Method for inducing peripheral blood CX3CR1+ potentially tumor-reactive t cells

Abstract

The present invention provides a method for inducing peripheral blood CX3CR1+ potentially tumor-reactive T cells. The present invention provides an optimized hot and cold ablation method, which effectively increases the level of potentially tumor-reactive T cells in the peripheral blood of a test animal, thereby improving anti-tumor immune response. The present invention further provides a tumor-reactive T cell population prepared by means of a hot and cold ablation method.

Description

A method for inducing peripheral blood CX3CR1+ potential tumor reactive T cells Technical Field

[0001] This invention relates to the field of biomedicine. Specifically, this invention relates to a method for inducing peripheral blood CX3CR1+ potential tumor reactive T cells. Background Technology

[0002] For patients with intermediate-stage hepatocellular carcinoma (HCC), local treatment has been identified as the preferred treatment option. Ablation therapy is the primary non-surgical local treatment method. Currently, the most commonly used ablation techniques in clinical practice include cryoablation, radiofrequency ablation (RFA), and microwave ablation (MWA). Ablation therapy can induce local and systemic immune responses, thereby eliminating distant metastatic lesions; this phenomenon is known as the "distant effect." However, studies have shown that ablation-induced immune responses are often weak and insufficient to induce sustained protective anti-tumor immunity. Ablation-induced anti-tumor immunity depends on the release of antigens and damage-associated molecular patterns (DAMPs) resulting from tumor cell immune death. The type and amount of tumor antigens and DAMPs released are influenced by the ablation method and conditions, ultimately leading to the induction of different immune responses.

[0003] Therefore, it is necessary to optimize ablation methods and conditions to maximize the induced anti-tumor immune response.

[0004] Summary of the Invention

[0005] The purpose of this invention is to provide an ablation therapy method that enhances anti-tumor immune response.

[0006] Another object of the present invention is to provide peripheral blood potential tumor reactive T cells induced by an ablation method.

[0007] In a first aspect of the invention, a method for treating cancer in a mammal having cancerous tissue is provided, the method comprising the steps of:

[0008] (1) Cold treatment: Cold treatment of one or more cancerous tissues of the mammal, wherein the cold treatment includes reducing the temperature of the treated cancerous tissue to ≤-10°C and maintaining it for 2-20 minutes;

[0009] (2) Rewarming treatment: The cancerous tissue that had undergone cold treatment was heated to 2-20℃; and

[0010] (3) Heat treatment: The cancer tissue that has been reheated in the previous step is subjected to heat treatment, which includes raising the temperature of the cancer tissue to 45-60°C and maintaining it for 2-20 minutes.

[0011] In another preferred embodiment, the method further includes the step of:

[0012] (4) Repeat steps (1) to (3) once or multiple times.

[0013] In another preferred embodiment, the cancer treatment includes increasing the level of tumor-reactive T cells in the mammal.

[0014] In another preferred embodiment, the tumor-reactive T cells are CX3CR1+ T cells or CX3CR1+GPR56+ T cells.

[0015] In another preferred embodiment, the tumor-reactive T cells are CX3CR1+GPR56+CD8+ T cells.

[0016] In another preferred embodiment, the mammals include: humans, orangutans, monkeys, rats, mice, rabbits, or combinations thereof.

[0017] In another preferred embodiment, the cancerous tissue includes: a tumor, tissue infected with cancer cells, tumor metastases, and blood from a cancer patient.

[0018] In another preferred embodiment, the cancerous tissue is superficial cancerous tissue or cancerous tissue located within the body.

[0019] In another preferred embodiment, a non-invasive or minimally invasive method is used in the cold treatment or heat treatment step.

[0020] In another preferred embodiment, the method further includes monitoring the temperature of the cancerous tissue in steps (1) to (3).

[0021] In another preferred embodiment, the monitoring of the temperature of cancerous tissue includes contact temperature sensors or non-invasive temperature monitoring, such as temperature monitoring through infrared image analysis, nuclear magnetic resonance temperature detection, or ultrasonic temperature detection.

