Use of IFN - Γ in preparation of Anti-tumor adjuvant

Abstract

The present invention relates to an anti-tumor drug for assisting anti-tumor immunotherapy and application thereof.SOLUTION: The present invention provides use of IFN - γ in the preparation of an anti-tumor adjuvant, wherein the IFN - γ sensitizes tumor cells to enhance the killing effect of a T cell preparation on the tumor cells, the T cell preparation comprises non-genetically modified T cells and / or genetically modified T cells, and the IFN - γ comprises a full-length or fragment of wild-type or mutant IFN - γ. The present invention breaks through the recognition of IFN - γ in the prior art, and finds that IFN - γ activates the IFN - γ signaling pathway of tumor cells, thereby inhibiting the acquired immune resistance mediated by PD - L1 - PD-1 and enhancing the anti-tumor effect of immune therapy.SELECTED DRAWING: Figure 17

Description

[Technical Field]

[0001] [1] This application claims priority to a patent application entitled "Application of IFN-γ in the preparation of antitumor adjuvant drugs," China Patent Application Publication No. 202010159630.9, filed on March 9, 2020, and a patent application entitled "CAR expression vector and its application," China Patent Application Publication No. 202010487110.0, filed on June 1, 2020. All of these priority invention patent applications are incorporated by reference.

[0002] [2] The present invention relates to the field of biomedicine, and more particularly to an antitumor drug that assists antitumor immunotherapy and its application. [Background technology]

[0003] [3] Tumor immunotherapy is a groundbreaking antitumor treatment that harnesses the body's own immune system to fight tumors. CAR-T therapy belongs to the field of immunotherapy. Targeting CD19, CAR-T cells have shown remarkable results in the treatment of hematologic malignancies. As of April 2018, there were 2,513 tumor-targeting immunotherapies in China, including 382 and 227 in Phase III and IV clinical trials. Commercially available immunotherapies include Novartis' Kymriah for pediatric acute B-lymphoblastic leukemia and Kite's Yescarta for adult B-cell lymphoma. Kymriah has a complete remission (CR) rate of 82.5%, with a relapse rate of 75% at 6 months and 64% at 12 months. Yescarta, another FDA-approved CD19-specific CAR-T product, can achieve a CR rate of 43–52% in patients with relapsed or refractory DLBCL.

[0004] [4] However, CAR-T has not been effective in treating solid tumors. For example, in a phase I clinical trial of EGFR-CAR-T targeting epidermal growth factor receptor cells in the treatment of relapsed or refractory EGFR+ non-small cell lung cancer, only two of 11 cancer patients achieved a response and five achieved stable disease (SD). The feasibility and safety of administering HER2-CAR-T cells to patients with relapsed or refractory HER2-positive tumors was evaluated in a phase I / II dose-escalation clinical trial. However, the clinical benefit was limited, with only 4 / 17 evaluable patients achieving SD. In other words, the efficacy of CAR-T cell therapy for solid tumors is very limited. The reasons for the low efficacy of CAR-T cell therapy for solid tumors are not well understood, but several factors are suspected. For example, CAR-T cells have a low survival rate in vivo, and they die soon after administration, negatively affecting their killing effect. In addition, solid tumors have pluripotent properties in metabolism, immune evasion, and tissue formation, which create an immunosuppressive microenvironment, making it difficult for CAR-T cells to infiltrate into the tissue of solid tumors and exert their effects. Even if CAR-T cells enter solid tumors, they are inactivated and die because they restrict the immune system in various ways. Therefore, how to enhance and improve the therapeutic effect of CAR-T cells against solid tumors has become an urgent challenge in conventional technology.

[0005] [5]. PD-1 was first discovered as an inhibitory molecule in costimulatory signaling, with the primary function of inducing T cell apoptosis and thereby preventing the immune system from killing cancer cells. Furthermore, PD-1 can negatively regulate T cell-mediated immune responses by binding to its ligand, programmed death ligand 1 (PD-L1). Tumor cells "trick" T cells by binding PD-L1 to PD-1, thereby evading their recognition. 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, clinical development of PD-1 / PD-L1 inhibitors for cancer immunotherapy has become more active than ever. PD-1 / PD-L1 inhibitors are being tested 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, and Merkel cell carcinoma. To date, three PD-1 inhibitors, Pembrolizumab, Nivolumab, and Cemiplimab, are on the market overseas, and four, Toripalimab, Tislelizumab, Tyvyt, and Camrelizumab, are on the market in China. Now that PD-L1 / PD-1 therapy is being used for more solid tumors, how to improve the anti-tumor therapeutic effect of PD-L1 / PD-1 therapy has become an urgent issue.

[0006] [6] Adoptive cell therapy (ACT) is an effective anti-cancer strategy often referred to as a "living drug" and plays an important role in cancer immunotherapy. ACT involves isolating immune cells from tumor patients, expanding and functionally characterizing them in vitro, and then returning them to the patient to either directly kill tumors or stimulate the body's immune response to destroy them. Currently, ACT mainly includes TIL, LAK, CIK, DC, NK, TCR-T, and CAR-T.

[0007] [7] To enhance the therapeutic efficacy of CAR-T cells, current research focuses on further genetic modification or nano-modification of CAR-T cells themselves. For example, inserting multi-target CAR genes into CAR-T cells and optimizing the CAR structure effectively influences T cell proliferation and persistence. Alternatively, CAR-T cells can be engineered to express chemokines or directly modified with adhesion molecules or chemical groups to enhance CAR-T cell affinity and recognition toward tumor tissue. However, each approach has its own set of drawbacks. For example, in the case of multi-target CAR-T cells, inserting too many foreign genes may affect the intrinsic activity of T cells. Therefore, CAR-T cell modification must comprehensively consider both positive and negative factors, and it is necessary to compare and balance the positive and negative effects of the relevant modifications to ultimately select a more targeted and effective modification method.

[0008] [8] Previous studies have shown that the inhibitory signaling pathway, PD-1, is a factor that affects the efficacy of CAR-T therapy, and blocking the PD-1 pathway can improve the antitumor effect of CAR-T cells. Therefore, technological means that can inhibit PD-1 are considered a feasible method for modifying CAR-T.

[0009] [9]. Among the conventional technological routes, attempts to genetically modify CAR-T cells to secrete antibodies against PD-1 or PD-L1 have significantly improved the therapeutic efficacy against solid tumors, or knocking out the gene encoding PD-1 in CAR-T cells by gene editing has also improved the anti-tumor activity of CAR-T.

[0010]

[10] However, the above technological solutions and technological pathways focus primarily on improving the function of CAR-T cells themselves, and have little impact on the immunosuppressive microenvironment within tumor cells or solid tumors. In other words, current technological solutions and technological pathways focus solely on improving CAR-T cells themselves, and are unable to further enhance the antitumor effect of CAR-T cell therapy in terms of its action on tumor cells.

[0011]

[11] . Therefore, how to improve the antitumor effects of conventional techniques such as immune cell therapy, immune checkpoint inhibitor therapy, and / or adoptive cell therapy, and propose more optimal and effective antitumor treatments, is a very important issue in the current field of tumor immunotherapy, and is a technical issue that the present invention aims to address. Furthermore, the present invention aims to provide an optimal CAR-T cell treatment strategy by influencing not only tumor cells but also the immunosuppressive microenvironment within solid tumors. Summary of the Invention

[0012]

[12] The present invention addresses the above technical problems and provides an antitumor adjuvant mainly prepared from type II interferon (IFN-γ) and its application, and is composed of two subordinate inventions (i.e., Invention A and Invention B) and respective developments based on these two inventions.

