CN118165113A - Method for improving immune response cell function - Google Patents

Abstract

The present invention relates to a method for improving the function of an immune responsive cell. The present invention discloses an immunoresponsive cell that expresses at least one receptor capable of binding an antigen and a type I interferon. The immune response cell is applied to treating tumors, infectious diseases and other diseases, and has remarkable excellent capability of killing tumors or pathogens.

Description

A method for improving the function of immune response cells

This application is a divisional application of the invention patent application for "A method for improving the function of immune response cells" with application number 2017800217918 and application date 2017.4.26.

Technical Field

The present invention belongs to the field of immunology, and more specifically, the present invention relates to a method for improving the function of immune response cells.

Background Art

Chimeric antigen receptor (CAR) is an artificial recombinant receptor, which usually contains the antigen recognition domain of a monoclonal antibody located in the extracellular region, a transmembrane region, and an intracellular activation signal domain of an immune response cell. In recent years, clinical trials have achieved great success in the treatment of B-cell leukemia using CAR-modified T cells (CAR-T) targeting CD19. However, many leukemia patients will relapse. And not all blood tumors are very effective. In addition, CAR-T cells are not very effective in treating solid tumors. Therefore, it is still very important to improve the existing CAR-T cell technology and increase its anti-tumor activity.

Type I interferons were discovered more than half a century ago. Type I interferons include IFNα protein (a class of identical proteins encoded by 13 human genes from IFNA1 to IFNA13), IFNβ (encoded by a single human and mouse gene IFNB1) and other less studied interferons such as IFNε, IFNκand IFNω (2. Trinchieri, G. Type I interferon: friend or foe? J. Exp. Med. 207, 2053-2063 (2010). 3. Kaur, S. & Platanias, LCIFN-β-specific signaling via a unique IFNAR1 interaction. Nat. Immunol. 14, 884-885 (2013)). Type I interferons are produced by various types of cells after activation of pattern recognition receptors (PRRs). PRRs respond to viral or bacterial components and ectopic endogenous molecules (such as cytoplasmic DNA and extracellular DNA and RNA) (Kawai, T. & Akira, S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat. Immunol. ¹¹ , 373-384 (2010)). Type I interferons transmit signals through the homodimeric IFNα/β receptor 1 (IFNAR1) (which has a particularly high affinity for IFNβ) or the IFNAR1-IFNAR2 heterodimer (which can bind to all type I interferons). Activation of these receptors leads to upregulation of transcription of IFN-stimulated genes (ISGs), which triggers many immunostimulatory effects (Hervas-Stubbs, S. et al. Direct effects of type I interferons on cells of the immune system. Clin. Cancer Res. 17, 2619er Res. es. mmde Weerd, NA et al. Structural basis of a unique interferon-β signaling axis mediated via the receptor IFNAR1. Nat. Immunol. 14, 901ol. nol. is oMcNab, F., Mayer-Barber, K., Sher, A., Wack, A. & O A. &, A. Type I interferons in infectious disease. Nat. Rev. Immunol. 15, 87nol. Immun). Studies have shown that type I interferons have anticancer effects on some tumors, which may be attributed to their immunostimulatory function. However, systemic administration of type I interferon may cause immunosuppression (Lotrich, FEMajordepression during interferon-αtreatment:vulnerability andprevention.Dialogues Clin.Neurosci. ¹¹ , 417-425(2009)), and is accompanied by major adverse events, the most common of which are fatigue, anorexia, hepatotoxicity, flu-like symptoms and severe depression (Kreutzer, K., Bonnekoh, B., Franke, I., Ulrich, J. & Gollnick, H.Sarcoidosis, myasthenia gravis and anteriorischaemic optic neuropathy: severe side effects of adjuvant interferon-αtherapy in malignant melanoma?. J.Dtsch.Dermatol.Ges.2, 689-694(in German)(2004)), and such serious side effects severely limit its application.

Summary of the invention

The present invention overcomes the aforementioned problems and provides additional advantages.

1. An immune response cell expressing a receptor that binds an antigen; and exogenous type I interferon.

2. The immune response cells according to item 1, wherein the immune response cells include: T cells, natural killer cells, cytotoxic T lymphocytes, natural killer T cells, DNT cells, and/or regulatory T cells.

3. The immune response cell as described in any of the preceding items, wherein the antigen is a tumor antigen or a pathogen antigen.

4. The immune response cell as described in any of the preceding items, wherein the exogenous type I interferon is constitutively expressed or inducibly expressed; preferably, the promoter used to express the type I interferon includes: an immune cell inducible promoter; preferably, the immune cell inducible promoter is the NFAT6 promoter.

5. The immune response cell as described in any of the preceding items, which expresses an endogenous or recombinant antigen-binding receptor; preferably, the antigen-binding receptor comprises sequentially connected: an antibody that specifically binds to the antigen, a transmembrane region and an intracellular signaling region.

6. An immune response cell as described in any of the preceding items, wherein the intracellular signaling region of the immune response cell contains a T cell stimulatory signal molecule or a combination of a T cell stimulatory signal molecule and a T cell activation co-stimulatory molecule; preferably, the T cell stimulatory signal molecule is selected from: CD3ζ or FcεRIγ; more preferably CD3ζ; or the T cell activation co-stimulatory molecule is selected from: CD27, CD28, CD137, CD134, the intracellular signaling region of ICOS protein, or a combination thereof.

7. The immune response cell according to any of the preceding items, wherein the amino acid sequence of the antigen-binding receptor is at least 90% identical to one of the following sequences:

SEQ ID NO:49; SEQ ID NO:50; SEQ ID NO:51; SEQ ID NO:54; SEQ ID NO:55; SEQ ID NO:56; SEQ ID NO:61; SEQ ID NO:62; SEQ ID NO :63; SEQ ID NO:64; SEQ ID NO:65; SEQ ID NO:66; SEQ ID NO:67; SEQ ID NO:68; SEQ ID NO:69; SEQ ID NO:70; SEQ ID NO:71; SEQ ID NO:72; SEQ ID NO:73; SEQ ID NO:74; SEQ ID NO:75; and SEQ ID NO:77.

8. The immune response cell as described in item 7, wherein the receptor that specifically binds to the antigen and the exogenous type I interferon are encoded by a nucleotide sequence that is at least 90% identical to SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60 or SEQ ID NO:76.

9. The immune response cell according to any of the preceding items, which does not contain exogenous co-stimulatory ligands.

10. The immune response cell according to any of the preceding items, wherein the type I interferon comprises: IFNα or IFNβ.

11. The immune response cell according to any of the preceding items, wherein the antigen-binding receptor and/or type I interferon are constitutively or inducibly expressed on the surface of the immune response cell.

12. The immune response cell according to any one of the preceding items, comprising an expression construct, the expression construct comprising: an expression cassette of the antigen-binding receptor; and an expression cassette of the type I interferon.

13. The immune response cell as described in any of the preceding items, wherein the antigen-binding receptor and/or type I interferon are expressed using a viral vector; preferably, the viral vector is a retroviral vector; more preferably, the viral vector includes: a lentiviral vector, a retroviral vector or an adenoviral vector.

14. An immune response cell as described in any of the preceding items, wherein the antigen-binding receptor recognizes and binds to pathogenic microorganisms.

15. An immune response cell as described in item 14, wherein the pathogenic microorganism includes a virus, a bacterium, a fungus, a protozoa or a parasite; more preferably, the pathogenic microorganism is a virus; or more preferably, the pathogenic microorganism is selected from cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus and influenza virus.

16. The immune response cell as described in any of the preceding items, wherein the antigens include: prostate specific membrane antigen (PSMA), carcinoembryonic antigen (CEA), IL13Ralpha, HER-2, CD19, NY-ESO-1, HIV-1Gag, Lewis Y, MART-1, gp100, tyrosinase, WT-I, hTERT, mesothelin, EGFR, EGFRvIII, phosphatidylinositol proteoglycan 3, EphA2, HER3, EpCAM, MUC1, MUC16, CLDN18.2, folate receptor, CLDN6, CD30, CD138, ASGPR1, CDH16, GD2, 5T4, 8H9, αvβ6 integrin, B cell maturation antigen (BCMA), B7-H3, B7-H6, CAIX, CA9, CD20, CD22, kappa light chain (kappa light chain), CD33, CD38, CD44, CD44v6, CD44v7/8, CD70, CD123, CD171, CSPG4, EGP2, EGP40, ERBB3, ERBB4, ErbB3/4, FAP, FAR, FBP, embryonic AchR, GD2, GD3, HLA-AI MAGE A1, MAGE3, HLA-A2, IL11Ra, KDR, Lambda, MCSP, NCAM, NKG2D ligand, PRAME, PSCA, PSC1, ROR1, Sp17, SURVIVIN, TAG72, TEM1, TEM8, VEGRR2, HMW-MAA, VEGF receptor, and/or fibronectin, tenascin, or oncofetal variants of tumor necrosis areas.

17. An expression construct comprising sequentially linked: an expression cassette for an antigen-binding receptor; and an expression cassette for a type I interferon; wherein the antigen-binding receptor and the type I interferon are as defined in one of the preceding items.

18. A method for increasing the viability of immune response cells administered to an individual, the immune response cells expressing an antigen-binding receptor as described in any one of items 1 to 16, and wherein the method comprises administering to the individual the immune response cells and an effective amount of exogenous type I interferon.

19. A method as described in claim 18, wherein the exogenous type I interferon and the immune response cells expressing the receptor that binds the antigen are administered sequentially or simultaneously.

20. A method as described in item 18 or 19, wherein the exogenous type I interferon is administered to the patient simultaneously with the immune response cells by co-expression in the immune response cells.

21. A method as described in any one of items 18 to 20, wherein the immune response cells include T cells, natural killer cells, cytotoxic T lymphocytes, natural killer T cells, DNT cells, and/or regulatory T cells.

22. A method as described in any one of items 18 to 21, wherein the method increases the sum of the number of cytotoxic T cells and helper T cells in the peripheral blood of the individual by at least 50% after administering the immune response cells to the individual, compared to the absence of the exogenous type I interferon.

23. A method as described in any one of items 18 to 22, wherein the method is such that about 5 days after the immune response cells are administered to the individual, the sum of the numbers of cytotoxic T cells and helper T cells in the individual's peripheral blood is greater than 15,000 cells/μL; about 7 days after the immune response cells are administered, the sum of the numbers of cytotoxic T cells and helper T cells in the individual's peripheral blood is greater than 500 cells/μL; or about 10 days after the immune response cells are administered, the sum of the numbers of cytotoxic T cells and helper T cells in the individual's peripheral blood is greater than 50 cells/μL.

24. Use of the immune response cells described in any one of items 1 to 16 in the preparation of a pharmaceutical composition for treating tumors, pathogen infections, or enhancing the immune tolerance of an individual in need thereof.

25. The use according to item 24, wherein the tumor comprises: pancreatic cancer, liver cancer, lung cancer, gastric cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, kidney cancer, leukemia, myeloma, ovarian cancer, cervical cancer, ovarian cancer, cervical cancer or glioma; or

The pathogens include: viruses, bacteria, fungi, protozoa or parasites; preferably, the viruses include: cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus or influenza virus.

26. The use of item 24 or 25, wherein the medicament reduces the tumor by at least 30% as measured by computed tomography.

27. The use of item 24 or 25, wherein the drug causes complete disappearance of the tumor as measured by computed tomography.

28. A pharmaceutical composition, comprising:

The immune response cell of any one of items 1 to 16; and

Pharmaceutically acceptable carrier or excipient.

29. A kit comprising:

The immune response cell as described in any one of items 1-16; and

Instructions directing how to administer the immune response cells to an individual.

According to one aspect of the present invention, the present invention provides an immune response cell, wherein the cell expresses a receptor that binds to an antigen; and exogenous type I interferon.

In some embodiments, the immune response cells of the present invention include T cells, natural killer cells, cytotoxic T lymphocytes, natural killer T cells, DNT cells, and/or regulatory T cells.

In some embodiments, the antigen-binding receptor is endogenous. In some embodiments, the antigen-binding receptor is recombinant. In some embodiments, the antigen-binding receptor is a chimeric antigen receptor. In some embodiments, the antigen-binding receptor includes an extracellular antigen binding region, a transmembrane region, and an intracellular signal region connected in sequence. In some embodiments, the antigen binding unit is an antibody or fragment thereof that specifically binds to the antigen. In some embodiments, the intracellular signal region may contain a signal motif of a known immunoreceptor tyrosine activation motif (ITAM). In some embodiments, examples of the ITAM containing cytoplasmic signaling sequences include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

In some embodiments, the intracellular signaling region of the antigen-binding receptor includes one or more costimulatory domains. In some embodiments, the costimulatory domain is selected from one or more of those listed in Table 1. In some embodiments, the costimulatory domain is selected from one or more of CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a/CD18), 4-1BBL, MyD88, and 4-1BB. In some embodiments, the costimulatory domain is selected from two of CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a/CD18), 4-1BBL, MyD88, and 4-1BB.

In some embodiments, the amino acid sequence of the antigen-binding receptor is at least 90% identical to one of SEQ ID NO:49; SEQ ID NO:50; SEQ ID NO:51; SEQ ID NO:54; SEQ ID NO:55; SEQ ID NO:56; SEQ ID NO:61; SEQ ID NO:62; SEQ ID NO:63; SEQ ID NO:64; SEQ ID NO:65; SEQ ID NO:66; SEQ ID NO:67; SEQ ID NO:68; SEQ ID NO:69; SEQ ID NO:70; SEQ ID NO:71; SEQ ID NO:72; SEQ ID NO:73; SEQ ID NO:74; SEQ ID NO:75; and SEQ ID NO:77.

In some embodiments, the antigen-binding receptor is encoded by a nucleotide sequence that is at least 90% identical to SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, or SEQ ID NO:76.

In some embodiments, the immune response cells do not contain exogenous co-stimulatory ligands.

In some embodiments, the antigen that can be bound by the antigen-binding receptor includes a tumor antigen or a pathogen antigen. In some embodiments, the tumor antigen is selected from prostate-specific membrane antigen (PSMA), carcinoembryonic antigen (CEA), IL13Ralpha, HER-2, CD19, NY-ESO-1, HIV-1Gag, Lewis Y, MART-1, gp100, tyrosinase, WT-I, hTERT, mesothelin, EGFR, EGFRvIII, phosphatidylinositol proteoglycan 3, EphA2, HER3, EpCAM, MUC1, MUC16, CLDN18.2, folate receptor, CLDN6, CD30, CD138, ASGPR1, CDH16, GD2, 5T4, 8H9, αvβ6 integrin, B cell maturation antigen (BCMA), B7-H3, B7-H6, CAIX, CA9, CD20, CD22, kappa light chain (kappa light chain), CD33, CD38, CD44, CD44v6, CD44v7/8, CD70, CD123, CD171, CSPG4, EGP2, EGP40, ERBB3, ERBB4, ErbB3/4, FAP, FAR, FBP, embryonic AchR, GD2, GD3, HLA-AI MAGE A1, MAGE3, HLA-A2, IL11Ra, KDR, Lambda, MCSP, NCAM, NKG2D ligand, PRAME, PSCA, PSC1, ROR1, Sp17, SURVIVIN, TAG72, TEM1, TEM8, VEGRR2, HMW-MAA, VEGF receptor, and/or fibronectin, tenascin, or oncofetal variants of tumor necrosis areas.

In some embodiments, the pathogen antigen includes a viral antigen, a bacterial antigen, a fungal antigen, or a parasitic antigen. In some embodiments, the pathogen is a virus. The virus includes cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus, and influenza virus.

In some embodiments, the expression of the type I interferon is constitutive expression. In some embodiments, the expression of the type I interferon is inducible expression. In some embodiments, the type I interferon is expressed on the surface of the immune response cell. In some embodiments, the type I interferon includes: IFNα or IFNβ.

According to one aspect of the present invention, the present invention provides an expression construct comprising: an expression cassette of an antigen-binding receptor of the present invention; and an expression cassette of a type I interferon, connected in sequence. In some embodiments, the expression of the type I interferon is constitutive expression. In some embodiments, the expression of the type I interferon is inducible expression. In some embodiments, the expression of the type I interferon is inducible expression, and the expression of the type I interferon is an inducible promoter. In some embodiments, the inducible promoter used to express the type I interferon is a NFAT6 promoter. In some embodiments, the NFAT6 promoter comprises a nucleic acid sequence as described in SEQ ID NO: 78.

According to one aspect of the present invention, the present invention provides a vector that expresses an antigen-binding receptor and/or type I interferon of the present invention. In some embodiments, the viral vector is a lentiviral vector, a retroviral vector, or an adenoviral vector. In some embodiments, the viral vector is a retroviral vector.

According to one aspect of the present invention, the present invention provides a method for improving the viability of immune response cells administered to an individual, wherein the immune response cells express the antigen-binding receptors of the present invention, and wherein the method comprises administering the immune response cells and an effective amount of exogenous type I interferon to the individual. In some embodiments, the exogenous type I interferon and the immune response cells expressing the antigen-binding receptors are administered sequentially or simultaneously. In some embodiments, the exogenous type I interferon is co-expressed in the immune response cells and administered to the patient simultaneously with the immune response cells.

According to one aspect of the present invention, the present invention provides the use of the immune response cells of the present invention in the preparation of a pharmaceutical composition for treating tumors, pathogen infections, or enhancing the immune tolerance of an individual in need thereof. The present invention also provides a method for treating tumors or pathogen infections of an individual, or for enhancing the immune tolerance of an individual, comprising administering the immune response cells of the present invention to an individual in need thereof.

In some embodiments, the method of the present invention increases the sum of the number of cytotoxic T cells and helper T cells in the peripheral blood of the individual by at least 50% after administering the immune response cells to the individual, compared to the absence of the exogenous type I interferon. In some embodiments, the method causes the sum of the number of cytotoxic T cells and helper T cells in the peripheral blood of the individual to be greater than 15,000 cells/μL about 5 days after administering the immune response cells to the individual; the sum of the number of cytotoxic T cells and helper T cells in the peripheral blood of the individual to be greater than 500 cells/μL about 5 days after administering the immune response cells; or the sum of the number of cytotoxic T cells and helper T cells in the peripheral blood of the individual to be greater than 50 cells/μL about 5 days after administering the immune response cells.

In some embodiments, the tumor includes pancreatic cancer, liver cancer, lung cancer, gastric cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, kidney cancer, leukemia, myeloma, ovarian cancer, cervical cancer, ovarian cancer, cervical cancer or glioma. In some embodiments, the pathogen includes viruses, bacteria, fungi, protozoa or parasites; preferably, the virus includes cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus or influenza virus.

In some embodiments, the tumor of the individual is reduced by at least 30% as measured by computed tomography after treatment by the methods of the invention. In some embodiments, the tumor of the individual is completely eliminated as measured by computed tomography after treatment by the methods of the invention.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition comprising the immune response cell of the present invention and a pharmaceutically acceptable carrier or excipient.

According to one aspect of the present invention, the present invention provides a kit comprising the immune response cell of the present invention and instructions for instructing how to administer the immune response cell to an individual.

Incorporated by Reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings further illustrate the novel features disclosed in this specification. The features and advantages disclosed in this specification can be better understood with reference to these drawings, but it should be understood that these drawings are only used to illustrate specific implementations of the principles disclosed herein, and are not intended to limit the scope of the appended claims.

FIG1 shows a schematic diagram of the structure of the recombinant lentiviral vector pRRL-EF-1α-92-CAR ( FIG1A ), and the construction of the 92-28Z-NFAT6-IFN-β plasmid ( FIG1B ).

FIG2 shows the positive rate detection graph of PBMC infected with lentivirus.

FIG3 shows the positive rate detection graph of PBMC infected with lentivirus.

FIG4 shows a comparison of cytokine release by GPC3 CAR-T cells containing IFN and without IFN. FIG4A shows the induced expression of IFN-β by GPC3-28Z-IFN and GPC3-28Z CAR T cells. The results showed that IFN-β was expressed only when GPC3-28Z-IFN was co-incubated with Huh7 cells, indicating that after GPC3-28Z-IFN was activated by the target antigen, IFNβ could be successfully induced to express and secreted outside the cells. FIG4B shows a comparison of INF-γ induced expression by GPC3-28Z-IFN and GPC3-28Z CAR. FIG4C shows a comparison of IL-2 release caused by GPC3-28Z-IFN and GPC3-28Z CAR T cells in vitro. The results showed that GPC3-28Z-IFN could more effectively cause cytokine release, indicating that CAR T cells containing IFNβ could be more effectively activated.

Figure 5 shows a comparison of 85-28Z T cells and 85-28Z-IFN T cells inducing cytokine release in vitro in different cell lines. The results show that CAR T cells containing IFNβ can be activated more effectively.

FIG6 shows a comparison of the killing efficiency of GPC3-28Z CAR T cells containing IFNβ and GPC3 CAR T cells not containing IFNβ against various cell lines ( FIG6A : Huh7; FIG6B : Hep3B; FIG6C : PLC/PRR/5; FIG6D : Hep G2; FIG6E : SK-hep-1) in vitro.

FIG. 7 shows the killing activity of CLD18A2 CAR-T cells with and without IFN.

