CN116253794A

CN116253794A - Antibody for CAR-T cell regulation and control and application thereof

Info

Publication number: CN116253794A
Application number: CN202210999396.XA
Authority: CN
Inventors: 陈志国; 赵宇
Original assignee: Xuanwu Hospital
Current assignee: Xuanwu Hospital
Priority date: 2022-08-19
Filing date: 2022-08-19
Publication date: 2023-06-13

Abstract

The invention relates to an antibody for regulating and controlling CAR-T cells and application thereof, wherein the antibody can identify CAR receptor molecules containing specific artificial antigen epitopes, and the antibody can realize the directional expansion of the CAR-T cells and improve the proportion of CAR positive cells in a final product by adding the antibody in the process of preparing the corresponding CAR-T; meanwhile, the CAR positive cells can be induced to differentiate into Tcm subgroups, so that the anti-tumor durability of the final product is improved. The CAR-T cells prepared by the antibody have higher in-vivo anti-tumor activity; in addition, the number of CAR-T cells in the body can also be regulated by the antibody after administration of CAR-T.

Description

Antibody for CAR-T cell regulation and control and application thereof

Technical Field

The invention relates to the field of gene therapy and cell therapy, in particular to an antibody for CAR-T cell regulation and application thereof.

Background

Chimeric antigen receptor-modified T cells (CAR-T) have achieved remarkable clinical efficacy in the treatment of B cell tumors, and two CAR-T preparations have been marketed with approval by the us FDA. However, CAR-T still presents a number of technical bottlenecks in clinical applications, which have hampered its further popularization. How to realize the preparation of stable high-purity CAR-T, and reduce the large variation caused by the difference between different batches of T cells and lentivirus batches; and how to avoid serious clinical side reactions caused by uncontrollable amplification in vivo after CAR-T feedback, etc., which are main technical problems not solved at present.

Although studies have reported that the use of inactive EGFR receptors, after co-expression with CARs, can serve as a screening marker molecule for CAR-T; the fusion CAR molecule is constructed by combining a small molecular compound with an iCas9 protein original, so that in-vivo regulation and control of the CAR-T after infusion can be realized. However, the achievement of the technical effect described above necessitates that different protein functional elements be co-expressed with the CAR receptor when constructing the CAR expression vector. According to the technical scheme, on one hand, the difficulty of constructing the CAR vector is increased, and on the other hand, the uncertainty of the modified T cell function is increased due to the fact that too many macromolecular originals are introduced. Therefore, how to achieve the preparation of high purity CAR positive T cells using a simpler method and in vivo control after CAR-T infusion remains a technical challenge in the art.

Disclosure of Invention

In order to solve the technical problems, the invention provides an antibody for CAR-T cell regulation and application thereof. The antibody can identify the CAR receptor molecules containing specific artificial antigen epitopes, and in the process of preparing the corresponding CAR-T, the antibody can realize the directional amplification of the CAR-T cells and improve the proportion of CAR positive cells in the final product; meanwhile, the CAR positive cells can be induced to differentiate into Tcm subgroups, so that the anti-tumor durability of the final product is improved.

To this end, in a first aspect, the invention provides an antibody comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR 3;

wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are selected from the group consisting of:

HCDR1：TYAMN(SEQ ID NO：5)，HCDR2：RIRSKSNNYATFYGESVRG(SEQ ID NO：6)，HCDR3：DYFGSYADY(SEQ ID NO：7)；

alternatively, HCDR1: TFAMN (SEQ ID NO: 15), HCDR2: RMRSKNNYYTTFYADSVKD (SEQ ID NO: 16), HCDR3: DYFGFNNAVDY (SEQ ID NO: 17);

the amino acid sequences of LCDR1, LCDR2 and LCDR3 are selected from the group consisting of:

LCDR1：RSSKSLLHSNGNTYLY(SEQ ID NO：8)，LCDR2：RMSNLAS(SEQ ID NO：9)，LCDR3：MQYLEYPYT(SEQ ID NO：10)；

alternatively, LCDR1: RASKSVSTSGYSYMH (SEQ ID NO: 18), LCDR2: LANLES (SEQ ID NO: 19), LCDR3: QHSRELPWT (SEQ ID NO: 20).