[0013]

[13] . One aspect of the present invention provides the use of IFN-γ in the preparation of an antitumor adjuvant drug, a kit for detecting the antitumor effect of an immune checkpoint inhibitor, an antitumor drug that adjuvantizes antitumor immunotherapy, the use of IFN-γ in the preparation of a tumor immunotherapy drug in combination with a T cell preparation, the use of IFN-γ in the preparation of a tumor immunotherapy drug in combination with an immune checkpoint inhibitor, the use of IFN-γ in the preparation of a tumor immunotherapy drug in combination with a T cell preparation and an immune checkpoint inhibitor, and combination drugs.

[0014]

[14] Another aspect of the present invention also provides a novel IFN-γ-modified CAR expression vector and its construction strategy. [Effects of the Invention]

[0015]

[15] .Beneficial effects

[16] The present invention breaks the conventional understanding of IFN-γ and finds that IFN-γ activates the IFN-γ signaling pathway in tumor cells, for example, by upregulating ICAM-1 expression in tumor cells, thereby sensitizing tumor cells to immunotherapy (T cell preparations and / or PD-L1 / PD-1 inhibitors), thereby suppressing PD-L1-PD-1-mediated acquired immune resistance and enhancing the anti-tumor effect of immunotherapy. This provides a technical proposal for using IFN-γ as an adjuvant to immunotherapy to fight tumors.

[0016]

[17] . Interferons (IFNs) are a group of secreted proteins. The IFN protein family is divided into three types: type I, type II, and type III interferons, based on their gene sequences, chromosomal location, and receptor specificity. Among them, type II interferons consist of a single gene family, IFN-γ, also known as immune interferons. Under normal conditions, IFN-γ is present at low levels intracellularly, but its expression significantly increases upon induction by viruses or other interferon inducers. IFN-γ is the primary cytokine involved in antitumor immunity and is an immune-activating cytokine released by CAR-T cells upon antigen contact. To avoid damage caused by an activated immune response, normal tissues induce multiple regulatory pathways to promote the reestablishment of cellular homeostasis in the presence of inflammation. Previous studies have suggested that tumor cells exploit these protective regulatory pathways for immune evasion. For example, tumor cells upregulate PD-L1 expression by releasing IFN-γ, thereby inhibiting T cell killing. This adaptive immune resistance is thought to be one of the reasons why CAR-T therapy effectively kills solid tumors. Because IFN-γ-induced upregulation of PD-L1 expression is thought to induce adaptive immune resistance in tumor cells, traditional immunotherapy solutions have not fully utilized IFN-γ. IFN-γ antitumor treatments that initially entered clinical trials were not commercially available due to their ineffectiveness. It is currently believed that IFN-γ upregulation of PD-1 expression reduces the antitumor effect, and that IFN-γ must be combined with a PD-L1 inhibitor to neutralize the adverse effects of IFN-γ-induced PD-L1 upregulation. However, this is merely theoretical speculation and has not been confirmed by solid research results.

[0017]

[18] . The experimental results of the present invention showed that IFN-γ (which activates the IFN-γ signaling pathway by pretreating tumor cells) upregulates PD-L1 expression on tumor cells, while also affecting the expression of other factors (e.g., upregulating ICAM-1 expression). As a result, IFN-γ significantly enhanced the cytotoxicity of CAR-T cells (or other types of genetically modified T cells) or non-genetically modified T cells against tumor cells, and also improved the durability of the tumor-killing ability of CAR-T cells. In other words, IFN-γ sensitizes tumor cells to T cells, i.e., increases the susceptibility of tumor cells to T cells. Furthermore, the experimental results of the present invention revealed that disruption of the IFN-γ signaling pathway significantly affected the tumor-killing effect of CAR-T cells.

[0018]

[19] Based on the above experimental results and rational reasoning, the present invention proposes a technical solution for using IFN-γ as an adjuvant in combination with T cell preparations to treat solid tumors, and the application of IFN-γ as an adjuvant in antitumor therapy. Activation of tumor cell signaling pathways using IFN-γ can enhance the antitumor effect of T cell preparations (including non-genetically modified T cells or genetically modified T cells (CAR-T, TCR-T, etc.)). Since the core of the present invention is the activation of the IFN-γ signaling pathway in tumor cells, any IFN-γ, whether wild-type or mutant, full-length or fragmented, can be applied to the present invention as long as it has the effect of activating the IFN-γ signaling pathway in tumor cells and is within the scope of the technical solution of the present invention.

[0019]

[20] Furthermore, because the principle of action of PD-L1 inhibitors and / or PD-1 inhibitors is to activate exhausted T cells and restore their anti-tumor effects, the anti-tumor effects of PD-L1 inhibitors and / or PD-1 inhibitors are also essentially mediated by T cells. Therefore, the present invention also proposes a technical solution for suppressing tumors by enhancing PD-L1 inhibitors and / or PD-1 inhibitors with IFN-γ, and a technical solution for inhibiting tumors by enhancing the combined effect of PD-L1 inhibitors and / or PD-1 inhibitors and T cell preparations with IFN-γ. It should be emphasized here that the technical solution proposed in the present invention utilizes IFN-γ to sensitize tumor cells, thereby helping to enhance 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 PD-L1 downregulation in tumor cells caused by activation of the IFN-γ signaling pathway. Furthermore, the former treatment strategy uses a PD-L1 inhibitor and / or a PD-1 inhibitor as the main drug, while the latter treatment strategy uses a PD-L1 inhibitor and / or a PD-1 inhibitor as an adjuvant drug.

[0020]

[21] Furthermore, experiments of the present invention show that defects in the IFN-γ signaling pathway affect the anti-tumor effect of CAR-T cells. In conjunction with research results showing that defects in the IFN-γ signaling pathway affect the anti-tumor effect of PD-L1 inhibitors and / or PD-1 inhibitors, the present invention further provides a method for preliminarily determining whether T cell therapy and / or PD-L1 inhibitors and / or PD-1 inhibitors are suitable as anti-tumor treatment options by detecting activation / defect of the IFN-γ signaling pathway (e.g., detecting the expression of ICAM-1 or IFN-γR2 on tumor cells).

[0021]

[22] .As mentioned above, one of the contributions of the technical solution of the present invention is to break the conventional understanding of IFN-γ, and propose an immunotherapy adjuvant centered on IFN-γ, which can enhance the antitumor effect of cell therapy and the antitumor effect of immune checkpoint inhibitors combined with cell therapy, and further enhance the antitumor effect of conventional immunotherapy.

[0022]

[23] . Meanwhile, the experimental results of the present invention demonstrate that IFN-γ can enhance the sensitivity of tumor cells to T cells. For example, IFN-γ upregulates PD-L1 expression on tumor cells by pre-treatment, ultimately significantly enhancing the cytotoxicity of CAR-T cells (or other types of genetically modified T cells, such as TCR-T cells) or non-genetically modified T cells against tumor cells. In other words, IFN-γ sensitizes tumor cells to T cells and enhances their sensitivity to T cells.

[0023]

[24] Based on this, the present invention proposes a technical solution of suppressing tumor cells using T cells with high IFN-γ expression (including natural T cells or T cells genetically modified to express high levels of IFN-γ, as well as genetically modified T cells with high levels of IFN-γ expression (CAR-T cells, TCR-T cells)).

[0024]

[25] Specifically, the present invention proposes a novel CAR-T cell engineered solution that not only has the function of activating CAR-T cells, but more importantly, alters the immunosuppressive microenvironment within solid tumors, thereby sensitizing tumor cells and enabling CAR-T cell therapy to exert a strong antitumor effect.