Figure 8 shows a comparison of the number of surviving cells in the peripheral blood of mice between CLD18A2 CAR-T cells containing IFNβ and CLD18A2 CAR-T cells not containing IFNβ after 5 days of reinfusion (Figure 8A), 7 days (Figure 8B) and 10 days (Figure 8C). The results showed that at all time points, the number of surviving cells of CLD18A2 CAR-T containing IFNβ was significantly higher than that of the CLD18A2 CAR-T cell group not containing IFNβ.

Figure 9 shows a comparison of the effects of GPC3-28Z CAR T cells containing IFNβ and GPC3-28Z CAR T cells not containing IFNβ on tumor volume over time in a mouse tumor model (Figure 9A) and a comparison of tumor photos (Figure 9B). The results showed that GPC3-28Z CAR T cells containing IFNβ were able to significantly reduce tumor volume compared with GPC3-28Z CAR T cells not containing IFNβ and the control group.

Figure 10 shows a comparison of the effects of CLD18A2 CAR-T cells containing IFNβ and CLD18A2 CAR-T cells not containing IFNβ on tumor volume over time in a mouse BGC-823-A2 cell subcutaneous transplantation tumor model (Figure 10A) and a comparison of tumor photos (Figure 10B). The results showed that CLD18A2 CAR-T cells containing IFNβ were able to significantly reduce tumor volume compared with CLD18A2 CAR-T cells not containing IFNβ and the control group.

Figure 11 shows a comparison of the anti-tumor activity of CLD18A2 CAR-T cells containing IFN and those without IFN in subcutaneous transplanted tumors of gastric cancer PDX models. The results showed that the tumor of one mouse in the treatment group containing IFN completely regressed.

Figure 12 shows a comparison of tumor infiltration in vivo by GPC3 CAR-T (92-28Z) cells containing IFN and without IFN. Figure 12A is a histochemical picture, and Figure 12B is a T cell number picture. The results showed that there were no obvious infiltrating CD3+ cells in the tumor tissue of the control group, and the number of CD3+T cells in the INFβ-CAR-T treatment group was higher than that in the 28Z CART group.

Figure 13 shows the comparison of immunohistochemistry of tumor infiltration in vivo by CLD18A2 CAR-T cells with and without IFN. The results showed that almost no T cell infiltration was observed around the tumor tissue for Mock T cells, 85-28Z and 85-2-28Z CAR T cells were seen at the edge of the tumor tissue, and 85-2-28Z-IFN T cells were observed to have some infiltration inside the tumor tissue.

FIG. 14 shows a schematic diagram of the structure of EGFR-CAR with and without IFN.

FIG. 15 shows the infection positive rate of T lymphocytes of mice infected with retrovirus.

Figure 16 shows a comparison of the ability of EGFR CAR T cells containing IFN and those without IFN to secrete mIFNβ in vitro. The results show that after stimulation of target cells, mCAR-806-mIFNβ can be successfully activated and induced to express mIFNβ, while no expression of mIFNβ was detected in the control group.

FIG. 17 is a comparison of cytokine release induced in vitro by EGFR CAR-T cells containing IFN and those without IFN ( FIG. 17A : mIL-2; FIG. 17B : mIFN-γ; FIG. 17C : mTNF-α).

Figure 18 shows a comparison of in vitro toxicity tests of EGFR CAR-T cells containing IFN and those without IFN. The results showed that EGFR-CAR and EGFR-CAR-IFN had a strong killing effect on target-positive CT26VIII cells compared with UT cells, and the difference was significant (***P < 0.001), and the killing percentage was dose-dependent, while untransfected UT cells had no killing effect on CT26 and CT26VIII, and EGFR-CAR and EGFR-CAR-IFN CAR-T cells had no killing effect on target-negative CT26 cells.

Figure 19 shows a comparison of in vivo toxicity tests of EGFR CAR-T cells with and without IFN. The results showed that the tumor size of EGFR-CAR-T cells was basically the same as that of the control group, and no inhibitory effect was observed. After the EGFR-CAR-IFN cells were reinfused, the phenomenon of tumor growth inhibition began to appear on the 7th day, with a tumor inhibition rate of 5.9%, reaching the highest level on the 10th day, which was 18.5%. By the 17th day, the tumor inhibition rate could still reach 12.4%, which was significantly better than the EGFR-CAR-T cell group.

DETAILED DESCRIPTION

The following detailed description shows the embodiments disclosed herein in detail. It should be understood that this specification is not intended to be limited to the specific embodiments disclosed herein, but may be changed. It will be understood by those skilled in the art that the contents disclosed in this specification may have a variety of changes or variations, all of which are covered within the disclosed scope and principles. Unless otherwise stated, each embodiment may be arbitrarily combined with any other embodiment.

Certain embodiments disclosed herein include numerical ranges, and certain aspects of the present invention may be described in a range manner. Unless otherwise stated, it should be understood that numerical ranges or the manner of range description are only for the purpose of brevity and convenience, and should not be considered as a strict limitation of the scope of the present invention. Therefore, the description using the range manner should be considered to specifically disclose all possible sub-ranges and all possible specific numerical points within the range, as these sub-ranges and numerical points have been clearly written in this article. For example, the description of the range from 1 to 6 should be considered to specifically disclose sub-ranges from 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and specific numerical points within these ranges, such as 1, 2, 3, 4, 5, 6. Regardless of the width of the numerical value, the above principles are equally applicable. When using a range description, the range includes the endpoints of the range.

In order to overcome the defects in the prior art, the present invention has conducted in-depth research and found that making CAR-T cells inducibly express type I interferon can effectively increase the anti-tumor activity of CAR-T cells and reduce their toxic side effects. On this basis, the present invention provides an immune response cell that expresses at least one receptor that can bind to an antigen (such as a tumor antigen or an antigen from a pathogen) and type I interferon, which is used to treat tumors, infectious diseases and other diseases, and has a significantly excellent ability to kill tumors or pathogens.

The terms "activation" and "activation" used herein are used interchangeably, and they and other grammatical forms thereof may refer to the process by which cells are transformed from a quiescent state to an active state. The process may include responses to phenotypic or genetic changes in antigen, migration and/or functional activity states. For example, the term "activation" may refer to the process by which T cells are gradually activated. For example, T cells may require at least two signals to be fully activated. The first signal may occur after the antigen-MHC complex engages the TCR, and the second signal may occur by the engagement of a co-stimulatory molecule (see the co-stimulatory molecules listed in Table 1). In vitro, anti-CD3 can simulate the first signal, and anti-CD28 can simulate the second signal. For example, engineered T cells can be activated by expressed CAR. T cell activation or T cell triggering used herein may refer to the state of T cells that have been sufficiently stimulated to induce detectable cell proliferation, cytokine production and/or detectable effector function.

As used herein, the term "co-stimulatory ligand" includes molecules on antigen presenting cells (e.g., aAPC, dendritic cells, B cells, etc.) that specifically bind to the same co-stimulatory molecule on a T cell, thereby providing a signal that mediates T cell responses together with the first signal provided by, for example, the binding of the TCR/CD3 complex to the MHC molecule loaded with the peptide, including but not limited to proliferation, activation, differentiation, etc. Co-stimulatory ligands may include but are not limited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecules (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Co-stimulatory ligands also specifically include antibodies that specifically bind to co-stimulatory molecules present on T cells, such as but not limited to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

The term "costimulatory molecule" as used herein refers to an identical binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as but not limited to proliferation. Costimulatory molecules include but are not limited to MHC class I molecules, BTLA and Toll ligand receptors.

As used herein, a "co-stimulatory signal" refers to a signal that, in combination with a first signal, such as TCR/CD3 binding, results in T cell proliferation and/or up-regulation or down-regulation of key molecules.

The term "antigen binding unit" as used herein refers to immunoglobulin molecules and immunologically active parts of immune molecules, i.e., molecules containing antigen binding sites that specifically bind to antigens ("immunoreactions") . The term "antigen binding unit" also includes immunoglobulin molecules of various species origin, including invertebrates and vertebrates. Structurally, the simplest naturally occurring antibodies (e.g., IgG) contain four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Immunoglobulins represent a large family of molecules including several types of molecules, such as IgD, IgG, IgA, IgM, and IgE. The term "immunoglobulin molecule" includes, for example, hybrid antibodies or altered antibodies and fragments thereof. It has been shown that the antigen binding function of an antibody can be performed by fragments of naturally occurring antibodies. These fragments are collectively referred to as "antigen binding units". The term "antigen binding unit" also includes any molecular structure containing polypeptide chains with a specific shape that matches and recognizes an epitope, wherein one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope.

An antigen binding unit "specifically binds" or is "immunoreactive" with an antigen if it binds the antigen with greater affinity or avidity than it binds to other reference antigens (including polypeptides or other substances).

As used herein, "antigen" refers to a substance that is recognized and specifically bound by an antigen binding unit. Antigens can include peptides, proteins, glycoproteins, polysaccharides and lipids, portions thereof and combinations thereof. Non-limiting exemplary antigens include tumor antigens or pathogen antigens. "Antigen" can also refer to a molecule that triggers an immune response. This immune response may involve antibody production or activation of specific immunologically-competent cells, or both. Those skilled in the art will appreciate that any macromolecule, including virtually all proteins or peptides, can serve as an antigen.

The term "immunoglobulin" or "Ig" as used herein may refer to a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as chimeric antigen receptors or antigen receptors. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE, of which IgG is the most common circulating antibody. It is the most effective immunoglobulin in agglutination, complement fixation, and other antibody reactions, and is important in defending against bacteria and viruses. For example, tumor cell antigens (or "tumor antigens") or pathogen antigens can be recognized by CAR.

As used herein, the term "autologous" and its grammatical alternatives may refer to being from the same source. For example, a sample (e.g., a cell) may be removed, processed, and administered to the same individual (e.g., a patient) at a later time. An autologous process is distinguished from an allogeneic process in which the donor and recipient are different individuals.

As used herein, "xenotransplantation" and its grammatical other forms may include any procedure in which cells, tissues or organs are transplanted, implanted or infused into a recipient in which the recipient and the donor are of different species. Transplantation of cells, organs and/or tissues described herein may be used for xenotransplantation into humans. Xenotransplantation includes, but is not limited to, vascularized xenografts, partially vascularized xenografts, non-vascularized xenografts, xenogeneic dressings, xenogeneic bandages, and xenogeneic structures, etc.

As used herein, "allogeneic transplantation" and its grammatical other forms (e.g., allogeneic transplantation) can include any procedure in which cells, tissues, or organs are transplanted, implanted, or infused into a recipient in which the recipient and donor are of the same species but different individuals. Transplantation of cells, organs, and/or tissues described herein can be used for allogeneic transplantation into the human body. Allogeneic transplantation includes, but is not limited to, vascularized allogeneic transplantation, partially vascularized allogeneic transplantation, non-vascularized allogeneic transplantation, allogeneic dressings, allogeneic bandages, and allogeneic structures.

As used herein, "autologous transplantation" and its grammatical other forms (e.g., autologous transplantation) can include any procedure in which cells, tissues, or organs are transplanted, implanted, or infused into a recipient in which the recipient and donor are the same individual. Transplantation of cells, organs, and/or tissues described herein can be used for autologous transplantation into the human body. Autologous transplantation includes, but is not limited to, vascularized autologous transplantation, partially vascularized autologous transplantation, non-vascularized autologous transplantation, autologous dressings, autologous bandages, and autologous structures.

The term "chimeric antigen receptor" or "CAR" as used herein refers to an engineered molecule that can be expressed by immune cells including but not limited to T cells. CAR is expressed in T cells and can redirect T cells to induce killing of target cells with specificity determined by artificial receptors. The extracellular binding domain of CAR can be derived from a mouse, humanized or fully human monoclonal antibody.

The term "epitope" and its grammatical alternatives used herein may refer to a portion of an antigen that can be recognized by an antibody, B cell, T cell, or engineered cell. For example, an epitope may be a tumor epitope or pathogen epitope recognized by a TCR. Multiple epitopes within an antigen may also be recognized. An epitope may also mutate.

The term "engineering" and its grammatical other forms used herein can refer to one or more changes in nucleic acids, such as nucleic acids in the genome of an organism. The term "engineering" can refer to changes, additions and/or deletions of genes. Engineered cells can also refer to cells with added, deleted and/or altered genes.

The term "cell" or "engineered cell" and other grammatical forms thereof used herein may refer to cells of human or non-human animal origin. Engineered cells may also refer to cells expressing CAR.

The term "transfection" as used herein refers to the introduction of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection and biolistics.

The term "stable transfection" or "stably transfected" refers to the introduction and integration of foreign nucleic acid, DNA or RNA, into the genome of the transfected cell. The term "stable transfectant" refers to a cell that has stably integrated foreign DNA into its genomic DNA.

As used herein, the terms "nucleic acid molecule encoding", "coding DNA sequence" and "coding DNA" refer to the sequence or order of deoxyribonucleotides along a deoxyribonucleic acid chain. The order of these deoxyribonucleotides determines the order of amino acids along a polypeptide (protein) chain. Thus, a nucleic acid sequence encodes an amino acid sequence.

The term "subject" as used herein refers to any animal, such as a mammal or marsupial. The subject of the present invention includes, but is not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and poultry of any kind.

The term "peripheral blood lymphocyte" (PBL) and its grammatical other forms used herein may refer to lymphocytes circulating in the blood (e.g., peripheral blood). Peripheral blood lymphocytes may refer to lymphocytes that are not restricted to an organ. Peripheral blood lymphocytes may include T cells, NK cells, B cells, or any combination thereof.

The term "immune response cell" or "immunoreactive cell" as used herein may refer to a cell that can initiate an immune response, including but not limited to T cells, B cells and NKT cells, their respective precursor cells and their progeny. Immune response cells may also refer to cells of lymphoid or myeloid lineages.

The term "T cell" and its grammatical other forms used herein can refer to T cells of any origin. For example, the T cell can be a primary T cell such as an autologous T cell, etc. The T cell can also be human or non-human.

The term "T cell activation" or "T cell triggering" used herein and its grammatical other forms may refer to the state of T cells that are fully stimulated to induce detectable cell proliferation, cytokine production and/or detectable effector function. In some embodiments, "complete T cell activation" may be similar to the cytotoxicity of triggering T cells. Various assays known in the art can be used to measure T cell activation. The assay may be an ELISA, ELISPOT, a flow cytometry assay (CD107) for measuring cytokine secretion, a flow cytometry assay for measuring intracellular cytokine expression, a flow cytometry assay for measuring proliferation, and a cytotoxicity assay (51Cr release assay) for determining target cell elimination. The assay is typically compared with a control (non-engineered cell) and an engineered cell (CAR T) to determine the relative activation of the engineered cell compared to the control. In addition, the assay may be compared with engineered cells incubated or contacted with target cells that do not express the target antigen. For example, the comparison may be a comparison of CD19-CART cells incubated with target cells that do not express CD19.

When used to refer to a nucleotide sequence, the term "sequence" as used herein and its grammatical other forms may include DNA or RNA, and may be single-stranded or double-stranded. Nucleic acid sequences may be mutated. Nucleic acid sequences may have any length, such as a length of 2 to 1,000,000 or more nucleotides (or any integer value therebetween or above) nucleic acids, such as a length of about 100 to about 10,000 nucleotides or about 200 to about 500 nucleotides.

As used herein, the term "effective amount" refers to an amount that provides a therapeutic or prophylactic benefit.

The term "expression vector" as used herein refers to a vector comprising a recombinant polynucleotide, which comprises an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be provided by a host cell or an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

The term "slow virus" as used herein refers to the genus of the Retroviridae family. Retroviruses are unique among retroviruses in being able to infect non-dividing cells; they can deliver large amounts of genetic information into the DNA of host cells, so they are one of the most effective methods for gene delivery vectors. HIV, SIV, and FIV are all examples of slow viruses. Vectors derived from slow viruses provide a means of achieving significant levels of gene transfer in vivo.

As used herein, the term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Typically, operably linked DNA sequences are contiguous and, if necessary, connect two protein coding regions in the same reading frame.

The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell or introduced synthetic machinery required to initiate specific transcription of a polynucleotide sequence.

The term "vector" as used herein is a composition comprising an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted as including non-plasmids and non-viral compounds that promote nucleic acid transfer into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

The term sequence "identity" as used herein is determined by comparing two sequences that are optimally matched over a comparison window (e.g., at least 20 positions) to determine the percentage of identity, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps), such as 20% or less gaps (e.g., 5 to 15%, or 10 to 12%) for the two sequences that are optimally matched compared to a reference sequence (which does not include additions or deletions). Percentages are usually calculated by determining the number of positions at which the same nucleic acid base or amino acid residue occurs in the two sequences to produce the number of correctly matched positions, dividing the number of correctly matched positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to produce the percentage of sequence identity.

The term "type I interferon" used herein includes IFNα, IFNβ, and IFN-ε, IFN-κ and IFN-ω, etc. All type I interferons bind to specific cell surface receptors (the so-called IFN-α/β receptors) composed of two chains, IFNAR1 and IFNAR2. In some embodiments, the term "type I interferon" used herein is IFNα or IFNβ. In some embodiments, the term "type I interferon" used herein is IFNβ. In some embodiments, the type I interferon used herein includes human, mouse, or synthetic type I interferon. In some embodiments, the term "interferon α" used herein can be a polypeptide having a sequence shown in NCBI aaa52724.1 or aaa52716.1 or aaa52725.1, or a polypeptide having at least 85% identity with these sequences. In some embodiments, the term "interferon beta" (INF-β) used herein may be a protein having at least 85% identity with NCBI aac41702.1 or np_002167.1 or aah96152.1p41273 or NP 001552, or a fragment thereof having tumor necrosis factor (TNF) ligand function.

In some embodiments, the element used to construct the antigen-binding receptor or the type I interferon can be naturally occurring, such as being isolated or purified from a mammal; or artificially prepared, such as being able to produce recombinant elements or type I interferon according to conventional genetic engineering recombination techniques. Preferably, the present invention can use recombinant elements or type I interferon.

Amino acid sequences formed by substitution, deletion or addition of one or more amino acid residues based on the above elements or type I interferon polypeptide sequences are also included in the present invention. Appropriate replacement of amino acids is a technique known in the art, which can be easily implemented and ensures that the biological activity of the resulting molecule is not changed. These techniques have made those skilled in the art realize that, in general, changing a single amino acid in a non-essential region of a polypeptide will not substantially change the biological activity.

The biologically active fragments of the polypeptides of each element or type I interferon can be applied to the present invention. Here, the meaning of the biologically active fragment is a polypeptide that, as a part of the full-length polypeptide, can still maintain all or part of the functions of the full-length polypeptide. Usually, the biologically active fragment maintains at least 50% of the activity of the full-length polypeptide. Under more preferred conditions, the active fragment can maintain 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the full-length polypeptide.

Based on the above elements or type I interferon polypeptide sequences, modified or improved polypeptides can also be applied to the present invention, for example, polypeptides modified or improved to improve their half-life, effectiveness, metabolism, and/or efficacy of the polypeptide can be used. In other words, any variation that does not affect the biological activity of the polypeptide can be used in the present invention.

The terms "disease", "condition", "disorder", etc. as used herein refer to any change or disorder that damages or interferes with the normal function of cells, tissues or organs. For example, the "disease" includes, but is not limited to, tumors, pathogen infection, autoimmune diseases, T cell dysfunction diseases, or immune tolerance defects (such as transplant rejection).

The term "tumor" as used herein refers to a disease characterized by pathological proliferation of cells or tissues, and their subsequent migration or invasion of other tissues or organs. Tumor growth is usually uncontrolled and progressive, and does not induce or inhibit normal cell proliferation. Tumors can affect a variety of cells, tissues or organs, including but not limited to those selected from the bladder, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph node, nervous tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testicles, thymus, thyroid, trachea, urethra, ureter, urethra, uterus, vaginal organs, or tissues or corresponding cells. Tumors include cancers such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells). The tumor described in the present invention may include, but is not limited to, leukemia (such as acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute granulocytic leukemia, acute promyelocytic leukemia, acute granulocytic-monocytic leukemia, acute monocytic leukemia, acute leukemia, chronic leukemia, chronic granulocytic leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphoma (Hodgkin's disease, non-Hodgkin's disease), primary macroglobulinemia, heavy chain disease, solid tumors such as sarcoma and cancer (such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endotheliosarcoma, lymphangiosarcoma, angiosarcoma, lymphangioendotheliosarcoma, synovial vioma, mesothelioma, Ewing's Tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, carcinoma, bronchial carcinoma, medullary carcinoma, renal cell carcinoma, liver cancer, Nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, retinoblastoma), esophageal cancer, gallbladder cancer, kidney cancer, multiple myeloma. Preferably, the "tumor" includes but is not limited to: pancreatic cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, kidney cancer, leukemia, multiple myeloma, ovarian cancer, cervical cancer and glioma.

The types of tumor antigens mentioned in the present invention may also be tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are unique to tumor cells and do not occur on other cells in the body. TAA-related antigens are not unique to tumor cells, but are expressed on normal cells under conditions where an immune tolerance state to the antigen cannot be induced. The expression of antigens on tumors can occur under conditions that enable the immune system to respond to the antigens. TAAs may be antigens expressed on normal cells during fetal development when the immune system is immature and unable to respond, or they may be antigens that are usually present at very low levels on normal cells but expressed at higher levels on tumor cells.

Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens such as MART-1/MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2 and tumor-specific multicentric antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens caused by chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens, such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7, etc. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

In some embodiments, the “tumor antigen” includes but is not limited to: prostate specific membrane antigen (PSMA), carcinoembryonic antigen (CEA), IL13Ralpha, HER-2, CD19, NY-ESO-1, HIV-1Gag, Lewis Y, MART-1, gp100, tyrosinase, WT-I, hTERT, mesothelin, EGFR, EGFRvIII, phosphatidylinositol proteoglycan 3, EphA2, HER3, EpCAM, MUC1, MUC16, CLDN18.2, folate receptor, CLDN6, CD30, CD138, ASGPR1, CDH16, GD2, 5T4, 8H9, αvβ6 integrin, B cell maturation antigen (BCMA), B7-H3, B7-H6, CAIX, CA9, CD20, CD22, kappa light chain (kappa light chain), CD33, CD38, CD44, CD44v6, CD44v7/8, CD70, CD123, CD171, CSPG4, EGP2, EGP40, ERBB3, ERBB4, ErbB3/4, FAP, FAR, FBP, embryonic AchR, GD2, GD3, HLA-AI MAGE A1, MAGE3, HLA-A2, IL11Ra, KDR, Lambda, MCSP, NCAM, NKG2D ligand, PRAME, PSCA, PSC1, ROR1, Sp17, SURVIVIN, TAG72, TEM1, TEM8, VEGRR2, HMW-MAA, VEGF receptor, and/or fibronectin, tenascin, or oncofetal variants of tumor necrosis areas.

The term "pathogen" as used herein refers to protozoa that can cause disease, including viruses, bacteria, fungi or parasites. The "viral antigen" refers to a polypeptide expressed by a virus that can induce an immune response.

Typical viruses include, but are not limited to, Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also known as HDTV-III, LAVE or HTLV-III/LAV, or HIV-III; and other strains, such as HIV-LP); Picornaviruses (e.g., poliovirus, hepatitis A virus; human enteroviruses, coxsackieviruses, rhinoviruses, echoviruses); Caliciviruses (e.g., strains causing gastroenteritis); Togaviruses (e.g., equine encephalitis virus, rubella virus); Flaviviridae (e.g., dengue virus, Japanese encephalitis virus, yellow fever virus); Coronaviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviruses (e.g., influenza virus); Subviridae ( (e.g., Hantavirus, some viruses, sandfly, and Nairovirus); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviruses, and rotaviruses); Binaviridae; Hepatoviruses (hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papillomaviruses, polyomaviruses); Adenoviridae (most adenoviruses); Herpesviruses (herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpes simplex virus); Poxviruses (smallpox virus, vaccinia virus, poxviruses); Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., hepatitis B virus with a defective satellite TE), non-A, non-B hepatitis agents, (Class 1 = internal transmission; Class 232 = parenteral transmission (i.e., hepatitis C); Norwalk and related viruses, and Astroviruses).

Typical bacteria include, but are not limited to, Pasteurella, Staphylococcus aureus, Streptococcus, Escherichia coli, Salmonella and Pseudomonas aeruginosa. Specific examples of infectious bacteria include, but are not limited to, Helicobacter pylori, spirochetes, Legionella pneumophila, Mycobacterium SPS (such as Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium endo, M. Kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B ... Fecal Streptococcus, Bovine Streptococcus, Streptococcus (anaerobic SPS), Streptococcus pneumoniae, Campylobacter, Enterococcus, Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium, Erysipelothrix, Clostridium, Clostridium perfringens, Clostridium tetani, Enterobacter cloacae, Klebsiella pneumoniae, Pasteurella multocida, Bacteroides, Fusobacterium nucleatum, Moniliform chain, Treponema pallidum, Treponema yawsii, Leptospira, Rickettsia, Actinomyces israelii.

In some embodiments, the immune response cells of the present invention are capable of recognizing and binding to parasite antigens. The parasites include internal parasites and external parasites. The internal parasites include protozoa, helminths, roundworms, and flukes. In some embodiments, the parasite antigen is, for example, an antigen from a species of the following families: Entamoeba histolytica; Babesia B.divergens, B.bigemina, B.equi, B.microfti, B.duncani; Balantidium coli; Blastocystis spp.; Trypanosoma cruzi; Cryptosporidium spp.; Cyclosporacayetanensis; Dientamoeba fragilis; Giardia lamblia; Balamuthia mandrillaris; Acanthamoeba spp.; Isospora belli; Leishmania spp.; Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale curtisi, Plasmodium ovale wallikeri, Plasmodium malariae, Plasmodium knowlesi; Naegleria fowleri; Rhinosporidiumseeberi; Sarcocystis bovihominis,Sarcocystis suihominis;Trypanosoma brucei;Toxoplasma gondii;Trichomonas vaginalis;Taenia saginata;Bertiella mucronata,Bertiella studeri;Taenia solium;Diphyllobothrium latum;Echinococcusgranulosus,Echinococcus multilocularis,E.vogeli,E.oligarthrus;Hymenolepisnana ymenolepis diminuta;Spirometra erinaceieuropaei;Cestoda,Taeniamulticeps;Clonorchis sinensis;Clonorchis viverrini;Dicrocoelium dendriticum;Fasciola hepatica,Fasciola gigantica;Fasciolopsis buski;Gnathostomaspinigerum,Gnathostoma hispidum;Metagonimusmus yokogawai; Metorchis conjunctus; Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis; Paragonimus westermani; Paragonimus africanus; Paragonimus caliensis; Paragonimus kellicotti; osoma haematobium; Schistosoma japonicum; Schistosoma mekongi; Echinostoma echinatum; Trichobilharzia regenti, Schistosomatidae; Ancylostoma duodenale, Necatoramericanus; Angiostrongylus costaricensis; Anisakis; Ascaris sp.Ascarislumbricoides; Baylisascaris procyonis; Brugia malayi, Brugia timori; Dioctophymerenale; Dracunculus medinensis; Enterobius vermicularis, Enterobius gregorii; Halicephalobus gingivalis; Loa loa filaria; Mansonella streptocerca; Onchocercavolvulus; Strongyloides stercoralis; Thelazia californiensis, Thelazia callipaeda; Tox ocara canis,Toxocara cati;Trichinella spiralis,Trichinellabritovi,Trichinella nelsoni,Trichinella nativa;Trichuris trichiura,Trichurisvulpis;Wuchereria bancrofti;Archiacanthocephala,Moniliformis moniliformis;Linguatula Serrata; Oestroidea; Calliphoridae, Sarcophagidae; Cochliomyiahominivorax; Tunga penetrans; Cimex lectularius; Dermatobia hominis; Pediculushumanus; Pediculus humanus corporis; Pthirus pubis; Demodex folliculorum/brevis/canis; Sarcoptes scabiei; Trombiculidae; Pulex irritans; odidae and Argasidae.

The term "autoimmune disease" as used herein is defined as a condition caused by an autoimmune response. Autoimmune diseases are the result of inappropriate and excessive reactions to autoantigens. Examples of autoimmune diseases include, but are not limited to, appendicitis, alopecia, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (type I), malnutrition epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, etc.

"Tolerance" or "immune tolerance" is the failure of the immune system to produce a defensive immune response to a specific antigen. Tolerance can be natural or autologous, in which the body does not attack its own proteins and antigens, or can be induced by the manipulation of the immune system. Central tolerance occurs during lymphocyte development and works in the thymus and bone marrow. During this process, T lymphocytes and B lymphocytes that recognize self-antigens are deleted before they develop into fully immunocompetent cells. This process is most active during fetal development, but continues throughout life with the generation of immature lymphocytes. Peripheral T cell tolerance refers to the functional anergy to self-antigens present in peripheral tissues and occurs after T and B cells mature and enter the periphery. These processes include the suppression of autoreactive cells by "regulatory" T cells, and the generation of low reactivity (anergy) in lymphocytes that encounter antigens in the absence of co-stimulatory signals that accompany inflammation. "Acquired" or "induced tolerance" refers to the adaptation of the immune system to external antigens, characterized by the specific non-reactivity of lymphoid tissues to a given antigen, which may otherwise induce cell-mediated or humoral immunity. In adults, tolerance can be induced clinically by repeated administration of very large doses of antigen or small doses below the threshold required to stimulate an immune response (e.g., by intravenous or sublingual administration of soluble antigens). Antigens that induce immune tolerance are called tolerogens. Immunosuppression is also beneficial for inducing tolerance. The destruction of self-tolerance can lead to autoimmunity.

Immune recognition of non-self antigens usually complicates the transplantation and transplantation of foreign tissues from organisms of the same species (allografts), leading to transplant rejection. Lymphocytes, particularly T lymphocytes, play a key role in allograft rejection, transplant failure, and GVHD. There are usually two situations in which allografts can be accepted. One is when cells or tissues are transplanted to immune-exempt sites (such as in the eyes or testicles) that are separated from the immune surveillance system, or with strong molecular signals to prevent dangerous inflammation (such as in the brain). The second situation is when a state of tolerance has been induced, whether previously exposed to donor antigens in a manner that causes immune tolerance rather than sensitization of the recipient, or after chronic rejection. Successful allogeneic transplantation requires a certain degree of immune tolerance to alloantigens. The realization of immune tolerance can prevent host-versus-graft reactions that lead to transplant rejection and failure, and prevent graft-versus-host reactions (GVHD).

The term "enhancing immune response cell function" used herein includes, for example, enhancing T cell function. Taking T cells as an example, enhancing T cell function includes inducing, causing or stimulating T cells to have continuous or enhanced biological functions, or renewing or reactivating exhausted (exhausted) or inactive T cells. Examples of enhancing T cell function include: relative to the level before intervention, CD8+T cells secrete increased interferon, increased proliferation, and increased antigen reactivity (e.g., virus or pathogen clearance). In one embodiment, the enhancement level is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. The way to measure this enhancement is known to those of ordinary skill in the art.

The term "T cell dysfunctional disease" as used herein includes disorders or conditions of T cells characterized by reduced responsiveness to antigen stimulation. In some embodiments, the T cell dysfunctional disorder is a disorder specifically associated with inappropriately increased signaling through PD-1. In some embodiments, the T cell dysfunctional disease is a disease in which T cells are anergic or have a reduced ability to secrete cytokines, proliferate, or perform cytolytic activity. Examples of T cell dysfunctional diseases characterized by T cell dysfunction include unabsorbed acute infections, chronic infections, and tumor immunity.

As used herein, the term "exogenous" refers to a nucleic acid molecule or polypeptide that is not endogenously expressed in a cell, or that is expressed at a level insufficient to achieve the function that it would have if overexpressed. Thus, "exogenous" includes recombinant nucleic acid molecules or polypeptides expressed in a cell, such as exogenous, heterologous, and overexpressed nucleic acid molecules and polypeptides.

As used herein, the term "receptor" refers to a polypeptide, or portion thereof, that selectively binds one or more ligands on a cell membrane.

In some embodiments, an antigen-binding receptor of the invention specifically binds to an antigen capable of binding thereto.

In some embodiments, the antigen-binding receptor of the present invention is a chimeric antigen receptor. The term "chimeric antigen receptor (CAR)" used herein refers to a tumor antigen binding domain fused to an intracellular signal transduction domain that can activate T cells. Commonly, the extracellular binding domain of CAR is derived from a mouse or humanized or human monoclonal antibody.

Chimeric antigen receptors generally include an extracellular antigen binding region or antigen binding unit. In some embodiments, the extracellular antigen binding region may be fully human. In other cases, the extracellular antigen binding region may be humanized. In other cases, the extracellular antigen binding region may be mouse-derived, or the chimera in the extracellular antigen binding region may be composed of amino acid sequences from at least two different animals. In some embodiments, the extracellular antigen binding region may be non-human.

A variety of antigen binding regions can be designed. 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. In some embodiments, the extracellular antigen binding region may comprise scFv, Fab, or natural ligands, and any derivatives thereof. The extracellular antigen binding region may refer to a molecule other than a complete antibody, which may comprise a portion of a complete antibody and may bind to an antigen to which the complete antibody binds. 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); and multispecific antibodies formed by antibody fragments.

The extracellular antigen binding region, such as scFv, Fab or natural ligand, can be a part of the CAR that determines the antigen specificity. The extracellular antigen binding region can bind to any complementary target. The extracellular antigen binding region can be derived from an antibody of a known variable region sequence. The extracellular antigen binding region can be obtained from an antibody sequence obtained from an available mouse hybridoma. Alternatively, the extracellular antigen binding region can be obtained from the full exonucleation sequencing of tumor cells or primary cells such as tumor infiltrating lymphocytes (TIL).

In some embodiments, the binding specificity of the extracellular antigen binding region can be determined by a complementary determining region or CDR, such as a light chain CDR or a heavy chain CDR. In many cases, the binding specificity can be determined by a light chain CDR and a heavy chain CDR. Compared with other reference antigens, a given combination of a heavy chain CDR and a light chain CDR can provide a given binding pocket, which can give an antigen (eg, GPC3) greater affinity and/or specificity. For example, a CDR specific for glypican-3 can be expressed in the extracellular binding region of CAR, so that a CAR targeting GPC3 can target immune response cells to tumor cells expressing GPC3.

In certain aspects of any of the embodiments disclosed herein, an extracellular antigen binding region, such as scFv, may include a light chain CDR specific for an antigen. A light chain CDR may be a complementary determining region of an antigen binding unit such as a scFv light chain of a CAR. A light chain CDR may include a continuous sequence of amino acid residues, or two or more continuous sequences of amino acid residues separated by a non-complementary determining region (e.g., a framework region). In some embodiments, a light chain CDR may include two or more light chain CDRs, which may be referred to as light chain CDR-1, CDR-2, etc. In some embodiments, a light chain CDR may include three light chain CDRs, which may be referred to as light chain CDR-1, light chain CDR-2, and light chain CDR-3, respectively. In some examples, a group of CDRs present on a common light chain may be collectively referred to as light chain CDRs.

In certain aspects of any embodiment disclosed herein, the extracellular antigen binding region, such as scFv, may include a heavy chain CDR specific for an antigen. The heavy chain CDR may be a heavy chain complementary determining region of an antigen binding unit such as scFv. The heavy chain CDR may include a continuous sequence of amino acid residues, or a continuous sequence of two or more amino acid residues separated by a non-complementary determining region (e.g., a framework region). In some embodiments, the heavy chain CDR may include two or more heavy chain CDRs, which may be referred to as heavy chain CDR-1, CDR-2, etc. In some embodiments, the heavy chain CDR may include three heavy chain CDRs, which may be referred to as heavy chain CDR-1, heavy chain CDR-2, and heavy chain CDR-3, respectively. In some embodiments, a group of CDRs present on a common heavy chain may be collectively referred to as heavy chain CDRs.

By using genetic engineering, the extracellular antigen binding region can be modified in various ways. In some embodiments, the extracellular antigen binding region can be mutated so that the extracellular antigen binding region can be selected to have a higher affinity for its target. In some embodiments, the affinity of the extracellular antigen binding region to its target can be optimized for targets that can be expressed at low levels on normal tissues. This optimization can be performed to minimize potential toxicity. In other cases, the clone of the extracellular antigen binding region with a higher affinity for the membrane-bound form of the target can be superior to its soluble counterpart. This modification can be performed because different levels of soluble forms of the target can also be detected, and their targeting can cause undesirable toxicity.

In some embodiments, the extracellular antigen binding region includes a hinge or a spacer. The terms hinge and spacer can be used interchangeably. The hinge can be considered as a part of a CAR for providing flexibility to the extracellular antigen binding region. In some embodiments, the hinge can be used to detect the CAR on the cell surface of the cell, particularly when the antibody detecting the extracellular antigen binding region does not work or is available. For example, the length of the hinge derived from an immunoglobulin may need to be optimized, depending on the position of the epitope on the extracellular antigen binding region targeting target.

In some embodiments, the hinge may not belong to an immunoglobulin, but to another molecule, such as the natural hinge of a CD8α molecule. The CD8α hinge may contain cysteine and proline residues known to play a role in the interaction of CD8 co-receptors and MHC molecules. The cysteine and proline residues may affect the performance of the CAR.

The CAR hinge can be size-adjustable. This morphology of the immune synapse between the immune response cell and the target cell also defines the distance that cannot be functionally bridged by the CAR due to the membrane distal epitope on the cell surface target molecule, even with a short hinge CAR, the synaptic distance cannot reach the approximate value at which the signal can be conducted. Similarly, the membrane proximal CAR target antigen epitope is only observed to output signals in the context of a long hinge CAR. The hinge can be adjusted according to the extracellular antigen binding region used. The hinge can be of any length.

The transmembrane domain can anchor the CAR to the plasma membrane of the cell. The natural transmembrane portion of CD28 can be used for CAR. In other cases, the natural transmembrane portion of CD8α can also be used in CAR. "CD8" can be a protein with at least 85, 90, 95, 96, 97, 98, 99 or 100% identity to NCBI reference number: NP_001759 or a fragment thereof having stimulatory activity. "CD8 nucleic acid molecule" can be a polynucleotide encoding a CD8 polypeptide, in some cases, the transmembrane region can be a natural transmembrane portion of CD28, and "CD28" can refer to a protein with at least 85, 90, 95, 96, 97, 98, 99 or 100% identity to NCBI reference number: NP_006130 or a fragment thereof having stimulatory activity. "CD28 nucleic acid molecule" can be a polynucleotide encoding a CD28 polypeptide. In some embodiments, the transmembrane portion can include a CD8α region.

The (thin) intracellular signaling region of CAR can be responsible for activating at least one of the effector functions of the immune response cells in which CAR has been placed. CAR can induce the effector function of T cells, for example, the effector function is cytolytic activity or auxiliary activity, including the secretion of cytokines. Therefore, the term intracellular signaling region refers to the protein portion that transduces the effector function signal and guides the cell to perform a specific function. Although the entire intracellular signaling region can usually be used, in many cases, it is not necessary to use the entire chain of the signaling domain. In some embodiments, a truncated portion of the intracellular signaling region is used. In some embodiments, the term intracellular signaling region is therefore intended to include any truncated portion of the intracellular signaling region sufficient to transduce the effector function signal.

Preferred examples of signaling domains used in CARs may include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act synergistically to initiate signal transduction following target-receptor binding, as well as any derivative or variant sequences thereof and any synthetic sequences of these sequences having the same functionality.

In some embodiments, the intracellular signaling region may contain a signal motif of a known immunoreceptor tyrosine-based activation motif (ITAM). Examples of ITAMs containing cytoplasmic signaling sequences include those derived from TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, and CD66d. However, in a preferred embodiment, the intracellular signaling domain is derived from the CD3 ζ chain.

An example of a T cell signaling domain containing one or more ITAM motifs is the CD3ζ domain, also known as the T cell receptor T3ζ chain or CD247. This domain is part of the T cell receptor-CD3 complex and plays an important role in combining antigen recognition of several intracellular signal transduction pathways with the primary effector activation of T cells. As used herein, CD3ζ refers primarily to human CD3ζ and its isoforms, as known from Swissprot entry P20963, including proteins with substantially the same sequence. As part of a chimeric antigen receptor, it is reiterated that the full T cell receptor T3ζ chain is not required, and any derivative thereof comprising the signaling domain of the T cell receptor T3ζ chain is suitable, including any functional equivalent thereof.

The intracellular signaling domain can be selected from any one of the domains in Table 1. In some embodiments, the domain can be modified so that the identity to the reference domain can be from about 50% to about 100%. Any one of the domains in Table 1 can be modified so that the modified form can contain about 50, 60, 70, 80, 90, 95, 96, 97, 98, 99 or up to about 100% identity.

The intracellular signaling region of CAR may further include one or more costimulatory domains. The intracellular signaling region may include a single costimulatory domain, such as a ζ chain (first generation CAR) or it and CD28 or 4-1BB (second generation CAR). In other examples, the intracellular signaling region may include two costimulatory domains, such as CD28/OX40 or CD28/4-1BB (third generation).

Together with intracellular signaling domains such as CD8, these co-stimulatory domains can produce downstream activation of kinase pathways, thereby supporting gene transcription and functional cellular responses. The co-stimulatory domains of CARs can activate proximal signaling proteins associated with the CD28 (phosphatidylinositol-4,5-bisphosphate 3-kinase) or 4-1BB/OX40 (TNF-receptor-associated factor adaptor protein) pathways as well as MAPK and Akt activation.

In some cases, the signal generated by CAR may be combined with auxiliary or costimulatory signals.For costimulatory signal domains, chimeric antigen receptor-like complexes can be designed to include several possible costimulatory signal domains.As well known in the art, in naive T cells, the single engagement of T cell receptors is not enough to induce the complete activation of T cells as cytotoxic T cells.Complete productive T cell activation requires a second costimulatory signal.It has been reported that several receptors for providing costimulation for T cell activation, including but not limited to CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a/CD18), 4-1BBL, MyD88 and 4-1BB.The signal transduction pathways used by these costimulatory molecules can all synergize with the main T cell receptor activation signal.The signals provided by these costimulatory signal transduction regions can synergize with the main effector activation signals derived from one or more ITAM motifs (such as CD3zeta signal transduction domains), and the requirements for T cell activation can be completed.