[0022] In another preferred embodiment, in the cold treatment in step (1), the cold treatment is to bring a cold source or its delivery device into contact with the epidermis or insert it into the tumor center.

[0023] In another preferred embodiment, the cold source is liquid nitrogen.

[0024] In another preferred embodiment, the heat treatment in step (3) is:

[0025] Contacting the heat source or its transmission device with the epidermis or inserting it into the center of the tumor; or

[0026] High-frequency electromagnetic field heating.

[0027] In another preferred embodiment, the delivery device has a contact head with a contact surface for adhering to the cancerous tissue. The cold source cools the cancerous tissue through the contact head, and / or the heat source warms the cancerous tissue through the contact head.

[0028] In another preferred embodiment, the delivery device includes a treatment probe that can be image-guided to penetrate the center of the tumor. A cold source cools the cancerous tissue through the contact head, and / or a heat source warms the cancerous tissue through the contact head.

[0029] In another preferred embodiment, the cancerous tissue carried by the mammal includes metastatic cancerous tissue.

[0030] In another preferred embodiment, the tumor includes malignant solid tumors and benign tumors.

[0031] In another preferred embodiment, the cancerous tissue is superficial cancerous tissue, cavity tumor, or deep tumor.

[0032] In another preferred embodiment, the cancer is selected from the group consisting of: liver cancer, colorectal cancer, melanoma, lung cancer, breast cancer, and pancreatic cancer.

[0033] In another preferred embodiment, in step (1), during the cold treatment process, the cancerous tissue is cooled to -10°C to -30°C and maintained for 5 min to 15 min.

[0034] In another preferred embodiment, in step (1), during the cold treatment process, the cancerous tissue is cooled to -15°C to -25°C; preferably, the cancerous tissue is cooled to -18°C to -20°C.

[0035] In another preferred embodiment, in step (1), the time for maintaining the low temperature is 5 min to 10 min; preferably, the time for maintaining the low temperature is 5 min to 7 min.

[0036] In another preferred embodiment, in step (2), the cold-treated cancerous tissue is heated to 4-10°C; preferably, the cancerous tissue is heated to 6-8°C.

[0037] In another preferred embodiment, in step (2), the rewarming process is natural rewarming.

[0038] In another preferred embodiment, in step (3), during the heat treatment process, the cancerous tissue is heated to 45°C to 55°C; preferably, the cancerous tissue is heated to 50°C to 55°C.

[0039] In another preferred embodiment, in step (3), the high temperature is maintained for 5 to 15 minutes during the heat treatment process. Preferably, the high temperature is maintained for 10 to 15 minutes.

[0040] In another preferred embodiment, in step (3), during the heat treatment process, the cancerous tissue is heated to 50°C to 55°C and maintained for 10 min to 15 min.

[0041] In another preferred embodiment, the method further includes: after the heat treatment in step (3), measuring the level of CX3CR1+ T cells.

[0042] In another preferred embodiment, the method further includes: after the heat treatment in step (3), measuring the level of GPR56+T cells.

[0043] In another preferred embodiment, the method further includes: after the heat treatment in step (3), detecting and evaluating cancerous tissue (especially metastatic lesions).

[0044] In a second aspect of the invention, a method for preparing a population of tumor-reactive T cells is provided, the method comprising the steps of:

[0045] (1) Cold treatment: A mammal with one or more cancerous tissues is provided, and the cancerous tissues of the mammal are subjected to cold treatment, the cold treatment comprising reducing the temperature of the treated cancerous tissues to ≤-10°C and maintaining it for 2-20 minutes;

[0046] (2) Rewarming treatment: The cancerous tissue that had undergone cold treatment was heated to 2-20℃; and

[0047] (3) Heat treatment: The cancer tissue that has been reheated in the previous step is subjected to heat treatment, wherein the heat treatment includes raising the temperature of the cancer tissue to 45-60°C and maintaining it for 2-20 minutes;

[0048] (4) Collect peripheral blood from the mammal to obtain a population of tumor-reactive T cells.

