CN117731760A

CN117731760A - Application of IFN-gamma in preparing antitumor auxiliary medicine

Info

Publication number: CN117731760A
Application number: CN202311622393.5A
Authority: CN
Inventors: 杨寒朔; 董娥; 岳小竺
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2020-03-09
Filing date: 2020-03-09
Publication date: 2024-03-22
Also published as: CN113368217B; CN113368217A

Abstract

The utility model belongs to the field of biological medicine, and in particular relates to an application of IFN-gamma in preparing an antitumor auxiliary drug, wherein the antitumor auxiliary drug is used for assisting a T cell preparation, and is characterized in that the IFN-gamma enhances the killing effect of the T cell preparation on tumor cells by sensitizing the tumor cells; the T cell formulations include T cells that are not genetically modified and/or T cells that are genetically engineered (e.g., CAR-T cells, TCR-T cells). Through the principles of activating tumor cell signal paths by INF-gamma and the like, the utility model not only can prolong the cytotoxicity of the T cell preparation to tumor cells and has better inhibition effect than the single use of the T cell preparation, but also can promote the sustainable inhibition of the T cell preparation to tumor.

Description

Application of IFN-gamma in preparing antitumor auxiliary medicine

This application will likely be the basis of priority for subsequent patent applications including, but not limited to, chinese utility model patent application, chinese utility model application, PCT application, foreign application based on paris convention.

Technical Field

The utility model belongs to the field of biological medicine, and in particular relates to an anti-tumor medicine for assisting anti-tumor immunotherapy and application thereof.

Background

Tumor immunotherapy is a revolutionary advance in anti-tumor therapy by mobilizing the body's own immune system to combat the tumor. CAR-T therapy belongs to the field of immunotherapy. CAR-T cell therapy achieves surprising anti-tumor effects in hematological tumors that target CD 19. By month 4 of 2018, there were a total of 2513 immunotherapies against tumors, 382 in china, 227 in clinical stages III and IV. Kymeriah from the nowa company and yescanta from the Kite company have been marketed for the treatment of pediatric acute B-cell leukemia and adult B-cell lymphoma, respectively. Wherein the total remission rate (CR) of Kymriah is 82.5%, the recurrence probability is 75% at 6 months and 64% at 12 months. Another CD19 specific CAR-T product, yescanta, approved by the FDA, achieves 43-52% CR in patients with recurrent or refractory DLBCL.

However, CAR-T is not as effective as treatment for solid tumors, e.g. EGFR-CAR-T in phase I clinical studies of epidermal growth factor receptor cells for the treatment of recurrent/refractory egfr+ non-small cell lung cancer, with only 2 responders and 5 patients with Stable Disease (SD) among 11 cancer patients. The feasibility and safety of HER2-CAR-T cells administration to patients with recurrent or refractory HER2 positive tumors was evaluated in phase I/II studies in dose escalation clinical trials. However, its clinical benefit is limited, with only 4/17 of the evaluable patients exhibiting SD. That is to say that CAR-T cell therapy against solid tumors is of limited efficacy. How to enhance and improve the therapeutic effect of the CAR-T cells on solid tumors is a problem to be solved in the prior art.

PD-1 was first discovered as an inhibitory molecule in costimulatory signals, the primary function being to induce T cell apoptosis. PD-1 negatively regulates T-cell mediated immune responses through engagement with its ligand, programmed death ligand 1 (PD-L1), and thus blocking this signaling pathway is considered an effective cancer immunotherapy. Since the first approval of the 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 an unprecedented increase. PD-1 and PD-L1 inhibitors are useful 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, and the like. Up to now, inhibitors against PD-1 have been marketed in three ways abroad: pembrolizumab, nivolumab, cemiplimab; there are 4 on the market in China: toripalimab, tislelizumab, tyvyt and Camrelizumab. Inhibitors of PD-L1 have been marketed as: atezolizumab, avelumab and Durvalumab. When PD-L1/PD-1 therapies are applied to more solid tumors, how to make PD-L1/PD-1 antibody therapies produce better anti-tumor therapeutic effects is a problem that is currently in need of resolution.

In summary, how to improve the anti-tumor effect of the existing immunotherapy such as immune cell therapy and/or immune checkpoint inhibitor therapy, and propose a more optimized and more effective anti-tumor therapeutic scheme, which is a very important problem in the field of current tumor immunotherapy, and is also a technical problem to be solved in an attempt of the utility model.

