Virus particles for engineered T cell preparation and uses thereof

Abstract

The invention relates to a method for packaging CAR-T cells through a one-step method, engineered cells used for virus packaging in the method and application of the engineered cells.

Description

Technical Field

[0001] The present application relates to optimized methods for preparing engineered T cells and related tools, and in particular to viral particles used for preparing engineered T cells and methods for using the viral particles to shorten the time and process of preparing engineered T cells. Background Art

[0002] The current CAR-T preparation technology includes four steps: (1) T cell isolation, (2) T cell activation, (3) CAR lentiviral transfection, and (4) CAR-T cell expansion. The preparation cycle is about 7-14 days. Among them, T cell activation, CAR gene transfer and in vitro expansion are the key steps in the preparation of CAR-T cells, which affect the final therapeutic effect of CAR-T cells. A good preparation process is the basis for obtaining excellent CAR-T cells. Since resting T cells cannot be genetically transfected, additional additives such as CD3 / CD28 antibodies or magnetic beads containing CD3 / CD28 antibodies are required for activation. The use of additives not only increases the preparation cost, but also increases the number of steps to remove the additives. The long-term in vitro expansion of CAR-T cells not only requires a long waiting period for patients, but also leads to the differentiation of CAR-T cells, making the infused CAR-T cells less persistent in vivo. Therefore, it is necessary to conduct in-depth research and optimization on the key steps of CAR-T preparation. On the one hand, it is necessary to reduce the in vitro differentiation of T cells and maintain better initial functional characteristics to improve their therapeutic efficacy; on the other hand, it is necessary to shorten the preparation cycle, reduce the number of operating steps, and reduce production costs to quickly promote its clinical industrial transformation. SUMMARY OF THE INVENTION

[0004] The present application provides a one-step method for preparing engineered T cells. The method combines T cell activation and viral transduction steps into one, thereby greatly saving time costs. Moreover, unexpectedly, the engineered T cells prepared by this method have stronger in vivo effectiveness than the same engineered T cells prepared by conventional methods.

[0005] This application relates to:

[0006] Item 1. Engineered cells, characterized in that the engineered cells express one or more proteins, and the one or more proteins are localized in the cell membrane of the engineered cells and interact with surface molecules of immune cells.

[0007] Item 2. An engineered cell according to Item 1, wherein the one or more proteins do not include one or more of MHC-I class molecules and MHC-II class molecules, and wherein the absence of one or more of the MHC-I class molecules and MHC-II class molecules results in loss of function of the MHC-I complex and / or MHC-II complex.

[0008] Item 3. The engineered cells according to Item 1 or 2, which are derived from 293 cells, 293T cells or their derivative cells.

[0009] Item 4. An engineered cell according to any one of Items 1 to 3, wherein the one or more proteins are selected from one or more of the following groups:

[0010] Proteins that stimulate the CD3 / TCR complex, proteins that stimulate cell surface co-stimulatory molecules, proteins that bind to molecules with endocytosis function on the surface of immune cells, proteins that bind to specific molecules on the surface of immune cells, and proteins that bind to cytokine receptors on the surface of immune cells.

[0011] Item 5. The engineered cell according to Item 4, wherein the immune cell is a T cell, and the one or more proteins are selected from:

[0012] (1) antibodies or antigen-binding fragments of the antibodies, or ligands or functional domains of the ligands for the following proteins: CD3, CD5, CD137, CD81, CD28, CD7, CD4, CD8, CD34, and CD56; and

[0013] (2) The following proteins or their functional domains: IL15, IL7, IL21 and IL15Ra.

[0014] Item 6. An engineered cell according to Item 4 or 5, wherein the antigen binding fragment is a single-chain variable region fragment (scFv), Fab, (Fab)2 or Fv.

[0015] Item 7. An engineered cell according to Item 5 or 6, wherein the one or more proteins comprise: (1) a CD3 ligand or a functional domain of a CD3 ligand, or an anti-CD3 antibody or an antigen-binding fragment of an anti-CD3 antibody; and (2) a CD86 protein or a functional domain thereof.

[0016] Item 8. An engineered cell according to Item 7, wherein (1) and (2) are directly or indirectly connected and expressed on the cell membrane of the engineered cell, or are independently expressed on the cell membrane of the engineered cell.

[0017] Item 9. An engineered cell according to any one of Items 5 to 8, wherein the CD3 ligand or the functional domain of the CD3 ligand, or the anti-CD3 antibody or the antigen-binding fragment of the anti-CD3 antibody is sequentially connected to the CD8 hinge region and the CD8 transmembrane region.

[0018] Item 10. The engineered cell according to any one of Items 5 to 9, wherein the one or more proteins further comprise one or more proteins selected from the following:

[0019] (1) IL15 protein or its functional domain;

[0020] (2) a ligand of CD137 or a functional domain thereof; and

[0021] (3) Anti-CD137 antibodies or antigen-binding fragments of anti-CD137 antibodies.

[0022] Item 11. An engineered cell according to Item 10, wherein the IL15 protein or its functional domain is connected to a transmembrane domain.

[0023] Item 12. An engineered cell according to Item 11, wherein the transmembrane domain is the transmembrane domain of CD4.

[0024] Item 13. A method according to Item 12, wherein the IL15 protein or its functional domain is connected to the transmembrane domain of CD4 via the hinge region of IgG4 and the CH2 and CH3 of the IgG4 heavy chain constant region in sequence.

[0025] Item 14. According to the engineered cell of Item 10, the one or more proteins further comprise an IL15 receptor protein or its functional domain, and the L15 receptor protein or its functional domain is directly or indirectly connected to the IL15 protein or its functional domain.

[0026] Item 15. An engineered cell according to Item 14, wherein the IL15 receptor protein or its functional domain is connected to the IL15 protein or its functional domain via a GS linker.

[0027] Item 16. An engineered cell according to Item 14 or 15, wherein the IL15 receptor protein is IL15Ra.

[0028] Item 17. An engineered cell according to any one of Items 5 to 16, wherein the one or more proteins further comprise a ligand of CD7 or a functional domain thereof; or an anti-CD7 antibody or an antigen-binding fragment of an anti-CD7 antibody.

[0029] Item 18. An engineered cell according to Item 17, wherein the CD7 ligand or its functional domain, or anti-CD7 antibody or antigen-binding fragment of anti-CD7 antibody is further connected to a transmembrane domain.

[0030] Item 19. An engineered cell according to Item 18, wherein the CD7 ligand or its functional domain, or anti-CD7 antibody or antigen-binding fragment of anti-CD7 antibody is connected to the transmembrane domain of CD8.

[0031] Item 20. An engineered cell according to Item 5, wherein the one or more proteins are selected from the following structures:

[0032] Anti-CD3scFv-CD86;

[0033] Anti-CD3scFv-8h-8TM-2A-CD86;

[0034] Anti-CD3scFv-8h-8TM-2A-CD86&B-137L-1;

[0035] Anti-CD3scFv-8h-8TM-2A-CD86&IL15-GSlinker-IL15Ra;

[0036] Anti-CD3scFv-8h-8TM-2A-CD86&IL15-IgG4-4TM; and

[0037] The expression of Anti-CD3scFv-CD86&ScFv(CD7)-8h-8TM gene is achieved by transducing engineered cells with a vector.

[0038] Item 21. An engineered cell according to any one of Items 1-20, wherein the one or more proteins comprise one or more amino acid sequences selected from SEQ ID NO: 1 to 25, conservatively substituted variants thereof, or amino acid sequences having more than 80% sequence identity therewith.

[0039] Item 22. An engineered viral particle having an envelope comprising one or more proteins according to any one of Items 1 to 21.

[0040] Item 23. The engineered virus particle according to Item 22, which is packaged with a nucleic acid molecule or is a virus empty shell particle.

[0041] Item 24. According to the engineered virus particle of Item 23, the nucleic acid molecule comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR), and the nucleic acid sequence of the CAR is the nucleic acid sequence described in 201710301492.1 and 201710613317.6, or any CAR nucleic acid sequence such as patents 201510304389.3, 201710301492.1, The CAR nucleic acid sequences described in 201710613317.6, 201810207761.2, 201811125906.0, 201910407001.0, 201910407027.5, 202010254451.3, 202110556154.9, 202111239065.8, 202311451763.3, etc.

[0042] Item 25. An engineered viral particle according to any one of Items 22-24, which is a lentiviral particle.

[0043] Item 26. A method for preparing engineered T cells, comprising: contacting the viral particles according to any one of Items 22-25 with T cells.

[0044] Item 27. A method according to Item 26, wherein the T cells are T cells isolated from peripheral blood mononuclear cells (PBMC) or T cells in peripheral blood mononuclear cells (PBMC) that have not been separated.

[0045] Item 28. A method according to Item 27, wherein when the viral particles contact the T cells, the density of PBMCs is 1.00E+06 to 3.00E+07 cells / ml, preferably 2.00E+06 cells / ml.

[0046] Item 29. The method according to any one of Items 26 to 28, wherein the virus particle is a virus particle packaged with a nucleic acid molecule, further comprising:

[0047] The engineered T cells are harvested after the viral particles are in contact with the T cells for 10 minutes to 72 hours, for example, 24 to 48 hours.

[0048] Item 30. According to the method described in Item 29, the nucleic acid molecule contains a coding sequence for CAR, and the engineered T cells are CAR-T cells.

[0049] Item 31. According to the method described in any one of Items 26 to 28, the viral particles are empty viral capsid particles, and the method further comprises transducing nucleic acid molecules into the T cells 24 to 48 hours after the viral particles contact the T cells.

[0050] Item 32. A method according to Item 31, wherein the transduction is electrical transduction.

[0051] Item 33. The method according to Item 31 or 32, further comprising adding IL7, IL15 and / or IL21 to the cell culture medium in which the T cells are located after electrotransduction, and allowing the T cells to remain stationary under culture conditions for a period of time.

[0052] Item 34. A method according to Item 33, wherein the T cells are allowed to rest under culture conditions for 30 to 60 minutes, for example 40 minutes.

[0053] Item 35. A method according to any one of Items 32 to 34, wherein the nucleic acid molecule comprises a coding sequence for CAR.

[0054] Item 36. A method according to Item 35, wherein the nucleic acid molecule is contained in a PB transposase plasmid.

[0055] Item 37. According to any one of Items 26 to 36, while or after transducing the nucleic acid molecule, further comprises knocking out one or more target genes of the T cell, wherein the knockout is achieved by transducing Cas9 protein and sgRNA targeting the one or more target genes into the T cell.

[0056] Item 38. The method according to Item 37, wherein the one or more target genes are TRAC gene, B2M gene and / or CIITA gene. The CDS sequence number and gene ID of the TRAC gene, B2M gene and / or CIITA gene sequence in NCBI are: CDS sequence number AH002619.2 of B2M, CIITA gene ID: 4261, TRAC gene ID: 28755, and the specific sequence is disclosed in patent 201710983276.X, which is introduced into this application.

[0057] Item 39. Engineered T cells prepared using the method described in any one of Items 26 to 38.

[0058] Item 40. A method for packaging viral particles using the engineered cells described in any one of Items 1-21, wherein the viral particles have an envelope.

[0059] Item 41. A method according to Item 40, wherein the viral particles are lentiviral particles.

[0060] Item 42. A nucleic acid molecule comprising a nucleic acid sequence encoding one or more proteins according to any one of Items 1-21.

[0061] Item 43. One or more proteins encoded by the nucleic acid molecule according to Item 42.

[0062] Item 44. Use of the nucleic acid molecule according to Item 42 or the protein according to Item 43 for preparing engineered cells.

[0063] Item 45. The use according to Item 44, wherein the engineered cells are derived from 293 cells, 293T cells or their derivative cells.

[0064] Item 46. The use according to Item 44, wherein the engineered cells are CAR-T cells.

[0065] Specifically, the first aspect of the present application provides an engineered cell, the engineered cell expresses one or more proteins, the biomacromolecules are located in the cell membrane of the engineered cell, and can interact with immune cell surface molecules. In some embodiments, the immune cell is selected from, for example: T cells, B cells, NK cells, macrophages, etc. In some embodiments, the one or more proteins do not include one or more of MHC-I class molecules and MHC-II class molecules, wherein the loss of one or more of the MHC-I class molecules and MHC-II class molecules can lead to loss of function of the MHC-I complex and / or MHC-II complex. In some embodiments, the engineered cell is a human cell, so the MHC-I class molecule is HLA-I, and the MHC-II class molecule is HLA-II. In some embodiments, the engineered cell is derived from 293 cells, 293T cells, or derived cells thereof. In some embodiments, the derived cells include, but are not limited to, 293T cells, 293F cells, HEK293 cells, and 293T / 17SF cells. In some embodiments, the engineered cell is derived from a 293 cell, a 293T cell, or a derivative thereof, and the engineered cell does not comprise β2 microglobulin.

[0066] In some embodiments, the one or more proteins expressed by the engineered cells include: proteins that stimulate the CD3 / TCR complex, proteins that stimulate cell surface co-stimulatory molecules, molecules that promote lentiviral infection of the immune cells, cytokines and / or cytokine receptors. In some embodiments, the one or more proteins are selected from:

[0067] Antibodies or antigen-binding fragments of the antibodies, or ligands or functional domains of the ligands of the following proteins: CD3, CD5, CD137, CD81, CD28, CD7, CD4, CD8, CD34 and CD56; and

[0068] The following proteins or their functional domains: IL15, IL7, IL21 and IL15Ra.

[0069] In some embodiments, the antigen-binding fragment is selected from the group consisting of: single-chain variable region fragment (scFv), Fv, Fab, Fab', Fab'-SH, F(ab')2, diabody and VHH. In some embodiments, the antigen-binding fragment is scFv.

[0070] In some embodiments, the one or more proteins expressed by the engineered cells include: CD3 ligands or functional domains of CD3 ligands, or anti-CD3 antibodies or antigen-binding fragments of anti-CD3 antibodies. In some embodiments, the one or more proteins include CD3 ligands or functional domains of CD3 ligands, or anti-CD3 antibodies or antigen-binding fragments of anti-CD3 antibodies; and CD28 antibodies or antigen-binding fragments of the antibodies, or CD28 ligands or functional domains of the ligands. In some embodiments, the one or more proteins include CD3 ligands or functional domains of CD3 ligands, or anti-CD3 antibodies or antigen-binding fragments of anti-CD3 antibodies; and CD86 protein or its functional domain. In some embodiments, the one or more proteins include CD3 ligands or functional domains of CD3 ligands, or anti-CD3 antibodies or antigen-binding fragments of anti-CD3 antibodies; and do not include CD28 antibodies and antigen-binding fragments of the antibodies, nor do they include CD28 ligands and functional domains of the ligands. In some embodiments, the one or more proteins comprise a structure of "anti-CD3 antibody scFv-CD86" from the N-terminus to the C-terminus, wherein "anti-CD3 antibody" may also be written as "Anti-CD3" or "Anti-CD3 antibody" in this application. In some embodiments, the one or more proteins comprise an amino acid sequence as described in SEQ ID NO: 1 or 2, or a conservatively substituted variant of the amino acid sequence as described in SEQ ID NO: 1 or 2, or an amino acid sequence having more than 80% sequence identity with the amino acid sequence as described in SEQ ID NO: 1 or 2. In some embodiments, the CD3 ligand or the functional domain of the CD3 ligand, or the anti-CD3 antibody or the antigen-binding fragment of the anti-CD3 antibody is directly or indirectly connected to the CD86 protein or its functional domain, or is independently expressed on the cell membrane of the engineered cell. In some embodiments, the CD3 ligand or the functional domain of the CD3 ligand, or the C-terminus of the anti-CD3 antibody or the antigen-binding fragment of the anti-CD3 antibody is directly or indirectly connected to the N-terminus of the CD86 protein or its functional domain. In some embodiments, from the N-terminus to the C-terminus, the CD3 ligand or the functional domain of the CD3 ligand, or the anti-CD3 antibody or the antigen-binding fragment of the anti-CD3 antibody is sequentially connected to the CD8 hinge region and the CD8 transmembrane region. In some embodiments, the CD3 antibody comprises OKT3 or an antigen-binding fragment of OKT3. In some embodiments, the CD3 antibody is an scFv of OKT3. In some embodiments, the CD3 antibody is an antigen-binding fragment of OKT3. In some embodiments, the CD3 antibody is an scFv of OKT3. In some embodiments, the CD3 antibody comprises UCHT1 or an antigen-binding fragment of UCHT1.In some embodiments, the CD3 antibody is UCHT1 or an antigen-binding fragment of UCHT1. In some embodiments, the CD3 antibody is a scFv of UCHT1. In some embodiments, the one or more proteins comprise an amino acid sequence as described in any one of SEQ ID NOs: 4-8, such as SEQ ID NO: 6, or a conservatively substituted variant of an amino acid sequence as described in any one of SEQ ID NOs: 4-8, such as SEQ ID NO: 6, or an amino acid sequence having more than 80% sequence identity with an amino acid sequence as described in any one of SEQ ID NOs: 4-8, such as SEQ ID NO: 6.

