WO2021237456A1

WO2021237456A1 - Anti-tumor pharmaceutical composition and use thereof

Info

Publication number: WO2021237456A1
Application number: PCT/CN2020/092328
Authority: WO
Inventors: 万晓春; 章桂忠; 程建; 刘曌; 逯晓旭; 邓湉
Original assignee: 深圳先进技术研究院
Priority date: 2020-05-26
Filing date: 2020-05-26
Publication date: 2021-12-02

Abstract

Disclosed in the present invention are an anti-tumor pharmaceutical composition and the use thereof. The pharmaceutical composition comprises a CD317 inhibitor and an engineered immune cell. The CD317 inhibitor in the present invention can greatly improve the anti-tumor effect of immune cells. The mode of action thereof is to weaken the resistance of tumor cells with respect to immune killing. More importantly, independent use of the inhibitor does not cause apoptosis, so the potential safety risk is lower than that of a traditional chemotherapy drug.

Description

An anti-tumor pharmaceutical composition and its application

Technical field

The invention belongs to the technical field of tumor treatment, and relates to an anti-tumor pharmaceutical composition and its application.

Background technique

With the continuous deepening of research in the fields of tumor immunology, molecular and cell biology technology, tumor immunotherapy has flourished in preclinical research and clinical applications, showing more and more potential, becoming a successor to surgery, chemotherapy and radiotherapy. The fourth effective way to treat tumors.

Tumor immunotherapy is a treatment method that allows the body to produce a tumor-specific immune response through active or passive means, and exert its function of inhibiting and killing tumors. Tumor immunotherapy mainly includes immune checkpoint inhibitors (ICB), adoptive cell transfer therapy (ACT), tumor-specific vaccines, and small molecule immune drugs.

ACT is an individualized tumor treatment method, by separating immunocompetent cells from the patient's body, inducing differentiation, transformation, and expansion in vitro, and then returning to the patient's body, targeting antigen-specific tumor cells to inhibit and kill tumors. At present, CAR-T therapy (chimeric antigen receptor T cell therapy), CAR-NK and TCR-T therapy (T cell receptor chimeric T cell therapy) are more effective therapies, which are used in the treatment of malignant hematological tumors. It shows strong and long-lasting lethality, but the effect in the treatment of solid tumors is not satisfactory.

