Application of IFN-gamma (interferon-gamma) in preparation of anti-tumor adjuvant drug

Abstract

The invention provides an application of IFN-gamma in preparation of an anti-tumor adjuvant drug. The IFN-gamma enhances the killing effect of a T cell preparation on tumor cells by sensitizing the tumor cells; the T cell preparation comprises T cells without gene modification and / or T cells modified by genetic engineering means; the IFN-gamma comprises a wild type or mutant IFN-gamma full length or an IFN-gamma fragment. According to the application disclosed by the invention, cognition on IFN-gamma in the prior art is broken through, and it is found that the IFN-gamma inhibits PD-L1-PD-1 mediated acquired immune resistance by activating an IFN-gamma signal channel in tumor cells, and the anti-tumor effect of immunotherapy is enhanced.

Description

[0001] This application is a divisional application based on the Chinese invention patent application with application number 202180019514.X, application date March 3, 2021, and invention name “Application of IFN-γ in the preparation of anti-tumor adjuvant drugs”.

[0002] Priority application

[0003] This application claims the priority of the Chinese invention patent [202010159630.9] filed on March 9, 2020, entitled “Application of IFN-γ in the preparation of anti-tumor adjuvant drugs”, and the priority of the Chinese invention patent [202010487110.0] filed on June 1, 2020, entitled “A CAR expression vector and its application”, the entire text of which is incorporated by reference. Technical Field

[0004] The present invention belongs to the field of biomedicine, and specifically relates to an anti-tumor drug for assisting anti-tumor immunotherapy and its application. Background Art

[0005] Tumor immunotherapy is a revolutionary advance in anti-tumor therapy, which fights tumors by mobilizing the body's own immune function. CAR-T therapy belongs to the field of immunotherapy. CAR-T cells have achieved surprising results in the treatment of blood tumors targeting CD19. As of April 2018, there were a total of 2,513 immunotherapy treatments for tumors, 382 of which were in China, and 227 of which entered clinical phases III and IV. Novartis' Kymriah and Kite's Yescarta have been launched, which are used to treat childhood acute B-cell leukemia and adult B-cell lymphoma, respectively. Among them, Kymriah has an overall remission rate (CR) of 82.5%, and the probability of relapse is 75% at 6 months and 64% at 12 months. Yescarta, another CD19-specific CAR-T product approved by the FDA, can achieve 43-52% CR in the treatment of patients with relapsed or refractory DLBCL.

[0006] However, the therapeutic effect of CAR-T on solid tumors is not good. For example, in a phase I clinical study of EGFR-CAR-T on epidermal growth factor receptor cells for the treatment of relapsed / refractory EGFR+ non-small cell lung cancer, only 2 responders and 5 patients with stable disease (SD) were found among 11 cancer patients. In a phase I / II study in a dose-escalation clinical trial, the feasibility and safety of HER2-CAR-T cells in patients with relapsed or refractory HER2-positive tumors were evaluated. However, its clinical benefit was limited, with only 4 / 17 evaluable patients showing SD. In other words, the therapeutic effect of CAR-T cells on solid tumors is very limited. The reasons why CAR-T cells are not effective against solid tumors are not very clear at present, but it is speculated that there may be many reasons, such as poor persistence of CAR-T cells in the body, and they die quickly after being injected into the body, which affects the killing effect; solid tumors will form an immunosuppressive microenvironment due to their multipotency in metabolism, immune escape, and tissue formation, making it difficult for CAR-T cells to infiltrate into solid tumor tissues to exert their effectiveness. Even if they enter the solid tumor, CAR-T cells will be inactivated and die due to restrictions on the immune system in many aspects. Therefore, how to enhance and improve the therapeutic effect of CAR-T cells against solid tumors is an urgent problem to be solved in the existing technology.

[0007] As an inhibitory molecule in the costimulatory signal, PD-1 was first discovered to induce T cell apoptosis, thereby preventing the immune system from killing cancer cells. In addition, PD-1 can negatively regulate T cell-mediated immune responses by binding to its ligand, programmed death ligand 1 (PD-L1), that is, tumor cells "deceive" T cells and escape T cell recognition by binding to PD-L1. Therefore, blocking this signaling pathway is considered to be an effective cancer immunotherapy. Since the approval of the first PD-1 inhibitors pembrolizumab and nivolumab in 2014, the clinical development of PD-1 / PD-L1 inhibitors as a form of cancer immunotherapy has seen unprecedented growth. PD-1 and PD-L1 inhibitors can be used in clinical trials for a variety of cancer types, including: advanced melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, Hodgkin lymphoma, liver cancer, head and neck squamous cell carcinoma, urothelial carcinoma, Merkel cell carcinoma, etc. So far, three PD-1 inhibitors have been launched overseas: Pembrolizumab, Nivolumab, and Cemiplimab; and four have been launched in China: Toripalimab, Tislelizumab, Tyvyt, and Camrelizumab. The PD-L1 inhibitors that have been launched are: Atezolizumab, Avelumab, and Durvalumab. When PD-L1 / PD-1 therapy is applied to more solid tumors, how to make PD-L1 / PD-1 therapy produce better anti-tumor therapeutic effects is an urgent problem to be solved.

[0008] Adoptive cell therapy (ACT) is an effective anti-cancer strategy, known as a "living drug", and plays an important role in cancer immunotherapy. This therapy involves isolating immune-active cells from tumor patients, amplifying and functionally identifying them in vitro, and then returning them to the patient to directly kill the tumor or stimulate the body's immune response to kill the tumor. Currently, ACT mainly includes TIL, LAK, CIK, DC, NK, TCR-T, and CAR-T.

[0009] In order to enhance the therapeutic effect of CAR-T cells, the current ideas are mainly focused on further genetic engineering or nano-modification of CAR-T cells themselves. For example, inserting multi-target CAR genes into CAR-T cells, optimizing the CAR structure to effectively affect T cell proliferation and persistence; or making CAR-T cells express chemokines, or directly modifying adhesion molecules or chemical groups to enhance the tendency and recognition of CAR-T cells to tumor tissues. However, these technical means all have some problems and shortcomings to a greater or lesser extent. Taking multi-target CAR-T cells as an example, too many inserted exogenous genes may affect the original activity of T cells. Therefore, the modification of CAR-T cells must comprehensively consider multiple positive and negative factors, and it is necessary to compare and balance the positive and negative effects of the relevant modifications, and finally choose a modification method with stronger targeting and better effect.

[0010] Studies have shown that PD-1, as an inhibitory signaling pathway, is an important factor affecting the therapeutic effect of CAR-T cells, and blocking the PD-1 pathway can enhance the anti-tumor effect of CAR-T cells. Therefore, technical means that can inhibit PD-1 are considered to be a feasible direction for CAR-T modification.

[0011] In the existing technical routes, attempts to make CAR-T cells secrete PD-1 or PD-L1 antibodies through genetic engineering can significantly improve the therapeutic effect on solid tumors, or knocking out the PD-1 coding gene in CAR-T cells through gene editing can also improve the anti-tumor activity of CAR-T.

[0012] However, the ideas of the above-mentioned technical solutions and technical routes are all aimed at improving the function of CAR-T cells themselves, and have little effect on tumor cells and the immunosuppressive microenvironment in solid tumors. In other words, the current technical solutions and technical routes only focus on improving CAR-T cells themselves, but do not further enhance the anti-tumor effect of CAR-T cell therapy from the perspective of acting on tumor cells.

