CN113801228A

CN113801228A - CD 38-targeting single-chain antibody, fully human chimeric antigen receptor, preparation method and application

Info

Publication number: CN113801228A
Application number: CN202010555320.9A
Authority: CN
Inventors: 何玫瑰; 闵晨雨
Original assignee: Changzhou Velox Pharmaceutical Science & Technology Co ltd
Current assignee: Changzhou Velox Pharmaceutical Science & Technology Co ltd
Priority date: 2020-06-17
Filing date: 2020-06-17
Publication date: 2021-12-17
Anticipated expiration: 2040-06-17
Also published as: CN113801228B

Abstract

The invention discloses a single-chain antibody targeting CD38, a fully human chimeric antigen receptor, a preparation method and application. The chimeric antigen receptor comprises: the kit comprises a promoter, a leader peptide, a single-chain antibody specifically recognizing CD38 antigen, a tag peptide segment, a hinge region, a transmembrane region, an intracellular signal region, a self-cutting peptide and a puromycin gene which are connected in sequence. The scFv sequence with moderate affinity for recognizing CD38 is selected, can specifically and efficiently recognize target cells over-expressing CD38 antigen, does not show obvious toxic or side effect on normal cells and tissues under low expression CD38, has high safety and has wide clinical application prospect.

Description

CD 38-targeting single-chain antibody, fully human chimeric antigen receptor, preparation method and application

Technical Field

The invention relates to the field of cellular immunology, in particular to a single-chain antibody targeting CD38, a fully human chimeric antigen receptor, a preparation method and application.

Background

Adoptive Cell Therapy (ACT) is one of the biological treatment technologies, and is a method of expanding autoimmune cells (mainly T cells) in vitro and then returning them to tumor patients for treatment, which is considered as the 4 th treatment mode following surgery, radiotherapy and chemotherapy and is widely used in clinical treatment. The main major applications of adoptive cell therapy are: the combination of lymphokine activated killer cells (LAK) and IL-2 has certain curative effect on treating late malignant tumor; the Tumor Infiltrating Lymphocytes (TIL) clinical test has better effect on treating metastatic melanoma; cytokine induced killer Cells (CIK) are currently subjected to many clinical tests in China, and have remarkable effects on tumors such as liver cancer and lung cancer. However, the three treatment methods need to activate T cells, and the T cell activation needs two activation signals, namely a first signal for the T cell surface TCR-CD3 to be combined with MHC-I molecules to be activated so as to determine the killing activity of the T cells on tumor cells; the binding of costimulatory molecules on the surface of T cells to the corresponding ligands is the second signal for activation, determining T cell proliferation. However, the tumor cells and the tumor microenvironment can down regulate MHC and ligand molecules, thereby inhibiting the killing activity of T cells on the tumor cells. Therefore, there are two main ways to perform genetic modification: gene transduction TCRs (T cell receptors) and Chimeric Antigen Receptors (CARs).

Chimeric Antigen Receptors (CAR), which are artificial receptors that mimic TCR function, are composed of an antigen recognition domain, a hinge region, and a transmembrane region, which are connected in sequence, and an intracellular signaling domain, which is usually a CD3 zeta chain, or is linked to one or more costimulatory molecules, such as 4-1BB, CD 28. When an antigen (receptor) on the surface of a tumor cell is bound to an antibody (ligand) of a chimeric antigen receptor, a signal is transmitted into the cell through a hinge region and a transmembrane region, an intracellular signal region converts the signal into an activation signal, an effector cell is activated, and the effector cell proliferates to produce a cytokine, thereby killing the tumor cell. Although CAR-T treatment of tumors has shown some tumor clearance, and CD19-CAR treatment in particular has made a breakthrough in the treatment of hematological tumors, CAR-T treatment still has a series of problems that need to be further explored: 1. lack of an ideal tumor antigen as a target; 2. tumor-mediated immunosuppression; 3. serious toxic side effects of CAR-T therapy.

