STRENGTHENED RECEPTOR FOR IMPROVING IMMUNE CELL FUNCTION

Abstract

The present invention provides an enhanced receptor for improving the function of an immune cell, and a composition and a cell related thereto. The enhanced receptor is a transmembrane protein, which is a fusion protein consisting of an extracellular domain and an intracellular domain, wherein the extracellular domain is capable of binding to a target cell and activating the signaling function of the intracellular domain, thereby increasing the activation level of the immune cell, and overcoming the inhibition of the target cell microenvironment on the immune cell, thus enhancing the effect of immunotherapy.

Claims

1. An enhanced receptor for improving the function of an immune cell comprising an enhanced transmembrane receptor protein expressed across a membrane of the immune cell, wherein the receptor protein comprises an extracellular domain (ECD) and an intracellular domain (ICD).

2. The enhanced receptor of claim 1, wherein the immune cell is any one of a T cell, a NK cell, a B cell, or a macrophage.

3. The enhanced receptor of claim 1, wherein the intracellular domain (ICD) comprises a costimulatory molecule that elicits an immune cell activating signal, and the ICD is capable of eliciting an immune cell activation signal.

4. The enhanced receptor of claim 1, wherein the intracellular domain (ICD) comprises a costimulatory molecule.

5. The enhanced receptor of claim 1, wherein the extracellular domain (ECD) (i) is capable of binding to a target cell of the immune cell; (ii) is a receptor, a ligand, or an antibody of a membrane protein of the target cell of the immune cell; or (iii) is a complete sequence structure or a portion comprising a binding domain thereof of any substance that is capable of binding to the target cell.

6. The enhanced receptor of claim 1, wherein the extracellular domain (ECD) comprises any one of PD1, CTLA4, NKG2D, SIRPα, LAG3, or other T cell surface protein.

7. The enhanced receptor of claim 1, wherein the extracellular domain (ECD) is an anti-PDL1 monoclonal antibody, an anti-B7 monoclonal antibody, an anti-HER2 monoclonal antibody, an anti-EGFR monoclonal antibody, an anti-CD47 monoclonal antibody, other antibody capable of binding to a membrane protein of a target cell, or a partial structure of said antibodies.

8. The enhanced receptor of claim 1, wherein: 1) the ECD is PD1, and the ICD is CD28, 41BBz or ICOS, in which the sequences of the corresponding enhanced receptors are as set forth in SEQ ID NO: 1 (PD1/CD28), SEQ ID NO: 2 (PD1/41BBz), and SEQ ID NO: 3 (PD1/ICOS), respectively; 2) the ECD is an anti-PDL1 monoclonal antibody, and the ICD is CD28, 41BBz or ICOS, in which the sequences of the corresponding enhanced receptors are as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; 3) the ECD is an anti-HER2 monoclonal antibody K4D5-628, K4D5-728 or K4D5-828, and the ICD is CD28, 41BBz or ICOS, in which the sequences of the corresponding enhanced receptors are as set forth in SEQ ID NO: 7 (K4D5-628/CD28), SEQ ID NO: 8 (K4D5-728/CD28), SEQ ID NO: 9 (K4D5-828/CD28), SEQ ID NO: 10 (K4D5-628/41BBz), SEQ ID NO: 11 (K4D5-728/41BBz), SEQ ID NO: 12 (K4D5-828/41BBz), SEQ ID NO: 13 (K4D5-628/ICOS), SEQ ID NO: 14 (K4D5-728/ICOS), and SEQ ID NO: 15 (K4D5-828/ICOS), respectively; 4) the ECD is NKG2D, and the ICD is CD28, 41BBz or ICOS, in which the sequences of the corresponding enhanced receptors are as set forth in SEQ ID NO: 16 (NKG2D/CD28), SEQ ID NO: 17 (NKG2D/41BBz), and SEQ ID NO: 18 (NKG2D/ICOS), respectively; 5) the ECD is an anti-CD47 monoclonal antibody, and the ICD is CD28, 41BBz or ICOS, in which the sequences of the corresponding enhanced receptors are as set forth in SEQ ID NO: 19 (CD47 monoclonal antibody/CD28), SEQ ID NO: 20 (CD47 monoclonal antibody/41BBz), and SEQ ID NO: 21 (CD47 monoclonal antibody/ICOS), respectively; or 6) the ECD is SIRPα, and the ICD is CD28, 41BBz or ICOS, in which the sequences of the corresponding enhanced receptors are as set forth in SEQ ID NO: 22 (SIRPα/CD28), SEQ ID NO: 23 (SIRPα/41BBz), and SEQ ID NO: 24 (SIRPα/ICOS), respectively.

9. The enhanced receptor of claim 1, wherein a target cell of the immune cell is a cell harmful to a human body.

10. The enhanced receptor of claim 1, wherein a target cell of the immune cell is a cell harmless or harmless in a short period of time to a human body.

11. A composition comprising (i) the enhanced receptor of claim 1, and (ii) a naturally unmodified T cell receptor (TCR), an artificially modified engineered TCR, or a CAR (chimeric antigen receptor).

12. An engineered T cell comprising a T cell concurrently expressing a naturally unmodified TCR, an artificially modified TCR or a CAR, and the enhanced receptor of claim 1, wherein the immune cell is the engineered T cell.

13. The engineered T cell of claim 12, wherein the intracellular domain (ICD) comprises a costimulatory molecule that elicits an immune cell activating signal, and the ICD is capable of eliciting an immune cell activation signal, and the immune cell activation signal is activated by a binding of the ECD of the enhanced receptor to a membrane protein of a target cell, thereby activating a function of the engineered T cell, so that the engineered T cell is capable of resisting an inhibition of the target cell microenvironment on the engineered T cell.

14. The engineered T cell of claim 13, wherein the inhibition of the target cell microenvironment on the engineered T cell is derived from any one or more of PDL1/2, CTLA4, TIM3, TIGIT, or PDGFβ.

15. The engineered T cell of claim 12, wherein the engineered T cell (1) is a single clone characterized by the naturally modified TCR, the artificially modified TCR or the CAR capable of recognizing only a single target, or (2) is a mixture of a plurality of T cell clones capable of recognizing multiple targets.

16. The engineered T cell of claim 12, wherein the T cell is derived from tumor-infiltrating T lymphocytes (TILs), and is positive or negative for one or more markers.

17. The engineered T cell of claim 12, wherein the T cell is derived from T cells that are from peripheral blood mononuclear cells (PBMCs) and positive or negative for one or more markers.

18. The engineered T cell of claim 12, wherein the engineered T cell has dual recognition functions, that is, a first target cell of the engineered T cell is a cell harmful to a human body, a second target cell of the engineered T cell is a cell harmless or harmless in a short period of time to a human body, the first target cell is recognized by the naturally unmodified TCR, the artificially modified TCR or the CAR, and concurrently the second target cell is recognized by the naturally unmodified TCR, the artificially modified TCR or the CAR.

19. The engineered T cell of claim 18, wherein the engineered T cell has dual recognition functions, that is, the engineered T cell is capable of recognizing and killing the first target cell to achieve disease treatment, and the engineered T cell is capable of being activated by the second target cell to expand to enhance efficacy.

20. The engineered T cell of claim 12, wherein the engineered T cell further comprises other gene modifications.

21. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0159] FIG. 1 shows an in vitro experiment comparing the improvement on T cell function (IFNγ secretion) of various enhanced receptors.

[0160] FIG. 2 shows an in vitro experiment comparing the improvement on T cell function (IL2 secretion) of various enhanced receptors.

[0161] FIG. 3 shows an animal experiment comparing the improvement on tumor inhibition by T cells of various enhanced receptors.

DETAILED DESCRIPTION OF EMBODIMENTS

[0162] For a more complete understanding and application of the present invention, the present invention will be described in detail hereinafter with reference to the examples and the accompanying drawings, which examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The scope of the present invention is specifically defined by the appended claims.

Example 1. Related Example of Enhanced Receptor-PD1/CD28

[0163] In this example, an enhanced receptor consisting of PD1 as the extracellular segment and CD28 as the intracellular segment, represented by PD1/CD28 or ER (Enhanced Receptor), was constructed and tested.