Disclosure of Invention

In view of the above, the present utility model aims to provide an application of IFN-gamma in preparing antitumor auxiliary drugs; a kit for detecting an anti-tumor effect of an immune checkpoint inhibitor; an antitumor drug for assisting antitumor immunotherapy; an application of IFN-gamma and T cell preparation in preparing medicines for tumor immunotherapy; use of IFN- γ in combination with an immune checkpoint inhibitor for the preparation of a medicament for tumour immunotherapy; use of an IFN- γ, a T-cell preparation and an immune checkpoint inhibitor in combination for the preparation of a medicament for tumour immunotherapy; a combination of the above.

In order to achieve the above purpose, the technical scheme of the utility model is as follows:

the application of IFN-gamma in preparing antitumor auxiliary medicine for auxiliary T cell preparation, and features that IFN-gamma enhances the killing effect of T cell preparation on tumor cell by sensitization of the tumor cell; the T cell preparation comprises T cells without genetic modification and/or T cells modified by genetic engineering means; the IFN-gamma includes wild-type or mutant IFN-gamma full length or IFN-gamma fragments.

Further, the genetically modified T cells include CAR-T cells and/or TCR-T cells.

Further, the IFN-gamma sensitizes the tumor cells by activating IFN-gamma signaling pathways of the tumor cells.

Further, IFN-gamma signaling activation of the tumor cells up-regulates ICAM-1 to enhance the killing effect of the T cell preparation on the tumor cells.

Further, the tumor type used by the antitumor auxiliary drug is solid tumor.

Further, the tumor type used by the antitumor auxiliary drug 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.

The utility model also provides application of IFN-gamma in preparing an antitumor auxiliary medicament, wherein the antitumor auxiliary medicament is used for assisting an immune checkpoint inhibitor, and is characterized in that the IFN-gamma assists the antitumor effect of the immune checkpoint inhibitor through sensitized tumor cells, and the immune checkpoint inhibitor comprises a PD-L1 inhibitor and/or a PD-1 inhibitor; the IFN-gamma includes wild-type or mutant IFN-gamma full length or IFN-gamma fragments.

Further, the PD-L1 inhibitor comprises 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.

Further, the tumor type used by the antitumor auxiliary drug is solid tumor.

Further, the antitumor auxiliary drug is characterized in that the tumor type used for the antitumor auxiliary drug 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.

The utility model also provides application of IFN-gamma in preparing an antitumor auxiliary medicament, wherein the antitumor auxiliary medicament is used for assisting a T cell preparation and an immune checkpoint inhibitor, and is characterized in that the IFN-gamma assists the antitumor effect of the T cell preparation and the immune checkpoint inhibitor by sensitizing tumor cells; the immune checkpoint inhibitor comprises a PD-L1 inhibitor and/or a PD-1 inhibitor; the T cell preparation comprises T cells without genetic modification and/or T cells modified by genetic engineering means; the IFN-gamma includes wild-type or mutant IFN-gamma full length or IFN-gamma fragments.

Further, stimulation of tumor cells by the helper drug to produce ICAM-1 enhances killing of tumor cells by the T cell preparation.

Further, the tumor is of the type of solid tumor.

Further, 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 cancer.

The utility model also provides a kit for detecting the anti-tumor effect of the immune checkpoint inhibitor, wherein the immune checkpoint inhibitor comprises a PD-L1 inhibitor and/or a PD-1 inhibitor, and the kit is characterized by comprising detection of the IFN-gamma signal pathway smoothness of tumor cells.

Further, the kit includes detection of ICAM-1 or IFN- γR2 expression levels in the tumor cells. It should be emphasized that the detection of ICAM-1 or IFN- γR2 expression levels is aimed at detecting IFN- γ signaling pathway patency, and that it is within the scope of the present utility model to be able to effectively detect IFN- γ signaling pathway patency, whether it be full length or a fragment of wild-type or mutant ICAM-1 and IFN- γR 2.

Further characterized in that the tumor is of the type solid tumor.

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

The utility model also provides an anti-tumor drug for assisting anti-tumor immunotherapy, which is characterized in that the anti-tumor drug comprises wild type or mutant IFN-gamma or IFN-gamma fragments, and the anti-tumor effect of the T cell preparation and/or the immune checkpoint inhibitor is assisted by sensitized tumor cells.