[0049] In another preferred embodiment, steps (1) to (3) are repeated once or more, followed by collection of peripheral blood from the mammal.

[0050] In another preferred embodiment, the proportion of CX3CR1+ T cells in the tumor reactive T cell population is ≥20%, more preferably ≥40%, and even more preferably ≥70%.

[0051] In another preferred embodiment, the proportion of CX3CR1+GPR56+ T cells in the total number of T cells in the tumor reactive T cell population is ≥20%, preferably ≥40%, and more preferably ≥70%.

[0052] In another preferred embodiment, the mammals include: humans, orangutans, monkeys, rats, mice, rabbits, or combinations thereof.

[0053] In another preferred embodiment, the cancerous tissue includes: a tumor, tissue infected with cancer cells, tumor metastases, and blood from a cancer patient.

[0054] In another preferred embodiment, the cancerous tissue is superficial cancerous tissue, cavity tumor, or deep tumor.

[0055] In another preferred embodiment, a non-invasive or minimally invasive method is used in the cold treatment or heat treatment step.

[0056] In another preferred embodiment, the method further includes monitoring the temperature of the cancerous tissue in steps (1) to (3).

[0057] In another preferred embodiment, the monitoring of the temperature of cancerous tissue includes contact temperature sensors or non-invasive temperature monitoring, such as temperature monitoring through infrared image analysis, nuclear magnetic resonance temperature detection, or ultrasonic temperature detection.

[0058] In another preferred embodiment, in the cold treatment in step (1), the cold treatment is to bring a cold source or its delivery device into contact with the epidermis or insert it into the tumor center.

[0059] In another preferred embodiment, the cold source is liquid nitrogen.

[0060] In another preferred embodiment, the heat treatment in step (3) is:

[0061] Contacting the heat source or its transmission device with the epidermis or inserting it into the center of the tumor; or

[0062] High-frequency electromagnetic field heating.

[0063] In another preferred embodiment, the delivery device has a contact head with a contact surface for adhering to the cancerous tissue. The cold source cools the cancerous tissue through the contact head, and / or the heat source warms the cancerous tissue through the contact head.

[0064] In another preferred embodiment, the delivery device includes a treatment probe that can be image-guided to penetrate the center of the tumor. A cold source cools the cancerous tissue through the contact head, and / or a heat source warms the cancerous tissue through the contact head.

[0065] In another preferred embodiment, the cancerous tissue carried by the mammal includes metastatic cancerous tissue.

[0066] In another preferred embodiment, the tumor includes malignant solid tumors and benign tumors.

[0067] In another preferred embodiment, in step (1), during the cold treatment process, the cancerous tissue is cooled to -10°C to -30°C and maintained for 5 min to 15 min.

[0068] In another preferred embodiment, in step (1), during the cold treatment process, the cancerous tissue is cooled to -15°C to -25°C; preferably, the cancerous tissue is cooled to -18°C to -20°C.

[0069] In another preferred embodiment, in step (1), the time for maintaining the low temperature is 5 min to 10 min; preferably, the time for maintaining the low temperature is 5 min to 7 min.

[0070] In another preferred embodiment, in step (2), the cold-treated cancerous tissue is heated to 4-10°C; preferably, the cancerous tissue is heated to 6-8°C.

[0071] In another preferred embodiment, in step (2), the rewarming process is natural rewarming.

[0072] In another preferred embodiment, in step (3), during the heat treatment process, the cancerous tissue is heated to 45°C to 55°C; preferably, the cancerous tissue is heated to 50°C to 55°C.

[0073] In another preferred embodiment, in step (3), the high temperature is maintained for 5 to 15 minutes during the heat treatment process. Preferably, the high temperature is maintained for 10 to 15 minutes.

[0074] In another preferred embodiment, in step (3), during the heat treatment process, the cancerous tissue is heated to 50°C to 55°C and maintained for 10 min to 15 min.

