CN114591443A

CN114591443A - Chimeric receptor CSR based on scTv and application thereof

Info

Publication number: CN114591443A
Application number: CN202210215387.7A
Authority: CN
Inventors: 徐振宇; 何伟杰
Original assignee: First Affiliated Hospital of Wannan Medical College
Current assignee: First Affiliated Hospital of Wannan Medical College
Priority date: 2022-03-07
Filing date: 2022-03-07
Publication date: 2022-06-07

Abstract

The invention discloses a chimeric receptor CSR based on scTv and application thereof, wherein the CSR comprises a single-chain TCR antibody scTv and an IL-15 complex containing a suicide gene, the scTv comprises a V alpha fragment with an alpha chain variable region and a V beta fragment with a beta chain variable region, and the amino acid sequence of the V alpha fragment is shown in SEQ ID NO: 1, and the amino acid sequence of the V beta fragment is shown as SEQ ID NO: 2, respectively. The CSR constructed by the invention not only retains the advantages of TCR recognition target spots, but also has a CAR signal path, and is combined with NK cells, so that the limitations of TCR-T, CAR-T, CAR-NK and TCR-NK can be effectively solved, and the CSR is a cell therapy product with wide target spot range, strong universality, safety, effectiveness and low cost.

Description

Chimeric receptor CSR based on scTv and application thereof

Technical Field

The invention belongs to the technical field of chimeric receptors, and particularly relates to a chimeric receptor CSR based on scTv and application thereof.

Background

CD 19-targeted CAR-T has made a major breakthrough in B cell hematologic tumors in recent years, and 6 CAR-T drugs have been approved for marketing worldwide. Compared with the huge achievement in blood tumor, when the immune technology is applied to the solid tumor with higher morbidity, strong heterogeneity, more complex pathogenic mechanism and tumor microenvironment, the immune technology such as CAR-T and the like cannot produce good effect, and the breakthrough of the target is undoubtedly important in a plurality of technical bottlenecks.

At present, the traditional CAR technology can only recognize cell membrane proteins, and the existing solid tumors lack effective membrane protein targets, but the problem of heterogeneity of the solid tumors cannot be completely solved only by one or a plurality of membrane protein targets, namely the tumor relapse is caused by the lack of the targets. Compared with the CAR, the TCR can theoretically recognize all polypeptides (proteins) in tumor cells, the antigen recognition range of the TCR is far wider than that of the CAR, and particularly, more individualized and multi-target tumor neoantigens (tumor specific antigens, TSA) are identified by combining a high-pass side sequencing-based technology in recent years, so that the TCR technology is brought back into the field of researchers.

Compared with a T cell vector, the NK cell has more advantages in tumor treatment as a targeted killing vector. On 11/2021, the national drug administration officially approved the clinical Implication (IND) of targeted Mesothelin (MSLN) chimeric antigen receptor NK cell (CAR-NK) injection for advanced epithelial ovarian cancer treatment of the first CAR-NK in our country. The traditional autologous CAR-T has the advantages of obtaining the curative effect equivalent to that of the traditional autologous CAR-T for treating B cell tumor, having the characteristics of no immunological rejection, no serious CRS reaction and the like, realizing the effectiveness, safety and universality of clinical treatment, and greatly reducing the production cost and the preparation risk. Based on the numerous advantages mentioned above, CAR-NK technology is currently replacing CAR-T in a step-by-step manner.

Because NK cells lack the complete CD3 signaling pathway, TCRs cannot be "mounted" directly into NK cell vectors, and in order to solve the above-mentioned problems, there is a report in the literature that NK cells are engineered with CD3 molecules, i.e., the four subunits CD3 ζ, CD3 γ, CD3 ∈, CD3 δ of CD3 are "mounted" at the same time as the TCR molecules are "mounted", but such engineered structures have large molecular weights, which are not conducive to transduction, and lack the second signaling pathway. It has also been reported in the literature that a mutant sc-TCRVaVbCb is constructed by modifying TCR, and the sc-TCRVaVbCb gene is linked to CD3-z to induce heterodimerization and T cell activation of the chimeric TCRab gene. Although the a-chain of the TCR is optimized, the molecular weight and second signaling pathway problems still remain. Chandran SS et al reported that the a chain of the TCR was truncated in its TM region, a cysteine was added to its constant domain, and the two chains were connected by a 2A peptide sequence. CD28 acts as a transmembrane domain (TM) and CD3 and CD28 are linked as a second stimulation signal. Although the second signaling pathway problem is solved, the molecular weight is still large and the problem of unfavorable transduction is not solved.

