High-affinity fusion protein, nucleic acid molecule, pharmaceutical composition and application thereof

Abstract

The invention belongs to the technical field of medical products, and particularly relates to a high-affinity fusion protein, a nucleic acid molecule, a pharmaceutical composition and application thereof. The high-affinity fusion protein HAPD-1-CD28 is obtained by carrying out local replacement of amino acid on the basis of a PD-1-CD28 fusion protein; the PD-1-CD28 fusion protein is composed of an extracellular structural domain, a transmembrane structural domain and an intracellular structural domain, the extracellular domain comprises a polypeptide derived from PD-1 located at the N-terminal of the extracellular domain; the transmembrane structural domain is a transmembrane structural domain derived from PD-1 (programmed death-1); the intracellular domain comprises a polypeptide derived from CD28 at its C-terminus. On the basis, the long-acting anti-tumor activity of CAR-T cells or TCR-T cells is further enhanced by combined application with an LSD1 inhibitor. The invention aims to solve the problem that the curative effect of CAR-T and TCR-T is limited due to T cell depletion at present, and can enhance the plasticity of a T cell skeleton and enhance the long-acting anti-tumor activity of the T cell skeleton.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical products, and specifically relates to a high-affinity fusion protein, a nucleic acid molecule, a pharmaceutical composition and uses thereof. Background Art

[0002] Adoptive T cell therapy (ACT), including tumor infiltrating lymphocyte (TIL) therapy, chimeric antigen receptor T cells, NK cells and macrophages (CAR-T, CAR-NK, CAR-M) therapy, AbTCR-T, TCR-T therapy have achieved good results in the treatment of melanoma, blood tumors, infectious diseases, autoimmune diseases and solid tumors. Although CAR-T and TCR-T cell therapy have shown significant efficacy in tumor treatment, they still face many challenges in clinical application, especially the poor treatment effect of solid tumors and the high recurrence rate of patients after initial response. Studies have shown that T cells are prone to terminal differentiation and T cell exhaustion when they are exposed to chronic antigen stimulation, metabolic competition and lack of co-stimulatory signals in the tumor microenvironment for a long time. This is one of the important reasons restricting the clinical application of CAR-T and TCR-T.

[0003] During the process of T cell exhaustion, programmed cell death protein 1 (PD-1) acts as a key inhibitory receptor. By binding to its ligand PD-L1 (programmed cell death ligand 1), it inhibits TCR signaling and T cell activation, thereby hindering T cells from recognizing and killing tumor cells.

[0004] At present, chemotherapy combined with immune checkpoint inhibitors (such as PD-1 / PD-L1 inhibitors, CTLA-4 inhibitors) are often used clinically to treat a variety of solid tumors and blood tumors. Although the efficacy is significant, it is easy to develop drug resistance. In addition, combination therapy may enhance the side effects of monotherapy and limit its effect on tumor-reactive T cells. The PD-1-CD28 fusion gene can convert PD1 intracellular inhibitory signals into CD28 co-stimulatory signals, thereby reversing T cell exhaustion and promoting CAR-T and TCR-T anti-tumor activity, which has been confirmed in multiple patents, papers and clinical trials. The relevant research team further sorted out the optimal sequence length of PD-1 in the PD-1-CD28 fusion gene. However, the effect of ordinary PD-1-CD28 fusion protein on enhancing the anti-tumor activity of CAR-T / TCR-T cells is limited. Summary of the invention

[0005] In order to solve the problem that the common PD-1-CD28 fusion protein in the prior art has limited effect in enhancing the anti-tumor activity of CAR-T / TCR-T cells, the present invention provides a high-affinity fusion protein, a nucleic acid molecule, a pharmaceutical composition and its use. The high-affinity fusion protein provided by the present invention significantly enhances the binding of PD-1-CD28 fusion protein and tumor cell PD-L1, thereby enhancing its long-term anti-tumor activity. To achieve the above purpose, the present invention adopts the following technical solution.

[0006] The present invention provides a high-affinity fusion protein HAPD-1-CD28. The high-affinity fusion protein HAPD-1-CD28 is obtained by partially replacing amino acids on the basis of the PD-1-CD28 fusion protein.

[0007] The PD-1-CD28 fusion protein consists of an extracellular domain, a transmembrane domain and an intracellular domain.

[0008] The extracellular domain comprises a polypeptide derived from PD-1 at its N-terminus.

[0009] The transmembrane domain is derived from the transmembrane domain of PD-1.

[0010] The intracellular domain comprises a CD28-derived polypeptide at its C-terminus.

[0011] The partial replacement of amino acids refers to replacing the amino acid at any site in the extracellular domain of the PD-1-CD28 fusion protein with any of the following amino acids: Glycine, alanine, valine, leucine, isoleucine, methionine (methionine), tryptophan, tyrosine, asparagine, glutamine, threonine, glutamic acid, lysine, arginine and histidine.