In some embodiments, the addition of a co-stimulatory domain to the chimeric antigen receptor-like complex can enhance the efficacy and durability of the engineered cells. In another embodiment, the T cell signaling domain and the co-stimulatory domain are fused to each other to form a signaling region.

Table 1. Co-stimulatory domains

Chimeric antigen receptors bind to target antigens. When T cell activation is measured in vitro or in vitro, target antigens can be obtained or isolated from various sources. Target antigens used herein are antigens or immune epitopes on antigens that are essential for immune recognition and ultimately eliminating or controlling pathogenic factors or disease states in mammals. Immune recognition can be cells and/or body fluids. In the case of intracellular pathogens and cancers, immune recognition can be, for example, T lymphocyte responses.

The target antigen can be derived or isolated from, for example, a viral microorganism such as an antigen of a virus described hereinabove. In some embodiments, the chimeric antigen receptor of the present invention binds to, for example, HIV (Korber et al., eds HIV Molecular Immunology Database, Los Alamos National Laboratory, Los Alamos, N.Mex. 1977), influenza, herpes, herpes simplex human papillomavirus (U.S. Pat. No. 5,719,054), hepatitis B (U.S. Pat. No. 5,780,036), hepatitis C (U.S. Pat. No. 5,709,995), EBV, cytomegalovirus (CMV) and the like.

The target antigen can also be derived from or isolated from the pathogenic bacteria described herein above. In some embodiments, the chimeric antigen receptor of the present invention binds antigens such as those from Chlamydia (U.S. Pat. No. 5,869,608), Mycobacterium, Legionella, Meningitis, Group A Streptococcus, Salmonella, Listeria, Haemophilus influenzae (U.S. Pat. No. 5,955,596), etc.

In some embodiments, the target antigen can be derived or isolated from pathogenic yeasts, such as Aspergillus, Candida invasives (U.S. Pat. No. 5,645,992), Nocardia, histoplasmosis, cryptosporidiosis, and the like.

In some embodiments, the target antigen can be derived or isolated from, for example, pathogenic protozoa and pathogenic parasites, including but not limited to Pneumocystis carinii, Trypanosomiasis, Leishmania (U.S. Pat. No. 5,965,242), Plasmodium (U.S. Pat. No. 5,589,343), and Asxoplasma gondii, among others.

In some embodiments, the target antigen includes an antigen associated with a precancerous or proliferative state. The target antigen may also be associated with or caused by cancer. For example, in some embodiments, the chimeric antigen receptor of the present invention recognizes and binds to tumor antigens including TSAs and TAAs described herein above.

As used herein, the term "modulate" refers to a positive or negative change. Examples of modulation include 1%, 2%, 10%, 25%, 50%, 75%, or 100% change.

The term "treatment" as used herein refers to clinical intervention in an attempt to change the disease process caused by an individual or a cell, which can be either preventive or intervention in the clinical pathological process. The therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the progression of the disease, improving or alleviating the condition, alleviating or improving the prognosis, etc.

As used herein, the term "immunocompromised" refers to a subject having an immune deficiency that makes them susceptible to infection. Organisms that cause opportunistic infections do not normally cause illness in people with healthy immune systems, but can infect people with a weakened or suppressed immune system.

As used herein, the term "constitutive expression" refers to expression under all physiological conditions.

The term "induced expression" as used herein refers to expression under certain conditions, such as when T cells bind to antigens. Those skilled in the art know how to perform conventional "induced expression".

In some embodiments, the invention provides an immune response cell that expresses an antigen-binding receptor and exogenous type I interferon.

In some embodiments, the immune response cells of the present invention can target antigens expressed on cancer. Its antigens or epitopes may be expressed on cancer or cancer-related tissues. In some cases, the target antigen may be overexpressed on cancer and have reduced or no expression on normal tissues. In some cases, cancer-specific antigens and their epitopes can be targeted with the immune response cells of the present invention. Antigens can be derived from a variety of tumor antigens, such as tumor antigens produced by mutations, shared tumor-specific antigens, differentiation antigens, and antigens overexpressed in tumors. Just to name a few examples, antigens that can be targeted or bound by the immune response cells of the present invention may be or are derived from, including but not limited to: folate receptor α, 707-AP, adipophilin, AFP, AIM-2, ALDH1A1, Annexin II, ART-4, ARTC1, BAGE, BAGE-1, BCLX (L), BCMA, BING-4, BRACHYURY (IVS7 T/C polymorphism), BRACHYURY (TIVS7-2, polymorphism), BRACHYURY, B-RAF, CAMEL, CAR-ABL fusion protein (b3a2), CASP-5, CASP-8, Cdc27, CDC27/m, CDK4, CDK-4/m, CDKN2A, CEA, COA-1, CPSF, Cyp-B, DAM-6, -10, dek-can fusion protein, DKK1, EFTUD2, EGFR, ELF2M, ENAH (hMena), EP-CAM, EphA3, ESO-1/LAGE-2, ETV6-AML1 fusion protein, ETV6/AML, EZH2, FGF5, FLT3-ITD, FN1, G250, G250/MN/CAIX, Gage 3,4,5,6,7,GAGE-1,2,8,GAGE-3,-4,-5,-6,-7B,GnT-V,GnTVf,Gp100,gp100/Pmel17,GPC3,GPNMB,Her2/neu,Her3,HERV-K-MEL,HLA-A1ld,HLA-A2d,HPV E6,HPV E7,hsp70-2,HSP70-2M,HST-2,hTERT,hTRT,iCE,IL13Rα2,KIAA0205,KK-LC-1,KM-HN-1,K-ras,LDLR/FUT,LDLR-fucosyltransferase fusion protein,MAGE-A1,MAGE-A10,MAGE-A12,MAGE-A2,MAGE-A3,MAGE-A4,MAGE-A6,MAGE-A9,MAGE-C2,MART-1,MART2,MC1R,M-CSFT,MCSP,mdm-2,ME1,Melan-A/MART-1,MMP-2,MUC1(VNTR polymorphism)-c,MUC1, MUC1-n, MUC2, MUM-1, MUM-1f, MUM-2, MUM-3, NA-88, NA88-A, NFYC, N-ras, NY-BR-1, NY-ESO-1, OA1, OGT, OS-9, P15, p53, PAP, PBF, Pml/RARα and TEL/AML1pml-RARα fusion protein, PRAME, PRDX5, PSA, PSCA, PS MA,PTPRK,RAB38/NY-MEL-1,RAGE,RAGE-1,RBAF600,RGS5,RNF43,RU1,RU2,RU2AS,SAGE,SART-1,SART-2,SART-3,secernin 1, SIRT2, SNRPD1, SOX10, Sp17, SSX-2, SSX-4, STEAP, STEAP1, SYT-SSX1- or -SSX2 fusion protein, TBRACHYURY, T, TAG-1, TAG-2, TGF-βRII, TPI/mbcr-abl, TRAG-3, TRP-2, TRP-1, TRP-1/gp75, TRP-2, TRP-2/INT2, TRP2-INT2g, VEGF and/or WT1, XA GE-1b, α-actinin-4, β-catenin, β-catenin/m, triphosphate isomerase, mammaglobulin-A, human papillomavirus (HPV), human epidermal growth factor receptor 2 (HER2/neu), human epidermal growth factor receptor 3 (HER3), elongation factor 2, prostate-specific antigen (PSA), survivin, neo-PAP, kallikrein 4, alpha-fetoprotein, telomerase, mucin, cyclin D1, myosin/m, myosin I, intestinal carboxylesterase, caspase-8/m, tyrosinase.

In addition, the immune response cells of the present invention can target tumor-associated antigens. Tumor-associated antigens can be antigens that are not normally expressed by the host, which may be mutated, truncated, misfolded or otherwise abnormally expressed by the molecules expressed by the host; they can be the same as the molecules that are normally expressed but expressed at abnormally high levels; or they can be expressed in abnormal environments. Tumor-associated antigens can be, for example, proteins or protein fragments, complex carbohydrates, gangliosides, haptens, nucleic acids, other biological molecules or any combination thereof. In some cases, the antigen can be a new antigen (neo-antigen). The new antigen can be derived from somatic mutations of cancer cells. For example, the new antigen can be a mutant form of triphosphate isomerase (TPI). Mutated fibronectin (FN) is another example of a new antigen that can be targeted with the immune response cells of the present invention. The new antigen can be identified by a screening platform, such as biochemistry, full external cleavage sequencing, genetically targeted expression (GTE) or a combination thereof.

In some cases, the target that can be bound by the immune response cells of the present invention may be associated with the cancer stroma. The cancer stroma may be associated with the tumor microenvironment. The antigen may be a matrix antigen. For example, matrix antigens and epitopes may be present on, including but not limited to, tumor endothelial cells, tumor vasculature, tumor fibroblasts, tumor pericytes, tumor stroma and/or tumor mesenchymal cells. Those antigens may be selected, for example, from CD34, MCSP, FAP, CD31, PCNA, CD117, CD40, MMP4 and/or tenascin.

The tissue expression of the antigen can be measured by immunohistochemistry (IHC) analysis and/or flow cytometry. Tissue expression can also be measured by the copy number obtained by quantitative PCT (qPCR). In some cases, the target antigen can be expressed on the surface of the cancer cell. In some cases, the antigen that can be targeted may not be expressed in the environment of MHC or HLA. The immune response cells of the present invention can target cell surface antigens in a non-MHC restricted manner. In some cases, compared with its expression on normal tissues, the antigen that can be targeted by CAR-T may be overexpressed. Measured by IHC, qPCR or flow cytometry, overexpression can be about 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or up to 100 times of the expression on normal tissues.

In some embodiments, the antigen-binding receptor comprises an antigen-binding domain (extracellular binding region) and an intracellular signaling domain that can activate immune response cells. Preferably, a transmembrane region is also included between the antigen-binding domain and the intracellular signaling domain (intracellular signaling region). As an embodiment, the extracellular binding region comprises an antibody against an antigen, and the antigen is a tumor antigen or a pathogen antigen. The antigen-binding receptor is expressed on the surface of immune response cells, so that the immune response cells have a highly specific cytotoxic effect on tumor cells or pathogens expressing the antigen.

In some embodiments, the antigen-binding receptor of the present invention comprises a single-chain antibody connected to a transmembrane region, and the transmembrane region is followed by an intracellular signaling region.

In some embodiments, the antigen binding domain of the present invention is a binding domain that binds to a tumor antigen. In some embodiments, the tumor antigen is a differentiation antigen selected from MART-1/MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, and tumor-specific multicentric antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens caused by chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens such as Epstein Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7, etc. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and one or more of TPS. In some embodiments, the tumor antigen is selected from prostate specific membrane antigen (PSMA), carcinoembryonic antigen (CEA), IL13Ralpha, HER-2, CD19, NY-ESO-1, HIV-1Gag, Lewis Y, MART-1, gp100, tyrosinase, WT-I, hTERT, mesothelin, EGFR, EGFRvIII, phosphatidylinositol proteoglycan 3, EphA2, HER3, EpCAM, MUC1, MUC16, CLDN18.2, folate receptor, CLDN6, CD30, CD138, ASGPR1, CDH16, GD2, 5T4, 8H9, αvβ6 integrin, B cell maturation antigen (BCMA), B7-H3, B7-H6, CAIX, CA9, CD20, CD22, kappa light chain (kappa lightchain), CD33, CD38, CD44, CD44v6, CD44v7/8, CD70, CD123, CD171, CSPG4, EGP2, EGP40, ERBB3, ERBB4, ErbB3/4, FAP, FAR, FBP, embryonic AchR, GD2, GD3, HLA-AI MAGE A1, MAGE3, HLA-A2, IL11Ra, KDR, Lambda, MCSP, NCAM, NKG2D ligand, PRAME, PSCA, PSC1, ROR1, Sp17, SURVIVIN, TAG72, TEM1, TEM8, VEGRR2, HMW-MAA, VEGF receptor, and/or one or more of fibronectin, tenascin, or oncofetal variants of tumor necrosis. In some embodiments, the tumor antigen is selected from one or more of prostate-specific membrane antigen, carcinoembryonic antigen, IL13Ralpha, HER-2, CD19, NY-ESO-1, HIV-1Gag, Lewis Y, MART-1, gp100, tyrosinase, WT-I, hTERT, mesothelin, EGFR, EGFRvIII, phosphatidylinositol proteoglycan 3, EphA2, HER3, EpCAM, MUC1, MUC16, claudin 18.2, folate receptor, claudin 6, CD30, CD138, MAGE3, ASGPR1 and CDH16.

In some embodiments, the transmembrane region of the antigen-binding receptor can be selected from the transmembrane region of proteins such as CD8 or CD28. The human CD8 protein is a heterodimer composed of two chains of αβ or γδ. In some embodiments, the transmembrane region is selected from the transmembrane region of CD8α or CD28. In addition, the CD8α hinge region is a flexible region, so CD8 or CD28 and the transmembrane region plus the hinge region are used to connect the target recognition domain scFv of the antigen-binding receptor CAR and the intracellular signaling region.

The intracellular signaling domain of the present invention can be selected from CD3ζ, FcεRIγ, CD28 costimulatory signaling domain, CD137 costimulatory signaling domain, and combinations thereof. The CD3 molecule consists of five subunits, of which the CD3ζ subunit (also known as CD3zeta, referred to as Z) contains three ITAM motifs, which are important signal transduction regions in the TCR-CD3 complex. In addition, as previously mentioned, CD28 and CD137 are costimulatory signaling molecules, and the costimulatory effects produced by their intracellular signaling segments after binding to their respective ligands cause sustained proliferation of immune response cells (mainly T lymphocytes), and can increase the levels of cytokines such as IL-2 and IFN-γ secreted by immune response cells, and increase the survival cycle and anti-tumor effects of CAR immune response cells in vivo. In some embodiments, the intracellular signaling domain is a combination of a CD3ζ signaling domain or a CD3ζ signaling domain with other costimulatory signals such as CD28.

In some embodiments, the immune response cell of the present invention may include an expression construct, wherein the expression construct contains elements connected in the following order: an antibody, a CD28 costimulatory signaling domain, CD3ζ, and NFAT6 and type I interferon expression units connected in reverse to the aforementioned elements. Preferably, the antibody and the CD28 costimulatory signaling domain are connected via a CD8α transmembrane region and a CD8α hinge region.

In some embodiments, NFAT (Nuclear factor of activated T cells) plays an important role in the transcriptional expression of cytokines during T cell activation. Based on this consideration, the inventors placed the IFN-beta coding sequence under the regulation of the NFAT6 promoter, so that only when CAR-T cells contact antigens to trigger T cell activation, IFN-beta can be expressed at a high level.

The NFAT6 promoter is a promoter composed of six NFAT binding sites and the minimal promoter of IL2 in series (Hooijberg E, Bakker AQ, Ruizendaal JJ, Spits H. NFAT-controlled expression of GFP permits visualization and isolation of antigen-stimulated primary human Tcells. Blood. 2000 Jul 15; 96 (2): 459-66), which can be used to regulate the expression of cytokines such as IL12 in T lymphocytes such as TCR-T (Zhang L, Kerkar SP, Yu Z, Zheng Z, Yang S, Restifo NP, Rosenberg SA, Morgan RA. Improving adoptive T cell therapy by targeting and controlling IL-12 expression to the tumor environment. Mol Ther. 2011 Apr; 19 (4): 751-9).

According to one aspect of the present invention, the present invention also includes nucleic acids encoding the receptors that bind to antigens. The present invention also relates to variants of the above polynucleotides, which encode polypeptides or polypeptide fragments, analogs and derivatives having the same amino acid sequence as the present invention.

The present invention also provides a vector comprising the above nucleic acid encoding the antigen-binding receptor protein expressed on the surface of the immune response cell. In a specific embodiment, the vector used in the present invention is a lentiviral plasmid vector pRRLSIN-cPPT.PGK-GFP.WPRE. It should be understood that other types of viral vectors and non-viral vectors can also be used.

The present invention also includes viruses comprising the above-mentioned vectors. The viruses of the present invention include viruses with infectious power after packaging, and also include viruses to be packaged that contain the necessary components for packaging into infectious viruses. Other viruses known in the art that can be used to transduce foreign genes into immune response cells and their corresponding plasmid vectors can also be used in the present invention.

The immune response cells of the present invention are transduced with a construct capable of expressing a receptor for binding to an antigen and exogenous type I interferon, or an expression vector, or a virus comprising the plasmid. Conventional nucleic acid transduction methods in the art, including non-viral and viral transduction methods, can be used in the present invention.

The immune response cells of the present invention may further carry coding sequences of exogenous cytokines; the cytokines include but are not limited to IL-12, IL-15 or IL-21. These cytokines have further immunomodulatory or anti-tumor activity, can enhance the function of effector T cells and activated NK cells, or directly exert anti-tumor effects. Therefore, those skilled in the art can understand that the use of these cytokines helps the immune response cells to function better.

The immune response cells of the present invention may also express another antigen-binding receptor in addition to the above-mentioned antigen-binding receptors.

The immune response cells of the present invention may also express chemokine receptors, including but not limited to CCR2. Those skilled in the art will appreciate that the CCR2 chemokine receptors may allow CCR2 in the body to competitively bind to them, which is beneficial for blocking tumor metastasis.

The immune response cells of the present invention may also express siRNA that can reduce the expression of PD-1 or a protein that blocks PD-L1. It will be appreciated by those skilled in the art that competitively blocking the interaction between PD-L1 and its receptor PD-1 is beneficial for restoring anti-tumor T cell responses, thereby inhibiting tumor growth.

The immune response cells of the present invention may also express a safety switch; preferably, the safety switch comprises: iCaspase-9, Truancated EGFR or RQR8.

In some embodiments, the immune response cells of the invention do not express co-stimulatory ligands such as 4-1BBL.

A transgene encoding a receptor or CAR for a target binding antigen can be incorporated into a cell. For example, a transgene can be incorporated into an immune response cell, such as a T cell. When inserted into a cell, the transgene can be a complementary DNA (cDNA) fragment, which is a copy of a messenger RNA (mRNA); or the gene itself (with or without introns) located in the original region of its genomic DNA.

Nucleic acids such as DNA encoding transgenic sequences can be randomly inserted into the chromosomes of cells. Random integration can be produced by any method for introducing nucleic acids (e.g., DNA) into cells. For example, the method can include, but is not limited to, electroporation, ultrasound, use of a gene gun, lipofection, calcium phosphate transfection, use of dendrimers, microinjection, and use of viral vectors including adenovirus, AAV, and retroviral vectors, and/or type II ribozymes.

The DNA encoding the transgene can also be designed to include a reporter gene so that the presence of the transgene or its expression product can be detected by activation of the reporter gene. Any reporter gene can be used, such as those described above. By selecting cells in cell culture in which the reporter gene has been activated, cells containing the transgene can be selected.

The expression of CAR can be verified by expression assays, such as qPCR or by measuring the level of RNA. The expression level can also indicate the copy number. For example, if the expression level is very high, this can indicate that more than one copy of CAR is integrated into the genome. Alternatively, high expression can indicate that the transgene is integrated in a highly transcribed region, such as near a highly expressed promoter. Expression can also be verified by measuring protein levels, such as by Western blotting.

In some embodiments, the immune response cells of the present invention may include one or more transgenes. The one or more transgenes may express a CAR protein, and the CAR protein recognizes and binds to at least one epitope on an antigen or a mutant epitope on a binding antigen. CAR may be a functional CAR. In some embodiments, the immune response cells of the present invention may include one or more CARs, or they may include a single CAR and a secondary engineered receptor.

In some embodiments, the transgene may encode a suicide gene. As demonstrated by many effective treatments for cancer patients, CAR immune response cells cause tumor regression but may be accompanied by toxicity. In some embodiments, when the target antigen is shared in normal tissues and tumor cells, the CAR immune response cells may not be able to distinguish between tumors and normal tissues ("on-target/off-target toxicity"). In other cases, systemic perturbations of the immune system may occur, referred to as cytokine release syndrome (CRS). The CRS may include systemic inflammatory response syndrome or cytokine storm, which may be the consequence of rapid expansion of CAR immune response cells in vivo. CRS is a disease characterized by fever and hypotension, and severe cases may lead to multiple organ failure. In most cases, the toxicity is associated with the in vivo amplification of the infused CAR immune response cells, which may cause overall perturbations of the immune system, as well as release of high levels of proinflammatory cytokines, such as TNFα and IL-6. Suicide genes can induce the elimination of CAR immune reactive cells. Suicide genes can be any gene that induces apoptosis in the CAR immune reactive cells. Suicide genes can be encoded in viral vectors together with the antigen-binding receptors. Encoding of suicide genes can, in certain circumstances, alleviate or completely terminate the toxicity caused by the in vivo expansion of infused CAR immune response cells.

In some embodiments, CAR immunoreactive cells for antigens present in normal tissues can be produced so that they transiently express CAR, for example after electroporation of mRNA encoding the receptor. In addition, the significant efforts to further enhance CAR immunoreactive cells by including a safety switch can greatly eliminate CAR immunoreactive cells in the case of severe on-target toxicity. The vector encoding CAR can be combined with a safety switch such as an inducible caspase-9 gene (activated by a dimerization chemical inducer) or a truncated form of the EGF receptor R (activated by the monoclonal antibody cetuximab) or RQR8.