[0071] In some embodiments, the one or more proteins contained in the engineered cells further include:

[0072] IL15 protein or its functional domain; and / or

[0073] A ligand or its functional domain of CD137, or an antibody or an antigen-binding fragment of an anti-CD137 antibody. That is, the one or more proteins include the above-mentioned CD3 ligand or the functional domain of CD3 ligand, or an anti-CD3 antibody or an antigen-binding fragment of an anti-CD3 antibody; and an IL15 protein or its functional domain or a ligand or its functional domain of CD137, or an anti-CD137 antibody or an antigen-binding fragment of an anti-CD137 antibody. Alternatively, in some embodiments, the one or more proteins include the above-mentioned CD3 ligand or the functional domain of CD3 ligand, or an anti-CD3 antibody or an antigen-binding fragment of an anti-CD3 antibody; and an IL15 protein or its functional domain or a ligand or its functional domain of CD137, or an anti-CD137 antibody and an antigen-binding fragment of an anti-CD137 antibody. In some embodiments, the one or more proteins include an IL15 protein and an antibody or an antigen-binding fragment of CD137. In some embodiments, the one or more proteins comprise an amino acid sequence as described in SEQ ID NO: 9 and / or 10, or a conservatively substituted variant of an amino acid sequence as described in SEQ ID NO: 9 and / or 10, or an amino acid sequence having more than 80% sequence identity with an amino acid sequence as described in SEQ ID NO: 9 and / or 10. In some embodiments, the one or more proteins further comprise an IL15 protein and a ligand of CD137. In some embodiments, the one or more proteins comprise an anti-CD3 antibody or an antigen-binding fragment of an anti-CD3 antibody; an IL15 protein; and an antibody or an antigen-binding fragment of CD137. In some embodiments, the one or more proteins comprise an anti-CD3 antibody or an antigen-binding fragment of an anti-CD3 antibody; an IL15 protein; and a ligand of CD137. In some embodiments, the IL15 protein or its functional domain is connected to a transmembrane domain. In some embodiments, the transmembrane domain is a transmembrane domain of CD4. In some embodiments, the IL15 protein or its functional domain is connected to the transmembrane domain of CD4 in sequence through the hinge region of IgG4 and the CH2 and CH3 of the constant region of the heavy chain of IgG4. In some embodiments, the IL15 protein contained in one or more proteins has a structure of "IL15-IgG4 hinge region-IgG4CH2-IgG4CH3-CD4 transmembrane domain" from N-terminus to C-terminus, wherein "-" indicates direct or indirect connection. In some embodiments, the "IL15-IgG4 hinge region-IgG4CH2-IgG4CH3-CD4 transmembrane domain" structure comprises an amino acid sequence as described in SEQ ID NO: 13, or a conservatively substituted variant of the amino acid sequence as described in SEQ ID NO: 13, or an amino acid sequence having more than 80% sequence identity with the amino acid sequence as described in SEQ ID NO: 13.

[0074] In some embodiments, the one or more proteins further comprise an IL15 receptor protein or a functional domain thereof, which is connected to the IL15 protein or its functional domain. In some embodiments, the IL15 receptor protein or its functional domain is directly or indirectly connected to the IL15 protein or its functional domain. In some embodiments, the one or more proteins comprise an amino acid sequence as described in SEQ ID NO: 11 or 12, or a conservatively substituted variant of an amino acid sequence as described in SEQ ID NO: 11 or 12, or an amino acid sequence having more than 80% sequence identity with an amino acid sequence as described in SEQ ID NO: 11 or 12. In some embodiments, the IL15 receptor protein or its functional domain is connected to the IL15 protein or its functional domain via a GS linker. In some embodiments, the C-terminus of the IL15 protein is connected to the N-terminus of the IL15 receptor via a GS linker. In some embodiments, the IL15 receptor protein is IL15Ra. In some embodiments, the one or more proteins comprise a CD3 ligand or a functional domain of a CD3 ligand, or an anti-CD3 antibody or an antigen-binding fragment of an anti-CD3 antibody; an IL15 protein and an IL15 receptor protein.

[0075] In some embodiments, the one or more proteins further comprise a ligand or a functional domain thereof of CD7, or a CD7 antibody or an antigen-binding fragment of a CD7 antibody. In some embodiments, the one or more proteins comprise a scFv of a CD7 antibody. In some embodiments, the CD7 antibody or the antigen-binding fragment of a CD7 antibody comprises an amino acid sequence as shown in SEQ ID NO: 24 or SEQ ID NO: 25, a conservative substitution variant of an amino acid sequence as shown in SEQ ID NO: 24 or SEQ ID NO: 25, or an amino acid sequence having more than 80% sequence identity with an amino acid sequence as shown in SEQ ID NO: 24 or 25. In some embodiments, the one or more proteins comprise a CD3 ligand or a functional domain of a CD3 ligand, or an anti-CD3 antibody or an antigen-binding fragment of an anti-CD3 antibody; and a CD7 ligand or a functional domain thereof, or a CD7 antibody or an antigen-binding fragment of a CD7 antibody. In some embodiments, the CD7 ligand or its functional domain, or an anti-CD7 antibody or an antigen-binding fragment of an anti-CD7 antibody is further connected to a transmembrane domain. In some embodiments, the ligand or functional domain of CD7, or anti-CD7 antibody or antigen-binding fragment of anti-CD7 antibody is connected to the transmembrane domain of CD8. In some embodiments, the C-terminus of the ligand or functional domain of CD7, or anti-CD7 antibody or antigen-binding fragment of anti-CD7 antibody is connected to the N-terminus of the transmembrane domain of CD8. In some embodiments, the one or more proteins are sequentially arranged from N-terminus to C-terminus as the structure of "CD7scFv-CD8 hinge region-CD8 transmembrane domain". In some embodiments, the one or more proteins comprise an amino acid sequence as shown in SEQ ID NO: 14 or SEQ ID NO: 15, a conservatively substituted variant of the amino acid sequence as shown in SEQ ID NO: 14 or SEQ ID NO: 15, or an amino acid sequence having more than 80% sequence identity with the amino acid sequence as shown in SEQ ID NO: 14 or 15.

[0076] In some embodiments, the one or more proteins contained in the engineered cells are molecules that interact with immune cell surface molecules, and the immune cell surface molecules are any combination of the following:

[0077] 1) CD3 and CD28;

[0078] 2) CD3, CD28 and CD137;

[0079] 3) CD3, CD28 and IL15Rb (IL15 receptor β chain); and

[0080] 4)CD3, CD28 and CD7.

[0081] In some embodiments, the one or more proteins contained in the engineered cells are molecules that interact with immune cell surface molecules, and the molecules that interact with immune cell surface molecules are any one of the following combinations:

[0082] 1) an anti-CD3 antibody or an antigen-binding fragment of the antibody, or a CD3 ligand or a functional domain of a CD3 ligand, and an anti-CD28 antibody or an antigen-binding fragment of the antibody, or a CD28 ligand or a functional domain of a CD28 ligand;

[0083] 2) an antibody or an antigen-binding fragment of the antibody against CD3, or a ligand of CD3, or a functional domain of a ligand of CD3, an antibody or an antigen-binding fragment of the antibody against CD28, or a ligand of CD28, or a functional domain of a ligand of CD28, and an antibody or an antigen-binding fragment of the antibody against CD137, or a ligand of CD137, or a functional domain of a ligand of CD137;

[0084] 3) an antibody or an antigen-binding fragment of said antibody, or a ligand of CD3 or a functional domain of a CD3 ligand, an antibody or an antigen-binding fragment of said antibody, or a ligand of CD28 or a functional domain of a CD28 ligand, and an antibody or an antigen-binding fragment of said antibody, or a ligand of IL15Rb or a functional domain of a IL15Rb ligand; and

[0085] 4) Antibodies against CD3 or antigen-binding fragments thereof, or ligands of CD3 or functional domains of CD3 ligands, antibodies against CD28 or antigen-binding fragments thereof, or ligands of CD28 or functional domains of CD28 ligands, and antibodies against CD7 or antigen-binding fragments thereof, or ligands of CD7 or functional domains of CD7 ligands.

[0086] In some embodiments, the one or more proteins contained in the engineered cells are molecules that interact with immune cell surface molecules, and the molecules that interact with immune cell surface molecules are any one of the following combinations:

[0087] 1) an anti-CD3 antibody or an antigen-binding fragment of said antibody, and CD86 or its functional domain;

[0088] 2) an anti-CD3 antibody or an antigen-binding fragment of said antibody, CD86 or its functional domain, and a ligand of CD137 or a functional domain of a CD137 ligand;

[0089] 3) Antibodies against CD3 or antigen-binding fragments thereof, CD86 or its functional domains, and IL15;

[0090] 4) Antibodies or antigen-binding fragments thereof against CD3, CD86 or its functional domains, IL15 and IL15Ra; and

[0091] 5) Antibodies against CD3 or antigen-binding fragments thereof, CD86 or its functional domains, and antibodies against CD7 or antigen-binding fragments thereof.

[0092] In some embodiments, the one or more proteins contained in the engineered cells are molecules that interact with immune cell surface molecules, and the molecules that interact with immune cell surface molecules are any one of the following combinations:

[0093] 1) Anti-CD3 antibody scFv (Anti-CD3-scFv), and CD86;

[0094] 2) Anti-CD3-scFv, CD86, and CD137 ligand (B-137L-1);

[0095] 3) Anti-CD3-scFv, CD86, and IL15;

[0096] 4) Anti-CD3-scFv, CD86, IL15 and IL15Ra; and

[0097] 5) Anti-CD3-scFv, CD86, and scFv of anti-CD7 antibody (Anti-CD7-scFv).

[0098] In some embodiments, the one or more proteins contained in the engineered cells are molecules that interact with immune cell surface molecules, and the molecules that interact with immune cell surface molecules are any one of the following combinations:

[0099] 1) Anti-CD3-scFv, and CD86;

[0100] 2) (Anti-CD3scFv)-8h-8TM, and CD86;

[0101] 3) Anti-CD3-scFv, CD86, and B-137L-1;

[0102] 4) (Anti-CD3scFv)-8h-8TM, CD86, and B-137L-1;

[0103] 5) Anti-CD3-scFv, CD86 and IL15-IgG4-4TM;

[0104] 6) (Anti-CD3scFv)-8h-8TM, CD86 and IL15-IgG4-4TM;

[0105] 7) Anti-CD3-scFv, CD86, and IL15-GSlinker-IL15Ra;

[0106] 8)(Anti-CD3scFv)-8h-8TM, CD86, and IL15-GSlinker-IL15Ra;

[0107] 9) Anti-CD3-scFv, CD86, and (Anti-CD7-scFv)-8h-8TM; and

[0108] 10) (Anti-CD3 scFv)-8h-8TM, CD86, and (Anti-CD7-scFv)-8h-8TM.

[0109] In some embodiments, the one or more proteins are structured as follows:

[0110] 1)(Anti-CD3scFv)-CD86;

[0111] 2) (Anti-CD3scFv)-8h-8TM-2A-CD86;

[0112] 3) (Anti-CD3scFv)-8h-8TM-2A-CD86&B-137L-1;

[0113] 4) (Anti-CD3scFv)-8h-8TM-2A-CD86&IL15-GSlinker-IL15Ra;

[0114] 5) (Anti-CD3scFv)-8h-8TM-2A-CD86&IL15-IgG4-4TM; and

[0115] 6) The expression of (Anti-CD3scFv)-CD86&(Anti-CD7-scFv)-8h-8TM gene vector was transduced into engineered cells.

[0116] Wherein, the "-" outside the brackets indicates direct or indirect connection, "&" indicates not connected to each other, 8h indicates CD8 hinge region, 8TM indicates CD8 transmembrane region, IgG4-4TM indicates the structure of "IgG4 hinge region-IgG4CH2-IgG4-CH3" from N-terminus to C-terminus, GSlinker indicates any suitable GS linker available in the art, B-137L-1 indicates the full-length CD137 ligand; and in some embodiments thereof, "-2A-" may be replaced by "&"; or in some embodiments thereof, "2A" may be P2A or T2A.

[0117] Specific:

[0118] (Anti-CD3scFv)-CD86 is a CD3-recognizing ScFv directly concatenated with the gene coding region of CD86 protein. The sequence is a gene sequence corresponding to the protein sequence shown in SEQ ID NO.4 or SEQ ID NO.5. The above gene sequence is used to construct an expression vector, and ultimately a fusion protein is formed on the surface of engineered cells for expression. The sequence of the fusion protein is shown in SEQ ID NO.4 or SEQ ID NO.5.

[0119] (Anti-CD3scFv)-8h-8TM-2A-CD86 is a ScFv gene coding region that recognizes CD3, a gene coding region derived from the hinge region of human CD8, and a gene coding region derived from the transmembrane region of human CD8 molecules, which are directly connected in series to form an open reading expression region of a fusion protein. The sequence is a gene sequence corresponding to the protein sequence shown in SEQ ID NO.6. The above gene sequence is connected to the gene sequence corresponding to the protein sequence shown in SEQ ID NO.3 through the gene of the 2A self-cleaving peptide and the CD86 gene sequence, and is constructed on the same expression vector and expressed on the surface of engineered cells. During the expression process, the 2A self-cleaving peptide undergoes a "cleavage reaction" in vivo to cut the (Anti-CD3scFv)-8h-8TM and CD86 molecules. Finally, the fusion protein sequence of (Anti-CD3scFv)-8h-8TM is shown in SEQ ID NO.6, and the CD86 protein molecule sequence is shown in SEQ ID NO.6, respectively forming two molecules expressed on the surface of engineered cells.

[0120] (Anti-CD3scFv)-8h-8TM-2A-CD86&B-137L-1 is a CD3-recognizing ScFv gene coding region, a human CD8 hinge region gene coding region, and a human CD8 molecule transmembrane region gene coding region, which are directly connected in series to form an open reading expression region of a fusion protein. The sequence is a gene sequence corresponding to the protein sequence shown in SEQ ID NO.6. The above gene sequence is connected to the gene sequence corresponding to the protein sequence shown in SEQ ID NO.3 through the gene of the 2A self-cleavage peptide and constructed on the same expression vector; the full-length CD137 ligand B-137L-1 sequence is a gene sequence corresponding to the protein sequence shown in SEQ ID NO.9, and is expressed on the second expression vector. The two expression vectors modify the engineered cells simultaneously or separately, and the engineered cells finally express the fusion protein sequence of (Anti-CD3scFv)-8h-8TM on the surface as shown in SEQ ID NO.6, and the CD86 protein molecule sequence as shown in SEQ ID NO.3 and the full-length CD137 ligand sequence is shown in SEQ ID NO.9.

[0121] (Anti-CD3scFv)-8h-8TM-2A-CD86&IL15-GSlinker-IL15Ra is a CD3-recognizing ScFv gene coding region, a human CD8 hinge region gene coding region, and a human CD8 molecule transmembrane region gene coding region, which are directly connected in series to form an open reading expression region of a fusion protein. The sequence is a gene sequence corresponding to the protein sequence shown in SEQ ID NO.6. The above gene sequence is connected to the gene sequence corresponding to the protein sequence shown in SEQ ID NO.3 through the gene of the 2A self-cleavage peptide and the CD86 gene sequence, and is constructed on the same first expression vector; the IL-15 gene editing sequence is connected to the IL15Ra gene sequence through the GS linker GSlinker to form an open reading expression region of a fusion protein. The sequence is a gene sequence corresponding to the protein sequence shown in SEQ ID NO.12. The gene sequence is constructed on the second expression vector. The two expression vectors modify the engineered cells simultaneously or separately, and the engineered cells finally express the fusion protein sequence of (Anti-CD3scFv)-8h-8TM on the surface of the engineered cells. NO.6, the CD86 protein molecule sequence is shown in SEQ ID NO.3 and the IL15-GSlinker-IL15Ra fusion protein sequence is shown in SEQ ID NO.12.