[0025]

[26] . The technical solution of the present invention is to prepare CAR-T cells capable of secreting the interferon cytokine IFN-γ by inserting an IFN-γ gene segment into a CAR expression vector. IFN-γ itself is an important cytokine involved in anti-tumor immunity. It not only activates CAR-T cells themselves (contact with tumor cells further activates CAR-T, releasing more cytokines, such as IL-2 and IFN-γ), but also sensitizes tumor cells, increasing their susceptibility to killing by CAR-T, when secreted into the tumor microenvironment. More importantly, molecules such as ICAM-1, newly expressed at high levels on IFN-γ-sensitized tumor cells, sensitize tumor cells to immunotherapy (T cell formulations and / or PD-L1 / PD-1 inhibitors), thereby suppressing the inhibition of CAR cell activity by PD-L1 / PD-1. Therefore, the optimization technology provided by the present invention can further improve the therapeutic efficacy of CAR-T against solid tumors.

[0026]

[27] In order to more clearly describe the technical solutions in the embodiments of the present invention or the prior art, the following briefly introduces the accompanying drawings used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention, and those skilled in the art can also obtain other drawings from these drawings without creative efforts. [Brief explanation of the drawings]

[0027] [Figure 1]

[28] . Figure 1 shows the activation of CAR-T cells and the expression of PD-L1 and PD-1 during CAR-T killing of solid tumor cells. (a) Epidermal growth factor receptor 2 (HER2)-CAR cells and mesothelin (MSLN)-CAR cells show upregulated PD-1 expression on CAR-T cells in the presence of antigen stimulation. (b) Tumor cells highly expressing PD-L1. (c) Effective tumor cell killing by CAR-T cells highly expressing Granzyme B and CD107a. (d) Three cases: mock, HER2-CAR minus, and HER2-CAR plus.) [Figure 2]

[29] Figure 2 shows a schematic diagram of an experiment in which CAR-T cells continuously encounter and kill tumor cells, simulating an anti-tumor environment in vivo. [Figure 3]

[30] . Figure 3 shows the enhanced cytotoxicity and cytokine release capacity of CAR-T cells. [Figure 4]

[31] . Figure 4 shows the high expression of PD-L1 on tumor cells. [Figure 5]

[32] . Figure 5 shows that when CAR-T cells encountered tumor cells that were pretreated with IFN-γ and highly expressed PD-L1, the CAR-T cells had higher potential cytolytic activity and IFN-γ secretion ability. [Figure 6]

[33] . Figure 6 shows that neutralization of IFN-γ with anti-IFN-γ antibody significantly reduces the cytotoxicity of CAR-T cells. [Figure 7]

[34] . Figure 7 shows the ability of CAR-T cells to kill tumor cells pretreated with IFN-γ despite neutralizing IFN-γ. [Figure 8]

[35] . Figure 8 shows that knockout-IFN-γR2 tumor cells lost PD-L1 expression when treated with IFN-γ (three concentrations: 0, 5, and 10 ng / ml). [Figure 9]

[36] . Figure 9 shows that the killing effect of CAR-T cells against tumor cells in which IFN-γR2 was knocked out was significantly reduced, and IFN-γ treatment did not improve the killing effect of CAR-T cells against tumor cells in which IFN-γR2 was knocked out. [Figure 10]

[37] . Figure 10 shows that overexpression of PD-L1 in tumor cells does not inhibit the killing ability of tumor cells by CAR-T, nor does it inhibit the enhancement of CAR-T cytotoxicity by IFN-γ pretreatment. [Figure 11]

[38] . Figure 11 shows that high expression of PD-L1 in knockout-IFN-γR2 tumor cells significantly inhibits tumor cell killing by CAR-T cells. [Figure 12]

[39] . Figure 12 shows the effect of CAR-T cells on tumor cells pretreated with IFN-γ in continuous in vitro killing detection. [Figure 13]

[40] . Figure 13 shows that high expression of ICAM-1 in tumor cells with INF-γR2 gene knockout enhances the killing effect of CAR-T cells. [Figure 14]

[41] . Figure 14 shows that knocking out the INF-γR2 gene in tumor cells and pretreating the tumor cells with IFN-γ did not enhance the killing effect of CAR-T cells. [Figure 15]

[42] . Figure 15 shows the effect of IFN-γ on enhancing the killing ability of conventional T cells. [Figure 16]

[43] . Figure 16 is a schematic diagram showing the establishment of an NSG mouse abdominal ovarian cancer model and the combined use of IFN-γ and CAR-T therapy. [Figure 17]

[44] . Figure 17 shows the effect of combined IFN-γ and CAR-T treatment in an established NSG mouse abdominal ovarian cancer model. [Figure 18]

[45] . Figure 18 shows that CAR-T cells exhibited higher potential cytolytic activity and IFN-γ secretion when they encountered tumor cells pretreated with IFN-γ and highly expressing PD-L1. (In Figure a, the CAR-T cells were HER2-CAR, and in Figure b, the CAR-T cells were MSLN-CAR.) [Figure 19]

[46] . Figure 19 shows that neutralization of IFN-γ with anti-IFN-γ antibody significantly reduces the cytotoxicity of CAR-T cells. [Figure 20]

[47] . Figure 20 shows the ability of CAR-T cells to kill tumor cells pretreated with IFN-γ despite the neutralization of IFN-γ secreted by the cells. [Figure 21]

[48] . Figure 21 shows the effect of IFN-γ-sensitized tumor cells on enhancing the killing ability of conventional T cells. [Figure 22]

[49] Figure 22 shows the backbone of the plasmid vector (22a is a reference diagram showing the structure and positional relationship of the components on the expression cassette, and 22b is a reference diagram of the backbone of the plasmid vector). [Figure 23]

[50] . Figure 23 shows the detection of the killing effect of CAR-T cells on tumor cells. [Figure 24]

[51] . Figure 24 shows the detection of secretion of the cytokine IFN-γ when CAR-T cells kill tumor cells. DETAILED DESCRIPTION OF THE INVENTION

[0028]

[52] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with accompanying drawings. It is obvious that the described embodiments are only a part of the embodiments of the present invention, but not all of them. All other embodiments that can be obtained by those skilled in the art based on the embodiments of the present invention without any inventive efforts are within the protection scope of the present invention.

[0029]

[53] The examples mentioned are for the purpose of better illustrating the present invention, but the content of the present invention is not limited to these examples. Therefore, those skilled in the art may make non-essential improvements or adjustments to the embodiments according to the above content of the present invention, and these improvements or adjustments also fall within the protection scope of the present invention.

[0030]

[54] It should be noted that, as used herein, the terms "comprise," "include," or any other variations thereof are intended to cover a non-exclusive inclusion. Thus, a process, method, article, or apparatus comprising a list of elements includes not only those elements but also other elements not expressly listed or inherent in such process, method, article, or apparatus. Absent further limitations, an element defined by the phrase "comprises a ..." does not preclude the presence of additional identical elements in the process, method, article, or apparatus that comprises that element.

[0031]

[55] For example, as used herein, the term "about" typically refers to + / -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.

[0032]

[56] Certain embodiments may be disclosed herein in a range format. It should be understood that this "within a range" description is merely for convenience and brevity and should not be construed as an inflexible limitation on the disclosed range. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, a description of a range of 1 to 6 should be considered to have specifically disclosed 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers 1, 2, 3, 4, 5, and 6 within that range. The above rule applies regardless of the breadth of the range.

[0033]

[57] . Glossary:

[58] The term "adjuvant drug" as used herein refers to a drug used adjunctively to enhance the effect of the main therapeutic drug (i.e., the principal drug). For example, IFN-γ as used herein can be administered to a patient as a pretreatment drug before treatment with the principal drug to increase the sensitivity of tumor cells to the subsequent principal drug, thereby enhancing the antitumor effect of the subsequent principal drug. As a further example, IFN-γ as used herein can also be used as a pretreatment drug to upregulate ICAM-1 expression in tumor cells and promote tumor cell killing and cytokine release by CAR-T cells or non-genetically modified T cells. Of course, adjuvant drugs do not necessarily need to be used before treatment with the principal drug; they can also be used in parallel with the principal drug depending on the situation.