One or more transgenes used herein can be from different species. For example, one or more transgenes can include human genes, mouse genes, rat genes, pig genes, cattle genes, dog genes, cat genes, monkey genes, chimpanzee genes, or any combination thereof. For example, the transgene can be from a human with a human genetic sequence. One or more transgenes can include human genes. In some cases, one or more transgenes are not adenovirus genes.

As described above, transgenics can be inserted into the genome of immunoreactive cells in a random or site-specific manner. For example, transgenics can be inserted into random sites in the genome of immune cells. These transgenics can be functional, for example, fully functional when inserted anywhere in the genome. For example, transgenics can encode its own promoter, or can be inserted into a position controlled by its internal promoter. Alternatively, transgenics can be inserted into genes, such as introns of genes or exons, promoters or non-coding regions of genes. Transgenics can be inserted so that insertion of disruptive genes, such as endogenous immune checkpoints, can be performed.

In some embodiments, more than one copy of the transgenic can be inserted into multiple random sites within the genome. For example, multiple copies can be inserted into random sites in the genome. This may result in increased overall expression compared to a random insertion of the transgenic once. Alternatively, a copy of the transgenic can be inserted into a gene, and another copy of the transgenic can be inserted into a different gene. The transgenic can be targeted so that it can be inserted into a specific site in the genome of an immunoreactive cell.

In some embodiments, the polynucleic acid comprising a receptor sequence encoding a binding antigen may take the form of a plasmid vector. The plasmid vector may include a promoter. In some cases, the promoter may be constitutive. In some embodiments, the promoter is inducible. The promoter may be or may be derived from CMV, U6, MND or EF1a. In some embodiments, the promoter may be adjacent to the CAR sequence. In some embodiments, the plasmid vector further comprises a splice acceptor. In some embodiments, the splice acceptor may be adjacent to the CAR sequence. The promoter sequence may be a PKG or MND promoter. The MND promoter may be a synthetic promoter containing the U3 region of the MoMuLV LTR modified by the myeloproliferative sarcoma virus enhancer.

In some embodiments, polynucleic acids encoding target receptors can be designed to be delivered to cells by non-viral techniques. In some cases, the polynucleic acids can be good manufacturing practice (GMP) compatible reagents.

The expression of the polynucleic acid encoding the receptor or CAR for the target binding antigen can be controlled by one or more promoters. The promoter can be ubiquitous, constitutive (unrestricted promoter, allowing continuous transcription of related genes), tissue-specific promoter or inducible promoter. The expression of a transgene inserted adjacent to or close to the promoter can be regulated. For example, the transgene can be inserted near or next to a ubiquitous promoter. Some ubiquitous promoters can be CAGGS promoter, hCMV promoter, PGK promoter, SV40 promoter or ROSA26 promoter.

The promoter can be endogenous or exogenous. For example, one or more transgenes can be inserted adjacent to or near an endogenous or exogenous ROSA26 promoter. In addition, the promoter can be specific for immunoreactive cells. For example, one or more transgenes can be inserted adjacent to or near a porcine ROSA26 promoter.

Tissue-specific promoters or cell-specific promoters can be used to control the position of expression. For example, one or more transgenes can be inserted adjacent to or near a tissue-specific promoter. The tissue-specific promoter can be a FABP promoter, a Lck promoter, a CamKII promoter, a CD19 promoter, a keratin promoter, an albumin promoter, an aP2 promoter, an insulin promoter, a MCK promoter, a MyHC promoter, a WAP promoter, or a Col2A promoter.

Inducible promoters may also be used. If desired, these inducible promoters may be turned on and off by adding or removing an inducing agent. Inducible promoters contemplated may be, but are not limited to, Lac, tac, trc, trp, araBAD, phoA, recA, proU, cst-1, tetA, cadA, nar, PL, cspA, T7, VHB, Mx, and/or Trex.

The term "inducible promoter" used herein is a controlled promoter that does not express or low-expresses a gene operably connected to it before the expected conditions are not met, and expresses or high-levels of a gene operably connected to it when the expected conditions are met. For example, in some embodiments, the inducible promoter of the present application does not express or low-expresses a gene operably connected to it under normal or high oxygen content conditions in the cell, and in response to the reduced oxygen content in the cell, expresses or high-expresses a gene operably connected to it under hypoxic conditions. In some embodiments, the inducible promoter used herein includes hypoxia-inducible transcription factor-1α (Hypoxia-InducibleTranscription factor-1α, HIF-1α). In some embodiments, the term "inducible promoter" used herein refers to an "immune cell inducible promoter", which does not express or low-expresses a gene operably connected to it before the immune response cell contacts the antigen or when the immune response cell is not activated, and only when the immune response cell contacts the antigen or the immune response cell is activated, will it drive the gene operably connected to it to be expressed at a high level or under conditions such as hypoxia. In some embodiments, the "immune cell-inducible promoter" includes a NFAT (nuclear factor of activated T cells) type promoter.

As used herein, "NFAT-type promoter" refers to a type of promoter that regulates the expression of a gene operably linked thereto based on NFAT binding activity.

NFAT is a general term for a family of transcription factors that play an important role in immune responses. One or more members of the NFAT family are expressed in most cells of the immune system. NFAT is also involved in the development of the heart, skeletal muscle, and nervous system.

The NFAT transcription factor family consists of five members: NFAT1, NFAT2, NFAT3, NFAT4, and NFAT5. NFAT1 to NFAT4 are regulated by calcium signals. Calcium signals are essential for NFAT activation because calmodulin (CaM) activates the serine/threonine phosphatase calcineurin (CN). Activated CN rapidly dephosphorylates the serine-rich region (SRR) and SP repeats at the amino terminus of the NFAT protein, leading to conformational changes, exposing nuclear localization signals, and causing NFAT to be imported into the nucleus.

Based on the role of NFAT in the transcriptional expression of cytokines during T cell activation, it can be used to regulate the immune cell inducible promoter described herein, thereby expressing or expressing at high levels the genes operably linked thereto when immune response cells are activated by contact with antigens.

The nucleic acid of the present invention may comprise any suitable nucleotide sequence encoding a NFAT-type promoter (or a functional part or functional variant thereof). "NFAT-type promoter" as used herein refers to one or more NFAT response elements connected to the minimal promoter of any gene expressed by T cells. Preferably, the minimal promoter of the gene expressed by T cells is the minimal human IL-2 promoter. The NFAT response element may include, for example, NFAT1, NFAT2, NFAT3 and/or NFAT4 response elements. In some embodiments, more than one NFAT binding motif may be included in the "NFAT-type promoter" described herein. For example, the "NFAT-type promoter" may include 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NFAT binding motifs. In some embodiments, the "NFAT-type promoter" includes up to 12 NFAT binding motifs. In some embodiments, the "NFAT-type promoter" may be a promoter composed of a plurality of the NFAT binding motifs and a promoter such as the IL2 minimal promoter in series. In some embodiments, the NFAT-type promoter described herein includes 6 NFAT binding motifs, denoted as (NFAT) _6. For the purpose of convenience, the (NFAT) ₆ is also denoted as NFAT6. In some embodiments, the NFAT6 also represents 6 repeated NFAT binding motifs (SEQ ID NO: 78) in the NFAT-type promoter.

Additionally, although not essential for expression, the transgenic sequence may also include transcriptional or translational regulatory sequences, such as promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals.

In some embodiments, the transgenic encodes a receptor or CAR for a target antigen-binding, wherein the transgenic is inserted into a safe harbor so that the antigen-binding receptor is expressed. In some embodiments, the transgenic is inserted into the PD1 and/or CTLA-4 locus. In other cases, the transgenic is delivered to the cell with a lentivirus for random insertion, and the PD1- or CTLA-4 specific nuclease can be provided as mRNA. In some embodiments, the transgenic is delivered by a viral vector system such as a retrovirus, AAV or adenovirus and mRNA encoding a nuclease (e.g., AAVS1, CCR5, albumin or HPRT) specific for the safe harbor. Cells can also be treated with mRNA encoding PD1 and/or CTLA-4 specific nucleases. In some embodiments, the polynucleotide encoding CAR is provided by a viral delivery system together with mRNA encoding HPRT specific nucleases and PD1- or CTLA-4 specific nucleases. CARs that can be used with the methods and compositions disclosed herein can include all types of these chimeric proteins, including the first, second and third generation designs described hereinabove.

In some embodiments, retroviral vectors (γ-retrovirus or lentiviral vectors) can be used to introduce transgenes into immunoreactive cells. For example, the transgene encoding CAR or any receptor or variant or fragment thereof binding antigen can be cloned into a retroviral vector and can be driven by its endogenous promoter, retroviral long terminal repeats, or promoters specific to the target cell type. Non-viral vectors can also be used. Non-viral vector delivery systems can include DNA plasmids, naked nucleic acids, and nucleic acids compounded with delivery vectors such as liposomes or poloxamer.

Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of transgenes and their propagation in daughter cells. Lentiviral vectors have additional advantages over vectors derived from retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells. They also have the additional advantage of low immunogenicity. The advantage of adenoviral vectors is that they do not fuse into the genome of the target cell, thereby bypassing negative integration-related events.

Cells can be transfected with transgenics encoding the receptors for the antigen-binding proteins. Transgenic concentrations can be from about 100 picograms to about 50 micrograms. In some embodiments, the amount of nucleic acid (e.g., ssDNA, dsDNA, or RNA) introduced into cells can be changed to optimize transfection efficiency and/or cell viability. For example, 1 microgram of dsDNA can be added to each cell sample for electroporation. In some embodiments, the amount of nucleic acid (e.g., double-stranded DNA) required for optimal transfection efficiency and/or cell viability varies according to cell type. In some embodiments, the amount of nucleic acid (e.g., dsDNA) used for each sample can directly correspond to transfection efficiency and/or cell viability. For example, a series of transfection concentrations. Transgenics encoded by a vector can be integrated into the cell genome. In some embodiments, transgenics encoded by a vector are integrated forward. In other cases, reverse integration of transgenics encoded by a vector.

In some embodiments, the immunoreactive cell may be a stem memory T SCM cell composed of CD45RO (-), CCR7 (+), CD45RA (+), CD62L + (L-selectin), CD27 +, CD28 + and/or IL-7Rα +, and _the stem memory cell may also express CD95, IL-2Rβ, CXCR3 and/or LFA-1, and exhibit many functional attributes different from the stem memory cell. Alternatively, the immunoreactive cell may also be a central memory T _CM cell comprising L-selectin and CCR7, wherein the central memory cell may secrete, for example, IL-2, but not IFNγ or IL-4. The immunoreactive cell may also be an effector memory T _EM cell comprising L-selectin or CCR7, and produces, for example, effector cytokines such as IFNγ and IL-4.

The vector is delivered in vivo, usually by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial infusion) or topical application, as described below. Alternatively, the vector can be delivered ex vivo to cells, such as cells removed from an individual patient (e.g., lymphocytes, T cells, bone marrow aspirates, tissue biopsies), which are then typically reimplanted into the patient after selecting cells that have incorporated the vector. Before or after selection, the cells can be expanded.

Suitable immunoreactive cells for expressing a receptor that binds an antigen may be autologous or non-autologous to the individual in need thereof.

The source of suitable immune response cells can be obtained from an individual. In some cases, T cells can be obtained. The T cells can be obtained from many sources, including PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue and tissue from infection sites, ascites, pleural effusion, spleen tissue and tumors. In some cases, any number of techniques known to those skilled in the art, such as Ficoll ^TM separation, can be used to obtain T cells from blood collected from the individual. In one embodiment, cells from the circulating blood of an individual are obtained by single blood sampling. Single blood sampling products usually contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells and platelets. In one embodiment, the cells collected by single blood sampling can be washed to remove the plasma fraction and the cells are placed in a suitable buffer or culture medium for subsequent processing steps.

Alternatively, cells can be derived from healthy donors, from patients diagnosed with cancer, or from patients diagnosed with infection. In some embodiments, cells can be part of a mixed cell population with different phenotypic characteristics. Cell lines can also be obtained from transformed T cells according to the aforementioned methods. Cells can also be obtained from a cell therapy library. Modified cells resistant to immunosuppressive therapy can be obtained by any method described herein. Suitable cell populations can also be selected before modification. Engineered cell populations can also be selected after modification. Engineered cells can be used for autologous transplantation. Alternatively, cells can be used for allogeneic transplantation. In some embodiments, cells are administered to the same patient whose sample is used to identify cancer-related target sequences. In other cases, cells are administered to patients different from those whose samples are used to identify cancer-related target sequences.

In some embodiments, suitable primary cells include peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL) and other blood cell subsets, such as but not limited to T cells, natural killer cells, monocytes, natural killer T cells, monocyte precursor cells, hematopoietic stem cells or non-pluripotent stem cells. In some embodiments, the cell can be any immune cell, including any T cell such as tumor infiltrating cells (TIL), such as CD3+T cells, CD4+T cells, CD8+T cells or any other type of T cells. T cells can also include memory T cells, memory stem T cells or effector T cells. T cells can also be selected from a large number of populations, such as selecting T cells from whole blood. T cells can also be expanded from a large number of populations. T cells may also tend to specific populations and phenotypes. For example, T cells can be inclined to phenotypes including CD45RO (-), CCR7 (+), CD45RA (+), CD62L (+), CD27 (+), CD28 (+) and/or IL-7Rα (+). Suitable cells can be selected from one or more markers in the following list: CD45RO (-), CCR7 (+), CD45RA (+), CD62L (+), CD27 (+), CD28 (+) and/or IL-7Rα (+). Suitable cells also include stem cells, such as embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells and mesenchymal stem cells. Suitable cells can include any number of primary cells, such as human cells, non-human cells and/or mouse cells. Suitable cells can be progenitor cells. Suitable cells can be derived from the subject to be treated (e.g., patient).

The amount of therapeutically effective cells required in the patient can vary according to the viability of the cell and the efficiency of the genetic modification of the cell (e.g., the efficiency of the transgene being integrated into one or more cells, or the expression level of the protein encoded by the transgene). In some embodiments, the product of the cell viability after genetic modification (e.g., multiplication) and the efficiency of transgene integration can correspond to the therapeutic amount of cells that can be used to administer to the subject. In some embodiments, the increase in cell viability after genetic modification may correspond to a reduction in the amount of cells necessary for the treatment to be effective for the patient. In some embodiments, the increase in the efficiency of transgene integration into one or more cells can correspond to a reduction in the number of cells necessary for the treatment to be effective in the patient. In some embodiments, determining the amount of therapeutically effective cells required may include determining functions related to cell changes over time. In some embodiments, determining the amount of therapeutically effective cells required may include determining functions corresponding to changes in the efficiency of integrating transgenes into one or more cells according to time-related variables (e.g., cell culture time, electroporation time, cell stimulation time). In some embodiments, therapeutically effective cells may be a cell population that includes about 30% to about 100% of the expression of antigen-binding receptors on the cell surface. In some embodiments, therapeutically effective cells may express about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more than about 99.9% of the receptor that binds the antigen on the cell surface as measured by flow cytometry.

In some embodiments, when the antigen-binding receptor is present on the plasma membrane of the cell, and when activated by binding to a target, it can cause the toxicity of the cell with the target that the antigen-binding receptor can bind to for its cell surface expression. For example, in some cases, when the cell is present in the plasma membrane of the cell, the cell can be a cytotoxic cell (e.g., NK cell or cytotoxic T lymphocyte), a receptor for binding antigens as described herein, and when it is activated by binding to its target, the cytotoxic activity of cytotoxic cells to target cells can be increased. For example, in some embodiments, the antigen-binding receptor described herein, when activated by the binding of its target, can increase cytotoxicity by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 75%, at least 2 times, at least 2.5 times, at least 5 times, at least 10 times or more 10 times compared to the cytotoxicity of cells without binding to the target.

The immune response cells of the present invention can be used to prepare pharmaceutical compositions. In addition to an effective amount of immune response cells, the pharmaceutical compositions may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means that when the molecular entities and compositions are appropriately administered to animals or humans, they will not produce adverse, allergic or other adverse reactions.

Specific examples of some substances that can serve as pharmaceutically acceptable carriers or components thereof are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols, such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as Wetting agents, such as sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer, etc.

The composition of the present invention can be prepared into various dosage forms as required, and the physician can determine the dosage that is beneficial to the patient according to factors such as the patient type, age, weight, general disease condition, and administration method. The administration method can be, for example, parenteral administration (such as injection) or other treatment methods.

&quot;Parenteral&quot; administration of the immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.) or intrasternal injection or infusion techniques.

The preparation containing the immunoreactive cell population administered to an individual contains multiple immunoreactive cells that effectively treat and/or prevent a specific indication or disease. Therefore, a therapeutically effective population of immunoreactive cells can be administered to an individual. Typically, a preparation containing about 1×10 ⁴ to about 1×10 ¹⁰ immunoreactive cells is administered. In most cases, the preparation will contain about 1×10 ⁵ to about 1×10 ⁹ immunoreactive cells, about 5×10 ⁵ to about 5×10 ⁸ immunoreactive cells, or about 1×10 ⁶ to about 1×10 ⁷ immunoreactive cells. However, depending on the location, source, identity, degree and severity of the cancer, the age and physical condition of the individual to be treated, etc., the number of CAR immunoreactive cells administered to the individual will vary between a wide range. The doctor will ultimately determine the appropriate dose to be used.

In some embodiments, chimeric antigen receptors are used to stimulate immune cell-mediated immune responses. For example, a T cell-mediated immune response is an immune response involving T cell activation. Activated antigen-specific cytotoxic T cells can induce apoptosis in target cells that display exogenous antigen epitopes on their surfaces, such as cancer cells that display tumor antigens. In another embodiment, chimeric antigen receptors are used to provide anti-tumor immunity in mammals. Due to the T cell-mediated immune response, the subject will develop anti-tumor immunity.

In some cases, the method for treating a subject with cancer may involve administering one or more immune response cells of the present invention to a subject in need of treatment. The immune response cells may bind to tumor target molecules and induce cancer cell death. As described above, the present invention also provides a method for treating a pathogen infection in an individual, comprising administering a therapeutically effective amount of immune response cells of the present invention to the individual.

The frequency of administration of the immunoreactive cells of the present invention will be based on factors including the disease being treated, elements of the specific immunoreactive cells and the mode of administration. For example, administration can be 4 times, 3 times, 2 times or once a day, every other day, every three days, every four days, every five days, every six days, once a week, once every eight days, once every nine days, every ten days, once a week, or twice a month. As described herein, because the immune response cells of the present application have improved vitality, they can be administered not only with a lower therapeutically effective amount than similar immune response cells that do not express exogenous type I interferon, but can be administered with a lower frequency to obtain at least similar and preferably more significant therapeutic effects.

In some embodiments, the immune response cells of the present invention can be administered in combination with another therapeutic agent. In some embodiments, the other therapeutic agent is a chemotherapeutic agent. Chemotherapeutic drugs that can be used in combination with the immune response cells of the present invention include, but are not limited to, mitotic inhibitors (vinca alkaloids), including vincristine, vinblastine, vindesine and novibin (TM) (vinorelbine, 5'-dehydrogen sulfide); topoisomerase I inhibitors, such as camptothecin compounds, including Camptosar ^TM (irinotecan HCL), Hycamtin ^TM (topotecan HCL) and other compounds derived from camptothecin and its analogs; podophyllotoxin derivatives, such as etoposide, teniposide and midoxazolidinone; alkylating agents cisplatin, cyclophosphamide, nitrogen mustard, trimethylenethiophosphoramide, carmustine, busulfan, chlorambucil, brequizine, uracil mustard, chlorprofen and dacarbazine; antimetabolites, including cytarabine, fluorouracil, methotrexate, mercaptopurine, azathioprine and procarbazine; antibiotics, including but not limited to doxorubicin, bleomycin, dactinomycin, daunorubicin, mycoplasmin, mitomycin, sarcomamycin C and daunomycin; and other chemotherapeutic drugs, including but not limited to anti-tumor antibodies, dacarbazine, azacytidine, amsarcan, melphalan, ifosfamide and mitoxantrone.

In some embodiments, chemotherapeutic drugs that can be used in combination with the immune response cells of the present invention include, but are not limited to, anti-angiogenic agents, including anti-VEGF antibodies (including humanized and chimeric antibodies, anti-VEGF aptamers and antisense oligonucleotides) and other angiogenesis inhibitors, such as angiostatin, endostatin, interferon, interleukin 1 (including α and β) interleukin 12, retinoic acid, and tissue inhibitors of metalloproteinases-1 and -2.

In some embodiments, compositions can be isotonic, that is, they can have the same osmotic pressure as blood and tears. The expected isotonicity of the compositions of the present invention can be realized using sodium chloride or other pharmaceutically acceptable reagents such as glucose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. If necessary, the viscosity of the compositions can be maintained at a selected level using a pharmaceutically acceptable thickener. Suitable thickeners include, for example, methylcellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The preferred concentration of thickener will depend on selected reagent. Obviously, the selection of suitable carriers and other additives will depend on the properties of definite route of administration and specific dosage form, such as liquid dosage form.

