCR5 negative CD8+ T cells, CXCR5 positive CD8+ T cells are in closer contact with and interact with autologous tumor cells, and CXCR5+ CD8+ T cells are in more significant up-regulation of cytotoxic particle expression when acting with autologous tumor cells, so that the diffuse large B-cell lymphoma killing effect is better.

The invention combines the characteristics of anti-CD19 antibody specific targeting B cells and CXCL13 factor secretion of CXCL 5 specific tendency tumor, obtains anti-CD19 CAR-T cells with high expression CXCR5, and the modified T cells can directionally migrate and gather to a B cell lymphoma region with high expression CXCL13, so that the tumor can be better killed, thereby being expected to solve the problem that the B cell lymphoma treatment effect of the existing anti-CD19 CAR-T treatment method is poor, and improving the CR rate.

In a first aspect, embodiments of the invention provide a fusion protein comprising, connected in order from N-terminus to C-terminus, a chimeric antigen receptor targeting CD19, a linker element, and a CXCR5 protein.

In the present application, "Chimeric Antigen Receptor (CAR)" generally refers to a fusion protein comprising an extracellular domain capable of binding an Antigen and at least one intracellular domain. A CAR is a core component of a chimeric antigen receptor T cell (CAR-T), which can include an antigen (e.g., a tumor-specific antigen and/or a tumor-associated antigen) binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. CARs are engineered receptors, and any specific receptor can be implanted on immune effector cells, particularly T cells. In CAR, scFv fragments or VHH fragments of monoclonal antibodies that specifically recognize tumor antigens can be implanted on T cells or NK cells. The nucleic acid encoding the CAR can be introduced into a T cell, NK cell, or NKT cell using, for example, a retroviral vector. In this way, a large number of cancer-specific T cells, NK cells or NKT cells can be generated for adoptive cell transfer. In the present application, the CAR may be combined with a T cell receptor activating intracellular domain based on the antigen (e.g., CD19) specificity of the antibody. Genetically modified CAR-expressing T cells can specifically recognize and eliminate malignant cells that express a target antigen.

As used herein, a "region" or "domain" included in the chimeric antigen receptor refers to a region in a polypeptide that can fold into a particular structure independently of other regions. These "regions" or "domains" may be sequences of murine or other animal origin, such as human sequences.

In the present application, "antibody" generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a particular antigen. For example, an antibody may comprise an immunoglobulin of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any molecule comprising 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 (e.g., dabs), single chain antibodies (e.g., scFv), and antibody fragments that bind to an antigen (e.g., Fab', and (Fab)2 fragments). The term "antibody" also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof 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. The VH and VL regions can be further distinguished as hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with more conserved regions termed Framework Regions (FRs). Each VH and VL may be composed of three CDR and four FR regions arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from the amino terminus to the as-completed terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

In some embodiments, the amino acid sequence of the CD19 antibody is SEQ ID NO: 2, respectively.

In some embodiments, the amino acid sequence of the CXCR5 protein is SEQ ID NO: shown in fig. 8.

The main function of the "linker" or called "protein cleavage element" is to express 2 or more proteins on a vector, and the two proteins are not connected to form a fusion protein, but are separated to exert their respective functions, so that the above-mentioned element is inserted between the two proteins, and the ribosome translation is temporarily stopped, and the downstream protein is re-translated after crossing the sequence, which is similar to the "scissors" function.

Optionally, the linker element is selected from one of T2A, P2A, E2A, F2A, and IRES, or any combination of at least two of T2A, P2A, E2A, F2A, and IRESZ. Preferably T2A, and the amino acid sequence is SEQ ID NO: shown at 7.

In the present application, the term "transmembrane domain" generally refers to a sequence in a cell surface protein that spans the cell membrane. The transmembrane domain may be linked directly or indirectly to an intracellular signaling domain and serves to transmit a signal.

Optionally, the transmembrane domain comprises a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-l, TRAC, TRBC, CD3 epsilon, CD3 zeta, CTLA-4, LAG-3, CD5, ICOS, OX40, CD 2D, 2B4, CD244, epsilon Fc RI gamma, BTLA, CD30, GITR, HVEM, DAPI0, CD2, NKG2C, LIGHT, DAPI2, CD40L, TIMl, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD 39134, CD137, CD154 and SLAM.