[0012] The high-affinity fusion protein HAPD-1-CD28 provided by the present invention is a high-affinity fusion protein expressed by a viral vector, or PD-1 is replaced in situ with the high-affinity PD-1-CD28 fusion protein by gene editing.

[0013] In order to enhance the anti-tumor activity of CAR-T cells and TCR-T cells, the present application firstly replaced (modified) the amino acid sequence of the extracellular region of PD-1, so as to greatly enhance its ability to bind to PD-L1 of tumor cells. The results after modification showed that the modification of the amino acid sequence of the PD-1-CD28 fusion protein significantly enhanced the anti-tumor activity of CAR-T cells and TCR-T, and promoted the expression of PD-L1 in tumor cells. Because the affinity of the modified PD-1-CD28 fusion gene to PD-L1 was significantly increased, for the convenience of description, the high-affinity PD-1-CD28 was named as high-affinity fusion protein HAPD-1-CD28 (High affinity PD-1-CD28). The high-affinity fusion protein HAPD-1-CD28 provided by the present invention can solve the problem that the ordinary PD-1-CD28 fusion protein in the prior art has limited effect in enhancing the anti-tumor activity of CAR-T / TCR-T cells.

[0014] Among them, the high-affinity fusion protein HAPD-1-CD28 is referred to as HAPD-1-CD28.

[0015] PD-1-CD28 fusion protein is abbreviated as PD-1-CD28.

[0016] Preferably, the amino acid sequence of the PD-1-CD28 fusion protein is as shown in SEQ ID NO.1: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGT YLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGSG*.

[0017] Preferably, the amino acid sequence of the high-affinity fusion protein HAPD-1-CD28 is shown in at least one of SEQ ID NO.2 to SEQ ID NO.20, but is not limited to the following sequence: Mutation-1, as shown in SEQ ID NO.2: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKESLRAELRVTERRAEVPTAHCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGSG*。

[0018] Mutation-2, as shown in SEQ ID NO.3: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFKVQWQRQSPSQQTDRLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYQCGVISLAPKMQIKESLRAELRVTERRAEVPTAHCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGSG*。

[0019] Mutation-3, as shown in SEQ ID NO.4: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFRLQWQRQSPSQQTDRLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYQCGVISLAPKMQIKESLRAELRVTERRAEVPTAHCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGSG*。

[0020] Mutation-4, as shown in SEQ ID NO.5: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFKVQWQRQSPSQQTDRLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYQCGVISLAPKMQIKESLRAELRVTERRAEVPTAHCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGSG*。

[0021] Mutation-5, as shown in SEQ ID NO.6: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFRVQWQRQSPSQQTDRLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYQCGVISLAPKMQIKESLRAELRVTERRAEVPTAHCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGSG*。

[0022] Mutation-6, as shown in SEQ ID NO.7: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVIWQRQSPSQQTDRLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYQCGVISLAPKMQIKESLRAELRVTERRAEVPTAHCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGSG*。

[0023] Mutation-7, as shown in SEQ ID NO.8: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVIWQRQSPSQQTDRLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKAQIKESLRAELRVTERRAEVPTAHCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGSG*。

[0024] Mutation-8, as shown in SEQ ID NO.9: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVIWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYQCGVISLAPKMQIKESLRAELRVTERRAEVPTAHCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGSG*。

[0025] Mutation-9, as shown in SEQ ID NO.10: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFKLQWQRQSPSQQTDRLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYQCGVISLAPKMQIKESLRAELRVTERRAEVPTAHCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGSG*。

[0026] Mutation-10, as shown in SEQ ID NO.11: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFRLQWQRQSPSQQTDRLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYQCGVISLAPKMQIKESLRAELRVTERRAEVPTAHCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGSG*。

[0027] Mutation-11, as shown in SEQ ID NO.12: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFKLQWQRQSPSQQTDRLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGSG*。

[0028] Mutation-12, as shown in SEQ ID NO.13: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFRLQWQRQSPSQQTDRLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGSG*。

[0029] Mutation-13, as shown in SEQ ID NO.14: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVIWKRESPSRQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGVISLAPKAQIKESLRAELRVTERRAEVPTAHCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGSG*。

[0030] Mutation-14, as shown in SEQ ID NO.15: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFGVWWRRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYKCGVISLAPKAQIKESLRAELRVTERRAEVPTAHCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGSG*。

[0031] Mutation-15, as shown in SEQ ID NO.16: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFGVVWYRMSPSNQTDQLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYRCGVISLAPKAQIKESLRAELRVTERRAEVPTAHCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGSG*。

[0032] Mutation-16, as shown in SEQ ID NO.17: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWQRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYQCGVISLAPKMQIKESLRAELRVTERRAEVPTAHCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGSG*。

[0033] Mutation-17, as shown in SEQ ID NO.18: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWYRQSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYVCGIISLAPKMQIKESLRAELRVTERRAEVPTAHCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGSG*。

[0034] Mutation-18, as shown in SEQ ID NO.19: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWYRMSPQNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYVCGIISLAPKMQIKESLRAELRVTERRAEVPTAHCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGSG*。

[0035] Mutation-19, as shown in SEQ ID NO.20: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWQRQSPQNQTDQLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGT YVCGIISLAPKMQIKESLRAELRVTERRAEVPTAHCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGSG*.

