CN115137830A

CN115137830A - Attenuated salmonella and immune checkpoint inhibitor combined medicine and application thereof

Info

Publication number: CN115137830A
Application number: CN202210181926.XA
Authority: CN
Inventors: 华子春; 包斐斐; 吴乐阳
Original assignee: Targetpharma Laboratories Jiangsu Co ltd
Current assignee: Targetpharma Laboratories Jiangsu Co ltd
Priority date: 2022-02-25
Filing date: 2022-02-25
Publication date: 2022-10-04

Abstract

The invention discloses an attenuated salmonella and immune checkpoint inhibitor combined drug and application thereof. The attenuated salmonella and immune checkpoint inhibitor combined drug is combined by attenuated salmonella typhimurium and an immune checkpoint inhibitor. The invention discloses attenuated salmonella which can increase the effects of resisting malignant tumor growth and inhibiting tumor metastasis of a tumor immune checkpoint inhibitor in a synergistic action mode, and greatly improves the treatment effectiveness of the immune checkpoint inhibitor as an anti-tumor medicament. The combined preparation method is simple, easy to operate and has good application prospect.

Description

Attenuated salmonella and immune checkpoint inhibitor combined medicine and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a combined drug of attenuated salmonella and an immune checkpoint inhibitor and application thereof.

Background

Immune checkpoint blockers are hot spots in the development of current international anti-malignant drugs, and as exemplified by PD-1/PD-L1 antibody inhibitors, PD-1 or PD-L1 inhibitors can relieve the immunosuppressive state of tumors by binding to the PD-1 receptor on the surface of T cells or blocking the binding of PD-L1 protein to cancer cells, thereby effectively inhibiting and alleviating the progression of malignant tumors (Sharma et al, science 2015, 62348 (30): 56-61). Tumor Immune checkpoint inhibitor treatment regimens (Immunocheckpoint inhibitor: ICI) also require that tumors be highly infiltrated by T cells (Sznol et al, cancer J2014, 20 (4): 290-295), surface expression of high levels of inhibitory checkpoints such as PD-1 (Fleming et al, nat Rev Drug Discov 2012,11 (8): 601), PD-L1, CTLA-4 (Rowshanravan et al, blood 2018,131 (1): 58-67), TIM-3 (Das et al, immunol Rev 2017,276 (1): 97-111), LAG3 (Maruhashi et al, J Immunotherther Cancer 2020, 8), and the like. Many malignancies lack infiltrating T cells and have a low response to ICI, resulting in inefficient clinical use of immune checkpoint blockers, such as PD-1/PD-L1 inhibitors, of only 10-30% (Sznol et al, cancer J2014, 20 (4): 290-295). Therefore, how to induce more T cells to appear at the tumor site, the initiation of the T cell response of the tumor, is a key factor for immune checkpoint blocker therapy including PD-1/PD-L1 inhibitors.

Scientists have recently discovered the presence of bacteria in tumors and bacterial therapy of malignant tumors is emerging as an anti-tumor therapy, including tumor therapy with attenuated salmonella. The anti-tumor ability of bacteria is not only related to its own inherent anti-tumor activity, but also to the immune response of the host, including inflammatory and T cell-dependent immune responses (Lee et al, appl Microbiol Biotechnol 2011,92 (6): 1251-1260 Lee et al, J Immunother 2009, 32 (4): 376-388. In addition, bacterial accumulation in tumor tissues can increase the proportion of host immune cells (e.g., neutrophils, natural Killer (NK) cells, macrophages, dendritic Cells (DCs), B cells, CD8 ⁺ T cells) (Duon et al, exp Mol Med 2019,51 (12): 1-15; lou et al, nanomicro Lett 2021, 13 (1): 37), the details of which vary from bacterium to bacterium.

Thus, it is possible to treat by combination with attenuated salmonella, by increasing infiltration of immune cells in malignant tumors, enhancing the tumor therapeutic effect of immune checkpoint blockers including PD-1/PD-L1 inhibitors. The present invention provides an effective method of improving the effectiveness of immune checkpoint blockade therapy.

Disclosure of Invention

The invention aims to provide a combined drug of attenuated salmonella and an immune checkpoint inhibitor and application thereof, and provides a scheme for treating malignant tumors by combined application of salmonella typhimurium and an immune checkpoint blocker.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows: the invention relates to an attenuated salmonella and immune checkpoint inhibitor combined medicine, which is characterized in that: the attenuated salmonella typhimurium and the immune checkpoint inhibitor combined drug are combined by attenuated salmonella typhimurium and an immune checkpoint inhibitor, and the attenuated salmonella typhimurium is the bacterium which is attenuated salmonella typhimurium VNP20009 and a genetically modified strain thereof; the blocking agent of the immune checkpoint comprises but is not limited to an antibody and a nano antibody, and the immune checkpoint inhibitor is a blocking agent which can block the immune checkpoint and can be CTLA-4, TIM-3, LAG3, nivolumab or Ceriluzumab or the nano antibody.