Preferably derived from the transmembrane domain of the CD8 protein and having the amino acid sequence of SEQ ID NO: 4, respectively.

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

Optionally, the co-stimulatory domain comprises a co-stimulatory domain derived from one or more proteins from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc RI γ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligands for CD83, CD40, and MyD 88;

preferably 4-1BB, and the amino acid sequence is SEQ ID NO: 5, respectively.

In the present application, a "signaling domain" generally refers to a domain that is located inside a cell and is capable of transducing a signal. In the present application, the intracellular signaling domain may conduct a signal into a cell. Generally, a signaling domain is any contiguous stretch of amino acids that is used to direct a protein to a target.

Optionally, the intracellular signaling domain comprises an intracellular signaling domain derived from one or more proteins from the group consisting of CD3 ζ, CD3 γ, CD3E, CD79a, CD79b, FceRI γ, FceRI β, and FcyRIIa;

preferably from the intracellular signaling domain of the CD3 zeta protein. CD3 ζ can form a T cell receptor-CD 3 complex with T cell receptor subunits and CD3-gamma, -delta, and l-epsilon. CD3 ζ contains three ITAM motifs, the ITAM sequences mediating intracellular signaling activation of the TCR. The zeta chain is a protein tyrosine kinase substrate activated by receptor, and can be quickly subjected to tyrosine phosphorylation after a TCR receptor is combined with a polypeptide MHC complex to participate in the transduction of lymphocyte activation signals.

In some embodiments, the intracellular signaling domain amino acid sequence of the CD3 ζ protein is SEQ ID NO: and 6.

In this application, a "hinge region" generally refers to the junction region between an extracellular domain (e.g., a CD19 targeting moiety) and a transmembrane region.

In some embodiments, the chimeric antigen receptor further comprises a hinge region linked between the CD19 antibody and the transmembrane domain. The hinge region comprises one or more.

Preferably, the amino acid sequence of the hinge region is SEQ ID NO: 3, respectively.

In some embodiments, the CD19 antibody is further provided with a signal peptide at the N-terminus;

preferably, the amino acid sequence of the signal peptide is SEQ ID NO: 1 is shown.

It will be appreciated that the "regions" or "domains" or "functional fragments", or other polypeptides (e.g. linker and signal peptides) to which the invention relates may be selected from the exemplary sequences corresponding to each of them, for example SEQ ID NOs: 1-9, or a sequence that is at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical thereto, and that retains a substantially similar function of the corresponding desired function.

Substantially similar sequences also retain the desired activity of the polypeptide. Substitutions which are generally regarded as conservative substitutions are substitutions for one another in the aliphatic amino acids Ala, Val, Leu and Ile, for the hydroxyl residues Ser and Thr, for the acidic residues Asp and Glu, for the amide residues Asn and Gln, for the basic residues Lys and Arg and for the aromatic residues Phe, Tyr. Further, substantially similar sequences also include modified sequences that do not differ significantly from the desired function, such as phosphorylation, glycosylation, ubiquitination, and the like.

In a second aspect, embodiments of the invention provide an isolated nucleic acid that is expressed to obtain a fusion protein as described in any of the above embodiments.

In a third aspect, embodiments of the invention provide a vector comprising a nucleic acid as described in any one of the above embodiments.

The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). In some embodiments, the vectors of the invention comprise regulatory elements commonly used in genetic engineering, such as enhancers, promoters, Internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

The vector may also be a composition, e.g., different segments of different nucleic acids may be located on different vectors.

In some specific embodiments of the present disclosure, the vector is selected from a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector or a CRISPR/CAS plasmid.

In a fourth aspect, embodiments of the invention provide a cell comprising a nucleic acid as described in any one of the above embodiments or a vector as described in any one of the above embodiments, or a cell membrane having expressed on its surface the chimeric antigen receptor and the CXCR5 protein of a fusion protein as described in any one of the above embodiments.

Alternatively, the gene of the fusion protein is transferred into the cell in a manner that: lentivirus, retrovirus, general plasmid vector, episomal vector, nano-delivery system, electrical transduction, transposon, or other delivery system.

In some embodiments, the cell is an immune cell.

The immune cell may be selected from any one of helper T cell, cytotoxic T cell, memory T cell, regulatory T cell, MAIT cell, γ δ T cell, NK cell, CIK cell.