In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from the group consisting of:

HCDR1：TYAMN(SEQ ID NO：5)，HCDR2：RIRSKSNNYATFYGESVRG(SEQ ID NO：6)，HCDR3：DYFGSYADY(SEQ ID NO：7)；LCDR1：RSSKSLLHSNGNTYLY(SEQ ID NO：8)，LCDR2：RMSNLAS(SEQ ID NO：9)，LCDR3：MQYLEYPYT(SEQ ID NO：10)；

alternatively, HCDR1: TFAMN (SEQ ID NO: 15), HCDR2: RMRSKNNYYTTFYADSVKD (SEQ ID NO: 16), HCDR3: DYFGFNNAVDY (SEQ ID NO: 17); LCDR1: RASKSVSTSGYSYMH (SEQ ID NO: 18), LCDR2: LANLES (SEQ ID NO: 19), LCDR3: QHSRELPWT (SEQ ID NO: 20).

In some embodiments, the heavy chain variable region has an amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO: 11.

In some embodiments, the light chain variable region has an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO: shown at 12.

In one embodiment, the amino acid sequence of the heavy chain variable region is SEQ ID NO:1, the amino acid sequence of the light chain variable region is SEQ ID NO:2.

in one embodiment, the amino acid sequence of the heavy chain variable region is SEQ ID NO:11, the amino acid sequence of the light chain variable region is SEQ ID NO:12.

in some embodiments, the antibody is a full length antibody, fab fragment, F (ab) ₂ Fragments, double-chain Fv fragments or single-chain Fv fragments

In some embodiments, the antibody is a monoclonal antibody.

In some embodiments, the antibody further comprises a heavy chain constant region and a light chain constant region; the heavy chain constant region comprises the amino acid sequence as set forth in SEQ ID NO:3 or SEQ ID NO:13, an amino acid sequence shown in seq id no; the light chain constant region comprises the amino acid sequence as set forth in SEQ ID NO:4 or SEQ ID NO:14, and a polypeptide having the amino acid sequence shown in seq id no.

In one embodiment, the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO:3, and the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide having the amino acid sequence shown in (a) and (b).

In one embodiment, the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO:13, and the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO:14, and a polypeptide having the amino acid sequence shown in seq id no.

In some embodiments, the antibody has a tag protein and/or signal peptide fused to the carboxy-terminus and/or amino-terminus.

In some embodiments, the signal peptide comprises the amino acid sequence as set forth in SEQ ID NO:21 or SEQ ID NO: 22.

In a second aspect, the invention provides a nucleic acid molecule encoding an antibody according to the invention.

In a third aspect, the invention provides a vector comprising a nucleic acid molecule according to the invention.

In a fourth aspect, the invention provides a host cell expressing an antibody according to the invention, or comprising a nucleic acid molecule according to the invention, or comprising a vector according to the invention.

In a fifth aspect, the present invention provides a method of producing the antibody, comprising the steps of: culturing a host cell according to the invention under conditions allowing expression of the antibody; and isolating and purifying the antibody from the resulting culture.

In a sixth aspect, the invention also provides the use of the antibody in the preparation of chimeric antigen receptor-modified T (CAR-T) cells, the chimeric antigen receptor having a sorting domain with an amino acid sequence of SEQ ID NO:27.

in some embodiments, the method of making a chimeric antigen receptor-modified T cell comprises: the activated T cells are infected with a virus encoding a chimeric antigen receptor, and then the antibody according to the first aspect of the invention is added to the culture system.