Multiple Myeloma (MM), is a malignant tumor of abnormal proliferation of plasma cells, an incurable hematological malignancy, and can affect the production of white blood cells, red blood cells, and stem cells in the bone marrow. Plasma cells that have become cancerous in the bone marrow are called myeloma cells, and they proliferate in large quantities in the bone marrow, replace normal cells in the bone marrow, destroy the normal immune system, inhibit the normal hematopoietic function, and further damage the functions of bones and soft tissues (nerves, muscles, etc.). In the united states, multiple myeloma is the second largest malignancy of the blood system following non-hodgkin's lymphoma, accounting for approximately 1% of all malignancies, 13% of hematologic malignancies, and 2% of deaths due to malignancies. The mean age of onset is 62 years in men, 61 years in women, and only 2% of patients under 40 years of age.

CD38 is uniformly highly expressed on multiple myeloma cells, but is poorly expressed on normal lymphocytes and bone marrow cells as well as on some tissues of non-hematopoietic origin, and is a specific antigen for multiple myeloma. Because of the high expression of CD38 on MM, CD 38-targeted immunotherapy theoretically has a safer and more effective therapeutic effect, and the development of CD 38-targeted drugs such as (e.g., Daratumumab, Isatuximab, MOR202) has been carried out abroad, but the efficacy is limited and safety problems still occur; therefore, there is a need to find new therapies targeting CD38 with better specificity and more effectiveness, and although some target CARs are being tested, there are relatively few CARs for treating MM, further modification tests are still needed in terms of stability and safety of CAR performance, and how to select more stable and suitable scFv (single chain antibody) is also an unsolved key problem in CAR research, so that the development and preparation of CD38-CAR targeting CD38 with better safety and effectiveness are necessary.

Disclosure of Invention

The invention aims to solve the problems of stability and safety of a targeted drug targeting CD38 in the prior art.

In order to achieve the above object, the present invention provides a single-chain antibody that recognizes CD38 antigen moderately and specifically, the single-chain antibody comprising: a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises three complementarity determining regions: HCDR1, HCDR2 and HCDR3, said HCDR1 having an amino acid sequence comprising SEQ ID NO: 2; the amino acid sequence of the HCDR2 comprises SEQ ID NO: 3; the amino acid sequence of the HCDR3 comprises SEQ ID NO: 4; the light chain variable region comprises three complementarity determining regions: LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the LCDR1 comprises SEQ ID NO: 5; the amino acid sequence of LCDR2 comprises SEQ ID NO: 6; the amino acid sequence of LCDR3 comprises SEQ ID NO: 7, or a sequence shown in the figure.

The invention also provides a fully human chimeric antigen receptor targeting CD38, which comprises a single-chain antibody specifically recognizing CD38 antigen, wherein the amino acid sequence of the single-chain antibody comprises the amino acid sequence shown in SEQ ID NO: 1, or a variant of SEQ ID NO: 1 with 95-99% identity.

Preferably, the chimeric antigen receptor comprises: the promoter, the leader peptide, the single-chain antibody specifically recognizing the CD38 antigen, the tag peptide segment, the hinge region, the transmembrane region and the intracellular signal region are connected in sequence.

Preferably, the promoter is derived from EF1 alpha, and the nucleic acid sequence is shown as SEQ ID NO: 8 is shown in the specification; the leader peptide is derived from GM-CSF, and the amino acid sequence is shown as SEQ ID NO: 9 is shown in the figure; the amino acid sequence of the tag peptide segment is shown as SEQ ID NO: 10 is shown in the figure; the hinge region and the transmembrane region are CD 8-derived hinge region and transmembrane region, and the amino acid sequences are respectively shown as SEQ ID NO: 11 and SEQ ID NO: 12 is shown in the specification; the intracellular signaling domain comprises: CD28, 4-1BB and CD3 zeta, the amino acid sequences are respectively shown in SEQ ID NO: 13. SEQ ID NO: 14 and SEQ ID NO: shown at 15.