[0164] Preparation of Lentiviral Vector for PD1/CD28 Enhanced Receptor

[0165] The lentiviral vector for PD1/CD28 enhanced receptor (or referred to as Switch for short hereinafter) employed a safer fourth-generation lentiviral vector system. The main vector PD1/CD28, packaging vector pMDL-gag, Rev, and envelope vector pMD2.G were co-transfected with calcium phosphate or liposome PEI into 293T cells, the supernatant was collected after 48 hours, and the lentiviruses were concentrated by ultracentrifugation.

[0166] Titer detection of the lentiviral vector for PD1/CD28 enhanced receptor: Using 3-fold dilution, 293T cells were infected with 50 ul dilution for 48 hours to 72 hours, 293T cells were collected for PD1 staining, the ratio of PD1+ cells was analysed by flow cytometry, and the titer was calculated according to the following formula:

Titer (TU/ml)=40000-45000(the number of starting 293T cells)*% PD1.sup.+ cells*fold of dilution*20(calculated by taking the first ratio of PD1.sup.+ cells<20%).

[0167] Similarly, lentiviral vectors comprising the following gene sequences were constructed: [0168] 1) non-fusion expression of PD1/CD28 and an artificial TCR targeting tumor target NY-ESO-1, referred to as ER-TCR for short; and [0169] 2) non-fusion expression of PD1/CD28 and a CAR targeting CD19, referred to as ER-CAR for short.

[0170] The titer of each lentiviral vector was calculated, and those with a titer of >3×10.sup.7 can be used for the next step.

[0171] Construction of Other Groups of Lentiviral Expression Vectors

[0172] Similar to the above steps, lentiviral vectors loaded with the following gene sequences were constructed, respectively: [0173] 1) an artificial TCR targeting tumor target NY-ESO-1; [0174] 2) a CAR targeting CD19; [0175] 4) non-fusion expression of PD1/CD28 and an artificial TCR targeting tumor target NY-ESO-1, referred to as ER-TCR for short; and [0176] 5) non-fusion expression of PD1/CD28 and a CAR targeting CD19, referred to as ER-CAR for short.

[0177] Preparation of Several Groups of Tumor-Recognizing T Cells

[0178] Four kinds of tumor-recognizing T cells were prepared as follows.

[0179] Preparation of TCR-T Cells (NY-ESO-1-TCR-T or TCR-T) Targeting Tumor Target NY-ESO-1

[0180] The patient's leukocytes with tumor cells being NY-ESO-1 positive and of HLA-A:0201 type were collected by apheresis, PBMCs were separated with Ficoll lymphocyte separation solution, and allowed to grow adherently for 2 hours, and the suspension cells-mainly T cells-were obtained. TCR lentiviral vectors recognizing NY-ESO-1 were added, lentiviruses were added at MOI (number of lentiviruses/number of cells)=1, and TCR-T cells were cultured and expanded. The expression rate of TCR was 68.7% by flow cytometry.

[0181] At MOI=1, ER lentiviruses were added to the TCR-T to obtain ER-TCR-T.

[0182] Preparation of CAR-T Cells (CD19 CAR-T or CAR-T) Targeting CD19 and CAR-T (ER-CAR-T) Transfected with Enhanced Receptor

[0183] The patient's leukocytes were collected by apheresis, PBMCs were separated with Ficoll lymphocyte separation solution, and allowed to grow adherently for 2 hours, and the suspension cells-mainly T cells-were obtained. CAR lentiviral vectors recognizing CD19 were added, lentiviruses were added at MOI (number of lentiviruses/number of cells)=1, and CAR-T cells were cultured and expanded.

[0184] At MOI=1, ER lentiviruses were added to the CAR-T to obtain ER-CAR-T.

[0185] Preparation of TIL Cells from Fresh Tumor Tissues and TIL (ER-TIL) Transfected with Enhanced Receptor

[0186] Cells of tumor tissues were isolated from 10 g or more of the patient's tumor tissues by enzymatic digestion, CD3-positive T cells-TIL-were separated and purified with CD3 magnetic beads, and the remaining cells were cultured adherently to obtain tumor cells autologous to the patient.

[0187] At MOI=1, ER lentiviruses were added to the TIL to obtain ER-TIL.

[0188] Preparation of Neoantigen-Reactive T Cells and neoT (ER-neoT) Transfected with Enhanced Receptor

[0189] Firstly, whole-exomic sequencing or RNA transcriptome sequencing was performed on the patient's peripheral blood or surgically removed tumor tissues, the mutation sites were selected, affinity peptide prediction was made in combination with the HLA typing of the patient, and about 20 mutations were selected as Neoantigen. The Neoantigen was genetically synthesized, and was transcribed into RNA in vitro. Again, the patient's peripheral blood was collected by apheresis or directly drawn, PBMCs were isolated and grown adherently for 2 hours. To the adherent mononuclear cells, related cytokines were added to promote the differentiation and maturation of DC cells, and the RNA was transferred into the cells by electroporation. The suspension cells-mainly T cells-were co-cultured with the DC cells transferred by electroporation, CD137+-positive T cells were isolated to obtain neoantigen-reactive T cells (or referred to as neoT for short hereinafter), and the cells were expanded.

[0190] At MOI=1, ER lentiviruses were added to the neoT to obtain ER-neoT.

[0191] In Vitro Functional Experiment of PD1/CD28 Enhanced Receptor in TCR-T Targeting NY-ESO-1

[0192] In order to verify the role of PD1/CD28 enhanced receptor in the TCR-T targeting NY-ESO-1, a tumor cell line, J82-NY-ESO-1-PDL1, with its HLA typing as A:0201 was firstly constructed. Lentiviruses carrying PDL1 and NY-ESO-1 were added to J82 cells at MOI=5. After 72 h, G418 and Puromycin were added to screen positive cells. After about 2 weeks, the expression of PDL1 and NY-ESO-1 was detected by flow cytometry. The flow cytometry results showed that up to 95% of J82 cells expressed PDL1 and NY-ESO-1 at the same time, indicating that the cell construction was successful.

[0193] In order to verify the function of ER, the expression of CD107a and cytokine secretion of T cells were detected by co-culturing tumor cells and T cells. As shown in FIG. 6a, the expression of CD107a in the ER-TCR-T cells was higher than that in ordinary TCR-T. The secretion of IFN-γ and IL-2 shown in the ER-TCR-T was also higher than that in TCR-T. The results of the in vitro experiment showed that the ability of TCR-T to kill tumors is significantly improved after transfection with ER.

[0194] In Vitro Functional Experiment of TIL Expressing PD1/CD28 Enhanced Receptor

[0195] In order to verify the role of Switch in the TIL, tumor cells isolated from the patient's fresh tumor tissues were co-cultured with the TIL and cells for 24 hours, respectively. The secretion of IFN-γ and IL-2 was detected by ELISA. The results showed that the killing ability of the ER-TIL was significantly higher than that of TIL not transfected with the enhanced receptor. The results of the in vitro experiment showed that the ability of TIL to kill tumors is significantly improved after transfection with ER.

[0196] In Vitro Functional Experiment of Neoantigen-Reactive T Cells Expressing PD1/CD28 Enhanced Receptor

[0197] In order to verify the role of Switch in the neoT, tumor cells isolated from the patient's fresh tumor tissues were co-cultured with the neoT and Switch-neoT cells for 24 hours, respectively. The secretion of IFN-γ and IL-2 was detected by ELISA. The results showed that the killing ability of the ER-neoT was significantly higher than that of neoT not transfected with the enhanced receptor. The results of the in vitro experiment showed that the ability of neoT to kill tumors is significantly improved after transfection with ER.

[0198] In Vitro Functional Experiment of CAR-T Cells Expressing PD1/CD28 Enhanced Receptor

[0199] A tumor cell line expressing CD19 was firstly constructed. The tumor cells were co-cultured with ordinary CD19CAR-T cells not transfected with the enhanced receptor and the ER-CAR-T for 24 hours, respectively. The results of the secretion of IFN-γ and IL-2 by ELISA showed that the killing ability of the ER-CAR-T is significantly higher than that of ordinary CAR-T not transfected with the enhanced receptor.

[0200] Animal Experiment of NY-ESO-1 TCR-T Cells Expressing PD1/CD28 Enhanced Receptor

[0201] Inoculation: 1×10.sup.6 J82-NY-ESO-1-PDL1 tumor cells were subcutaneously inoculated into NSG mice. The blank control group was subcutaneously injected with PBS (A0). The mice injected with tumor cells began to form tumors about two weeks later. The tumor size was measured on Day 23, and 30 mice were selected for recruitment.