The immune cells can be prepared into a preparation which is a pharmaceutically acceptable carrier, diluent or excipient. Administration of the formulations of the invention may be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation.

In a fifth aspect, embodiments of the invention provide a pharmaceutical composition comprising a cell according to any one of the embodiments above.

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable carrier" includes any material that, when combined with an active component, allows the component to retain biological activity and not react with the immune system of a subject. Examples include, but are not limited to, standard pharmaceutical carriers such as phosphate buffered saline solution, water, emulsions such as oil/water emulsions, and any of various types of wetting agents. Exemplary diluents for aerosol or parenteral administration are Phosphate Buffered Saline (PBS) or physiological (0.9%) saline. Compositions comprising such carriers are formulated by well-known conventional methods (see, e.g., Remington's Pharmaceutical Sciences, 18 th edition, A.Gennaro eds., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 21 st edition, Mack Publishing, 2005).

In a sixth aspect, embodiments of the invention provide use of a cell according to any one of the above embodiments in the manufacture of a medicament for the treatment of a B-cell lymphoma.

In some embodiments, the B cell lymphoma is derived from mature B cell lymphocytes, the B cell lymphoma is non-hodgkin's lymphoma, or the non-hodgkin's lymphoma is selected from Small Lymphocytic Lymphoma (SLL), mantle cell lymphoma, burkitt's lymphoma, burkitt-like lymphoma, follicular central cell lymphoma, follicular lymphoma, marginal zone B-cell lymphoma, nodal marginal zone B cell lymphoma, extranodal marginal zone B cell lymphoma, splenic marginal zone B cell lymphoma, lymphoplasmacytic lymphoma, lymphoblastic B cell lymphoma, diffuse large B cell lymphoma, mediastinal large B cell lymphoma, and waldenstrom's macroglobulinemia.

In some embodiments, the B cell lymphoma is a B cell leukemia, or the B cell leukemia is selected from B cell acute lymphocytic leukemia (B-ALL), precursor B cell acute lymphocytic leukemia, B cell chronic lymphocytic leukemia (B-CLL), precursor B-lymphoblastic leukemia, B cell prolymphocytic leukemia, hairy cell leukemia and burkitt cell leukemia.

In some embodiments, the B cell lymphoma is selected from the group consisting of plasma cell myeloma, plasmacytoma, primary effusive lymphoma, diffuse mixed B cell lymphoma, and undifferentiated B cell lymphoma.

According to a further aspect of the invention, it also relates to a method of treating a tumor in a patient in need thereof, which method comprises administering to the patient a therapeutically effective amount of a cell or a pharmaceutical composition as described above.

"patient" refers to an animal. Preferably, the animal is a mammal. Patients also refer to, for example, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, fish, birds, rodents (including mice, rats, guinea pigs), and the like.

Embodiments of the present invention will be described in detail with reference to examples.

Preparation of CAR T used in the following examples:

first, each fusion protein structure was constructed according to the ligation order shown in FIG. 1. Included in FIG. 1 are 3 fusion protein structures, CD19-CAR, CD19-CXCR5-CAR, CD19-CD28T & I-CXCR 5-CAR. CD19-CAR, CD19-CXCR5-CAR employ the CD8 transmembrane region and the 4-1BB co-stimulatory domain. CD19-CD28T & I-CXCR5-CAR employs a CD28 transmembrane region and a costimulatory domain.

Each fusion protein structure was constructed in the same vector plasmid, as shown in FIGS. 2-4. The vectors were then transfected into T cells by lentiviruses, resulting in CD19-CAR T, CD19-CXCR5-CAR T, CD19-CD28T & I-CXCR5-CAR T.

Wherein the amino acid sequence of the signal peptide (Leader) is SEQ ID NO: 1 is shown. The amino acid sequence of the CD19 antibody (VL-VH) is SEQ ID NO: 2, respectively. The Hinge region (CD8Hinge) amino acid sequence is SEQ ID NO: 3, respectively. The TM amino acid sequence is SEQ ID NO: 4, respectively. The amino acid sequence of the 4-1BB co-stimulation domain is SEQ ID NO: 5, respectively. The amino acid sequence of the intracellular signaling domain of CD3 ζ is SEQ ID NO: and 6. The amino acid sequence of the T2A linker element is SEQ ID NO: shown at 7. The amino acid sequence of CXCR5 is SEQ ID NO: shown in fig. 8. The CD28T & I amino acid sequence is SEQ ID NO: shown at 9.

