TW202328440A - Preparation method for universal car-t cells, and application thereof - Google Patents

Abstract

The present invention relates to the technical field of CAR-T cell therapy, and provides a preparation method for universal CAR-T cells, and an application thereof. A VH structural domain and a VL structural domain of a [beta]2M binding molecule provided comprise CDR sequences, which can specifically bind to [beta]2M. The binding molecule is added to the CAR-T cells, and the binding molecule can specifically bind to the CAR-T cells without knockout of a [beta]2M gene, so that the CAR-T cells with the [beta]2M gene knocked out is separated from the CAR-T cells without knockout of the [beta]2M gene, and purification of the universal CAR-T cells is achieved.

Description

Methods for preparing universal CAR-T cells and their applications

[Priority Statement]

This invention requires the priority of the Chinese invention patent application with the application number 202111253059.8, the filing date being October 27, 2021, and the invention title being "A method of preparing universal CAR-T cells and its application", the entire content of which is obtained by Incorporated into this article by reference.

The present invention relates to the technical field of CAR-T cell therapy, and in particular to a method for preparing universal CAR-T cells and its application.

Since August 2017, the U.S. FDA has approved two CAR-T products targeting CD19 (kyriamh from Novartis Pharmaceuticals and yescarta from Kate Pharmaceuticals). Therefore, the demand for CAR-T cells in the treatment of hematological tumors continues to increase. However, the current adoptive cell therapy is still based on the infusion of autologous cells. Each patient has to prepare CAR-T cells individually. The operation is complicated and the production and preparation cost is very expensive. The listing price of Novartis Pharma's kyriamh product is 475,000 per case. US dollars, Kate Pharmaceuticals’ yescarta also costs US$375,000. Most cancer patients have undergone repeated treatments in other ways before CAR-T cell therapy, especially radiotherapy and chemotherapy. This has resulted in very few T lymphocytes obtained from their peripheral blood, and these T lymphocytes are very rare in vitro. It is difficult to expand and prepare autologous CAR-T cells. In addition, many infant patients and critically ill patients are unable to obtain sufficient T lymphocytes from the patient's peripheral blood before preparing CAR-T cells. Therefore, allogeneic CAR-T cells (i.e., universal CAR-T cells) can not only greatly reduce the production cost of CAR-T cells, but also provide treatment opportunities for patients who cannot prepare autologous CAR-T cells. Universal CAR-T cell therapy has natural advantages over autologous CAR-T cell therapy, so its research and development has attracted increasing attention.

With the continuous improvement of gene editing technology, the efficiency of knocking out the β2M gene of T cells has become increasingly higher. However, gene editing operations have a great impact on the status and growth rate of T cells. Generally, the higher the knockout efficiency of the β2M gene, the worse the status of the T cells after gene editing and the slower the cell growth rate. Therefore, in order to balance the two, the β2M gene knockout efficiency will be controlled to a certain extent during the preparation process of universal CAR-T cells. Therefore, if the prepared universal CAR-T cells are to be used for clinical cell therapy, T cells that have not deleted the β2M gene must be removed. Otherwise, graft-versus-host disease (GvHD) or host-versus-graft disease (HvGD) may occur during clinical application.

In view of this, the present invention is proposed.

The object of the present invention is to provide a β2M binding molecule. This binding molecule can be used to remove T cells that have not deleted the β2M gene from universal CAR-T cells and achieve purification of universal CAR-T cells, thereby alleviating or avoiding graft-versus-host disease (GvHD) or host-versus-graft. disease (HvGD).

Another object of the present invention is to provide the application of the above-mentioned β2M binding molecule in detecting β2M and purifying universal CAR-T cells, and in preparing products for detecting β2M or purifying universal CAR-T cells. The products include preparations or kits that can be commercialized.

Another object of the present invention is to provide a method for universal CAR-T cell purification using the above preparation or kit. This method has good purification effect, is easy to operate and is suitable for promotion.

Another object of the present invention is to provide a method for preparing universal CAR-T cells using the above-mentioned β2M binding molecules. It is hoped that universal CAR-T cells that meet clinical purity requirements can be directly obtained, avoiding the cumbersome purification process before application.

In order to solve the above technical problems and achieve the above objects, the present invention provides the following technical solutions.

In a first aspect, the present invention provides a β2M binding molecule, which includes module a that specifically binds to β2M; The module a includes a VH structural domain, and the VH structural domain includes CDR-H1 having the amino acid sequence shown in SEQ ID No. 1, CDR-H2 having the amino acid sequence shown in SEQ ID No. 2, and CDR-H3 having the amino acid sequence shown in SEQ ID No. 3.

In an optional embodiment, the module a also includes a VL domain, the VL domain includes CDR-L1 with the amino acid sequence shown in SEQ ID No. 4, and CDR-L1 with the amino acid sequence of AAA -L2 and CDR-L3 having the amino acid sequence shown in SEQ ID No. 5.

On the other hand, the present invention also provides a β2M binding molecule, which includes module a that specifically binds to β2M; The module a includes a VH structural domain and a VL structural domain, and the VH structural domain includes CDR-H1, CDR-H2 and CDR-H3; The VL domain includes CDR-L1, CDR-L2 and CDR-L3; Wherein, the amino acid sequences of the CDR-H1, the CDR-H2 and the CDR-H3 include the complementarity determining region sequence of SEQ ID No. 6; The amino acid sequences of the CDR-L1, the CDR-L2 and the CDR-L3 include the complementarity determining region sequence of SEQ ID No. 7.

In an alternative embodiment, the amino acid sequences of the above-mentioned CDR-H1, the above-mentioned CDR-H2 and the above-mentioned CDR-H3 are defined by SEQ ID No. 6 according to the Kabat, Chothia, or AbM numbering system; The amino acid sequences of the above-mentioned CDR-L1, the above-mentioned CDR-L2 and the above-mentioned CDR-L3 are defined by SEQ ID No. 7 according to the Kabat, Chothia, or AbM numbering system.

In an optional embodiment, the VH domain of module a has the amino acid sequence shown in SEQ ID No. 6.