[0202] Administration: The blank control group (A0) was injected with PBS via tail vein. The mice inoculated with tumor cells were divided into 5 groups: no drug-PBS group (A1); T cell group (A2); ER-T cell group (A3); TCR-T cell group (A4); and 5. ER-TCR-T cell group (A5), respectively. The administrations were all performed by intravenous infusion of 1×10.sup.7 cells via tail vein.

[0203] After administration, the tumor size was measured every 2-3 days and the mouse state was observed. Tumor volume=½*long diameter*short diameter*short diameter. The observation was continued for 30 days.

[0204] Change in Tumor Burden:

[0205] The tumor volume of tumor-bearing mice continued to increase one week after administration. On Day 7-12, the tumors in A4 and A5 groups were detected to begin to shrink, but the tumors in A4 group began to rebound on Day 15-25, while the tumors in A5 group continued to shrink to almost disappear. The other three groups of tumors maintained natural growth.

[0206] Change in Total Amount of Infused T Cells In Vivo:

[0207] In the three groups of administration, on Day 10 after administration, one mouse was randomly selected from groups 3, 4 and 5, tumor tissues were removed, T cells (i.e., TIL) were isolated therefrom and counted, and the TIL density, that is, the total number of TIL per unit weight of tumor tissue, was calculated. The results showed that the TIL density in the tumors of A5 group was far higher than that of other groups.

[0208] Animal Experiment of CAR-T Cells Expressing PD1/CD28 Enhanced Receptor

[0209] Inoculation: 1×10.sup.6 CD19-expressing tumor cells were subcutaneously inoculated into NSG mice. The blank control group was subcutaneously injected with PBS (A0). The mice injected with tumor cells began to form tumors about two weeks later. The tumor size was measured on Day 23, and 30 mice were selected for recruitment.

[0210] Administration: The blank control group (A0) was injected with PBS via tail vein. The mice inoculated with tumor cells were divided into 5 groups: no drug-PBS group (A1); T cell group (A2); ER-T cell group (A3); CAR-T cell group (A4); and ER-CAR-T cell group (A5), respectively. The administrations were all performed by intravenous infusion of 1×10.sup.7 cells via tail vein.

[0211] After administration, the tumor size was measured every 2-3 days and the mouse state was observed. Tumor volume=½*long diameter*short diameter*short diameter. The observation was continued for 30 days.

[0212] Change in Tumor Burden:

[0213] The tumor volume of tumor-bearing mice in each group continued to increase one week after administration. The trend of A1-3 groups was basically the same, and the tumor volume continued to increase, until death occurred on Day 30-40. The tumors in A4 and A5 groups were detected to begin to shrink from Week 2, but the tumors in A4 group began to rebound in Week 3-4, while the tumors in A5 group continued to shrink to almost disappear.

[0214] Change in Total Amount of Infused T Cells In Vivo:

[0215] In the three groups of administration, on Day 10 after administration, one mouse was randomly selected from groups 3, 4 and 5, tumor tissues were removed, T cells (i.e., TIL) were isolated therefrom and counted, and the TIL density, that is, the total number of TIL per unit weight of tumor tissue, was calculated. The results showed that the TIL density in the tumors of A5 group was far higher than that of other groups.

Example 2. Related Example of Enhanced Receptor-PD1/41BBz

[0216] In this example, an enhanced receptor consisting of PD1 as the extracellular segment and 41BBz as the intracellular segment, represented by PD1/41BBz or ER (Enhanced Receptor), was constructed and tested. The difference between the enhanced receptor of this example and the enhanced receptor of Example 1 was that the costimulatory molecules of the intracellular segment were different.

[0217] The experimental design was the same as that of Example 1, and the only difference was that the enhanced receptors were different.

[0218] In Vitro Functional Experiment of NY-ESO-1-Targeting TCR-T Expressing PD1/41BBz Enhanced Receptor

[0219] The experimental results showed that the expression of CD107a, and the secretion of IFN-γ and IL-2 in the ER-TCR-T cells were both higher than those in ordinary TCR-T cells not transfected with the enhanced receptor.

[0220] In Vitro Functional Experiment of TIL Expressing PD1/41BBz Enhanced Receptor

[0221] The results of the in vitro experiment showed that the ability of TIL to kill tumors is significantly improved after transfection with ER.

[0222] In Vitro Functional Experiment of Neoantigen-Reactive T Cells Expressing PD1/41BBz Enhanced Receptor

[0223] The results showed that the killing ability of the ER-neoT is significantly higher than that of neoT.

[0224] In Vitro Functional Experiment of CAR-T Cells Expressing PD1/41BBz Enhanced Receptor

[0225] The results showed that the killing ability of the ER-CAR-T is significantly higher than that of ordinary CAR-T not transfected with the enhanced receptor.

[0226] Animal Experiment of NY-ESO-1 TCR-T Cells Expressing PD1/41BBz Enhanced Receptor

[0227] The experimental design was exactly the same as that of the TCR-T animal experiment of Example 1, and the only difference was that the enhanced receptors used were different. The experimental conclusions were also similar to those of the TCR-T animal experiment of Example 1, that is, in the ER-TCR-T group, the tumors were eliminated more thoroughly than those in the TCR-T group not transfected with the enhanced receptor (reflecting the property of the ER to enhance activation), the TIL density in the tumor tissue was higher than that of other groups (reflecting that the ER can help TCR-T cells to better recruit into tumors), and the tumors in the non-tumor-recognizing T cell (ER-T) group transfected with the enhanced receptor were not killed (reflecting the safety of the ER, that is, when T cells do not recognize the target cells, the ER will not independently kill PDL1-positive target cells).

[0228] Animal Experiment of CD19 CAR-T Cells Expressing PD1/41BBz Enhanced Receptor

[0229] The experimental design was exactly the same as that of the CAR-T animal experiment of Example 1, and the only difference was that the enhanced receptors used were different. The experimental conclusions were also similar to those of the CAR-T animal experiment of Example 1, that is, in the ER-CAR-T group, the tumors were eliminated more thoroughly than those in the CAR-T group not transfected with the enhanced receptor (reflecting the property of the ER to enhance activation), the TIL density in the tumor tissue was higher than that of other groups (reflecting that the ER can help CAR-T cells to better recruit into tumors), and the tumors in the non-tumor-recognizing T cell (ER-T) group transfected with the enhanced receptor were not killed (reflecting the safety of the ER, that is, when T cells do not recognize the target cells, the ER will not independently kill PDL1-positive target cells).

Example 3. Related Example of Enhanced Receptor-PD1/ICOS

[0230] In this example, an enhanced receptor consisting of PD1 as the extracellular segment and ICOS as the intracellular segment, represented by PD1/ICOS or ER (Enhanced Receptor), was constructed and tested. The difference between the enhanced receptor of this example and the enhanced receptor of Example 1 was that the costimulatory molecules of the intracellular segment were different.

[0231] The experimental design was the same as that of Example 1, and the only difference was that the enhanced receptors were different.

[0232] In the in vitro experiment, the experimental conclusion was also similar to that of Example 1, that is, as compared to natural unmodified TCR-T, CAR-T, TIL and neoT, all of the TCR-T, CAR-T, TIL and neoT expressing the ER showed a stronger killing response to target cells, respectively.

[0233] In the animal experiment, the experimental conclusions were also similar to those of Example 1, that is, in the TCR-T and CAR-T groups expressing the ER, the tumors were eliminated more thoroughly than those in the TCR-T and CAR-T groups not transfected with the enhanced receptor (reflecting the property of the ER to enhance activation), the TIL density in the tumor tissue was higher than that of other groups (reflecting that the ER can help TCR-T and CAR-T cells to better recruit into tumors), and the tumors in the non-tumor-recognizing T cell (ER-T) group transfected with the enhanced receptor were not killed (reflecting the safety of the ER, that is, when T cells do not recognize the target cells, the ER will not independently kill PDL1-positive target cells).