[0013] In summary, how to improve the anti-tumor effects of existing technologies such as immune cell therapy and / or immune checkpoint inhibitor therapy and / or adoptive cell therapy, and propose more optimized and more effective anti-tumor treatment plans, is a very important issue in the current field of tumor immunotherapy, and is also a technical problem that the present invention attempts to solve. In addition, the present invention also aims to affect tumor cells and the immunosuppressive microenvironment in solid tumors, and provide an optimized CAR-T cell therapy strategy. Summary of the invention

[0014] In response to the above technical problems, the present invention is centered around type II interferon (IFN-γ), provides anti-tumor adjuvant drugs prepared therefrom and other applications, and includes two sub-inventions (i.e., invention A and invention B), as well as respective expansions based on these two inventions.

[0015] One aspect of the present invention provides an application of IFN-γ in the preparation of an auxiliary anti-tumor drug; a kit for detecting the anti-tumor effect of an immune checkpoint inhibitor; an anti-tumor drug for auxiliary anti-tumor immunotherapy; an application of IFN-γ in combination with a T cell preparation in the preparation of a tumor immunotherapy drug; an application of IFN-γ in combination with an immune checkpoint inhibitor in the preparation of a tumor immunotherapy drug; an application of IFN-γ, a T cell preparation and an immune checkpoint inhibitor in the preparation of a tumor immunotherapy drug; and a combination drug combination.

[0016] Another aspect of the present invention also provides a novel CAR expression vector modified with IFN-γ and a construction strategy thereof.

[0017] Beneficial Effects

[0018] The present invention breaks through the prior art understanding of IFN-γ and finds that IFN-γ activates the IFN-γ signaling pathway in tumor cells, for example by upregulating the expression of ICAM-1 in tumor cells, thereby sensitizing tumor cells to immunotherapy (T cell preparations and / or PD-L1 / PD-1 inhibitors), inhibiting PD-L1-PD-1-mediated acquired immune resistance and enhancing the anti-tumor effect of immunotherapy. A technical solution is then proposed for using IFN-γ as an adjuvant drug for immunotherapy in anti-tumor treatment.

[0019] Interferon (IFN) is composed of a cluster of secreted proteins. The IFN protein family is divided into three types based on their gene sequence, chromosomal location and receptor specificity, namely type I, type II and type III interferons. Among them, type II interferons are composed of the single gene family IFN-γ, also known as immune interferons. Under normal circumstances, the intracellular content of IFN-γ is very low, but the expression level is significantly increased after induction by viruses or other interferon inducers. IFN-γ is the most important cytokine involved in anti-tumor immunity and is an immune activation cytokine released after CAR-T contacts antigens. In order to avoid damage from activated immune responses, normal tissues will induce multiple regulatory pathways to promote the reconstruction of the balance of cells in the body under inflammatory conditions. Existing studies believe that tumor cells will use these protective regulatory pathways for immune escape. For example, tumor cells will upregulate PD-L1 expression by releasing IFN-γ, thereby inhibiting T cells from killing them. This acquired immune resistance is considered to be one of the possible reasons that restrict CAR-T from effectively killing solid tumors. Because IFN-γ upregulates PD-L1 expression and induces acquired immune resistance in tumor cells, the existing immunotherapy technology schemes have not fully utilized IFN-γ. Even the IFN-γ anti-tumor treatment scheme that entered clinical trials in the early years failed to become an anti-tumor drug on the market due to poor results. It is now believed that it is because IFN-γ upregulates PD-1 expression, resulting in poor anti-tumor effects. Therefore, it is believed that if IFN-γ is to produce an anti-tumor effect, PD-L1 inhibitors must be used at the same time to neutralize the negative effects of IFN-γ upregulating PD-L1. However, this is only a theoretical speculation, and no definite research results have yet to confirm it.

[0020] The experimental results of the present invention show that although IFN-γ (activating the IFN-γ signaling pathway by pre-treating tumor cells) can upregulate the expression of PD-L1 in tumor cells, IFN-γ also affects the expression of other factors (for example, upregulating the expression of ICAM-1). The result is that IFN-γ significantly enhances the cytotoxicity of CAR-T cells (or other types of genetically modified T cells) or non-genetically modified T cells to tumor cells, and IFN-γ can also enhance the sustained killing ability of CAR-T cells to tumor cells. In other words, IFN-γ can sensitize tumor cells to T cells, that is, it enhances the sensitivity of tumor cells to T cells. Not only that, the experimental results of the present invention also found that the damage of the IFN-γ signaling pathway significantly affects the killing effect of CAR-T on tumor cells.

[0021] Based on the above experimental results and reasonable inferences, the present invention proposes a technical solution for treating solid tumors with IFN-γ as an auxiliary drug combined with T cell preparations, as well as the application of IFN-γ as an auxiliary drug in anti-tumor treatment. Activating the tumor cell signaling pathway using INF-γ can enhance the anti-tumor effect of T cell preparations (including non-genetically modified T cells or T cells modified by genetic engineering means (for example, CAR-T, TCR-T)). Because the core of the present invention is to activate the IFN-γ signaling pathway of tumor cells, therefore, whether it is wild-type or mutated IFN-γ, whether it is full-length or fragmented IFN-γ, as long as it can effectively achieve the activation of the IFN-γ signaling pathway of tumor cells, it can be applied to the present invention and within the technical solution of the present invention.

[0022] In addition, because the working principle of PD-L1 inhibitors and / or PD-1 inhibitors is to activate exhausted T cells to restore their anti-tumor effects, the anti-tumor effects of PD-L1 inhibitors and / or PD-1 inhibitors are essentially mediated by T cells. Therefore, the present invention also proposes a technical solution for inhibiting tumors by enhancing PD-L1 inhibitors and / or PD-1 inhibitors through IFN-γ, as well as a technical solution for inhibiting tumors by enhancing PD-L1 inhibitors and / or PD-1 inhibitors combined with T cell preparations through IFN-γ. It should be emphasized that the technical solution proposed by the present invention is to use IFN-γ to sensitize tumor cells, thereby assisting in enhancing the anti-tumor effect of immune checkpoint inhibitors (PD-L1 inhibitors and / or PD-1 inhibitors), which is different from the technical solution of using PD-L1 inhibitors and / or PD-1 inhibitors to inhibit the downregulation of PD-L1 in tumor cells caused by the activation of the IFN-γ signaling pathway. The positioning of the main drug and auxiliary drugs is also different.

[0023] In addition, the experiments of the present invention also show that defects in the IFN-γ signaling pathway can affect the anti-tumor effect of CAR-T cells. Combined with the research results that defects in the IFN-γ signaling pathway can affect the anti-tumor effects of PD-L1 inhibitors and / or PD-1 inhibitors, the present invention also provides a method for detecting the activation / defect status of the IFN-γ signaling pathway (for example, detecting the expression of ICAM-1 or IFN-γR2 on tumor cells) to determine in advance whether T cell therapy and / or PD-L1 inhibitors and / or PD-1 inhibitors are suitable anti-tumor treatment options.

[0024] In summary, one of the contributions of the technical solution of the present invention is to break through the existing understanding of IFN-γ and propose an immunotherapy adjuvant drug with IFN-γ as the core, which enhances the anti-tumor effect of cell therapy and immune checkpoint inhibitor combined with cell therapy, and further improves the anti-tumor effect of existing immunotherapy schemes.

[0025] On the other hand, the experimental results of the present invention show that IFN-γ can increase the sensitivity of tumor cells to T cells. For example, although IFN-γ can upregulate the expression of PD-L1 in tumor cells by pre-treating tumor cells, the final result is that IFN-γ significantly enhances the cytotoxicity of CAR-T cells (or other types of genetically modified T cells, such as TCR-T cells) or unmodified T cells to tumor cells. In other words, IFN-γ can sensitize tumor cells to T cells and increase the sensitivity of tumor cells to T cells CR.

[0026] Based on this, the present invention proposes a technical solution for inhibiting tumor cells by using T cells that highly express IFN-γ (including: natural or genetically engineered T cells that highly express IFN-γ, genetically engineered T cells that highly express IFN-γ (CAR-T cells, TCR-T cells)).