The present invention also provides a kit comprising immune response cells of the present invention. The kit can be used to treat or prevent cancer, pathogen infection, immune disorder or allogeneic transplantation. In one embodiment, the kit may include a therapeutic or preventive composition containing an effective amount of immune response cells containing one or more unit dosage forms. In some embodiments, the kit contains a sterile container that can contain a therapeutic or preventive composition; such a container can be a box, an ampoule, a bottle, a vial, a tube, a bag, a blister pack, or other suitable container forms known in the art. Such a container can be made of plastic, glass, laminated paper, metal foil or other materials suitable for holding drugs. In some embodiments, immunoreactive cells, such as CAR T cells, and instructions for administering CAR immune-reactive cells to subjects at risk of developing cancer, pathogen infection, immune disorder or allogeneic transplantation can be provided. The instructions will generally include information about the composition for treating or preventing cancer, pathogen infection, immune disease or allogeneic transplantation. In some embodiments, the kit may include about 1×10 ⁴ cells to about 1×10 ⁶ cells. In some embodiments, the kit can include at least about 1×10 ⁵ cells, at least about 1×10 ⁶ cells, at least about 1×10 ⁷ cells, at least about 4×10 ⁷ cells, at least about 5×10 ⁷ cells, at least about 6×10 ⁷ cells, at least about 6×10 ⁷ cells, 8×10 ⁷ cells, at least about 9×10 ⁷ cells, at least about 1×10 ⁸ cells, at least about 2×10 8 cells, at least about 3×10 ⁸ cells, at least about 4×10 ⁸ cells, at least about 5×10 ⁸ cells, at least about 6×10 ⁸ cells, at least about 6×10 ⁸ cells, at least about 8×10 ⁸ cells, at least about 9×10 ⁸ cells, at least about 1×10 ⁹ cells, at least about 2×10 ⁹ cells, at least about 3×10 ⁹ cells, at least about 4×10 8 cells ^{10 9} cells, at least about 5 x 10 ⁹ cells, at least about 6 x 10 ⁹ cells, at least about 8 x 10 ⁹ cells, at least about 9 x 10 ⁹ cells, at least about 1 x 10 ¹⁰ cells, at least about 2 x 10 ¹⁰ cells, at least about 3 x 10 ¹⁰ cells, at least about 4 x 10 ¹⁰ cells, at least about 5 x 10 ¹⁰ cells, at least about 6 x 10 ¹⁰ cells, at least about 9 x ^{10 10 cells} , at least about 1 x 10 ¹¹ cells, at least about 2 x 10 ¹¹ cells, at least about 3 x 10 ¹¹ cells, at least about 4 x 10 ¹¹ cells, at least about 5 x 10 ¹¹ cells, at least about 8 x 10 ¹¹ cells, at least about 9 x 10 ¹¹ cells, or at least about 1 x 10 ¹² cells. For example, approximately 5×10 ¹⁰ cells can be included in the kit. In another example, the kit can include 3×10 ⁶ cells; the cells can be expanded to approximately 5×10 ¹⁰ cells and administered to a subject.

In some embodiments, the kit may include allogeneic cells. In some embodiments, the kit may include cells that may include genomic modifications. In some embodiments, the kit may include "off-the-shelf" cells. In some embodiments, the kit may include cells that may be expanded for clinical use. In some cases, the kit may include contents for research purposes.

In some embodiments, the instructions include at least one of the following: a description of the therapeutic agent; dosage regimens and administration for treating or preventing tumors, pathogen infections, immune diseases, or allogeneic transplants or symptoms thereof; precautions, warnings, contraindications, overdose information, adverse reactions, animal pharmacology, clinical studies, and/or references. The instructions can be printed directly on the container (if any), or as a label on the container, or as a separate paper, brochure, card, or folder provided in or in the container. In some embodiments, the instructions provide methods for administering the immune response cells described in the present invention for treating or preventing tumors, pathogen infections, immune diseases, or allogeneic transplants or symptoms thereof. In some cases, the instructions provide methods for administering the immune response cells of the present invention before, after, or simultaneously with the administration of a chemotherapeutic agent.

According to one aspect of the present invention, the present invention also provides a method for treating tumors or pathogen infections in individuals, or for enhancing individual immune tolerance. In some embodiments, the method includes administering the immune response cells of the present invention to individuals in need thereof, wherein the immune cells express the antigen-binding receptors and exogenous type I interferon. In some embodiments, the method includes administering the antigen-binding receptors of the present invention and exogenous type I interferon to individuals in need thereof. In some embodiments, the exogenous type I interferon is administered sequentially or simultaneously with the immune response cells expressing the antigen-binding receptors. In some embodiments, the exogenous type I interferon is administered to the patient simultaneously with the immune response cells by co-expression in the immune response cells.

The present invention provides a method for improving the viability of immune response cells administered to an individual, wherein the immune response cells express the antigen-binding receptors of the present invention, and wherein the method comprises administering the immune response cells and an effective amount of exogenous type I interferon to the individual. In some embodiments, the exogenous type I interferon and the immune response cells expressing the antigen-binding receptors are administered sequentially or simultaneously. In some embodiments, the exogenous type I interferon is co-expressed in the immune response cells and administered to the patient simultaneously with the immune response cells.

In some embodiments, due to the improved viability of the immune response cells of the present invention, the immune response cells of the present invention can be administered at a lower dose and/or a lower frequency compared to when exogenous type I interferon is not administered or the immune response cells do not co-express the exogenous type I interferon.

In some embodiments, the amount of the immune response cells of the present invention administered to an individual in need thereof is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to when no exogenous type I interferon is administered or when the immune response cells do not co-express the exogenous type I interferon. In some embodiments, the frequency of the immune response cells of the present invention administered to an individual in need thereof is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to when no exogenous type I interferon is administered or when the immune response cells do not co-express the exogenous type I interferon. Alternatively, when the immune response cells of the present invention are administered multiple times to an individual in need thereof, the interval between each administration is extended by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, 200%, 500%, 750%, 1000% compared to when exogenous type I interferon is not administered or when the immune response cells do not co-express the exogenous type I interferon.

In some embodiments, the method of the present invention is such that after administering the immune response cells to the individual, the sum of the number of cytotoxic T cells and helper T cells in the individual's peripheral blood is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, 200%, 500%, 750%, 1000% compared to the absence of the exogenous type I interferon. In some embodiments, the method is such that about 5 days after administering the immune response cells to the individual, the sum of the number of cytotoxic T cells and helper T cells in the individual's peripheral blood is greater than 5,000/μL, 10,000/μL, 15,000/μL, 20,000/μL, 25,000/μL; about 7 days after administering the immune response cells, the sum of the number of cytotoxic T cells and helper T cells in the individual's peripheral blood is greater than 100/μL, 200/μL, 300/μL, 400/μL, 500/μL, 600/μL, 700/μL, 800/μL. /μL, 900/μL, 1,000/μL, 1,500/μL, 2,000/μL, 2,500/μL, 3,000/μL, 3,500/μL, 4,000/μL, 4,500/μL, or 5,000/μL; or about 10 days after administration of the immune response cells, the sum of the number of cytotoxic T cells and helper T cells in the individual's peripheral blood is greater than 10/μL, 20/μL, 30/μL, 40/μL, 50/μL, 60/μL, 70/μL, 80/μL, 90/μL, or 100/μL.

The present invention also provides a method for regulating an immune response in an individual, the method comprising administering to the individual an effective amount of any one of the immune response cells of the present invention.

The present invention also provides a method for strengthening individual immune tolerance, the method comprising administering to the individual an effective amount of the immune response cell of the present invention, the cell comprising a receptor that binds to a tumor antigen and a vector encoding type I interferon. Preferably, the method can prevent or reduce autoimmune diseases or diseases associated with allogeneic transplantation.

The present invention also provides a method for treating or preventing an individual from being infected by a pathogen, the method comprising administering an effective amount of immune response cells, the cells comprising a receptor that binds to a viral antigen and a vector encoding type I interferon.

Autologous lymphocyte infusion can be used for treatment. Autologous peripheral blood mononuclear cells (PBMC) can be collected from patients in need of treatment, and T cells can be activated and amplified using methods described herein and known in the art, and then injected into the patient. In other cases, allogeneic cells can be used to treat patients.

The methods disclosed herein may include transplantation. Transplantation may refer to adoptive transplantation of a cell product. Transplantation may be an autologous transplantation, an allogeneic transplantation, a xenotransplantation, or any other transplantation. For example, a transplantation may be a xenotransplantation. A transplantation may also be an allogeneic transplantation.

In some embodiments, the subject may be given an immunoreactive cell, wherein the immunoreactive cell that can be administered may be about 1 to about 35 days old. For example, the cells administered may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or up to about 40 days. The age of the CAR immunoreactive cell can be calculated from the time of stimulation. The age of the immunoreactive cell can be calculated from the time of blood collection. The age of the immunoreactive cell can be calculated from the time of transduction. In some embodiments, the immunoreactive cell that can be administered to the subject is about 10 to about 14 or about 20 days old. In some embodiments, the "age" of the immunoreactive cell can be determined by telomere length. For example, "young" immunoreactive cells can have longer telomere lengths than "exhausted" or "old" immunoreactive cells. Without being bound by a particular theory, it is believed that immunoreactive cells lose an estimated telomere length of about 0.8 kb per week in culture, and that young immunoreactive cell cultures may have telomeres that are about 1.4 kb longer than immunoreactive cells that are about 44 days old. Without being bound by a particular theory, it is believed that longer telomere length may correlate with positive objective clinical responses in patients and persistence of cells in vivo.

Before, after and/or during transplantation, cell (for example, engineered cell or engineered primary T cell) can be functional.For example, the cell of transplantation can be at least about 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,6,27,28,29,30,40,50,60,70,80,90 or 100 days after transplantation work.Transplanted cells can work at least about 1,2,3,4,5,6,7,8,9,10,11 or 12 months after transplantation.Transplanted cells can work at least about 1,2,3,4,5,6,7,8,9,10,15,20,25 or 30 years after transplantation.In some embodiments, transplanted cells can work during the life span of recipient.

Furthermore, the transplanted cells can function at 100% of their normal intended function. The transplanted cells can also function at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, , 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or up to about 100% functionality.

The transplanted cells may also function at more than 100% of their normal intended function. For example, the transplanted cells may function at about 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, or up to about 5000% of their normal intended function.

Transplantation can be by any type of transplantation. Local can include but not limited to the subcapsular space under the liver, the subcapsular space under the spleen, the subcapsular space under the kidney, the omentum, the stomach or intestinal submucosal layer, the small intestinal vascular segment, the venous sac, the testis, the brain, the spleen or the cornea. For example, the transplantation can be a subcapsular transplantation. The transplantation can also be an intramuscular transplantation. The transplantation can be a portal vein transplantation.

Compared to when one or more wild-type cells are transplanted into a recipient, transplant rejection can be improved after treatment with the immune response cells of the present invention. For example, transplant rejection can be a hyperacute rejection. Transplant rejection can also be an acute rejection. Other types of rejection may include chronic rejection. Transplant rejection can also be a cell-mediated rejection or a T-cell-mediated rejection. Transplant rejection can also be a natural killer cell-mediated rejection.

Improving engraftment may mean alleviating hyperacute rejection, which may include reducing, alleviating or decreasing adverse effects or symptoms. Transplantation may refer to adoptive transplantation of a cell product.

Another indication of a successful transplant can be the number of days that the recipient does not require immunosuppressive therapy. For example, after providing the immune response cells of the invention, the recipient may not require immunosuppressive therapy for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days. This can indicate a successful transplant. This can also indicate that the transplanted cells, tissues and/or organs were not rejected.

In some cases, the recipient does not require immunosuppressive therapy for at least 1 day. The recipient may also not require immunosuppressive therapy for at least 7 days. The recipient does not require immunosuppressive therapy for at least 14 days. The recipient does not require immunosuppressive therapy for at least 21 days. The recipient does not require immunosuppressive therapy for at least 28 days. The recipient does not require immunosuppressive therapy for at least 60 days. In addition, the recipient may not require immunosuppressive therapy for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years.

Another sign of successful transplantation may be the number of days of immunosuppressive therapy that the recipient needs to reduce. For example, after the treatment provided herein, the recipient may need at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days of reduced immunosuppressive therapy. This can indicate successful transplantation. This can also indicate that the transplanted cells, tissues and/or organs have no or only minimal rejection.

For example, a recipient may require at least 1 day of reduced immunosuppressive therapy. A recipient may also require at least 7 days of reduced immunosuppressive therapy. A recipient may require at least 14 days of reduced immunosuppressive therapy. A recipient may require at least 21 days of reduced immunosuppressive therapy. A recipient may require at least 28 days of reduced immunosuppressive therapy. A recipient may require at least 60 days of reduced immunosuppressive therapy. In addition, a recipient may require at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years of reduced immunosuppressive therapy.

Reduced immunosuppressive therapy can refer to less immunosuppressive therapy than is required when one or more wild-type cells are transplanted into a recipient.

Immunosuppressive therapy can include any treatment that suppresses the immune system. Immunosuppressive therapy can help alleviate, reduce, or eliminate a patient's transplant rejection. For example, immunosuppressants can be used before, during, and/or after a transplant, including MMF (mycophenolate mofetil (Cellcept)), ATG (anti-thymocyte globulin), anti-CD154 (CD4OL), anti-CD40 (2C10), immunosuppressive drugs, anti-IL-6R antibodies (tocilizumab, Actemra), anti-IL-6 antibodies (sarilumab, olokizumab), CTLA4-Ig (Abatacept/Orencia), anti-IL-6 antibodies (ASKP1240, CCFZ533X2201), amphetamines (Campath), Anti-CD20 (rituximab), bevacizumab (LEA29Y), sirolimus (Rapimune), everolimus, tacrolimus (Prograf), dactizumab (Ze-napax), basiliximab (Similect), infliximab (Remicade), cyclosporine, deoxyprotamine, soluble complement receptor 1, cobra toxin, anti-C5 antibody (eculizumab/Soliris), methylprednisolone, FTY720, everolimus, leflunomide, anti-IL-2R-Ab, rapamycin, anti-CXCR3 antibody, anti-ICOS antibody, anti-OX40 antibody and anti-CD122 antibody. In addition, one or more immunosuppressants/drugs can be used together or sequentially. One or more immunosuppressants/drugs can be used for induction therapy or maintenance therapy. The same or different drugs can be used in the induction and maintenance phases. In some cases, daclizumab (Zenapax) can be used for induction therapy, and tacrolimus (Prograf) and sirolimus (Rapimune) can be used for maintenance therapy. Non-drug regimens can also be used to achieve immunosuppression, including but not limited to total body irradiation, thymic irradiation, and total and/or partial splenectomy. These techniques can also be used in combination with one or more immunosuppressive drugs.

Example

The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd edition, Science Press, 2002, or according to the conditions recommended by the manufacturer.

In the following embodiments of the present invention, when constructing the antigen-binding receptor or CAR, the CD28 costimulatory signal domain is referred to as 28; CD3ζ is referred to as Z; 4-1BB or CD137 is referred to as BB. For example, a chimeric antigen receptor constructed by using the scFv coded as 85-2 and the CD3ζ and CD28 costimulatory signal domains as intracellular signal domains can be recorded as 85-2-28Z. This is the case for the construction of CARs for different antigens.

1. Experimental Materials

Hepatocellular carcinoma cell lines SK-HEP-1 and PLC/PRF/5 were purchased from ATCC cell bank, and Huh-7 was purchased from RIKEN cell bank in Japan.

PBMCs were obtained from Shanghai Blood Center.

AIM V Medium: CTS, Cat#1665773.

Human T-Activator CD3/CD28: Life technologies, Cat#11161D.

Fetal calf serum (FCS): Gibco, Cat# 10099-141.

IL-2: Shanghai Huaxin, recombinant human interleukin-2 for injection.

Goat anti-human F(ab') ₂ antibody: Jackson ImmunoResearch, Cat# 109-066-006.

PE-Streptavidin: BD pharmingen, Cat#554061.

CytoTox Non-radioactive cytotoxicity assay: Promega, Cat#G1780.

2. Experimental Methods

2.1 Lentiviral vector construction

2.1.1 Construction of pRRL-EF1α-92-CAR lentiviral vector

As an example, the vector system used to construct the lentiviral plasmid vector of the present invention belongs to the third generation self-inactivating lentiviral vector system, which has four plasmids: the packaging plasmid pMDLg RRE (purchased from addgene) encoding the protein Gag/Pol, the packaging plasmid pRSV-REV (purchased from addgene) encoding the Rev protein, the envelope plasmid pCMV-VSV-G (purchased from addgene) encoding the VSV-G protein, and the recombinant expression vector encoding the target gene CAR based on the empty vector pRRLSIN-cPPT.PGK-GFP.WPRE (purchased from addgene). The system can effectively reduce the risk of forming replicable lentiviral particles.

In this system, the inventors first transformed the empty vector pRRLSIN-cPPT.PGK-GFP.WPRE by conventional molecular cloning technology, replaced the promoter of the original vector with the promoter of elongation factor-1α (EF-1α), and added an MluI restriction site between the promoter and the CD8αsp signal peptide. Specifically, the vector pWPT-EGFP (purchased from addgene) was double-digested with ClaI/SalI (purchased from NEB), and a 1.1Kb DNA fragment was recovered and connected to the vector pRRLSIN-cPPT.PGK-GFP.WPRE double-digested with ClaI/SalI using T4 DNA ligase, and transformed into the host bacteria TOP10, and the clones were picked to identify the positive clones by colony PCR and confirmed by sequencing to obtain the recombinant plasmid pRRLSIN-cPPT.EF-1α-EGFP.WPRE.

Chinese patent 201510481235.1 describes an antibody 92 that has been humanized and can specifically recognize human GPC3 protein. In order to construct the 92-CAR lentiviral plasmid, the heavy chain variable region fragment was amplified using the plasmid containing the 92 heavy chain variable region (SEQ ID NO: 80 in patent 201510481235.1) as a template, and the upstream primer 5'-ctccacgccgccaggccggaggtgcagctggtgcag-3' (SEQ ID NO: 1) and the downstream primer 5'-GCGGTGTCCTCGCTCCGCAGGCTGCTCAGCTCCATGTAGGCGGTG-3' (SEQ ID NO: 2); the plasmid containing the 92 light chain variable region (SEQ ID NO: 79 in patent 201510481235.1) was used as a template, and the upstream primer 5'-GCGGAGCGAGGACACCGCCGTGTACTACTGCGCCCGGTTCTACAGCTAC-3' (SEQ ID NO:3) and downstream primer 5'-CGGCGCTGGCGTCGTGGTACGTTTGATCTCCAGCTTGGTG-3' (SEQ ID NO:4) to amplify the light chain variable region fragment. The heavy chain and light chain variable region primers were further amplified by bridge PCR to obtain a 92scFv fragment (SEQ ID NO:5) containing the upstream CD8α signal peptide and the downstream hinge region repeating sequence, named fragment 1, with a size of 765bp. The PCR amplification conditions were pre-denaturation: 94℃, 4min; denaturation: 94℃, 40s; annealing: 58℃, 40s; extension: 68℃, 40s; 25 cycles, followed by extension at 68℃ for 10min. The PCR amplification bands were confirmed to be consistent with the expected fragment size by agarose gel electrophoresis.

Using the upstream primer 5'-gcaggggaaagaatagtagaca-3' (SEQ ID NO: 6) and the downstream primer 5'-CGGCCTGGCGGCGTGGAG-3' (SEQ ID NO: 7), the vector plasmid pRRLSIN-cPPT.EF-1α-EGFP.WPRE constructed in this example was used as a template to amplify the EF-1α promoter (SEQ ID NO: 8) containing the CD8α signal peptide (containing the MluI restriction site), named fragment 2, with a size of 442 bp. The PCR amplification conditions were pre-denaturation: 94°C, 4min; denaturation: 94°C, 30s; annealing: 53°C, 30s; extension: 68°C, 30s; 25 cycles, and then total extension 68°C, 10min. The PCR amplification band was confirmed by agarose gel electrophoresis to be consistent with the expected fragment size.

Using the upstream primer 5'-accacgacgccagcgccg-3' (SEQ ID NO: 9) and the downstream primer 5'-aatccagaggttgattgtcgacctagcgagggggcagggcctgc-3' (SEQ ID NO: 10), pWPT-eGFP-F2A-GPC3-BBZ, pWPT-eGFP-F2A-GPC3-28Z and pWPT-eGFP-F2A-GPC3-28BBZ were used as templates (for details, refer to Chinese patent CN 104140974 A) to amplify fragment 3 containing Hinge-BBZ (SEQ ID NO: 11), fragment 4 of Hinge-28Z (SEQ ID NO: 12) and fragment 5 of Hinge-28BBZ (SEQ ID NO: 13) (all containing Sal I restriction site), with sizes of 694 bp, 703 bp and 829 bp, respectively. The PCR amplification conditions were pre-denaturation: 94°C, 4 min; denaturation: 94°C, 30 s; annealing: 60°C, 30 s; extension: 68°C, 30 s; 25 cycles, and then total extension at 68°C, 10 min. The PCR amplification bands were confirmed to be consistent with the expected fragment size by agarose gel electrophoresis.