Example 4. Related Example of Enhanced Receptor-Anti-HER2 Monoclonal Antibody/CD28

[0234] In this example, an enhanced receptor consisting of an anti-HER2 monoclonal antibody (referred to as aHER2mAb for short) as the extracellular segment and CD28 as the intracellular segment was constructed and tested. K4D5 was usually used to represent the antibody Fab segment of HER2. This first enhanced receptor was represented by aHER2mAb/CD28 or K4D5/CD28.

[0235] Amino Acid Sequences of Three Anti-HER2 Antibodies K4D5 with Different Affinities

[0236] K4D5-6.28, K4D5-7.28 and K4D5-8.28 were three antibody Fab segments that specifically recognize HER2 from low to high affinity. K4D5-7.28 and K4D5-6.28 only differed in the amino acid residue at position 76, while K4D5-8.28 and K4D5-6.28 only differed in the amino acid residue at position 250.

[0237] Construction of Three K4D5/CD28 and K4D5/CD28-CD19 CAR Lentiviral Expression Plasmids

[0238] Three K4D5/CD28 and K4D5/CD28-CD19 CAR lentiviral expression plasmids were constructed. K4D5/CD28 and CD19 CAR were non-fusion expression.

[0239] Preparation of K4D5/CD28 and K4D5/CD28-CD19 CAR Lentiviral Vectors

[0240] The three K4D5/CD28 and K4D5/CD28-CD19 CAR lentiviral vectors employed a safer fourth-generation lentiviral vector system. The main vector K4D5/CD28 or K4D5/CD28-CD19 CAR, packaging vector pMDL-gag, Rev, and envelope vector pMD2.G in each group were co-transfected with lipofectamine 2000 into 293T cells, the supernatant was collected after 48 hours, and the lentiviruses were concentrated by ultracentrifugation.

[0241] Titer detection of the three K4D5/CD28 and K4D5/CD28-CD19 CAR lentiviral vectors: Using 3-fold dilution, 293T cells were infected with 50 ul dilution for 48 hours to 72 hours, 293T cells were collected and labeled and stained with HER2 recombinant protein, the ratio of HER2-positive cells was analysed by flow cytometry, and the titer was calculated according to the following formula:

[0242] Titer (TU/ml)=number of starting 293T cells*% HER2.sup.+ cells*fold of dilution*20 (calculated by taking the first ratio of HER2.sup.+ cells <20%). Taking K4D5-8.28/CD28 as an example, the titer of each lentiviral vector in each group was calculated, and those with a titer of >1×10.sup.7 can be used for the next step.

[0243] Preparation of CAR-T Cells Transfected with K4D5/CD28 Enhanced Receptor

[0244] 30 ml peripheral blood from a healthy individual was collected via vein, PBMCs were separated with Ficoll lymphocyte separation solution, and allowed to grow adherently for 2 hours, and the suspension cells-mainly T cells-were obtained. Three groups of K4D5/CD28-CD19 CAR lentiviral vectors with different affinities were added, respectively; and unmodified CD19 CAR without the enhanced receptor was added as control to another group of T cells, respectively. Lentiviruses were added at MOI (number of lentiviruses/number of cells)=2, and enhanced CAR-T cells were cultured and expanded.

[0245] Detection of K4D5/CD28 Function by OKT3/HER2 Protein Costimulation

[0246] In order to verify the function of K4D5/CD28, T cells were firstly activated by OKT3 and HER2 protein costimulation, and the secretion of cytokines was detected by ELISA. A 96-well plate was co-coated with HER2 protein and OKT3 protein, and then T cells in each group were inoculated into the 96-well plate at a density of 1×10{circumflex over ( )}5 cells/well. After 48 hours of culture, the culture supernatant was collected for ELISA. The results showed that IFN-γ secreted by the three groups of K4D5/CD28-T cells and K4D5/CD28-CAR-T cells with different affinities was far higher than that secreted by CAR-T cells not transfected with the enhanced receptor. According to the comparison among K4D5/CD28-CAR-T cells with different affinities, the stronger the affinity, the more IFN-γ secreted. The same results were obtained for the detection of IL-2. The results of the OKT3/HER2 protein costimulation experiment showed that T cells expressing K4D5/CD28 can effectively respond to the activating signal of CD3. When the signal of CD3 is weak, K4D5 can effectively enhance the activating signal by binding to HER2 protein, thereby further enhancing the function of T cells, and this enhancement effect is positively correlated with the affinity for HER2 protein, that is, the stronger the affinity, the stronger the enhancement effect.

[0247] Detection of Activation Function of CAR-T Cells Transfected with K4D5/CD28 Enhanced Receptor

[0248] In order to verify the role of K4D5/CD28 in the CAR-T cells, a tumor cell line, J82-CD19, expressing CD19 was firstly constructed. Lentiviruses carrying CD19 were added to J82 cells at MOI=5. After 72 h, the expression of CD19 was detected by flow cytometry. The flow cytometry results showed that 90% of J82 cells expressed CD19, indicating that the cell construction was successful.

[0249] In order to verify the function of K4D5/CD28, the cytokine secretion of T cells were detected by co-culturing tumor cells and T cells. The results showed that IFN-γ secreted by the three groups of K4D5/CD28-CAR-T cells with different affinities after co-culturing with J82-CD19 tumor cells was each higher than that of CAR-T cells not transfected with the enhanced receptor. According to the comparison among the three groups of K4D5/CD28-CAR-T cells with different affinities, the weaker the affinity, the more IFN-γ secreted. The basically consistent results were obtained for the detection of IL-2. The results of the in vitro experiment showed that K4D5/CD28 can effectively enhance the tumor killing effect of CAR-T cells, and this enhancement effect is negatively correlated with the affinity for K4D5.

Example 5. Related Example of Enhanced Receptor-Anti-HER2 Monoclonal Antibody/41BBz

[0250] In this example, an enhanced receptor consisting of an anti-HER2 monoclonal antibody (referred to as aHER2mAb for short) as the extracellular segment and 41BBz as the intracellular segment was constructed and tested. K4D5 was usually used to represent the antibody Fab segment of HER2. In this example, the enhanced receptor was represented by aHER2mAb/41BBZ or K4D5/41BBZ or ER (Enhanced Receptor). The difference between the enhanced receptor of this example and the enhanced receptor of Example 3 was that the costimulatory molecules of the intracellular segment were different.

[0251] Amino Acid Sequences of Three Anti-HER2 Antibodies K4D5 with Different Affinities

[0252] See Example 3.

[0253] Construction of Three K4D5/41BBZ and K4D5/41BBZ-CD19 CAR Lentiviral Expression Plasmids

[0254] Three K4D5/41BBZ and K4D5/41BBZ-CD19 CAR lentiviral expression plasmids were constructed. K4D5/41BBZ and CD19 CAR were non-fusion expression.

[0255] Preparation of K4D5/41BBZ and K4D5/41BBZ-CD19 CAR Lentiviral Vectors

[0256] See Example 3 for the preparation process.

[0257] Preparation of CAR-T Cells Transfected with K4D5/41BBZ Enhanced Receptor

[0258] See Example 3 for the preparation process.

[0259] Detection of K4D5/41BBZ Function by OKT3/HER2 Protein Costimulation

[0260] See Example 3 for the experimental design. The experimental results showed that T cells expressing K4D5/41BBZ can effectively respond to the activating signal of CD3. When the signal of CD3 is weak, K4D5 can effectively enhance the activating signal by binding to HER2 protein, thereby further enhancing the function of T cells, and this enhancement effect is positively correlated with the affinity for HER2 protein, that is, the stronger the affinity, the stronger the enhancement effect.

[0261] Detection of Activation Function of CAR-T Cells Transfected with K4D5/41BBZ Enhanced Receptor

[0262] See Example 3 for the experimental design. The results of the in vitro experiment showed that K4D5/41BBZ can effectively enhance the tumor killing effect of CAR-T cells, and this enhancement effect is negatively correlated with the affinity for K4D5.

Example 6. Related Example of Enhanced Receptor-Anti-HER2 Monoclonal Antibody/ICOS

[0263] In this example, an enhanced receptor consisting of an anti-HER2 monoclonal antibody (referred to as aHER2mAb for short) as the extracellular segment and ICOS as the intracellular segment was constructed and tested. K4D5 was used to represent the antibody Fab segment of HER2. In this example, the enhanced receptor was represented by aHER2mAb/ICOS or K4D5/ICOS or ER (Enhanced Receptor). The difference between the enhanced receptor of this example and the enhanced receptor of Example 3 was that the costimulatory molecules of the intracellular segment were different.