Further, the anti-tumor drug comprises a targeting vector that delivers the wild-type or mutated IFN-gamma or IFN-gamma fragment to the tumor cells.

Further, the anti-tumor agent is used in combination with the T cell preparation and/or the immune checkpoint inhibitor.

Further characterized in that the antitumor drug further comprises any pharmaceutically acceptable carrier and/or adjuvant.

The utility model also provides an anti-tumor drug, which comprises an anti-tumor composition: wild-type or mutant IFN-gamma and T cell preparations.

The utility model also provides an anti-tumor composition comprising a wild-type or mutant IFN-gamma and PD-L1 inhibitor and/or PD-1 inhibitor and a T cell preparation.

The beneficial effects of the utility model are that

The utility model breaks through the cognition of IFN-gamma in the prior art, discovers that IFN-gamma activates an IFN-gamma signal channel in tumor cells, and enables the tumor cells to sensitize (sensitize) to immunotherapy (T cell preparation and/or PD-L1/PD-1 inhibitor) by, for example, up-regulating the expression of ICAM-1 in the tumor cells, inhibits the acquired immune resistance mediated by PD-L1-PD-1, enhances the anti-tumor effect of immunotherapy, and further provides a technical scheme for applying IFN-gamma as an immunotherapy auxiliary medicament in anti-tumor.

Interferon (IFN) consists of a cluster of secreted proteins. The IFN protein family is classified into type 3, i.e., type I, type II and type III interferons based on their gene sequences, chromosome localization and receptor specificity. Among them, type II interferon is composed of monogenic family IFN-gamma, also called immune interferon. Under normal conditions, IFN-gamma is very low in intracellular content, and the expression level is obviously improved after induction by viruses or other interferon inducers. IFN-gamma is the most important cytokine involved in anti-tumor immunity and is an immunocompetent cytokine released after CAR-T contacts antigen. Normal tissues, in order to avoid damage from the activated immune response, promote the reconstitution of the balance of cells in the body under inflammatory conditions by inducing various regulatory pathways. Prior studies suggest that tumor cells may utilize these protective regulatory pathways for immune escape, e.g., tumor cells may up-regulate PD-L1 expression by releasing IFN- γ, thereby inhibiting T cell killing, and this acquired immune resistance is considered one of the possible reasons for restricting CAR-T from effectively killing solid tumors. Because it is considered that IFN-gamma up-regulation of PD-L1 expression induces tumor cells to develop acquired immune resistance, the existing immunotherapy technical scheme fails to fully utilize IFN-gamma. Even IFN-gamma antitumor treatment schemes which enter clinical trials in the early years cannot be marketed as antitumor drugs because of poor effects. It is now believed that IFN-gamma up-regulates PD-1 expression, resulting in poor anti-tumor effects, and therefore, it is believed that the use of a PD-L1 inhibitor in combination with IFN-gamma up-regulates PD-L1 is required to neutralize the negative effects of IFN-gamma. However, this is only a theoretical assumption, and no actual results have yet been confirmed.

The experimental results of the present utility model show that IFN-gamma (through pretreatment of tumor cells, IFN-gamma signaling pathway is activated) can up-regulate the expression of PD-L1 by tumor cells, but the IFN-gamma also affects the expression of other factors (such as up-regulating the expression of ICAM-1), and the result shows that the IFN-gamma significantly enhances the cytotoxicity of CAR-T cells (or other types of T cells which are modified by genes) or T cells which are not modified by genes to the tumor cells (figure 12), and the IFN-gamma can also promote the continuous killing capacity of the CAR-T cells to the tumor cells. In other words, IFN-gamma sensitizes tumor cells to T cells, increasing their sensitivity to T cells. Furthermore, the experimental result of the utility model also shows that the killing effect of the CAR-T on tumor cells is obviously affected after the IFN-gamma signal channel is damaged.