In an optional embodiment, the VL domain of module a has the amino acid sequence shown in SEQ ID No. 7.

In an alternative embodiment, the β2M binding molecule is selected from scFv molecules, Fv molecules, Fab molecules or intact antibody molecules that specifically bind to β2M antigen.

On the other hand, the present invention provides the use of the β2M binding molecule described in any of the preceding embodiments in the preparation of β2M detection products, or the use in in vitro detection of β2M not for the purpose of disease diagnosis or treatment.

On the other hand, the present invention provides the use of the β2M binding molecule according to any one of the preceding embodiments in purifying universal CAR-T cells or preparing products for purifying universal CAR-T cells.

On the other hand, the present invention provides a preparation for detecting β2M or a preparation for purifying universal CAR-T cells. The preparation includes the β2M binding molecule according to any one of the preceding embodiments, and the β2M binding molecule is preferably coupled. Contains biotin.

In an optional embodiment, the preparation for purifying universal CAR-T cells further includes a second binding molecule that specifically binds to universal CAR-T cells.

In an alternative embodiment, the second binding molecule is preferably coupled with biotin.

In an optional embodiment, the second binding molecule is selected from other molecules capable of specifically binding to TCR-positive CAR-T cells.

As for the dosage ratio of the second binding molecule and the β2M binding molecule, those skilled in the art can make routine selections based on actual needs.

In an optional embodiment, the target protein that specifically binds to the second binding molecule is TCR or CD3.

In an alternative embodiment, the second binding molecule includes a module c that specifically binds to CD3; the module c includes a VH domain having the amino acid sequence shown in SEQ ID No. 8, and A VL domain having the amino acid sequence shown in SEQ ID No. 9.

On the other hand, the present invention provides a kit for purifying universal CAR-T cells, which kit includes the preparation for purifying universal CAR-T cells described in the previous embodiment and optional consumables.

On the other hand, the present invention provides a kit for detecting β2M, which kit includes the preparation for detecting β2M described in the previous embodiment and optional consumables.

On the other hand, the present invention provides a preparation for purifying universal CAR-T cells as described in any of the preceding embodiments or purifying universal CAR-T cells using a kit for purifying universal CAR-T cells as described in any of the preceding embodiments. A method for CAR-T cells, which includes: culturing the universal CAR-T cells to be purified in a preparation for purifying universal CAR-T cells, and then using anti-biotin-coupled magnetic beads for magnetic adsorption. , take the cell suspension and obtain purified universal CAR-T cells.

In the present invention, the term "antibiotin-coupled magnetic beads" refers to magnetic beads coupled with a material capable of specifically binding biotin, such as magnetic beads coupled with avidin.

On the other hand, the present invention provides a method for preparing universal CAR-T cells, including the following steps: (1) Preparing the β2M binding molecule and optional anti-CD3 antibody described in the previous embodiment; (2) The β2M binding molecule and anti-CD3 antibody are respectively coupled to biotin; (3) Knock out the β2M gene and TCR gene of CAR-T cells; (4) Biotin-conjugated β2M binding molecules and anti-CD3 antibodies were co-cultured with gene-knocked-out CAR-T cells; (5) Use anti-biotin-coupled magnetic beads for magnetic adsorption, and take the cell suspension to obtain purified universal CAR-T cells.

In a ninth aspect, the present invention provides a method for purifying universal CAR-T cells, which includes the step of using the β2M binding molecule as described above to contact the universal CAR-T cells to be purified.

The VH domain and VL domain of the β2M binding molecule provided by the invention contain specific CDR sequences and can achieve specific binding with β2M. The binding molecule is added to CAR-T cells with incomplete β2M gene knockout. The binding molecule can specifically bind to CAR-T cells without β2M gene knockout, thereby separating CAR-T cells with β2M gene knockout from CAR-T cells without β2M gene knockout to achieve universal CAR-T cells. of purification.

The invention provides a method for purifying universal CAR-T cells using preparations or kits containing the above-mentioned β2M binding molecules. After the universal CAR-T cells are purified by the β2M binding molecules provided by the invention, the universal CAR-T cells can be significantly improved. T cell purity. Especially when used in combination with the second binding molecule whose target protein is CD3, the purity of the universal CAR-T cells can be as high as 99.96%, and the characterization of the tumor killing activity in vitro and in vivo has proved that the universal CAR-T cells provided by the present invention have The purification method did not cause adverse effects on universal CAR-T cells.

The invention also provides a method for preparing universal CAR-T cells. The preparation method uses the β2M binding molecule provided by the invention and optional anti-CD3 antibodies. After knocking out the β2M gene and the TCR gene, in the CAR-T cells Without separation, they can be directly co-cultured with them and then isolated to obtain universal CAR-T cells, eliminating the need for an independent purification process and reducing the labor intensity of front-line staff.

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments These are some embodiments of the present invention, rather than all embodiments. The elements of the embodiments of the invention generally described and illustrated in the drawings herein may be arranged and designed in a variety of different configurations.

Therefore, the following detailed description of the embodiments of the invention provided in the drawings is not intended to limit the scope of the claimed invention, but rather to represent selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

It should be noted that similar symbols and letters represent similar items in the following figures, therefore, once an item is defined in one figure, it does not require further definition or explanation in subsequent figures.

The term "amino acid" refers to naturally occurring carboxy alpha-amino acids. Naturally occurring amino acids include alanine (three-letter code: Ala, one-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D ), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), iso Leucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro , P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

The term "universal CAR-T cells" generally refers to T cells obtained from healthy volunteers whose related genes (mainly genes related to GVHD and HvGD diseases, such as those encoding TCR (such as alpha chain, beta chain)) have been knocked out. gene), HLA (such as the gene coding for β2 microglobulin (β2M gene)), or the coding gene for molecules such as CD52), and CAR-T cells made after transferring the CAR (chimeric antigen receptor) gene.