Preferably, the chimeric antigen receptor is further connected with a self-cleaving peptide T2A and a puromycin gene, and the amino acid sequence of the self-cleaving peptide T2A is shown as SEQ ID NO: 16 is shown in the figure; the amino acid sequence of the puromycin gene is shown as SEQ ID NO: shown at 17.

The invention also provides a polynucleotide encoding the fully human chimeric antigen receptor targeting CD38, wherein the chimeric antigen receptor comprises a single-chain antibody specifically recognizing CD38 antigen, and the nucleic acid sequence of the single-chain antibody comprises the nucleotide sequence shown in SEQ ID NO: 18, or a variant of SEQ ID NO: 18 has 95-99% identity.

The invention also provides a preparation method of the CD 38-targeted fully human chimeric antigen receptor based on a pMD19-T vector, which comprises the following steps:

(1) synthesizing a nucleotide sequence for encoding the chimeric antigen receptor to obtain an EF1 alpha-SP-CD 38-CAR-T2A-Puro gene segment; the gene fragment comprises a promoter, a leader peptide, a single-chain antibody specifically recognizing CD38 antigen, a tag peptide fragment, a hinge region, a transmembrane region, an intracellular signal domain, T2A and a puromycin gene;

(2) carrying out double enzyme digestion on the EF1 alpha-SP-CD 38-CAR-T2A-Puro gene segment by using restriction enzymes BamH-I and SalI to obtain a target segment; and (2) digesting the pMD19-T vector by using restriction enzymes BamH-I and SalI, and then connecting the digested target fragment and the pMD19-T vector by using T4 ligase to obtain a pMD19-T plasmid capable of expressing the chimeric antigen receptor.

The invention also provides a pMD19-T plasmid capable of expressing the chimeric antigen receptor, and the pMD19-T plasmid is prepared by the preparation method of the CD 38-targeted fully human chimeric antigen receptor based on the pMD19-T vector.

The invention also provides a T cell expressing the fully human chimeric antigen receptor targeting CD 38.

The invention also provides application of the CD 38-targeted fully human chimeric antigen receptor in preparation of drugs for inhibiting or treating malignant hematological diseases or solid tumors capable of expressing CD 38.

Compared with the prior art, the invention has the beneficial effects that:

(1) the scFv sequence capable of recognizing CD38 with moderate affinity is selected, the target cell of high expression CD38 can be specifically and efficiently recognized, and meanwhile, the scFv has no obvious toxic or side effect on the cell and tissue of low expression CD38, and has good immune tolerance and high safety.

(2) In the traditional preparation process, the CAR-T cells used for treatment are often in the terminal differentiation stage, have short survival time and are difficult to exert long-acting killing function, so the treatment effect is poor. The co-stimulation signals of CD28 and 41BB adopted by the invention obviously inhibit terminal differentiation of T cells and promote generation of memory T cells with longer survival time. The transgenic modified CAR-T cell has stronger and more durable killing capability and is expected to improve the treatment effect of solid tumors.

(3) The invention ensures that the extracellular recognition region and the intracellular signal region are well combined by utilizing the sequences of the CD8 hinge region and the transmembrane region, and ensures the function of the CAR-T cell.

(4) The non-viral vector pMD19-T electrotransfection system is adopted, potential safety hazards caused by using viral vectors are avoided, and the method has higher safety and effectiveness and is more suitable for clinical treatment.

Drawings

FIG. 1 is a block diagram of a fully human CD38-CAR (hCD38 CAR).

FIG. 2 is a flow chart of transfection efficiency of fully human CD38-CAR-T cells.

FIG. 3 is the results of the cytotoxicity of fully human CD38-CAR-T cells against CD 38-overexpressing RPMI-8226 and Daudi tumor cells.

FIG. 4 is the cytotoxicity assay of fully human CD38-CAR-T cells on normal PBMC cells naturally expressing CD 38.

FIG. 5 shows the control of mouse tumor growth by fully human CD38-CAR-T cells.

Detailed Description

The technical solution of the present invention is further described below with reference to the accompanying drawings and examples.