In view of the solid tumor dilemma of ACT, the strategies currently under research mainly include three aspects: 1) Transforming adoptively transferred immune cells, such as CAR-T, TCR-T or CAR-NK cells, to make them have stronger killing ability; 2) Combine with immune checkpoint blocking therapy; 3) Combine with other anti-tumor drugs, such as chemotherapy drugs. Each of these three schemes has its shortcomings. First of all, engineered immune cells have too strong killing function, not only cannot improve the anti-tumor effect, but it is more likely to accidentally injure healthy tissues due to off-target toxicity, causing serious side effects [see: 1. Tan MP, Gerry AB, Brewer JE, Melchiori L, Bridgeman JS, Bennett AD, Pumphrey NJ, Jakobsen BK, Price DA, Ladell K, et al. T cell receptor binding affinity governments the functional profile of cancer-specific CD8 + T cells. Clin Exp Immunol. 2015; 180(2): 255 -70; 2. Zhong S, Malecek K, Johnson LA, Yu Z, Vega-Saenz de Miera E, Darvishian F, McGary K, Huang K, Boyer J, Corse E, et al. T-cell receptor affinity and avidity definitions antitumor response and autoimmunity in T-cell immunotherapy. Proc Natl Acad Sci U S A. 2013; 110(17): 6973-8; 3. Chmielewski M, Hombach A, Heuser C, Adams GP, Abken HT cell activation by antibody- like immunoreceptors: increase in affinity of the single-chain fragment domain above threshold does not increase T cell activation against antigen-positive target cells but decreases selectivity.JImmunol.2004;173(12):7647–53HG Hurton LV, Najjar A, Rushworth D, Ang S, Olivares S, Mi T, Switzerland K, Singh H, Huls H, et al. Tuning sensitivity of CAR to EGFR d ensity limits recognition of normal tissue while maintaining potent antitumor activity. Cancer Res. 2015; 75(17): 3505-18; 5. Liu X, Jiang S, Fang C, Yang S, Olalere D, Pequignot EC, Cogdill AP, Li N, Ramones M, Granda B, et al. Affinity-tuned ErbB2or EGFR chimeric antigen receptor T cells exhibit an increased therapeutic index against tumors in mice. Cancer Res. 2015; 75(17): 3596–607]. The structural design of Juno’s JCAR015 is similar to Kite’s KTEC019. The difference is that the former uses scFv derived from SJ25C1 and the latter uses the same FMC63scFv as Novatis. In the end, JCAR015 terminated the experiment due to severe neurotoxicity and suffered heavy losses, while Kite’s KETC019 was successfully obtained. Batch. Although there is no direct evidence that the affinity of SJ25C1 is higher than that of FMC63, according to the horizontal comparison of the clinical remission rate of the two companies' products (91% vs 61%), the reason for Juno's failure is inseparable from the high affinity of SJ25C1 ( See: Zhenguang Wang1, Zhiqiang Wu1, Yang Liu2 and Weidong Han. New development in CAR-T cell therapy. Journal of Hematology&Oncology(2017) 10:53). Therefore, blindly enhancing the killing function of engineered immune cells is not a favorable way to enhance its anti-solid tumor effect. Second, the combination with immune checkpoint blocking therapy is currently limited to research. Immune checkpoint blocking therapy itself has serious complications. Most patients have inflammatory damage to tissues such as the digestive tract, endocrine glands, skin, liver, central nervous system, and cardiovascular system [see: 1. Michael A. Postow, MD , Robert Sidlow, MD, and Matthew D. Hellmann, MDImmune-Related Adverse Events Associated with Immune Checkpoint Blockade. N Engl J Med 2018; 378: 158-68; 2. Ravneet Bajwa, Anmol Chemir, Taimo, Khan, Alireza Anju Paul, Saira Chaughtai, Shrinil Patel, Tejas Patel, Joshua Bramson, Varsha Gupta, Michael Levitt, Arif Asif, Mohammad A. Hossain. Adverse Effects of Immune Checkpoint Inhibitors-Inhibitor-Inhibitor-Inhibitor-Inhibitor-Inhibitor- ): Results of a Retrospective Study. J Clin Med Res. 2019; 11(4):225-236]. There is currently no good control method for these complications [see: Michael A. Postow, MD, Robert Sidlow, MD, and Matthew D. Hellmann, MDImmune-Related Adverse Events Associated with Immune Checkpoint Blockade. N Engl J Med2018; 378:158-68.] Therefore, when it is used in combination with immune cell therapy such as CART, not to mention whether the therapeutic effect is substantially improved, its side effects are the major safety hazards of primary consideration. Different from the first two options, the third option combines immune cell therapy with anti-tumor drugs to indirectly enhance the therapeutic effect by weakening the resistance of tumor cells. This may be a mainstream direction for future research. However, it is currently in the exploratory stage, and there is no way to avoid the side effects of radiotherapy and chemotherapy drugs. More importantly, it cannot directly cooperate with immunotherapy, so that the activation level of immune cells is within a safe range to form a good killing effect on tumor cells.

Existing strategies for enhancing immune cell therapy may have uncontrollable side effects, or cannot form a direct synergistic anti-tumor effect with immune cells. Therefore, there is currently no truly safe and reliable combination of tumor cell immunotherapy.

Summary of the invention

In order to solve the above-mentioned background art problems, the purpose of the present invention is to provide an anti-tumor pharmaceutical composition and its application.

In order to achieve the above-mentioned objective, the technical scheme adopted by the present invention is as follows:

One aspect of the present invention provides an application of a CD317 inhibitor in enhancing the sensitivity of tumor cells to immune killing.

Another aspect of the present invention provides an application of a CD317 inhibitor in the preparation of a drug that enhances the sensitivity of tumor cells to immune killing.

Another aspect of the present invention provides an application of a CD317 inhibitor in enhancing the therapeutic effect of tumor immune cells.

Another aspect of the present invention provides the application of a CD317 inhibitor in the preparation of drugs for enhancing the therapeutic effect of tumor immune cells.

Another aspect of the present invention provides the application of a CD317 inhibitor in the preparation of a synergistic drug that enhances the anti-tumor efficacy of engineered immune cells.

Another aspect of the present invention provides an application of a combination of a CD317 inhibitor and engineered immune cells to enhance the therapeutic effect of tumor immune cells.

Another aspect of the present invention provides an application of a combination of a CD317 inhibitor and engineered immune cells in the preparation of anti-tumor drugs.

Another aspect of the present invention provides an anti-tumor pharmaceutical composition, including a CD317 inhibitor and engineered immune cells.