In a seventh aspect, the invention provides the use of said antibody in the preparation of a medicament according to a) or B):

a) A medicament that modulates the number of chimeric antigen receptor-modified T (CAR-T) cells in vivo;

b) A medicament for modulating anti-tumor activity of chimeric antigen receptor-modified T (CAR-T) cells in vivo.

In previous studies, the inventors of the present invention reported a CAR molecule with an artificial epitope, and can efficiently sort target cells expressing CAR positivity by a secondary sorting method to increase the proportion of positive CAR-T cells in the final product (CN 108017717 a). Based on the research, the inventor surprisingly found that, in the preparation process, the specific activation of the T cells introduced with the CAR by using the anti-artificial epitope antibody can not only improve the proportion of the CAR positive T cells in the final product, but also remarkably improve the proportion of the Tcm in the CAR positive T cells, thereby bringing unexpected technical effects. In the research technology, the invention further develops an antibody which can specifically recognize the artificial antigen epitope E-tag and can effectively activate the corresponding CAR-T cell. It has further been found that the antibody not only can exert the above-mentioned effects in the preparation stage of CAR-T cells, but also CAR-T cells prepared by the antibody have higher in vivo antitumor activity; in addition, the number of CAR-T cells in the body can also be regulated by the antibody after administration of CAR-T.

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

1. the antibody provided by the invention can be used in the preparation of the CAR-T cells, can effectively improve the proportion of the CAR positive T cells in the product, can realize the in-vitro specific amplification of the Tcm subgroup in the CAR positive T cells, and can obviously improve the proportion of the Tcm in the final product.

2. The antibody provided by the invention is used for preparing the CAR-T cells, so that the technical problem that the Tcm cannot be cultured for a long time in vitro is effectively solved, the long-time culture of the Tcm in vitro is realized, and the yield of the Tcm is effectively improved; the Tcm proportion starts to drop from the culture of the prior art to about day 6, but the Tcm proportion can still be maintained at a higher level when the Tcm proportion is cultured to 12 days in the technical scheme of the invention.

3. The CAR-T technology has the following problems in clinical applications: how to regulate and control the CAR-T cells after reinfusion in vivo to improve the safety of the CAR-T cells; how to effectively regulate the activity of the CAR-T in vivo, and further improve the clinical anti-tumor activity of the CAR-T. The experiment proves that the antibody provided by the invention can be used for in-vivo regulation and control after CAR-T feedback, and the safety and controllability of the CAR-T technology in clinical application are improved.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. In the drawings:

fig. 1: SDS-PAGE identification results of the antibodies prepared by the invention;

fig. 2: in the preparation process of the CAR-T cells, the antibody provided by the invention is adopted to perform specific activation, and the analysis result of proliferation capacity of each group of cells is obtained;

fig. 3: in the preparation process of the CAR-T cells, the antibody provided by the invention is adopted for specific activation, and the proportion of the CAR positive T cells in the prepared cell product is calculated;

fig. 4: in the preparation process of the CAR-T cells, the antibody provided by the invention is adopted for specific activation, and the proportion of Tcm in the CAR positive T cells in the prepared cell product;

fig. 5: in the preparation process of the CAR-T cells, the antibody provided by the invention is adopted for specific activation, and the in-vitro killing activity of the prepared cells on target cells is detected;

fig. 6: the CAR-T cells prepared by the antibody provided by the invention are subjected to specific activation to obtain an evaluation result of the anti-tumor activity of the CAR-T cells in vivo;

fig. 7: the antibody provided by the invention is used for regulating and controlling the number of the CAR-T cells in the body, and the relative multiple of the CAR-T cells in peripheral blood changes along with time.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

The specific techniques and conditions are not specified in the examples, and the techniques and conditions described in the literature in the art are followed (for example, refer to J. Sam Brooks et al, edited by molecular cloning Experimental guidelines, fourth edition, scientific Press; J. E. Coli et al, edited by Cao Xuetao, edited by scientific Press, etc.) or the specifications of the products.