In order to eliminate CAR-T cells that have not deleted the β2M gene, the present invention uses β2M protein as an antigen and starts from the antibody structure to construct a binding molecule that specifically binds to the β2M protein, that is, a β2M binding molecule.

In a first aspect, in a specific embodiment, the present invention provides a module (a1) that specifically binds to β2M. The module (a1) includes a VH structural domain, and the VH structural domain includes a component with SEQ ID No. .CDR-H1 having the amino acid sequence shown in SEQ ID No. 1, CDR-H2 having the amino acid sequence shown in SEQ ID No. 2 and CDR-H3 having the amino acid sequence shown in SEQ ID No. 3.

Among them, SEQ ID No. 1-3 are defined by the heavy chain variable region sequence according to the IMGT numbering system.

In another specific embodiment, the present invention provides a module (a2) that specifically binds to β2M. The module (a2) includes a VL domain, and the VL domain includes a component with the protein shown in SEQ ID No. 4. CDR-L1 has the amino acid sequence, CDR-L2 has the amino acid sequence AAA, and CDR-L3 has the amino acid sequence shown in SEQ ID No. 5.

Among them, SEQ ID No. 4, AAA of CDR-L2, and SEQ ID No. 5 are defined by the light chain variable region sequence according to the IMGT numbering system.

In another specific embodiment, the present invention provides a module (a3) that specifically binds to β2M. The module (a3) includes a VH domain and a VL domain. The VH domain includes a protein having SEQ ID No. .CDR-H1 having the amino acid sequence shown in SEQ ID No. 1, CDR-H2 having the amino acid sequence shown in SEQ ID No. 2 and CDR-H3 having the amino acid sequence shown in SEQ ID No. 3, the VL The structural domain includes CDR-L1 having the amino acid sequence shown in SEQ ID No. 4, CDR-L2 having the amino acid sequence AAA and CDR-L3 having the amino acid sequence shown in SEQ ID No. 5.

On the other hand, in another specific embodiment, the amino acid sequences of the CDR-H1, the CDR-H2 and the CDR-H3 include the complementarity determining region sequence of SEQ ID No. 6; The amino acid sequences of the CDR-L1, the CDR-L2 and the CDR-L3 include the complementarity determining region sequence of SEQ ID No. 7.

In another specific embodiment, the above-mentioned CDR-H1, CDR-H2 and CDR-H3 can also be defined by SEQ ID No. 6 according to the Kabat, Chothia, or AbM numbering system.

In another specific embodiment, the amino acid sequences of the above-mentioned CDR-L1, the above-mentioned CDR-L2 and the above-mentioned CDR-L3 can also be defined by SEQ ID No. 7 according to the Kabat, Chothia, or AbM numbering system.

The above complementarity determining regions are defined according to the sequence of Kabat, Chothia, or AbM numbering system, as shown in the following table: Heavy chain variable region: SEQ ID No.6 Light chain variable region: SEQ ID No.7 CDR-H1 CDR-H2 CDR-H3 CDR-L1 CDR-L2 CDR-L3 Kabat SYVLH (SEQ ID No.12) YFNPYNDGTKYNEKFKG (SEQ ID No.15) RGNTYDNFDY (SEQ ID No.18) LASQTIGTWLA (SEQ ID No.19) AAASLAD(SEQ ID No.20) QQLYSSPLT（SEQ ID No.21） Chothia GYTFSSY（SEQ ID No.13） NPYNDG (SEQ ID No.16) RGNTYDNFDY (SEQ ID No.18) LASQTIGTWLA (SEQ ID No.19) AAASLAD(SEQ ID No.20) QQLYSSPLT（SEQ ID No.21） ikB GYTFSSYVLH（SEQ ID No.14） YFNPYNDGTK (SEQ ID No.17) RGNTYDNFDY (SEQ ID No.18) LASQTIGTWLA (SEQ ID No.19) AAASLAD(SEQ ID No.20) QQLYSSPLT（SEQ ID No.21）

IMGT is based on the numbering system of the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.

Kabat immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991 ).

Chothia Immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying CDR region boundaries based on the position of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. Man (1989) Nature 342:878‑883).

The definition of AbM comes from Martin's related research (Martin ACR, Cheetham JC, Rees AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268–9272). This definition method integrates Kabat and Chothia Partial definition of both.

Combined with the amino acid sequences containing CDRs in each module described in the first aspect, in the second aspect, in a specific implementation, the present invention provides the following modules: Module (a11) includes a VH domain having the amino acid sequence shown in SEQ ID No. 6. Module (a21) includes a VL domain having the amino acid sequence shown in SEQ ID No. 7. The module (a31) includes a VH domain having the amino acid sequence shown in SEQ ID No. 6 and a VL domain having the amino acid sequence shown in SEQ ID No. 7.

In combination with each module provided in the second aspect, in the third aspect, the present invention provides a scFV molecule, which includes a VH domain and a VL domain connected by an elastic linker peptide (Linker); the VH domain has SEQ The amino acid sequence shown in ID No. 6, the VL domain has the amino acid sequence shown in SEQ ID No. 7.

The amino acid residue composition and length of the Linker can be obtained by those skilled in the art through conventional means according to actual needs, including but not limited to glycine (glycine) with a length of 15 to 25 amino acid residues. Gly) and serine (Ser).

Exemplary Linkers include: (GGGGS)n (SEQ ID No. 22), (GGGS)n (SEQ ID No. 23), (GGS)n (SEQ ID No. 24), or (GS)n (SEQ ID No. 25) where n is selected from 1, 2, 3, 4, 5 or 6. In a fourth aspect, the present invention provides an Fv molecule, which includes a VH domain and a VL domain connected by a short peptide; the VH domain has the amino acid sequence shown in SEQ ID No. 6, and the VL The structural domain has the amino acid sequence shown in SEQ ID No. 7.

The short peptides can be obtained by those skilled in the art through conventional means according to actual needs, including but not limited to short peptide chains composed of 3 to 9 amino acid residues.