[0034]

[59] In the present invention, "sensitization" means that an adjuvant increases the sensitivity of tumor cells to the main drug (immunotherapy), further enhancing the action of the main drug and killing tumor cells more effectively. For example, the present invention uses IFN-γ to activate the IFN-γ signaling pathway in tumor cells, thereby increasing the sensitivity of tumor cells to T cells (including in vivo T cells and adoptively administered T cell preparations) and immune checkpoint inhibitors (PD-L1 inhibitors and / or PD-1 inhibitors), thereby achieving better results from immunotherapy using T cells and / or immune checkpoint inhibitors.

[0035]

[60] . In addition, "sensitization" as used herein also refers to the modification of the immunosuppressive microenvironment within tumors by tumor cell sensitizing factors (e.g., wild-type or mutant IFN-γ) to increase the sensitivity of tumor cells to immunotherapy, thereby enabling more effective killing of tumor cells.

[0036]

[61] .A Invention: Application of IFN-γ to the preparation of antitumor adjuvant drugs

[62] .The present invention relates to the field of biomedicine, and more particularly to an antitumor drug that assists antitumor immunotherapy and its application.

[0037]

[63] . Tumor immunotherapy is a groundbreaking antitumor treatment that harnesses the body's own immune system to fight tumors. CAR-T therapy belongs to the field of immunotherapy. Targeting CD19, CAR-T cells have shown remarkable results in the treatment of hematologic malignancies. As of April 2018, there were 2,513 tumor-targeting immunotherapies in China, including 382 in Phase III and IV clinical trials. Commercially available immunotherapies include Novartis' Kymriah for pediatric acute B-lymphoblastic leukemia and Kite's Yescarta for adult B-cell lymphoma. Kymriah has a complete remission (CR) rate of 82.5%, with a relapse rate of 75% at 6 months and 64% at 12 months. Yescarta, another FDA-approved CD19-specific CAR-T product, can achieve a CR rate of 43–52% in patients with relapsed or refractory DLBCL.

[0038]

[64] However, CAR-T has not been effective in treating solid tumors. For example, in a phase I clinical trial of EGFR-CAR-T targeting epidermal growth factor receptor cells in the treatment of relapsed or refractory EGFR+ non-small cell lung cancer, only two of 11 cancer patients achieved a response and five achieved stable disease (SD). The feasibility and safety of administering HER2-CAR-T cells to patients with relapsed or refractory HER2-positive tumors was evaluated in a phase I / II dose-escalation clinical trial. However, the clinical benefit was limited, with only 4 / 17 evaluable patients achieving SD. In other words, the efficacy of CAR-T cell therapy against solid tumors is very limited. Enhancing and improving the therapeutic efficacy of CAR-T cells against solid tumors is an urgent issue in the prior art.

[0039] PD-1 was first discovered as an inhibitory molecule in costimulatory signaling, with its primary function inducing T cell apoptosis. Furthermore, PD-1 negatively regulates T cell-mediated immune responses by binding to its ligand, programmed death ligand 1 (PD-L1), helping tumor cells 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, clinical development of PD-1 / PD-L1 inhibitors as cancer immunotherapies has become more active than ever. PD-1 / PD-L1 inhibitors are being tested 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, and Merkel cell carcinoma. To date, there are three PD-1 inhibitors on the market overseas: Pembrolizumab, Nivolumab, and Cemiplimab, and four PD-1 inhibitors on the market in China: Toripalimab, Tislelizumab, Tyvyt, and Camrelizumab. Now that PD-L1 / PD-1 therapy is being used to treat more solid tumors, how to improve the anti-tumor therapeutic effect of PD-L1 / PD-1 therapy has become an urgent issue.

[0040]

[66] . From the above, how to improve the antitumor effects of conventional immunotherapies such as immune cell therapy and / or immune checkpoint inhibitor therapy and propose more optimal and effective antitumor treatment methods is currently a very important issue in the field of tumor immunotherapy, and is the technical issue that Invention A aims to alleviate.

[0041]

[67] .The object of the invention is to provide an application of IFN-γ to the preparation of an antitumor adjuvant drug, a kit for detecting the antitumor effect of immune checkpoint inhibitors, an antitumor drug that adjuvant antitumor immunotherapy, an application of IFN-γ in combination with a T cell preparation to prepare a tumor immunotherapeutic drug, an application of IFN-γ in combination with an immune checkpoint inhibitor to prepare a tumor immunotherapeutic drug, an application of IFN-γ in combination with a T cell preparation and an immune checkpoint inhibitor to prepare a tumor immunotherapeutic drug, and a combination of compound drugs.

[0042]

[68] .To achieve the above, the technical solution of invention A is as follows:

[69] . An application of IFN-γ to the preparation of an antitumor adjuvant drug that supports a T cell preparation, wherein the IFN-γ enhances the killing effect of the T cell preparation against tumor cells by sensitizing the tumor cells, the T cell preparation comprising non-genetically modified T cells and / or genetically modified T cells, and the IFN-γ comprising a full-length or fragment of wild-type or mutant IFN-γ.

[70] .Furthermore, the genetically modified T cells comprise CAR-T cells and / or TCR-T cells.

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

[72] . Furthermore, IFN-γ signal activation of the tumor cells upregulates ICAM-1, enhancing the killing effect of the T cell preparation against the tumor cells.

[73] . Furthermore, the tumor for which the antitumor adjuvant is used is a solid tumor.

[74] . Furthermore, the tumors for which the antitumor adjuvant is used are 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 cancer.

[0043]

[75] The present invention further provides an application of IFN-γ to the preparation of an antitumor adjuvant drug that adjuvantizes an immune checkpoint inhibitor, wherein the IFN-γ adjuvantizes the antitumor effect of the immune checkpoint inhibitor by sensitizing tumor cells, the immune checkpoint inhibitor is a PD-L1 inhibitor and / or a PD-1 inhibitor, and the IFN-γ includes a full-length or fragment of wild-type or mutant IFN-γ.

[76] .Furthermore, the PD-L1 inhibitor is one or more selected from Atezolizumab, Avelumab, and Durvalumab, and the PD-1 inhibitor is one or more selected from Pembrolizumab, Nivolumab, Cemiplimab, Toripalimab, Tislelizumab, Tyvyt, and Camrelizumab.

[77] . Furthermore, the tumor for which the antitumor adjuvant is used is a solid tumor.

[78] .Furthermore, the tumor for which the antitumor adjuvant drug is used is characterized in that it 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 cancer.

[0044]

[79] .A The present invention further provides an application of IFN-γ to the preparation of an antitumor adjuvant drug that adjuvantizes a T cell preparation and an immune checkpoint inhibitor, wherein the IFN-γ adjuvantizes the antitumor effects of the T cell preparation and the immune checkpoint inhibitor by sensitizing tumor cells, the immune checkpoint inhibitor comprising a PD-L1 inhibitor and / or a PD-1 inhibitor, the T cell preparation comprises non-genetically modified T cells and / or genetically modified T cells, and the IFN-γ comprises a full-length or fragment of wild-type or mutant IFN-γ.

[80] .Furthermore, the genetically modified T cells comprise CAR-T cells and / or TCR-T cells.

[81] .Furthermore, the PD-L1 inhibitor is one or more selected from Atezolizumab, Avelumab, and Durvalumab, and the PD-1 inhibitor is one or more selected from Pembrolizumab, Nivolumab, Cemiplimab, Toripalimab, Tislelizumab, Tyvyt, and Camrelizumab.

[82] Furthermore, the adjuvant stimulates the production of ICAM-1 by tumor cells, enhancing the killing effect of the T cell preparation on the tumor cells.

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

[84] 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 cancer.