Based on the experimental results and reasonable deduction, the utility model provides a technical scheme for treating solid tumors by using IFN-gamma as an auxiliary drug in combination with a T cell preparation, and application of IFN-gamma as an auxiliary drug in anti-tumor treatment. The anti-tumor effect of T cell preparations (including T cells which are not modified by genes or T cells modified by genetic engineering means (for example, CAR-T, TCR-T)) can be improved by the principles of activating tumor cell signaling pathway by INF-gamma and the like. Since the core of the utility model is to activate the IFN-gamma signal pathway of the tumor cells, whether wild type or mutant, whether full length or fragment of IFN-gamma can be applied to the utility model, so long as the IFN-gamma signal pathway of the tumor cells can be effectively activated, and the utility model is within the technical scheme.

In addition, because the principle of action of PD-L1 inhibitors and/or PD-1 inhibitors is that activated depleted T cells restore their anti-tumor effects, the anti-tumor effects of PD-L1 inhibitors and/or PD-1 inhibitors are also mediated by T cells in nature. Thus, the present utility model also provides therapeutic regimens for enhancing the anti-tumor effect of PD-L1 inhibitors and/or PD-1 inhibitors by IFN- γ, and therapeutic regimens for enhancing the anti-tumor effect of PD-L1 inhibitors and/or PD-1 inhibitors in combination with T cell preparations by IFN- γ. It should be emphasized that the technical solution proposed in the present utility model is to utilize IFN- γ sensitized tumor cells to further enhance the anti-tumor effect of auxiliary immune checkpoint inhibitors (PD-L1 inhibitors and/or PD-1 inhibitors), which is different from the technical solution that utilizes PD-L1 inhibitors and/or PD-1 inhibitors to inhibit the up-regulation of tumor cells PD-L1 generated by activation of IFN- γ signaling pathway. The main medicine and the auxiliary medicine are also positioned differently.

In addition, the experiments of the utility model show that the defect of IFN-gamma signal pathway can affect the anti-tumor effect of CAR-T cells, and the combination of the defect of IFN-gamma signal pathway can affect the anti-tumor effect of PD-L1 inhibitor and/or PD-1 inhibitor, the utility model also provides a method for judging whether T cell treatment and/or PD-L1 inhibitor and/or PD-1 inhibitor is a proper anti-tumor treatment choice or not in advance by detecting the activation/defect condition of IFN-gamma signal pathway (for example, detecting ICAM-1 or IFN-gamma R2 expression on tumor cells).

In summary, the technical scheme provided by the utility model breaks through the established cognition of IFN-gamma in the prior art, and the provided immunotherapy auxiliary drug taking IFN-gamma as a core enhances the anti-tumor effect of cell therapy and immune checkpoint inhibitor combined cell therapy, and further improves the anti-tumor effect of the existing immunotherapy scheme.

Drawings

In order to more clearly illustrate the embodiments of the present utility model or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are of some embodiments of the utility model and that other drawings may be derived from these drawings without inventive faculty.

FIG. 1 shows the activation of CAR-T cells and the expression of PD-L1 and PD-1 when the CAR-T cells kill solid tumor cells (a is HER2 (Epidermal Growth Factor Receptor 2) -CAR cells and MSLN (Mesothelin) -CAR cells, the expression of PD-1 on the CAR-T cells is up-regulated under the condition of antigen stimulation, B is the high expression of PD-L1 by tumor cells, c is the effective killing of the tumor cells by the CAR-T cells, granzyme B and CD107a are high expression, and d is 3 cases: MOCK, HER2-CAR minus and HER2-CAR plus).

Figure 2 is a schematic of an experiment simulating CAR-T cells in an in vivo anti-tumor environment continuously encountering and killing tumor cells.

Figure 3 shows that CAR-T cells show increasingly higher cytotoxicity and cytokine release capacity.

FIG. 4 shows the high expression of PD-L1 on tumor cells.

Figure 5 is a graph of 5.CAR-T cells showing more potential cytolytic activity and IFN- γ secretion capacity when CAR-T cells encounter tumor cells pretreated with IFN- γ and highly expressing PD-L1.

FIG. 6 shows that when IFN-gamma is neutralized by anti-IFN-gamma antibodies, the cytotoxicity of the CAR-T cells is significantly reduced.

FIG. 7 is a graph showing that CAR-T cells still exhibit killing ability against IFN-gamma pretreated tumor cells when IFN-gamma is neutralized.

FIG. 8 shows that tumor cells from which IFN-. Gamma.R 2 was knocked out were treated with IFN-. Gamma.at three concentrations of 0,5,10ng/ml and did not express PD-L1.