In a fifth aspect, the invention provides Fab molecules, which include the VH domain, the VL domain, a light chain constant region (CL) and a heavy chain constant region (CH1); the VH domain It has the amino acid sequence shown in SEQ ID No. 6, and the VL domain has the amino acid sequence shown in SEQ ID No. 7.

For CH1 and CL, those skilled in the art can select according to actual needs. For example, the CH1 is selected from any one or more of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE or IgM, and the CL is selected from kappa chain or lambda chain. In addition, those skilled in the art can also adjust the sequences and modifications of the above selected CH1 and CL by conventional means according to actual needs.

In a sixth aspect, the present invention also provides a complete antibody molecule, the complete antibody molecule includes 2 identical heavy chains and 2 identical light chains, the heavy chain includes a VH domain and a heavy chain constant region, and the light chain The chain includes a VL domain and a light chain constant region; the VH domain has the amino acid sequence shown in SEQ ID No. 6, and the VL domain has the amino acid sequence shown in SEQ ID No. 7.

For the specific sequence composition of the heavy chain constant region and the light chain constant region, those skilled in the art can choose according to actual needs. For example, the heavy chain constant region can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE or Any one or more of IgM, the light chain constant region can be selected from kappa chain or lambda chain.

In a specific embodiment, the heavy chain constant region and light chain constant region of the intact antibody molecule are selected from murine IgG.

In addition, those skilled in the art can also adjust the sequences or modifications of the above-selected heavy chain constant regions and light chain constant regions by conventional means according to actual needs.

In the seventh aspect, the present invention provides the application of each module, scFV molecule, Fv molecule, Fab molecule or complete antibody molecule described in the aforementioned embodiments in the preparation of β2M detection products, or in the in vitro detection of β2M not for the purpose of disease diagnosis. applications in.

In the eighth aspect, the present invention provides each module, scFV molecule, Fv molecule, Fab molecule or complete antibody molecule described in the aforementioned embodiments for purifying universal CAR-T cells or preparing products for purifying universal CAR-T cells. applications in.

In a specific embodiment, the genes knocked out in the universal CAR-T cells include at least the β2M gene.

In a specific embodiment, the genes knocked out in the universal CAR-T cells also include TCR genes. For example, it may be the gene encoding the α chain of the TCR molecule or the gene encoding the β chain thereof.

In a ninth aspect, in a specific embodiment, the present invention provides a preparation for purifying universal CAR-T cells, which preparation includes each module, scFV molecule, Fv molecule, Fab molecules or complete antibody molecules; each module, scFV molecule, Fv molecule, Fab molecule or complete antibody molecule is coupled with biotin. It can be understood that the conjugated biotin is a magnetic bead adsorption separation method commonly used in this field.

In a tenth aspect, in a specific embodiment, the present invention provides a kit for purifying universal CAR-T cells, which kit includes the preparations and consumables described in the previous embodiment. It can be understood that the consumables include conventional kit components in the art, such as well plates, reaction vessels, liquid extraction devices, etc.

In an eleventh aspect, in a specific embodiment, when the gene knocked out of the universal CAR-T cell also includes a TCR gene; the preparation or the kit further includes a second binding molecule, The second binding molecule is coupled to biotin. The second binding molecule can specifically bind to TCR-positive CAR-T cells.

The target protein of the second binding molecule is TCR or CD3, and can be selected from CD3 antibodies or TCR antibodies, for example. As for the dosage ratio of the second binding molecule and the β2M binding molecule, those skilled in the art can make routine selections based on actual needs.

In another specific embodiment, the target protein that specifically binds to the second binding molecule is TCR.

In another specific embodiment, the target protein that specifically binds to the second binding molecule is CD3, and the second binding molecule includes module c that specifically binds to CD3.

The module c includes a VH domain having an amino acid sequence shown in SEQ ID No. 8, and a VL domain having an amino acid sequence shown in SEQ ID No. 9.

In a twelfth aspect, the present invention provides a method for purifying universal CAR-T cells in a specific embodiment. The method uses the above-mentioned preparation or kit to place the universal CAR-T cells in the preparation and culture them. , use anti-biotin-coupled magnetic beads for magnetic adsorption, take the cell suspension, and obtain purified universal CAR-T cells.

In another specific embodiment, the present invention provides a method for preparing universal CAR-T cells, including the following steps: (1) Preparing the β2M binding molecule and optional anti-CD3 antibody described in the previous embodiment; (2) The β2M binding molecule and anti-CD3 antibody are respectively coupled to biotin; (3) Knock out the β2M gene and TCR gene of CAR-T cells; (4) Biotin-conjugated β2M binding molecules and anti-CD3 antibodies were co-cultured with gene-knocked-out CAR-T cells; (5) Use anti-biotin-coupled magnetic beads for magnetic adsorption, and take the cell suspension to obtain purified universal CAR-T cells.

In a thirteenth aspect, in a specific embodiment, the present invention provides a method for purifying universal CAR-T cells, which includes: using any of the above modules, Fv molecules, Fab molecules, complete antibody molecules, or preparations and to-be- Purify universal CAR-T cells to isolate and remove CAR-T cells without β2M gene knockout.

In another specific embodiment, when the desired universal CAR-T cell also has the TCR gene knocked out, the purification method may also include using a second binding molecule to contact the universal CAR-T cell to be purified to separate and remove it. CAR-T cells without TCR gene knockout (i.e., CAR-T cells with positive TCR expression).

In another specific embodiment, the target protein of the second binding molecule can be TCR or CD3. On CAR-T cells that have not deleted the TCR gene, TCR and CD3 exist in the form of a complex. The use of binding molecules that bind TCR or CD3 can achieve the removal of CAR-T cells that have not deleted the TCR gene.

In another embodiment, the sequence of use of the second binding molecule can be after, before, or both at the same time as using any of the above-mentioned modules, Fv molecules, Fab molecules, intact antibody molecules, or preparations.