[0045]

[85] .A The present invention further provides a kit for detecting the antitumor effect of an immune checkpoint inhibitor consisting of a PD-L1 inhibitor and / or a PD-1 inhibitor, the kit detecting the patency of the IFN-γ signaling pathway in tumor cells.

[86] Furthermore, the kit detects the expression level of ICAM-1 or IFN-γR2 in the tumor cells. Since the purpose of detecting the expression level of ICAM-1 or IFN-γR2 is to detect the patency of the IFN-γ signaling pathway, it should be emphasized that full-length or fragments of ICAM-1 and IFN-γR2, whether wild-type or mutant, fall within the scope of protection of the present invention as long as they are effective in detecting the patency of the IFN-γ signaling pathway.

[87] .Furthermore, the PD-L1 inhibitor is one or more selected from Atezolizumab, Avelumab, and Durvalumab, and the PD-1 inhibitor is one or more selected from Pembrolizumab, Nivolumab, Cemiplimab, Toripalimab, Tislelizumab, Tyvyt, and Camrelizumab.

[88] .Furthermore, the tumor is characterized in that it is a solid tumor.

[89] .Furthermore, the tumor is characterized in that it 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 cancer.

[90] .A The present invention further provides an antitumor drug for supporting antitumor immunotherapy, the antitumor drug comprising a full-length or fragment of wild-type or mutant IFN-γ, and supporting the antitumor effect of a T cell preparation and / or an immune checkpoint inhibitor by sensitizing tumor cells.

[91] . Furthermore, the antitumor drug comprises a targeting vector, which delivers the full-length or fragment of the wild-type or mutant IFN-γ to the tumor cells.

[92] . Furthermore, the antitumor drug is combined with the T cell preparation and / or the immune checkpoint inhibitor.

[93] .Furthermore, the antitumor drug is characterized in that it further comprises any pharmaceutically acceptable vector and / or adjuvant.

[0046]

[94] The present invention further provides an antitumor drug comprising an antitumor composition comprising wild-type or mutant IFN-γ and a T cell preparation.

[95] .A The present invention further provides an antitumor composition comprising wild-type or mutant IFN-γ, a PD-L1 inhibitor and / or a PD-1 inhibitor, and a T cell preparation.

[0047]

[96] .A specific embodiment of the invention:

[97] . Our experiments revealed that upregulation of PD-L1 and PD-1 expression levels coexists with CAR-T cell activation, tumor cell killing, and cytokine secretion during tumor cell killing by CAR-T cells (Figure 1). In Figure 1a, two types of CAR-T cells, epidermal growth factor receptor 2 (HER2)-CAR cells and mesothelin (MSLN)-CAR cells, were used. PD-1 expression was upregulated in both CAR-T cells in the presence of antigen stimulation. In this case, the antigen was tumor cells expressing HER2 or MSLN. In Figure 1b, CAR-T cells were co-cultured with tumor cells, and CAR-T cells were activated to kill tumor cells. Cell debris was removed by centrifugation to obtain the supernatant. This supernatant served as a microenvironment in which CAR-T cells could be activated and act on other tumor cells. Figure 1c shows that the addition of CAR-T cells increased the tumor cell lysis rate and upregulated both Granzyme B and OD107a+ in CAR-T cells, indicating that CAR-T cells were activated and played a role in tumor cell killing. Figure 1d shows the experiment, which included three groups: MOCK, HER2-CAR minus, and HER2-CAR plus. MOCK was a control T cell group that did not express the CAR construct. Figure 1c shows the co-culture of CAR-T and tumor cells. After 24 hours, CAR-T cells killed all tumor cells. The supernatant was collected by centrifugation and then used to culture new tumor cells. PD-L1 expression on tumor cells was then detected.

[0048]

[98] . T cell exhaustion markers TIM-3 and LAG-3 were significantly increased in CAR-T cells. In a stress experiment simulating the in vivo antitumor environment in which CAR-T cells repeatedly encountered and killed tumor cells (Figure 2), CAR-T cells exhibited enhanced cytotoxicity and cytokine release, despite high PD-L1 expression on tumor cells (Figure 3). These results suggest that CAR-T itself exerts a PD-L1-PD-1 inhibitory effect in CAR-T-mediated tumor cell killing.

[0049]

[99] . Subconcentrations of IFN-γ induce high expression of PD-L1 on tumor cells. However, when CAR-T cells encountered tumor cells pretreated with IFN-γ and highly expressing PD-L1, they exhibited higher potential cytolytic activity and IFN-γ secretion (Figure 5). On the other hand, neutralization of IFN-γ with an anti-IFN-γ antibody significantly reduced CAR-T cytotoxicity (Figure 6), indicating that IFN-γ is important for CAR-T activity.

[0050]

[0100] In the IFN-γ pretreatment test, to exclude the direct effect of IFN-γ on CAR-T cells, the medium was replaced with fresh medium so that residual IFN-γ was undetectable (<10 pg / ml).

[0101] In this study, tumor cells were pretreated with a low concentration of IFN-γ, and CAR-T cytotoxicity was measured in fresh medium containing sufficient anti-IFN-γ antibody to neutralize CAR-T IFN-γ. Unlike the case where CAR-T cells lost their tumor-killing effect against tumor cells not pretreated with IFN-γ (Figure 6), even after IFN-γ was neutralized, CAR-T cells still exhibited the ability to kill tumor cells pretreated with low concentrations of IFN-γ (Figure 7). Therefore, IFN-γ is thought to act on tumor cells to enhance the killing ability of CAR-T. In other words, pretreatment with IFN-γ increased the sensitivity of tumor cells to CAR-T cells.

[0051]

[0102] We then used the CRISPR-Cas9 system to block the IFN-γ signaling pathway and inhibit the expression of the IFN-γR2 (IFNGR2) gene on tumor cells. While IFN-γR1 is ubiquitous in mammalian cells, IFN-γR2 expression is more dynamic and is thought to determine the degree of IFN-γ-induced signaling in specific cell populations. Knockout-IFN-γR2 (IFN-γR2-null) tumor cells proliferated normally, similar to control cells. Knockout-IFN-γR2 tumor cells no longer expressed PD-L1 when treated with IFN-γ (three concentrations: 0, 5, and 10 ng / ml) (Figure 8). However, knockout-IFN-γR2 tumor cells were more resistant to lysis by CAR-T cells (Figure 9). In both tumor cell lines (SK-OV-3 and A549), the enhancement of CAR-T cytotoxicity by IFN-γ pretreatment was significantly suppressed (Figure 9), i.e., the tumor cell sensitization effect of IFN-γ pretreatment was suppressed. These results suggest that the action of IFN-γ on tumor cells is an essential mechanism by which CAR-T maintains or enhances its cellular activity (and antitumor effect) despite the expression of PD-L1 and PD-1.

[0052]

[0103] To directly characterize the inhibitory effect of IFN-γ on PD-L1-PD-1 signaling, our experimental group performed CAR-T activity detection on tumor cells stably expressing PD-L1. Overexpression of PD-L1 on tumor cells did not render tumor cells resistant to CAR-T killing, nor did it inhibit the enhanced cytotoxicity of CAR-T cells against tumor cells pretreated with IFN-γ (Figure 10). Thus, the enhanced sensitivity of tumor cells to CAR-T cells induced by IFN-γ pretreatment was not inhibited by PD-L1. However, in tumor cells with IFN-γR2 knockout, high PD-L1 expression significantly suppressed the killing effect of CAR-T cells (Figure 11). These results suggest that IFN-γ signaling overcomes the inhibitory effect of PD-L1-PD-1 signaling, enhancing tumor cell sensitivity to CAR-T cells and enhancing CAR-T cell activity.