[0036] The present invention also provides a nucleic acid molecule, which comprises a nucleotide sequence encoding the high-affinity fusion protein HAPD-1-CD28.

[0037] The present invention also provides a pharmaceutical composition, which comprises a raw material composition of the high-affinity fusion protein HAPD-1-CD28 or a pharmaceutically acceptable salt of the high-affinity fusion protein HAPD-1-CD28.

[0038] Preferably, the pharmaceutical composition is prepared with the high-affinity fusion protein HAPD-1-CD28 as an active ingredient, and pharmaceutically acceptable excipients or auxiliary ingredients; or the pharmaceutical composition is prepared with a raw material composition of a pharmaceutically acceptable salt of the high-affinity fusion protein HAPD-1-CD28 as an active ingredient, and pharmaceutically acceptable excipients or auxiliary ingredients.

[0039] Preferably, the pharmaceutical composition further comprises a small molecule compound that promotes the expression of PD-L1 in tumor cells.

[0040] Preferably, the high-affinity fusion protein HAPD-1-CD28 can be directly expressed in CAR-T / TCR-T cells via a viral vector, and used in combination with the small molecule compound that promotes the expression of PD-L1 in tumor cells; or the endogenous PD-1 can be directly replaced with PD-1-CD28 or HA PD-1-CD28 by gene editing, and used in combination with the small molecule compound that promotes the expression of PD-L1 in tumor cells.

[0041] Preferably, the small molecule compound that promotes the expression of PD-L1 in tumor cells includes any one or more of an LSD1 inhibitor, a UHRF1 inhibitor, and a cytokine that can promote the expression of PD-L1 in tumor cells using IFN-γ.

[0042] On this basis, the present invention combines CAR-T or TCR-T cells containing PD-1-CD28 and HAPD-1-CD28 with LSD1 inhibitors or other small molecule compounds that can promote tumor cells to express PD-L1. This combined use can significantly enhance the anti-tumor activity of T cells. Since HAPD-1-CD28 can further promote the expression of endogenous PD-1 in T cells, the present invention knocks out endogenous PD-1 while overexpressing the HAPD-1-CD28 fusion gene, or directly replaces endogenous PD-1 with HAPD-1-CD28 in situ, which can further enhance the anti-tumor activity of T cells.

[0043] The present invention also provides the use of the high-affinity fusion protein HAPD-1-CD28 or the pharmaceutical composition in preparing a drug for preventing and / or treating solid tumors and leukemia tumors. The present invention also provides use of the high-affinity fusion protein HAPD-1-CD28 or the pharmaceutical composition in preparing a drug for preventing and / or treating autoimmune diseases.

[0044] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a high-affinity fusion protein HAPD-1-CD28. The high-affinity fusion protein HAPD-1-CD28 provided by the present invention is obtained by modifying the amino acid sequence of the PD-1-CD28 fusion protein. The modification refers to replacing the amino acid at any site in the extracellular domain of the PD-1-CD28 fusion protein with any amino acid.

[0045] The high-affinity fusion protein HAPD-1-CD28 provided by the present invention enhances the binding of PD-1-CD28 fusion protein and tumor cell PD-L1, enhances the long-term anti-tumor activity of T cells, inhibits T cell exhaustion and enhances the plasticity of T cell skeleton. The high-affinity fusion protein HAPD-1-CD28 provided by the present invention can solve the problem that the ordinary PD-1-CD28 fusion protein in the prior art has limited effect in enhancing the anti-tumor activity of CAR-T / TCR-T cells.

[0046] 2. The present invention provides a high-affinity fusion protein, which is named high affinity PD-1-CD28 fusion protein (HAPD-1-CD28 fusion protein). After amino acid sequence optimization, the affinity of PD-1 and PD-L1 is improved, and then the N-terminus of HAPD-1 and the intracellular domain of CD28 are fused. HAPD-1 contains an optimized PD-1 extracellular domain and / or transmembrane domain.