Further, the engineered immune cells are immune cells that function through the perforin granzyme pathway. Preferably, the engineered immune cells are CAR-T, CAR-NK, or TCR-T.

Further, the tumor is a solid tumor.

Further, the solid tumor is a solid tumor with high expression of CD317;

Preferably, the solid tumors include liver cancer, lung cancer, breast cancer, colon cancer, kidney cancer, and cervical cancer.

Further, the CD317 inhibitor is CD317siRNA, CD317shRNA, shRNA encoding DNA, CD317sgRNA, PROTAC, antibody or blocking peptide.

Further, the CD317 inhibitor is CD317 siRNA, and the CD317 siRNA is designed according to the CD317 gene transcript NM_004335.3;

Preferably, the CD317 siRNA includes the sense strand of the nucleotide sequence shown in SEQ ID NO: 1 and the antisense strand of the nucleotide sequence shown in SEQ ID NO: 2, and/or the core of SEQ ID NO: 3 The sense strand of the nucleotide sequence and the antisense strand of the nucleotide sequence shown in SEQ ID NO: 4.

The beneficial effects of the present invention are: the CD317 inhibitors of the present invention can significantly enhance the immune cell therapy effect of tumors, and provide a new potential program for clinical tumor treatment. Using siRNA to inhibit the expression of CD317 in tumor cells does not directly lead to cell death, but can significantly enhance the sensitivity of cancer cells to immune killing.

The CD317 inhibitor disclosed in the present invention can greatly improve the anti-tumor effect of immune cells, and its mode of action is by weakening the resistance of tumor cells to immune killing. More importantly, the use of inhibitors alone will not cause cell death, and the potential safety hazard is lower than that of traditional chemotherapy drugs. Subsequent optimization of dosage forms/administration methods is expected to develop a safe and effective regimen to enhance tumor immune cell therapy.

Description of the drawings

Figure 1 is a diagram showing the effect of FACS detecting CD317 interference in embodiment 2 of the present invention;

Figure 2 shows the effect of CD317 inhibitor (siRNA) on YTS cell-mediated HeLa cell death in Example 3 of the present invention; a is the FACS detection of the effect of CD317siRNA on YTS-mediated tumor cell killing; b is the analysis and statistics result;

Figure 3 shows the effect of CD317 inhibitor (siRNA) on YTS NK cell activation in Example 4 of the present invention; the left is the FACS detection of the effect of tumor cell CD317 on YTS NK cell activation; the right is the result of statistical analysis;

Figure 4 shows that the CD317 inhibitor (siRNA) in Example 5 of the present invention enhances the killing sensitivity of HepG2 and MCF-7 cells to CAR-YTS; a is the LDH method to detect the effect of CD317 inhibitor on the killing effect of CAR-YTS on HepG2 cells; b To detect the effect of CD317 inhibitor on the killing effect of CAR-YTS on MCF7 cells by LDH method.

Detailed ways

The present invention will be described in detail below through specific embodiments. The following embodiments are only used to illustrate the present invention, but not to limit the scope of the present invention. The test methods in the following examples, unless otherwise specified, are conventional methods, and the experimental materials used are all commercially available products.

Example

1. siRNA design

According to the basic principles of siRNA target sequence, two 21-nucleotide siRNA sequences were designed for the human CD317 gene transcript (NM_004335.3), namely si317-1 and si317-2, including the sense strand and the antisense strand, and their bases The sequence is as follows:

si317-1

Sense chain: 5’-CCAGGUCUUAAGCGUGAGAdTdT-3’, SEQ ID NO:1

Antisense strand: 5’-UCUCACGCUUAAGACCUGGdTdT-3’; SEQ ID NO: 2

si317-2

Sense chain: 5’-UCGCGGACAAGAAGUACUAdTdT-3’, SEQ ID NO: 3

Antisense strand: 5’-UAGUACUUCUUGUCCGCGAdTdT-3’, SEQ ID NO: 4

The base sequence of the negative control (NC) siRNA selected in this example is as follows:

Sense chain: 5’-UUCUCCGAACGUGUCACGUdTdT-3’, SEQ ID NO: 5

Antisense strand: 5’-ACGUGACACGUUCGGAGAAdTdT-3’; SEQ ID NO: 6

In this application, si317 is an equal volume mixture of si317-1 and si317-2.