[0053]

[0104] Next, we investigated how IFN-γ overcomes the inhibitory effect of PD-L1-PD-1 on CAR-T. IFN-γ not only induces PD-L1 expression, but also induces the expression of HLA molecules by tumor cells. HLA is a natural ligand that activates TCRs to activate T cells, but blocking HLA-ABC molecules does not affect the enhancing effect of IFN-γ on CAR-T cells.

[0054]

[0105] Intercellular adhesion molecule 1 (ICAM-1, also known as CD54) is a cell surface glycoprotein on antigen-presenting cells (APCs) and plays an important role in effective immune responses. In this study, we demonstrated that knockout of ICAM-1 in tumor cells with functional IFN-γ receptors almost completely abolished the enhanced CAR-T killing toxicity induced by IFN-γ pretreatment of tumor cells (Figure 14), predicting the importance of ICAM-1 for CAR-T activity. At the same time, IFN-γ stimulated tumor cell production of ICAM-1, enhancing the killing ability of T cells against tumor cells (Figure 15). On the other hand, overexpression of ICAM-1 in tumor cells with a normal IFN-γ signaling pathway did not significantly enhance specific tumor cell lysis by CAR-T (Figure 13). We believe this is due to the upregulation of ICAM-1 triggered by IFN-γ released from activated CAR-T cells. Therefore, in this study, we investigated the overexpression of ICAM-1 in IFN-γR2 knockout cells and found that the tumor cells became more sensitive to killing by CAR-T cells (Figure 13). These results suggest that IFN-γ-induced ICAM-1 expression suppresses PD-L1-PD-1 function and enhances the antitumor activity of CAR-T and T cells.

[0055]

[0106] Therefore, in this study, we investigated whether IFN-γ pretreatment contributes to improved therapeutic outcomes of CAR-T cell formulations for solid tumors. Continuous in vitro killing assays demonstrated that CAR-T cells maintained their killing activity against IFN-γ-pretreated tumor cells up to round 5, but only maintained their killing activity against non-IFN-γ-pretreated tumor cells up to round 3 (Figure 12). In other words, pretreatment with IFN-γ (as an adjuvant) enhanced the sensitivity of tumor cells to CAR-T cells, enhancing the therapeutic efficacy of CAR-T cell formulations as first-line treatments for solid tumors.

[0056]

[0107] Next, in our experiments, we established an intraperitoneal ovarian cancer model in NSG mice by injecting IFN-γ twice before intraperitoneal injection of CAR-T cells (Figure 16). Bioluminescence imaging revealed that IFN-γ did not significantly suppress tumor growth, and CAR-T alone only delayed tumor growth. However, the combination of IFN-γ pretreatment and CAR-T cells resulted in persistent tumor disappearance in three of five mice, and in the remaining two mice, tumors were significantly smaller than those in control mice (Figure 17). However, IFN-γR2-deficient tumors were more resistant to the combination therapy (Figure 17). These in vitro and in vivo results suggest that sequential administration of IFN-γ (as a sensitizing adjuvant) and CAR-T (as the primary drug) is an effective approach for solid tumor treatment.

[0057]

[0108] In summary, the experiments of the present invention revealed the following:

[0109] 1) After CAR-T cells contacted tumor cells, the inhibitory molecules PD-1 and PD-L1 were highly expressed on CAR-T cells and tumor cells, respectively. At the same time, activation markers such as granzyme B, perforin, and cytokine release were upregulated in CAR-T cells, leading to efficient tumor cell killing by CAR-T.

[0110] 2) IFN-γ pretreatment upregulated PD-L1 expression on tumor cells, but simultaneously promoted (but did not inhibit) tumor cell killing and cytokine release by CAR-T. (IFN-γ was administered at a low concentration and did not significantly inhibit tumor cells.)

[0111] 3) In a system in which CAR-T cells were co-cultured with tumor cells, neutralizing IFN-γ using an IFN-γ neutralizing antibody significantly reduced the killing function of CAR-T cells. Knocking out the IFN-γ receptor gene (IFNγR2) in tumor cells resulted in the tumor cells no longer expressing PD-L1, but the killing function of CAR-T cells was reduced.

[0112] 4) Overexpression of PD-L1 on tumor cells did not significantly inhibit CAR-T activity, but overexpression of PD-L1 on tumor cells with the IFNγ receptor gene (IFNγR2) knocked out significantly inhibited the killing function of CAR-T.

[0113] 5) IFN-γ induced high expression of ICAM-1 in tumor cells. After ICAM-1 was knocked out in tumor cells, IFN-γ pretreatment could no longer enhance the killing activity of CAR-T.

[0114] .6) Overexpression of ICAM-1 molecules in IFNγR2-deficient tumor cells promoted the killing function and cytokine release ability of CAR-T.

[0115] .7)IFN-γ can enhance the therapeutic effect of CAR-T cells in a mouse ovarian cancer peritoneal tumor model.

[0116] .8) Pretreatment of tumor cells with IFN-γ promoted the expression of ICAM-1 on tumor cells and improved the killing ability of conventional T cells against tumor cells.

[0058]

[0117] .B Invention: CAR expression vector and its application

[0118] The present invention belongs to the field of biomedicine, and more particularly relates to CAR expression vectors and their applications.

[0119] Adoptive cell therapy (ACT) is an effective anti-cancer strategy often referred to 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 shown remarkable success in the treatment of hematological malignancies. However, their effectiveness in the treatment of solid tumors remains unsatisfactory and significant challenges remain. The reasons for the poor efficacy of CAR-T cell therapy against solid tumors are unclear, but several factors are suspected. For example, CAR-T cells have low survival rates in vivo, and die soon after administration, negatively impacting their killing effect. In addition, solid tumors create an immunosuppressive microenvironment due to their pluripotency in metabolism, immune evasion, and tissue formation, making it difficult for CAR-T cells to infiltrate and exert their effects within solid tumor tissues. Even if they do enter solid tumors, CAR-T cells are inactivated and die due to various immune-limiting mechanisms. Therefore, how to improve the killing effect of CAR-T cells and related drug formulations against solid tumors is an urgent issue in this field.

[0059]

[0120] To enhance the therapeutic efficacy of CAR-T cells, current research focuses on further genetically modifying or nanomodifying CAR-T cells themselves. For example, multi-target CAR genes can be inserted into CAR-T cells, chemokine expression can be induced in CAR-T cells, or adhesion molecules or chemical groups can be directly modified to enhance CAR-T cell affinity and recognition toward tumor tissue. However, each approach has its own set of drawbacks. For example, in the case of multi-target CAR-T cells, inserting too many foreign genes can affect the intrinsic activity of T cells. Chemical modification typically reduces cell activity and also reduces motility and migration. Therefore, CAR-T cell modification must comprehensively consider both positive and negative factors, ultimately leading to the selection of a more targeted and effective modification method.

[0060]

[0121] Among the conventional technological routes, modifying CAR-T cells to inhibit PD-1 is considered a feasible approach. PD-1 negatively regulates T cell-mediated immune responses by binding to its ligand, programmed death ligand 1 (PD-L1), so blocking the PD-L1-PD-1 signaling pathway is an effective strategy. Therefore, blocking the PD-L1-PD-1 signaling pathway is an effective antitumor immunotherapy strategy and has shown favorable clinical results. Several antibody drugs targeting PD-L1 / PD-1 are currently on the market, including PD-1 antibodies such as pembrolizumab, nivolumab, and cemiplimab, and PD-L1 antibodies such as atezolizumab, avelumab, and durvalumab.

[0061]

[0122] Inhibitory signaling pathways, such as PD-1, are also important factors that determine the efficacy of CAR-T therapy. Studies have shown that blocking the PD-1 pathway can improve the antitumor efficacy of CAR-T cells. Therefore, traditional technological approaches have attempted to significantly improve the therapeutic efficacy against solid tumors by genetically engineering CAR-T cells to secrete antibodies against PD-1 and PD-L1. Alternatively, gene editing can be used to knock out the gene encoding PD-1 in CAR-T cells, potentially improving the antitumor activity of CAR-T.