The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental reagents and materials involved are conventional biochemical reagents and materials unless otherwise specified.

Definition of terms

An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains that are linked to each other by disulfide bonds, or an antigen-binding portion thereof. Antibodies include single chain antibodies.

The "heavy chain" consists of a heavy chain variable region (VH) and a heavy chain constant region.

The "light chain" consists of a light chain variable region (VL) and a light chain constant region.

Single-chain antibody fragment (scFv): the antibody heavy chain variable region and the antibody light chain variable region are connected through a short peptide (linker) with 15-20 amino acids.

The "heavy chain variable region" and "light chain variable region" can be further subdivided into hypervariable regions, termed "complementarity determining regions" (CDRs), interspersed with more conserved regions termed "framework regions" (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRI, CDRl, FR2, CDR2, FR3, CDR3, FR4, the variable regions of the heavy and light chains contain binding domains that can interact with antigens. In the present invention, CDRl, CDR2, CDR3 of the heavy chain variable region are represented as HCDR1, HCDR2 and HCDR3, respectively; the CDRl, CDR2, CDR3 of the light chain variable region are denoted LCDR1, LCDR2 and LCDR3, respectively.

"CAR-T" refers to a chimeric antigen receptor T cell. The scFv for recognizing tumor-associated antigen and an intracellular signal domain, namely an immunoreceptor tyrosine activation motif, are subjected to in vitro gene recombination to generate recombinant plasmids, the recombinant plasmids are transfected into T cells of a patient in vitro by a transfection technology to enable the T cells of the patient to express tumor antigen receptors, and the transfected T cells are purified and amplified in a large scale and are called chimeric antigen receptor T cells.

"95-99% identity" refers to homology or similarity of greater than 95% (optimally greater than 98%). It is known to those skilled in the art that mutation of one or a few amino acids or nucleotides in a sequence does not, in some cases, substantially alter the biological function of the sequence. The mutation of amino acids or nucleotides also includes modification, and the amino acids or nucleotides modified sequence, biological function and the sequence provided by the invention is the same or equivalent, and this type of sequence is within the protection scope of the invention.

The nucleic acid sequence for coding scFv provided by the invention is shown as SEQ ID NO: 18, the amino acid sequence of the corresponding scFv is set forth in SEQ ID NO: 1 is shown. SEQ ID NO: 2 to SEQ ID NO: 7 is SEQ ID NO: 1.

Specific screening methods for the sequences of the present invention can be found in CN201911234429.6 (title: fully human antibody or antibody fragment against CD19, chimeric antigen receptor, and application). The following examples are for polypeptides comprising SEQ ID NO: 1 (scFv), and the results of the experiments for verifying the chimeric antigen receptor of the single-chain antibody (scFv).

A, CAR structure

The basic structure of CD38-CAR (human chimeric antigen receptor targeting CD 38) is composed of EF1 alpha promoter (promoter), leader peptide (SP), single chain antibody (scFv) specifically recognizing CD38 antigen, tag peptide segment (Myc-tag), Hinge region of human CD8a molecule (CD8 Hinge), transmembrane region (CD8 TM), intracellular region of human CD28 molecule (CD28), intracellular region of human 4-1BB molecule (4-1BB), intracellular region of human CD3 zeta molecule (CD3 zeta), 2A self-cleaving peptide (T2A) and puromycin (puromycin) gene which are connected in series in sequence, and the structure formed by connecting in series is shown as hCD38 CAR in FIG. 1. The tandem structure is denoted hereinafter by EF1 α -SP-CD 38-CAR-T2A-Puro.

II, CAR sequence synthesis and vector construction

The CAR coding sequence is synthesized by Kingsry of Jiangsu, and the obtained EF1 alpha-SP-CD 38-CAR-T2A-Puro structural DNA sequence (shown in figure 1) is inserted into a pMD19-T plasmid through enzyme digestion connection to construct a non-viral electrotransformation expression plasmid pMD19-T-EF1 alpha-SP-CD 38-CAR-T2A-Puro, namely a pMD19-T plasmid capable of expressing a chimeric antigen receptor.