About 50 ng of fragment 2, fragment 1 and fragment 3 were spliced by PCR respectively, and the splicing conditions were as follows: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 40 s; annealing at 60°C for 40 s; extension at 68°C for 140 s, for 5 cycles, and then total extension at 68°C for 10 min. DNA polymerase and upstream primer 5'-gcaggggaaagaatagtagaca-3' (SEQ ID NO: 6) and downstream primer 5'-aatccagaggttgattgtcgacctagcgagggggcagggcctgc-3' (SEQ ID NO: 10) were supplemented, and PCR amplification was performed for 25 cycles. The amplification conditions were as follows: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 40 s; annealing at 60°C for 40 s; extension: 68°C for 140 s, and total extension at 68°C for 10 min. The theoretical size of the amplified 92-BBZ DNA fragment (SEQ ID NO: 14) was 1865 bp, and the amplified product was confirmed to be consistent with the theoretical size by agarose electrophoresis.

Equimolar amounts of about 50 ng of fragment 2, fragment 1 and fragment 4 were subjected to splicing PCR, respectively, and the same splicing reaction conditions were used to amplify the 92-28Z DNA fragment (SEQ ID NO: 15) with a theoretical size of 1874 bp. The amplified product was confirmed to be consistent with the theoretical size by agarose electrophoresis.

Equimolar amounts of about 50 ng of fragment 2, fragment 1, and fragment 5 were subjected to splicing PCR, respectively, and the same splicing reaction conditions were used to amplify the 92-28BBZ DNA fragment (SEQ ID NO: 16), with a theoretical size of 2000 bp. The amplified product was confirmed to be consistent with the theoretical size by agarose electrophoresis.

The above-mentioned vector plasmid pRRLSIN-cPPT.EF-1α-EGFP.WPRE and fragments 92-BBZ, 92-28Z and 92-28BBZ were digested with restriction endonucleases Mlu I and SalI (purchased from NEB), respectively. Connected by T4 ligase (purchased from NEB), transformed into TOP10, cloned for PCR identification of positive bacteria, and sent to Invitrogen for sequencing to confirm that the sequence is correct, thereby obtaining pRRL-EF-1α-92-BBZ, pRRL-EF-1α-92-28Z and pRRL-EF-1α-92-28BBZ.

2.1.2 Construction of 92-CAR lentiviral vector co-expressing 4-1BBL

In addition, to construct a plasmid for co-expressing 92-28Z and 41BBL, the pRRL-EF-1α-92-28Z plasmid constructed above was used as a template, and fragment 6 was first amplified by PCR using the upstream primer 5’-gcaggggaaagaatagtagaca-3’ (SEQ ID NO: 6) and the downstream primer 5’-TCAGAAGGTCAAAATTCAAAGTCTGTTTCACGCGAGGGGGCAGGGCCTGCA TGTGAA-3’ (SEQ ID NO: 17). To obtain the F2A-41BBL fragment, plasmid HG15693-G (purchased from Beijing Sino Biological Biotechnology Co., Ltd., the 562nd base of the 41BBL gene contains a mutation from G to A) was used as a template, and the upstream primer 5'-gagacgttgagtccaaccctgggcccatggaatacgcctctgacgc-3' (SEQ ID NO: 18) and the downstream primer 5'-TCGGAGGAGGCGGGTGGCAGGTCCACGGTC-3' (SEQ ID NO: 19) were used to amplify fragment 7 by PCR; the upstream primer 5'-ctgccacccgcctcctccgaggctcggaa-3' (SEQ ID NO: 20) and the downstream primer 5'-TGATTGTCGACTTATTCCGACCTCGGTGAAGGGA-3' (SEQ ID NO:21), fragment 8 was amplified by PCR, and then fragments 7 and 8 were spliced in equal moles, and then PCR amplified with primers (SEQ ID NO:18 and SEQ ID NO:21) to obtain fragment 9. Finally, equimolar fragments 6 and 9 were spliced and amplified with primers (SEQ ID NO:6 and SEQ ID NO:21) to obtain 92-28Z-F2A-41BBL (SEQ ID NO:22). This fragment was digested with Mlu I and Sal I, and inserted into the vector pRRLSIN-cPPT.EF-1α-EGFP.WPRE that was digested with the same enzymes using the same method as above, and confirmed to be correct by sequencing to obtain plasmid pRRL-EF-1α-92-28Z-F2A-41BBL.

2.1.3 Construction of 92-CAR lentiviral vector capable of regulating co-expression of IFN

In order to construct a plasmid that can co-express 92-28Z and IFN beta (and at the same time achieve controllable expression by inserting the NFAT element in front of IFN beta), fragment 10 was first synthesized by primer bridging using primers (SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35), and then fragment 11 was amplified using the pWPT-EGFP plasmid as a template and the upstream primer (SEQ ID NO:36) and the downstream primer (SEQ ID NO:37). Fragments 10 and 11 were mixed in equal moles, amplified by bridge PCR and primer pair (SEQ ID NO:35 and SEQ ID NO:38), and then digested with ClaI and SalI. The fragment was inserted into the vector pRRLSIN-cPPT-PGK-EGFP.WPRE that had been digested with the same enzymes using the same method as above. The correctness was confirmed by sequencing to obtain the vector pRRLSIN-NFAT3-EGFP-PA2 containing three NFAT repeat sequences. Using pRRLSIN-NFAT3-EGFP-PA2 as a template, the primer pairs (SEQ ID NO:39 and SEQ ID NO:40) and primer pairs (SEQ ID NO:41 and SEQ ID NO:42) were used to amplify fragments 12 and 13, respectively. Then, by bridge PCR, the primer pairs (SEQ ID NO:39 and SEQ ID NO:42) were used to amplify fragment 14, which was then digested with Mlu I and Sal I, ligated to the pRRLSIN-NFAT3-EGFP-PA2 digested with the same enzymes, and sequenced to obtain the vector pRRLSIN-NFAT6-EGFP-PA2 containing six NFAT repeat sequences. Using the vector pGMT-IFN-β (purchased from Beijing Sino Biological Biotechnology Co., Ltd.) as a template, the primer pairs (SEQ ID NO:43 and SEQ ID NO:44) were used to amplify fragment 15. Using this fragment as a template, PCR amplification was performed using a primer pair (SEQ ID NO:43 and SEQ ID NO:38), and the amplified product was digested with Mlu I and Cla I, connected to the vector pRRLSIN-NFAT6-EGFP-PA2 digested with the same enzymes, and confirmed to be correct by sequencing to obtain the pRRLSIN-NFAT6-huIFNβ-PA2 plasmid. Using the constructed pRRLSIN-NFAT6-huIFNβ-PA2 plasmid as a template, the EGFP fragment 16 with the NdeI restriction site was amplified by a primer pair (SEQ ID NO:45 and SEQ ID NO:46); using the above-constructed plasmid pRRLSIN-NFAT6-EGFP-PA2 plasmid as a template, the NFAT6 fragment 17 with the NdeI restriction site was amplified by a primer pair (SEQ ID NO:47 and SEQ ID NO:48) to obtain the NFAT6 fragment 17 with the NdeI restriction site (it is necessary to amplify 6 units of the fragment and eliminate the SalI restriction site at the same time). Fragments 16 and 17 were mixed in equal moles, amplified using a primer pair (SEQ ID NO: 45 and SEQ ID NO: 48), and the fragments were double-digested with EcoRI and KpnI, ligated to the vector pRRLSIN-cPPT.EF-1α-EGFP.WPRE that was also digested with the same enzymes, and the plasmid pRRLSIN-EF1α-EGFP-NFAT6-huIFNβ-PA2 was obtained after sequencing verification. Finally, the plasmid pRRL-EF-1α-92-28Z was double-digested with MluI and SalI to obtain the 92-28Z fragment, which was ligated to the pRRLSIN-EF1α-EGFP-NFAT6-huIFNβ-PA2 vector that was also double-digested with the same enzymes to obtain the plasmid pRRLSIN-EF1α-92-28Z-NFAT6-hu IFNβ-PA2 that was correctly sequenced.

The above five plasmids pRRL-EF-1α-92-BBZ, pRRL-EF-1α-92-28Z, pRRL-EF-1α-92-28BBZ, pRRL-EF-1α-92-28Z-F2A-41BBL and pRRL-EF-1α-92-28Z-NFAT6-huIFNβ-PA2 are collectively referred to as pRRL-EF-1α-92-CAR ( Figure 1 ). The amino acid sequences corresponding to 92-BBZ, 92-28Z, 92-28BBZ and 92-28Z-F2A-41BBL are SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 and SEQ ID NO:52, respectively. The amino acid sequence expressed by 92-28Z-NFAT6-IFN-β comprises two segments, namely, the CAR constructed as 92-28Z as shown in SEQ ID NO:50 and the IFN as shown in SEQ ID NO:53, and its vector construction is shown in FIG1B .

2.2 Virus preparation

1) 293T cells were seeded at a density of 4.5×10 ⁶ in a 10 cm culture dish and cultured overnight at 37°C, 5% CO ₂ to prepare for virus packaging. The culture medium was DMEM supplemented with 10% fetal bovine serum.

2) Dissolve lentiviral shuttle vector pRRL-92-28Z-NFAT6-IFN-β5.2ug and packaging plasmid pRsv-REV 6.2μg, pRRE-PMDLg 6.2μg, and VSVg 2.4μg into 800μL serum-free DMEM culture medium and mix well;

3) Dissolve 60 μg PEI (1 μg/μl) in 800 μl serum-free DMEM medium, mix gently (or vortex at 1000 rpm for 5 seconds), and incubate at room temperature for 5 min;

4) Formation of transfection complex: Add the plasmid mixture to the PEI mixture, vortex or gently mix immediately after addition, and incubate at room temperature for 20 minutes;

5) Add 1.6 ml of the transfection complex dropwise into a 10 cm culture dish containing 11 ml of DMEM medium. After 4-5 hours, replace with fresh medium.

6) After 72 hours, collect the virus supernatant.

2.3 Virus Concentration

1) Preparation of 5X PEG8000 NaCl: weigh 8.766 g NaCl and 50 g PEG8000 and dissolve them in 200 ml Milli-Q pure water. Sterilize with wet heat at 121°C for 30 min. Cool to room temperature and store in a refrigerator at 4°C.

2) Filter the collected viral supernatant using a 0.45 μm filter, add 7.5 ml of 1/4 5X PEG-8000NaCl stock solution, and mix by inverting;

3) Mix once every 20-30 minutes, for a total of 3-5 times;

4) Place at 4℃ overnight;

5) Centrifuge at 4000 g for 60 min at 4°C;

6) After removing the supernatant, add an appropriate amount of AIM V medium (containing 2% AB serum) to dissolve and resuspend the virus precipitate;

7) The concentrated lentiviral suspension is divided into 50 μl portions, stored in finished tubes, and stored at -80°C; monotropic retroviruses are unstable and need to be used as soon as possible after packaging. Cryopreservation at -80°C is not recommended.

2.4 Lentivirus titer determination

293T cells were seeded in 12-well culture plates at a cell number of 1×10 ⁵ ;

The concentrated lentivirus was added to the cell suspension at 1uL, 0.2uL, and 0.04uL, respectively, and polybrene was added to a final concentration of 6ug/mL;

After overnight culture at 37°C, 5% CO ₂ , fresh culture medium was replaced;

72 h after infection, 293T cells were trypsinized, and after adding an equal amount of culture medium, the cells were pipetted evenly and the cell suspension was transferred into a 1.5 mL centrifuge tube;

Centrifuge at 400 g for 5 min, discard the supernatant, and wash once with PBS + 2% FBS solution;

6) Take an appropriate amount of cells, add Biotin-labeled goat anti-human Fab antibody at a dilution ratio of 1:50, and incubate on ice for 30 minutes;

7) After washing once with 1 mL of PBS + 2% FBS solution, PE-labeled streptavidin was added at a dilution ratio of 1:50 and incubated on ice for 30 min;

8) After washing twice with PBS + 2% FBS solution, add an appropriate volume of PBS + 2% FCS solution to resuspend the cells and transfer them to a flow cytometry tube;

9) After flow cytometry detection, it is appropriate to take a cell sample with a positive rate of 5-20% and calculate the titer (TU/mL) = number of cells (10 ⁵ ) × positive rate/virus volume (mL).

2.5 Preparation of lentiviral-transduced T lymphocytes-CAR-T lymphocytes

1) T lymphocyte activation: Human PBMCs were obtained from Shanghai Blood Center. The culture medium was AIM V + 2% AB serum + IL-2 (500 U/mL). The PBMC density was adjusted to 1×10 ⁶ /mL, and magnetic beads coated with anti-human CD3 and CD28 antibodies were added at a ratio of 1:1 for activation for 48 h.

2) Retronectin coated 48-well plate: add 160 μl retronectin solution (5 μg/mL) to each well and incubate at 4°C overnight;

3) Discard the Retronectin solution in the 48-well plate and wash twice with 1 ml PBS;

4) The cells were inoculated in a 48-well plate coated with Retronectin, with 3×10 ⁵ cells per well, lentivirus was added at an MOI of 10, and the culture medium was supplemented to 300 μL;

5) Centrifuge at 32°C, 1800 rpm for 40 min, then transfer to a cell culture incubator and continue culturing for 24 h;

6) Replace with fresh culture medium, adjust the cell density to 5×10 ⁵ /mL, and perform subculturing every 2-3 days.

2.6 Expression of chimeric antigen receptor on T lymphocytes

1) 7 days after infection, take 4×10 ⁵ T cells, centrifuge at 4°C, 400g for 5 min, discard the supernatant, and wash once with PBS + 2% FCS;

2) Add 50 μL PBS + 2% FCS to resuspend the cells, add 1 μL Biotin-labeled goat anti-human Fab antibody, and incubate on ice for 30 min;

3) After washing twice with PBS + 2% FCS, add 50 μL PBS + 2% FCS to resuspend the cells, add 1 μL PE-labeled Streptavidin, and incubate on ice for 30 min;

4) After washing twice with PBS+2% FCS, 400 μL PBS+2% FCS was added to resuspend the cells, and the cells were transferred to a flow cytometer, and the infection efficiency was detected by flow cytometry.

2.7 In vitro toxicity test

Target cells

The target cells corresponding to 92-CAR are SK-HEP-1 (GPC3-) and Huh-7 (GPC3+);

Adjust the target cell concentration to 1×10 ⁶ /mL, and take 100 μL to inoculate into a 96-well plate;

Effector cells: CAR-T cells and control T cells were added to 96-well plates at effector-target ratios of 0.3:1, 1:1, and 3:1;

Each group was set up with 5 replicate wells, and the average value of the 5 replicate wells was taken.

The experimental groups and control groups are as follows:

Each experimental group: each target cell + CTL expressing different chimeric antigen receptors;

Control group 1: maximum release of LDH by target cells;

Control group 2: spontaneous release of LDH by target cells;

Control group 3: effector cells spontaneously released LDH;

Detection method: After the effector cells and target cells were co-cultured for 18 hours, the CytoTox 96 non-radioactive cytotoxicity detection kit (Promega) was used for detection. This method is based on colorimetry and can replace the ⁵¹ Cr release method. The degree of cell lysis is reflected by detecting the content of lactate dehydrogenase (LDH). LDH is a stable cytoplasmic enzyme that is released when cells are lysed, and its release mode is basically the same as the release mode of ⁵¹ Cr in radioactive analysis. The released LDH in the culture medium supernatant can be detected by a 30-minute coupled enzyme reaction, in which LDH can convert a tetrazolium salt (INT) into red formazan. The amount of red product generated is proportional to the number of lysed cells. For details, please refer to the instructions of CytoTox 96 Non-Radioactive Cytotoxicity Detection Kit.

The cytotoxicity calculation formula is:

3. Results

Example 1. Expression of 92-CAR on human T lymphocytes

Human T lymphocytes were stimulated with magnetic beads coated with anti-CD3 and CD28 antibodies for 48 hours and then centrifuged with high titer lentivirus at MOI = 10. The positive rate of lentivirus-infected T lymphocytes was detected by flow cytometry on the 7th day after infection. The infection efficiency of T cells expressing 92-28Z (SEQ ID NO: 15, the encoding nucleotide sequence is shown in SEQ ID NO: 57) (GPC3-CD28Z in Figure 2) was 35.1%, the infection efficiency of T cells expressing 92-28Z-NFAT6-IFN-β (GPC3-CD28Z-IFN in Figure 2) was 19.2%, and the infection efficiency of the control vector MOCK was 49%, as shown in Figure 2.

Example 2. In vitro anti-tumor activity of 92-CAR T cells

After the infection positive rate was detected, the 92-CAR T cells expressing 92-28Z-NFAT6-IFN-β, 92-28Z and empty vector MOCK were used to detect the in vitro killing effect on liver cancer cell lines SK-HEP-1 (GPC3 ^- ) and Huh-7 (GPC3 ⁺ ) and PLC/PRF/5 (GPC3 ⁺ ) at the effect-target ratio of 0.3:1, 1:1, and 3:1, respectively. After co-culture for 18 hours, the LDH content in the supernatant was detected. The results show that the 92-28Z CAR-T cells specifically kill GPC3-positive Huh-7 and PLC/PRF/5 cells, but not GPC3-negative SK-HEP-1 cells; the killing ability of CAR-T cells co-expressing huIFN-β is higher than that of 92-28Z CAR-T cells with the same effect-target ratio, as shown in Table 2.

Table 2. Detection of toxicity and killing of target cells by 92-CAR T cells

The inventors further compared 92-28Z-NFAT6-IFN-β with other GPC3-BBZ, GPC3-28BBZ and GPC3-41BBL (co-expressed 4-1BBL on the basis of CD28Z) CAR-T cells constructed by the same exogenous antigen binding unit 92 (SEQ ID NO: 5). First, FACS detected the expression of various CARs (see Figure 3), and the expression ratio of various CARs was basically around 40%.

Then, the in vitro killing effects of these CAR-T cells and empty vector MOCK T lymphocytes on liver cancer cell lines SK-HEP-1 (GPC3 ^- ) and Huh-7 (GPC3 ⁺ ) and PLC/PRF/5 (GPC3 ⁺ ) were detected at effector-target ratios of 0.3:1, 1:1, and 3:1, respectively. After 18 hours of co-culture, the LDH content in the supernatant was detected. The results show that cells expressing 92CAR-T specifically kill GPC3-positive Huh-7 and PLC/PRF/5 cells, but not GPC3-negative SK-HEP-1 cells. At an effector-target ratio of 1:1, CAR-T cells co-expressing IFN-β have a higher killing ability against two GPC3-positive liver cancer cells than all other CAR-T cells, as shown in Table 3.

Table 3. Toxicity detection of various GPC3 CAR-T cells on target cells

The above studies show that adding exogenously expressed IFN-beta to CD28-Z CAR T cells can enhance anti-tumor activity. Moreover, after co-expression of IFN-beta by CD28Z-expressing CAR-T cells, they have better tumor killing ability than immune cells expressing CD28Z-41BBL in certain effector-target ratios.

Example 3. In vitro cytokine release assay of GPC3 CAR-T cells containing IFN and without IFN

The cytokines released by untransfected T cells, 92-28Z T cells, and 92-28Z-IFN T cells were detected respectively. The three types of T cells that grew well within 1-2 weeks after lentiviral infection were collected and inoculated into 24-well plates at 5×10 ⁴ /200μL (positive cell number). Huh7 cells were inoculated in the same way at 5×10 ⁴ /200μL/24 wells. After incubation with CAR T cells for 24 hours, the supernatant was collected and the concentrations of IFN-β, IFN-γ, and IL-2 were detected. The results are shown in Figures 4A-4C.

According to Figure 4A, IFNβ was expressed only when GPC3-28Z-IFN T cells were co-cultured with Huh7 cells, indicating that after GPC3-28Z-IFN T cells were activated by target antigens, IFNβ could be successfully induced to express and secreted outside the cells. According to the detection of in vitro cytokines in Figures 4B and 4C, the results showed that GPC3-28Z-IFN T cells could be more effectively activated in a variety of GPC3-positive cells, such as Huh7, PLC\PRF\5, Hep-3B and other cells.

Example 4. In vitro cytokine release assay of CLD18A2 CAR-T cells containing IFN and without IFN

Construction of chimeric antigen receptor 85-28Z and 85-2-28Z plasmids:

Using PRRLSIN-cPPT.EF-1α as a vector, lentiviral plasmids expressing the second-generation chimeric antigen receptors 85-28Z (SEQ ID NO: 55) and 85-2-28Z (SEQ ID NO: 54) of antibodies 85 and 85-2 were constructed, respectively. The sequence of 85-28Z consists of CD8α signal peptide, 85scFV, CD8 hinge, CD28 transmembrane region and intracellular signal transduction domain, and CD3ξ intracellular segment; the sequence of 85-2-28Z consists of CD8α signal peptide, hu8E5-2IscFV, CD8 hinge, CD28 transmembrane region and intracellular signal transduction domain, and CD3ξ intracellular segment.

Construction of chimeric antigen receptor 85-28Z-IFN and 85-2-28Z-IFN plasmids:

Based on 85-28Z and 85-2-28Z, 85-28Z-IFNb CAR expressing IFNb cytokine was constructed (the encoding nucleotide sequence is shown in SEQ ID NO: 58), and based on 85-2-28Z CAR, 85-2-28Z-IFNb CAR expressing IFNb cytokine was constructed (the encoding nucleotide sequence is shown in SEQ ID NO: 59).

In order to verify that the constructed 85-28Z T cells and 85-28Z-IFN T cells can also be effectively activated under the stimulation of target cells, we detected the secretion of cytokines by 85-28Z T and 85-28Z-IFN T cells after co-incubation with target cells.