[0062]

[0123] However, current technological solutions and pathways focus on improving the function of CAR-T cells themselves, with little impact on tumor cells or the immunosuppressive microenvironment within solid tumors. In other words, current technological solutions and pathways focus solely on improving CAR-T cells themselves, limiting their potential to enhance the antitumor effect of CAR-T cell therapy from the perspective of interacting with tumor cells.

[0063]

[0124] Therefore, the invention B aims to provide a novel CAR-T cell preparation that can affect not only tumor cells but also the immunosuppressive microenvironment within solid tumors, thereby optimizing the anti-tumor effect of the CAR-T cell preparation.

[0125] One of the aims of the present invention is to provide novel CAR expression vectors and construction strategies.

[0126] In order to achieve the above objective, the technical solution of invention B is as follows:

[0127] A CAR expression vector comprising 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 nucleic acid encoding a full-length or fragment of wild-type or mutant IFN-γ.

[0128] Further, the nucleic acid encoding the CAR is linked to the nucleic acid encoding the tumor cell sensitizing factor by a sequence encoding a self-cleaving peptide.

[0129] Further, the nucleic acid encoding the CAR comprises a nucleic acid encoding a single-chain antibody variable region (ScFv) targeting a tumor-specific antigen, a CD8a signal peptide, a CD8 hinge region, a CD8a transmembrane domain, a 4-1BB intracellular costimulatory element, and a CD3ζ intracellular domain.

[0130] Furthermore, the tumor-specific antigen is one or more selected from HER2, Mesothelin, GPC-3, EGFRvIII, MUC1, CD19, CD30, BCMA, EGFR, CD123, CD133, PSCA, GD2, and LewisY.

[0131] Furthermore, the sequence encoding the self-cleaving peptide is a P2A sequence.

[0132] Furthermore, the nucleic acid sequence encoding the CAR is represented by SEQ ID NO: 1, the nucleic acid sequence encoding the IFN-γ is represented by SEQ ID NO: 2, and the sequence encoding the self-cleaving peptide is represented by SEQ ID NO: 3.

[0133] A further object of the present invention is to provide a lentivirus.

[0134] A lentivirus comprising the CAR expression vector.

[0135] In a preferred embodiment of the present invention, the lentivirus is pWPXLd. However, lentivirus expression plasmids commonly used in the art, including psPAX2, pMD2.G, pVSVG, etc., may also be included in the technical embodiments of the present invention.

[0136] Another object of the present invention is to provide novel CAR-T cells and preparations thereof, as well as novel T cells and preparations thereof.

[0137] CAR-T cells into which a vector shown in (a) or (b) below has been introduced: (a) the CAR expression vector, or (b) a CAR expression vector containing a nucleic acid encoding a CAR and a nucleic acid encoding IFN-γ.

[0138] An anti-tumor drug comprising the CAR-T cells and pharmaceutically acceptable auxiliary materials and / or adjuvants.

[0139] Furthermore, the tumor is a solid tumor.

[0140] Further, 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 cancer.

[0141] .T cells expressing CAR and IFN-γ.

[0142] Further, the T cells comprise a nucleic acid encoding a CAR and a nucleic acid encoding IFN-γ.

[0143] An antitumor drug comprising T cells expressing CAR and IFN-γ and pharmaceutically acceptable auxiliary materials and / or adjuvants.

[0144] Furthermore, the tumor is a solid tumor.

[0145] Further, 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 cancer.

[0146] A method for preparing T cells that express CAR and IFN-γ, comprising reintroducing a nucleic acid encoding CAR and a nucleic acid encoding IFN-γ into the T cells using a vector.

[0064]

[0147] .B Specific embodiments of the invention

[0148] .B Invention Example 1: IFN-γ enhances tumor cell sensitivity to immunotherapy

[0149] Tumor cells were induced to express high levels of PD-L1 with subcytotoxic concentrations of IFN-γ (i.e., tumor cells were pretreated with IFN-γ). Next, when CAR-T cells (HER2-CAR, MSLN-CAR) were co-administered with IFN-γ-pretreated tumor cells with high levels of PD-L1 expression, the CAR-T cells exhibited higher potential cytolytic activity and IFN-γ secretion (Figure 18). To rule out a direct effect of IFN-γ on CAR-T cells, the IFN-γ pretreatment test confirmed that residual IFN-γ was undetectable (<10 pg / ml) before replacing the medium with fresh medium.

[0065]

[0150] In addition, we pretreated tumor cells with a low concentration of IFN-γ and measured the cytotoxicity of CAR-T (HER2-CAR) in fresh medium containing sufficient anti-IFN-γ antibody to neutralize IFN-γ of CAR-T. Unlike the case where the killing effect of CAR-T cells against tumor cells not pretreated with IFN-γ was significantly reduced (Figure 19), even after IFN-γ was neutralized, CAR-T cells still exhibited the ability to kill tumor cells pretreated with a low concentration of IFN-γ (Figure 20).

[0151] These experiments demonstrated that IFN-γ plays a crucial role in tumor cell killing by CAR-T, and that IFN-γ acts on tumor cells to enhance the killing ability of CAR-T. In other words, pretreatment with IFN-γ increased the susceptibility of tumor cells to CAR-T cells.

[0066]

[0152] Next, our experimental team established an intraperitoneal ovarian cancer model in NSG mice, in which IFN-γ was injected twice before intraperitoneal injection of CAR-T cells (Figure 16). Bioluminescence imaging revealed that IFN-γ did not significantly suppress tumor growth, and CAR-T cells alone only delayed tumor growth. However, the combination of IFN-γ pretreatment and CAR-T cells resulted in persistent tumor disappearance in three of five mice, and in the remaining two mice, tumors were significantly smaller than those in control mice (Figure 17). However, when the IFN-γ signaling pathway in tumor cells was blocked (e.g., by knocking out IFN-γR2 in the experiment shown in Figure 17), the tumors became more resistant to CAR-T cell therapy combined with IFN-γ pretreatment (Figure 17).

[0153] Furthermore, our experiments demonstrated that IFN-γ is not only important for tumor cell killing by CAR-T, but also enhances the tumor cell-killing effect of conventional T cells (i.e., non-genetically modified T cells) (Figure 21).

[0154] These in vitro and in vivo experimental results demonstrate that both genetically modified and conventional T cells exhibit enhanced killing effects (i.e., the IFN-γ signaling pathway is activated) when they encounter tumor cells pretreated with IFN-γ. IFN-γ is suggested to enhance the tumor cell-killing ability of both conventional T cell lines and genetically modified T cells (e.g., various types of CAR-T and / or TCR-T).

[0155] In addition to increasing the susceptibility of tumor cells to T cells by IFN-γ pretreatment, the present invention proposes a technical solution to genetically modify T cells (e.g., CAR-T cells) so that the genetically modified T cells themselves secrete IFN-γ to sensitize tumor cells and enhance their tumor cell-killing effectiveness. This may provide a more effective and convenient therapeutic strategy than IFN-γ pretreatment of tumor cells.

[0067]

[0156] .B Invention Example 2: Novel CAR Expression Vector

[0157] Example 2 of the present invention provides a novel CAR expression vector comprising 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 nucleic acid encoding the full-length or fragment of wild-type or mutant IFN-γ.