[0027] Specifically, the present invention proposes a novel CAR-T cell modification scheme. In addition to activating CAR-T cells, this scheme can more importantly change the immunosuppressive microenvironment in solid tumors, thereby sensitizing tumor cells and making CAR-T cell therapy produce a stronger anti-tumor effect.

[0028] The technical solution of the present invention inserts an IFN-γ gene into the CAR expression vector, and the prepared CAR-T cells can secrete the interferon cytokine IFN-γ. IFN-γ itself is an important cytokine involved in anti-tumor immunity. It can not only activate the CAR-T cells themselves (making the CAR-T more activated after contacting tumor cells, releasing more cytokines, such as IL-2 and IFN-γ), but also sensitize tumor cells and enhance the sensitivity of tumor cells to CAR-T killing when IFN-γ is secreted into the tumor microenvironment. More importantly, the newly highly expressed molecules on IFN-γ-sensitized tumor cells, such as ICAM-1, sensitize tumor cells to immunotherapy (T cell preparations and / or PD-L1 / PD-1 inhibitors), inhibiting the inhibition of PD-L1-PD-1 on the activity of CAR-T cells. Therefore, the optimization technology provided by the present invention can further improve the therapeutic effect of CAR-T on solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art descriptions. Obviously, the drawings described below are some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor.

[0030] Figure 1CAR-T cell activation and PD-L1 and PD-1 expression when CAR-T kills solid tumor cells (a is HER2 (Epidermal Growth Factor Receptor 2)-CAR cells and MSLN (Mesothelin)-CAR cells. In the presence of antigen stimulation, the expression of PD-1 on CAR-T cells is upregulated; b is tumor cells highly expressing PD-L1; c is CAR-T cells effectively killing tumor cells, highly expressing Granzyme B and CD107a; d is 3 situations: MOCK, HER2-CAR minus, HER2-CAR plus);

[0031] Figure 2 Schematic diagram of the experiment simulating the in vivo anti-tumor environment in which CAR-T cells continuously encounter and kill tumor cells;

[0032] Figure 3 CAR-T cells show increasingly higher cytotoxicity and cytokine release capabilities;

[0033] Figure 4 To highly express PD-L1 on tumor cells;

[0034] Figure 5 CAR-T cells showed more potential cytolytic activity and IFN-γ secretion ability when they encountered tumor cells pretreated with IFN-γ and highly expressing PD-L1;

[0035] Figure 6 When IFN-γ is neutralized by anti-IFN-γ antibodies, the cytotoxicity of CAR-T cells is significantly weakened;

[0036] Figure 7 When IFN-γ is neutralized, CAR-T cells still show the ability to kill tumor cells pretreated with IFN-γ;

[0037] Figure 8 After the tumor cells with knockout IFN-γR2 were treated with IFN-γ (three concentrations: 0, 5, and 10 ng / ml), they no longer expressed PD-L1;

[0038] Fig. 9 The killing effect of CAR-T cells on tumor cells with IFN-γR2 knocked out was significantly weakened, and IFN-γ treatment could not enhance the killing effect of CAR-T cells on tumor cells with IFN-γR2 knocked out;

[0039] Fig.10Overexpression of PD-L1 in tumor cells did not inhibit tumor cells from killing CAR-T cells, nor did it inhibit the enhancement of CAR-T cell cytotoxicity by IFN-γ pretreatment;

[0040] Fig.11 In order to overexpress PD-L1 in tumor cells with IFN-γR2 knockout, it can significantly inhibit the killing of tumor cells by CAR-T cells;

[0041] Fig.12 The effect of CAR-T cells on tumor cells pretreated with IFN-γ in continuous in vitro killing experiments;

[0042] Fig.13 To highly express ICAM-1 in tumor cells with INF-γR2 gene knocked out, thereby enhancing the killing effect of CAR-T cells;

[0043] Fig.14 To knock out the INF-γR2 gene in tumor cells, IFN-γ pretreatment of tumor cells could not enhance the killing effect of CAR-T cells;

[0044] Fig.15 This is the effect diagram of IFN-γ enhancing the killing ability of ordinary T cells;

[0045] Fig.16 Schematic diagram of the IFN-γ combined with CAR-T treatment regimen to establish the NSG mouse peritoneal ovarian cancer model;

[0046] Fig.17 To establish the NSG mouse model of peritoneal ovarian cancer and the effect of IFN-γ combined with CAR-T therapy;

[0047] Fig.18 When CAR-T cells encounter tumor cells that have been pretreated with IFN-γ and highly express PD-L1, CAR-T cells show more potential cytolytic activity and IFN-γ secretion ability. (In Figure a, CAR-T cells are HER2-CAR; in Figure b, CAR-T cells are MSLN-CAR);

[0048] Fig.19 When IFN-γ is neutralized by anti-IFN-γ antibodies, the cytotoxicity of CAR-T cells is significantly weakened;

[0049] Fig. 20 When IFN-γ secreted by CAR-T cells is neutralized, CAR-T cells still show the ability to kill tumor cells that have been pre-treated with IFN-γ;

[0050] Fig.21 The effect diagram of IFN-γ sensitized tumor cells to enhance the killing ability of ordinary T cells;

[0051] Figure 22 is a diagram of a plasmid vector skeleton (Figure 22a is a reference diagram of the structure and positional relationship of each element on the expression cassette; Figure 22b is a reference diagram of a plasmid vector skeleton);

[0052] Fig.23 To detect the killing effect of CAR-T cells on tumor cells;

[0053] Fig.24 To detect the secretion of cytokine IFN-γ when CAR-T cells kill tumor cells. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0056] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0057] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0058] In this specification, certain embodiments may be disclosed in a format of being in a certain range. It should be understood that such description of "being in a certain range" is merely for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0059] Definition of noun:

[0060] The "adjuvant drug" described in the present invention refers to a drug used to assist in enhancing the efficacy of the main therapeutic drug (i.e., the main drug). For example, the IFN-γ described in the present invention can be administered to the patient in the form of a pretreatment drug before the main drug is used for treatment, thereby increasing the sensitivity of tumor cells to the subsequent main drug, thereby enhancing the anti-tumor effect of the subsequent main drug. For another example, the IFN-γ described in the present invention can also be used as a pretreatment drug to upregulate the expression of ICAM-1 in tumor cells, and enhance the killing of tumor cells and cytokine release by CAR-T cells or non-genetically modified T cells. Of course, the use of auxiliary drugs is not necessarily before the main drug treatment, and can also be used simultaneously with the main drug according to actual conditions.

[0061] The "sensitization" mentioned in the present invention refers to increasing the sensitivity of tumor cells to the main drug (immunotherapy) through auxiliary drugs, which is more conducive to the main drug to play a role and more effectively kill tumor cells. For example, the present invention uses IFN-γ to activate the IFN-γ signaling pathway in tumor cells, enhance the sensitivity of tumor cells to T cells (including T cells in the body and adoptively infused T cell preparations), and to immune checkpoint inhibitors (PD-L1 inhibitors and / or PD-1 inhibitors), so that immunotherapy based on T cells and / or immune checkpoint inhibitors can achieve better results.

[0062] The "sensitization" mentioned in the present invention may also refer to changing the immunosuppressive microenvironment in the tumor through tumor cell sensitizing factors (for example, wild-type or mutant IFN-γ), increasing the sensitivity of tumor cells to immunotherapy, and thus killing tumor cells more effectively.

[0063] Invention A: Application of IFN-γ in the preparation of anti-tumor adjuvant drugs

[0064] Invention A belongs to the field of biomedicine, and specifically relates to an anti-tumor drug for auxiliary anti-tumor immunotherapy and its application.