Example 1: ELISA (enzyme-Linked immuno sorbent assay) for detecting the binding condition of anti CD19-scFv (CD19 antibody) and CD19 antigen

The 96-well Nunc-ImmunoTM enzyme standard plates were pre-coated overnight with 10. mu.g/mL recombinant streptavidin 100. mu.L/well. The biotin-labeled recombinant human CD19 (rhCD19), recombinant human BCMA (rhBCMA), recombinant human CD27(rhCD27) and recombinant human CD40 (rhCD40) proteins purchased from Beijing Bethes Biotech, Inc. were diluted with PBS to a working solution of 5. mu.g/mL. After treatment, the ELISA plate is washed by PBST, 50 mu L/hole of the recombinant human protein working solution is added into the corresponding hole, and the plate is sealed and incubated overnight at room temperature. And (3) washing the incubated enzyme label plate by using PBST, then sealing the enzyme label plate by using 5% BSA at 37 ℃ in an incubator for 1 hour, washing and spin-drying for later use. Mouse anti-human anti CD19-scFv antibodies were serially diluted with 1% BSA in a gradient. Add 100. mu.L/well of the above gradient solution of anti-CD19 scFv to the corresponding wells, with 4 duplicate wells per gradient. Rabbit anti-human BCMA, rabbit anti-human CD27, and rabbit anti-human CD40 antibodies were additionally used as positive controls for rhBCMA, rhCD27, and rhCD40 proteins. After incubating at 37 ℃ for 1 hour, the cells were washed and spun dry. Adding 100 μ L of 0.25 μ g/mL secondary HRP-mouse anti-His antibody per well in the sample incubated with anti-CD19 scFv; mu.L of 0.081. mu.g/mL HRP-goat anti-rabbit IgG secondary antibody (Polyclonal, GeneTex.) was added to each well of the sample co-incubated with rabbit anti-human antibody, and incubated at 37 ℃ for 1 hour. After washing and spin-drying, 100. mu.L of TMB substrate developing solution is added into each hole, the reaction is carried out for 20 minutes at 37 ℃ in a dark place, and the OD value of 450nm wavelength is read by an Infinite F50 microplate reader (TECAN) immediately after the reaction is stopped by adding 100. mu.L/hole stop solution. As shown in fig. 5, the results show that the anti-CD19 scFv has good target specificity and can specifically bind to the recombinant human CD19 protein, and the OD450 value of the combination of the minimum dose of the anti-CD19 scFv (40pg/mL) and the recombinant human CD19 protein is also significantly higher than that of the experimental group of recombinant human BCMA, CD27 and CD40 proteins, thus indicating that the anti-CD19 scFv has higher affinity to the recombinant human CD19 protein and does not bind to other proteins.

Example 2: membrane protein Array (Membrane protein Array) validation of non-target binding interactions of antibodies

Membrane Proteome Array (MPA) analysis. MPA is a platform to analyze whether antibodies and other human membrane protein ligands are targeted, and can be used to determine the specificity of antibody targets. Plasmids containing about 6000 membrane protein clones (94% or more of the human membrane protein group) were transfected into HEK-293T cells (ATCC, CRL-3216; or QT6 cells (ATCC, CRL-1708; 384 well cell culture plates (Corning,3764), 18000 cells/well), respectively, after incubation for 36 hours, test antibodies were added to the membrane protein group array substrate plates at a predetermined concentration, and binding of the target antibody anti CD19-scFv-hFc to about 6000 membrane protein expressing cells was directly detected using flow cytometry.

As shown in fig. 6, the membrane protein array results show: the anti CD19-scFv-hFc can specifically bind to human CD19, but not to other non-target proteins.

Example 3: flow detection of anti CD19-scFv binding to target cells

K562, K562-CD19 and Raji cells were stained with PE-anti-CD 19 antibody (HIB19, Biolegend) and K562-BCMA was stained with PE-anti-BCMA antibody (19F2, Biolegend). K562-CD19 and Raji cells were confirmed to be positive for CD19, K562-BCMA cells positive for BCMA, and K562 cells negative for CD19, BCMA. Next, based on the characteristics of the above cells, the specific binding ability of anti CD19-scFv to CD19 positive and negative cells was tested.