[0158] .Vector structure

[0159] A plasmid vector containing an expression cassette expressing a chimeric antigen receptor (CHIR) capable of specifically secreting IFN-γ was constructed. The structure and positional relationship of the elements on the expression cassette are shown in Figure 22a, and the plasmid vector backbone is shown in Figure 22b. The specific steps are as follows: The lentiviral vector pWPXLd contains a first expression cassette targeting the CHIR in HER2-positive tumor cells. The first expression cassette (HER2-CAR) contains the CD8a signal peptide, the HER2 single-chain antibody variable region, the CD8 hinge region, the CD8a extended membrane domain, the 4-1BB intracellular costimulatory element, and the CD3ζ intracellular domain. Primers were designed to amplify a specific fragment of human IFN-γ from T cell cDNA for the second expression cassette. The second IFN-γ expression cassette and the first expression cassette were co-expressed in the lentiviral vector pWPXLd via the P2A sequence (a self-cleaving sequence). The entire recombinant plasmid was designated the "HER2-CAR-IFN-γ plasmid."

[0068]

[0160] In this embodiment, a plasmid vector targeting HER2 was constructed, but the method provided by the present invention is not limited to this. The method provided by the present invention also enables the construction of 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.

[0161] The methods provided by the present invention can also be used to construct recombinant plasmids with similar functions using other molecular biology tools, such as using other promoters and / or linking a first expression cassette to a second expression cassette using T2A or IRES.

[0162] Construction of CAR-T cells expressing chimeric antigen receptors and IFN-γ

[0163] Peripheral blood mononuclear cells (PBMCs) were isolated from lymphocyte isolates and activated using CD3 and CD28 magnetic beads. The virus was then added at a set MOI to infect activated T cells. The resulting two CAR-T cells were named HER2-CART cells and HER2-IFNG-CART cells, respectively, and the expression level of CAR in T cells was detected by flow cytometry.

[0069]

[0164] .B Invention Example 3

[0165] CAR-T killing function and cytokine secretion detection

[0166] (1) SK-OV-3, a target cell line expressing HER2 positive, was divided into a MOCK group (MOCK is a T cell line without a CAR expression construct), a HER2-CAR group, a HER2-IFNG-CAR group, a maximum release group, and a spontaneous release group, and 1 × 10 cells were placed in a 96-well plate. 4 After cell attachment, CAR-T cells were added at the corresponding effector:target (E:T) ratio and co-cultured for 24 hours. The killing level of CAR-T was detected using an LDH (lactate dehydrogenase) kit, and the cell supernatant was frozen and stored at -80°C for cytokine detection.

[0070]

[0167] (2) Cytokine detection

[0168] IFN-γ expression was detected using an ELISA kit from Biolegend according to the manufacturer's instructions.

[0169] Figures 23 and 24 summarize the results of CAR-T tumor-killing function and cytokine secretion detection. As shown in Figure 23, CAR-T cells expressing the CAR expression vector of the present invention (containing IFN-γ nucleic acid) were confirmed to have a significantly higher tumor cell-killing effect than CAR-T cells expressing only HER2-CAR. As shown in Figure 24, the cytokine IFN-γ secretion ability of CAR-T cells expressing the CAR expression vector of the present invention (containing IFN-γ nucleic acid) was also significantly improved compared to CAR-T cells expressing only HER2-CAR.

[0170] The above experiments of the present invention demonstrate that genetic modification of T cells to increase their IFN-γ secretion can alter the tumor cell microenvironment and improve the tumor cell sensitivity to T cell immunotherapy. Furthermore, in addition to the CAR expression vectors constructed according to the present invention and CAR-T cells expressing these vectors, natural T cells that naturally express high levels of IFN-γ and T cells (e.g., TCR-T cells) that express high levels of IFN-γ through other genetic modifications are also likely to be more effective at killing tumors.

[0171] Although the embodiments of the present invention have been described above with reference to the accompanying drawings, the present invention is merely exemplary and not limiting, and there are many forms that a person skilled in the art can create by being inspired by the present invention without departing from the purpose of the present invention and the scope protected by the claims, all of which fall within the scope of protection of the present invention.

Claims

1. A kit for detecting the antitumor effect of an immune checkpoint inhibitor consisting of a PD-L1 inhibitor and / or a PD-1 inhibitor, for detecting the patency of the IFN-γ signaling pathway in tumor cells.

2. The kit according to claim 1, for detecting the expression level of ICAM-1 or IFN-γR2 in tumor cells.

3. The PD-L1 inhibitor is one or more selected from atezolizumab, avelumab, and durvalumab, and The PD-1 inhibitor is one or more selected from pembrolizumab, nivolumab, cemiplimab, toripalimab, tislelizumab, sintilimab (Tyvyt™), and camrelizumab; The kit according to claim 1 or 2.

4. The kit according to any one of claims 1 to 3, wherein the tumor is a solid tumor.

5. The kit according to any one of claims 1 to 4, wherein 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 cancer.

6. An antitumor drug for supporting antitumor immunotherapy, comprising full-length or fragments of wild-type or mutant IFN-γ, and for supporting the antitumor effect of T cell preparations and / or immune checkpoint inhibitors by sensitizing tumor cells.

7. The antitumor drug according to claim 6, further comprising a targeting vector for delivering full-length or fragments of wild-type or mutant IFN-γ to the tumor cells.

8. The antitumor drug according to claim 6 or 7, which is used in combination with a T cell preparation and / or an immune checkpoint inhibitor.

9. The antitumor drug according to any one of claims 6 to 8, further comprising any pharmaceutically acceptable vector and / or adjuvant.

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

11. The CAR expression vector of claim 10, wherein the nucleic acid encoding the CAR is linked to the nucleic acid encoding the tumor cell sensitizer by a sequence encoding a self-cleaving peptide.

12. The CAR expression vector according to claim 10, wherein the nucleic acid encoding the CAR consists of nucleic acids encoding a single-chain antibody variable region (ScFv) targeting a tumor-specific antigen, a CD8a signal peptide, a CD8 hinge region, a CD8a transmembrane domain, a 4-1BB intracellular costimulatory element, and a CD3ζ intracellular domain.

13. The CAR expression vector according to claim 12, wherein the tumor-specific antigen is one or more selected from HER2, mesothelin, GPC-3, EGFRvIII, MUC1, CD19, CD30, BCMA, EGFR, CD123, CD133, PSCA, GD2, and Lewis Y.

14. The CAR expression vector of claim 10, wherein the sequence encoding the self-cleaving peptide is a P2A sequence.

15. The CAR expression vector according to claim 10, wherein the nucleic acid sequence encoding the CAR is represented by SEQ ID NO: 1, the nucleic acid sequence encoding IFN-γ is represented by SEQ ID NO: 2, and the sequence encoding the self-cleaving peptide is represented by SEQ ID NO:

3.

16. A lentivirus comprising the CAR expression vector according to any one of claims 10 to 15.

17. The lentivirus of claim 16, wherein the lentivirus is pWPXLd.

18. (a) or (b) below: (a) the CAR expression vector according to any one of claims 10 to 15, or (b) a CAR expression vector comprising a nucleic acid encoding a CAR and a nucleic acid encoding IFN-γ; CAR-T cells into which

19. An antitumor drug comprising the CAR-T cells of claim 18 and pharmaceutically acceptable auxiliary materials and / or adjuvants.

20. T cells expressing CAR and IFN-γ.

21. The T cell of claim 20, comprising a nucleic acid encoding a CAR and a nucleic acid encoding IFN-γ.

22. 22. An antitumor drug comprising the T cells of claim 20 or 21 and pharmaceutically acceptable secondary ingredients and / or adjuvants.

23. The antitumor drug according to claim 19 or 22, wherein the tumor is a solid tumor.

24. The antitumor drug according to claim 19 or 22, wherein 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 cancer.

25. A method for preparing T cells expressing a CAR and IFN-γ, the method comprising reintroducing a nucleic acid encoding a CAR and a nucleic acid encoding N-γ into the T cells using a vector.

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