[0122] (Anti-CD3scFv)-8h-8TM-2A-CD86&IL15-IgG4-4TM is a CD3-recognizing ScFv gene coding region, a human CD8 hinge region gene coding region, and a human CD8 molecule transmembrane region gene coding region, which are directly connected in series to form an open reading expression region of a fusion protein. The sequence is a gene sequence corresponding to the protein sequence shown in SEQ ID NO.6. The above gene sequence is connected to the gene sequence corresponding to the protein sequence shown in SEQ ID NO.3 through the gene of the 2A self-cleavage peptide and the CD86 gene sequence, and is constructed on the same first expression vector; the IL-15 gene sequence is linked to the transmembrane region sequence derived from the human CD4 molecule through the IgG4 linker sequence to form an open reading expression region of a fusion protein. The sequence is as shown in SEQ ID The gene sequence corresponding to the protein sequence shown in NO.13 forms an open reading expression region of a fusion protein, and the gene sequence is constructed on a second expression vector. The two expression vectors modify the engineered cells simultaneously or separately, and finally the engineered cells express three protein molecules on the surface of (Anti-CD3scFv)-8h-8TM, including the fusion protein sequence as shown in SEQ ID NO.6, the CD86 protein molecule sequence as shown in SEQ ID NO.3, and the IL15-IgG4-4TM fusion protein sequence as shown in SEQ ID NO.13.

[0123] (Anti-CD3scFv)-CD86&(Anti-CD7-scFv)-8h-8TM is a CD3-recognizing ScFv and CD86 protein gene coding region directly connected in series, and the sequence is the gene sequence corresponding to the protein sequence shown in SEQ ID NO.4 or SEQ ID NO.5. The above gene sequence is used to construct the first expression vector; the CD7-recognizing ScFv sequence is the gene sequence corresponding to the protein sequence shown in SEQ ID NO.24 or SEQ ID NO.25, and the gene coding region derived from the hinge region gene coding region of human CD8 and the gene coding region derived from the transmembrane region of human CD8 molecule are directly connected in series to form an open reading expression region of a fusion protein, and the sequence is the gene sequence corresponding to the protein sequence shown in SEQ ID NO.14 or SEQ ID NO.15. The gene sequence is constructed on the second expression vector, and the two expression vectors are used to modify the engineered cells simultaneously or separately, and the final engineered cell surface expression sequence is the (Anti-CD3scFv)-CD86 fusion protein shown in SEQ ID NO.4 or SEQ ID NO.5, and the expression sequence is the (Anti-CD3scFv)-CD86 fusion protein shown in SEQ ID NO.14 or SEQ ID NO.15. There are two protein molecules of (Anti-CD7-scFv)-8h-8TM fusion protein shown in NO.15.

[0124] In some embodiments, the one or more proteins comprise one or more amino acid sequences selected from SEQ ID NOs: 1 to 25, conservatively substituted variants thereof, or amino acid sequences having greater than 80% sequence identity therewith.

[0125] The second aspect of the present application provides an engineered virus particle having an envelope, the envelope comprising one or more proteins, the one or more proteins being the same as the one or more proteins contained in the engineered cell of the aforementioned first aspect. In some embodiments, the engineered virus particle is packaged with a nucleic acid molecule or is a virus empty shell particle. The virus empty shell particle is a virus particle that does not contain a nucleic acid molecule, and the virus empty shell particle comprises a capsid and a capsule, or may also only comprise a capsule. In some embodiments, the nucleic acid molecule contained in the virus particle comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR). In some embodiments, the virus particle is a lentiviral particle.

[0126] The third aspect of the present application also provides a method for preparing an engineered T cell, which comprises: contacting the virus particle of the second aspect, or one or more proteins identical to the one or more proteins contained in the engineered cell of the first aspect with the T cell. In some embodiments, the method further comprises: while or after the virus particle or the engineered cell contacts the T cell, transducing a nucleic acid molecule to the T cell. The transduction can be carried out by liposome, electroporation, viral infection or any other method that can be used to introduce a nucleic acid molecule into a cell. In some embodiments, the nucleic acid molecule is packaged in the virus particle of the second aspect, and the nucleic acid molecule is introduced into the cell by infecting the T cell with the virus particle. In some embodiments, the nucleic acid molecule is packaged in another virus particle, the other virus particle is not the virus particle of the second aspect, and the nucleic acid molecule is introduced into the cell by infecting the T cell with the other virus particle. In some embodiments, the virus particle of the second aspect is a virus empty shell particle, the nucleic acid molecule is packaged in another virus particle, and the nucleic acid molecule is introduced into the cell by infecting the T cell with the other virus particle. In some embodiments, the virus particle of the second aspect is a virus empty shell particle, and the nucleic acid molecule is introduced into the T cell by liposome, electrotransfection, calcium phosphate transfection, etc. In some embodiments, the method further comprises harvesting the T cell after the virus particle or the engineered cell contacts the T cell for a period of time, the T cell is an engineered T cell, the contact period is 10 minutes to 72 hours, for example 30 minutes, 24 hours, 30 hours, 48 ​​hours or 72 hours, and in the case of comprising the step of "transducing nucleic acid molecules to the T cell", the transduction occurs before the harvest.

[0127] In some embodiments, the T cells are human T cells; in some embodiments, the T cells are isolated from PBMCs; in some embodiments, the T cells remain in the population of PBMCs throughout the method and are not isolated, that is, the method comprises:

[0128] 1) contacting the viral particles according to the second aspect or the engineered cells according to the first aspect with PBMC;

[0129] 2) transducing nucleic acid molecules to the PBMCs simultaneously or after step 1), wherein the nucleic acid molecules are packaged in the virus particles according to the second aspect, or are isolated nucleic acid molecules, or are packaged in other virus particles, and the other virus particles are not the virus particles according to the second aspect;

[0130] 3) harvesting the PBMCs after a period of time starting from step 1), the time of harvesting the PBMCs is later than step 2), and the period of time is 10 minutes to 72 hours, for example, 30 minutes, 24 hours, 30 hours, 48 ​​hours or 72 hours;

[0131] In step 1) to step 3), PBMCs are cultured in culture medium.

[0132] In some embodiments, the density of PBMC in step 1) is 1.00E+06 to 3.00E+07 cells / ml; preferably 2.00E+06 cells / ml.

[0133] In some embodiments, the culture medium is a serum-free culture medium. In some embodiments, the culture medium comprises 2% to 10% PBMC donor-derived plasma and immune cell basal culture medium. In some embodiments, the culture medium comprises 2% to 10% fetal bovine serum (FBS) and immune cell basal culture medium. In some embodiments, the immune cell basal culture medium is 1640 culture medium.

[0134] In some embodiments, the transduction is electrotransfection. In some embodiments, the virus empty shell particles are contacted with T cells that have not been separated from PBMC during the electrotransfection, and the contacted T cells are activated by incubation in the culture medium for 24-48 hours. In some embodiments, the activated T cells are mixed with a vector containing a target gene such as a CAR gene and / or a PB transposase plasmid for electrotransfection, and the electrotransfection uses a Lonza 4D nucleofector electrotransfection system, and the electrotransfection procedure is EO-115. In some embodiments, the method further comprises adding IL7, IL15 and / or IL21 to the electrotransfection cup after electrotransfection, and 10% PBS, and allowing the PBMC to stand for a period of time under culture conditions. The culture conditions are known in the art for maintaining or amplifying T cells in vitro, and the culture conditions include conditions such as temperature, humidity, carbon dioxide and oxygen concentration. In some embodiments, the stationary state is stationary state in a cell culture incubator at 37° C., and necessary maintenance medium, such as AM-V Serum Free Medium (Tianjin Haoyang; ANDLTBD) is added to the electroporation cuvette.

[0135] In some embodiments, the method for preparing engineered T cells is a method for preparing CAR-T cells, that is, the engineered T cells are CAR-T cells. In some embodiments, the CAR in the CAR-T cells targets CD19, CEA, CD70 and / or CD70.

[0136] In some embodiments, the method for preparing engineered T cells is a method for preparing universal T cells. In some embodiments, the method for preparing engineered T cells is a method for preparing universal CAR-T cells. In some embodiments, the method for preparing engineered T cells further comprises knocking out one or more target genes of the T cells while or after transducing the nucleic acid molecule. In some embodiments, the nucleic acid molecules transduced in the method for preparing engineered T cells include nucleic acid molecules for knocking out one or more target genes of the T cells. In some embodiments, the method for preparing engineered T cells further comprises knocking out one or more target genes of the T cells while or after transducing the nucleic acid molecules, and the knockout is achieved by transducing Cas9 protein and sgRNA targeting the one or more target genes to the T cells, and the molar ratio of the Cas9 protein to the sgRNA is 1: 2. In some embodiments, the one or more target genes are TRAC genes and B2M genes.

[0137] In some embodiments, the T cells for knocking out one or more target genes are CAR-T cells prepared by the above-mentioned method for preparing engineered T cells. In some embodiments, the T cells for knocking out one or more target genes are T cells prepared by the above-mentioned method for preparing engineered T cells. In some embodiments, the T cells for knocking out one or more target genes are prepared by contacting the viral particles according to the aforementioned second aspect with T cells, wherein the viral particles are virus empty shell particles. In some embodiments, the T cells for knocking out one or more target genes are prepared by contacting the viral particles according to the aforementioned second aspect with T cells, wherein the viral particles are viral particles packaged with nucleic acid molecules encoding CAR.

[0138] In addition, the present application also provides engineered T cells prepared using the method of the third aspect above. In some embodiments, the T cell is a TCR-T cell. In some embodiments, the T cell is a CAR-T cell. In some embodiments, the T cell is a universal CAR-T cell. In some embodiments, the T cell is a T cell with reduced immunogenicity. In some embodiments, the T cell is a T cell that does not recognize MHC-presented antigens. In some embodiments, the T cell is a T cell that has reduced immunogenicity and does not recognize MHC-presented antigens.

[0139] The present application also provides a method for packaging virus particles using the engineered cells according to the first aspect above, wherein the virus particles have an envelope. In some embodiments, the virus particles are lentiviral particles.

[0140] The present application also provides a nucleic acid molecule comprising a nucleic acid sequence encoding one or more proteins, wherein the one or more proteins are the same as the one or more proteins contained in the engineered cell described in the first aspect. The present application also provides a protein encoded by the nucleic acid molecule, wherein the protein is the same as the one or more proteins contained in the engineered cell described in the first aspect.

[0141] The application also provides the use of the aforementioned nucleic acid molecules or the aforementioned one or more proteins for preparing engineered cells, comprising contacting the aforementioned nucleic acid molecules or the aforementioned one or more proteins with cells. In some embodiments, the engineered cells are for enveloped viruses, such as viruses packaged by lentiviruses, which are derived from 293 cells, 293T cells, or cells derived therefrom. In some embodiments, the engineered cells are the engineered cells of the aforementioned first aspect. In some embodiments, the engineered cells are engineered immune cells, such as CAR-T cells or TCR-T cells. In some embodiments, the engineered cells are engineered T cells prepared using the method of the third aspect above. BRIEF DESCRIPTION OF THE DRAWINGS

[0142] Figure 1 Schematic diagram showing the expression pattern of T cell-related molecules on the surface of engineered cell lines.

[0143] Figure 2 Schematic diagram showing the plasmid structure and inserted sequence of the lentiviral system.

[0144] Figure 3 Schematic diagram showing the plasmid structure and insertion sequence of the PB system.

[0145] Figure 4 shows that the one-step virus addition can better complete the activation of resting T cells 24 hours later. Figure 4A , control T cells; Figure 4B , add C-1-50 lentivirus; Figure 4C , adding one-step C-1-50 (293T-1) lentivirus; Figure 4D , add one-step C-1-50 (293T-2) lentivirus.

[0146] Figure 5 shows that the one-step virus can better complete CAR transfection and stable expression. Figure 5A , CAR positive rate detection; Figure 5B , CAR copy number detection.

[0147] Figure 6 shows that the CAR-T prepared by the one-step method has good anti-tumor effect in vivo. Fig. 6A , C-1-50 (activated) and C-1-50 (293T-1) effectiveness verification in vivo; Figure 6B , in vivo effectiveness verification of C-1-50(293T-2) and C-1-50(293T-1).

[0148] FIG. 7 shows that the addition of empty viral capsid particles can promote conventional lentivirus to complete T cell transduction. Fig. 7A For the positive rate test; Figure 7B For copy number detection.

[0149] Figure 8 The next step shows the in vivo effectiveness of CAR-T cells under the action of co-stimulatory signaling.

[0150] Fig. 9 One-step CAR-T CAR expression detection of 293T-29 and 293T-31 prepared by CD7 (ScFv) alone on CD7 molecule stimulation.

[0151] Fig.10 Shows one-step CAR-T cell activation detection prepared by 293T-40 and 293T-41.

[0152] Fig.11 Shows one-step CAR-T cell activation detection prepared by 293T-36 and 293T-37.

[0153] Fig.12 One-step CAR-T activation assay showing one-step virus preparation expressing full-length OKT3 fusion protein.

[0154] Fig.13 Shows the in vivo effectiveness comparison between one-step CAR-T and CAR-T prepared by traditional activation protocol.

[0155] Fig.14 The subcutaneous tumor-bearing model was used to verify the in vivo effectiveness of one-step CAR-T with different starting materials.

[0156] Fig.15 The peritoneal tumor-bearing model was used to verify the in vivo effectiveness of one-step CAR-T with different starting materials.

[0157] Fig.16 The viability of universal CAR-T cells prepared by different preparation schemes is shown.

[0158] Fig.17 The CAR transduction efficiency of CAR-T prepared by electroporation schemes under different activation schemes is shown.

[0159] Fig.18 Shows the activity rate of CAR-T cells prepared by electroporation under different activation schemes. DETAILED DESCRIPTION OF THE INVENTION

[0161] The present application provides a method for efficiently packaging pseudoviruses, an envelope glycoprotein used therein, and pseudoviruses packaged using the method. The method saves pseudovirus packaging steps and improves packaging efficiency. It provides a usable viral vector for cells that are difficult to transduce, such as immune cells, stem cells, primary cells, etc.

[0162] definition

[0163] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this application belongs. The following references provide general definitions of many terms used in this application for technical personnel: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al (ed.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise indicated, the following terms have the meanings assigned to them.

[0164] As used herein, "immune cell surface molecule" refers to an immune cell membrane protein, which can change the state of the membrane protein when it binds to its ligand or a suitable antibody, thereby sending a signal to the cell and changing the state of the membrane protein and / or the immune cell. For example, the membrane protein is changed from an inhibitory state to an activated state or from an activated state to an inhibitory state, or the conformation of the membrane protein is changed; in some embodiments, when the membrane protein binds to its ligand or a suitable antibody, it can promote the adsorption, invasion, uncoating, replication and / or release of lentivirus. The ligand here can be any protein or other molecule that can bind to the membrane protein, as long as the binding of the protein or other molecule to the membrane protein can change the state of the membrane protein. In some embodiments, the "immune cell surface molecule" is a "T cell surface molecule", in some embodiments, the immune cell surface molecule is any molecule that can constitute a CD3 / TCR complex; in some embodiments, the immune cell surface molecule is a molecule specifically expressed on the surface of T cells. In some embodiments, the T cell surface molecule can be an indicator molecule of T cell activation such as CD69; in some embodiments, the T cell surface molecule can be a molecule expressed by T cells at different differentiation stages such as CD27; in some embodiments, the T cell surface molecule can be expressed in a variety of immune cells and is also mainly expressed in T cells, such as CD7. In some embodiments, the "immune cell surface molecule" includes but is not limited to: CD3, CD28, CD2 (LFA-2), CD7, CD25, CD69, CD45, MHC-peptide complex, MHC Class I molecules, MHC II molecules, CD58 (LFA-3), CD54, ECM, ICAM, TCR molecules, CD3 / TCR complex, CD137, CD81, cytokine receptors such as IL7R, IL15R, IL21R, IL2R, IL4R, IL6R, etc., CD5, CD4, CD8, CD34, ICOS, CD226, CD86, NGFR / TNFR superfamily members such as OX40, CD27, CD40, GITR (TNFRSF18 / CD357 / AITR), 4-1BB, etc., chemokines or chemokine receptors such as CXCR5, CXCR4, CCR5 and / or CD56, etc., which can be expressed on the surface of immune cells such as T cells (including multiple phenotypic classifications such as αβT cells, γδT cells, Treg, etc.), NK cells, macrophages, and DC cells. In some embodiments, the molecules that "interact with immune cell surface molecules" are molecules that "interact with T cell surface molecules", which include proteins that stimulate the CD3 / TCR complex, proteins that stimulate cell surface co-stimulatory molecules, molecules that promote lentiviral infection of the immune cells, cytokines, and cytokine receptors.In some embodiments, the molecule "interacting with immune surface molecules" is an antibody or antigen binding fragment thereof, or a ligand or a functional domain of the ligand of an immune cell surface molecule. In some embodiments, the molecule "interacting with immune cell surface molecules" is a membrane-bound polypeptide modified by a cytokine that can recognize a cytokine receptor.