For example, use any of the above modules, Fv molecules, Fab molecules, complete antibody molecules or preparations, and the second binding molecule to contact the universal CAR-T cells to be purified at the same time; For example, after using any of the above modules, Fv molecules, Fab molecules, intact antibody molecules or preparations to contact the universal CAR-T cells to be purified, then continue to use the second binding molecule to contact the universal CAR-T cells to be purified; For example, after using the second binding molecule to contact the universal CAR-T cells to be purified, then use any of the above modules, Fv molecules, Fab molecules, complete antibody molecules or preparations to contact the universal CAR-T cells to be purified.

In another specific embodiment, the second binding molecule includes module c that specifically binds to CD3; The module c includes a VH domain having the amino acid sequence shown in SEQ ID No. 8, and a VL domain having the amino acid sequence shown in SEQ ID No. 9. Some embodiments of the present invention will be described in detail below with reference to the drawings. The following embodiments and features in the embodiments may be combined with each other without conflict.

The modules, scFV molecules, Fv molecules, Fab molecules and complete antibody molecules provided in the above first aspect can be obtained through artificial synthesis. The complete antibody molecules can also be obtained by constructing hybridoma cells and expressing and secreting them.

Example 1

This embodiment provides a method for preparing an anti-human β2M antibody. The specific steps are as follows.

Mix equal amounts of recombinant β2M protein antigen (produced by Shenzhen Feipeng Biotechnology Co., Ltd., Cat. No. BA-PAB-MU0001) with Freund's adjuvant (sigma, Cat. No. F5881) for emulsification. Healthy BALB/C mice (Guangdong Experimental Animal Center, 6 weeks old, female) were injected subcutaneously at multiple points on the back; 14 days later, the intraperitoneal immunization was boosted; after four boosted immunizations, mouse tail blood was collected for titer testing, and the efficacy was selected. The mouse spleen with the highest price was used for cell fusion with mouse myeloma cells SP2/0.

On the third day after the final immunization of the mouse, remove the spleen of the mouse under sterile conditions, place it in a petri dish, rinse it once with RPMI1640 (gibco, Cat. No. 12633012) basic culture medium, place it on a nylon mesh in a small beaker and grind it to make a cell suspension. . Centrifuge, discard the supernatant, and resuspend in RPMI1640 culture medium. Mix the spleen cells and the mouse myeloma cells SP20 prepared in advance at a ratio of 10:1, then use 1ml of 50% PEG1500 (sigma, product number 81210) for cell fusion. After 1 minute of fusion, add 15ml of RPMI1640 complete culture medium Terminate cell fusion. Centrifuge at 1000rpm for 5 minutes, discard the supernatant, gently resuspend in 50ml of RPMI1640 screening culture medium, divide equally into 10 96-well plates, 100μl/well, culture at 37°C, 5% _CO2 . On the 6th day of culture, the HAT culture medium was changed twice. The next day, the cell supernatant was taken and detected by ELISA using human β2M antigen. The cells corresponding to the positive wells were single-selected and colonized using the limiting dilution method; after three sub-selections, multiple cell lines that could stably secrete monoclonal antibodies were screened, one of which was named β2M-2B1 cell line.

Total RNA was extracted from 1 to 5 million subselected hybridoma cells of the β2M-2B1 cell line using the QIAGEN RNAeasy Mini kit, and then the SuperScript III RT kit (Invitrogen) was used to generate first-strand cDNA. Conventional primers for mouse IgG heavy chain (IgG1, IgG2a, IgG2b) and light chain (κ or λ) constant regions are used to react and amplify the double-stranded cDNA of the anti-human β2M IgG variable region by PCR. The PCR cycle conditions are 95°C. 1 cycle per minute; 25 cycles at 95°C for 1 minute, 63°C for 1 minute, and 72°C for 1 minute. The obtained PCR products were cloned into T vector (Invitrogen) and sequenced.

The sequencing results are as follows: Anti-human β2M antibody β2M-2B1 belongs to the mouse IgG1 subtype, and the light chain is a kappa chain; The sequence of its heavy chain variable region is as follows: EVQLQQSGPELVKPGASVRMSCKASGYTFSSYVLHWVKQKPGQGLEWIGYFNPYNDGTKYNEKFKGKATLTSDKSSSTAYMEFSSLTSEDSAVYYCARRGNTYDNFDYWGQGTTLTVSS (SEQ ID No. 6).

Among them, according to the definition of the IMGT numbering system, it includes the heavy chain complementarity determining region 1 (CDR-H1): GYTFSSYV, which is SEQ ID No. 1, and the heavy chain complementarity determining region 2 (CDR-H2): FNPYNDGT, which is SEQ ID No. .2 and heavy chain complementarity determining region 3 (CDR-H3): ARRGNTYDNFDY, which is SEQ ID No. 3. The sequence of its light chain variable region is as follows: DVQMTQSPASQSASLGESVTITCLASQTIGTWLAWYQQKPGKSPQLLIYAAASLADGVPSRFSGSGSGTKFSFKISSLQAEDFVSYYCQQLYSSPLTFGGGTMLEIKR (SEQ ID No. 7).

Among them, according to the definition of IMGT numbering system, it includes light chain complementarity determining region 1 (CDR-L1): QTIGTW, which is SEQ ID No. 4, light chain complementarity determining region 2 (CDR-L2): AAA and light chain complementarity determining region Area 3 (CDR-L3): QQLYSSPLT, which is SEQ ID No. 5.

Example 2

In this example, the anti-human β2M antibody β2M-2B1 obtained in Example 1 is coupled with biotin to prepare a β2M binding preparation. The specific steps are as follows.

Dilute anti-human β2M antibody β2M-2B1 to 1mg/ml with 0.1mol/L sodium bicarbonate (pH8.0), dissolve 1mg of BNHS in 1ml of DMSO (dimethylsulfoxide), and add 1ml of antibody (i.e. 1mg) 120 μl BNHS solution (that is, containing 120 μg BNHS), mix biotin and antibody at a mass ratio of 1:8.3, stir at room temperature (20~25°C) for 2 hours (the stirring time should not be too long, as the antibody will be inactivated), mix Put the solution into a dialysis bag, dialyze with 0.01 mol/L, pH=7.4 PBS solution at 4°C overnight, and then dialyze again for 4 to 6 hours the next day to remove free biotin. Add an equal volume according to the final volume of the dialyzed mixture. The buffer solution should be stored in the dark at 4°C. The buffer solution ratio (volume ratio) is: PBS + 0.01% preservative + 1% BSA.