[0264] The experimental design was the same as that of Example 3, and the only difference was that the enhanced receptors were different.

[0265] In the in vitro experiment, the experimental conclusion was also similar to that of Example 1, that is, as compared to natural unmodified CAR-T, the CAR-T expressing the ER showed a stronger killing response to target cells.

[0266] In the animal experiment, the experimental conclusions were also similar to those of Example 1, that is, in the CAR-T group expressing the ER, the tumors were eliminated more thoroughly than those in the CAR-T group not transfected with the enhanced receptor (reflecting the property of the ER to enhance activation), the TIL density in the tumor tissue was higher than that of other groups (reflecting that the ER can help CAR-T cells to better recruit into tumors), and the tumors in the non-tumor-recognizing T cell (ER-T) group transfected with the enhanced receptor were not killed (reflecting the safety of the ER, that is, when T cells do not recognize the target cells, the ER will not independently kill PDL1-positive target cells).

Example 7. Related Example of Enhanced Receptor-Anti-PDL1 Monoclonal Antibody/CD28

[0267] In this example, an enhanced receptor consisting of an anti-PDL1 monoclonal antibody (referred to as aPDL1mAb for short) as the extracellular segment and CD28 as the intracellular segment was constructed and tested. In this example, the enhanced receptor was represented by aPDL1mAb/CD28 or ER (Enhanced Receptor). Moreover, aPDL1mAb protein transmembrane expressed was used as a control for the enhanced receptor.

[0268] Preparation of Lentiviral Vectors for aPDL1mAb/CD28 Enhanced Receptor and Transmembrane aPDL1mAb

[0269] 1. Lentiviral vectors for transmembrane expressed aPDL1mAb and without CD28 were constructed.

[0270] 2. Lentiviral vectors for aPDL1mAb/CD28 enhanced receptor were constructed. See Example 1 for the construction process.

[0271] Construction of Other Groups of Lentiviral Expression Vectors

[0272] Similar to the above steps, lentiviral vectors loaded with the following gene sequences were constructed, respectively: [0273] 1) an artificial TCR targeting tumor target NY-ESO-1; [0274] 2) a CAR targeting CD19; [0275] 4) non-fusion expression of PD1/CD28 and an artificial TCR targeting tumor target NY-ESO-1, referred to as ER-TCR for short; and [0276] 5) non-fusion expression of PD1/CD28 and a CAR targeting CD19, referred to as ER-CAR for short.

[0277] Preparation of Several Groups of Tumor-Recognizing T Cells

[0278] Four kinds of tumor-recognizing T cells were prepared as follows.

[0279] Preparation of TCR-T Cells (NY-ESO-1-TCR-T or TCR-T) Targeting Tumor Target NY-ESO-1

[0280] The patient's leukocytes with tumor cells being NY-ESO-1 positive and of HLA-A:0201 type were collected by apheresis, PBMCs were separated with Ficoll lymphocyte separation solution, and allowed to grow adherently for 2 hours, and the suspension cells-mainly T cells-were obtained. TCR lentiviral vectors recognizing NY-ESO-1 were added, lentiviruses were added at MOI (number of lentiviruses/number of cells)=1, and TCR-T cells were cultured and expanded. The expression rate of TCR was 68.7% by flow cytometry.

[0281] At MOI=1, ER lentiviruses were added to the TCR-T to obtain ER-TCR-T.

[0282] Preparation of CAR-T Cells (CD19 CAR-T or CAR-T) Targeting CD19 and CAR-T (ER-CAR-T) Transfected with Enhanced Receptor

[0283] The patient's leukocytes were collected by apheresis, PBMCs were separated with Ficoll lymphocyte separation solution, and allowed to grow adherently for 2 hours, and the suspension cells-mainly T cells-were obtained. CAR lentiviral vectors recognizing CD19 were added, lentiviruses were added at MOI (number of lentiviruses/number of cells)=1, and CAR-T cells were cultured and expanded.

[0284] At MOI=1, ER lentiviruses were added to the CAR-T to obtain ER-CAR-T.

[0285] Preparation of TIL Cells from Fresh Tumor Tissues and TIL (ER-TIL) Transfected with Enhanced Receptor

[0286] Cells of tumor tissues were isolated from 10 g or more of the patient's tumor tissues by enzymatic digestion, CD3-positive T cells-TIL-were separated and purified with CD3 magnetic beads, and the remaining cells were cultured adherently to obtain tumor cells autologous to the patient.

[0287] At MOI=1, ER lentiviruses were added to the TIL to obtain ER-TIL.

[0288] Preparation of Neoantigen-Reactive T Cells and neoT (ER-neoT) Transfected with Enhanced Receptor

[0289] Firstly, whole-exomic sequencing or RNA transcriptome sequencing was performed on the patient's peripheral blood or surgically removed tumor tissues, the mutation sites were selected, affinity peptide prediction was made in combination with the HLA typing of the patient, and about 20 mutations were selected as Neoantigen. The Neoantigen was genetically synthesized, and was transcribed into RNA in vitro. Again, the patient's peripheral blood was collected by apheresis or directly drawn, PBMCs were isolated and grown adherently for 2 hours. To the adherent mononuclear cells, related cytokines were added to promote the differentiation and maturation of DC cells, and the RNA was transferred into the cells by electroporation. The suspension cells-mainly T cells-were co-cultured with the DC cells transferred by electroporation, CD137+-positive T cells were isolated to obtain neoantigen-reactive T cells (or referred to as neoT for short hereinafter), and the cells were expanded.

[0290] At MOI=1, ER lentiviruses were added to the neoT to obtain ER-neoT.

[0291] In Vitro Functional Experiment of NY-ESO-1-Targeting TCR-T Expressing aPDL1mAb/CD28 Enhanced Receptor

[0292] 1) ELISA Plate Experiment

[0293] In order to verify the role of aPDL1mAb/CD28 enhanced receptor in ordinary T cells and NY-ESO-1-targeting TCR-T, ELISA plates respectively coated with 0.5 ug OKT3 (an anti-CD3 antibody) protein, 1 ug PDL1-Fc segment+0.5 ug OKT3 protein, and 1 ug PDL1-Fc segment+0.1 ug OKT3 protein were firstly prepared. Then, the following T cells were respectively added, and IFNγ (FIG. 1) and IL2 (FIG. 2) were detected after 48 hours: [0294] NOTD, referring to ordinary T cells that were not engineered; [0295] PDL1-T, referring to T cells with aPDL1mAb-CD28; [0296] PD1-T, referring to T cells with PD1-CD28; [0297] PDL1-TCR-T, referring to TCR-T cells with aPDL1mAb-CD28; and [0298] PD1-TCR-T, referring to TCR-T cells with PD1-CD28, [0299] wherein PDL1 referred to aPDL1mAb-CD28; and PD1 referred to PD1-CD28.

[0300] As can be seen in FIGS. 1 and 2, after encountering PDL1 protein (both 1 ug PDL1-Fc segment+0.5 ug OKT3 protein, and 1 ug PDL1-Fc segment+0.1 ug OKT3 protein groups) that inhibited T cell function, IFNγ and IL2 secreted by the T cells and TCR-T cells with aPDL1mAb enhanced receptor were both higher than those secreted by T cells and TCR-T cells without aPDL1mAb enhanced receptor.

[0301] 2) Cell Experiment

[0302] In order to verify the role of aPDL1mAb/CD28 enhanced receptor in the TCR-T targeting NY-ESO-1, a tumor cell line, J82-NY-ESO-1-PDL1, with its HLA typing as A:0201 was firstly constructed. Lentiviruses carrying PDL1 and NY-ESO-1 were added to J82 cells at MOI=5. After 72 h, G418 and Puromycin were added to screen positive cells. After about 2 weeks, the expression of PDL1 and NY-ESO-1 was detected by flow cytometry. The flow cytometry results showed that most J82 cells expressed PDL1 and NY-ESO-1 at the same time, indicating that the cell construction was successful.