[0065] Tumor immunotherapy is a revolutionary advance in anti-tumor therapy. It fights tumors by mobilizing the body's own immune function. CAR-T therapy belongs to the field of immunotherapy. CAR-T cell therapy has achieved surprising anti-tumor effects in blood tumors targeting CD19. As of April 2018, there were a total of 2,513 immunotherapy treatments for tumors, 382 of which were in China, and 227 of which entered clinical phases III and IV. Novartis' Kymriah and Kite's Yescarta have been launched, which are used to treat childhood acute B-cell leukemia and adult B-cell lymphoma, respectively. Among them, Kymriah has an overall remission rate (CR) of 82.5%, and the probability of relapse is 75% at 6 months and 64% at 12 months. Yescarta, another CD19-specific CAR-T product approved by the FDA, can achieve 43-52% CR in patients with relapsed or refractory DLBCL.

[0066] However, the therapeutic effect of CAR-T on solid tumors is not good. For example, in a phase I clinical study of EGFR-CAR-T on epidermal growth factor receptor cells for the treatment of relapsed / refractory EGFR+ non-small cell lung cancer, only 2 responders and 5 patients with stable disease (SD) were found among 11 cancer patients. The feasibility and safety of HER2-CAR-T cells in patients with relapsed or refractory HER2-positive tumors were evaluated in a phase I / II study in a dose-escalation clinical trial. However, its clinical benefit was limited, with only 4 / 17 evaluable patients showing SD. In other words, the therapeutic effect of CAR-T cells on solid tumors is very limited. How to enhance and improve the therapeutic effect of CAR-T cells on solid tumors is an urgent problem to be solved in the existing technology.

[0067] As an inhibitory molecule in the costimulatory signal, PD-1 was first discovered to induce T cell apoptosis. PD-1 negatively regulates T cell-mediated immune responses by binding to its ligand, programmed death ligand 1 (PD-L1), helping tumor cells to "escape." Therefore, blocking this signaling pathway is considered an effective cancer immunotherapy. Since the approval of the first PD-1 inhibitors pembrolizumab and nivolumab in 2014, there has been an unprecedented growth in clinical research on PD-1 / PD-L1 inhibitors as a form of cancer immunotherapy. PD-1 and PD-L1 inhibitors can be used in clinical trials for a variety of cancer types, including: advanced melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, Hodgkin's lymphoma, liver cancer, head and neck squamous cell carcinoma, urothelial carcinoma, Merkel cell carcinoma, etc. So far, three PD-1 inhibitors have been launched overseas: Pembrolizumab, Nivolumab, and Cemiplimab; and four have been launched in China: Toripalimab, Tislelizumab, Tyvyt, and Camrelizumab. The PD-L1 inhibitors that have been launched are: Atezolizumab, Avelumab, and Durvalumab. When PD-L1 / PD-1 therapy is applied to more solid tumors, how to make PD-L1 / PD-1 antibody therapy produce better anti-tumor therapeutic effects is an urgent problem to be solved.

[0068] In summary, how to improve the anti-tumor effects of existing immunotherapies such as immune cell therapy and / or immune checkpoint inhibitor therapy, and propose more optimized and more effective anti-tumor treatment plans, is a very important issue in the current field of tumor immunotherapy, and is also the technical problem that invention A attempts to solve.

[0069] The purpose of the invention is to provide an application of IFN-γ in the preparation of anti-tumor auxiliary drugs; a kit for detecting the anti-tumor effect of immune checkpoint inhibitors; an anti-tumor drug for auxiliary anti-tumor immunotherapy; an application of IFN-γ in combination with T cell preparations in the preparation of tumor immunotherapy drugs; an application of IFN-γ in combination with immune checkpoint inhibitors in the preparation of tumor immunotherapy drugs; an application of IFN-γ, T cell preparations and immune checkpoint inhibitors in the preparation of tumor immunotherapy drugs; and a combination of IFN-γ, T cell preparations and immune checkpoint inhibitors in the preparation of tumor immunotherapy drugs;

[0070] To achieve the above purpose, the technical solution of invention A is:

[0071] The use of IFN-γ in the preparation of an anti-tumor auxiliary drug, wherein the anti-tumor auxiliary drug is used to assist a T-cell preparation, wherein the IFN-γ enhances the killing effect of the T-cell preparation on the tumor cells by sensitizing the tumor cells; the T-cell preparation comprises non-genetically modified T cells and / or T cells modified by genetic engineering means; the IFN-γ comprises a wild-type or mutant full-length IFN-γ or an IFN-γ fragment.

[0072] Furthermore, the T cells modified by genetic engineering include CAR-T cells and / or TCR-T cells.

[0073] Furthermore, the IFN-γ sensitizes the tumor cells by activating the IFN-γ signaling pathway of the tumor cells.

[0074] Furthermore, the IFN-γ signal activation of the tumor cells upregulates ICAM-1 to enhance the killing effect of the T cell preparation on the tumor cells.

[0075] Furthermore, the anti-tumor adjuvant drug is used for solid tumors.

[0076] Furthermore, the anti-tumor adjuvant drug is used for tumor types including ovarian tumors, breast cancer, brain gliomas, gastric cancer, colon cancer, melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, liver cancer or urothelial carcinoma.

[0077] Invention A also provides the use of IFN-γ in the preparation of anti-tumor adjuvant drugs, wherein the anti-tumor adjuvant drugs are used to assist immune checkpoint inhibitors, wherein the IFN-γ assists the anti-tumor effect of immune checkpoint inhibitors by sensitizing tumor cells, wherein the immune checkpoint inhibitors include PD-L1 inhibitors and / or PD-1 inhibitors; and the IFN-γ includes wild-type or mutant full-length IFN-γ or IFN-γ fragments.

[0078] Furthermore, the PD-L1 inhibitor includes one or more of Atezolizumab, Avelumab or Durvalumab; the PD-1 inhibitor includes one or more of Pembrolizumab, Nivolumab, Cemiplimab, Toripalimab, Tislelizumab, Tyvyt or Camrelizumab.

[0079] Furthermore, the anti-tumor adjuvant drug is used for solid tumors.

[0080] Furthermore, it is characterized in that the anti-tumor adjuvant drug is used for tumor types such as ovarian tumors, breast cancer, brain gliomas, gastric cancer, colon cancer, melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, liver cancer or urothelial carcinoma.

[0081] Invention A also provides the use of IFN-γ in the preparation of anti-tumor adjuvant drugs, wherein the anti-tumor adjuvant drugs are used to assist T cell preparations and immune checkpoint inhibitors, and the IFN-γ assists the anti-tumor effects of the T cell preparations and the immune checkpoint inhibitors by sensitizing tumor cells; the immune checkpoint inhibitors include PD-L1 inhibitors and / or PD-1 inhibitors; the T cell preparations include non-genetically modified T cells and / or T cells modified by genetic engineering; the IFN-γ includes wild-type or mutated full-length IFN-γ or IFN-γ fragments.

[0082] Furthermore, the T cells modified by genetic engineering include CAR-T cells and / or TCR-T cells.

[0083] Furthermore, the PD-L1 inhibitor includes one or more of Atezolizumab, Avelumab or Durvalumab; the PD-1 inhibitor includes one or more of Pembrolizumab, Nivolumab, Cemiplimab, Toripalimab, Tislelizumab, Tyvyt or Camrelizumab.

[0084] Furthermore, the auxiliary drug stimulates tumor cells to produce ICAM-1 to enhance the killing of tumor cells by the T cell preparation.

[0085] Furthermore, the type of the tumor is a solid tumor.

[0086] Furthermore, the type of the tumor is ovarian tumor, breast cancer, brain glioma, gastric cancer, colon cancer, melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, liver cancer or urothelial carcinoma.