[0047] After sequence optimization, it can be seen that compared with ordinary T cells (expressing GFP only) and T cells overexpressing PD-1-CD28 (overexpressing PD-1-CD28-T2A-GFP), the affinity of T cells overexpressing HAPD-1-CD28-T2A-GFP and tumor cells expressing PD-L1-T2A-mCherry is significantly increased, and the morphology of T cells overexpressing HAPD-1-CD28 is significantly changed. This phenomenon provides a solution to the current problem that T cells cannot infiltrate into solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 In the present invention, overexpression of PD-1-CD28 and HA PD-1-CD28 in T cells or CAR-T cells can promote the binding of T cells and tumor cells and promote the release of cytokines by T cells; wherein, Figure 1 Figure A is a picture of PD-1 / PD-L1 binding. After T cells were infected with EGFP, PD-1-CD28-T2A-EGFP or HAPD-1-CD28-T2A-EGFP virus through retrovirus, each group of T cells was co-cultured with solid tumor cells (A673) for 5 hours, and the binding of different groups of T cells and tumor cells was analyzed by microscopy. Figure 1 Panel B is a picture of T cells overexpressing normal affinity PD-1-CD28-T2A-EGFP or HAPD-1-CD28-T2A-EGFP by retrovirus co-cultured with A673 cells expressing mCherry-PD-L1 (10X); Figure 1 Figure C in Figure 1 is a picture of Figure B in which T cells overexpressing normal affinity PD-1-CD28-T2A-EGFP or HAPD-1-CD28-T2A-EGFP were co-cultured with A673 cells expressing mCherry-PD-L1 (40X). It can be seen that after co-culture with A673 cells expressing mCherry-PD-L1, the morphology of T cells overexpressing HAPD-1-CD28-T2A-EGFP changed significantly; Figure 1 Figure D shows the binding rate of different cells to tumor cells after T cells overexpressing EGFP, normal affinity PD-1-CD28-T2A-EGFP or HAPD-1-CD28-T2A-EGFP were co-cultured with A673 cells expressing mCherry-PD-L1. Among them, the binding rate of HAPD-1-CD28-T2A-EGFP T cells to A673 cells expressing mCherry-PD-L1 was the highest; Figure 1Figure E is a picture of T cells overexpressing normal affinity PD-1-CD28-T2A-EGFP or HAPD-1-CD28-T2A-EGFP co-cultured with K562 cells expressing mCherry-PD-L1 (10X); Figure 1 Figure F is a picture of T cells overexpressing normal affinity PD-1-CD28-T2A-EGFP or HAPD-1-CD28-T2A-EGFP co-cultured with K562 cells expressing mCherry-PD-L1 (40X). It can be seen that after co-culture with K562 cells expressing mCherry-PD-L1, the morphology of T cells overexpressing HAPD-1-CD28-T2A-EGFP changed significantly; Figure 1 Figure G in the figure shows the binding rate of different cells to tumor cells after T cells overexpressing EGFP, normal affinity PD-1-CD28-T2A-EGFP or HAPD-1-CD28-T2A-EGFP were co-cultured with K562 cells expressing mCherry-PD-L1. Among them, the binding rate of HAPD-1-CD28-T2A-EGFP T cells to K562 cells expressing mCherry-PD-L1 was the highest; Figure 1 Figure H shows the extent of TNF-α release detected by ELISA after nonspecific T cells (Unspecific T), CAR-T cells (anti-CD22), CAR-T cells overexpressing PD-1-CD28 (anti-CD22), and CAR-T cells overexpressing HA PD-1-CD28 (anti-CD22) were co-cultured with tumor cells for 16 hours.

[0049] Figure 2 The HA PD-1-CD28 in the present invention can significantly enhance the anti-tumor activity of CAR-T / TCR-T cells and promote the further expression of PD-L1 in tumors; wherein, Figure 2 Figure A shows the nonspecific T cells from the first donor. After TCR-T, PD-1-CD28 TCR-T or HA PD-1-CD28 TCR-T cells were co-cultured with tumor cells for 24 hours, the relative activity and number of tumors were detected by SRB. Figure 2 Figure B shows the nonspecific T cells from the second donor. After TCR-T, PD-1-CD 28TCR-T or HA PD-1-CD 28 TCR-T cells were co-cultured with tumor cells for 24 hours, the relative activity and number of tumors were detected by SRB; Figure 2Figure C shows nonspecific T cells from the second donor. After CAR-T, PD-1-CD28 CAR-T or HAPD-1-CD 28 CAR-T cells were co-cultured with tumor cells for 24 hours, the relative activity and number of tumors were detected by SRB. Among them, the anti-tumor activity of HA PD-1-CD 28 TCR-T or HA PD-1-CD 28 CAR-T cells was significantly increased; Figure 2 Figure D shows that after nonspecific T cells, CAR-T cells (anti-CD22), CAR-T cells overexpressing PD-1-CD28 (anti-CD22), and CAR-T cells overexpressing HA PD-1-CD28 (anti-CD22) were co-cultured with tumor cells for 16 hours, the expression of PD-L1 in tumor cells was detected by flow cytometry. Among them, HA PD-1-CD28 CAR-T cells can significantly increase the expression of PD-L1 in tumor cells.