As shown in FIG. 7, the data show that anti-CD19 scFv can bind to CD19 positive cell lines, including K562-CD19 and Raji, and the fluorescence intensity is enhanced with increasing dose of anti-CD19 scFv, with significant dose dependence. In contrast, fluorescence intensity of CD19 negative cells (K562-BCMA, K562) after incubation with anti-CD19 scFv was maintained at background level, indicating that anti-CD19 scFv can specifically recognize CD19 positive cells, but does not substantially bind to CD19 negative cells. In conclusion, the anti-CD19 scFv has good target specificity and can specifically bind to the recombinant human CD19 protein and CD19 positive cells.

In FIG. 7, A,4 tumor cell lines (K562-CD19, K562-BCMA, K562, Raji) were incubated with 0.2-20000pg/mL anti-CD19 scFv for 1 hour, and then the PE-tagged anti-His was used to tag the anti-CD19 scFv bound to the cell surface. B, flow analysis was performed to detect CD19 expression of K562, K562-CD19, Raji cells after staining with PE-anti-CD 19 antibody (HIB19), respectively. C, flow analysis was performed to detect K562, K562-BCMA, which was expressed by BCMA after staining with PE-anti-BCMA antibody (19F2), respectively.

Example 4: CAR-T cell migration ability assay

Transfected CD19-CAR-T and CD19-CXCR5-CAR-T cells were starved overnight in serum-free medium prior to chemotaxis assay. Will be 1x10⁵Each cell/well was placed in the upper chamber (5mm pore size; Costar Transwell, Corning, N.Y.) of a 24-well membrane migration chamber containing 6.5-mm diameter polycarbonate wells, and the lower chamber contained 600ml of serum-free medium containing CXCL13 factor at concentrations of 0, 1, and 5ug/ml, respectively. Chemokine-free medium without serum was used as a negative control. CAR-T cells at 37 ℃ and 5% CO₂The conditioned chamber was migrated and the migrated cells were collected from the lower chamber after 4h, 8h and 16h of migration, respectively. Counting beads (Invitrogen) were added, cells were then resuspended in 100ul volumes and cell number was detected on a flow machine. The results are shown in FIGS. 8A and 8B.

Example 5: ELISA (enzyme-Linked immuno sorbent assay) detection of killing effect of CAR-T cells on normal human body cells

anti-CD 19-CXCR5-CAR T cells were diluted in serum-free medium (RPMI-1640,2mM GlutaMAX,10mM HEPES, 100U/mL penicillin, 100. mu.g/mL streptomycin) and then treated at a ratio of 10:1, 3:1, 1: the target ratio is respectively compared with 1X105 positive control target cells (Raji), Human Pulmonary Microvascular Endothelial Cells (HPMEC), Human Pulmonary Artery Endothelial Cells (HPAEC) and Human Pulmonary Artery Smooth Muscle Cells (HPASMC),co-culture in 96-well round bottom plates, 3 wells in duplicate, at 37 ℃ and 5% CO₂Incubate in incubator for 16 hours, and take 50 μ L supernatant per well for IFN-gamma and IL-2 detection by ELISA. As shown in fig. 9A-9B, the results show that anti-CD 19-CXCR5-CAR T has a strong killing function on CD19 positive tumor cells and no killing effect on CD19 negative human primary cells.

Example 6: ELISA detection of secretion of IFN gamma and IL2 when CAR-T cells kill tumor target cells

anti-CD 19-CAR T, anti-CD 19-CXCR5-CAR T, anti-CD 19-CD28TM-CXCR5-CAR T, and T mock cells were diluted in serum-free medium (RPMI-1640,2mM GlutaMAX,10mM HEPES, 100U/mL penicillin, 100 μ g/mL streptomycin) and then the ratio was 30: 1: and (3) CO-culturing the target ratio with 1X106 positive control target cells (Raji), K562-CD19 cells and CD19 negative cells K562 respectively in a 96-well round bottom plate, incubating for 16 hours at 37 ℃ in a 5% CO2 incubator, and taking 50 mu L of supernatant per well for detecting IFN-gamma and IL-2 by an enzyme-linked immunosorbent assay. As shown in fig. 10A, 10B, 10C results, anti-CD 19-CXCR5-CAR T secreted more IFN- γ and IL-2 to CD19 positive tumor cells, suggesting a stronger killing ability. In each figure, A is IFN-gamma and IL-2 secretion after incubation of Raji and T cell effective target ratio 30: 1; b is IFN-gamma and IL-2 secretion after co-incubation of k562-CD19 and T cell effective target ratio of 30: 1; c is the IFN-gamma and IL-2 secretion condition after the co-incubation of k562 and T cell effective target ratio of 30: 1.