Example 1 preparation of chimeric antigen receptor-targeted sortable peptide monoclonal antibodies

(1) The artificial antigen epitope polypeptide and the ovalbumin are coupled by a carbodiimide (EDC) method to prepare a fusion antigen:

5mg of the target antigen polypeptide (SEQ ID NO:27, KPLPEVTDEY) and 2.5mg of ovalbumin (available from Soy Co., ltd., cat. No. A8040) were dissolved in 250. Mu.l of TE buffer (pH 8.0), and the mixture was thoroughly shaken to dissolve the mixture, and 79mg of EDC. HCl was further thoroughly dissolved in 250. Mu.l of TE buffer (pH 8.0). The mixed solution of the target antigen peptide and the ovalbumin is added into EDC solution dropwise while shaking, and the mixture reacts for more than 2 hours in a shaking table at 37 ℃. The coupled product and unbound target antigen peptide small molecules are separated by a Sephadex column, dialyzed and purified, and 0.5ml of the solution is dialyzed in PBS for 3 days, and the solution is changed twice a day, and 200ml of the solution is changed each time. The dialysis product is the prepared fusion antigen, and is split-packed in small dose and stored at-20 ℃ for standby.

(2) According to the conventional technical method for preparing monoclonal antibodies, the fusion antigen prepared in the step (1) is immunized on 5 Bal b/c mice. After the immune cycle is finished, the antigen polypeptide (amino acid sequence: KPLPEVTDEY) is taken as an antigen, ovalbumin is taken as a negative control, the titer of the serum antibody of the mice is detected, and the detection result is shown in table 1. Mice No. 4 with highest serum titer were sacrificed, spleen cells were isolated from spleen, and fused with S/P20 myeloma cell line to prepare hybridoma cells. After subcloning and screening, two positive cell strains are obtained in total, wherein the positive cell strains are respectively as follows: MM02H (SmAb-1) and MM04H (SmAb-2).

Table 1 immune mice serum titer assay

EXAMPLE 2 identification of monoclonal antibodies

Two positive cell lines obtained in example 1 were amplified and then injected into the abdominal cavity of Bal b/c mice, and the mice were sacrificed 10 days after injection to obtain ascites. And (3) centrifuging the ascites at 1200rpm, and purifying the supernatant by using a Protein-A gel column to obtain the monoclonal antibody. After precipitation, the antibodies were reconstituted in PBS and stored at-80℃for later experiments. The obtained antibody is identified by denaturing polyacrylamide gel electrophoresis (SDS-PAGE), the detection result is shown in figure 1, wherein the bands with larger molecular weight are antibody heavy chains, the bands with smaller molecular weight are antibody light chains, and the purity of the antibody can reach more than 95%. The titer was determined by ELISA, and the results are shown in Table 2, and the binding titer of both antibodies to the antigen can reach 1:1000.

Table 2 antibody titer detection

Antibody clone number	Dilution ratio	OD450 detection value-1	OD450 detection value-2	Average OD450 value
					MM02H
	1∶1000	2.734	2.632	2.683
					MM04H	1∶1000	2.911	2.764	2.836

Genome extraction is carried out on two hybridoma cells expressing the monoclonal antibody, and the sequence of the antibody coding sequence and the protein sequence are sequenced, so that the sequence information of the two monoclonal antibodies is obtained.

According to the detection result, the heavy chain amino acid sequence of MM02H (SmAb-1) is shown as SEQ ID NO:23, wherein the 1 st to 20 th positions are signal peptide, the 21 st to 140 th positions are heavy chain variable region, and the 141 st to 466 th positions are heavy chain constant region; the light chain amino acid sequence is shown in SEQ ID NO:24, wherein the 1 st to 19 th positions are signal peptide, the 20 th to 131 th positions are light chain variable region, and the 132 th to 237 th positions are light chain constant region. Specifically, the amino acid sequence of the heavy chain variable region of MM02H (SmAb-1) is shown in SEQ ID NO:1, the amino acid sequence of the light chain variable region is shown as SEQ ID NO:2 is shown in the figure; the heavy chain constant region has an amino acid sequence shown in SEQ ID NO:3, the amino acid sequence of the constant region of the light chain is shown as SEQ ID NO: 4. The variable region CDR region sequences are shown in table 3.