[0303] In order to verify the function of ER, the expression of CD107a and cytokine secretion of T cells were detected by co-culturing tumor cells and T cells. The results showed that the expression of CD107a in the ER-TCR-T cells was higher than that in ordinary TCR-T. The secretion of IFN-γ and IL-2 shown in the ER-TCR-T was also higher than that in TCR-T. The results of the in vitro experiment showed that the ability of TCR-T to kill tumors is significantly improved after transfection with ER switch.

[0304] In order to compare the function of the transmembrane aPDL1mAb, the expression of CD107a and cytokine secretion of T cells were detected by co-culturing tumor cells and T cells. The results showed that the expression of CD107a, IFN-γ and IL-2 in the aPDL1mAb-NY-ESO-1-TCR-T cells was similar to that in ordinary TCR-T, neither promoting activation nor inhibiting activity.

[0305] In Vitro Functional Experiment of TIL Expressing aPDL1mAb/CD28 Enhanced Receptor

[0306] In order to verify the role of ER in the TIL, tumor cells isolated from the patient's fresh tumor tissues were co-cultured with the TIL and cells for 24 hours, respectively. The secretion of IFN-γ and IL-2 was detected by ELISA. The experimental results showed that the killing ability of the ER-TIL was significantly higher than that of TIL. The results of the in vitro experiment showed that the ability of TIL to kill tumors is significantly improved after transfection with ER switch.

[0307] In order to compare the function of the transmembrane aPDL1mAb, the expression of CD107a and cytokine secretion of T cells were detected by co-culturing tumor cells and T cells. The results showed that the expression of CD107a, IFN-γ and IL-2 in the aPDL1mAb-TIL cells was similar to that in ordinary TIL, neither promoting activation nor inhibiting activity.

[0308] In Vitro Functional Experiment of Neoantigen-Reactive T Cells Expressing aPDL1mAb/CD28 Enhanced Receptor

[0309] In order to verify the role of ER in the neoT, tumor cells isolated from the patient's fresh tumor tissues were co-cultured with the neoT and ER-neoT cells for 24 hours, respectively. The secretion of IFN-γ and IL-2 was detected by ELISA. The results showed that the killing ability of the ER-neoT was significantly higher than that of neoT. The results of the in vitro experiment showed that the ability of neoT to kill tumors is significantly improved after transfection with ER switch.

[0310] In order to compare the function of the transmembrane aPDL1mAb, the expression of CD107a and cytokine secretion of T cells were detected by co-culturing tumor cells and T cells. The results showed that the expression of CD107a, IFN-γ and IL-2 in the aPDL1mAb-neoT cells was similar to that in ordinary neoT, neither promoting activation nor inhibiting activity.

[0311] In Vitro Functional Experiment of CAR-T Cells Expressing aPDL1mAb/CD28 Enhanced Receptor

[0312] A tumor cell line expressing CD19 was firstly constructed. The tumor cells were co-cultured with ordinary CD19CAR-T cells not transfected with the enhanced receptor and the ER-CAR-T for 24 hours, respectively. The results of the secretion of IFN-γ and IL-2 by ELISA showed that the killing ability of the ER-CAR-T is significantly higher than that of ordinary CAR-T not transfected with the enhanced receptor.

[0313] In order to compare the function of the transmembrane aPDL1mAb, the expression of CD107a and cytokine secretion of T cells were detected by co-culturing tumor cells and T cells. The results showed that the expression of CD107a, IFN-γ and IL-2 in the aPDL1mAb-CAR-T cells was similar to that in ordinary CAR-T, neither promoting activation nor inhibiting activity.

[0314] Animal Experiment of NY-ESO-1 TCR-T Cells Expressing aPDL1mAb/CD28 Enhanced Receptor

[0315] Inoculation: 1×10.sup.6 J82-NY-ESO-1-PDL1 tumor cells were subcutaneously inoculated into NSG mice. The blank control group was subcutaneously injected with PBS (A0). The mice injected with tumor cells began to form tumors about two weeks later. The tumor size was measured on Day 23, and 30 mice were selected for recruitment.

[0316] The results of the animal experiment of the combination of ER and TCR-T are shown in FIG. 3. In the legend, PDL1/CD28 referred to aPDL1mAb-CD28 enhanced receptor; and PD1/CD28 referred to PD1/CD28 enhanced receptor.

[0317] Administration: The blank control group (A0) was injected with PBS via tail vein. The mice inoculated with tumor cells were divided into 5 groups: no drug-PBS group (A1); non-engineered ordinary T cell group (NOTD, A2); TCR-T cell group (A3); ER-TCRT cell group (A4); and PD1/CD28-TCRT group (A5), respectively. The administrations were all performed by intravenous infusion of 1×10.sup.7 cells via tail vein.

[0318] After administration, the tumor size was measured every 2-3 days and the mouse state was observed. Tumor volume=1/2*long diameter*short diameter*short diameter. The observation was continued for 30 days.

[0319] Change in Tumor Burden:

[0320] The tumor volume of tumor-bearing mice in each group continued to increase one week after administration. The trend of A1-2 groups was basically the same, and the tumor volume continued to increase, until death occurred on Day 40. The tumors in A3, A4 and A5 groups were detected to begin to shrink from Day 10, but the tumors in A3 group began to rebound on Day 15, while the tumors in A4 and A5 groups continued to shrink to almost disappear.

[0321] It can be seen that the TCR-T cells with PD1/CD28 or aPDL1mAb/CD28 enhanced receptor had significantly enhanced inhibition on tumors than TCR-T cells without the enhanced receptor.

[0322] Change in Total Amount of Infused T Cells In Vivo:

[0323] In the three groups of administration, on Day 10 after administration, one mouse was randomly selected from groups 3, 4 and 5, tumor tissues were removed, and TILs were isolated therefrom. The total amount of cells reinfused was detected by flow cytometry. It can be seen that the total amount of T cells reinfused in the tumors of the A5 group far exceeded that of other groups.

[0324] Animal Experiment of CAR-T Cells Expressing aPDL1mAb/CD28 Enhanced Receptor

[0325] Inoculation: 1×10.sup.6 CD19- and PDL1-expressing tumor cells were subcutaneously inoculated into NSG mice. The blank control group was subcutaneously injected with PBS (A0). The mice injected with tumor cells began to form tumors about two weeks later. The tumor size was measured on Day 23, and 30 mice were selected for recruitment.

[0326] Administration: The blank control group (A0) was injected with PBS via tail vein. The mice inoculated with tumor cells were divided into 5 groups: no drug-PBS group (A1); T cell group (A2); ER-T cell group (A3); CAR-T cell group (A4); and ER-CAR-T cell group (A5), respectively. The administrations were all performed by intravenous infusion of 1×10.sup.7 cells via tail vein.

[0327] After administration, the tumor size was measured every 2-3 days and the mouse state was observed. Tumor volume=1/2*long diameter*short diameter*short diameter. The observation was continued for 30 days.

[0328] Change in Tumor Burden:

[0329] The tumor volume of tumor-bearing mice in each group continued to increase one week after administration. The trend of A1-3 groups was basically the same, and the tumor volume continued to increase, until death occurred on Day 30-40. The tumors in A4 and A5 groups were detected to begin to shrink from Week 2, but the tumors in A4 group began to rebound in Week 3-4, while the tumors in A5 group continued to shrink to almost disappear.

[0330] Change in Total Amount of Infused T Cells In Vivo:

[0331] In the three groups of administration, on Day 10 after administration, one mouse was randomly selected from groups 3, 4 and 5, tumor tissues were removed, T cells (i.e., TIL) were isolated therefrom and counted, and the TIL density, that is, the total number of TIL per unit weight of tumor tissue, was calculated. The results showed that the TIL density in the tumors of A5 group was far higher than that of other groups.

Example 8. Related Example of Enhanced Receptor-aPDL1mAb/41BBz

[0332] In this example, an enhanced receptor consisting of an anti-PDL1 monoclonal antibody (referred to as aPDL1mAb for short) as the extracellular segment and 41BBz as the intracellular segment was constructed and tested. In this example, the enhanced receptor was represented by aPDL1mAb/41BBz or ER (Enhanced Receptor). Moreover, aPDL1mAb protein transmembrane expressed was used as a control for the enhanced receptor. The difference between the enhanced receptor of this example and the enhanced receptor of Example 7 was that the costimulatory molecules of the intracellular segment were different.

[0333] The experimental design was the same as that of Example 7, and the only difference was that the enhanced receptors were different.