Disclosure of Invention

The purpose of the invention is as follows: in view of the above technical problems, the present invention provides a chimeric receptor CSR based on scTv and its use in immune cell therapy. The CSR modified by the invention not only has the dual characteristics of multi-target recognition and signal conduction, but also has the advantages of large molecular weight and small size. Membrane anchored IL-15 complexes can enable NK cells to continuously kill tumor cells. When necessary, the immune cells can start a 'suicide' switch to ensure the safety of the cell therapy product.

The technical scheme is as follows: in order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

a scTv-based chimeric receptor CSR comprising a single chain TCR antibody scTv comprising a fragment having an alpha chain variable region V α and a fragment having a beta chain variable region V β, the amino acid sequence of said V α fragment being as set forth in SEQ ID NO: 1, and the amino acid sequence of the V beta fragment is shown as SEQ ID NO: 2, respectively.

Preferably, the V alpha segment and the V beta segment are connected through a Linker, and the amino acid sequence of the Linker is shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

Preferably, the IL-15 complex comprises a suicide gene CD20 epitope polypeptide, an IL-15 mutant and a truncated CD4 molecular scaffold, and the amino acid sequences of the amino acid sequences are respectively shown as SEQ ID NO: 5. SEQ ID NO: 6 and SEQ ID NO: shown at 7.

Preferably, the CSR comprises a single chain TCR antibody, scTv, a hinge region, a transmembrane domain, an intracellular signaling domain, an intracellular costimulatory domain, and an IL-15 complex comprising a suicide gene, linked in sequence.

Further preferably, the hinge region is a CD8 a hinge region; the transmembrane domain is selected from CD8 alpha, CD28, CD16, NKp44, NKp46, NKG2C or NKG2D domain; the intracellular costimulatory domain is selected from the CD3 zeta, CD28, 4-1BB, 2B4, DAP10, DAP12 or CD137 domain; the intracellular signaling domain is selected from the CD3 zeta domain. More preferably, the hinge region and transmembrane domain are selected from the group consisting of a CD8 α hinge region and transmembrane region, the amino acid sequence of which is as set forth in SEQ ID NO: 8 is shown in the specification; the intracellular costimulatory domain is 4-1BB, and the amino acid sequence of the intracellular costimulatory domain is shown as SEQ ID NO: shown at 9.

Further preferably, the IL-15 complex is linked to the intracellular costimulatory domain by a 2A linker peptide; preferably, the 2A linker peptide is P2A, and its amino acid sequence is as shown in SEQ ID NO: shown at 10.

Preferably, the CSR is further linked to a signal peptide; more preferably, the signal peptide is a CD8 signal peptide, the amino acid sequence of which is as set forth in SEQ ID NO: shown at 11.

A nucleic acid molecule encoding said scTv-based chimeric receptor CSR.

A vector comprising said nucleic acid molecule. Preferably, the vector is a lentiviral vector.

A host cell comprising said nucleic acid molecule or said vector.

Preferably, the host cell includes an immune cell such as a T cell, an NK cell, a γ δ T cell, or an NKT cell.

The invention finally provides the chimeric receptor CSR based on the scTv or the application of the host cell in the preparation of antitumor drugs.

In view of the problems of the prior art, the present invention refers to the fact that the TCR is highly similar in structure to the antibody molecule (the TCR is composed of two chains), and the single-chain TCR, i.e., the scTv, is constructed by referring to the single-chain antibody structure. The modified scTv only contains a variable region fragment (V alpha) of alpha and a variable region fragment (V beta) of beta, greatly shortens the length of a plasmid and is beneficial to the transduction of the plasmid. Meanwhile, by taking the molecular structure of CAR as a reference, a novel chimeric receptor CSR based on scTv is established. Not only retains the advantages of TCR recognition target, but also has the signal path of CAR. CSR can modify T cells, NK cells and other immune cells, and provides a treatment strategy for solving the heterogeneity and complexity of solid tumors. NK cells have a short life span, relatively low toxicity risk, and a wide range of NK cell sources, such as NK92 cell line, Peripheral Blood Mononuclear Cells (PBMCs), Umbilical Cord Blood (UCB), Induced Pluripotent Stem Cells (iPSCs), etc., can provide a "ready-to-use" product, eliminating the need for personalized and patient-specific products that plague current CAR-T cell therapies. Therefore, CSR-NK is the most ideal solid tumor cell treatment product at present, realizes no dead angle killing of tumor cells, and benefits a large number of tumor patients.