Example 7: efficacy of anti-CD 19-CXCR5-CAR T cells in a mouse model of human Burkitt's lymphoma (Raji) bearing tumors.

As shown in FIG. 11, after Raji-Luc (luciferase-labeled human Burkitt lymphoma Raji cells) cells were prepared, female B-NDG mice were used, and based on body weights measured 1 day (D-1) before administration, animals were randomly divided into 3 groups by body weight section using a computer system, and 1X10 cells were inoculated into the tail vein of each animal⁶And Raji-luc tumor cells. On day 10 after tumor cell inoculation, 3 × 106 total cells per mouse were injected via tail vein with corresponding CAR-T cells (anti-CD 19-CAR T cells, anti-CD 19-CXCR5-CAR T cells) and non-transduced T cells (Mock control). On

days

1, 4, 7 and 14 after receiving the treatment, the photogenic substrate D-luciferi was injected intraperitoneallyn, in vivo imaging and measurement of tumor size.

As shown in fig. 12, the results show that: Raji-Luc tumor growth was significantly inhibited in mice treated with anti-CD 19-CAR T or anti-CD 19-CXCR5-CAR T cells.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the patent protection scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims, and the description should be used for explaining the contents of the claims.

Sequence listing

<110> Guangzhou Bai-and-Gen-Tech Co Ltd

<120> fusion protein and use thereof

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<212> PRT

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Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 2

<211> 242

<212> PRT

<213> Artificial Sequence

<400> 2

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile

180 185 190

Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln

195 200 205

Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val

225 230 235 240

Ser Ser

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<211> 45

<212> PRT

<213> Artificial Sequence

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Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 4