[0165] As used herein, "proteins that stimulate CD3 / TCR complexes" can be used to refer to any antibody, ligand or any small molecule compound that can bind to any one or more subunits of CD3 or TCR, as long as its binding to the CD3 / TCR complex can phosphorylate the tyrosine residues in the ITAM (immunoreceptor tyrosine activation motif) of CD3, thereby causing downstream pathway activation, which is included in the scope of the term "proteins that stimulate CD3 / TCR complexes". In some embodiments, the "proteins that stimulate CD3 / TCR complexes" bind to any one or more chains of CD3. In some embodiments, the "proteins that stimulate CD3 / TCR complexes" bind to one, two or more of the ε chain, γ chain and δ chain of CD3. In some embodiments, the "proteins that stimulate CD3 / TCR complexes" are selected from one or more of antibodies, small molecules, ligands (e.g., naturally occurring ligands, recombinant ligands, or chimeric ligands). In some embodiments, the "proteins that stimulate CD3 / TCR complexes" are monoclonal antibodies or polyclonal antibodies against CD3. In some embodiments, the "protein that stimulates the CD3 / TCR complex" is a monospecific antibody or multispecific antibody against CD3. In some embodiments, the monospecific antibody or multispecific antibody comprises an antigen-binding fragment that specifically binds to the ε chain, γ chain or δ chain of CD3.

[0166] As used herein, a "protein that stimulates a cell surface co-stimulatory molecule" is a protein that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof. In some embodiments, the "protein that stimulates cell surface costimulatory molecules" is an antibody, ligand, or a functional domain of the ligand that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof, as long as its combination with CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2 and / or CD226 can produce a costimulatory signal of the CD3 / TCR complex, which is included in the term "protein that stimulates cell surface costimulatory molecules" within the scope of coverage. For example, the "protein that stimulates cell surface costimulatory molecules" that stimulates CD28 includes CD86 or its functional domain. As used herein, the "functional domain of a ligand" refers to a fragment of the ligand, which can bind to the receptor of the ligand and change the state of the receptor, for example, changing the receptor from an inhibitory state to an activated state, or changing the receptor from an activated state to an inhibitory state, and the change is consistent with the change caused by the binding of the ligand to the receptor.

[0167] As used herein, "cytokines" can be used to refer to any small molecule protein with a wide range of biological activities synthesized and secreted by immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) after stimulation. In some embodiments, cytokines regulate cell growth, differentiation and effects by binding to corresponding receptors to regulate immune responses. In some embodiments, cytokines include low molecular weight soluble proteins produced by cells induced by immunogens, mitogens or other stimulants. In some embodiments, cytokines have functions such as regulating innate immunity and adaptive immunity, hematopoiesis, cell growth, APSC multipotent cells, and damaged tissue repair. In some embodiments, cytokines cover interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemokines, growth factors, etc. In some specific embodiments, the "cytokines" of the present application include cytokines required for T cell in vitro culture or maintenance of a good state. As used in this application, the "functional domain of a cytokine or a cytokine receptor" refers to a fragment of a cytokine or a cytokine receptor, which fragment can play the same or substantially the same function as the cytokine or the cytokine receptor.

[0168] As used herein, the term "antibody" has its broadest meaning, specifically including full-length monoclonal antibodies, polyclonal antibodies, and, unless otherwise specified or contradictory to the context, antigen-binding fragments, antibody variants and multispecific molecules thereof, as long as they exhibit the desired biological activity (i.e., the activity of binding to its antigen or its ligand). In general, a "full-length antibody" is a glycoprotein comprising at least two heavy chains and two light chains, interconnected by disulfide bonds. The term antibody refers to an immunoglobulin molecule and an immunologically active fragment of an immunoglobulin molecule, i.e., a molecule containing an antigen binding site. Immunoglobulin molecules can be of any type (such as IgG, IgE, IgM, IgD, IgA and IgY), category (such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. Therefore, an "antibody" can also be a single variable domain (VHH) on a heavy chain antibody, also known as a single heavy chain antibody (HcAb), which has no light chain and can be naturally produced by camels or sharks. The antigen-binding portion of HcAb consists of a VHH fragment. As used herein, the term "antigen-binding fragment" refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to an antigen (e.g., CD3), including the antigen-binding site or variable region of the intact antibody, and in some cases, excluding the constant heavy chain domains (i.e., CH2, CH3, and / or CH4, depending on the isotype of the antibody) of the Fc region of the intact antibody. Examples of antibody fragments include antibody fragments such as Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; polypeptides consisting of an uninterrupted sequence of continuous amino acid residues in an antibody; and diabodies, etc. The term "antigen-binding fragment" includes, but is not limited to: (1) single-chain Fv (scFv) molecules; (2) single-chain polypeptides containing only one light chain variable domain, or fragments thereof containing three CDRs of the light chain variable domain, without a heavy chain molecule; and (3) single-chain polypeptides containing only one heavy chain variable region, or fragments thereof containing three CDRs of the heavy chain variable region, without a light chain molecule; and multispecific or multivalent structures formed by antibody fragments.

[0169] As used herein, "vector" refers to a vector by which a polynucleotide sequence (e.g., a target gene sequence) can be introduced into a host cell to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, bacteriophages, viruses, artificial nanoparticles, etc.

[0170] As used herein, "target gene" refers to a gene or its coding sequence contained in a vector, such as a viral vector, which is intended to be expressed in a target cell by introducing the vector into the target cell.

[0171] The virus described in the present application is a virus with an envelope component. The viral vector particle with an envelope according to the present application comprises at least the following components: (i) an envelope component (in the present application, "envelope" and "enveloped" are used interchangeably), which is composed of a phospholipid bilayer bound to an envelope protein, wherein the envelope protein comprises at least a chimeric or modified glycoprotein as defined above, and the envelope surrounds (ii) a core component composed of structural proteins for assembling virus-like particles, and the core itself surrounds (iii) a genome component generally composed of ribonucleic acid (RNA) and (iv) an enzyme component. The biological material may be present in the envelope, in the core and / or in the genome component.

[0172] In some embodiments, the virus with envelope components may be a retrovirus, such as a lentivirus, a γ-retrovirus, etc.; in some embodiments, the virus with envelope components may be a recombinant adenovirus (Adenovirus, Ad), such as a replication-deficient human recombinant adenovirus V5 (HadV5). This technical solution has the same technical principle in all viruses with envelope components, so it does not differ due to the difference in viruses with envelope components, and is applicable to all viruses with envelope components. Here, taking lentivirus as an example, the specific decomposition of this technical solution is carried out:

[0173] The construction method of lentiviral vector is known in the art and is specifically described in Naldini et al. (2000) Adv. Virus. Res. 55: 599 609 and Negre et al. (2002) Biochimie 84: 1161-1171. In general, the lentiviral vector particles according to the present application include at least the following components: (i) an envelope component (in this application, "envelope" and "envelope" are used interchangeably), which is composed of a phospholipid bilayer bound to an envelope protein, wherein the envelope protein at least comprises a chimeric or modified glycoprotein as defined above, and the envelope surrounds (ii) a core component composed of gag protein binding, and the core itself surrounds (iii) a genome component generally composed of ribonucleic acid (RNA) and (iv) an enzyme component (pol). The biological material may be present in the envelope, in the core and / or in the genome component. For example, by following the general guidance provided by Sandrin et al. (2002) Biood 100: 823 832, a person skilled in the art can easily prepare a lentiviral vector. In short, lentiviral vector particles can be generated by co-expressing packaging elements (i.e., core and enzyme components), genome components, and capsule components in production cells (e.g., 293T human embryonic kidney cells or cells derived therefrom). Three to four plasmids can usually be used, but the number of plasmids can also be more depending on the extent to which the lentiviral components are decomposed into separate elements. In some embodiments, the partial components, such as capsule components, enzyme components, etc., can be inserted into the production cell genome, and the production cells are used to package the viral vector. In some embodiments, the packaging elements and capsule components can be present in plasmids, wherein the plasmid containing the viral genome component, the plasmid containing the capsule component, and the components containing the enzyme component and / or the core component protein coding sequence are respectively referred to as transfer plasmids, capsule plasmids, and packaging plasmids. Commonly used lentiviral packaging plasmids include psPAX2, as well as pMDlg / pRRE and pRSV-Rev, which are components of the second and third generation lentiviral packaging systems, respectively. The psPAX2 plasmid also contains the coding sequences of gag, pol, rev and tat, the pMDlg / pRRE plasmid contains the coding sequences of gag and pol, and the pRSV-Rev contains the coding sequence of rev.

[0174] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a group of engineered polypeptides or proteins that, when in an immune effector cell, bind to a specific antigen contained on a target cell, and generate an intracellular signal after recognizing the specific antigen, activating the downstream pathway of the cell where the receptor is located, so as to initiate the killing of the target cell by the immune effector cell. The immune effector cells include but are not limited to NK cells, macrophages, neutrophils, T cells, etc. CAR generally includes at least one extracellular antigen binding fragment, a transmembrane domain, and a cytoplasmic signaling domain. The extracellular antigen binding fragment can specifically recognize antigens, and non-limiting examples include single-chain variable fragments (scFv) derived from antibodies, fragment antigen binding regions (Fab) selected from libraries, single domain fragments, or natural ligands that engage their cognate receptors, artificially designed target-specific recognition domains that recognize specific targets, such as fibronectin type III (FN3) domain combinations, target-specific anchor protein repeat proteins (designed ankyrin repeat proteins, DARPins) with recognition of specific targets. In some embodiments, the extracellular antigen binding fragment may comprise scFv, Fab or natural ligand, and any derivatives thereof. The extracellular antigen binding region may refer to a molecule other than an intact antibody, which may comprise a portion of an intact antibody and may bind to an antigen bound by the intact antibody. Examples of antibody fragments may include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bifunctional antibodies, linear antibodies; single-chain antibody molecules (e.g., scFv), ScFv may be ScFv of a murine antibody or a fully human antibody or a human-mouse chimeric antibody, or may be a single domain antibody such as a shark, alpaca or camel antibody; and multispecific antibodies formed by antibody fragments. The "signal transduction domain" generally comprises an immune-receptor tyrosine-based activation motif (ITAM), the basic composition of which is: YXXL / V. Wherein Y is tyrosine, L / V refers to leucine or valine, and X may be any amino acid. When the receptor binds to the corresponding ligand, the tyrosine in the ITMA connected to it can be phosphorylated under the action of a class of protein tyrosine kinases PTK connected to the cell membrane, thereby recruiting other free protein kinases or adapter proteins in the cell to transmit activation signals into the cell. In some embodiments, the "signal transduction domain" uses the intracellular signal transduction domain of TCRζ (CD3ζ) or FcεRIγ. As used herein, the "co-stimulatory domain" is also called the "co-stimulatory signal domain", which is mainly used to provide co-stimulatory signals to enhance the ability of immune cells, including, for example, enhancing the proliferation, survival and / or development of memory cells.In some embodiments, the "costimulatory domain" is selected from the intracellular domain of CD28, 4-1BB (CD137), OX40 (CD134), etc. As used herein, the "transmembrane domain", also known as the "transmembrane region", refers to a protein structure region anchored in the cell membrane with thermodynamic stability. The transmembrane domain can be obtained from a natural protein, such as a transmembrane domain derived from a T cell receptor (TCR). In some embodiments, the transmembrane domain is selected from the transmembrane domain of CD4, CD8α, CD28 and CD3ζ.

[0175] In some embodiments, the extracellular antigen binding fragment of the "chimeric antigen receptor" or "CAR" structure can recognize target molecules expressed on the surface of solid tumors and hematological tumor cells / tissues, and the target molecules include but are not limited to: CD19, CD20, CD22, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEACAM5, CEACAM6, CEACAM7, Mesothelin, MUC1, CLDN18.2, CDH17, Trop2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, AFP, FOLR1, c-Met, LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma-associated antigen 1) or TAG-72, etc.

[0176] In some embodiments, the structure of "chimeric antigen receptor" or "CAR" comprises: an antigen recognition domain (such as ScFv), a hinge structure (such as 8hinge derived from human CD8), a transmembrane structure (such as CD8TM derived from human CD8 transmembrane) and an intracellular signaling domain, wherein the intracellular signaling domain can be a primary intracellular signaling domain containing only an immunoreceptor tyrosine-based activation motif or ITAM: Examples of primary cytoplasmic signaling sequences containing ITAM include, but are not limited to, those derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, ICOS (CD278), FcεRI, CD66d, DAP10 and DAP12, etc., and can also include, in addition to the primary intracellular signaling domain, one or two or more costimulatory signaling domains, and the costimulatory signaling domains can be selected from the following Any one or more of the following molecules and functional variants derived therefrom: CD28, 41BB, OX40, CD27, DAP10, 2B4 (SLAMF4, CD244), CD3γ, CD3δ, FcεRI, CD2, CD16, TCRζ, FcRβ, CD30, CD40, ICOS, LFA-1, IL-2 receptor, Fcγ receptor, KIRDS2, SLAMF7, NKp80 (KLRF1), signaling lymphocyte activation molecule (SLAM protein), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, DAP12, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, LFA-1 (CD11a / CD18), GITR, BAFFR, LIGHT, HVEM (LIGHTR), etc. The derived functional variants are amino acid or nucleic acid sequences having at least about 80%, 85%, 90%, 95% or 99% sequence identity with the above molecules.

[0177] "Chimeric antigen receptor" or "CAR" can have a variety of structures, such as containing cytokines and antibody gene sequences that can be secreted or expressed in the membrane; such as containing structures that can be regulated for activation or inactivation, the regulated activation or inactivation structures include: suicide switch such as inducible caspase-9 (iCasp9), thymidine kinase in herpes simplex virus (HSV-TK) and suicide epitope, truncated EGFR (EGFRt), Fas-FasL apoptosis structure; inducible CAR (Inducible CAR) structure: peptide new epitope (PNE), fluorescein (FITC), 10 amino acids (5B9-tag), FITC-HM-3 bifunctional molecule (FHBM) and scFv connected to antibody, streptavidin 2 (mSA2) biotin-binding domain, leucine ZipFv, VIPER CAR induction structure, biotin-targeting immune receptor (biotin-bidingimmune receptor, BBIR) system; "logic gate" regulatory system combined with SynNotch receptor, etc.

[0178] In some embodiments, the structure described in "chimeric antigen receptor" or "CAR" may also include a chimeric fusion protein, which includes an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain. The extracellular antigen recognition domain of the chimeric fusion protein can be a complete and continuous extracellular segment of the cell membrane of the expressed molecule / polypeptide, or it can be a fusion form of the expressed molecule and other polypeptides such as polypeptides derived from human CD8 and CD4.

[0179] The artificial / engineered TCR receptor described in this article refers to a specific receptor on the surface of a natural or engineered T cell, which mainly relies on affinity-optimized or purely natural TCRs to recognize antigens presented by tumor MHC molecules and transmit stimulation signals to the cell through the TCR-CD3 complex.

[0180] The construction method of the above-mentioned CAR structure and artificial / engineered TCR receptor does not affect the use protected by this application. Based on the contents disclosed in this specification, technicians in this field can use any disclosed or undisclosed chimeric antigen receptor (CAR) and artificial / engineered TCR receptor to prepare related viruses through the cells for packaging viruses disclosed in this application. Therefore, no matter what kind of CAR structure is based on the above-mentioned, it can be applied to the virus preparation described in this article, including but not limited to T cells, NK cells, macrophages and other immune cells.