Two deoxyribonucleotides in a single-stranded suspension structure are added to the 3'end of the interference fragment of the present invention to enhance the stability of siRNA in vivo and in vitro and prevent degradation. The siRNA was synthesized by Shanghai Gima Company.

According to a preferred embodiment of the present invention, Lipofectamine 3000, which is a more common cationic liposome, is selected as the transfection reagent.

2. Cell transfection and effect verification

1) According to the siRNA synthesis report, add an appropriate amount of DEPC water (DEPC, Diethyl pyrocarbonate, diethyl pyrocarbonate) to prepare a 20μM stock solution;

2) Inoculate Hela, HepG2 or MCF-7 cells in a 6-well plate at a density such that the cell confluence after overnight culture reaches 60%-80%;

3) Dilute 4μL Lipofectamine 3000 transfection reagent with 50μL Opti-MEM medium, mix well, and let it stand at room temperature for 5 minutes;

4) Dilute 2μL of siRNA with 50μL of Opti-MEM medium, mix well, and let stand at room temperature for 5 minutes;

5) Mix the diluent from step 3) and step 4), mix well and let stand at room temperature for 10 minutes. At this time, the ratio of siRNA to Lipofectamine3000 in the mixed solution is 1:2;

6) Add the transfection mixture of step 5) dropwise to the cell culture wells, and then mix them evenly with a cross method;

7) The cells were cultured for 36 hours, and some cells were taken to verify the interference effect by flow cytometry, and other cells were used for subsequent experiments.

8) Interference effect verification: 36 hours after Hela cells were transfected with siRNA, the cells were collected, washed twice with PBS, added 5μL APC anti-human CD317 antibody (Biolegend) or isotype control antibody (Isotype), stained on ice or 4℃ for 15 min; FACS Detect the APC channel signal, the more the signal peak goes to the right, the higher the CD317 expression level. As shown in Figure 1, compared with the control cells (siNC), the APC signal of the siRNA transfected cells (si317) shifted to the left, which was close to the signal of the isotype control antibody. It shows that siRNA-mediated CD317 interference is successful.

3. Flow cytometry to detect the effect of CD317 siRNA on enhancing the sensitivity of tumor cells (Hela) to immune killing

1) Collect the transfected tumor cells, wash them twice with PBS, and then label the tumor cells with CFSE (1.25μM×10min);

2) Terminate with complete medium, wash with PBS to remove residual CFSE, and then count and dilute CFSE-labeled tumor cells to 1×10 ⁵ cells/mL;

3) Pipette 50μL into a round-bottom 96-well plate, and then add YTS-NK cell line or CAR-YTS cell according to the corresponding effective target ratio, and set 3 multiple wells for each effective target ratio; set to add only target cells at the same time Group to determine the proportion of naturally dead cells;

4) After mixing, place it in an incubator for 4-6 hours;

5) Transfer the cells to a 1.5mL EP tube (observed under the microscope, if a large number of cells adhere to the wall, trypsinization is required), add 3μl 7-AAD to each tube, pipette to mix, and place on ice, protected from light Incubate for 10min;

6) Analyze the ratio of 7-AAD ⁺ ^{cells in CFSE+} cells by flow cytometry, which is the ratio of dead target cells; the specific killing rate is the ratio of 7-AAD ⁺ cells of each effective target ratio minus the background death of cells Level (the ratio of 7-AAD ⁺ cells in the 0:1 group with effective target ratio). ^{As shown in Figure 2, the ratio of 7-AAD+} cells with an effective target ratio of 1:1 and 0:1 in the si317 group were 29.9% and 13%, respectively, so the specific killing rate of an effective target ratio of 1:1 was 29.9%- 13%=16.9%; in the same way, the specific killing rate of the si317 group with a medium-efficiency target ratio of 5:1 is 39.4; the specific killing rate of the siNC group with a medium-efficiency target ratio of 1:1 is 5.6%, and the effective-target ratio is 5:1 The specific killing rate was 12.9%. The above results can indicate that CD317siRNA has the effect of enhancing the sensitivity of tumor cells to immune killing.