TABLE 3 MM02H (SmAb-1) variable region CDR region sequences

According to the detection result, the heavy chain amino acid sequence of MM04H (SmAb-2) is shown as SEQ ID NO:25, wherein the signal peptide is at positions 1-20, the heavy chain variable region is at positions 21-142, and the heavy chain constant region is at positions 143-468; the light chain amino acid sequence is shown in SEQ ID NO:26, wherein the signal peptide is at positions 1-19, the light chain variable region is at positions 20-129, and the light chain constant region is at positions 130-236. Specifically, the amino acid sequence of the heavy chain variable region of MM04H (SmAb-2) is shown in SEQ ID NO:11, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown at 12; the heavy chain constant region has an amino acid sequence shown in SEQ ID NO:13, the amino acid sequence of the constant region of the light chain is shown as SEQ ID NO: 14. The variable region CDR region sequences are shown in table 4.

TABLE 4 MM04H (SmAb-2) variable region CDR region sequences

EXAMPLE 3 preparation of chimeric antigen receptor-encoding viruses

This example prepares lentiviruses encoding CD19sCAR, CD19CAR and EGFP, respectively. The method comprises the following specific steps:

(1) Construction of expression vectors for chimeric antigen receptors:

the chimeric antigen receptor CD19sCAR can target the CD19 antigen and comprises an artificial antigen epitope E-tag (SEQ ID NO: 27), and the amino acid sequence of the CD19sCAR is shown in SEQ ID NO:28, the nucleotide sequence is shown in SEQ ID NO:29.

meanwhile, a control chimeric antigen receptor CD19CAR which does not contain artificial antigen epitope is arranged, and the amino acid sequence of the control chimeric antigen receptor CD19CAR is shown in SEQ ID NO:30, the nucleotide sequence is shown in SEQ ID NO:31.

the nucleotide sequences of the CD19sCAR and the CD19CAR are respectively subjected to full sequence synthesis by using a chemical synthesis method, and meanwhile, restriction enzyme cutting sites are added at two ends of the nucleotide sequences, and the synthesized sequences are respectively inserted into the downstream of the CMV promoter of the lentivirus expression vector pLenti6.4 containing the CMV promoter by using a gene cloning method. A lentiviral expression vector for CD19sCAR and a lentiviral expression vector for CD19CAR were obtained.

(2) Preparation of chimeric antigen receptor-encoding viruses

Preparation of chimeric antigen receptor-encoding viruses was performed using HEK293T as packaging cell. HEK293T cells in the logarithmic growth phase were digested, centrifuged at 800rpm for 5min, and the medium was discarded and resuspended in DMEM medium containing 10% FBS. After cell counting, the density of the cell suspension was adjusted to 3.6X10 ⁶ And/ml, placing in a cell incubator at 37 ℃ for later use.

Transfection of virus packaging plasmid used Lipofectamine 3000 kit (Thermo Fisher Co.) and was performed according to the kit instructions. Three plasmids required by lentiviral packaging, including a lentiviral expression vector (a CD19sCAR lentiviral expression vector, a CD19CAR lentiviral expression vector and a lentiviral vector pLenti6.4-CMV-EGFP containing EGFP coding genes prepared in the step 1), a plasmid psPAX2 encoding viral nucleocapsid proteins Gag/Pol and Rev and a plasmid pVSVG encoding viral envelope proteins, are mixed with Lipofectamine 3000 according to the recommended proportion of the specification to prepare a DNA liposome compound, and are stood for 15min at room temperature. After the standing, a 6-hole culture plate is taken, the DNA-liposome complex is added into the 6-hole plate, 1ml of each hole is added, the HEK293T cell suspension prepared before is gently mixed evenly, and then the mixture is added into the 6-hole plate and evenly mixed with the liposome complex. The culture plates were placed in an incubator for further culture, and culture supernatants containing the viruses were collected at 24h and 48h, respectively. After the last collection of the supernatant, 2000g of the supernatant is centrifuged for 10min and filtered by a filter membrane with the diameter of 0.45 mu m, so that the slow viruses respectively encoding the CD19sCAR, the CD19CAR and the EGFP are prepared, and the slow viruses are frozen at the temperature of minus 80 ℃ for standby after split charging.