[0181] As used herein, the term "linker" is a short peptide used to connect multiple domains or components in a protein or polypeptide. "Directly connected" or "directly connected" means that there are no other amino acid residues between the domains or components.

[0182] In the present application, "indirect connection" or "indirect connection" means that the two structures also contain one or more amino acid residues, and the peptide segment formed by the connection, such as a linker. In some embodiments, the fusion protein can be a bispecific or multispecific antibody that recognizes different epitopes of a molecule or two or more different molecules described in the present application, such as a bispecific antibody against CD3 and CD86, a bispecific antibody against CD3 and IL15, a bispecific antibody against CD28 and CD86, etc.

[0183] "Independent expression" as described herein means that when two or more molecules are expressed on the cell surface, they are not connected to each other by any structure. When two or more molecules are "independently expressed", the two or more molecules can be expressed by different or the same vector, and when they are expressed using the same vector, the coding sequences of the two or more molecules are connected by a self-cleaving peptide. In some embodiments, the "self-cleaving peptide" is a 2A peptide, such as P2A, T2A, E2A and / or F2A. In some embodiments, the amino acid sequence of the "self-cleaving peptide" is selected from: SEQ ID NO: 18-19.

[0184] In some embodiments, one or more gene sequences of polypeptides that recognize the cell surface molecules can be constructed in the same expression vector for expression, or multiple expression vectors can be constructed to express the cells separately or simultaneously; in some embodiments, one or more gene sequences of polypeptides that recognize the cell surface molecules, where the gene sequence can be an RNA sequence or a DNA sequence, can be expressed in the cells by non-viral vectors such as transposon, physical electroporation, and circular RNA. Regardless of which of the above methods is used, the cells for packaging viruses protected by this application can be constructed in the end.

[0185] The cell line described in the present application can be a HEK-293T cell line, or a cell line derived / derived from the 293T cell line, such as Platinum-E cells, HEK-293FT cells, Plat-A cell line, Plat-GP packaging cell line, 293A human embryonic kidney cells, etc. The above cells can be used to prepare the cells for packaging viruses described in the present application.

[0186] In this application, "293T cells" refers to HEK 293T cells, which are an immortalized cell line derived from human embryonic kidneys. HEK 293T cells are a cell line derived from HEK 293 cells through genetic technology. They are transfected with adenovirus E1A gene from HEK 293 cells, can stably express SV40 large T antigen, and contain SV40 replication start point and promoter region. HEK 293 cells and all other HEK 293 cell derivatives are classified as "293T cell derivatives" in this application, including but not limited to 293T / 17 cells, 293F cells, HEK293 cells, and 293T / 17SF cells.

[0187] As used herein, in some embodiments, the “non-expression” can use gene editing methods to knock out or inactivate genes that are desired not to be expressed, and the gene editing methods include but are not limited to: ZFN (zinc finger nuclease) system, TALEN (transcription activator-like effector nuclease) system, CRISPR / Cas system and other schemes to inactivate HLA; in some embodiments, the non-expression can use TAP protein or lysosome to downregulate the expression of HLA molecules, and the relevant functional proteins are selected from: HSV, BHV-1, EHV-1 / 4, PRV, HSV-1 / 2, VZV, EBV, hCMV, mCMV, RhCMV, HHV-6 / 7, KSHV, MHV-68, vaccinia virus and adenovirus that can directly target and degrade HLA-I, and can be degraded by T AP protein to downregulate the functional protein expressed by HLA-I class molecules and the functional protein that can downregulate the expression of HLA-I class molecules via lysosomes; in some embodiments, the non-expression can be the use of short molecule RNA (shRNA) to interfere with the gene that is desired not to be expressed, so as to downregulate its expression; in some embodiments, the non-expression can be the use of endoplasmic reticulum blocking peptides and inhibitors of target molecules to block the expression of target genes such as the HLA molecules, and the inhibitor can be an antigen binding polypeptide, a ligand, a fusion protein of the antigen binding polypeptide, etc.; in some embodiments, the non-expression can be all technical solutions that ultimately lead to the non-expression, inactivation or partial inactivation of the target gene, which can be deduced and designed by those skilled in the art based on the technical features disclosed in the present application, and can be used as tools or means for preparing the cells disclosed in the present application.

[0188] As used herein, the percentage of "identity", such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% identity, refers to 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% similarity between amino acid sequences or between nucleotide sequences determined by sequence alignment. For example, the proportion of the number of positions with the same base or amino acid residue to the total number of positions determined after two sequences have the same residue at as many positions as possible by introducing spaces, etc. The percentage of "identity" can be determined using software programs known in the art, preferably using default parameters for comparison. A preferred comparison program is BLAST. Preferred programs are BLASTN and BLASTP. Details of these programs can be found on the Internet at ncbi.nlm.nih.gov / cgi-bin / BLAST. In the present application, more than 80% sequence identity clearly includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.10%, 99.20%, 99.30%, 99.40%, 99.50%, 99.60%, 99.70%, 99.80%, or 99.90% and above sequence identity.

[0189] As used herein, a "conservative substitution variant" of a protein, polypeptide or amino acid sequence refers to one in which one or more amino acid residues undergo amino acid substitution without changing the overall conformation and function of the protein or enzyme, including but not limited to replacing the amino acids in the amino acid sequence of the parent protein in the manner described by the aforementioned "conservative substitution". Therefore, the similarity of two proteins or amino acid sequences with similar functions may be different. For example, a similarity (identity) of 70% to 99% based on the MEGALIGN algorithm. "Conservative substitution variants" also include polypeptides or enzymes with more than 60% amino acid identity as determined by the BLAST or FASTA algorithm, preferably more than 75%, preferably more than 85%, and even more than 90% as the best, and have the same or substantially similar properties or functions compared to the native or parent protein or enzyme.

[0190] In this application, the term "contacting" is used in its simple ordinary sense to refer to the process of allowing at least two different substances (e.g., contact between a virus particle and a cell, contact between a nucleic acid molecule and a cell) to become close enough to react, interact, or physically contact. It should be understood that the resulting reaction product can be produced directly by the reaction between the added reagents, or by an intermediate of one or more added reagents, which can be produced in the reaction mixture. The term "contacting" can include allowing two substances to react, interact, or physically contact, wherein the two substances can be, for example, a nucleic acid and a cell as provided herein. In some embodiments, contacting includes, for example, allowing a nucleic acid as described herein to enter a cell, allowing a virus particle as described herein to enter a cell, etc.

[0191] As used herein, the term "engineered cell" can be used to refer to any bioengineered cell, such as a cell transduced with an exogenous nucleic acid molecule or containing an exogenous protein. Depending on the context, the engineered cell can be a cell (e.g., 293T cell or its derivative cell) for virus (e.g., lentivirus) packaging, or an immune cell (e.g., CAR-T cell).

[0192] As used herein, the term "protein" has the broadest meaning, and includes any peptide (including dipeptides or polypeptides) composed of amino acids by dehydration condensation, substances with a certain spatial structure formed by the coiled folding of dipeptides and / or polypeptides, and organic compounds or complexes with certain functions formed by combining multiple dipeptides and / or polypeptides in a specific manner.

[0193] As used herein, the term "virus particle" includes an empty virus shell and a virus particle that packages a nucleic acid molecule. The term "empty virus shell" may also be referred to as an "empty virus", which at least includes a viral capsid, in which no nucleic acid molecule is packaged. In some embodiments, the "empty virus shell" includes a viral capsid and a capsule, wherein the protein contained in the capsule may be derived from the same virus or a different virus as the capsid.

[0194] The beneficial effects of this application are:

[0195] 1) The present application provides a packaging cell line, which does not contain HLA, and the virus packaged by it is not immunogenic. The packaged lentivirus has the function of activating T cells in addition to the gene transfer function, so that T cell activation and gene transfer are completed in one step, saving preparation time and operation links.

[0196] 2) The present application provides a special lentivirus that can simultaneously complete the activation and transfection of T cells without the need for additional activation stimulation reagents, thereby saving preparation costs and reducing the addition of exogenous substances.

[0197] 3) The present application provides a virus empty shell particle, which can effectively stimulate the activation of resting T cells and promote the completion of lentivirus gene transfer.

[0198] 4) The present application provides a method for rapidly preparing CAR-T cells, which can complete the preparation of CAR-T cells within 10 minutes, 30 minutes, 24 hours, 30 hours, 48 ​​hours, or 72 hours. The preparation of CAR-T cells can be completed in a relatively short period of time. The CAR-T cell culture time is short, which can effectively reduce the in vitro differentiation of T cells, maintain better initial functional characteristics, and improve the in vivo anti-tumor efficacy of CAR-T cells.

[0199] It should be understood that the present application includes various aspects, embodiments and combinations of the aspects and / or embodiments described herein. The above description and subsequent examples are intended to illustrate rather than limit the scope of the present application. Other aspects, improvements and modifications within the scope of the present application will be apparent to those skilled in the art to which the present application belongs. Therefore, those of ordinary skill in the art should recognize that the scope of the present application also includes the improvements and modifications to the aspects and embodiments. Example

[0200] The exemplary embodiments of the present application are described below in conjunction with the accompanying drawings, including various details of the embodiments of the present application to facilitate understanding, which should be considered to be merely exemplary. Therefore, it should be appreciated by those of ordinary skill in the art that various changes and modifications may be made to the embodiments described herein without departing from the scope and spirit of the present application. Similarly, for the sake of clarity and brevity, descriptions of well-known features and structures are omitted in the following description.

[0201] Example 1: Transformation of 293T or 293T / 17 packaging cell line construction

[0202] The method for constructing packaging cells includes using gene editing methods to construct engineered cells of 293T / 17 (or 293T) that knock out HLA and express activation stimulation signals, and directly using retroviral vectors (such as lentiviral vectors) to construct 293T / 17 (or 293T) stable cell lines that express activation stimulation signals. The gene editing method can directly use the CRISPR-Cas9 system, or it can use a transposon system: such as the Sleeping Beauty transposon system (also known as the SB transposon system), the PiggyBac transposon system (also known as the PB transposon system), and other gene editing schemes known to those skilled in the art. Specifically, we use the PB transposon system combined with the new construction scheme of the CRISPR-Cas9 system to construct our target cells of "293T / 17 (or 293T) cells that knock out HLA and express activation stimulation signals"; use a conventional lentiviral system to construct a 293T / 17 (or 293T) stable cell line that expresses activation stimulation signals:

[0203] 1.1. Construction of 293T / 17 (or 293T) stable cell lines expressing activation stimulation signals using lentiviral system 1.1.1 Vector construction

[0204] The antibody ScFv (clone numbers include OKT3, UCHT1, etc.) for T cell recognition molecules: activation molecule CD3, antibody ScFv or ligand (such as CD86) corresponding to co-stimulatory signals (CD28, CD137, CD81, etc.), cytokine (IL7, IL15, IL21, etc.) proteins or their functional domains are connected in two different forms (such as Figure 1 The lentiviral vector was constructed (as shown in the figure), and after virus packaging, 293T / 17 (or 293T) cells were infected, and positive selection was performed to obtain cell lines 293T-1, 293T-2 and other cell lines with a positive rate of >90%.

[0205] 1.1.2 Lentiviral system transformation 293T or 293T / 17

[0206] Through PCR, different clone numbers of anti-CD3ScFv, CD86, CD137L, IL15, IL15Ra or different clone numbers of anti-CD5ScFv and other target fragments were obtained, and the activation signal or targeting molecule nucleic acid sequence was inserted into the lentiviral vector by restriction endonuclease digestion, and transformed into E.coli TOP10. After correct sequencing, the plasmid was extracted and purified using the plasmid purification kit of Omega Company to obtain the plasmid of the recombinant expression vector. The structure of the viral vector and the vector expressing the target gene is as follows Figure 2 , 293T or 293T / 17 expressing the vector gene constructed based on the above scheme is the packaging cell described in the present application, and the characteristics are shown in the following Table 1:

[0207] Table 1 The modified 293T or 293T / 17 cells described in this application

[0208]

[0209]

[0210] Note: In the above table, unless otherwise specified, OKT3 and UCHT1 represent the scFv of OKT3 and the scFv of UCHT1, respectively. GMR-OKT3 represents the constant region of IgM and OKT3ScFv to construct a full-length antibody, IgG4-4TM refers to the Hinge+CH2+CH3 of IgG4 and then the transmembrane of CD4; OKT3 (CL-hIgMCH) and CL-mIgMCH are the structural combinations of the constant regions of mouse IgM and human IgM and OKT3ScFv, respectively.

[0211] The virus was prepared by calcium phosphate-mediated plasmid transfection into eukaryotic cells, and then PEG and NaCl were added to concentrate the virus. The concentrated virus was gradiently infected into CHO cells to calculate the virus titer.

[0212] The cells (the cells can be unmodified wild-type 293T cells or 293T cells with HLA knockout based on the gene editing method of Scheme 1) are cultured to the logarithmic growth phase, inoculated in a 24-well plate, and the virus is added at a certain multiplicity of infection (MOI) (generally 1-5MOI), and the culture is continued. The cells are collected, the corresponding antibodies are added for labeling, and the positive cells are sorted by flow cytometry to obtain high-expressing cell lines. The expression of each target gene is shown in Table 2 below:

[0213] Table 2 Target gene expression detection in modified 293T packaging cells

[0214]

[0215] The cell line expresses activation stimulatory molecules that can activate immune cells such as T cells and NK cells, or protein molecules that help mediate lentiviral infection of specific immune cells. Among them, T cell activation stimulatory molecules include but are not limited to ligands or antibodies such as CD3, CD28, CD137, CD81, and proteins such as IL7, IL15, IL21 or their functional domains, and various combinations of the stimulatory molecules. Protein molecules that help mediate lentiviral infection of specific immune cells include but are not limited to ligands or antibodies such as CD5, CD7, CD4, CD8, CD34, CD56, and various combinations thereof.

[0216] 1.2. Construction of 293T / 17 (or 293T) cells with HLA knockout and expression of activation stimulation signals in the PB system

[0217] 293T / 17 (or 293T) cells with HLA knockout and expression of anti-CD3 antibody and / or CD86 co-stimulatory polypeptide were constructed: 293T-PB1, 293T / 17-PB5, 293T / 17-PB6.

[0218] Specifically, nucleic acid sequences of anti-CD3 ScFv (OKT3 or UCHT1) and CD86 fragments of different clone numbers were obtained by PCR, and the sequences were inserted into a transposon vector by restriction endonuclease digestion, and transformed into E. coli TOP10. After sequencing was correct, the plasmid was extracted and purified using a plasmid purification kit from QIAGEN to obtain Figure 3 The plasmids PB-3-1, PB-3-6, and PB-3-10 of the recombinant expression vectors shown. 293T or 293T / 17 expressing the vector genes constructed based on the above scheme is the packaging cell described in this application, and its characteristics are shown in Table 3 below:

[0219] Table 3 293T or 293T / 17 cells modified using the PB system

[0220] Serial number Cell Name Plasmid number Plasmid construction 01 293T-PB1 PB-3-1 OKT3-8h-8TM-BBZ 02 293T / 17-PB5 PB-3-6 OKT3-CD86 03 293T / 17-PB6 PB-3-10 UCHT1-CD86

[0221] Use Lonza electroporation instrument or other electroporation instruments such as Neon, select appropriate electroporation program, and transfer Cas9 protein and sgRNA (sgRNA targeting HLA knockout) or / and target plasmid (below) at a molar ratio of 1:2 or other suitable ratios. Figure 3 The vector plasmid in the expression vector expresses the scFv of CD3 monoclonal antibody OKT3 and CD86), and the plasmid for HLA knockout or / and expression of T cell-related molecules is constructed by electroporation. 72 hours after electroporation, the flow cytometry antibody with detectable fluorescent marker HLA-I (B2M-PE, Biolegend, 316306) or HLA-II (HLA-DR-APC, Biolegend, 361714) can be incubated and the knockout efficiency can be detected by flow cytometry, and the expression of anti-CD3 antibody (OKT3 or UCHT1) and CD86 (packaged target plasmid) can be detected by CD3E & CD3G (Kaixia Bio, CD3-HM557B) and CD86 antibody (Biolegend, 305406), respectively. The monoclonal cell lines 293T / 17-PB5-D10, 293T / 17-PB5-H10, 293T / 17-PB6-A2, and 293T / 17-PB6-H11, which were negative for HLA-I and HLA-II and positive for CD86, were selected by flow cytometry and cultured for later viral packaging. The molecular detection of the constructed and modified packaging cells is shown in Table 4 below.