[0334] In the in vitro experiment, the experimental conclusion was also similar to that of Example 7, that is, as compared to natural unmodified TCR-T, CAR-T, TIL and neoT, all of the TCR-T, CAR-T, TIL and neoT expressing the ER showed a stronger killing response to target cells, respectively.

[0335] In the animal experiment, the experimental conclusions were also similar to those of Example 7, that is, in the TCR-T and CAR-T groups expressing the ER, the tumors were eliminated more thoroughly than those in the TCR-T and CAR-T groups not transfected with the enhanced receptor (reflecting the property of the ER to enhance activation), the TIL density in the tumor tissue was higher than that of other groups (reflecting that the ER can help TCR-T and CAR-T cells to better recruit into tumors), and the tumors in the non-tumor-recognizing T cell (ER-T) group transfected with the enhanced receptor were not killed (reflecting the safety of the ER, that is, when T cells do not recognize the target cells, the ER will not independently kill PDL1-positive target cells).

Example 9. Related Example of Enhanced Receptor-aPDL1mAb/ICOS

[0336] In this example, an enhanced receptor consisting of an anti-PDL1 monoclonal antibody (referred to as aPDL1mAb for short) as the extracellular segment and ICOS as the intracellular segment was constructed and tested. In this example, the enhanced receptor was represented by aPDL1mAb/ICOS or ER (Enhanced Receptor). Moreover, aPDL1mAb protein transmembrane expressed was used as a control for the enhanced receptor. The difference between the enhanced receptor of this example and the enhanced receptor of Example 7 was that the costimulatory molecules of the intracellular segment were different.

[0337] The experimental design was the same as that of Example 7, and the only difference was that the enhanced receptors were different.

[0338] In the in vitro experiment, the experimental conclusion was also similar to that of Example 7, that is, as compared to natural unmodified TCR-T, CAR-T, TIL and neoT, all of the TCR-T, CAR-T, TIL and neoT expressing the ER showed a stronger killing response to target cells, respectively.

[0339] In the animal experiment, the experimental conclusions were also similar to those of Example 7, that is, in the TCR-T and CAR-T groups expressing the ER, the tumors were eliminated more thoroughly than those in the TCR-T and CAR-T groups not transfected with the enhanced receptor (reflecting the property of the ER to enhance activation), the TIL density in the tumor tissue was higher than that of other groups (reflecting that the ER can help TCR-T and CAR-T cells to better recruit into tumors), and the tumors in the non-tumor-recognizing T cell (ER-T) group transfected with the enhanced receptor were not killed (reflecting the safety of the ER, that is, when T cells do not recognize the target cells, the ER will not independently kill PDL1-positive target cells).

Example 10. Related Example of Enhanced Receptor-NKG2D/CD28

[0340] In this example, an enhanced receptor consisting of NKG2D as the extracellular segment and CD28 as the intracellular segment was constructed and tested. In this example, the enhanced receptor was represented by NKG2D/CD28 or ER (Enhanced Receptor). Moreover, NKG2D protein transmembrane expressed was used as a control for the enhanced receptor.

[0341] The experimental design was the same as that of Example 7, and the only difference was that the enhanced receptors were different.

[0342] In the in vitro experiment, the experimental conclusion was also similar to that of Example 7, that is, as compared to natural unmodified TCR-T, CAR-T, TIL and neoT, all of the TCR-T, CAR-T, TIL and neoT expressing the ER showed a stronger killing response to target cells, respectively.

[0343] In the animal experiment, the experimental conclusions were also similar to those of Example 7, that is, in the TCR-T and CAR-T groups expressing the ER, the tumors were eliminated more thoroughly than those in the TCR-T and CAR-T groups not transfected with the enhanced receptor (reflecting the property of the ER to enhance activation), the TIL density in the tumor tissue was higher than that of other groups (reflecting that the ER can help TCR-T and CAR-T cells to better recruit into tumors), and the tumors in the non-tumor-recognizing T cell (ER-T) group transfected with the enhanced receptor were not killed (reflecting the safety of the ER, that is, when T cells do not recognize the target cells, the ER will not independently kill PDL1-positive target cells).

Example 11. Related Example of Enhanced Receptor-NKG2D/41BBz

[0344] In this example, an enhanced receptor consisting of NKG2D as the extracellular segment and 41BBz as the intracellular segment was constructed and tested. In this example, the enhanced receptor was represented by NKG2D/41BBz or ER (Enhanced Receptor). Moreover, NKG2D protein transmembrane expressed was used as a control for the enhanced receptor. The difference between the enhanced receptor of this example and the enhanced receptor of Example 10 was that the costimulatory molecules of the intracellular segment were different.

[0345] The experimental design was the same as that of Example 7, and the only difference was that the enhanced receptors were different.

[0346] In the in vitro experiment, the experimental conclusion was also similar to that of Example 7, that is, as compared to natural unmodified TCR-T, CAR-T, TIL and neoT, all of the TCR-T, CAR-T, TIL and neoT expressing the ER showed a stronger killing response to target cells, respectively.

[0347] In the animal experiment, the experimental conclusions were also similar to those of Example 7, that is, in the TCR-T and CAR-T groups expressing the ER, the tumors were eliminated more thoroughly than those in the TCR-T and CAR-T groups not transfected with the enhanced receptor (reflecting the property of the ER to enhance activation), the TIL density in the tumor tissue was higher than that of other groups (reflecting that the ER can help TCR-T and CAR-T cells to better recruit into tumors), and the tumors in the non-tumor-recognizing T cell (ER-T) group transfected with the enhanced receptor were not killed (reflecting the safety of the ER, that is, when T cells do not recognize the target cells, the ER will not independently kill PDL1-positive target cells).

Example 12. Related Example of Enhanced Receptor-NKG2D/ICOS

[0348] In this example, an enhanced receptor consisting of NKG2D as the extracellular segment and ICOS as the intracellular segment was constructed and tested. In this example, the enhanced receptor was represented by NKG2D/ICOS or ER (Enhanced Receptor). Moreover, NKG2D protein transmembrane expressed was used as a control for the enhanced receptor. The difference between the enhanced receptor of this example and the enhanced receptor of Example 10 was that the costimulatory molecules of the intracellular segment were different.

[0349] The experimental design was the same as that of Example 7, and the only difference was that the enhanced receptors were different.

[0350] In the in vitro experiment, the experimental conclusion was also similar to that of Example 7, that is, as compared to natural unmodified TCR-T, CAR-T, TIL and neoT, all of the TCR-T, CAR-T, TIL and neoT expressing the ER showed a stronger killing response to target cells, respectively.

[0351] In the animal experiment, the experimental conclusions were also similar to those of Example 7, that is, in the TCR-T and CAR-T groups expressing the ER, the tumors were eliminated more thoroughly than those in the TCR-T and CAR-T groups not transfected with the enhanced receptor (reflecting the property of the ER to enhance activation), the TIL density in the tumor tissue was higher than that of other groups (reflecting that the ER can help TCR-T and CAR-T cells to better recruit into tumors), and the tumors in the non-tumor-recognizing T cell (ER-T) group transfected with the enhanced receptor were not killed (reflecting the safety of the ER, that is, when T cells do not recognize the target cells, the ER will not independently kill PDL1-positive target cells).

Example 13. Related Example of Enhanced Receptor-Anti-CD47 Monoclonal

[0352] Antibody/CD28

[0353] In this example, an enhanced receptor consisting of an anti-CD47 monoclonal antibody (referred to as aCD47mAb for short) as the extracellular segment and CD28 as the intracellular segment was constructed and tested. In this example, the enhanced receptor was represented by aCD47mAb/CD28 or ER (Enhanced Receptor). Moreover, aCD47mAb protein transmembrane expressed was used as a control for the enhanced receptor.

[0354] The experimental design was the same as that of Example 7, and the only difference was that the enhanced receptors were different.

[0355] In the in vitro experiment, the experimental conclusion was also similar to that of Example 7, that is, as compared to natural unmodified TCR-T, CAR-T, TIL and neoT, all of the TCR-T, CAR-T, TIL and neoT expressing the ER showed a stronger killing response to target cells, respectively.