[0087] Invention A also provides a kit for detecting the anti-tumor effect of immune checkpoint inhibitors, wherein the immune checkpoint inhibitors include PD-L1 inhibitors and / or PD-1 inhibitors, and the kit includes a test for the patency of the IFN-γ signaling pathway of tumor cells.

[0088] Furthermore, the kit includes detection of the expression level of ICAM-1 or IFN-γR2 in the tumor cells. It should be emphasized that the purpose of detecting the expression level of ICAM-1 or IFN-γR2 is to detect the smoothness of the IFN-γ signaling pathway, so whether it is the full length or fragment of wild-type or mutant ICAM-1 and IFN-γR2, as long as it can effectively detect the smoothness of the IFN-γ signaling pathway, it is within the protection scope of the present invention.

[0089] Furthermore, the PD-L1 inhibitor includes one or more of Atezolizumab, Avelumab or Durvalumab; the PD-1 inhibitor includes one or more of Pembrolizumab, Nivolumab, Cemiplimab, Toripalimab, Tislelizumab, Tyvyt or Camrelizumab.

[0090] Furthermore, it is characterized in that the type of the tumor is a solid tumor.

[0091] Further, it is characterized in that the type of the tumor is ovarian tumor, breast cancer, brain glioma, gastric cancer, colon cancer, melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, liver cancer or urothelial carcinoma.

[0092] A invention also provides an anti-tumor drug for auxiliary anti-tumor immunotherapy, wherein the anti-cancer drug includes wild-type or mutated IFN-γ or IFN-γ fragments, which assist the anti-tumor effect of the T cell preparation and / or the immune checkpoint inhibitor by sensitizing tumor cells.

[0093] Furthermore, the anti-tumor drug includes a targeting vector, which delivers the wild-type or mutant IFN-γ or IFN-γ fragment to the tumor cell.

[0094] Furthermore, the anti-tumor drug is used in combination with the T cell preparation and / or the immune checkpoint inhibitor.

[0095] Furthermore, it is characterized in that the anti-tumor drug also includes any pharmaceutically acceptable carrier and / or adjuvant.

[0096] A invention also provides an anti-tumor drug, which comprises an anti-tumor composition: wild-type or mutant IFN-γ and T cell preparation.

[0097] A invention also provides an anti-tumor composition, which comprises wild-type or mutant IFN-γ, a PD-L1 inhibitor and / or a PD-1 inhibitor and a T cell preparation.

[0098] A specific embodiment of the invention:

[0099] The present invention found that during the process of CAR-T cells killing tumor cells, the upregulation of PD-L1 and PD-1 expression coexists with CAR-T cell activation, tumor cell killing and cytokine secretion ( Figure 1 ). Figure 1 Two types of CAR-T cells were used in a: HER2 (Epidermal Growth Factor Receptor 2)-CAR cells and MSLN (Mesothelin)-CAR cells. In the presence of antigen stimulation, the expression of PD-1 on both CAR-T cells was upregulated. Among them, antigen refers to tumor cells expressing HER2 or MSLN. Figure 1 b. Co-culture CAR-T and tumor cells, activate CAR-T to kill tumor cells, and centrifuge to remove cell debris to obtain the "supernatant". This supernatant is the "microenvironment" that acts on other tumor cells after CAR-T activation. Figure 1 c After adding CAR-T cells, the tumor cell lysis rate was higher; Granzyme B and OD107a+ on CAR-T cells were upregulated, indicating that CAR-T cells were activated and played a role in killing tumor cells. Figure 1 d's experiment has three groups: MOCK, HER2-CARminus, and HER2-CAR plus; MOCK is a control T cell that does not express the CAR structure. Figure 1 CAR-T and tumor cells were co-incubated. After 24 hours, CAR-T killed all tumor cells. The supernatant was taken by centrifugation and used to culture new tumor cells. The expression of PD-L1 on the tumor cells was then detected.

[0100] T cell exhaustion markers TIM-3 and LAG-3 were significantly increased in CAR-T cells. Stress experiments simulating the in vivo anti-tumor environment in which CAR-T cells continuously encounter and kill tumor cells ( Figure 2 ), despite high expression of PD-L1 on tumor cells, CAR-T cells showed increasing cytotoxicity and cytokine release capacity ( Figure 3 ). These results indicate that CAR-T itself has an inhibitory effect on PD-L1-PD-1 in CAR-T killing tumor cells.

[0101] Subtoxic concentrations of IFN-γ induce high expression of PD-L1 on tumor cells. However, when CAR-T cells encounter tumor cells pretreated with IFN-γ and highly expressing PD-L1, CAR-T cells show more potential cytolytic activity and IFN-γ secretion capacity ( Figure 5). In contrast, when IFN-γ was neutralized by anti-IFN-γ antibodies, the cytotoxicity of CAR-T was significantly weakened ( Figure 6 ), indicating that IFN-γ is very important for CAR-T activity.

[0102] In the IFN-γ pretreatment assay, the culture medium was replaced with fresh medium to ensure that the residual IFN-γ was undetectable (less than 10 pg / ml) to exclude the direct effect of IFN-γ on CAR-T cells.

[0103] The present invention uses low concentrations of IFN-γ to pre-treat tumor cells, and then detects the cytotoxicity of CAR-T in fresh culture medium, which contains sufficient anti-IFN-γ antibodies to neutralize the IFN-γ in CAR-T. After IFN-γ is neutralized, CAR-T cells still show the ability to kill tumor cells pre-treated with low concentrations of IFN-γ ( Figure 7 ), unlike tumor cells that have not been pretreated with IFN-γ, CAR-T cells lose the effect of killing tumor cells ( Figure 6 ). Therefore, it is believed that IFN-γ enhances the killing ability of CAR-T by acting on tumor cells. In other words, pretreatment with IFN-γ enhances the sensitivity of tumor cells to CAR-T cells.

[0104] Then, the IFN-γ signaling pathway was blocked using the CRISPR-Cas9 system to interrupt the expression of the IFN-γR2 (IFNGR2) gene on tumor cells. Because IFN-γR1 is ubiquitous in mammalian cells, but the expression of IFN-γR2 is more dynamic, it is thought to determine the extent of IFN-γ-induced signaling in a specific cell population. Knockout-IFN-γR2 (IFN-γR2-null) tumor cells grew normally like control cells, and knockout-IFN-γR2 tumor cells no longer expressed PD-L1 after treatment with IFN-γ (three concentrations: 0, 5, 10 ng / ml) Figure 8 However, IFN-γR2 knockout tumor cells were more resistant to lysis by CAR-T cells ( Fig. 9 In both tumor cells (SK-OV-3, A549), the enhancement effect of IFN-γ pretreatment on CAR-T cell cytotoxicity was significantly inhibited ( Fig. 9 ), that is, the effect of IFN-γ pretreatment on sensitized tumor cells was inhibited. These results together indicate that the effect of IFN-γ on tumor cells is an intrinsic mechanism for CAR-T to maintain or enhance its cell activity (and anti-tumor effect) under the expression of PD-L1 and PD-1.

[0105] In order to directly characterize the inhibitory effect of IFN-γ on PD-L1-PD-1, this experimental group performed CAR-T activity detection on tumor cells that stably expressed PD-L1. Overexpression of PD-L1 on tumor cells does not make tumor cells resistant to CAR-T killing, nor does it inhibit the enhancement of CAR-T cell toxicity to tumor cells pretreated with IFN-γ ( Fig.10 ). In other words, IFN-γ pretreatment enhances the sensitivity of tumor cells to CAR-T cells and is not inhibited by PD-L1. However, in tumor cells with IFN-γR2 knockout, high expression of PD-L1 can significantly inhibit the killing effect of CAR-T cells ( Fig.11 ). These results indicate that IFN-γ signaling can overcome the inhibitory effects of PD-L1-PD-1, enhance the sensitivity of tumor cells to CAR-T cells, and enhance the activity of CAR-T cells.