[0222] Table 4 Detection of positive rates of HLA I, HLA II and CD86 in monoclonal cell lines

[0223]

[0224] Example 2 One-step virus and virus empty shell particle packaging

[0225] 2.1 Virus Empty Shell Particle Packaging

[0226] The cell line constructed in Example 1 is used for lentiviral packaging. When packaging the virus, a transfer target plasmid (RRE+REV+2G+transfer target plasmid) containing a transfer target gene (such as CAR) can be added to package virus particles with both activation stimulation and target gene transfer, or a transfer plasmid target plasmid (RRE+REV+2G) can be not added to package virus empty shell particles with activation stimulation function.

[0227] The modified 293T or 293T / 17 cells were expanded for virus packaging using the calcium phosphate-mediated plasmid transfection eukaryotic cell method. Fresh culture medium was added before virus packaging, Cacl2 (2.5M) was mixed with RRE and REV plasmids, and the mixture was allowed to stand for 3-5 minutes, then added to the culture medium and mixed up and down for 3-5 minutes, and then added to the packaging cells for transfection. After 48 hours, the cell supernatant was collected, centrifuged at 3000rpm for 15 minutes to remove cell debris, and then PEG and Nacl were added to concentrate the virus, and the virus empty shell particle stock solution was concentrated to obtain a sample for analysis of the virus empty shell particle P24 and particle size.

[0228] 2.2 One-step virus packaging

[0229] Using the calcium phosphate-mediated plasmid transfection method for eukaryotic cells, three packaging plasmids (RRE, REV, 2G) and the target plasmid (taking the CAR structure targeting CD19 as an example) were mixed and allowed to stand for 3-5 minutes. The mass ratio was RREREV: 2G: target plasmid = 4:3:6:15, and the mixture was fully mixed and added to the modified 293T or 293T / 17 cells for cell culture. After 4-6 hours, the cells were replaced with a fresh medium containing 10ml 5% FBS, and the cell supernatant was collected after 48 hours. The cell fragments were removed by centrifugation at 3000rpm for 15 minutes, and PEG and Nacl were added for virus concentration. The concentrated virus was gradient infected with CHO cells. After 48 hours, the cells were collected for flow cytometry, and the virus titer was calculated according to the proportion of positive cells. The virus titer is shown in Table 5.

[0230] Table 5 One-step virus titer

[0231] One-step packaging cells Flow titer RT titer C-1-50(293T-1) 6.72E+08 3.77E+09 C-1-50(293T-2) 5.96E+08 5.50E+09

[0232] Example 3 Preparation of one-step CAR-T

[0233] Peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation, cultured overnight with 1640+10% FBS medium, adherent cells were removed and suspended cells were collected. After centrifugation and resuspending, the cells were counted and the cell density was adjusted to 2.00E+06cell / ml and inoculated into well plates (500ul for 24 wells, 1ml for 12 well plates, and 2ml for 6 well plates).

[0234] Fresh PBMCs were separated or frozen PBMCs were resuscitated, and viral infection was performed in a medium without or with cytokines. Special viral particles with activation stimulation signals could be directly added during infection, or empty viral shell particles could be added at the same time as normal lentivirus to complete transfection, and the cells were cultured statically at 37°C and 5% CO2 incubator. The day of virus transfection was recorded as Day 0. The amount of virus added was calculated according to the flow titer at 5MOI, and the one-step virus obtained above or the empty viral shell particles were added at the same time as the conventional virus, and the cells in each well were mixed and cultured statically at 37°C and 5% CO2 incubator. CAR-T cells were collected and washed 10 minutes, 30 minutes, 24 hours, 30 hours, 48 ​​hours, and 72 hours after infection with the virus.

[0235] A small amount of prepared CAR-T cells (about 1.00E+06) was taken to mark the activation markers (CD3 / CD69 / CD25) to detect cell activation, and the prepared CAR-T cells were cultured continuously until the 5th day for flow cytometry positive rate and copy number detection.

[0236] In another scheme, peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation, cultured overnight with 1640+10% FBS medium, adherent cells were removed and suspended cells were collected, CD3 positive cells were sorted using anti-CD3 positive magnetic beads or antibodies, or CD4 and CD8 single positive cells were sorted using anti-CD4, CD8 positive magnetic beads or antibodies for merging. The density of T cells collected by the above scheme was adjusted to 2.00E+06cell / ml and inoculated into a well plate (24 wells 500ul, 12 well plate 1ml, 6 well plate 2ml). The amount of virus added was calculated according to the flow titer at 5MOI, and the one-step virus obtained above or the virus empty shell particles were added while adding the conventional virus, and the cells in each well were mixed and cultured in a 37°C, 5% CO2 incubator. CAR-T cells were collected and washed after 10 minutes, 30 minutes, 24 hours, 30 hours, 48 ​​hours, and 72 hours of virus infection.

[0237] Example 4 CAR-T performance verification

[0238] 4.1 Preparation of one-step CAR-T

[0239] The structure and preparation method of C-1-50 are detailed in patent CN 201710613317.6, which is a CAR structure targeting CD19. The transmembrane domain of the CAR uses the transmembrane domain of CD8, and the intracellular domains of CD3Z and 4-1BB are used as the intracellular signal transduction domains of the CAR.

[0240] illustrate:

[0241] 1.C-1-50 is the C-1-50 virus packaged in conventional 293T cells (conventional C-1-50 lentivirus)

[0242] 2. CT is the unactivated PBMC control group

[0243] 3. C-1-50+viral empty shell particles are prepared by conventional virus plus virus empty shell particles

[0244] 4. The other groups are one-step viruses packaged by different modified packaging cells

[0245] The lentiviruses in 3 and 4 above can both be referred to as one-step C-1-50 lentiviruses.

[0246] The preparation of one-step CAR-T was completed using the preparation method of Example 3. Conventional preparation method: Lymphocyte separation by gradient centrifugation; mononuclear cells (PBMC cells) or T lymphocytes were obtained, and the obtained PBMC cells or T lymphocytes were activated by anti-CD3, CD28 monoclonal antibodies or anti-CD3 / CD28 magnetic beads, and the activated PBMC or T cells were infected at a certain multiplicity of infection (MOI), and the positive rate of CAR-T was detected on the 8th day of viral infection. The detection method was flow cytometry, and the antibody was: Protein-L-PE, Protein-L can recognize the antibody light chain, and the light chain of the ScFv sequence in the CAR antigen recognition region can be recognized by Protein-L, so Protein-L can be used to detect the CAR positive rate and CAR expression intensity. The specific method for T cell activation detection is: take 5×10^5-1×10^6 cells, centrifuge at 300g for 5 minutes, discard the supernatant and add PBS to wash once, resuspend and dilute the cells with 100μl PBS containing 1-2% FBS, add labeling reagent, incubate at 4℃ in the dark for 30 minutes, wash twice with PBS and resuspend the cells with 200μl PBS containing 1-2% FBS for detection. The antibody information is shown in the table below. CD69 is one of the earliest markers upregulated after T cell activation, and CD25 is the α chain of IL2 receptor, which is upregulated within 24 hours after T cell activation and remains elevated for several days. Therefore, CD69 and CD25 can be used to detect the activation state of T cells. Related reagents are shown in Table 6.

[0247] Table 6 T cell and CAR-T cell activation index detection reagents

[0248]

[0249] 4.2 One-step C-1-50 lentivirus-infected T cells with high expression of activation signal CD25 and CD69 molecules

[0250] After adding conventional C-1-50 lentivirus and one-step C-1-50 lentivirus and culturing for 24 hours, it was found that T cells infected with one-step C-1-50 lentivirus had higher expression of activation signal CD25 and CD69 molecules. As shown in Figure 5 and Table 1. C-1-50 (conventional) means that monocytes (PBMC cells) or T lymphocytes are directly added with conventional C-1-50 virus without being activated by anti-CD3, CD28 monoclonal antibodies or anti-CD3 / CD28 magnetic beads; C-1-50 (activated) means that monocytes (PBMC cells) or T lymphocytes are activated by anti-CD3, CD28 monoclonal antibodies or anti-CD3 / CD28 magnetic beads (see the above conventional preparation method section) and then conventional C-1-50 virus is added; C-1-50 (2 C-1-50 (293T-2) refers to monocytes (PBMC cells) or T lymphocytes that have not been activated with anti-CD3, CD28 monoclonal antibodies or anti-CD3 / CD28 magnetic beads and are directly added to one-step viruses packaged using the 293T-1 packaging cell line; C-1-50 (293T-2) refers to monocytes (PBMC cells) or T lymphocytes that have not been activated with anti-CD3, CD28 monoclonal antibodies or anti-CD3 / CD28 magnetic beads and are directly added to one-step viruses packaged using the 293T-2 packaging cell line. The detection of one-step CAR-T cell activation markers prepared by different one-step viruses is shown in Figure 4: the horizontal axis of Figure 4 is the expression of CD25, the vertical axis is the expression of CD69, and Figures AD are control T cells, C-1-50 (conventional), C-1-50 (293T-1), and C-1-50 (293T-2), respectively. It can be seen that the one-step CAR-T prepared by the 293T-1 / 293T-2 one-step virus in 24h: C-1-50 (293T-1), C-1-50 (293T-2) have high activation. The specific detection values ​​corresponding to Figure 4 are shown in Table 7:

[0251] Table 7 Activation ratio of one-step CAR-T cells prepared by 24h one-step virus

[0252] <![CDATA[CD69 + (%)]]> <![CDATA[CD25 + (%)]]> <![CDATA[CD69 + CD25 + (%)]]> Control T cells 2.00 4.22 0.17 C-1-50(conventional) 1.63 3.98 0.10 C-1-50(293T-1) 87.06 49.36 48.17 C-1-50(293T-2) 85.96 42.82 41.50

[0253] It can be seen that the lentivirus (one-step C-1-50) prepared by the engineered cells described in the present application can complete the activation of resting T cells. The one-step cell preparation process can be performed.

[0254] 4.3 One-step method for high expression of CD25 and CD69 molecules, activation signals of T cells, by C-1-50 (293T-1) and C-1-50 (293T-2) viruses

[0255] After adding C-1-50 conventional lentivirus and one-step C-1-50 lentivirus and culturing for 48 hours, it was found that T cells added with one-step C-1-50 (293T-1) and C-1-50 (293T-2) viruses had higher expression of activation signal CD25 and CD69 molecules. However, the C-1-50 lentivirus group had no expression of activation signal molecules as shown in Table 8.

[0256] Table 8 Activation ratio of one-step CAR-T cells prepared by one-step virus at 48 h

[0257] <![CDATA[CD69 + (%)]]> <![CDATA[CD25 + (%)]]> <![CDATA[CD69 + CD25 + (%)]]> Control T cells 2.64 13.86 0.95 C-1-50(conventional) 2.89 11.54 1.24 C-1-50(293T-1) 93.64 97.35 92.31 C-1-50(293T-2) 88.35 96.23 86.60

[0258] The results showed that the one-step viruses C-1-50 (293T-1) and C-1-50 (293T-2) prepared using the engineered cells described in this application can be used for the preparation of the one-step CAR-T cells described in this application.

[0259] 4.4 Detection of CAR positivity and copy number of one-step CAR-T

[0260] After collecting the cells, the cells were cultured continuously. The CAR expression positivity rate was detected by flow cytometry and the CAR copy number was detected by RT on the 8th and 19th days, respectively. The one-step virus preparation can effectively complete the infection and stably express. Its CAR expression positivity rate and copy number are comparable to those of the conventional preparation method using anti-CD3 / CD28 magnetic beads (Beads / activated magnetic beads), while the C-1-50 conventional virus cannot complete the infection. In order to more accurately detect the CAR positivity rate of one-step CAR-T, in this experiment, one-step CAR-T C-1-50 (293T-1) and C-1-50 (293T-2) were prepared and harvested 24 hours after mixing with the one-step virus. The harvested one-step CAR-T cells were centrifuged and cultured using the same protocol as the conventional protocol. The CAR expression positivity rate and copy number were as follows: Figure 5A-5B and as shown in Table 9. Figure 5A-5B The horizontal axis represents different CAR-T preparation schemes. Figure 5A The vertical axis represents the positive rate of CAR expression. Figure 5B The vertical axis represents the copy number detection. Table 9 shows the specific detection values, where "——" means that the detection is below the detectable line.

[0261] Table 9 CAR expression of CAR-T cells prepared by different preparation schemes

[0262]

[0263] The results showed that the lentivirus C-1-50 prepared using the engineered cells described in the present application can be used for the preparation of the one-step CAR-T cells described in the present application. Both C-1-50 (293T-1) and C-1-50 (293T-2) have high CAR positive expression, which is comparable to the expression of C-1-50 (activated) prepared by the conventional activation scheme (see the above description).

[0264] 4.5 In vivo efficacy testing of viruses produced by different modified packaging cell lines

[0265] In order to further verify the in vivo efficacy of the virus prepared by the 293T-1 packaging cell line constructed with the OKT3-8h-8TM-2A-CD86 gene structure and the 293T-2 packaging cell line constructed with the OKT3-CD86 gene structure, we used the above two cell lines to complete the one-step virus preparation, collected the cells 48 hours after adding the virus, and used the traditional preparation method as a control. The traditional preparation method is to first activate T cells with activated magnetic beads, then use C-1-50 conventional virus infection, and then amplify and culture in vitro, and collect cells after 8 days of co-culture.

[0266] Female NCG mice aged 6-8 weeks were selected and Nalm6-Luc-GFP cells were intravenously injected into the tail of the mice at a volume of 5×10^5 cells / mouse to construct a tumor-bearing model. On the 3rd day of tumor formation, the above-prepared CAR-T cells were injected into the tail vein at a volume of 3×10^5CAR-T cells / mouse; the Control T group was infused with the same total number of T lymphocytes on the 3rd day. Figure 6A-6B As shown: Fig. 6A To verify the in vivo effectiveness of C-1-50 (activated) and C-1-50 (293T-1), Figure 6B To verify the in vivo effectiveness of C-1-50 (293T-2) and C-1-50 (293T-1), and to verify the in vivo effectiveness of one-step CAR-T prepared by one-step virus prepared by different modified packaging cell lines, tumor imaging was performed every 7 days after CAR-T transfusion. Fig. 6A It can be seen that the one-step virus preparation of the one-step CAR-T C-1-50 (293T-1) has significantly better effectiveness than the traditional activation scheme to prepare C-1-50 (activated); on this basis Figure 6B The in vivo effectiveness of C-1-50 (293T-2) and C-1-50 (293T-1) one-step CAR-T prepared by one-step viruses expressing different fusion peptides was further verified. Figure 6BAs shown, the one-step preparation of CAR-T C-1-50 (293T-1) has a good and better anti-tumor effect in vivo, and is superior to the conventional preparation process and C-1-50 (293T-2) in vivo efficacy. Finally, the virus prepared by the 293T-1 packaging cell line constructed with the OKT3-8h-8TM-2A-CD86 gene structure is preferred for one-step virus preparation, which has a better effect; Figure 4B The in vivo efficacy of C-1-50 (293T-1) CAR-T cells prepared using the engineered cells described in this application is superior to that of C-1-50 (activated) CAR-T cells cultured using existing conventional activation protocols.

[0267] 293T-1 is a 293T cell expressing the OKT3-8h-8TM-2A-CD86 gene. The lentiviral particles prepared using 293T-1 can be empty virus shell particles that express the OKT3-8h-8TM fusion peptide and CD86 protein without the virus shell containing the CAR gene, or can be a one-step virus that expresses the OKT3-8h-8TM fusion peptide and CD86 protein with the virus shell containing CAR genes of different structures and different targets. OKT3-8h-8TM fusion peptide is used to stimulate CD3 molecules on the surface of T cells, and CD86 stimulates CD28 molecules on the surface of T cells. It can be seen from the above results that the virus prepared by 293T-1 of the present application can realize one-step CAR-T cell preparation, and the prepared one-step CAR-T cells further enhance the in vivo effectiveness of CAR-T cells on the basis of completing T cell activation. This is not only achieved by stimulating T cell CD3 and CD28 signaling pathways through anti-CD3, CD28 monoclonal antibodies or anti-CD3 / CD28 magnetic beads. Our CAR-T one-step preparation scheme based on the preparation of viruses by 293T-1 packaging cell line, and the prepared one-step CAR-T unexpectedly improves the effectiveness of CAR-T cells.