EXAMPLE 4 preparation of chimeric antigen receptor-modified T cells

The highest titer antibody (MM 04H, smAb-2) detected in example 2 was selected for its ability to selectively amplify CD19sCAR modified T cells in vitro.

Isolation of mononuclear cells (PBMC) from peripheral blood by Ficol density gradient centrifugation, and adjustment of the density to 0.5X10 after PMBC was resuspended in D-PBS ⁶ /mL. The magnetic beads were added according to the ratio of T cell absolute number to CD3/CD28 magnetic beads of 1:1, and gently mixed at room temperature for 20min. And (3) sorting the cell-magnetic bead mixed suspension after uniform mixing by using a sorting magnetic frame. T cell activation is carried out on the T cells after sorting: 1X 10 with X-VIVO15 Medium containing 50ng/mL OKT-3 and 500IU/mL IL-2 ⁶ The T cells are resuspended per mL, inoculated into a culture container and cultured under the conditions of placing 37 ℃ and 5 percent CO ₂ Is cultured in a saturated humidity incubator for 24 hours. After T cell activation, cells were collected in 50mL centrifuge tubes and counted to adjust the cell density to 3X 10 ⁶ /mL。

The activated T cells were grouped according to table 5, wherein SmAb was antibody MM04H (SmAb-2) prepared in example 2, and the negative control group was a treatment group in which T cells were infected with a lentivirus encoding EGFP.

TABLE 5 Experimental component groups

Sequence number	Packet numbering	Treatment and culture method
			1	Experimental group a1	Specific activation with SmAb after CD19sCAR infection
2	Experiment group a2	Post infection CD19sCARSpecific activation with SmAb
			3	Experiment group a3	Specific activation with SmAb after CD19CAR infection
4	Experiment group a4	Specific activation without SmAb after CD19CAR infection
			5	Experiment group a5	Negative control group

After grouping the activated T cells according to table 5, lentiviruses encoding CD19sCAR, CD19CAR and EGFP prepared in example 3 were infected, respectively. 24h after infection, the cells of each treatment group were transferred to X-VIVO15 medium containing 80ng/mL IL-7, 500IU/mL IL-2 and 20ng/mL IL-15 for culture at 37℃with 5% CO ₂ Is a saturated humidity incubator. On the 6 th day of cultivation, the experimental group a1 and the experimental group a3 were subjected to an activation treatment according to the treatment mode of the experimental group: cells of experimental group a1 and experimental group a3 were transferred to a culture vessel coated with 20. Mu.g/mL SmAb antibody, and culture was continued with X-VIVO15 medium containing 80ng/mL IL-7, 500IU/mL IL-2 and 20ng/mL IL-15 under the same conditions as above.

Cells were fed every 2-3 days during the culture, depending on the growth of the cells. Thereafter, each group of cells was further cultured until day 12, and cell preparations were harvested, respectively.

The experiments of 3 independent batches were counted and the following analytical tests were performed on the end products of each experimental group:

the proliferation capacity of the cells of each experimental group is analyzed, the analysis result is shown in fig. 2, and as can be seen from fig. 2, after the cells of the experimental group a1 are stimulated and activated by SmAb, the proliferation level is obviously better than that of the cells of other experimental groups; the final products of each experimental group are subjected to flow detection analysis, the detection result is shown in fig. 3, and as can be seen from fig. 3, the ratio of CAR positive T cells in the final products of the experimental group a1 is obviously higher than that in other experimental groups, which indicates that the SmAb is applied to the preparation of sCAR-T, so that the amplification of the CAR positive T cells can be promoted, and the ratio of the CAR positive T cells in the final products can be improved.