[0106] Next, the present invention also studies how IFN-γ overcomes the inhibitory effect of PD-L1-PD-1 on CAR-T. In addition to inducing PD-L1 expression, IFN-γ also induces tumor cells to express HLA molecules. HLA is the natural ligand of TCR to activate T cells, but blocking HLA-ABC molecules does not affect the enhancement effect of IFN-γ on CAR-T cells.

[0107] Intercellular adhesion molecule 1 (ICAM-1, also known as CD54) is a cell surface glycoprotein on antigen presenting cells (APCs) that plays an important role in effective immune responses. The present study found that knocking out ICAM-1 in tumor cells with functional IFN-γ receptors almost completely eliminated the enhancement of CAR-T cell cytotoxicity by IFN-γ pre-treated tumor cells ( Fig.14 ), indicating the importance of ICAM-1 to CAR-T activity. At the same time, IFN-γ stimulates tumor cells to produce ICAM-1, which can enhance the killing ability of T cells against tumor cells ( Fig.15 ). In contrast, overexpression of ICAM-1 in tumor cells with normal IFN-γ signaling pathways does not significantly promote the specific cytolysis of tumor cells by CAR-T ( Fig.13 ). We speculate that this is due to the increase in ICAM-1 induced by IFN-γ released by activated CAR-T cells. Therefore, the present invention studies overexpression of ICAM-1 in cells with IFN-γR2 gene knockout and finds that it can make tumor cells more susceptible to CAR-T cell killing ( Fig.13 ). These results collectively indicate that IFN-γ induces ICAM-1 expression to inhibit PD-L1-PD-1 function and enhance the anti-tumor activity of CAR-T and T cells.

[0108] Then, the present invention experiments tested whether IFN-γ pretreatment can help CAR-T cell preparations achieve better therapeutic effects on solid tumors. Continuous in vitro killing experiments showed that CAR-T cells maintained killing activity against tumor cells pretreated with IFN-γ until the fifth round, while the killing activity against tumor cells not pretreated with IFN-γ only lasted until the third round ( Fig.12 ). In other words, IFN-γ (as an adjuvant) pretreatment can enhance the sensitivity of tumor cells to CAR-T cells, which is beneficial to the therapeutic effect of CAR-T cell preparations as the main drug on solid tumors.

[0109] Then, the experiment of the present invention established the NSG mouse peritoneal ovarian cancer model, and injected IFN-γ twice before the peritoneal injection of CAR-T cells ( Fig.16 Bioluminescence imaging showed that IFN-γ had no significant inhibitory effect on tumor growth. The use of CAR-T alone could only delay tumor growth, while the combined use of IFN-γ pretreatment and CAR-T cells caused the tumors in 3 of the 5 mice to disappear continuously, and the tumors in the remaining two mice were significantly smaller than those in the control group mice ( Fig.17 However, tumors lacking IFN-γR2 were more resistant to the combined therapy ( Fig.17 ). These in vitro and in vivo experimental results show that sequential treatment with IFN-γ (as a sensitizing adjuvant) and CAR-T (as the main drug) is an effective method for treating solid tumors.

[0110] In summary, the experiments of the present invention show that:

[0111] 1) After CAR-T cells come into contact with tumor cells, CAR-T cells highly express the inhibitory molecule PD-1, and tumor cells highly express PD-L1. At the same time, the activation markers granzyme B, perforin, and cytokine release in CAR-T cells are all upregulated, and CAR-T efficiently kills tumor cells.

[0112] 2) IFN-γ pre-treated tumor cells. IFN-γ upregulated the expression of PD-L1 in tumor cells, but at the same time enhanced (rather than inhibited) CAR-T cell killing and cytokine release. (IFN-γ was at a low concentration and had no significant inhibitory effect on tumor cells).

[0113] 3) After using IFN-γ neutralizing antibodies to neutralize IFN-γ in the system where CAR-T cells and tumor cells were co-incubated, the killing function of CAR-T cells was significantly reduced. After knocking out the IFNγ receptor gene (IFNγR2) in tumor cells, tumor cells did not express PD-L1, but the killing function of CAR-T was reduced.

[0114] 4) Overexpression of PD-L1 on tumor cells had no significant inhibitory effect on CAR-T activity, but overexpression of PD-L1 on tumor cells in which the IFNγ receptor gene (IFNγR2) had been knocked out significantly inhibited the killing function of CAR-T.

[0115] 5) IFN-γ induces high expression of ICAM-1 in tumor cells. After knocking out ICAM-1 in tumor cells, IFN-γ pretreatment no longer enhances the killing activity of CAR-T.

[0116] 6) Overexpression of ICAM-1 molecules in tumor cells lacking IFNγR2 enhances the killing function and cytokine release ability of CAR-T.

[0117] 7) In the mouse ovarian cancer peritoneal tumor model, IFN-γ can enhance the therapeutic effect of CAR-T cells.

[0118] 8) IFN-γ pre-treats tumor cells, prompting them to express ICAM-1, which can enhance the killing ability of ordinary T cells against tumor cells.

[0119] Invention B: A CAR expression vector and its application

[0120] Invention B belongs to the field of bioengineering, and specifically relates to a CAR expression vector and its application.

[0121] Adoptive cell therapy is an effective anti-cancer strategy, known as a "living drug", and plays an important role in cancer immunotherapy. Chimeric antigen receptor T cells (CAR-T) are genetically engineered T cells that have achieved remarkable results in the treatment of malignant blood tumors, but the therapeutic effect on solid tumors is not satisfactory and still faces huge challenges. Although the reasons for the poor efficacy of CAR-T cells on solid tumors are not very clear at present, there are many reasons, such as poor persistence of CAR-T cells in the body, and they die quickly after being injected into the body, which affects the killing effect; solid tumors will form an immunosuppressive microenvironment due to their multipotency in metabolism, immune escape, and tissue formation, making it difficult for CAR-T cells to infiltrate into solid tumor tissues to exert their effectiveness. Even if they enter the solid tumor, they will be inactivated and die because of restrictions on the immune system in many aspects. Therefore, how to improve the killing effect of CAR-T cells and related drug preparations on solid tumors is an urgent problem to be solved in this field.

[0122] In order to enhance the therapeutic effect of CAR-T cells, the current ideas are mainly focused on further genetic engineering or nano-modification of CAR-T cells themselves. For example, inserting multi-target CAR genes into CAR-T cells, or making CAR-T cells express chemokines, or directly modifying adhesion molecules or chemical groups to enhance the tendency and recognition of CAR-T cells to tumor tissues. However, these technical means all have some problems and shortcomings to a greater or lesser extent. Taking multi-target CAR-T cells as an example, too many inserted exogenous genes may affect the activity of the T cells themselves. Chemical modification usually leads to reduced cell activity and impaired movement and migration ability. Therefore, the modification of CAR-T cells must comprehensively consider multiple positive and negative factors, and choose a modification method with stronger targeting and better effect.

[0123] In the existing technology route, modifying CAR-T from the perspective of inhibiting PD-1 is considered a feasible approach. PD-1 is an inhibitory molecule in an important co-stimulatory signal. PD-1 negatively regulates T cell-mediated immune responses by binding to its ligand programmed death ligand 1 (PD-L1). Therefore, blocking the PD-L1-PD-1 signaling pathway is an effective anti-tumor immunotherapy strategy, which has achieved good therapeutic effects in clinical practice. Currently, there are many antibody drugs targeting PD-L1 / PD-1 on the market, such as antibodies targeting PD-1: Pembrolizumab, Nivolumab, Cemiplimab, antibodies targeting PD-L1: Atezolizumab, Avelumab, Durvalumab.