[0356] In the animal experiment, the experimental conclusions were also similar to those of Example 7, that is, in the TCR-T and CAR-T groups expressing the ER, the tumors were eliminated more thoroughly than those in the TCR-T and CAR-T groups not transfected with the enhanced receptor (reflecting the property of the ER to enhance activation), the TIL density in the tumor tissue was higher than that of other groups (reflecting that the ER can help TCR-T and CAR-T cells to better recruit into tumors), and the tumors in the non-tumor-recognizing T cell (ER-T) group transfected with the enhanced receptor were not killed (reflecting the safety of the ER, that is, when T cells do not recognize the target cells, the ER will not independently kill PDL1-positive target cells).

Example 14. Related Example of Enhanced Receptor-aCD47mAb/41BBz

[0357] In this example, an enhanced receptor consisting of aCD47mAb as the extracellular segment and 41BBz as the intracellular segment was constructed and tested. In this example, the enhanced receptor was represented by aCD47mAb/41BBz or ER (Enhanced Receptor). Moreover, SIRPα protein transmembrane expressed was used as a control for the enhanced receptor. The difference between the enhanced receptor of this example and the enhanced receptor of Example 13 was that the costimulatory molecules of the intracellular segment were different.

[0358] The experimental design was the same as that of Example 7, and the only difference was that the enhanced receptors were different.

[0359] In the in vitro experiment, the experimental conclusion was also similar to that of Example 7, that is, as compared to natural unmodified TCR-T, CAR-T, TIL and neoT, all of the TCR-T, CAR-T, TIL and neoT expressing the ER showed a stronger killing response to target cells, respectively.

[0360] In the animal experiment, the experimental conclusions were also similar to those of Example 7, that is, in the TCR-T and CAR-T groups expressing the ER, the tumors were eliminated more thoroughly than those in the TCR-T and CAR-T groups not transfected with the enhanced receptor (reflecting the property of the ER to enhance activation), the TIL density in the tumor tissue was higher than that of other groups (reflecting that the ER can help TCR-T and CAR-T cells to better recruit into tumors), and the tumors in the non-tumor-recognizing T cell (ER-T) group transfected with the enhanced receptor were not killed (reflecting the safety of the ER, that is, when T cells do not recognize the target cells, the ER will not independently kill PDL1-positive target cells).

Example 15. Related Example of Enhanced Receptor-aCD47mAb/ICOS

[0361] In this example, an enhanced receptor consisting of aCD47mAb as the extracellular segment and ICOS as the intracellular segment was constructed and tested. In this example, the enhanced receptor was represented by aCD47mAb/ICOS or ER (Enhanced Receptor). Moreover, SIRPα protein transmembrane expressed was used as a control for the enhanced receptor. The difference between the enhanced receptor of this example and the enhanced receptor of Example 13 was that the costimulatory molecules of the intracellular segment were different.

[0362] The experimental design was the same as that of Example 7, and the only difference was that the enhanced receptors were different.

[0363] In the in vitro experiment, the experimental conclusion was also similar to that of Example 7, that is, as compared to natural unmodified TCR-T, CAR-T, TIL and neoT, all of the TCR-T, CAR-T, TIL and neoT expressing the ER showed a stronger killing response to target cells, respectively.

[0364] In the animal experiment, the experimental conclusions were also similar to those of Example 7, that is, in the TCR-T and CAR-T groups expressing the ER, the tumors were eliminated more thoroughly than those in the TCR-T and CAR-T groups not transfected with the enhanced receptor (reflecting the property of the ER to enhance activation), the TIL density in the tumor tissue was higher than that of other groups (reflecting that the ER can help TCR-T and CAR-T cells to better recruit into tumors), and the tumors in the non-tumor-recognizing T cell (ER-T) group transfected with the enhanced receptor were not killed (reflecting the safety of the ER, that is, when T cells do not recognize the target cells, the ER will not independently kill PDL1-positive target cells).

Example 16. Related Example of Enhanced Receptor-SIRPα/CD28

[0365] In this example, an enhanced receptor consisting of SIRPα as the extracellular segment and CD28 as the intracellular segment was constructed. In this example, the enhanced receptor was represented by SIRPα/CD28 or ER (Enhanced Receptor). Moreover, SIRPα protein transmembrane expressed was used as a control for the enhanced receptor.

[0366] The experimental design was the same as that of Example 7, and the only difference was that the enhanced receptors were different.

[0367] In the in vitro experiment, the experimental conclusion was also similar to that of Example 7, that is, as compared to natural unmodified TCR-T, CAR-T, TIL and neoT, all of the TCR-T, CAR-T, TIL and neoT expressing the ER showed a stronger killing response to target cells, respectively.

[0368] In the animal experiment, the experimental conclusions were also similar to those of Example 7, that is, in the TCR-T and CAR-T groups expressing the ER, the tumors were eliminated more thoroughly than those in the TCR-T and CAR-T groups not transfected with the enhanced receptor (reflecting the property of the ER to enhance activation), the TIL density in the tumor tissue was higher than that of other groups (reflecting that the ER can help TCR-T and CAR-T cells to better recruit into tumors), and the tumors in the non-tumor-recognizing T cell (ER-T) group transfected with the enhanced receptor were not killed (reflecting the safety of the ER, that is, when T cells do not recognize the target cells, the ER will not independently kill PDL1-positive target cells).

Example 17. Related Example of Enhanced Receptor-SIRPα/41BBz

[0369] In this example, an enhanced receptor consisting of SIRPα as the extracellular segment and 41BBz as the intracellular segment was constructed and tested. In this example, the enhanced receptor was represented by SIRPα/41BBz or ER (Enhanced Receptor). Moreover, SIRPα protein transmembrane expressed was used as a control for the enhanced receptor. The difference between the enhanced receptor of this example and the enhanced receptor of Example 16 was that the costimulatory molecules of the intracellular segment were different.

[0370] The experimental design was the same as that of Example 7, and the only difference was that the enhanced receptors were different.

[0371] In the in vitro experiment, the experimental conclusion was also similar to that of Example 7, that is, as compared to natural unmodified TCR-T, CAR-T, TIL and neoT, all of the TCR-T, CAR-T, TIL and neoT expressing the ER showed a stronger killing response to target cells, respectively.

[0372] In the animal experiment, the experimental conclusions were also similar to those of Example 7, that is, in the TCR-T and CAR-T groups expressing the ER, the tumors were eliminated more thoroughly than those in the TCR-T and CAR-T groups not transfected with the enhanced receptor (reflecting the property of the ER to enhance activation), the TIL density in the tumor tissue was higher than that of other groups (reflecting that the ER can help TCR-T and CAR-T cells to better recruit into tumors), and the tumors in the non-tumor-recognizing T cell (ER-T) group transfected with the enhanced receptor were not killed (reflecting the safety of the ER, that is, when T cells do not recognize the target cells, the ER will not independently kill PDL1-positive target cells).

Example 18. Related Example of Enhanced Receptor-SIRPα/ICOS

[0373] In this example, an enhanced receptor consisting of SIRPα as the extracellular segment and ICOS as the intracellular segment was constructed and tested. In this example, the enhanced receptor was represented by SIRPα/ICOS or ER (Enhanced Receptor). Moreover, SIRPα protein transmembrane expressed was used as a control for the enhanced receptor. The difference between the enhanced receptor of this example and the enhanced receptor of Example 16 was that the costimulatory molecules of the intracellular segment were different.

[0374] The experimental design was the same as that of Example 7, and the only difference was that the enhanced receptors were different.

[0375] In the in vitro experiment, the experimental conclusion was also similar to that of Example 7, that is, as compared to natural unmodified TCR-T, CAR-T, TIL and neoT, all of the TCR-T, CAR-T, TIL and neoT expressing the ER showed a stronger killing response to target cells, respectively.

[0376] In the animal experiment, the experimental conclusions were also similar to those of Example 7, that is, in the TCR-T and CAR-T groups expressing the ER, the tumors were eliminated more thoroughly than those in the TCR-T and CAR-T groups not transfected with the enhanced receptor (reflecting the property of the ER to enhance activation), the TIL density in the tumor tissue was higher than that of other groups (reflecting that the ER can help TCR-T and CAR-T cells to better recruit into tumors), and the tumors in the non-tumor-recognizing T cell (ER-T) group transfected with the enhanced receptor were not killed (reflecting the safety of the ER, that is, when T cells do not recognize the target cells, the ER will not independently kill PDL1-positive target cells).