[0050] Figure 3 In the present invention, knocking out or inhibiting LSD1 expression can promote the expression of tumor PD-L1, and TCR-T cells overexpressing PD-1-CD28 have synergistic anti-tumor activity with LSD1 inhibitors: wherein, Figure 3 Figure A shows the expression level of LSD1 detected by WB after silencing LSD1 expression; Figure 3 Figure B shows the expression levels of immune-related genes such as PD-L1 detected by RT-Q-PCR after silencing LSD1; Figure 3 Figure C shows that after silencing tumor LSD1 expression, PD-1-CD28 TCR-T cells were co-cultured with tumor cells for 24 hours, and tumor cell activity was detected by SRB; Figure 3 Figure D shows the expression levels of genes such as PD-L1 detected by RT-Q-PCR after LSD1 inhibited SP2577 and treated Ewing sarcoma cells (A673) with 1um and 2um, respectively; Figure 3 Figure E shows the expression levels of genes such as PD-L1 detected by RT-Q-PCR after LSD1 inhibited SP2577 at 1um and 2um to treat Ewing sarcoma cells (RD-ES); Figure 3 Figure F shows the activity of tumor cells detected by SRB 24 hours after the LSD1 inhibitor SP2577 was combined with nonspecific T cells (UnspecificT), CAR-T cells (anti-CD22), CAR-T cells overexpressing PD-1-CD28 (anti-CD22), and CAR-T cells overexpressing HA PD-1-CD28 (anti-CD22) to kill tumor cells.

[0051] Figure 4The anti-tumor activity of HAPD-1-CD28CAR-T cells in tumor-bearing mice in the present invention; wherein, Figure 4 Figure A in the middle shows the tumor volume recorded regularly after T cell injection; Figure 4 Figure B in the middle shows photos of tumors in different groups after the mice were dissected. It can be seen that HA PD-1-CD28 CAR-T cells significantly inhibited tumor proliferation in vivo; Figure 4 Figure C in the middle shows the tumor weights of different groups after the mice were dissected; Figure 4 Figure D in the middle shows the weight changes of mice before and after T cell injection; Figure 4 Figure E in the middle shows the analysis of organ coefficients of different organs after dissecting mice, and it was found that all groups of CAR-T cells had no organ toxicity. DETAILED DESCRIPTION

[0052] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0053] Materials and methods: 1. Tumor cell and packaging cell culture: Tumor cells include A673, RD-ES, Raji, A673 cells and Raji cells are from the cell bank of the Chinese Academy of Sciences, packaging cells 293 Vec -RD114 cells were provided by Professor Stefan Burdach of the Technical University of Munich; RD-ES cells were provided by Professor He Jinxue of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences and stored in this laboratory (Hematology Laboratory, The First Affiliated Hospital of Xi'an Jiaotong University). Culture conditions: A673, RD-ES and 293 Vec -RD114 cell culture system consists of DMEM medium (gibco, China), 10% fetal bovine serum (FBS) (Sigma, Germany), 5% L-glutamine / penicillin / streptomycin (New Cyme, China), and non-essential amino acids (Biosharp, China); Raji cell culture system consists of RPMI-1640 medium (gibco, China), 10% fetal bovine serum (FBS) (Sigma, Germany), 5% L-glutamine / penicillin / streptomycin (New Cyme, China), and non-essential amino acids (Biosharp, China).

[0054] 2. CAR-T / TCR-T cell preparation: 2.1. T cells were derived from healthy donors. PBMC (peripheral blood mononuclear cells) were separated from the peripheral blood of healthy donors by density gradient centrifugation. T cells were separated by CD3 beads (Miltenyi, Germany) and cultured in T cell culture medium. The incubator conditions were 37°C and 5% CO 2 .

[0055] Among them, the source of normal healthy donors is the mobilized peripheral blood of hematopoietic stem cell transplant donors.

[0056] PBMC separation from peripheral blood: 3 mL of normal healthy donor peripheral blood was diluted to 25 mL with purchased DPBS buffer (Biyuntian China), and the diluted peripheral blood was added to the 15 mL lymphocyte separation liquid surface after room temperature equilibrium along the inclined tube wall, and centrifuged at 2200 rpm for 30 minutes. The speed of the centrifuge was 9 up and 1 down. After the centrifugation, the buffy coat layer was collected, and DPBS was added to dilute it to 40 mL. The centrifuge was centrifuged at 700 rpm for 7 minutes. The speed of the centrifuge was 9 up and 9 down. After the centrifugation, the supernatant was aspirated, 15 mL of red blood cell lysis buffer (Solabo China) was added, and it was allowed to stand for 15 minutes. DPBS was added to mix and centrifuged, and the precipitate was collected, resuspended and counted to obtain PBMC.

[0057] The composition system of T cell culture medium is AIM-V (Life Techhology, Germany), 10% fetal bovine serum (FBS) (Sigma, Germany) by volume, 5% L-glutamine / penicillin / streptomycin (Sinosemi, China) by volume, non-essential amino acids (Biosharp, China), 100 IU / mL IL-2 (R&D, USA), 5 ng / mL IL-7 (R&D, USA) and 2 ng / mL IL-15 (R&D, USA).

[0058] 2.2. CAR-T / TCR-T cell preparation process: (1) Retroviral packaging: 1) Prepare to use 293 of 16 generations Vec -RD114 cells were transfected when the cell confluence reached 75%.

[0059] 2) This protocol uses a single plasmid system, using the pMP-71 vector (provided by Professor Stefan Burdach of the Technical University of Munich) to express the target gene PD-1-CD28 or HA PD-1-CD28. Use 250 μL of purchased Opti-MEM medium (gibco, USA) to dilute 2 ug of plasmid and 6 μL of TransIT. After standing at room temperature for 20 minutes, mix and stand for another 30 minutes.