The cytokines released by empty-transfected T cells (Mock), 85-28Z T cells, and 85-28Z-IFN T cells were detected respectively. The three types of T cells that grew well within 1-2 weeks after lentiviral infection were collected and inoculated into 24-well plates at 5×104/200μL (positive cell number), and 5×104/200μL/24-well target cells were inoculated at an effect-target ratio of 1:1. The target cells included 293T-A1, 293T-A2, AGS, AGS-A2, BGC-823, and BGC-823-A2 cells. The supernatant was collected after 24 hours of co-culture. The IFN-γ cytokine released in the supernatant during the co-culture of CAR T lymphocytes and target cells was detected by sandwich ELISA.

The experimental results are shown in Figure 5. The presence of IFN leads to increased secretion of IFN-γ cytokine when 85-28Z CAR T cells are co-incubated with target cells.

Example 5. Cytotoxicity of GPC3 CAR-T (92-28Z) cells with and without IFN

SK-HEP-1 is a GPC3-negative human hepatocellular carcinoma cell line, PLC/PRF/5 is a GPC3-positive human hepatocellular carcinoma cell line, HepG2 is a GPC3-positive human hepatocellular carcinoma cell line, and Hep3B is a GPC3-positive human hepatocellular carcinoma cell line, all of which were purchased from the American Type Culture Collection (ATCC); Huh-7 (also known as Huh7) is a GPC3-positive human hepatocellular carcinoma cell line purchased from the RIKEN Cell Bank in Japan.

Detection method: The CytoTox 96 non-radioactive cytotoxicity detection kit (Promega) was used for detection (the specific method can be referred to the instructions of the CytoTox 96 non-radioactive cytotoxicity detection kit) to detect the in vitro toxic killing effect of CAR T lymphocytes on Huh 7, Hep 3B, PLC/PRF/5, Hep G2 and SK-HEP-1 liver cancer cells.

Untransfected T cells, 92-28Z T cells, and 92-28Z-IFN T cells were co-cultured with tumor cells at effector-target ratios of 1:3, 1:1, and 3:1 for 18 h. The settings of each experimental group and each control group are as follows:

Experimental group settings: each target cell + T lymphocytes expressing different chimeric antigen receptors;

Control group 1: spontaneous LDH release from effector cells;

Control group 2: spontaneous LDH release from target cells;

Control group 3: Maximum LDH release from target cells

Control group 4: volume-corrected control;

Control group 5: culture medium background control.

Calculation of experimental results: The absorbance of all experimental groups, target cell spontaneous LDH release group and effector cell spontaneous LDH release group was subtracted from the mean absorbance of the culture medium background; the absorbance of the target cell maximum LDH release control was subtracted from the mean absorbance of the volume correction control; the corrected values obtained in the above steps were substituted into the following formula to calculate the cytotoxicity (%) generated by each effector-target ratio:

Calculation formula: % cytotoxicity = (experimental group - effector cell spontaneous group - target cell spontaneous group / target cell maximum - target cell spontaneous) * 100

The results are shown in Figures 6A-6E. The above data show that CAR-GPC3 T cells expressing IFN can not only specifically kill GPC3-positive cells, but also improve the killing activity of CAR-T cells expressing IFN (GPC3-28Z-IFN).

Example 6. Cytotoxicity of CLD18A2 CAR-T cells with and without IFN

293T-A1 and 293T-A2 cells are human renal epithelial cell lines stably expressing CLD18A1 and CLD18A2 constructed in vitro. AGS and BGC-823 are human gastric cancer cell lines, and on this basis, AGS-A2 and BGC-823-A2 cell lines stably expressing CLD18A2 were constructed.

Detection method: The CytoTox 96 non-radioactive cytotoxicity detection kit (Promega) was used for detection (the specific method can be referred to the instructions of the CytoTox 96 non-radioactive cytotoxicity detection kit) to detect the in vitro toxic killing effect of CAR T lymphocytes on 293T-A1, 293T-A2, AGS, AGS-A2, BGC-823, and BGC-823-A2 cells.

To compare the killing effects of 85-2-28Z and 85-2-28Z-IFN T cells on target cells, we

Mock T cells, 85-28Z T cells, and 85-28Z-IFN T cells were incubated with CLD18A2-positive 293T-A2, AGS-A2, and BGS-823A2 cells at effector-target ratios of 1:3, 1:1, and 3:1 for 18 h, respectively. CLD18A2-negative 293T-A1, AGS, and BGC-823 cells were used as controls.

Experimental group: each target cell + CAR T expressing different chimeric antigen receptors;

① Spontaneous LDH release from effector cells: Correction of LDH spontaneously released from effector cells;

② Spontaneous LDH release from target cells: Correction of LDH spontaneously released from target cells;

③ Maximum LDH release of target cells: This control is required to determine 100% LDH release during calculation;

④ Volume correction control: correct for volume changes caused by the addition of lysis buffer (10×);

⑤ Culture medium background control: Correct the LDH activity produced by the serum in the culture medium and the background absorption caused by phenol red.

Calculation formula: Cytotoxicity % = [(experimental group – effector cell control – target cell control) / (maximum target cell lysis amount – target cell control)] × 100. Before calculation, effector cell control, target cell control, experimental group minus medium control; maximum target cell lysis amount minus volume control.

In the in vitro toxicity experiment, CAR T lymphocytes expressing Mock, 85-2-28Z, and 85-2-28Z-IFN were co-cultured with tumor cells at effector-target ratios of 1:3, 1:1, and 3:1 for 18 h. The results of the killing activity of the two CART cells against CLD18A2-positive target cells are shown in Figure 7.

Example 7. Determination of GPC3 CAR-T cell survival time in vivo

Referring to the experimental operation of steps 1) to 3) in Example 3, CAR-T (GPC3-28ZT cells or GPC3-28Z-IFN T cells) cells were infused through the tail vein for 7 days, and the survival of CAR-T cells (GPC3-28Z T cells or GPC3-28Z-IFN T cells) in vivo was detected. The results are shown in Table 4. The number of T cells (CD3+) and CAR-T cells per μl of peripheral blood in the GPC3-28Z-IFN T cell group were higher than those in the GPC3-28ZT cell group and the Mock group.

Table 4. Number of surviving T cells in peripheral blood

CD3+ (cells/μL) CAR-T (cells/μL) Mock 193.1453 0 GPC3-28Z 375.802 232.2 GPC3-28Z-IFN 1034.315 439.5

Example 8. Determination of in vivo survival time of CLD18A2 CAR-T cells

Establishment of Gastric Cancer PDX Model:

Gastric cancer PDX tumors of about 2×2×2 mm in size were inoculated subcutaneously in the right axilla of NOD/SCID mice. The day of tumor cell inoculation was designated as day 0.

Adoptive transfer of T cells:

When the tumor volume was 100 mm ³ , 100 mg/kg of cyclophosphamide was injected intraperitoneally. 24 hours later, 1.0×10 ⁷ CAR-T cells (85-2-28Z T cells or 85-2-28Z-IFN T cells) were infused through the tail vein. Mock T cell group was used as control.

Peripheral blood was drawn from the saphenous vein of mice on D5, D7, and D10 days after CAR-T cell infusion to detect the survival of CAR-T cells (empty T cells (Mock), 85-2-28Z T cells, or 85-2-28Z-IFN T cells) in vivo.

The results are shown in Figures 8A-8C. The number of surviving T cells in the 85-2-28Z-IFN T cell treatment group was significantly higher than that in the 85-2-28Z T cell treatment group.

Example 9. In vivo killing activity of GPC3 CAR-T (92-28Z) cells with and without IFN

The anti-tumor therapeutic effect of untransfected T cells (Mock), GPC3-28Z T cells and GPC3-28Z-IFN T cells on Huh7 subcutaneous transplanted tumors was determined.

1) Experimental groups: 21 NOD-SCID mice aged 6-8 weeks were randomly divided into 3 groups, 7 mice in each group, including untransfected T cell group, GPC3-28Z T cell group and GPC3-28Z-IFN T cell group.

2) Inoculation of subcutaneous transplanted tumors: Huh7 cells in logarithmic growth phase and in good growth condition were collected and adjusted to a density of 1×10 ⁷ /mL with physiological saline. The cells were inoculated into NOD-SCID mice to prepare a mouse model. The injection volume was about 200 μL (2×10 ⁶ /mouse). The day of tumor cell inoculation was designated as day 0.

3) Adoptive transfer of T cells: When the tumor volume was 200-300 ^mm3 , 200 mg/kg of cyclophosphamide was injected intraperitoneally. 24 hours later, 1.4× ¹⁰⁷ CAR-T cells (GPC3-28Z T cells or GPC3-28Z-IFN T cells) were infused through the tail vein. The untransfected T cell group was used as a control to observe and measure the growth of subcutaneous transplanted tumors (Figure 9A). When the tumor size of the control group mice reached 2000 ^mm3 , they were killed and the tumors were isolated and photographed (Figure 9B).

The results are shown in Figures 9A and 9B. GPC3-28Z-IFN T cells were able to significantly inhibit tumor cell growth. On the 13th day after CART cell infusion, the tumor inhibition rate of GPC3-28Z CART cells was 66.5%, and the tumor inhibition rate of GPC3-28Z-IFN CART cells was 82.3%, indicating that GPC3-28Z-IFN T cells further enhanced the ability of CAR-T cells to inhibit tumor growth.

Example 10 In vivo killing activity of CLD18A2 CAR-T cells with and without IFN

The anti-tumor therapeutic effect of untransfected T cells (Mock), 85-28Z T cells and 85-2-28Z-IFN T cells on BGC-823-A2 cell subcutaneous transplanted tumors was determined.

1) Inoculation of BGC-823-A2 subcutaneous transplanted tumors: BGC-823-A2 cells in the logarithmic growth phase and in good growth condition were collected and adjusted to a density of 2.5×10 ⁷ /mL with physiological saline. 200 μL of the cell suspension (5×10 ⁶ /mouse) was injected subcutaneously in the right axilla of the mouse. The day of tumor cell inoculation was designated as day 0.

2) Experimental grouping: On the 11th day after tumor inoculation, the volume of BGC-823-A2 transplanted tumors was measured, and NOD-SCID mice were randomly divided into 4 groups, 6 mice in each group, namely, the untransfected T cell group, the 85-28Z T cell group, the 85-2-28Z cell group, and the 85-2-28Z-IFN T cell group.

3) Adoptive transfer of T cells: When the tumor volume was 100-150 mm3 (day ¹¹ ), 100 mg/kg of cyclophosphamide was injected intraperitoneally. 24 hours after the injection, 1× ¹⁰⁷ CAR T cells (Mock cells, 85-28Z T, 85-2-28Z T cells or 85-2-28Z-IFN cells) were infused through the tail vein. At the same time, the untransfected T cell group (Mock group) was used as a control to observe and measure the growth of subcutaneous transplanted tumors.

The results of animal experiments are shown in Figures 10A and 10B, which show that the therapeutic effect of 85-2-28Z-IFN CAR T cells on BGC-823-A2 transplanted tumors is better than that of 85-2-28Z CAR T cells.

Example 11 Anti-tumor test of CLD18A2 CAR-T cells with and without IFN in subcutaneous transplanted tumors of gastric cancer PDX model

The anti-tumor treatment experiment of untransfected T cells (UTD), 85-2-28Z T cells and 85-2-28Z-IFN T cells on subcutaneous transplanted tumors in gastric cancer PDX model was observed.

1) Establishment of gastric cancer PDX model: A gastric cancer PDX tumor mass of about 2×2×2 mm in size was inoculated subcutaneously in the right axilla of 6-8 week-old female NOD/SCID mice. The day of tumor cell inoculation was designated as day 0.

2) Experimental grouping: On D15 of tumor inoculation, NOD-SCID mice were randomly divided into 3 groups, 7 mice in each group, including non-transfected T cell group, 85-2-28Z T cell group and 85-2-28Z-IFN T cell group.

3) Adoptive transfer of T cells: When the tumor volume was 30 mm3, 100 mg/kg of cyclophosphamide was injected intraperitoneally, and 1.0×107 CAR-T cells (85-2-28Z T cells or 85-2-28Z-IFN T cells) were infused through the tail vein 24 hours after the injection. The untransfected T cell group was used as a control. The growth of gastric cancer PDX subcutaneous transplanted tumors was observed and measured.

The results are shown in FIG11 . In the 85-2-28Z-IFN treatment group, the tumor of one of the seven mice completely regressed.

Example 12 Effects of GPC3 CAR-T (92-28Z) cells with and without IFN on tumor infiltration in vivo

Referring to the animal model established in Example 9, 14 days after the two CAR-T cells GPC3-28Z and GPC3-28Z-IFN were reinfused, tumor tissue was taken, and CD3+ cells were detected by histochemistry. The results are shown in Figures 12A and 12B: 4-7 fields of view were taken for each sample, and the number of CD3-positive T cells was counted. The results showed that there were no obvious infiltration of CD3+ cells in the tumor tissue of the control group, and the number of CD3+T cells in the INFβ-CAR-T treatment group was higher than that in the 28ZCART group.

Example 13 Effects of CLD18A2 CAR-T cells with and without IFN on tumor infiltration in vivo

Referring to the animal model established in Example 10, 17 days after the reinfusion of Mock, 85-28Z, 85-2-28Z and 85-2-28Z-IFN cells, tumor tissues were obtained and CD3+ cells were detected by histochemistry.

The results are shown in Figure 13 . Mock T cells had almost no T cell infiltration observed around the tumor tissue, 85-28Z and 85-2-28Z CAR T cells could be seen at the edge of the tumor tissue, and 85-2-28Z-IFN T cells had a certain infiltration observed inside the tumor tissue.

Example 14. Construction of EGFR CAR with and without IFN

The structures of EGFR-CAR (806-28Z, SEQ ID NO: 56) and EGFR-CAR-IFN (encoded by the nucleic acid shown in SEQ ID NO: 60) are shown in FIG. 14 .

The retrovirus packaging system was used to package the EGFR-CAR and EGFR-CAR-IFN into retrovirus and then infected mouse T lymphocytes. The infection positive rates were 65.1% and 35.2%, respectively ( FIG. 15 ).

Example 15. Determination of the ability of EGFR CAR (806-28Z) T cells containing IFN and without IFN to secrete mIFNβ in vitro

In order to detect the function of EGFR-CAR-IFN inducing the secretion of mIFNβ, we co-cultured CAR-T cells with target cells CT26-VIII at 1:1 and 3:1 for 24 hours, took the supernatant, and detected the expression of mIFNβ by ELISA. At the same time, CT26 cells without targets were used as negative controls, and concanavalin A (ConA) was used as positive controls. The results showed that after stimulation of target cells, mCAR-806-mIFNβ could be successfully activated and induced to express mIFNβ, while no expression of mIFNβ was detected in the control group (Figure 16).

Example 16. Cytokine release by EGFR CAR (806-28Z)-T cells containing IFN and without IFN

In order to verify that the constructed EGFRCAR and EGFR-CAR-IFN can also be effectively activated under the stimulation of target cells, we detected the secretion of muCAR-T cytokines after co-incubation with target cells. Untransfected UT cells, EGFR-CAR and EGFR-CAR-IFN were co-incubated with target-positive CT26-VIII cells at a ratio of 1:1 for 24 hours, and the culture supernatant was taken to detect the secretion of cytokines mIL-2, mIFN-γ, and mTNF-α. Target-negative CT26 cells were used as controls. The results showed that EGFR-CAR and egfr-CAR-IFN secreted high concentrations of mIL-2, mIFN-γ, and mTNF-α after co-incubation with target cells (Figures 17A, 17B, and 17C), indicating that EGFR-CAR and EGFR-CAR-IFN can effectively activate both muCAR-T cells under the stimulation of target antigens.

Example 17 In vitro toxicity test of EGFR CAR (806-28Z)-T cells with and without IFN

To compare the in vitro killing activity of EGFR-CAR and EGFR-CAR-IFN on target cells, we co-incubated EGFR-CAR and EGFR-CAR-IFN with EGFR-positive CT26VIII cells at a ratio of 1:3, 1:1, or 3:1 for 18 h, respectively. Untransfected mouse UT cells were used as isotype controls, and CT26 was used as an EGFR-negative control.

The results showed that compared with UT cells, EGFR-CAR and EGFR-CAR-IFN had a strong killing effect on target-positive CT26VIII cells, and the difference was significant (***P < 0.001). The killing percentage was dose-dependent, while untransfected UT cells had no killing effect on CT26 and CT26VIII. Both EGFR-CAR and EGFR-CAR-IFN CAR-T cells had no killing effect on target-negative CT26 cells (Figure 18).

Example 18 In vivo toxicity test of EGFR CAR (806-28Z)-T cells with and without IFN

Using the mouse colon cancer CT26 cell line stably transfected with the human EGFRvIII-806 site, subcutaneous transplanted tumors were inoculated in Babl/c mice, and then EGFR-CAR T cells were given. The results are shown in Figure 19. The tumor size of the EGFR-CAR-T cells was basically the same as that of the control group, and no inhibitory effect was observed. After the EGFR-CAR-IFN cells were reinfused, the phenomenon of tumor growth inhibition began to appear on the 7th day, with a tumor inhibition rate of 5.9%. The strongest rate was 18.5% on the 10th day. By the 17th day, the tumor inhibition rate could still reach 12.4%, which was significantly better than the EGFR-CAR-T cell group.

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Table 5. Sequences used in this article

Claims (22)

1. An antibody that binds to CLD18A2, characterized in that the antibody has the amino acid sequence shown at positions 1-247 in SEQ ID NO: 54 or 55. 2. A receptor comprising the antibody according to claim 1. 3. The receptor according to claim 2, characterized in that the receptor comprises: the antibody according to claim 1, the transmembrane region and the intracellular signal region connected in sequence. 4. The receptor according to claim 3, characterized in that the intracellular signal region comprises: a T cell stimulatory signal molecule or a combination of a T cell stimulatory signal molecule and a T cell activation co-stimulatory molecule. 5. The receptor according to claim 3 or 4, wherein the intracellular signaling region is selected from the group consisting of: TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b and CD66d; or the co-stimulatory domain is selected from the group consisting of: intracellular signaling regions of CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a/CD18), 4-1BBL, MyD88 and 4-1BB, or a combination thereof. 6. The receptor according to any one of claims 3 to 5, wherein the transmembrane region is selected from the transmembrane region of CD8 or CD28 or a protein having at least 85, 90, 95, 96, 97, 98, 99 or 100% identity with the natural transmembrane region of CD8 or CD28. 7. The receptor according to any one of claims 2 to 6, characterized in that the intracellular signal region is selected from: CD3ζ, or the intracellular signal region of CD137 and CD3ζ, or CD28 and CD3ζ, or CD28, CD137 and CD3Z. 8. A nucleic acid encoding the antibody of claim 1 or the receptor of any one of claims 2 to 7. 9. A vector, characterized in that the vector comprises the nucleic acid according to claim 8. 10. The vector according to claim 9, which is an expression vector. 11. A virus comprising the nucleic acid of claim 8 or the vector of claim 9 or 10. 12. An engineered cell, characterized in that the engineered cell comprises the antibody according to claim 1, the receptor according to any one of claims 2 to 7, the nucleic acid according to claim 8, and/or the vector according to claim 9 or 10; or the cell is prepared by viral transduction according to claim 11. 13. The engineered cell of claim 12, wherein the engineered cell is an immune response cell. 14. The engineered cell according to claim 12 or 13, characterized in that the engineered cell is a T cell, a natural killer cell, a cytotoxic T lymphocyte, a natural killer T cell, a DNT cell, and/or a regulatory T cell. 15. The engineered cell according to any one of claims 12 to 14, characterized in that the engineered cell further carries a coding sequence of an exogenous cytokine; or further expresses another receptor that binds to an antigen; or further expresses a chemokine receptor; or further expresses an siRNA that can reduce the expression of PD-1 or a protein that blocks PD-L1; or further expresses a safety switch. 16. The engineered cell according to any one of claims 12 to 14, characterized in that the engineered cell also constitutively expresses or inducibly expresses exogenous interferon β. 17. A pharmaceutical composition, characterized in that it comprises the antibody of claim 1, the receptor of any one of claims 2-7, the nucleic acid of claim 8, the vector of claim 9 or 10, the virus of claim 11, and/or the engineered cell of any one of claims 12-16, and a pharmaceutically acceptable carrier or excipient. 18. Use of the antibody according to claim 1, the receptor according to any one of claims 2 to 7, the nucleic acid according to claim 8, the vector according to claim 9 or 10, the virus according to claim 11, the engineered cell according to any one of claims 12 to 16, and/or the pharmaceutical composition according to claim 17 in the preparation of a drug for treating a disease. 19. The use according to claim 18, characterized in that the drug is used for treating or preventing tumors, pathogen infection, enhancing immune tolerance, autologous transplantation or allogeneic transplantation. 20. The use according to claim 18 or 19, characterized in that the drug is used to induce cancer cell death. 21. The use according to any one of claims 18 to 20, characterized in that the drug is used to induce CLD18A2-positive cell death. 22. The use according to claim 19, characterized in that the tumor is selected from: pancreatic cancer, liver cancer, lung cancer, gastric cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, kidney cancer, leukemia, myeloma, ovarian cancer, cervical cancer, or glioma.