FIG. 9 shows that the killing effect of CAR-T cells on IFN- γR2 knocked-out tumor cells was significantly reduced, and that IFN- γ treatment also failed to enhance the killing effect of CAR-T cells on IFN- γR2 knocked-out tumor cells.

FIG. 10 is an illustration of over-expression of PD-L1 in tumor cells without inhibiting the inhibition of CAR-T killing by tumor cells nor the enhancement of CAR-T cytotoxicity by IFN-gamma pretreatment.

FIG. 11 shows that high expression of PD-L1 in tumor cells knocked out of IFN- γR2 can significantly inhibit killing of tumor cells by CAR-T cells.

FIG. 12 is the effect of CAR-T cells on IFN-gamma pre-treated tumor cells in a continuous in vitro killing experiment.

FIG. 13 shows that ICAM-1 is highly expressed in tumor cells knocked out of INF- γR2 gene, enhancing killing effect of CAR-T cells.

FIG. 14 shows that knocking out INF- γR2 gene in tumor cells, IFN- γ pretreatment of tumor cells did not enhance the killing effect of CAR-T cells.

FIG. 15 is a graph showing the effect of IFN-gamma on enhancing the killing ability of normal T cells.

FIG. 16 is a schematic of a combined IFN-gamma CAR-T treatment regimen for setting up a model of NSG mice celiac ovarian cancer.

FIG. 17 is the effect of IFN-gamma combination CAR-T treatment in setting up a model of NSG mice celiac ovarian cancer.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present utility model more clear, the technical solutions of the embodiments of the present utility model will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present utility model. It will be apparent that the described embodiments are some, but not all, embodiments of the utility model. All other embodiments, which can be made by those skilled in the art based on the embodiments of the utility model without making any inventive effort, are intended to be within the scope of the utility model.

The examples are presented for better illustration of the utility model, but the utility model is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the utility model.

The "auxiliary drug" as used herein refers to a drug for assisting in enhancing the efficacy of a primary therapeutic drug (i.e., a primary drug). For example, IFN-gamma of the present utility model can be administered to a patient prior to treatment with a primary drug in a manner that pre-treats the drug, increasing the sensitivity of tumor cells to the subsequent primary drug, thereby enhancing the anti-tumor effect of the subsequent primary drug. For another example, IFN-gamma as described herein can also be used as a pretreatment drug to up-regulate expression of ICAM-1 by tumor cells, enhance killing and cytokine release of tumor cells by CAR-T cells or T cells not genetically modified. Of course, the auxiliary drug is not necessarily used before the main drug treatment, and may be used together with the main drug according to the actual situation.

The sensitization refers to the increase of the sensitivity of tumor cells to a main drug (immunotherapy) by auxiliary drugs, which is more beneficial to the main drug to play a role and more effectively kill the tumor cells. For example, the present utility model utilizes IFN-gamma signaling pathways in IFN-gamma activated tumor cells to enhance the sensitivity of tumor cells to T cells (including T cells in vivo and adoptively infused T cell preparations), as well as to immune checkpoint inhibitors (PD-L1 inhibitors and/or PD-1 inhibitors), thereby allowing T cell and/or immune checkpoint inhibitor-based immunotherapy to be better effective.

The experiments of the utility model find that during the process of killing tumor cells by the CAR-T cells, the up-regulation of PD-L1 and PD-1 expression exists together with the activation of the CAR-T cells, the killing of tumor cells and the secretion of cytokines (figure 1). Two types of CAR-T cells were used in fig. 1 a: HER2 (Epidermal Growth Factor Receptor 2) -CAR cells and MSLN (Mesothelin) -CAR cells, in the presence of antigen stimulation, both up-regulate PD-1 expression on CAR-T cells. Wherein, the antigen refers to a tumor cell expressing HER2 or MSLN. FIG. 1b Co-cultures CAR-T and tumor cells, the CAR-T activates killer tumor cells, and the "supernatant" after centrifugation to remove cell debris. This supernatant is the "microenvironment" of the CAR-T after activation to act on other tumor cells. FIG. 1c shows a higher tumor cell lysis rate upon CAR-T cell addition; both Granzyme B and OD107a+ on CAR-T cells were upregulated, indicating activation of CAR-T cells, exerting a killing effect on tumor cells. In the experiment of fig. 1d, there are three groups: MOCK, HER2-CAR minus, HER2-CAR plus; wherein MOCK is a control T cell that does not express the CAR structure. In FIG. 1, the CAR-T and tumor cells were incubated together, and after 24 hours the CAR-T killed all tumor cells, and the supernatant (supernatant) was centrifuged to culture new tumor cells with the supernatant, and the expression of PD-L1 on tumor cells was detected.