Example 3

This example provides a method for preparing anti-human CD3 antibodies. The overall steps are the same as those in Example 1, except that the immunizing antigen is CD3. One of the selected cell lines was named CD3-2C1 cell line.

After sequencing, the CD3-2C1 antibody belongs to the mouse IgG1 subtype, and the light chain is a kappa chain; it has a VH domain with the amino acid sequence shown in SEQ ID No. 8, and a VH domain with the amino acid sequence shown in SEQ ID No. 9 VL domain.

Example 4

In this example, the anti-human CD3 antibody CD3-2C1 obtained in Example 3 was coupled with biotin to prepare a CD3-binding preparation. The coupling method was the same as that in Example 2.

Experimental example 1

In this experimental example, the anti-human β2M antibody β2M-2B1 prepared in the above Example 1 was used to conduct in vitro detection of the solution of recombinant human β2M. The detection method is as follows.

5μg/ml Ag (recombinant human β2M, His tag), mIgG (10μg/ml, 2×) and anti-human β2M antibody β2M-2B1 (10μg/ml, 2×) were co-cultured, 2-fold gradient diluted, and then HIS1K The biosensor monitors the binding of β2M-2B1 to β2M, and Octet HIS1K is used to determine the affinity of the anti-human β2M antibody β2M-2B1.

The experimental conditions are set as shown in Table 1:

[Table 1] Experimental conditions for HIS1K biosensor detection in Experimental Example 1 N Data Name Assay Time(sec) analysis time Flow RateFlow rate 1 Baseline 60 1000 2 Association(combination) 180 1000 3 Dissociation 300 1000 4 Baseline1(baseline1) 90 1000 5 Loading 120 1000 6 Custom(custom) 5 1000

Buffer: 1×DPBS + 0.02% Tween-20.

The kinetic affinity analysis fitting curve of anti-human β2M antibody β2M-2B1 after 2-fold gradient dilution is shown in Figure 2. The affinity experimental results of anti-human β2M antibody β2M-2B1 for human β2M are shown in Table 2. The results show: The anti-human β2M antibody β2M-2B1 has a high affinity for human β2M (KD＜1.0E-12 M), proving that the anti-human β2M antibody β2M-2B1 provided in Example 1 can be used to detect β2M, or be used to prepare and detect β2M products.

[Table 2] Affinity results of anti-human β2M antibody β2M-2B1 to human β2M Sample ID (test sample name) Loading Sample ID(sample name) KD(M) (affinity) KD Error (affinity error) kon(1/Ms)(binding rate) kon Error (binding rate error) kdis(1/s) (dissociation rate) kdis Error (dissociation rate error( Full X ² (X squared) Full R ² (R squared) β2M-2B1 β-2M <1.0E-12 2.34E-11 7.72E+05 1.37E+04 <1.0E-7 N/A 0.0223 0.9987

Experimental example 2

In this experimental example, the β2M-2B1 conjugated biotin preparation prepared in Example 2 and the CD3-2C1 conjugated biotin preparation prepared in Example 4 were used to purify universal CAR-T cells with knockout of TCR and β2M genes. , and investigate the purification effect.

1.1 TCR/β2M gene knockout

Infect T cells with the lentivirus prepared from the target plasmid in Figure 1 to obtain CART-CD19 cells (CD19-targeting CART cells). Spread 1×10 ⁶ CART-CD19 cells in the wells of a six-well plate, and add 1 μg of pX330-spCAS9-HF1-TRAC TRAC sgRNA expression plasmid (knockout of T cell receptor α gene (MHC) to 200 μL of OPTI-MEM Class II molecules), the nucleic acid sequence for expressing sgRNA is shown in SEQ ID NO.10), 1 μg pX330-spCAS9-HF1-β2M β2M sgRNA expression plasmid (knockout the β2 microglobulin gene of CAR-T cells (MHC class I Molecule), the nucleic acid sequence expressing sgRNA is shown in SEQ ID NO.11) and mix evenly. The pX330-spCAS9-HF1 plasmid map is shown in Figure 3. Then add 4μL PEI or 6μL lipo2000 reagent, mix evenly and let stand at room temperature for 15 minutes. , drop into the wells, and after 6 to 8 hours, replace the cells with fresh medium and culture them.

Among them, SEQ ID NO.10: acaaaacugugcuagacaug; SEQ ID NO. 11: cgcgagcacagcuaaggcca.

The TCR/β2M gene double knockout efficiency is shown in Figure 4. The results show that the proportion of TCR/β2M gene KO cells analyzed by flow cytometry (FACS) 48 hours after medium replacement was 71.35%. In addition, editing tools can also be introduced into cells in the form of sgRNA and RNP through electroporation and viral infection.

1.2 Purification of KO cells (UCAR-T cells)

Take the cells that have been cultured for 4 days after electroporation, centrifuge, resuspend in a certain amount of DPBS to a density of 1×10 ⁸ cells/ml, and pass through CD3-2C1-Biotin (biotin-conjugated anti-human CD3 antibody, from Example 4) and β2M-2B1-Biotin (biotin-conjugated anti-human β2M antibody, from Example 2) is purified in one step. The purification method is: add 100 μl/ml CD3-2C1-Biotin and 100 μl/ml β2M-2B1-Biotin and protect from light on ice. Incubate for 20 minutes, wash once with PBS, then add anti-Biotin Beads (purchased from Miltenyi Biotec, product number 130-090-485), incubate on ice for 15 minutes, then place in a magnet for 5 minutes to collect the cell suspension containing UCAR-T cells. 2×10 ⁵ cells/ml were taken and cultured with APC-anti-human TCRa/β (Biolegend, B259839) and PE-anti-human β2M (Biolegend, B226121) antibodies for 15 minutes to detect cell purity by flow cytometry.