[0075] In another preferred embodiment, step (4) further includes the step of collecting CX3CR1+GPR56+ T cells by flow cytometry or immunomagnetic bead sorting.

[0076] In a third aspect of the invention, a tumor-reactive T cell population is provided, which is prepared by the method described in the second aspect of the invention.

[0077] In another preferred embodiment, the proportion of CX3CR1+ T cells in the tumor reactive T cell population is ≥20%, more preferably ≥40%, and even more preferably ≥70%.

[0078] In another preferred embodiment, the proportion of CX3CR1+GPR56+ T cells in the total number of T cells in the tumor reactive T cell population is ≥20%, preferably ≥40%, and more preferably ≥70%.

[0079] In a fourth aspect of the invention, the use of tumor-reactive T cell populations as described in the third aspect of the invention in the preparation of medicaments for treating cancer is provided.

[0080] In another preferred embodiment, the tumor includes malignant solid tumors and benign tumors.

[0081] In another preferred embodiment, the cancer is selected from the group consisting of: liver cancer, colorectal cancer, melanoma, lung cancer, breast cancer, and pancreatic cancer.

[0082] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0083] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0084] Figure 1 shows the tumor reactivity assessment of CX3CR1+ or CX3CR1+GPR56+ T cells.

[0085] Figure 2 shows the assessment of the proportion of CX3CR1+ or CX3CR1+GPR56+ potential tumor reactive T cells in the peripheral blood of patients or animals before and after treatment. Detailed Implementation

[0086] Through extensive and in-depth research, the inventors have developed, for the first time, a method for inducing peripheral blood CX3CR1+ latent tumor-reactive T cells. This invention effectively increases the level of latent tumor-reactive T cells in the peripheral blood of test animals by optimizing the steps and conditions of cryoablation, thereby enhancing the anti-tumor immune response. Based on this, the invention was completed.

[0087] the term

[0088] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0089] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0090] As used in this article, the term "cryoablation" refers to a procedure that uses low temperatures to treat tumors; "radiofrequency ablation" refers to a method that kills tumor tissue by heating it with high-frequency electromagnetic waves.

[0091] As used herein, the term "CX3CR1" refers to C-X3-C Motif Chemokine Receptor 1, see accession numbers: HGNC:2558, NCBI Gene:1524, Ensembl:ENSG00000168329. 601470,UniProtKB / Swiss-Prot:P49238.

[0092] As used in this article, the term "GPR56" refers to Adhesion G Protein-Coupled Receptor G1, see accession number: HGNC:4512 NCBI Gene:9289 Ensembl:ENSG00000205336 604110 UniProtKB / Swiss-Prot:Q9Y653.

[0093] The main advantages of this invention include:

[0094] This invention combines cryoablation and radiofrequency ablation to precisely control the temperature of the ablation area, thereby stimulating a stronger immune response and promoting the expansion of CX3CR1+ or CX3CR1+GPR56+ potential tumor reactive T cells.

[0095] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0096] Example 1: Screening of tumor reactive T cell markers

[0097] Peripheral blood was collected from cancer patients, and peripheral blood mononuclear cells were separated by density gradient centrifugation using Ficoll-Paque Plus medium (GE Healthcare), followed by washing with calcium / magnesium-free phosphate buffer. Red blood cells were removed using erythrocyte lysis buffer.

[0098] Tumor tissue was collected from the patient using an 18G needle biopsy, washed three times with Hanks balanced salt solution, cut into small pieces, and then digested at 37°C for 15 minutes using 3 mL of tissue dissociation fluid and a tissue dissociation system. The cell suspension was collected and filtered through a 40-micron sterile filter. Red blood cells were then removed using red blood cell lysis buffer.

[0099] Single-cell RNA libraries and single-cell TCR libraries were constructed from peripheral blood and tumor tissues of patients and sequenced using Illumina HiSeq X.