[0268] 4.6 Infection efficiency of CAR viruses prepared under different conditions

[0269] Adding empty virus particles at the same time as conventional C-1-50 lentivirus can also complete T cell transduction well and obtain CAR-T cells with a high positive rate. The specific experimental plan is: isolate peripheral blood PBMC cells, spread a certain number of cells into a 12-well plate, add empty virus particles (empty virus particles packaged using 293T-) and conventional C-1-50 virus and mix them, change the medium after 24 hours and add cytokine-containing culture medium, continue to expand culture until Day 7, send for flow cytometry and copy testing, and evaluate the infection efficiency of CAR viruses under different preparation conditions. Figure 7A-7B and as shown in Table 10. Fig. 7A and Figure 7BThe ability of the one-step virus to transfect T cells with CAR was tested from two dimensions: CAR positive expression rate and CAR gene copy number.

[0270] Table 10 Preparation of one-step CAR-T cell CAR expression by different schemes

[0271]

[0272]

[0273] Figure 7A-7B The results in Table 9 show that the one-step virus empty shell particles can promote conventional lentivirus to complete T cell transduction, and the corresponding one-step CAR-T cells can be prepared in the form of one-step virus empty shell particles + CAR target plasmid (such as CAR vector targeting CD19, CAR vector targeting CD123, CAR vector targeting CEA, etc.).

[0274] Further, considering that OKT3 is an anti-CD3 antibody, whether CD86 can be replaced with an anti-CD28 antibody to achieve the same effectiveness, 293T-3 packaging cells were prepared in which CD86 in the OKT3-8h-8TM-2A-CD86 vector structure was replaced with the anti-CD28 antibody C28, and the construction method is shown in Examples 1 and 2. One-step CAR-T cells were prepared by the method of Example 3, and the cell activation degree was detected. The specific detection scheme and detection indicators are shown in the T cell activation degree detection part of Example 4. The results are shown in Table 11 below:

[0275] Table 11 Detection of 293T-3 one-step CAR-T cell activation degree

[0276] CD69+(%) CD25+(%) CD69+CD25+(%) Control T cells 1.23 9.03 0.62 C-1-50(activated) 92.15 96.01 90.47 C-1-50(293T-3) 17.19 10.18 2.47

[0277] Further increasing the dosage of C-1-50 (293T-3) virus did not significantly improve the activation level of T cells, as shown in Table 12 below:

[0278] Table 12 Detection of CAR-T cell activation degree with different doses of 293T-3 one-step virus

[0279] CD69+(%) CD25+(%) CD69+CD25+(%) C-1-50(293T-3)-5MOI 21.55 12.04 4.72 C-1-50(293T-3)-10MOI 21.38 10.15 4.05 C-1-50(293T-3)-20MOI 21.24 7.91 3.27

[0280] It can be seen from the above results that OKT3+C28 antibody (293T-3) can only activate T cells at a very low level, which is significantly lower than the activation level of OKT3+CD86 (293T-1). Therefore, in the scheme of the present application, expressing the OKT3-8h-8TM-2A-CD86 vector structure in 293T is the most preferred packaging cell line construction scheme.

[0281] 4.7 Detection of CAR expression positivity rate of CAR-T cells after adding co-stimulatory signals

[0282] The activation signal was further optimized and the costimulatory signal was added. The results showed that the one-step preparation was possible after adding CD137L (293T-6), IL15 (293T-8), and IL15Ra (293T-9), and the transfection efficiency was further improved compared with 293T-1. The results are shown in Table 13.

[0283] Table 13 CAR expression positive rate of viruses expressing CD137L, IL15, and IL15Ra in CAR-T cells prepared by one-step process

[0284] Positive rate C-1-50(293T-6) 61.02 C-1-50(293T-8) 69.38 C-1-50(293T-9) 66.01 C-1-50(293T-1) 55.63

[0285] 4.8 In vivo effectiveness testing of one-step CAR-T prepared with co-stimulatory signals

[0286] One-step virus was prepared, and cells were collected by culturing for 24 hours using the protocol of Example 3 to prepare one-step CAR-T cells C-1-50 (293T-1), C-1-50 (293T-6), C-1-50 (293T-8), and C-1-50 (293T-9). The conventionally prepared C-1-50 (activated) and T cells not expressing CAR were used as controls to verify the in vivo effectiveness of the one-step CAR-T prepared by increasing CD137L (293T-6), IL15 (293T-8), and IL15Ra (293T-9) signals. The results are as follows: Figure 8 As shown. Female NCG mice aged 6-8 weeks were selected, and Nalm6-Luc-GFP cells were intravenously injected into the tail of the mice at a volume of 5×10^5 cells / mouse to construct a tumor-bearing model. On the 3rd day of tumor formation, the above-prepared CAR-T cells were injected into the tail vein at a volume of 3×10^5CAR-T cells / mouse; the Control T group was re-infused with the same total number of T lymphocytes on the 3rd day.

[0287] The results showed that the one-step preparation of CAR-T after adding the costimulatory signal had the same in vivo efficacy as 293T-1, and was superior to the CAR-T cells prepared using the conventional preparation scheme (here the functional comparison of different preparation processes was performed using the C-1-50CAR structure). The viruses expressing anti-CD137L, IL15, IL15Ra, CD3, CD86 ScFv or their fusion protein form polypeptides on the viral envelope prepared by the engineered cells expressing CD137L (293T-6), IL15 (293T-8), IL15Ra (293T-9), 293T1, 293T2, etc. in the present application can be used in the one-step CAR-T cell preparation process of the present application.

[0288] 4.9 Effect of one-step CAR-T cells prepared by one-step viruses expressing different fusion proteins on T cell activation

[0289] In addition to the above signals, CD7 and CD5 are also molecules expressed on the surface of T cells. We further constructed 293T-29 and 293T-31 structures based on CD7, and studied the effects of one-step virus or virus empty shell particles prepared from the structures on T cell activation. The results are as follows Fig. 9 shown. Fig. 9 The horizontal axis represents the one-step CAR-T cells prepared by one-step viruses expressing different fusion proteins, and the vertical axis represents the CAR expression positive rate. The results showed that the one-step cells prepared by 293T-29 and 293T-31 with only CD7 signal activation did not detect CAR expression and could not complete T cell activation.

[0290] On the basis of the above, 293T-29 and 293T-31 were co-expressed with the vector expressed by 293T-2 to form 293T-40 and 293T-41, and 293T-40 and 293T-41 one-step viruses were prepared using 293T-40 and 293T-41 packaging cell lines. The activation of one-step CAR-T cells prepared using one-step viruses prepared by 293T-40 and 293T-41 (one-step CAR-T cells harvested 24 hours after the one-step virus was mixed with T cells) was detected, as shown in FIG. Fig.10 shown. Fig.10 The horizontal axis represents the one-step CAR-T cells prepared by different one-step viruses, and the columns from top to bottom on the vertical axis represent the proportions of CD69-CD25+, CD69+CD25+, CD69+CD25-, and CD69-CD25- cells under CD3 positive cells, respectively.

[0291] like Fig.10 As shown in the figure, it was unexpectedly found that the one-step virus prepared by 293T-40 and 293T-41, which co-express fusion proteins recognizing CD3, CD28, and CD7 molecules, can significantly increase T cell / CAR-T cell activation compared with 293T-2, which co-expresses CD3 and CD28 molecules. The above one-step CAR-T from three different donors was further verified, and the results are shown in Table 14 below:

[0292] Table 14 One-step CAR-T cell activation assay prepared by 293T-40 and 293T-41

[0293] CD69-CD25-% CD69+CD25-% CD69+CD25+% CD69-CD25+% C-1-50(293T-40) 23.61 28.25 47.01 1.14 C-1-50(293T-41) 30.05 41.29 27.99 0.68 C-1-50(293T-2) 33.82 45.09 20.71 0.37 Control T cells 85.05 5.57 7.77 1.61

[0294] In addition, the fusion molecules expressed by 293T-29 and 293T-31 were co-expressed with 293T-7 to verify whether the co-expression of the fusion peptide (OKT3-8h-8TM) that recognizes CD3 alone and the fusion peptide CD7 (ScFv)-8h-8TM that recognizes CD7 can also enhance the effect of one-step CAR-T cell activation. The results are as follows Fig.11 shown. Fig.11 The horizontal axis represents one-step CAR-T cells prepared by different one-step viruses, and the vertical axis columns from top to bottom represent the proportions of CD69-CD25+, CD69+CD25+, CD69+CD25-, and CD69-CD25- cells. The results showed that the co-expression of the fusion peptide (OKT3-8h-8TM) that recognizes CD3 alone and the fusion peptide CD7 (ScFv)-8h-8TM that recognizes CD7 did not enhance the activation of one-step CAR-T cells. The applicant verified the above-mentioned one-step CAR-T from 3 different donor sources, and the results are shown in Table 15 below:

[0295] Table 15 One-step CAR-T cell activation assay prepared by 293T-36 and 293T-37

[0296] CD69-CD25- CD69+CD25- CD69+CD25+ CD69-CD25+ C-1-50(293T-36) 28.2 53.09 18.02 0.68 C-1-50(293T-37) 28.21 49.6 21.07 1.12 C-1-50(293T-7) 38.65 51.71 9.05 0.59 Control T cells 89.78 7.94 0.53 1.74

[0297] The difference in activation between the one-step virus prepared by 293T-40 / 293T-41 is due to the introduction of the CD7 molecule recognition fusion peptide, but the one-step virus prepared by the packaging cell lines 293T-29 and 293T-31 containing only CD7(ScFv)-8h-8TM fusion protein cannot promote the activation of T cells / CAR-T cells, and the fusion peptide (OKT3-8h-8TM) recognizing CD3 alone and the fusion peptide CD7(ScFv)-8h-8TM recognizing CD7 co-expressed by 293T- The one-step virus prepared by 36 / 293T-37 also cannot enhance the activation of one-step CAR-T cells. Only the one-step virus prepared by 293T-40 / 293T-41, which co-expresses fusion proteins that recognize CD3, CD28, and CD7 molecules, has the effect of enhancing the activation of the prepared one-step CAR-T cells. The enhanced T cell / CAR-T cell activation caused by the OKT3-CD86 fusion peptide and CD7 (ScFv)-8h-8TM is an unpredictable effect beyond molecular recognition.

[0298] The above experiments also prove that only the viruses prepared by the engineered cells of the present application and applied to the CAR-T cells prepared by the one-step process of the present application have high CAR expression and activity and better in vivo effectiveness. Conventional viruses cannot successfully prepare functional CAR-T in the one-step preparation process. 4.10 Analysis of the application of viruses expressing full-length OKT3 in the envelope to CAR-T cells prepared by the one-step process

[0299] The constant regions of mouse IgM and human IgM were used with OKT3ScFv to construct full-length antibody models, and it was found that full-length antibodies can also activate and transduce T cells well. Fig.12 As shown in Table 16, Fig.12 The horizontal axis represents one-step CAR-T, and the vertical axis represents the proportions of CD69-CD25+, CD69+CD25+, CD69+CD25-, and CD69-CD25- cells from top to bottom.

[0300] Table 16 Expression of surface active molecules in CAR-T cells prepared by one-step process using viruses expressing full-length OKT3 on the envelope

[0301] CD69-CD25- CD69+CD25- CD69+CD25+ CD69-CD25+ 293T-14 16.17 29.07 51.96 2.8 CT 88.57 2.59 3.85 5

[0302] Further, the CAR expression detection of one-step CAR-T cells was performed, as shown in Table 17 below:

[0303] Table 17 CAR expression of CAR-T cells prepared by one-step process using viruses expressing full-length OKT3 on the envelope:

[0304] CAR positive rate 293T-14 65.99

[0305] The results showed that the one-step prepared 293T-14 had a higher expression of the activation marker CD69+CD25+. The OKT3 fusion protein expressed by the one-step virus and one-step virus prepared cells used the constant regions of mouse IgM and human IgM and OKT3ScFv to construct a full-length antibody model, and also had the ability to prepare one-step CAR-T cells. The prepared one-step CAR-T cells had good activation and CAR expression capabilities.

[0306] The above experiments prove that anti-CD3 full-length antibody or ScFv, CD86 and CD3 ScFv fusion protein form polypeptide, CD86 and CD3ScFv fusion protein form polypeptide with CD137L, IL15, IL15Ra added, wherein the fusion protein form polypeptide refers to the ScFv fusion protein form polypeptide, can all use the scheme described in this application to prepare one-step virus and then perform one-step CAR-T preparation.

[0307] 4.11 Analysis of the activity of CAR-T cells obtained by one-step CAR-T cell preparation using engineered cell lines expressing OKT3 (UCHT1) and CD86 molecules

[0308] The CAR-T cells prepared by one-step method can activate T cells well and achieve T cell transfection using monoclonal cell lines that knock out HLA expression OKT3 (UCHT1) and CD86. The CAR-T cells obtained by one-step method are higher than those prepared by conventional methods (C-1-50 (activation)). Based on the cell construction scheme of knocking out HLA expression by gene editing method, we constructed engineered cell lines expressing OKT3 (UCHT1) and CD86 molecules, obtained multiple cell lines, and verified that multiple cell lines have the ability to activate resting T cells. The CAR-T cells obtained by one-step CAR-T cell preparation (C-1-50CAR structure) are all functionally active. The results of CAR positivity test are shown in Table 18 below, and the degree of activation of one-step CAR-T cells is shown in Table 19:

[0309] Table 18 CAR expression of one-step C-1-50 CAR-T cells obtained by infecting T cells with C-1-50 viruses prepared from multiple engineered cell lines expressing OKT3 (UCHT1) and CD86 molecules using one-step CAR-T process

[0310] CAR positive rate 293T-PB5-D10 79.57 293T-PB5-H10 83.85 293T-PB6-A2 81.99 293T-PB6-H11 67.06 293T-1 78.42 Beads-1503 68.2

[0311] Table 19 Expression of surface activity markers of one-step C-1-50 CAR-T cells obtained by infecting T cells with C-1-50 viruses prepared from multiple engineered cell lines expressing OKT3 (UCHT1) and CD86 molecules using one-step CAR-T process

[0312] <![CDATA[CD69 - CD25 - (%)]]> <![CDATA[CD69 + CD25 - (%)]]> <![CDATA[CD69 + CD25 + (%)]]> <![CDATA[CD69 - CD25 + (%)]]> C-1-50(293T-PB5-D10) 3.14 9.81 85.87 1.17 C-1-50(293T-PB5-H10) 4.76 12.22 81.54 1.48 C-1-50(293T-PB6-A2) 2.13 6.71 90.28 0.87 C-1-50(293T-PB6-H11) 13.63 21.77 61.8 2.8 C-1-50(293T-1) 1.24 2.5 96.01 0.25 C-1-50(activated) 3.74 8.09 86.46 1.72 Magnetic beads activated T cells 6.62 8.45 81.68 3.25 Control T cells 88.57 2.59 3.85 5

[0313] As shown in Tables 18 and 19, the C-1-50 virus prepared by multiple engineered cell lines expressing OKT3 (UCHT1) and CD86 molecules, the one-step C-1-50CAR-T cell CAR expression and surface activity marker expression obtained by infecting T cells with the one-step CAR-T process all have high levels of CAR expression, and CD69 and CD25 expression are both higher than 50%, proving that the 293T packaging cells transformed by the present application, whether the 293T-X cell line is constructed by lentivirus or the one-step virus prepared by constructing the 293T-PBX cell line by gene editing or non-viral transfection, the one-step CAR-T prepared has high activation and CAR expression ability. It can be seen that although multiple one-step engineered cell lines expressing OKT3 (UCHT1) and CD86 molecules can be obtained through the gene editing scheme (their differences may be that they express OKT3 (UCHT1) and CD86 molecules to varying degrees), this does not affect their final application in the one-step process preparation.

[0314] 4.12 Analysis of the in vivo anti-tumor ability of CAR-T cells obtained by using engineered cell lines expressing OKT3 (UCHT1) and CD86 molecules in one-step CAR-T cell preparation

[0315] The CAR-T cells prepared in one step by packaging viruses using a monoclonal cell line that knocked out HLA expression OKT3 (UCHT1) and CD86 have good in vivo anti-tumor ability.