4. Flow cytometry to detect the effect of CD317 inhibitor on immune cell activation

1) Collect the transfected Hela cells, resuspend in complete medium, and count;

2) Take 1×10 ⁵ tumor cells (suspended in 100μL medium) and the same amount of YTS cells and mix well;

3) Place in an incubator and incubate for 30-60min;

4) Collect the cells, resuspend in 100μL PBS, and add anti-LFA-1 activated antibody;

5) Incubate at 4°C for 30 minutes, and then add anti-human CD56 antibody to label NK cells;

6) After washing with PBS, analyze the percentage of ^{CD56 +} LFA-1 ^{+ cells with a flow cytometer.} CD56 is a marker of NK cells, and LFA is a marker of NK cell activation. Therefore, the activation level of the cell population can be judged by the percentage of ^{CD56 +} LFA-1 ^{+ cells.} As shown in Figure 2, the percentages of CD56 ⁺ LFA-1 ⁺ cells in the siNC and si317 groups were 23.3% and 21.5%, respectively. There was no significant difference, indicating that knocking down tumor cells CD317 would not affect the activation of NK cells co-cultured with them. , To rule out the possibility that CD317 is used as an immune checkpoint or affects the immune checkpoint signal to inhibit the activation of effector cells. .

5. LDH method detects the effect of CD317 inhibitor on enhancing the sensitivity of tumor cells to immune cells

1) Preparation of target cells: Collect the transfected HepG2 or MCF7 cells, wash and resuspend them in phenol red-free KBM581 medium;

2) Inoculation: Inoculate 1×10 ⁴ cells/well into a U-bottom 96-well plate;

3) Preparation of effector cells: After washing CAR-YTS cells, resuspend them in phenol red-free KBM581 medium for use;

4) Add CAR-YTS to each well according to the effective target ratio, and set up separate control wells for effector cells and target cells;

5) Mix well, centrifuge at 300g×5min, then put it back into the incubator for 4-6h;

6) LDH detection kit (Biyuntian, C0017) was used to detect cell killing, as shown in Figure 4. It can be seen from a that under the same effective target ratio, the specific killing rate of CAR-YTS in killing HepG2 cells in the CD317 inhibitor group (Si317-1 and Si317-2) is higher than that in the SiNC group; It can be seen that under the same effective target ratio, the specific killing rate of CAR-YTS in killing MCF-7 cells in the CD317 inhibitor group (Si317-1 and Si317-2) is higher than that in the SiNC group. The above results can indicate that CD317 siRNA can enhance the sensitivity of tumor cells to immune cells.

In summary, in the present application, the use of siRNA to inhibit the expression of CD317 in tumor cells can significantly enhance the sensitivity of cancer cells to immune killing.

The above are only specific implementations of the present invention, not all implementations. Any equivalent changes made by those of ordinary skill in the art to the technical solutions of the present invention by reading the specification of the present invention are covered by the claims of the present invention. .

Claims

Application of CD317 inhibitor in the preparation of drugs that enhance the sensitivity of tumor cells to immune killing.
Application of CD317 inhibitor in the preparation of drugs for enhancing the therapeutic effect of tumor immune cells.
Application of CD317 inhibitors in the preparation of synergistic drugs that enhance the anti-tumor efficacy of engineered immune cells.
Application of CD317 inhibitor in combination with engineered immune cells in the preparation of anti-tumor drugs.
An anti-tumor pharmaceutical composition, which is characterized by comprising a CD317 inhibitor and engineered immune cells.
The application according to any one of claims 3-4 or the pharmaceutical composition according to claim 5, wherein the engineered immune cells are immune cells that function through the perforin granzyme pathway, preferably, the The engineered immune cells are CAR-T, CAR-NK, and TCR-T.
The application according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5, wherein the tumor is a solid tumor.
The application or pharmaceutical composition according to claim 7, wherein the solid tumor is a solid tumor with high expression of CD317;

Preferably, the solid tumors include liver cancer, lung cancer, breast cancer, colon cancer, kidney cancer, and cervical cancer.
The application of any one of claims 1-4 or the pharmaceutical composition of claim 5, wherein the CD317 inhibitor is CD317 siRNA, CD317 shRNA, shRNA encoding DNA, CD317 sgRNA, PROTAC, antibody Or blocking peptides.
The application or pharmaceutical composition according to claim 9, wherein the CD317 inhibitor is CD317 siRNA, and the CD317 siRNA is designed according to the CD317 gene transcript NM_004335.3;

Preferably, the CD317 siRNA includes the sense strand of the nucleotide sequence shown in SEQ ID NO: 1 and the antisense strand of the nucleotide sequence shown in SEQ ID NO: 2, and/or, the nucleotide sequence shown in SEQ ID NO: 3 The sense strand of the nucleotide sequence and the antisense strand of the nucleotide sequence shown in SEQ ID NO: 4.