The comparison of CAR-T cell purity before and after purification is shown in Figure 5. The results show: TCR/β2M gene knockout and cell purity, with a purity of 99.96%.

It should be noted that the above one-step purification method of CD3-2C1-Biotin and β2M-2B1-Biotin can also be replaced by two-step purification using CD3-2C1-Biotin and β2M-2B1-Biotin respectively, and the addition of two purified antibodies The order has no significant impact on the purification results.

Experimental example 3

Detect the priming activity of the universal CAR-T cells purified in step 1.2 of Experimental Example 2. The steps are as follows.

In this experimental example, the effector cells are UCAR-T cells, CAR-T cells and T cells (the latter two are controls). In a 24-well plate, 5.0×10 ⁵ cells are seeded in each well, with a volume of 500 μl, and a PHA-P concentration of 2.5 μg/ml, and the membrane surface activation molecules CD25 and CD69 were detected after 48 hours.

The comparison chart of the activation effect of CAR-T cells after purification is shown in Figure 6. The results show that the universal CAR-T cannot be activated again after two-step purification, and the control is obviously activated.

Experimental example 4

The in vitro tumor-killing activity of the purified universal CAR-T cells purified in step 1.2 of Experimental Example 2 is as follows.

This experiment uses K562 cells and K562-CD19 cells (denoted as K19) as target cells. The purified effector cell UCAR-T used in this experimental example is named UCART-19, and CAR-T cells without gene knockout are selected. (named CART-19) and T cells as controls, first use cytocalceinTM violet 550 to stain the target cells, and then adjust the effector cell density to 5×10 ⁶ /ml and the target cell density to 5×10 ⁵ /ml. Add the above three types of effector cells and target cells into the 96-well plate according to the quantity ratios of (0:1), (0.25:1), (1:1), (5:1) and (10:1) respectively and mix well. Co-culture for 6h and 24h, observe the apoptosis under the microscope during the above time period, and centrifuge the mixed cells. The supernatant is used to detect IL-2 using Human IL-2 Ready-SET-Go and Human IFN gamma ELISA Ready-SET-GoELISA kits. 2 and IFN-γ, resuspend the precipitated part in 100 μl binding buffer, centrifuge at 300 g for 5 min, add 1.2 μl APC-Annexin V and 1.2 μl PI dye, incubate in the dark for 15 min, add 100 μl binding buffer to detect apoptosis efficiency by flow cytometry.

The in vitro tumor killing effect of purified CAR-T cells and the comparison of cytokine release are shown in Figures 7 and 8. The results show that the in vitro tumor killing effect of purified universal CAR-T cells is equivalent to that of conventional CAR-T cells. .

Experimental example 5

The in vivo tumor killing activity of the purified universal CAR-T cells purified in step 1.2 of Experimental Example 2 is as follows.

20 NPG mice were injected with 5.0×10 ⁵ raji-luc cells. Five days later, they were imaged using a small animal in vivo fluorescence imager. According to the imaging results, they were divided into 5 groups and injected with PBS, T-cell, CAR-T, and UCART-19 respectively. 1# cells, UCART-19 2# cells, the number is 5.0×10 ⁶ , and then imaged weekly using a small animal in vivo fluorescence imager, as shown in Figure 9, the results show: CAR-T, UCART-19 1#, UCART The three groups of -19 2# have equivalent in vivo tumor killing effects; and the survival curves are drawn according to the survival of the mice, as shown in Figure 10. The results show: CAR-T, UCART-19 1#, UCART-19 2# these 3 groups The survival rates of mice in the two groups were the same; it can be seen that the in vivo tumor killing effect of purified universal CAR-T cells is equivalent to that of conventional CAR-T cells.

Comparative example 1

The anti-human β2M antibody β2M-2B1 in Example 2 was replaced with a commercially available anti-human β2M antibody (Biolegend, 395702), coupled to PE, to prepare a β2M binding preparation; in addition, the anti-human CD3 antibody CD3 in Example 4 was -2C1 was replaced with a commercially available anti-human CD3 antibody (Biolegend, 317302), coupled to PE, to prepare a CD3-binding preparation; the coupling method was the same as Example 2. Among them, PE and biotin work on the same principle.

The β2M binding preparation and CD3 binding preparation prepared above were used to purify the universal CAR-T cells with TCR and β2M gene knockout, and the purification effect was investigated.

The results show that the purification effect of the above β2M binding preparations and CD3 binding preparations on universal CAR-T cells is only 72.73%, indicating that compared with commercially available anti-human β2M antibodies and anti-human CD3 antibodies, the anti-human β2M antibody β2M- 2B1 and anti-human CD3 antibody CD3-2C1 have significant purification effects on universal CAR-T cells.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, but not to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: The technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features can be equivalently replaced; and these modifications or substitutions do not deviate from the essence of the corresponding technical solutions from the technical solutions of the embodiments of the present invention. Scope.

without.

In order to more clearly explain the specific embodiments of the present invention or the technical solutions in the prior art, the drawings that need to be used in the description of the specific implementations or the prior art will be briefly introduced below. Obviously, the drawings in the following description are: For some embodiments of the present invention, those of ordinary skill in the art can also obtain other drawings based on these drawings without exerting creative efforts.

Figure 1 is a map of the pCDHF plasmid used in Experimental Example 2 of the present invention.

Figure 2 is a kinetic affinity analysis fitting curve of anti-human β2M antibody β2M-2B1.

Figure 3 is a map of the pX330-spCAS9-HF1 plasmid used in Example 2 of the present invention.

Figure 4 shows the TCR/β2M gene double knockout efficiency in Experimental Example 2 of the present invention.

Figure 5 is a comparison chart of the purity of CAR-T cells before and after purification in Experimental Example 2 of the present invention.

Figure 6 is a comparison chart of the activation effects of purified CAR-T cells in Experimental Example 3 of the present invention.

Figure 7 is a diagram showing the in vitro tumor killing effect of purified CAR-T cells in Experimental Example 4 of the present invention.