Has the advantages that: compared with the prior art, the invention has the following advantages:

(1) by referring to the single-chain antibody structure, the single-chain variable region that the scTv only contains TCR is constructed, the molecular weight of the single chain is greatly reduced, and the advantage of TCR recognition target is retained. Meanwhile, the targeting property of the CSR is ensured by using the CAR molecular structure and possessing a CAR signal path.

(2) NK cells are not restricted by MHC recognition, and transplantation of allogeneic NK cells induces little graft-versus-host disease (GVHD) and does not cause CRS. And the level of secreted PD-1 is lower, and the immunosuppressive effect is smaller.

(3) The IL15 super complex can not only increase the activity of NK cells and keep the NK cells to have a continuous killing function, but also comprises a suicide gene 'CD 20', can induce the apoptosis of the NK cells when necessary and ensure the product safety.

(4) The cord blood NK has wide source and low cost, has the characteristic of universal type, and can be used as a product of 'spot type'.

Drawings

FIG. 1: schematic diagram of construction of scTv and CSR plasmids.

FIG. 2: infection efficiency of CSR-NK cells.

FIG. 3: killing efficiency of CSR-NK on K562.

FIG. 4: the expression level of IFN γ.

Detailed Description

In order that the invention may be more readily understood, the invention will now be further described with reference to specific examples.

The following examples use RAS (G12D) mutant and HLA-A1101 tumor cells, but the examples are not intended to limit the invention, but only to illustrate the invention.

Example 1 sequence selection of scTv and CSR

At present, the literature reports that the TCR-NK-based modification technology adopts a full-length TCR or a TCR of a partial constant region. The molecular weight of the modified technology is large, and the transduction is not facilitated. The invention screens out single-chain TCR (scTv) consisting of V alpha and V beta fragments through continuous groping experiments. CSR comprises a single-chain, membrane-anchored IL15 complex that recognizes the alpha and beta variable regions of the mTCR of a RAS (G12D) mutant polypeptide presented by HLA-a 1101. The membrane-anchored IL15 complex was linked to CAR via P2A, enabling independent expression of the IL15 complex in cells.

The sequence of CSR was chosen as follows, and ligated as shown in FIG. 1:

the amino acid sequence of the V alpha fragment is shown as SEQ ID NO: 1, the amino acid sequence of the V beta fragment is shown as SEQ ID NO: 2, respectively. The V alpha segment and the V beta segment are connected through a Linker, and the amino acid sequence of the Linker is shown as SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

The IL-15 complex comprises a suicide gene CD20 epitope polypeptide, an IL-15 mutant and a truncated CD4 molecular scaffold, and the amino acid sequences of the IL-15 complex are respectively shown as SEQ ID NO: 5. SEQ ID NO: 6 and SEQ ID NO: shown at 7.

The hinge region and the transmembrane domain are CD8 alpha hinge region and transmembrane region, and the amino acid sequence of the hinge region and the transmembrane domain is shown as SEQ ID NO: shown in fig. 8. The intracellular costimulatory domain is 4-1BB, and the amino acid sequence of the intracellular costimulatory domain is shown as SEQ ID NO: shown at 9. The intracellular signaling domain is selected from the CD3 zeta domain.

The IL-15 complex is linked to the intracellular costimulatory domain by a 2A linker peptide. The 2A connecting peptide is P2A, and the amino acid sequence of the connecting peptide is shown as SEQ ID NO: 10 is shown in the figure;

the CSR is also connected with a CD8 signal peptide, and the amino acid sequence of the CSR is shown as SEQ ID NO: shown at 11.

EXAMPLE 2 construction of the sequence vectors

And amplifying the nucleotide sequence of the CSR by using a PCR technology, cloning the CSR sequence into a vector, selecting positive clones, sequencing and identifying correct plasmids. After the amplification culture, plasmids are extracted to obtain the required plasmid vectors.

Example 3 preparation of lentivirus

(1) And (3) optimizing a lentivirus expression vector, and packaging lentivirus by adopting a suspension cell system on the basis of a third generation lentivirus packaging system.