[0316] NALM6-Luc-GFP was used as the positive target cell and injected into NCG mice through the tail vein at a dose of 5e5 / mouse for pre-tumor formation. The pre-tumor formation time was 3 days. The virus expressing OKT3 (UCHT1) and CD86 molecules in the envelope was prepared by a one-step engineered cell line that knocked out HLA molecules through a gene editing scheme. The one-step CAR-T cells prepared by the one-step process were reinfused through the tail vein at a dose of 5e5 effective cells / mouse. Conventionally prepared CAR-T cells were used as the control C-1-50 (conventional). The 8-day collection group, the results are as follows Fig.13 shown.

[0317] Fig.13 Among them, 293T-PB5-D10 and 293T-PB5-H10 are OKT3 clones, and 293T-PB6-H11 is UCHT1 clone. The results showed that 293T-PB5-D10, 293T-PB5-H10, 293T-PB6-H11 and 293T-1 prepared by one-step method had comparable in vivo efficacy, and were superior to C-1-50 (activated) CAR-T cells prepared by traditional activation scheme.

[0318] Example 5 Optimization of one-step CAR-T cell preparation scheme

[0319] Monocytes (PBMC cells, either fresh cells collected or cells frozen after separation) or T lymphocytes (either fresh cells after sorting or cells frozen after sorting) are prepared by using serum-free medium or serum-free medium + 2% to 10% autologous plasma or 1640 (or other immune cell culture medium) + 10% FBS, the cell density is adjusted to 2.00E+06 cell / ml, inoculated into a well plate (500ul for 24 wells, 1ml for 12 well plates, 2ml for 6 well plates), and the one-step virus prepared by packaging any of the above cell lines of 293T-1 / 293T-2 / 293T-6 / 293T-8 / 293T-9 / 293T-13 / 293T-14 / 293T40 / 293T-41, 293T-PB5, 293TPB6 and 293T-PB5, 293TPB6 daughter cell lines is added, and the virus is incubated at 37°C and 5% CO2 incubator, static culture, collection and washing after 10 minutes, 30 minutes, 24 hours, 30 hours, 48 ​​hours, and 72 hours after adding the one-step virus can all be called one-step CAR-T cells.

[0320] During the cell preparation process, mononuclear cells (PBMC cells, either fresh cells collected or cells frozen after separation) or T lymphocytes (either fresh cells after sorting or cells frozen after sorting) were compared as the starting cell raw materials for one-step CAR-T: PBMC or separated T lymphocytes were used as the starting cells for one-step CAR-T cells, respectively. Considering the individual differences of the human body, the above cells were selected from two donor sources #865 and #866 to more fully illustrate the effect of the starting cell composition on the function of one-step CAR-T. The specific steps are as follows:

[0321] 1) Use lymphocyte separation solution (Tianjin Haoyang Huake Biological, catalog number: LTS1077) to process whole blood samples from healthy donors, separate peripheral blood mononuclear cells (PBMCs), wash with physiological saline, and directly resuspend PBMCs in cryopreservation solution (cryopreservation solution composition is FBS:DMSO=9:1) and cryopreserve in different tubes, store in -80℃ refrigerator or liquid nitrogen tank, as PBMC starting cells. Use anti-CD3 antibody or Beads (also called magnetic beads or activated magnetic beads) to separate and purify T cells, and resuspend the harvested T cells in cryopreservation solution (cryopreservation solution composition is FBS:DMSO=9:1) and cryopreserve in different tubes, store in -80℃ refrigerator or liquid nitrogen tank as T cell starting scheme.

[0322] 2) Take out the frozen PBMCs from the -80°C refrigerator or liquid nitrogen tank, revive them in a 37°C water bath, inoculate them into immune cell culture medium containing 10% FBS, and let them adhere to the wall overnight in a 37°C, 5% CO2 incubator to remove some of the non-adherent T cells and restore the frozen T cells.

[0323] 3) Collect PBMC or T cells, resuspend and plate with RPMI 1640 medium containing 10% FBS (manufacturer: Gibco, catalog number: C11875500BT), and the cell concentration is 2.00E+06 cells / ml. Calculate the amount of lentiviral vector required for transduction according to the cell amount, virus titer, and virus infection multiplicity. Add the one-step lentiviral vector and the transfection agent (DEAE Dextran, 8μg / ml) to the transduction system at the same time, collect and wash the CAR-T cells after 10 minutes, 30 minutes, 24 hours, 30 hours, 48 ​​hours, and 72 hours of transduction and wash with washing buffer (such as PBS, saline, etc.). After transduction, a small amount of cells before harvesting are reserved and replaced with RPMI 1640 complete medium containing 10% FBS and cytokines (IL7, IL15, IL21) in a well plate or culture bottle for 6-10 days for subsequent CAR-T cell positive rate and functional evaluation.

[0324] 6-8 week old NCG female mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were used to construct a tumor model using the DLD1-CEA-Luc-GFP cell line (the DLD1-CEA-Luc-GFP cell line is constructed based on the DLD1 cell line with CEA antigen and Luc-GFP, and the positive expression rate of CEA and Luc-GFP is ≥90%). Method for constructing a tumor model: using DLD1-CEA-Luc-GFP cells, subcutaneous model: tumor-bearing dose 1.00E+06 cells / mouse, tumor-bearing for 12 days; peritoneal model: tumor-bearing dose 1.00E+06 cells / mouse, peritoneal tumor-bearing for 7 days; the day of CAR-T infusion was recorded as day 0, and the mice were grouped according to the subcutaneous tumor volume, and the test product was administered by intraperitoneal injection, with a dose of 5.00E+05 CAR-T cells / mouse. Observation after administration: body weight was measured once a week, tumor size was measured twice a week, and live imaging was performed once a week.

[0325] The results are as follows Fig.14 and Fig.15 As shown, the applicant unexpectedly discovered that in the one-step CAR-T preparation, PBMC as the starting material has better in vivo effectiveness, and this better effectiveness is because PBMC is used as the starting material, which is not related to the tumor-bearing method and the CAR-T reinfusion method.

[0326] Therefore, the preferred one-step CAR-T cell preparation scheme of the present application is:

[0327] 1) Preparation of one-step virus: see Examples 1 and 2 for details

[0328] 2) For mononuclear cells (PBMC cells, either fresh cells or cells frozen after separation), use serum-free medium or serum-free medium + 2%-10% autologous plasma or 1640 (or other immune cell culture medium) + 2%-10% FBS (or 2%-10% autologous plasma), and adjust the cell density to a suitable density, such as 2.00E+06 cell / ml, which can be 1.00E+06 cell / ml-3.00E+07 cell / ml;

[0329] 3) adding the one-step virus prepared in step 1) to the cells in step 2), mixing evenly, and culturing statically in a 37° C., 5% CO2 incubator;

[0330] 4) After step 3), one-step CAR-T cells can be obtained by collecting and washing at any time period between 10 minutes and 72 hours.

[0331] Example 6 One-step virus preparation of universal CAR-T

[0332] The general CAR-T preparation process of one-step virus is as follows:

[0333] Peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation, cultured overnight with 1640+10% FBS medium, adherent cells were removed and suspended cells were collected, and after centrifugation and resuspension, the cell density was adjusted to 2.00E+06cell / ml and inoculated into well plates (24 wells 500ul, 12 well plates 1ml, 6 well plates 2ml). Fresh PBMCs were separated or frozen PBMCs were resuscitated, and viral infection was performed in a medium without cytokines or with cytokines. During infection, special virus particles with activation stimulation signals can be directly added, or virus empty shell particles can be added while adding normal lentiviruses to complete transfection, and cultured statically at 37°C, 5% CO2 incubator. The day of virus transfection was recorded as Day0. The amount of virus added was calculated according to the flow cytometry titer at 5MOI, and the one-step virus obtained above or the virus empty shell particles were added while adding conventional viruses, and the cells in each well were mixed and cultured statically at 37°C, 5% CO2 incubator.

[0334] At 48 hours after virus infection, collect and wash the cells for electroporation. Use Lonza electroporation instrument or other electroporation instruments such as Neon, and select a suitable electroporation procedure for T cells. The molar ratio of Cas9 protein:sgRNA can be 1:2 or other suitable ratios. RNP needs to be incubated in vitro for several minutes, and mixed evenly with an appropriate amount of T cells resuspended in electroporation buffer and transferred into an electroporation cup for electroporation; after electroporation is completed, transfer to a culture bottle containing an appropriate amount of culture medium for continued culture.

[0335] The steps for preparing universal CAR-T using the traditional magnetic bead activation method are as follows:

[0336] Lymphocytes are separated by gradient centrifugation; mononuclear cells (PBMC cells) or T lymphocytes are obtained, and the obtained PBMC cells or T lymphocytes are activated by magnetic beads with anti-CD3, CD28 monoclonal antibodies or anti-CD3 / CD28 antibodies, and the activated PBMC or T cells are infected at a certain multiplicity of infection (MOI). At 48 hours of viral infection, the cells are collected and washed for electroporation. A Lonza electroporation instrument or other electroporation instruments such as Neon are used to select a suitable electroporation procedure for T cells. The molar ratio of Cas9 protein: sgRNA is 1:2 or other suitable ratios. RNP needs to be incubated in vitro for several minutes, and mixed evenly with an appropriate amount of T cells resuspended in electroporation buffer and transferred to an electroporation cup for electroporation; after electroporation, the cells are transferred to a culture flask containing an appropriate amount of culture medium for further culture. Among them, sgRNA is designed for one or more genes of TRAC, B2M, and CIITA. For specific sgRNA sequences, please refer to patent 201710983276.X.

[0337] Table 20 Source units and product numbers of main reagents

[0338]

[0339] The viable cells were analyzed using a cell counter 2 days before electroporation (-2), on the day of electroporation (0), and 1 day after electroporation (1), and the cell viability was calculated. The results are shown in Table 21 and Fig.16 As shown:

[0340] Table 21 Viability of universal CAR-T cells prepared by different preparation schemes

[0341]

[0342] The results showed that the cell viability of T cells activated by the one-step method after electroporation was significantly higher than that by the traditional magnetic bead activation method, and T cells activated by the one-step method were more tolerant to electroporation damage.

[0343] Example 7 Application of Virus Empty Shell Particles in Electroporation

[0344] Electroporation is also called electroporation transfection, which is a physical transduction method that can not only transfer DNA and RNA, but also antibodies, enzymes and other bioactive molecules into bacteria, yeast, animal cells and plant cells. Electroporation has the advantages of high efficiency, simplicity and wide spectrum application. At present, engineered immune cells such as CAR-T and CAR-NK have also begun to use the method of electroporation, but in the process of preparing CAR-T by electroporation, T cells need to be activated in advance, and the CD3 / 28 magnetic beads used in the activation process cannot be completely removed before electroporation, which greatly affects the stability of the electroporation system, and T cells prepared by different culture schemes have different tolerance to voltage intensity. In order to ensure the vitality of CAR-T cells after electroporation, the optimal voltage for CAR transduction conditions is often not used, which leads to the electroporation of CAR-T. The cell viability and positivity are often inferior to traditional processes. The applicant of this application found that the use of the virus shell particles of this application can significantly improve the CAR transduction efficiency of the electroporation scheme while ensuring the vitality of T cells.

[0345] The electroporation protocol using empty viral shell particles is as follows:

[0346] 1. PBMCs and virus empty shell particles were incubated in PRMI-1640 (Gibco; C1187500BT) complete medium at different MOIs for 24 or 48 hours; CD3 / 28 beads were incubated at a ratio of cells (PBMC): beads = 1:1 under the same conditions and time;

[0347] 2. After 24 or 48 hours, collect the activated cells and count them (for cells activated by traditional CD3 / 28Beads, it is necessary to remove the magnetic beads through a magnetic stand (Promega; Z5332) before counting);

[0348] 3. The cells under different activation conditions were mixed with 30ug of PBCAR19 plasmid and 10ug of PB transposase plasmid at a cell number of 1e7 / reaction, and the system was supplemented to 100uL with the electroporation buffer in P3Primary Cell 4D X Kit L (Lonza; V4XP-3024). The above system was added to the electroporation cup and electroporated by Lonza 4D nucleofector electroporation system. The electroporation program was EO-115.

[0349] 4. After electroporation, add 600uL of AM-V Serum Free Medium (Tianjin Haoyang; ANDLTBD) containing IL-7 / 15 / 21 (PeproTech; AF-200-07-250ug, AF-200-15-250ug, AF-200-21-250ug) and 10% FBS to the electroporation cup and recover in the cell culture incubator for 40 minutes;

[0350] 5. Use the pipette in the P3Primary Cell 4D X Kit L to transfer the recovered cells to a 6-well plate for continued culture;

[0351] 6. 5 and 9 days after electroporation, cells were harvested and labeled with Protein L-PE (Sino biological; 11044-H07E-P) and then flow cytometry was used to detect the CAR-T positive rate. The CAR positive rate results are shown in Table 22 and Fig.17 shown.

[0352] Table 22 CAR transduction efficiency of CAR-T prepared by electroporation scheme under different activation schemes

[0353]

[0354] The results showed that the positive rate of T cells activated by virus empty shell particles after electroporation was higher than that of T cells activated by traditional CD3 / 28Beads, and the positive rate of CAR-T was optimal when the virus empty shell particles were at an MOI of 2-4. We also conducted the above test for 48 hours and obtained consistent results.

[0355] We further compared the viability of CAR-T cells prepared by electroporation of T cells activated by virus empty shell particles with that of CAR-T cells activated by traditional CD3 / 28Beads. Cells were collected 1, 3, and 5 days after electroporation and the cell viability was detected by trypan blue staining. The results are shown in Table 23 and Fig.18 As shown:

[0356] Table 23 CAR-T activity rate (%) prepared by electroporation scheme under different activation schemes

[0357]

[0358] The results showed that there was no significant difference in the activity rate among the experimental groups at the same activation time.

[0359] In summary, the use of viral empty shell particle activation scheme can significantly improve the transduction efficiency of electroporated CAR without affecting the viability of T cells.

[0360] The sequences used in the above examples of the present application are shown in the following sequence table. It should be understood that the following sequences are only exemplary sequences of the embodiments of the present application, and are not any limitation to the embodiments of the present application.

[0361] Sequence Listing:

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

Claims

1. An engineered cell, characterized in that The engineered cells express one or more proteins, which are localized in the cell membrane of the engineered cells and interact with immune cell surface molecules.

2. The engineered cell according to claim 1, wherein the one or more proteins comprise one or more selected from the group consisting of: Proteins that stimulate the CD3 / TCR complex, proteins that stimulate co-stimulatory molecules on the cell surface, proteins that can bind to molecules with endocytosis function on the surface of immune cells, proteins that can bind to certain specific molecules on the surface of immune cells, and proteins that can bind to cytokine receptors on the surface of immune cells.

3. The engineered cell of claim 1, wherein the one or more proteins are selected from the group consisting of: Anti-CD3 scFv-CD86; Anti-CD3 scFv-8h-8TM-2A-CD86; Anti-CD3 scFv-8h-8TM-2A-CD86&B-137L-1; Anti-CD3 scFv-8h-8TM-2A-CD86&IL15-GSlinker-IL15Ra; Anti-CD3 scFv-8h-8TM-2A-CD86&IL15-IgG4-4TM; and The expression of Anti-CD3 scFv-CD86&ScFv(CD7)-8h-8TM gene is achieved by transducing engineered cells with a vector.

4. The engineered cell according to any one of claims 1-3, wherein the one or more proteins comprise one or more amino acid sequences selected from SEQ ID NOs: 1 to 25, conservatively substituted variants thereof, or amino acid sequences having more than 80% sequence identity therewith.

5. An engineered viral particle having an envelope comprising one or more proteins according to any one of claims 1-3.

6. The engineered viral particle of claim 5, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR).

7. A method for preparing engineered T cells, comprising: contacting the viral particle according to any one of claims 4-5 with T cells.

8. The method according to claim 7, wherein the T cells are T cells isolated from peripheral blood mononuclear cells (PBMC) or T cells in peripheral blood mononuclear cells that have not been separated.

9. A nucleic acid molecule comprising a nucleic acid sequence encoding one or more proteins according to any one of claims 1 to 3.

10. One or more proteins encoded by the nucleic acid molecule according to claim 9.

11. Use of the nucleic acid molecule according to claim 9 or the protein according to claim 10 for preparing an engineered cell.

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