Figure 8 is a comparative chart of the release of tumor-killing cytokines in vitro from purified CAR-T cells in Experimental Example 4 of the present invention.

Figure 9 is a diagram showing the in vivo tumor killing effect of purified CAR-T cells in Experimental Example 5 of the present invention.

Figure 10 is a survival curve graph drawn based on the survival of mice in Experimental Example 5 of the present invention.

TW202328440A_111140563_SEQL.xml

Claims (16)

A β2M binding molecule, characterized in that it includes module a that specifically binds to β2M; The module a includes a VH structural domain, and the VH structural domain includes CDR-H1 having the amino acid sequence shown in SEQ ID No. 1, CDR-H2 having the amino acid sequence shown in SEQ ID No. 2, and CDR-H3 having the amino acid sequence shown in SEQ ID No. 3; The module a also includes a VL domain, which includes CDR-L1 with the amino acid sequence shown in SEQ ID No. 4, CDR-L2 with the amino acid sequence AAA and SEQ ID No. .CDR-L3 of the amino acid sequence shown in .5. A β2M binding molecule, characterized in that it includes module a that specifically binds to β2M; The module a includes a VH structural domain and a VL structural domain, and the VH structural domain includes CDR-H1, CDR-H2 and CDR-H3; The VL domain includes CDR-L1, CDR-L2 and CDR-L3; Wherein, the amino acid sequences of the CDR-H1, the CDR-H2 and the CDR-H3 include the complementarity determining region sequence of SEQ ID No. 6; The amino acid sequences of the CDR-L1, the CDR-L2 and the CDR-L3 include the complementarity determining region sequence of SEQ ID No. 7; Preferably, wherein the amino acid sequences of the CDR-H1, the CDR-H2 and the CDR-H3 are defined by SEQ ID No. 6 according to the Kabat, Chothia, or AbM numbering system; The amino acid sequences of the CDR-L1, the CDR-L2 and the CDR-L3 are defined by SEQ ID No. 7 according to the Kabat, Chothia, or AbM numbering system. The β2M binding molecule according to claim 1 or 2, wherein the VH domain has the amino acid sequence shown in SEQ ID No. 6. The β2M binding molecule according to any one of claims 1 to 3, wherein the VL domain has the amino acid sequence shown in SEQ ID No. 7. The β2M binding molecule according to any one of claims 1 to 4, wherein the β2M binding molecule is selected from scFv molecules, Fv molecules, Fab molecules or intact antibody molecules that specifically bind to β2M antigen. The application of a β2M binding molecule as described in any one of claims 1 to 5 in the preparation of β2M detection products, or the application in β2M in vitro detection not for the purpose of disease diagnosis or treatment. The use of a β2M binding molecule as described in any one of claims 1 to 5 in purifying universal CAR-T cells or preparing products for purifying universal CAR-T cells. A preparation for detecting β2M or a preparation for purifying universal CAR-T cells, characterized in that the preparation includes a first binding molecule, and the first binding molecule is as in any one of claims 1 to 5 The β2M binding molecule; Preferably, the β2M binding molecule is coupled with biotin. The preparation for purifying universal CAR-T cells as described in claim 8, wherein when the genes knocked out of the universal CAR-T cells also include TCR genes; used for purifying the universal CAR-T cells The preparation of T cells also includes a second binding molecule that specifically binds to CAR-T cells that express positive TCR; Preferably, the target protein that specifically binds to the second binding molecule is TCR or CD3; Preferably, the second binding molecule is coupled with biotin. The preparation for purifying universal CAR-T cells as described in claim 9, wherein the second binding molecule includes module c that specifically binds to CD3; The module c includes a VH domain having an amino acid sequence shown in SEQ ID No. 8, and a VL domain having an amino acid sequence shown in SEQ ID No. 9. A kit for purifying universal CAR-T cells, characterized in that the kit includes the preparation for purifying universal CAR-T cells as described in any one of claims 8 to 10; and any Selected supplies. A kit for detecting β2M, wherein the kit includes the preparation for detecting β2M as described in claim 8; and optional consumables. A preparation for purifying universal CAR-T cells as described in any one of claims 8 to 10, or a kit for purifying universal CAR-T cells as described in claim 11 for purifying universal CAR-T cells. A method for type CAR-T cells, which is characterized in that after culturing the universal CAR-T cells to be purified in a preparation for purifying universal CAR-T cells, using anti-biotin-coupled magnetic beads for magnetic adsorption , take the cell suspension and obtain purified universal CAR-T cells. A method for preparing universal CAR-T cells, which includes the following steps: (1) Prepare the β2M binding molecule as described in any one of claims 1 to 5, and prepare anti-CD3 antibodies; (2) The β2M binding molecule and anti-CD3 antibody are respectively coupled to biotin; (3) CAR-T cells knock out β2M gene and TCR gene; (4) The biotin-conjugated β2M binding molecule and anti-CD3 antibody are co-cultured with the gene-knocked-out CAR-T cells; (5) Use anti-biotin-coupled magnetic beads for magnetic adsorption, and take the cell suspension to obtain purified universal CAR-T cells. A method for purifying universal CAR-T cells, which includes the step of using the β2M binding molecule as described in any one of claims 1 to 5 to contact the universal CAR-T cells to be purified. The method of claim 15, which includes the step of: using the β2M binding molecule and the second binding molecule to contact the universal CAR-T cells to be purified, or After using the second binding molecule to contact the universal CAR-T cells to be purified, the β2M binding molecule is then used to contact the universal CAR-T cells to be purified, or After using the β2M binding molecule to contact the universal CAR-T cell to be purified, a second binding molecule is then used to contact the universal CAR-T cell to be purified; Wherein, the second binding molecule specifically binds to TCR-positive CAR-T cells; Preferably, the target protein of the second binding molecule is TCR or CD3; Preferably, the second binding molecule includes module c that specifically binds to CD3; The module c includes a VH domain having an amino acid sequence shown in SEQ ID No. 8, and a VL domain having an amino acid sequence shown in SEQ ID No. 9.