[0124] Inhibitory signaling pathways such as PD-1 are also important factors affecting the therapeutic effect of CAR-T. Studies have shown that blocking the PD-1 pathway can enhance the anti-tumor effect of CAR-T cells. Therefore, the existing technical route attempts to use genetic engineering to make CAR-T cells secrete antibodies against PD-1 or PD-L1, which can significantly improve the therapeutic effect on solid tumors, or to knock out the coding gene of PD-1 in CAR-T cells through gene editing, which can also enhance the anti-tumor activity of CAR-T.

[0125] However, the current technical solutions and technical routes are all focused on improving the function of CAR-T cells themselves, and have little effect on tumor cells and the immunosuppressive microenvironment in solid tumors. In other words, the current technical solutions and technical routes are relatively limited, focusing only on improving CAR-T cells themselves, but not on improving the anti-tumor effect of CAR-T cell therapy from the perspective of acting on tumor cells.

[0126] Therefore, invention B attempts to provide a new type of CAR-T cell preparation that can affect tumor cells and the immunosuppressive microenvironment within solid tumors, thereby optimizing the anti-tumor effect of the CAR-T cell preparation.

[0127] B One of the purposes of the invention is to provide a new CAR expression vector and construction strategy.

[0128] To achieve the above purpose, the technical solution of invention B is:

[0129] A CAR expression vector comprises a nucleic acid encoding a chimeric antigen receptor (CAR) and a nucleic acid encoding a tumor cell sensitizing factor, wherein the nucleic acid encoding the tumor cell sensitizing factor is: a full-length or fragment of a wild-type or mutant IFN-γ nucleic acid.

[0130] Furthermore, the nucleic acid encoding CAR and the nucleic acid encoding the tumor cell sensitizing factor are connected via a sequence encoding a self-cleaving peptide.

[0131] Furthermore, the nucleic acid encoding CAR includes a single-chain antibody variable region (ScFv) encoding a targeting specific tumor antigen, a CD8a signal peptide encoding, a CD8 hinge region encoding, a CD8a transmembrane domain encoding, a 4-1BB intracellular co-stimulatory element encoding, and a CD3ζ intracellular domain encoding.

[0132] Furthermore, the encoded targeting specific tumor antigens include one or more of HER2, Mesothelin, GPC-3, EGFRvIII, MUC1, CD19, CD30, BCMA, EGFR, CD123, CD133, PSCA, GD2, and LewisY.

[0133] Furthermore, the sequence encoding the self-cleaving peptide is a P2A sequence.

[0134] Furthermore, the nucleic acid sequence encoding CAR is shown as SEQ ID NO.1; the nucleic acid sequence encoding IFN-γ is shown as SEQ ID NO.2; and the sequence encoding the self-cleaving peptide is shown as SEQ ID NO.3.

[0135] Another object of the present invention is to provide a lentivirus.

[0136] A lentivirus comprising the above-mentioned CAR expression vector.

[0137] As a preferred embodiment, the lentivirus in one embodiment of the present invention is pWPXLd. However, lentivirus expression plasmids commonly used in the art, including psPAX2, pMD2.G, pVSVG, etc., can also be included in the technical solution of the present invention.

[0138] The present invention also aims to provide a novel CAR-T cell and its preparation, as well as a novel T cell and its preparation.

[0139] CAR-expressing T cells, into which the vector shown in the following (a) or (b) is introduced: (a) the above-mentioned CAR expression vector; (b) a CAR expression vector containing a nucleic acid encoding CAR and a nucleic acid encoding IFN-γ.

[0140] An anti-tumor drug, comprising the above-mentioned CAR-T cells and pharmaceutically permitted excipients and / or adjuvants.

[0141] Furthermore, the tumor is a solid tumor.

[0142] Furthermore, the tumor is an ovarian tumor, breast cancer, brain glioma, gastric cancer, colon cancer, melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, liver cancer or urothelial carcinoma.

[0143] A T cell expressing CAR and IFN-γ.

[0144] Furthermore, the T cell contains a nucleic acid encoding CAR and a nucleic acid encoding IFN-γ.

[0145] An anti-tumor drug, comprising T cells expressing CAR and IFN-γ and pharmaceutically permitted excipients and / or adjuvants.

[0146] Furthermore, the tumor is a solid tumor.

[0147] Furthermore, the tumor is an ovarian tumor, breast cancer, brain glioma, gastric cancer, colon cancer, melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, liver cancer or urothelial carcinoma.

[0148] A method for producing T cells expressing CAR and IFN-γ, wherein the method uses a vector to reintroduce a nucleic acid encoding CAR and a nucleic acid encoding IFN-γ into T cells.

[0149] B. Specific embodiments of the invention

[0150] Example 1 of the invention: IFN-γ increases the sensitivity of tumor cells to immunotherapy

[0151] Subcytotoxic concentrations of IFN-γ were used to induce tumor cells to highly express PD-L1 (i.e., tumor cells were pretreated with IFN-γ). When CAR-T cells (HER2-CAR, MSLN-CAR) were co-acted with tumor cells pretreated with IFN-γ and highly expressing PD-L1, CAR-T cells showed more potential cytolytic activity and IFN-γ secretion ability ( Fig.18In the IFN-γ pretreatment assay, the culture medium was replaced with fresh culture medium to ensure that the residual IFN-γ was undetectable (less than 10 pg / ml) to exclude the direct effect of IFN-γ on CAR-T cells.

[0152] In addition, the present invention also uses low concentrations of IFN-γ to pre-treat tumor cells, and then detects the cytotoxicity of CAR-T (HER2-CAR) in a fresh culture medium containing sufficient anti-IFN-γ antibodies to neutralize the IFN-γ in CAR-T. After IFN-γ is neutralized, CAR-T cells still show the ability to kill tumor cells pre-treated with low concentrations of IFN-γ ( Fig. 20 ), unlike tumor cells that have not been pretreated with IFN-γ, the effect of CAR-T cells in killing tumor cells is significantly weakened ( Fig.19 ).

[0153] From the above experiments, we can see that IFN-γ has an important effect on CAR-T killing tumor cells. IFN-γ enhances the killing ability of CAR-T by acting on tumor cells. In other words, pretreatment with IFN-γ increases the sensitivity of tumor cells to CAR-T cells.

[0154] Then, the experimental team of the present invention established an NSG mouse peritoneal ovarian cancer model and injected IFN-γ twice before intraperitoneal injection of CAR-T cells ( Fig.16 Bioluminescence imaging showed that IFN-γ had no significant inhibitory effect on tumor growth. The use of CAR-T alone could only delay tumor growth, while the combined use of IFN-γ pretreatment and CAR-T cells caused the tumors in 3 of the 5 mice to disappear continuously, and the tumors in the remaining two mice were significantly smaller than those in the control group mice ( Fig.17 However, when the IFN-γ signaling pathway is blocked in tumor cells (e.g. Fig.17 In the experiment, by knocking out IFN-γR2), the tumor was more resistant to the treatment regimen of CAR-T cells combined with IFN-γ pretreatment ( Fig.17 ).

[0155] In addition, the experiments of the present invention show that IFN-γ not only has an important influence on CAR-T killing tumor cells, but also can enhance the killing effect of ordinary T cells (i.e., non-genetically modified T cells) on tumor cells ( Fig.21 ).

[0156] In summary, the in vitro and in vivo experimental results of the present invention show that whether it is genetically engineered T cells or ordinary T cells, when encountering tumor cells pretreated with IFN-γ (i.e., the IFN-γ signaling pathway is activated), its killing effect will be enhanced. This shows that whether it is based on ordinary T cells or genetically engineered T cells (for example, various types of CAR-T and / or TCR-T), IFN-γ can enhance the sensitivity of tumor cells to be killed by them.