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 4

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 5

<211> 42

<212> PRT

<213> Artificial Sequence

<400> 5

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 6

<211> 112

<212> PRT

<213> Artificial Sequence

<400> 6

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 7

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 7

Gly Ser Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210> 8

<211> 372

<212> PRT

<213> Artificial Sequence

<400> 8

Met Asn Tyr Pro Leu Thr Leu Glu Met Asp Leu Glu Asn Leu Glu Asp

1 5 10 15

Leu Phe Trp Glu Leu Asp Arg Leu Asp Asn Tyr Asn Asp Thr Ser Leu

20 25 30

Val Glu Asn His Leu Cys Pro Ala Thr Glu Gly Pro Leu Met Ala Ser

35 40 45

Phe Lys Ala Val Phe Val Pro Val Ala Tyr Ser Leu Ile Phe Leu Leu

50 55 60

Gly Val Ile Gly Asn Val Leu Val Leu Val Ile Leu Glu Arg His Arg

65 70 75 80

Gln Thr Arg Ser Ser Thr Glu Thr Phe Leu Phe His Leu Ala Val Ala

85 90 95

Asp Leu Leu Leu Val Phe Ile Leu Pro Phe Ala Val Ala Glu Gly Ser

100 105 110

Val Gly Trp Val Leu Gly Thr Phe Leu Cys Lys Thr Val Ile Ala Leu

115 120 125

His Lys Val Asn Phe Tyr Cys Ser Ser Leu Leu Leu Ala Cys Ile Ala

130 135 140

Val Asp Arg Tyr Leu Ala Ile Val His Ala Val His Ala Tyr Arg His

145 150 155 160

Arg Arg Leu Leu Ser Ile His Ile Thr Cys Gly Thr Ile Trp Leu Val

165 170 175

Gly Phe Leu Leu Ala Leu Pro Glu Ile Leu Phe Ala Lys Val Ser Gln

180 185 190

Gly His His Asn Asn Ser Leu Pro Arg Cys Thr Phe Ser Gln Glu Asn

195 200 205

Gln Ala Glu Thr His Ala Trp Phe Thr Ser Arg Phe Leu Tyr His Val

210 215 220

Ala Gly Phe Leu Leu Pro Met Leu Val Met Gly Trp Cys Tyr Val Gly

225 230 235 240

Val Val His Arg Leu Arg Gln Ala Gln Arg Arg Pro Gln Arg Gln Lys

245 250 255

Ala Val Arg Val Ala Ile Leu Val Thr Ser Ile Phe Phe Leu Cys Trp

260 265 270

Ser Pro Tyr His Ile Val Ile Phe Leu Asp Thr Leu Ala Arg Leu Lys

275 280 285

Ala Val Asp Asn Thr Cys Lys Leu Asn Gly Ser Leu Pro Val Ala Ile

290 295 300

Thr Met Cys Glu Phe Leu Gly Leu Ala His Cys Cys Leu Asn Pro Met

305 310 315 320

Leu Tyr Thr Phe Ala Gly Val Lys Phe Arg Ser Asp Leu Ser Arg Leu

325 330 335

Leu Thr Lys Leu Gly Cys Thr Gly Pro Ala Ser Leu Cys Gln Leu Phe

340 345 350

Pro Ser Trp Arg Arg Ser Ser Leu Ser Glu Ser Glu Asn Ala Thr Ser

355 360 365

Leu Thr Thr Phe

370

<210> 9

<211> 68

<212> PRT

<213> Artificial Sequence

<400> 9

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser

20 25 30

Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly

35 40 45

Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala

50 55 60

Ala Tyr Arg Ser

65

Claims

1. A fusion protein comprising a chimeric antigen receptor targeting CD19, a linker element, and a CXCR5 protein linked in sequence from N-terminus to C-terminus;

2. The fusion protein of claim 1, wherein the amino acid sequence of the CD19 antibody is SEQ ID NO: 2, respectively.

3. The fusion protein of claim 2, wherein the amino acid sequence of CXCR5 protein is SEQ ID NO: shown in fig. 8.

4. The fusion protein of claim 3, wherein the linker element is selected from one of T2A, P2A, E2A, F2A, and IRES, or any combination of at least two of T2A, P2A, E2A, F2A, and IRES; preferably T2A, and the amino acid sequence is SEQ ID NO: shown at 7.

5. The fusion protein of claim 4, wherein the transmembrane domain comprises a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-l, TRAC, TRBC, CD3 epsilon, CD3 zeta, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc epsilon RI gamma, BTLA, CD30, GITR, HVEM, DAPI0, CD2, NKG2C, LIGHT, DAPI2, CD40L, TIMl, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM;

6. The fusion protein of claim 4, wherein the costimulatory domain comprises a costimulatory domain derived from one or more proteins from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc RI γ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligands for CD83, CD40, and MyD 88;

preferably 4-1BB, and the amino acid sequence is SEQ ID NO: 5, respectively.

7. The fusion protein of any one of claims 1 to 6, wherein the intracellular signaling domain comprises an intracellular signaling domain derived from one or more proteins selected from the group consisting of CD3 ζ, CD3 γ, CD3E, CD79a, CD79b, FceRI γ, FceRI β, and FcyRIIa; preferably derived from the intracellular signaling domain of the CD3 ζ protein and having the amino acid sequence of SEQ ID NO: and 6.

8. The fusion protein of any one of claims 1 to 6, further comprising a hinge region linked between the CD19 antibody and the transmembrane domain; preferably, the amino acid sequence of the hinge region is SEQ ID NO: 3, respectively.

9. The fusion protein of any one of claims 1-6, wherein the CD19 antibody is further provided with a signal peptide at the N-terminus; preferably, the amino acid sequence of the signal peptide is SEQ ID NO: 1 is shown.

10. An isolated nucleic acid, which is expressed to obtain the fusion protein of any one of claims 1 to 9.

11. A vector comprising the nucleic acid of claim 10.

12. A cell comprising the nucleic acid of claim 10 or the vector of claim 11, or a cell membrane having the chimeric antigen receptor and the CXCR5 protein of the fusion protein of any one of claims 1 to 9 expressed on the surface thereof.

13. The cell of claim 12, wherein the cell is an immune cell; preferably, the immune cell is selected from any one of helper T cell, cytotoxic T cell, memory T cell, regulatory T cell, MAIT cell, γ δ T cell, NK cell and CIK cell.

14. A pharmaceutical composition comprising the cell of claim 12 or 13.

15. Use of a cell according to claim 12 or 13 in the manufacture of a medicament for the treatment of B-cell lymphoma.