Extraction of expression plasmid by electrotransformation

pMD19-T-EF1 alpha-SP-CD 38-CAR-T2A-Puro was transferred into E.coli and allowed to amplify in E.coli in large quantities. Then, plasmid DNA of Escherichia coli was extracted using an endotoxin-free plasmid macrodrawing kit.

Fourth, T cell transfection and purification

Separating lymphocytes from human serum, and adding T lymphocyte culture fluid [ CTS^TM AIM V^TMSfm (ruo), Thermo, cargo number: a3021002]And the irradiated PBMC cells, the OKT-3, the hIL-2 and endotoxin-free plasmid DNA are subjected to expansion culture, and the plasmid pMD19-T-EF1 alpha-SP-CD 38-CAR-T2A-Puro is transferred into the T cells by adopting an electrotransfection mode to obtain the T lymphocytes with EF1 alpha-SP-CD 38-CAR-T2A-Puro genes, namely the CAR-T cells.

Fifth, T cell CAR expression efficiency

After 7 days of T cell transfection, puromycin was added at a certain concentration to the T lymphocyte culture broth. After 20 days of T cell transfection, T cell surface CAR expression was examined by flow cytometry. The results are shown in FIG. 2. Figure 2 shows that the CAR expression positive rate reached about 100%, demonstrating the success of CAR expression plasmid construction and electroporation.

Sixth, long-lasting killing effect of CAR-T cells on tumor cells over-expressing associated antigens

To determine the better sustained killing of CD38-CAR-T cells, the killing assay was performed using target cells carrying the Luciference fluorescent signal. The ratio of effective target (effector cell: target cell, E: T) 4: 1 and 1: 1, T cells (effector cells) were co-incubated with CD38 positive target cells (Daudi-Fluc, RPMI-8226-Fluc) or CD38 negative target cells (K562-Fluc), respectively, for 72 hours (as shown in fig. 3). After the completion of the co-incubation, a substrate for fluorescence signal of luciferase (Perkinelmer, cat # 122799) was added, and then the target cell apoptosis was analyzed by a microplate reader. The result shows that the CAR-T cell has strong killing capacity to tumor Cells (CD38+ Cells) over expressing CD38, and has little influence on CD38 negative Cells, which indicates that the CAR-T cell has strong specific killing capacity.

Seventh, toxic side effects of CAR-T cells on normal cells that underexpress the relevant antigen

To further determine the toxic side effects of CD38-CAR-T cells (pMD-T-CD 38-scfv-003-CAR T cells in FIG. 4) on normal cells that underexpress CD38, we previously stained human Peripheral Blood Mononuclear Cells (PBMC) using the CellTrace (TM) Violet Cell Proliferation Kit (Thermo, cat # C34571). The ratio of effective target to target (effector cells: target cells, E: T) 4: 1. 1: 1T cells (effector cells) were incubated with normal PBMC cells that underexpress CD38 for 72 hours (as shown in FIG. 4). After the end of the co-incubation, the cells were harvested by centrifugation and co-incubated with an anti-CD 3 antibody and an anti-CD 38 antibody, and finally the PBMC cells were analyzed for CD38 positive apoptosis by flow cytometry. The results show that CAR-T cells have very low toxic side effects on normal cells that underexpress CD 38.

Eighthly, animal experiments verify the function of the CAR-T cells

Immunodeficient mice were inoculated via tail vein with 5X 10⁵7 days after each Daudi cell, 1X 10 cells were injected into the tail vein⁷Individual CAR-T cells were treated. At 0.7 days of treatment, tumor signals were observed using a small animal in vivo imaging device. As shown in FIG. 5, tumor signals were significantly reduced 7 days after CAR-T cell injection, suggesting that CAR-T cells have good tumor killing ability.