[0060] 3) Use 2.5 mL of DMEM medium containing 10% FBS by volume to feed 293 Vec -RD114 cells were replaced with Opti-MEM medium, and then 293 Vec -RD114 cells. The virus supernatant was collected / filtered at 48 hours and 72 hours, added to T cells, and cultured after centrifugation at 32°C and 2200 rpm.

[0061] 4) Flow cytometry (Agilent, USA) was used to detect the tag protein cmyc contained in the target gene to analyze the viral infection efficiency.

[0062] (2) Plasmid preparation: pMP-71 vector was used, and the relevant target gene sequences (EGFP, PD-1-CD28-T2A-EGFP, HAPD-1-CD28-T2A-EGFP, CAR, PD-1-CD28-T2A-CAR, HAPD-1-CD28-T2A-CAR, TCR, PD-1-CD28-T2A-TCR and HAPD-1-CD28-T2A-TCR) were placed in the restriction endonuclease Not I and EcoR I restriction site.

[0063] Among them, restriction endonucleases Not I and EcoR I were purchased from NEB.

[0064] DNA ladder was purchased from Thermo Corporation.

[0065] Plasmid extraction and gel recovery kits were purchased from Tiangen Biotechnology Co., Ltd.

[0066] Stbl3 competent Escherichia coli was purchased from Qingke Technology Biological Co., Ltd.

[0067] 3. SRB detection of tumor cell activity: (1) Detection principle: SRB is a water-soluble protein dye that can bind to basic amino acids of biological macromolecules. The amount of SRB bound to cells can reflect the total protein amount and thus the number of cells. The OD value at a wavelength of 515nm shows a good linear relationship with the number of living cells.

[0068] (2) Brief steps for SRB detection of A673 tumor cell activity: A673 tumor cells were plated and T cells were added; according to the growth rate of A673 tumor cells, adherent tumor cells in the logarithmic growth phase were inoculated into 96-well culture plates (Select, China) at 100 μL / well. After observing the adherent growth for 24 hours, different groups of T cells with different effector-target ratios were added at 100 μL / well, and 3 replicates were set for each concentration. Nonspecific T cells were used as the control group, and the group without T cells was used as the cell-free zero well. The cells were incubated at 37°C and 5% CO 2 Cultured under the conditions for 24 h.

[0069] 1) Fixation: Before fixation of A673 cells, the cells were first slowly washed three times with preheated PBS (pH 7.3 ± 0.1, Pronocell, China) to remove the culture medium, T cells and apoptotic A673 tumor cells. Then, 50 μL of 10% (m / v) trichloroacetic acid (TCA) (Aladdin, China) was added to each well to fix the cells and incubated at 4°C overnight.

[0070] Overnight refers to a culture time of ≥12 h.

[0071] 2) Washing: Discard the fixative, wash 5 times with distilled water, and dry naturally in the air.

[0072] 3) Staining: After drying in air, add 100 μL of lissamine rhodamine B (SRB) (Aladdin, China) solution to each well and leave at room temperature for 60 min.

[0073] 4) Washing: Remove the supernatant, wash five times with 1% by volume acetic acid, and air dry.

[0074] 5) Dissolution: Finally, add 150 μL / well of 10 mM Tris solution and shake on a plate shaker for 15 minutes.

[0075] 6) Measurement: Measure the OD value in an enzyme-linked immunosorbent assay (ELISA) and adjust the value to zero using a blank control. The wavelength used is 515 nm.

[0076] 7) Calculation: Calculate tumor cell activity and compare T cell anti-tumor activity according to the following formula: Tumor cell activity = [OD 515 Control well / OD 515 control wells] × 100%.

[0077] 4. PCR detection of tumor PD-L1 and other gene expressions: To detect the involvement of LSD1 in the expression of tumor immune-related genes, after silencing LSD1 expression, tumor cells were digested with 0.25% trypsin by mass volume, and RNA of tumor cells was extracted using an RNA extraction kit (Shanghai Feljet Biotechnology Co., Ltd., China).

[0078] Among them, the cell line with silenced LSD1 expression was constructed: Lentivirus packaging and transfection: 1) Prepare 293T cells at passage 13 and start virus packaging when the cell confluence reaches 80%.

[0079] 2) According to the three-plasmid system of lentiviral packaging, use the expression plasmid Tet-pLKO-puro-human- LSD1: packaging plasmid psPAX2: envelope plasmid pMD2.G = 1:1.2:1.2 mass ratio to prepare plasmid dilution solution, dilute 2ug plasmid and 4ul Lipo2000 with 250μL purchased Opti-MEM medium (gibco, USA). Let stand at room temperature for 5 minutes, mix, and let stand for another 15 minutes.