[0100] Barcodes and UMIs were extracted from R1 reads and corrected. Adaptor sequences and poly-A tails were trimmed from R2 reads, and the trimmed R2 reads were aligned to the GRCh38 transcriptome using STAR (v2.6.1b). Uniquely mapped reads were then assigned to exons using FeatureCounts (v2.0.1). Successfully assigned reads with the same cell barcodes, UMIs, and genes were grouped to generate a gene expression matrix for further analysis.

[0101] Using GRCh38 as a reference, TCR clonal type assignment was performed using the Cell Ranger (v4.0.0) vdj pipeline. A TCR diversity index containing clonal frequency and barcode information was obtained. Each unique TCR β chain was defined as a clonal type.

[0102] Gene expression matrices were analyzed and processed using the Seurat software package (version 4.4.0). Cells were screened based on UMI count, gene count, and mitochondrial content ratio before further analysis. Cells with 300 to 4,000 genes, a UMI count greater than 1,000, and mitochondrial genes comprising more than 35% of the total cells were retained. Gene expression was normalized and scaled using the NormalizeData and ScaleData functions, and 2000 hypervariable genes were selected for PCA analysis. Cells were divided into multiple groups using the first 15 principal components. Finally, the UMAP algorithm was used to visualize the cells in two-dimensional space.

[0103] Using the FindMarkers feature in Seurat v4.4.0, differentially expressed genes (DEGs) were identified for each cluster or group with default parameters. DEGs were defined as genes expressed in more than 10% of the cells. Cell types were then annotated based on DEGs.

[0104] TCR clones with more than 5 clones within tumor tissue were defined as potential tumor-reactive TCR clones. Single-cell RNA sequencing analysis revealed that most potential tumor-reactive T cells in peripheral blood were found to be in two subsets: CD8_GZMB and CD4_GZMB (Figure 1A). Differential gene analysis showed that T cells in the CD8_GZMB and CD4_GZMB subsets highly expressed CX3CR1 and GPR56, and were highly co-expressed (Figure 1B).

[0105] C57BL6 mice were subcutaneously injected with 5*10 6 A mouse hepatocellular carcinoma model was constructed using Hepa1-6 cells. Spleens from the hepatocellular carcinoma model mice were collected, and single-cell suspensions were prepared using mechanical grinding. Red blood cells were removed using red blood cell lysis buffer. The spleen single-cell suspension was co-incubated overnight with tumor cells at a 10:1 effector-to-target ratio. Brefeldin A (BFA) was added to inhibit protein transport, and the cells were cultured for 4-6 hours. Cell surface staining for CD3, CD8, CX3CR1, and intracellular interferon-γ was performed, and the results were detected by flow cytometry. The results are shown in Figure 1C, where CD8+ T cells expressing interferon-γ were tumor-reactive T cells. The results indicate that all tumor-reactive T cells expressed CX3CR1.

[0106] Example 2: Cold and hot ablation to induce tumor-reactive T cells

[0107] Subjects were drawn from patients with malignant tumors.

[0108] Tumor model construction method: tumor cells are subcutaneously inoculated to construct a tumor model.

[0109] The steps for inducing CX3CR1+GPR56+ latent tumor-reactive T cells by cryoablation are as follows:

[0110] (1) Insert the ablation needle into the center of the patient's tumor or attach it to the surface of the tumor model animal, and introduce liquid nitrogen to freeze the in vivo tumor tissue: rapidly cool the tumor tissue to -20°C and maintain it for 5 minutes;

[0111] (2) Natural rewarming: The tumor tissue of the tumor patient or tumor model animal is rewarmed to 6-8℃;

[0112] (3) Start radiofrequency therapy and heat the tumor tissue of the tumor patient or tumor model animal in vivo through a high-frequency electric field: rapidly heat the tumor tissue to 50°C and maintain it for 10 minutes.

[0113] (4) Seven days after treatment, peripheral blood from tumor patients or spleen and peripheral blood from tumor model animals were collected for flow cytometry analysis.