In conclusion, the scFv sequence or the chimeric antigen receptor provided by the invention has the capacity of recognizing CD38 with moderate affinity, can efficiently recognize target cells, and has low toxic and side effects on cells with low expression of CD38 and high safety.

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.

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Claims

1. A single chain antibody that recognizes CD38 antigen moderately and specifically, said single chain antibody comprising: a heavy chain variable region and a light chain variable region;

the heavy chain variable region comprises three complementarity determining regions: HCDR1, HCDR2 and HCDR3, said HCDR1 having an amino acid sequence comprising SEQ ID NO: 2;

the amino acid sequence of the HCDR2 comprises SEQ ID NO: 3;

the amino acid sequence of the HCDR3 comprises SEQ ID NO: 4;

the light chain variable region comprises three complementarity determining regions: LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the LCDR1 comprises SEQ ID NO: 5;

the amino acid sequence of LCDR2 comprises SEQ ID NO: 6;

the amino acid sequence of LCDR3 comprises SEQ ID NO: 7, or a sequence shown in the figure.

2. A fully human chimeric antigen receptor targeting CD38, said chimeric antigen receptor comprising a single chain antibody specifically recognizing CD38 antigen, said single chain antibody having an amino acid sequence comprising SEQ ID NO: 1, or a variant of SEQ ID NO: 1 with 95-99% identity.

3. The fully human chimeric antigen receptor targeting CD38 of claim 2, wherein said chimeric antigen receptor comprises: the promoter, the leader peptide, the single-chain antibody specifically recognizing the CD38 antigen, the tag peptide segment, the hinge region, the transmembrane region and the intracellular signal region are connected in sequence.

4. The fully human chimeric antigen receptor targeting CD38 of claim 3, wherein: the promoter is derived from EF1 alpha, and the nucleic acid sequence is shown as SEQ ID NO: 8 is shown in the specification; the leader peptide is derived from GM-CSF, and the amino acid sequence is shown as SEQ ID NO: 9 is shown in the figure; the amino acid sequence of the tag peptide segment is shown as SEQ ID NO: 10 is shown in the figure; the hinge region and the transmembrane region are CD 8-derived hinge region and transmembrane region, and the amino acid sequences are respectively shown as SEQ ID NO: 11 and SEQ ID NO: 12 is shown in the specification; the intracellular signaling domain comprises: CD28, 4-1BB and CD3 zeta, the amino acid sequences are respectively shown in SEQ ID NO: 13. SEQ ID NO: 14 and SEQ ID NO: shown at 15.

5. The fully human chimeric antigen receptor targeting CD38, according to claim 3, wherein said chimeric antigen receptor further comprises a self-cleaving peptide T2A and a puromycin gene, wherein the amino acid sequence of said self-cleaving peptide T2A is as shown in SEQ ID NO: 16 is shown in the figure; the amino acid sequence of the puromycin gene is shown as SEQ ID NO: shown at 17.

6. A polynucleotide encoding the chimeric antigen receptor of full human origin targeting CD38 according to any one of claims 2 to 5, wherein the chimeric antigen receptor comprises a single chain antibody specifically recognizing CD38 antigen, and the nucleic acid sequence of the single chain antibody comprises the amino acid sequence of SEQ ID NO: 18, or a variant of SEQ ID NO: 18 has 95-99% identity.

7. The method for preparing the chimeric antigen receptor of full human origin targeting CD38 based on the pMD19-T vector of any one of claims 2 to 5, characterized in that it comprises the following steps:

8. The pMD19-T plasmid capable of expressing the chimeric antigen receptor of any one of claims 2 to 5, wherein the pMD19-T plasmid is prepared by the method for preparing the CD 38-targeting fully human chimeric antigen receptor based on the pMD19-T vector of claim 7.

9. A T cell expressing the fully human chimeric antigen receptor targeting CD38 of any one of claims 2 to 5.

10. Use of the fully human chimeric antigen receptor targeting CD38 of any one of claims 2 to 5 in the manufacture of a medicament for inhibiting or treating a hematological malignancy or a solid tumor capable of expressing CD 38.