[0080] 3) Replace the 293T cells with 2.5 mL of DMEM medium containing 10% FBS by volume, then add the Opti-MEM medium dropwise to the 293T cells after standing for 15 minutes and place them in a cell culture incubator. Collect / filter the viral supernatant at 48 hours and 72 hours, and infect A673 cells.

[0081] 4) The infected A673 cells were cultured in DMEM complete medium containing puromycin (working concentration 1ug / mL, Beyotime, China) and 10% FBS. After 2 weeks of screening, all the empty A673 cells died under the action of puromycin. The infected cells were screened and amplified with a medium containing screening resistance (DMEM complete medium containing 1ug / mL puromycin). DMEM complete medium containing Doxycycline (working concentration 1ug / mL, Beyotime, China) was used to induce LSD1 silencing. The LSD1 expression level was detected by WB to obtain an A673 cell line with stable LSD1 silencing.

[0082] Wherein, the sequence of human-sh LSD1 is shown in SEQ ID NO.21: CCGGGCCTAGACATTAAACTGAATACTCGAGTATTCAGTTTAATGTCTAGGCTTTTTT.

[0083] The extracted RNA was reverse transcribed into cDNA using a reverse transcription kit (Takara, Japan). The expression of target genes (PD-L1, OAS2, IFITM1, IFIT2, IFIH1, B2M, HLA-A, HLA-B, HLA-C and DDX58, (primers were purchased from Qingke Biotechnology Co., Ltd.)) was detected respectively.

[0084] After A673 cells or RD-ES cells were treated with DMSO, 1uM or 2uM SP2577 for 48 hours, the cells were digested with 0.25% trypsin by mass volume ratio, and RNA was extracted using an RNA extraction kit, which was reverse transcribed into cDNA using a reverse transcription kit to detect the expression of the above target genes.

[0085] Among them, LSD1 inhibitor SP2577 was purchased from MCE.

[0086] 5. Tumor-bearing mouse experiment: 4-6 weeks old immunodeficient female NCG mice were purchased from Jicui Pharmaceutical (Nanjing, China) and raised in an SPF environment. All animal experiments were in accordance with the ARRIVE guidelines and were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

[0087] Subcutaneous inoculation of 1×10 5 Four days later, 5 × 10 Raji cells were injected via the tail vein. 6 Tumor growth was monitored 3 times a week by measuring tumor size with calipers.

[0088] The calculation formula for subcutaneous tumor size is as follows: Subcutaneous tumor size = d1 × (d2) 2 ×0.52; In the above formula, d1 is the longest diameter and d2 is the shortest diameter perpendicular to d1.

[0089] When mice lose weight significantly, tumors ulcerate, or tumors are larger than 1500 mm 3 euthanasia was performed on him.

[0090] Example 1: Preparation of high affinity fusion protein Use 293 Vec -RD114 packaging cells were used to prepare retrovirus expressing GFP or PD-1-CD28-GFP or HA PD-1-CD28-GFP, and the virus was used to infect T cells to express GFP protein, PD-1-CD28 and GFP protein or HA PD-1-CD28 and GFP protein, and the binding of T cells and tumor cells was observed under a fluorescence microscope.

[0091] Example 2: SRB staining, ELISA and flow cytometry to detect T cell anti-tumor activity In the in vitro study of whether HA PD-1-CD28 can enhance the anti-tumor activity of CAR-T and TCR-T cells in the present invention, strategies such as SRB staining, ELISA and flow cytometry were mainly used.

[0092] In order to analyze whether HAPD-1-CD28 enhances the anti-tumor activity of T cells, the present invention co-cultured T cells overexpressing HAPD-1-CD28-T2A-CAR with tumor cells in a 37°C incubator for 24 hours, collected the supernatant, and detected the release of TNF-α by ELISA. After T cells and tumor cells were co-cultured for 24 hours, the co-culture supernatant was collected, and the secretion level of TNF-α in the supernatant was detected. It was found that HAPD-1-CD28 can significantly enhance the release of TNF-α in T cells ( Figure 1 Figure H in the figure).

[0093] The above experimental results suggest that HAPD-1-CD28 can promote the anti-tumor activity of T cells. After co-culture with A673 tumor cells, we also observed that CAR-T cells overexpressing HAPD-1-CD28 can further promote the expression of PD-L1 by A673 tumor cells, and A673 tumor cells with high expression of PD-L1 can further enhance the anti-tumor activity of T cells ( Figure 2 ).

[0094] Example 3: TCR-T cells overexpressing PD-1-CD28 have synergistic anti-tumor activity with LSD1 inhibitors Considering that HA PD-1-CD28 can both reverse T cell exhaustion and enhance affinity with PD-L1, and promote the expression of PD-L1 in tumor cells, we believe that promoting overexpression of PD-L1 in tumor cells can enhance the anti-tumor activity of T cells. To test the feasibility of this approach, the following experiments were conducted: The present invention prepares a cell line for silencing LSD1 expression. It is found that silencing LSD1 can promote the expression of PD-L1 in tumor cells, and the use of LSD1 inhibitors can also promote the expression of PD-L1 in tumor cells.