[0157] In addition to enhancing the sensitivity of tumor cells to T cells by pre-treating tumor cells with IFN-γ, the present invention also proposes a technical solution for genetically engineering T cells (for example, CAR-T cells), so that the modified T cells themselves can secrete IFN-γ to sensitize tumor cells and enhance the effect of T cells in killing tumor cells. This provides a treatment plan that may be better and simpler than pre-treating tumor cells with IFN-γ.

[0158] Example 2 of B invention: Novel CAR expression vector

[0159] Embodiment 2 of the present invention provides a novel CAR expression vector, which includes a nucleic acid encoding a chimeric antigen receptor (CAR) and a nucleic acid encoding a tumor cell sensitizing factor, characterized in that the nucleic acid encoding the tumor cell sensitizing factor is: a full-length or fragment of a wild-type or mutant IFN-γ nucleic acid.

[0160] Vector construction

[0161] Construct a plasmid vector containing an expression cassette that expresses a chimeric antigen receptor and can secrete INF-γ. The structure and position relationship of each element on the expression cassette refer to Fig.22a , plasmid vector backbone reference Figure 22b The specific steps are as follows: the lentiviral vector pWPXLd contains the first expression cassette targeting the HER2-positive chimeric antigen receptor, and the first expression cassette (HER2-CAR) includes: CD8a signal peptide, HER2 single-chain antibody variable region, CD8 hinge region, CD8a membrane expansion domain, 4-1BB intracellular co-stimulatory element and CD3ζ intracellular domain; the second expression cassette amplifies the specific fragment of human IFN-γ from T cell cDNA by designing primers, and the IFN-γ second expression cassette and the first expression cassette are co-expressed in the lentiviral vector pWPXLd through the P2A sequence (i.e., self-cleavage sequence), and the entire recombinant plasmid is named "HER2-CAR-IFN-γ plasmid".

[0162] In this embodiment, a plasmid vector targeting HER2 is constructed, but the method provided by the present invention is not limited thereto. The directions provided by the present invention can also construct plasmid vectors targeting common targets, including but not limited to HER2, Mesothelin, GPC-3, EGFRvIII, MUC1, CD19, CD30, BCMA, EGFR, CD123, CD133, PSCA, GD2, LewisY, etc.

[0163] The direction provided by the present invention can also use other molecular biological tools to construct recombinant plasmids with similar functions, such as using other promoters, and / or using T2A or IRES to connect the first expression cassette and the second expression cassette.

[0164] Construction of CAR-T cells expressing chimeric antigen receptor and IFN-γ

[0165] Peripheral blood mononuclear cells (PBMCs) were separated by lymphocyte separation fluid and activated using CD3 and CD28 magnetic beads. The activated T cells were then infected with the virus at the set MOI value. The two types of CAR-T cells obtained were named HER2-CART and HER2-IFNG-CART cells, and the expression level of CAR in T cells was detected by flow cytometry.

[0166] Embodiment 3 of B invention

[0167] CAR-T killing function and cytokine secretion detection

[0168] (1) SK-OV-3 target cells expressing HER2 were plated at 1×104 in a 96-well plate, and MOCK group (MOCK is T cells without CAR structure expression), HER2-CAR group, HER2-IFNG-CAR group, maximum release group, and spontaneous release group were set. After the cells adhered to the wall, CAR-T cells with corresponding effector-target ratio were added and incubated for 24 hours. The killing level of CAR-T was detected using LDH (lactated dehydrogenase) kit, and the cell supernatant was frozen at -80℃ for cytokine detection.

[0169] (2) Cytokine detection

[0170] The expression of IFN-γ was detected using the ELISA kit of Biolegend Company, and the specific operation was carried out according to the instruction manual.

[0171] Fig.23 and Fig.24 This is a summary of the results of CAR-T cell killing function and cytokine secretion testing. Fig.23It can be seen that compared with CAR-T cells that only express HER2-CAR, the effect of CAR-T cells expressing the CAR expression vector of the present invention (including IFN-γ nucleic acid) on killing tumor cells is significantly enhanced. Fig.24 It can be seen that compared with CAR-T cells that only express HER2-CAR, the ability of CAR-T cells expressing the CAR expression vector of the present invention (including IFN-γ nucleic acid) to secrete cytokine IFN-γ is also significantly enhanced.

[0172] The above experiments of the present invention show that by genetically engineering, T cells can secrete more IFN-γ, which can change the tumor cell microenvironment and increase the sensitivity of tumor cells to T cell immunotherapy. In addition to the CAR expression vector constructed by the present invention and the CAR-T cells expressing the vector, natural T cells that naturally highly express IFN-γ or T cells that highly express IFN-γ by other genetic engineering (e.g., TCR-T cells) will also have a stronger effect in killing tumors.

[0173] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. Application of IFN-γ in the preparation of an anti-tumor auxiliary drug, wherein the anti-tumor auxiliary drug is used to assist T cell preparations, characterized in that: The IFN-γ enhances the killing effect of the T cell preparation on the tumor cells by sensitizing the tumor cells; the T cell preparation includes non-genetically modified T cells and / or T cells modified by genetic engineering; the IFN-γ includes wild-type or mutant full-length IFN-γ or IFN-γ fragments.

2. The use according to claim 1, characterized in that The IFN-γ signal activation of the tumor cells upregulates ICAM-1 to enhance the killing effect of the T cell preparation on the tumor cells.

3. Application of IFN-γ in the preparation of an anti-tumor adjuvant drug, wherein the anti-tumor adjuvant drug is used to assist an immune checkpoint inhibitor, characterized in that: The IFN-γ assists the anti-tumor effect of immune checkpoint inhibitors by sensitizing tumor cells. The immune checkpoint inhibitors include PD-L1 inhibitors and / or PD-1 inhibitors; the IFN-γ includes wild-type or mutant IFN-γ full-length or IFN-γ fragments.

4. Application of IFN-γ in the preparation of anti-tumor adjuvant drugs, wherein the anti-tumor adjuvant drugs are used to assist T cell preparations and immune checkpoint inhibitors, characterized in that: The IFN-γ assists the anti-tumor effects of the T cell preparation and the immune checkpoint inhibitor by sensitizing tumor cells; the immune checkpoint inhibitor includes a PD-L1 inhibitor and / or a PD-1 inhibitor; the T cell preparation includes non-genetically modified T cells and / or T cells modified by genetic engineering; the IFN-γ includes wild-type or mutant full-length IFN-γ or IFN-γ fragments.

5. The use according to claim 4, characterized in that The auxiliary drug stimulates tumor cells to produce ICAM-1 to enhance the killing of tumor cells by the T cell preparation.

6. A kit for detecting the anti-tumor effect of an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor comprises a PD-L1 inhibitor and / or a PD-1 inhibitor, characterized in that: The kit includes a method for detecting the smoothness of the IFN-γ signaling pathway of tumor cells.

7. The kit according to claim 6, characterized in that The kit includes the detection of the expression level of ICAM-1 or IFN-γR2 in the tumor cells.

8. The kit according to claim 6, characterized in that The PD-L1 inhibitor includes one or more of Atezolizumab, Avelumab and Durvalumab; the PD-1 inhibitor includes one or more of Pembrolizumab, Nivolumab, Cemiplimab, Toripalimab, Tislelizumab, Tyvyt and Camrelizumab.

9. The kit according to claim 6, characterized in that The type of tumor is a solid tumor.

10. The kit according to claim 6, characterized in that The type of the tumor is ovarian tumor, breast cancer, brain glioma, gastric cancer, colon cancer, melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, liver cancer or urothelial carcinoma.

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