The T cell depletion markers TIM-3 and LAG-3 were significantly increased in CAR-T cells. Stress experiments simulating that CAR-T cells continuously encounter and kill tumor cells in an in vivo anti-tumor environment (fig. 2), although PD-L1 is highly expressed on tumor cells, CAR-T cells show increasingly higher cytotoxicity and cytokine release capacity (fig. 3). These results indicate that CAR-T itself presents a mechanism in CAR-T killing tumor cells that has not yet been elucidated to inhibit the effects of PD-L1-PD-1.

IFN-gamma at sub-toxic concentrations induces 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, CAR-T cells showed more potential cytolytic activity and IFN- γ secretion capacity (fig. 5). In contrast, when IFN-gamma is neutralized by anti-IFN-gamma antibodies, the cytotoxicity of CAR-T is significantly reduced (FIG. 6), indicating that IFN-gamma is important for CAR-T activity.

In the IFN-gamma pretreatment assay, the medium was replaced with fresh medium to ensure that residual IFN-gamma was not detected (less than 10 pg/ml) to rule out the direct effect of IFN-gamma on CAR-T cells.

The present utility model pretreats tumor cells with low concentrations of IFN-gamma and then detects the cytotoxicity of CAR-T in fresh medium containing sufficient anti-IFN-gamma antibody to neutralize IFN-gamma in CAR-T. After IFN-gamma neutralization, CAR-T cells still exhibited killing ability against tumor cells pretreated with low concentration of IFN-gamma (FIG. 7), whereas CAR-T cells lost the effect of killing tumor cells when not pretreated with IFN-gamma (FIG. 6). IFN-gamma is therefore thought to enhance the killing capacity of CAR-T by acting on tumor cells. In other words, pretreatment with IFN-gamma enhances the sensitivity of tumor cells to CAR-T cells.

Then, the CRISPR-Cas9 system is used to block the IFN- γ signaling pathway to interrupt the expression of the IFN- γr2 (IFNGR 2) gene on tumor cells. Because IFN- γR1 is ubiquitous in mammalian cells, but IFN- γR2 expression is more dynamic, it is believed that the extent of IFN- γ -induced signaling in a particular cell population can be determined. knock-out-IFN- γR2 (IFN- γR2-null) tumor cells grew as normal as control cells, and knock-out-IFN- γR2 tumor cells no longer expressed PD-L1 after treatment with IFN- γ (three concentrations: 0,5,10 ng/ml) (FIG. 8). However, knockout-IFN- γr2 tumor cells were more resistant to CAR-T cell lysis (figure 9). In both tumor cells (SK-OV-3, A549), the enhancement of CAR-T cytotoxicity by IFN-gamma pretreatment was significantly inhibited (FIG. 9), i.e., the effects of IFN-gamma pretreatment on sensitized tumor cells were inhibited. Together, these results demonstrate that the effect of IFN- γ on tumor cells is an intrinsic mechanism by which CAR-T maintains or enhances its cellular activity (and thus the anti-tumor effect) in the case of PD-L1 and PD-1 expression.

In order to directly characterize the inhibition of PD-L1-PD-1 by IFN-gamma, the present experimental group performed CAR-T activity assays on tumor cells stably expressing PD-L1. Overexpression of PD-L1 on tumor cells did not confer resistance to CAR-T killing by tumor cells nor inhibited the enhancement of CAR-T cell toxicity to IFN- γ pretreated tumor cells (fig. 10). In other words, IFN-gamma pretreatment enhances the sensitivity of tumor cells to CAR-T cells and is not inhibited by PD-L1. However, in IFN- γR2 knockdown tumor cells, PD-L1 overexpression significantly inhibited the killing effect of CAR-T cells (FIG. 11). These results indicate that IFN-gamma signaling can overcome the inhibitory effect of PD-L1-PD-1, enhance the sensitivity of tumor cells to CAR-T cells, and enhance the activity of CAR-T cells

Next, the present utility model also investigated 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 a natural ligand for TCR activated T cells, however, blocking HLA-ABC molecules does not affect IFN-gamma enhancement of CAR-T cells.