[0095] After TCR-T / CAR-T cells, PD-1-CD28TCR-T / CAR-T cells and HA PD-1-CD28 TCR-T / CAR-T cells were successfully prepared using the above retroviral packaging and infection method, they were co-cultured with Ewing sarcoma tumor cells after doxcycline-induced LSD1 silencing in vitro. It was found that after LSD1 silencing, HA PD-1-CD28 TCR-T cells had the strongest anti-tumor activity compared with TCR-T cells ( Figure 3 Figure F in the figure).

[0096] Case study 4: HA PD-1-CD28 CAR-T has significant anti-tumor activity in vivo To further verify that HA PD-1-CD28 CAR-T has good anti-tumor activity in vivo, mice were subcutaneously tumor-bearing (Raji) for 4 days and then injected with PBS, CAR-T, PD-1-CD28 CAR-T or HA PD-1-CD28 CAR-T cell suspension (5x10 6 The tumor volume and mouse body weight were measured every 3 days. The tumor weight and organ coefficient of the mice were analyzed on the 36th day.

[0097] Among them, organ coefficient = (organ wet weight / mouse body weight) × 1000.

[0098] The results are as follows Figure 4 shown.

[0099] Compared with CAR-T cells and PD-1-CD28 CAR-T cells, HA PD-1-CD28 CAR-T cells can significantly inhibit tumor proliferation ( Figure 4 Figure A in Figure 4 C in Figure ). There was no significant change in organ coefficients and mouse weight during the treatment, indicating that PD-1-CD28 CAR-T cells are safe and effective ( Figure 4 Figure D in Figure 4 Figure E in the figure).

[0100] From the above experimental results, it can be seen that the use of PD-1-CD28 fusion protein can enhance the anti-tumor activity of CAR-T / TCR-T cells, and modifying the amino acid sequence of the PD-1 extracellular region to increase the affinity of PD-1-CD28 and PD-L1 can further reverse the exhaustion of T cells and enhance the anti-tumor activity of CAR-T / TCR-T cells; the PD-1-CD28 fusion gene with high affinity to PD-L1 has synergistic anti-tumor activity with the LSD1 inhibitor. The high-affinity fusion protein HAPD-1-CD28 provided by the present invention can solve the problem that the ordinary PD-1-CD28 fusion protein in the prior art has limited effect in enhancing the anti-tumor activity of CAR-T / TCR-T cells.

[0101] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0102] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. A high-affinity fusion protein HAPD-1-CD28, characterized in that: The high-affinity fusion protein HAPD-1-CD28 is obtained by performing partial replacement of amino acids on the basis of the PD-1-CD28 fusion protein; The PD-1-CD28 fusion protein consists of an extracellular domain, a transmembrane domain and an intracellular domain; The extracellular domain comprises a polypeptide derived from PD-1 at its N-terminus; The transmembrane domain is derived from the transmembrane domain of PD-1; The intracellular domain comprises a polypeptide derived from CD28 at its C-terminus; The partial replacement of amino acids refers to replacing the amino acid at any site in the extracellular domain of the PD-1-CD28 fusion protein with any of the following amino acids: Glycine, alanine, valine, leucine, isoleucine, methionine, tryptophan, tyrosine, asparagine, glutamine, threonine, glutamic acid, lysine, arginine and histidine.

2. The high-affinity fusion protein HAPD-1-CD28 according to claim 1, characterized in that: The amino acid sequence of the high-affinity fusion protein HAPD-1-CD28 is shown in at least one of SEQ ID NO.2 to SEQ ID NO.

20.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleotide sequence encoding the high-affinity fusion protein HAPD-1-CD28 as claimed in claim 1.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the high-affinity fusion protein HAPD-1-CD28 according to claim 1, or a raw material composition of a pharmaceutically acceptable salt of the high-affinity fusion protein HAPD-1-CD28.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition is prepared by using the high-affinity fusion protein HAPD-1-CD28 as an active ingredient and pharmaceutically acceptable excipients or auxiliary ingredients; or the pharmaceutical composition is prepared by using a raw material composition of a pharmaceutically acceptable salt of the high-affinity fusion protein HAPD-1-CD28 as an active ingredient and pharmaceutically acceptable excipients or auxiliary ingredients.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition also includes a small molecule compound that promotes the expression of PD-L1 in tumor cells.

7. The pharmaceutical composition according to claim 6, characterized in that The small molecule compound that promotes the expression of PD-L1 in tumor cells includes any one or more of LSD1 inhibitors, UHRF1 inhibitors, and cytokines that can promote the expression of PD-L1 in tumor cells using IFN-γ.

8. Use of the high-affinity fusion protein HAPD-1-CD28 according to claim 1 or the pharmaceutical composition according to claim 4 in the preparation of a drug for preventing and / or treating solid tumors and leukemia tumors.

9. Use of the high-affinity fusion protein HAPD-1-CD28 according to claim 1 or the pharmaceutical composition according to claim 4 in the preparation of a medicament for preventing and / or treating autoimmune diseases.

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