Example 5 Effect of SmAb on the in vitro differentiation ability of sCAR modified T cells

Subjecting each group of T cells obtained in example 4 after 12 days of culture to flow detection analysis, and detecting proportion of different T cell subsets in final product, including central memory type T cells (Tcm), effector memory type T cells (Tem), and initial type T cells

T) and terminally differentiated T cells (ttes). Molecular markers that sort different T cells include CD3, CD45RO, CD197, and sCAR. />

The detection result is shown in fig. 4, and the Tcm proportion in the end product obtained in the experimental group a1 is significantly higher than that in other experimental groups, which indicates that the application of SmAb in the preparation of sCAR-T can effectively promote the differentiation of CAR positive T cells to central memory type T cells and improve the Tcm proportion in the end product.

Example 6 Effect of SmAb on in vitro killing Activity of sCAR modified T cells

Human lymphoma cell line raji was used as target cell according to 5×10 ⁴ inoculating/mL to a U-shaped bottom 96-well plate, co-culturing CAR-T cells of each experimental group obtained in 12 days of culture in example 4 according to the ratio of effective target ratio (E/T) of 25:1, 12.5:1, 6.25:1 and 1:1, wherein the culture medium is RPMI1640 culture medium without serum, and the culture condition is 37 ℃ and 5% CO ₂ After culturing for 12 hours in a saturated humidity incubator, the killing activity of CAR-T cells of different experimental groups on target cells is detected by a lactate dehydrogenase releasing method (LDH method), and the detection result is shown in figure 5. As can be seen from the experimental results of fig. 5, the killing activity of the cell product obtained by applying SmAb to the preparation of sCAR-T was significantly better than that of the other experimental groups.

Example 7 modulation of sCAR modified T cell anti-tumor Activity by SmAb

Selection of 6-8 week old NOD/SCID IL2Rγc-/-immunodeficient mice human Raji cell line (Raji-Luci) stably expressing luciferase protein was used at 10 ⁶ A tumor-bearing mouse model was established by tail vein injection at a dose of 100. Mu.L/dose. 3 days after injection of tumor cells, the experimental groups were according to example 4, each group of experimental animals being according to 1X 10 through the tail vein ⁶ 100. Mu.L/injection of the CAR-T cell preparation prepared in example 4 alone. Tumor progression in experimental animals was observed weekly after injection using a small animal imaging system until all experimental animals died. The statistical analysis result of the experimental data is shown in fig. 6, and compared with other experimental groups, the CAR-T cells obtained in the experimental group a1 can effectively prolong the median survival time of tumor-bearing mice, delay the in-vivo progress of tumors, and have longer in-vivo anti-tumor activity, so that the application of SmAb to the preparation of sCAR-T can effectively improve the in-vivo anti-tumor activity of the prepared CAR-T cell products.

Example 8 in vivo modulation of sCAR-modified T cells by SmAb

(1) The SmAb antibody is coupled with a micropipe-like small molecular medicine Maytansinoid (DM 1) to prepare SmAb-DM1. The method comprises the following specific steps:

the MM04H (SmAb-2) antibody was dissolved at 1mg/mL in PBS containing 1.8N DTT and 1mM DTPA, and the buffer was adjusted to pH 8.0 with 50mM boric acid, and incubated at 37℃for 1 hour to effect reductive denaturation of the antibody. The reduced antibody solution was equilibrated to 4℃and purified by a G25 desalting column chromatography column. The purified denatured antibody, thioether linker-DM 1 was mixed in a ratio of 2.4:4.6, reacted at 4℃for 1 hour, and after the reaction, the reaction was terminated by adding 20 volumes of cysteine. The reaction mixture was purified by G25 desalting column, and the final product was SmAb conjugated with DM1, and SmAb-DM1 antibody was stored at 5mg/mL at 4deg.C for subsequent experiments.