Intercellular adhesion molecule 1 (ICAM-1, also known as CD 54) is a cell surface glycoprotein on Antigen Presenting Cells (APCs) that plays an important role in an effective immune response. The research of the utility model finds that IFN-gamma induces ICAM-1 to be obviously over-expressed in various solid tumor cells; and after CAR-T cells encounter tumor cells, their LFA-1 (ICAM-1 receptor) is significantly elevated. Knocking out ICAM-1 in tumor cells with functional IFN- γ receptor almost completely abrogated the potentiation of CAR-T on IFN- γ pretreated tumor cytotoxicity (fig. 14), suggesting the importance of ICAM-1 on CAR-T activity. Simultaneously, IFN-gamma stimulates tumor cells to produce ICAM-1, which can enhance the killing ability of T cells to tumor cells (FIG. 15). In contrast, overexpression of ICAM-1 in tumor cells with normal IFN- γ signaling pathways did not significantly promote specific cytolysis of tumor cells by CAR-T (fig. 13). We speculate that this is due to the increased ICAM-1 induced by IFN- γ released by activated CAR-T cells. Thus, the present utility model studies on overexpression of ICAM-1 in IFN- γR2 knockout cells, found that tumor cells were more susceptible to killing by CAR-T cells (FIG. 13). Together, these results indicate that IFN- γ -induced ICAM-1 expression is an important mechanism for IFN- γ to inhibit PD-L1-PD-1 function, enhancing CAR-T and T cell anti-tumor activity.

Then, the experiment of the utility model tests whether IFN-gamma pretreatment is helpful for CAR-T cell preparation to achieve better treatment effect on solid tumors. Continuous in vitro killing experiments showed that CAR-T cells maintained killing activity on IFN- γ pretreated tumor cells to round 5, while killing activity on tumor cells not pretreated with IFN- γ was maintained only to the third round (fig. 12). In other words, IFN-gamma (as an adjuvant) pretreatment can enhance the sensitivity of tumor cells to CAR-T cells, facilitating the therapeutic effect of CAR-T cell formulations as a primary drug on solid tumors.

Then, the experiments of the present utility model established a model of NSG mice celiac ovarian cancer, with IFN-gamma injection twice prior to celiac injection of CAR-T cells (FIG. 16). Bioluminescence imaging showed no significant inhibition of tumor growth by IFN- γ, but only delayed tumor growth by CAR-T alone, whereas 3 tumors in 5 mice remained by combined IFN- γ pretreatment and CAR-T cells, with the remaining two tumors also significantly smaller than tumors in the control group (fig. 17). However, IFN- γR2 deleted tumors were more resistant to combination therapy (FIG. 17). These in vitro and in vivo experimental results demonstrate that sequential treatment of IFN-gamma (as a sensitization adjuvant) and CAR-T (as a primary drug) is an effective method for treating solid tumors.

In summary, the experiments of the utility model show that:

1) After the CAR-T cells are contacted with tumor cells, the CAR-T cells highly express the inhibitory molecule PD-1, and the tumor cells highly express PD-L1. Meanwhile, the release of activation markers granzyme B, perforin and cytokines in the CAR-T cells are up-regulated, and the CAR-T efficiently kills tumor cells.

2) IFN-gamma pre-treatment of tumor cells, IFN-gamma upregulates tumor cell PD-L1 expression, but simultaneously enhances (rather than inhibits) killing of tumor cells by CAR-T and cytokine release. (IFN-gamma is at low concentration, no obvious inhibition to tumor cells).

3) After IFN-gamma neutralizing antibodies are used to neutralize IFN-gamma in a system in which the CAR-T cells and tumor cells are incubated together, the killing function of the CAR-T cells is obviously reduced. After knocking out the ifnγ receptor gene (ifnγr2) in tumor cells, the tumor cells do not express PD-L1, but the CAR-T killing function is reduced.

4) Over-expressing PD-L1 on tumor cells does not obviously inhibit the activity of CAR-T, but over-expressing PD-L1 on tumor cells knocked out of IFN gamma receptor gene (IFN gamma R2) obviously inhibits the killing function of CAR-T.