(2) The suspension cells which passed over 3 generations were adjusted to a density of 3.5-5.5X 10 24 hours before transfection⁶viable cells/ml. After 24 hours, the cells were directly diluted to 3.5X 10 with the medium⁶viable cells/ml are grown overnight, and the activity rate is more than or equal to 95 percent.

(3) Diluting the expanded cells to 4.7X 10 with fresh culture medium⁶viable cells/ml, then Supplement was added and the flask was gently swirled to mix well.

(4) DNA/transfection reagent complexes were prepared.

(5) The DNA/transfection reagent complex was slowly transferred to the shake flask, during which the flask was gently rotated.

(6) And (5) shaking culture in an incubator.

(7) 5-6h after transfection, 4% transfection enhancer is added

(8) After the virus is collected 48-55h after transfection, the collected supernatant culture medium is centrifuged at 5000rpm and 4 ℃ for 10min, the precipitate is discarded, the supernatant is left, and the supernatant is filtered by a filter membrane of 0.45 um.

(9) The virus supernatant was added to a centrifuge tube containing a sucrose solution, centrifuged at 25,000 rpm (82,700 g) for 2 hours at 4 ℃, and virus concentration was performed using an ultra-high speed centrifuge.

(10) And discarding the supernatant, and carrying out heavy suspension precipitation by using PBS (phosphate buffer solution) without calcium and magnesium to obtain the virus suspension. Subpackaging and freezing at-80 deg.C for use;

(11) and the virus titer is detected by using QPCR (quantitative polymerase chain reaction), and the virus titer can reach 8 th power of 10.

Example 4 preparation of CSR-NK cells

(1) Cord blood was obtained from healthy donors and PBMCs were isolated using lymphocyte apheresis.

(2) And (4) separating the cord blood NK cells by using an NK cell separation kit.

(3) Freshly isolated cord blood NK cells 2.0 x 10⁵The cells were cultured in 200ul of X-VIVO15 medium plus 5% FBS, and IL-2 at 40ng/mL, IL-15 at 10ng/mL, and IL-18 at 20 ng/mL for 2-3 days.

(4) Centrifuging to remove supernatant, resuspending in 200ul of X-VIVO15 medium, adding the lentivirus prepared above according to MOI value of 50 and 100, adding 1X lv-03 (12 ug/mL), 32 ℃ and 400g, centrifuging for 2h, and supplementing cytokines of IL-2 of 40ng/mL, IL-15 of 10ng/mL and IL-21 of 20 ng/mL after infection.

(5) 24 hours after infection, the medium was replaced with fresh X-VIVO15 plus 5% FBS, IL-2 at 40ng/mL, IL-15 at 10ng/mL, and IL-21 at 20 ng/mL to continue the culture.

(6) The fluid infusion is carried out every 1-2 days. Infection efficiency was measured 96 hours after infection by flow cytometry. And simultaneously, sorting CSR-NK positive cells in a flow mode and carrying out expanded culture. The color change, cell density, cell morphology of the culture medium were observed daily and recorded accordingly. As a result, as shown in FIG. 2, the infection efficiency of the virus was about 49.53%.

Example 5 evaluation of the killing Effect of CSR-NK on in vitro tumors

The killing effect of the CSR-NK cells on the target cells is detected by a luciferase method.

(1) Respectively culturing K562 cells and cord blood NK cells capable of co-expressing HLA-A1101 and RAS (G12D), growing to logarithmic growth state, centrifuging, precipitating, and counting;

(2) adding 1 × 10 parts into a 96-hole flat-bottom opaque white board⁴K562 cells co-expressing HLA-A1101 and RAS (G12D).

(3) After 24 hours, CSR-NK cells were co-cultured with K562 cells at 2:1, 5:1, 10: 1-effect target ratios.

(4) Setting a Mock cell group, and adding the NK cells which are not transduced with the virus, wherein the number of the NK cells is the same as that of the CAR-NK cells in the step (3);

(5) two controls were set simultaneously, the negative control being K562 cells co-expressing HLA-a 1101 and RAS (G12D); the positive control was the addition of 2.5% Triton-X100 to the medium, neither Mock cells nor CSR-NK cells, as the minimum and maximum background values for cell killing, i.e., Kmin and Kmax.