(2) NOD/SCID IL2 Ryc-/-immunodeficient mice were grouped according to Table 6, mice from different experimental groups were grouped according to 1X 10 ⁷ 200 μl/different CAR-T cell preparations were injected by tail vein alone, 2h after injection,mouse peripheral blood was collected as baseline data, and then SmAb 1mg/kg, smAb-DM 11 mg/kg or PBS was injected via the tail vein, respectively, according to the experimental group. Mouse peripheral blood was collected 24h after injection and CAR-T cells in the samples were detected using flow. The molecular markers used for detection were CD45, CD3 and CAR. The results of the assay are shown in FIG. 7, where the proportion of CAR-T cells in mice infused with CD19sCAR-T and administered SmAb-DM1 decreased significantly over the time of assay, indicating that SmAb-DM1 was effective in clearing sCAR-T cells in vivo.

TABLE 6 grouping of animal experiments

Sequence number	Packet numbering	Treatment and culture method
			1	Experimental group b1	Post-reinfusion CD19sCAR-T injection of SmAb-DM1
2	Experimental group b2	Post-reinfusion CD19sCAR-T injection of SmAb
			3	Experimental group b3	PBS was injected after reinfusion of CD19sCAR-T
4	Experiment group b4	Post-reinfusion CD19CAR-T injection of SmAb-DM1
			5	Experiment group b5	Post-reinfusion CD19CAR-T injection of SmAb
6	Experiment group b6	PBS was injected after reinfusion of CD19CAR-T
			7	Experimental group b7	Negative control group

The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. An antibody comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR 3;

2. The antibody of claim 1, wherein the heavy chain variable region has an amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO: 11;

preferably, the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO:2 or SEQ ID NO: shown at 12.

3. The antibody of claim 1, wherein said antibody is a full length antibody, fab fragment, F (ab) ₂ Fragments, double-chain Fv fragments or single-chain Fv fragments.

4. The antibody of claim 1, wherein the antibody is a monoclonal antibody.

5. The antibody of claim 1, further comprising a heavy chain constant region and a light chain constant region;

preferably, the heavy chain constant region comprises the amino acid sequence as set forth in SEQ ID NO:3 or SEQ ID NO:13, an amino acid sequence shown in seq id no;

preferably, the light chain constant region comprises the amino acid sequence as set forth in SEQ ID NO:4 or SEQ ID NO:14, and a polypeptide having the amino acid sequence shown in seq id no.

6. The antibody of claim 1, wherein the antibody has a tag protein and/or a signal peptide fused to the carboxy-terminus and/or amino-terminus of the antibody;

preferably, the signal peptide comprises the amino acid sequence as set forth in SEQ ID NO:21 or SEQ ID NO: 22.

7. A product, characterized in that the product is i), ii) or iii) below,

i) A nucleic acid molecule encoding the antibody of any one of claims 1-6;

ii) a vector comprising the nucleic acid molecule as described in i).

iii) A host cell expressing the antibody of any one of claims 1-6, or comprising the nucleic acid molecule of i), or comprising the vector of ii).

8. The method for producing an antibody according to any one of claims 1 to 6, comprising the steps of: culturing the host cell of claim 7 under conditions allowing expression of the antibody; and isolating and purifying the antibody from the resulting culture.

9. Use of the antibody of any one of claims 1-6 for the preparation of chimeric antigen receptor-modified T cells, said chimeric antigen receptor having a sorting domain with an amino acid sequence of SEQ ID NO:27.

10. use of the antibody of any one of claims 1-6 for the preparation of a) or B) medicament:

a) A medicament for regulating the number of chimeric antigen receptor-modified T cells in vivo;

b) A medicament for regulating the anti-tumor activity of chimeric antigen receptor modified T cells in vivo.