5) IFN-gamma induces tumor cells to highly express ICAM-1. IFN-gamma pretreatment no longer enhances the killing activity of CAR-T after ICAM-1 knockdown in tumor cells.

6) ICAM-1 molecules are overexpressed in tumor cells lacking IFNγR2, and the killing function and cytokine release capacity of CAR-T are enhanced.

7) IFN-gamma can enhance the treatment effect of CAR-T cells in a mouse ovarian cancer abdominal cavity tumor model.

8) IFN-gamma pretreatment of tumor cells promotes the expression of ICAM-1 by the tumor cells, and can enhance the killing power of common T cells on the tumor cells.

The embodiments of the present utility model have been described above with reference to the accompanying drawings, but the present utility model is not limited to the above-described embodiments, which are merely illustrative and not restrictive, and many forms may be made by those having ordinary skill in the art without departing from the spirit of the present utility model and the scope of the claims, which are to be protected by the present utility model.

Claims

The application of IFN-gamma in preparing antitumor auxiliary medicine for auxiliary T cell preparation, and features that IFN-gamma enhances the killing effect of T cell preparation on tumor cell via sensitization of tumor cell; the T cell preparation comprises T cells without genetic modification and/or T cells modified by genetic engineering means; the IFN-gamma includes wild-type or mutant IFN-gamma full length or IFN-gamma fragments.
2. The use of claim 1, wherein the genetically modified T cells comprise CAR-T cells and/or TCR-T cells.
3. The use of claim 1 or 2, wherein the IFN- γ sensitizes the tumor cell by activating the IFN- γ signaling pathway of the tumor cell.
4. The use of claim 3, wherein IFN- γ signaling activation of said tumor cells up-regulates ICAM-1 to enhance killing of said tumor cells by said T cell preparation.
5. The use according to any one of claims 1 to 4, wherein the anti-tumour auxiliary drug is for use in a tumour of the type of solid tumour.
6. The use according to any one of claims 1 to 4, wherein the anti-tumor adjuvant is used for the treatment of ovarian, breast, brain glioma, gastric, colon, melanoma, non-small cell lung, renal cell carcinoma, bladder, liver or urothelial carcinoma.
The application of IFN-gamma in preparing antitumor auxiliary medicines for assisting immune checkpoint inhibitors, which is characterized in that the IFN-gamma assists the antitumor effect of the immune checkpoint inhibitors by sensitization of tumor cells, and the immune checkpoint inhibitors comprise PD-L1 inhibitors and/or PD-1 inhibitors; the IFN-gamma includes wild-type or mutant IFN-gamma full length or IFN-gamma fragments.
8. The use of claim 7, wherein the PD-L1 inhibitor comprises 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.
9. The use according to claim 7 or claim 8, wherein the anti-tumour auxiliary drug is for use in a tumour of the type of solid tumour.
10. The use according to any one of claims 7 to 9, wherein the anti-tumour adjuvant is used in a tumour of the ovarian tumour, breast cancer, brain glioma, gastric cancer, colon cancer, melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, liver cancer or urothelial carcinoma.
The use of IFN- γ for the preparation of an antitumor adjuvant for the adjuvant of T cell preparations and immune checkpoint inhibitors, characterized in that said IFN- γ assists the antitumor effect of said T cell preparations and said immune checkpoint inhibitors by sensitizing tumor cells; the immune checkpoint inhibitor comprises a PD-L1 inhibitor and/or a PD-1 inhibitor; the T cell preparation comprises T cells without genetic modification and/or T cells modified by genetic engineering means; the IFN-gamma includes wild-type or mutant IFN-gamma full length or IFN-gamma fragments.
12. The use of claim 11, wherein the genetically modified T cells comprise CAR-T cells and/or TCR-T cells.
13. The use of claim 11 or 12, wherein the PD-L1 inhibitor comprises 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.
14. The use of any one of claims 11-13, wherein stimulation of tumor cell production by the helper drug ICAM-1 enhances killing of tumor cells by the T cell preparation.
15. The use according to any one of claims 11 to 14, wherein the tumour is of the type solid.
16. The use according to any one of claims 11 to 14, wherein the tumour is of the type ovarian, breast, brain glioma, gastric, colon, melanoma, non-small cell lung, renal cell carcinoma, bladder, liver or urothelial carcinoma.