[0114] Flow cytometry was used to assess the proportion of CX3CR1+GPR56+ potential tumor reactive T cells in the peripheral blood of patients or animals. Peripheral blood mononuclear cells were isolated from patient peripheral blood using density gradient centrifugation in Ficoll-Paque Plus medium (GE Healthcare) and washed with calcium / magnesium-free phosphate buffer. Red blood cells were removed using erythrocyte lysis buffer. In mouse tumor models, erythrocytes were directly removed from peripheral blood using erythrocyte lysis buffer. In mouse tumor models, spleen tissue was mechanically homogenized to prepare a single-cell suspension, and erythrocytes were removed using erythrocyte lysis buffer.

[0115] The prepared single-cell suspension was stained with CD3, CD4, CD8, CX3CR1, and GPR56, and the proportion of CX3CR1+GPR56+ T cells was detected by flow cytometry. The results are shown in Figure 2. After treatment, the number of CX3CR1+GPR56+ T cells in the peripheral blood of patients and different tumor model animals significantly increased. This result demonstrates that cryoablation therapy can effectively induce an increase in the level of anti-tumor reactive T cells in peripheral blood, thereby enhancing tumor-killing effects.

[0116] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for treating cancer in mammals in need, wherein, The mammal has cancerous tissue, and the method includes the steps of: (1) Cold treatment: Cold treatment of one or more cancerous tissues of the mammal, wherein the cold treatment includes reducing the temperature of the treated cancerous tissue to ≤-10°C and maintaining it for 2-20 minutes; (2) Warming treatment: The cancer tissue that has been cold-treated is heated to 2-20℃; and (3) Heat treatment: The cancer tissue that has been reheated in the previous step is subjected to heat treatment, which includes raising the temperature of the cancer tissue to 45-60°C and maintaining it for 2-20 minutes.

2. The method as described in claim 1, characterized in that, The cancer treatment includes increasing the level of tumor-reactive T cells in the mammal, wherein the tumor-reactive T cells are CX3CR1+ T cells or CX3CR1+GPR56+ T cells.

3. The method as described in claim 1, characterized in that, The cancers mentioned are selected from the following group: liver cancer, colorectal cancer, melanoma, lung cancer, breast cancer, and pancreatic cancer.

4. The method as described in claim 1, characterized in that, In step (1), during the cold treatment process, the cancerous tissue is cooled to -10℃ to -30℃ and maintained for 5min to 15min.

5. The method as described in claim 1, characterized in that, In step (3), during the heat treatment process, the cancerous tissue is heated to 50°C to 55°C and maintained for 10 min to 15 min.

6. A method for preparing a tumor-reactive T cell population, characterized in that, The method includes the following steps: (1) Cold treatment: A mammal with one or more cancerous tissues is provided, and the cancerous tissues of the mammal are subjected to cold treatment, the cold treatment comprising reducing the temperature of the treated cancerous tissues to ≤-10°C and maintaining it for 2-20 minutes; (2) Warming treatment: The cancer tissue that has been cold-treated is heated to 2-20℃; and (3) Heat treatment: The cancer tissue that has been reheated in the previous step is subjected to heat treatment, wherein the heat treatment includes raising the temperature of the cancer tissue to 45-60°C and maintaining it for 2-20 minutes; (4) Collect peripheral blood from the mammal to obtain a population of tumor-reactive T cells.

7. The method as described in claim 6, characterized in that, In the tumor reactive T cell population, CX3CR1+ T cells account for ≥20% of the total T cell count, preferably ≥40%, and more preferably ≥70%.

8. The method as described in claim 6, characterized in that, In the tumor reactive T cell population, CX3CR1+GPR56+ T cells account for ≥20% of the total T cell count, preferably ≥40%, and more preferably ≥70%.

9. A tumor-reactive T cell population, characterized in that, The tumor-reactive T cell population is prepared by the method described in claim 6.

10. Use of the tumor-reactive T cell population as described in claim 9 in the preparation of a medicament for treating cancer.

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