(6) After 24 hours of culture, the 96-well plate was centrifuged at 1500rpm for 5min, and the supernatant was collected for cytokine detection. Washing the precipitate with culture medium once and then re-suspending the cells;

(7) adding 0.5mM D-fluorescein into each hole, standing for 10min in a dark place, and detecting the fluorescence intensity on an enzyme labeling instrument;

(8) counting the fluorescence intensity value K of each well, comparing the killing efficiency of the CSR-NK and Mock NK cells to K562 cells, and calculating according to the formula: the results are shown in fig. 3, where the killing efficiency = (Kmin-K)/(Kmin-Kmax) × 100%. Compared with the Mock NK group, the killing efficiency of CSR-NK is obviously improved.

Example 6 cytokine detection assay

Multi-factor detection kit LEGENDplex using Dake^TMIFN-. gamma.was detected in the supernatant collected in example 5. As a result of analysis, as shown in FIG. 4, the amount of the CSR-NK cytokine released was about 1.5 times as much as that of NK.

The above embodiments are only for illustrating the present invention and are not to be construed as limiting the present invention. Although the present invention has been described in detail with reference to the embodiments, it should be understood by those skilled in the art that various combinations, modifications or equivalents may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and the technical solution of the present invention is covered by the claims of the present invention.

Sequence listing

<110> the first subsidiary Hospital of southern Anhui medical school (Yijieshan Hospital of southern Anhui medical school)

<120> chimeric receptor CSR based on scTv and application thereof

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Claims

1. A scTv-based chimeric receptor CSR comprising a single chain TCR antibody scTv comprising a va fragment having an alpha chain variable region and a ν β fragment having a β chain variable region, the amino acid sequence of said va fragment being as set forth in SEQ ID NO: 1, and the amino acid sequence of the V beta fragment is shown as SEQ ID NO: 2, respectively.

2. The scTv-based chimeric receptor CSR according to claim 1, wherein the V α segment and the V β segment are linked by a Linker, the amino acid sequence of which is as shown in SEQ ID NO: 3 and SEQ ID NO: 4 is shown in the specification; the IL-15 complex comprises a suicide gene CD20 epitope polypeptide, an IL-15 mutant and a truncated CD4 molecular scaffold, and the amino acid sequences of the amino acid sequence are respectively shown in SEQ ID NO: 5. SEQ ID NO: 6 and SEQ ID NO: shown at 7.

3. The scTv-based chimeric receptor CSR according to claim 1, wherein said CSR comprises a single chain TCR antibody scTv, a hinge region, a transmembrane domain, an intracellular signaling domain, an intracellular costimulatory domain, and an IL-15 complex comprising a suicide gene, connected in sequence.

4. The scTv-based chimeric receptor CSR according to claim 3, wherein the hinge region is selected from a CD8 a hinge region; the transmembrane domain is selected from the domains of CD8 alpha, CD28, CD16, NKp44, NKp46, NKG2C or NKG2D, preferably, the transmembrane domain is selected from the domains of CD8 alpha transmembrane, CD8 alpha hinge region and transmembrane region, and the amino acid sequence of the transmembrane region is shown in SEQ ID NO: 8 is shown in the specification; the intracellular costimulatory domain is selected from the CD3 zeta, CD28, 4-1BB, 2B4, DAP10, DAP12 or CD137 domain; the intracellular signaling domain is a CD3 zeta domain; preferably, the intracellular co-stimulatory domain is 4-1BB, and the amino acid sequence of the intracellular co-stimulatory domain is as shown in SEQ ID NO: shown at 9.

5. The scTv-based chimeric receptor CSR according to claim 3, wherein the IL-15 complex is linked to the intracellular costimulatory domain by a 2A linker peptide; preferably, the 2A linker peptide is P2A, and its amino acid sequence is as shown in SEQ ID NO: shown at 10.

6. The scTv-based chimeric receptor CSR according to claim 1, wherein the CSR is further linked to a signal peptide; preferably, the signal peptide is a CD8 signal peptide, and the amino acid sequence thereof is as shown in SEQ ID NO: shown at 11.

7. A nucleic acid molecule encoding the scTv-based chimeric receptor CSR of claim 1.

8. A vector comprising the nucleic acid molecule of claim 7.

9. A host cell comprising the nucleic acid molecule of claim 7 or the vector of claim 8; preferably, the host cell comprises a T cell, NK cell, γ δ T cell or NKT cell.

10. Use of a scTv-based chimeric receptor CSR according to claim 1 or a host cell according to claim 9 in the preparation of an anti-tumor medicament.