The combined drug of the attenuated salmonella and the immune checkpoint inhibitor is combined use of the attenuated salmonella typhimurium and the immune checkpoint inhibitor, compared with the theoretical value of the sum of tumor growth doubling time or growth delay time prolonging amplitude caused by the curative effect of each group after the attenuated salmonella and the immune checkpoint inhibitor are treated independently, the tumor growth doubling time prolonging amplitude of the combined drug is 1.47-1.87 times of the theoretical value of the sum of the curative effects of the two groups, and the tumor growth delay time prolonging amplitude of the combined drug is 1.57-2.33 times of the theoretical value of the sum of the curative effects of the two groups. Compared with the curative effect of singly treating and inhibiting tumor metastasis by using the attenuated salmonella and the immune checkpoint inhibitor, the combined medicament can reduce the lung tumor metastasis by 45.3 to 74.8 percent.

Further, htrA of the attenuated Salmonella typhimurium VNP20009 is a key gene for producing synergistic effect by combined treatment.

Further, the attenuated Salmonella typhimurium VNP20009 reduces PD1 ⁺ CD38 ^High CD8 ⁺ T cells remarkably improve the expression of PDL1 on the surface of tumor cells and remarkably improve the expression level of PDL1 on the surface of mononuclear cells in tumor tissues.

Furthermore, the time interval and times of administration of the salmonella typhimurium and the PD-1/PD-L1 immune checkpoint antibody blocker are specifically: nivolumab or carlixizumab or PD-1/PD-L1 nano antibody is administered at the fourth day after the bacteria, and the administration is carried out three times or more every two days; the administration mode of the PD-1/PD-L1 immune checkpoint antibody/nano antibody blocking agent is intraperitoneal or intravenous administration.

Further, different doses of attenuated salmonella VNP20009 and PD-1/PD-L1 immune checkpoint antibody blockers, VNP20009: 2x10 ⁴ ，5×10 ⁴ ，1×10 ⁵ ，2×10 ⁵ ，5×10 ⁵ ，1×10 ⁶ ，2×10 ⁶ The unit is CFU/only; nivolumab or carprilizumab or PD-1/PD-L1 nano antibody, the administration dosage is respectively as follows: 1.0,1.6,3.2,8, said units being mg/kg.

Furthermore, the attenuated salmonella is subjected to combined treatment by intravenous injection, intraperitoneal injection, oral administration, subcutaneous administration and intratumoral administration.

Furthermore, after the attenuated salmonella is combined with a PD-1/PD-L1 immune checkpoint antibody blocking agent Nivolumab or a Carrilizumab/nano antibody blocking agent, the tumor growth of the mouse is obviously inhibited, and the survival time is obviously prolonged.

The method for treating malignant tumor by combining attenuated salmonella and an immune checkpoint inhibitor is applied to preparing an anti-malignant tumor medicament.

The method for treating malignant tumor by combining attenuated salmonella and an immune checkpoint inhibitor is applied to preparing a medicine for resisting malignant tumor metastasis.

Has the advantages that: the invention discovers that the attenuated salmonella and the immune checkpoint blocking agent comprising the PD-1/PD-L1 antibody and the nano antibody are combined and applied to generate the obviously enhanced malignant tumor treatment effect, the tumor growth and the tumor metastasis of a tumor-bearing mouse are obviously inhibited, and the survival time is prolonged. The attenuated salmonella VNP20009 and the mutant strain thereof are used as low-toxicity and easily-cultured bacteria, have low cost and have the prospect of large-scale popularization and application.

Compared with the prior art, the invention has the following advantages:

(1) The facultative anaerobic bacterium salmonella typhimurium VNP20009 and the mutant strain thereof are selected because of good tumor targeting property and tumor inhibiting effect. Attenuated salmonella VNP20009 and its mutant strain were selected as potentiators of immune checkpoint inhibitors because attenuated salmonella VNP20009 is characterized by good safety.

(2) The attenuated salmonella VNP20009 was chosen as a potentiator for immune checkpoint inhibitors because VNP20009 can increase infiltration of immune cells in tumor bearing mouse tissues, increasing expression of immune checkpoint inhibitors such as PD-L1 in tumor tissues.

(3) The attenuated salmonella VNP20009 and the mutant strain thereof are combined with an immune checkpoint inhibitor, so that the good anti-tumor effect and anti-tumor metastasis curative effect of the attenuated salmonella VNP20009 are verified in a mouse model, and the application of the delta htrA strain proves that the synergistic effect of the VNP20009 on the immune checkpoint inhibitor is related to the gene htrA.

Drawings

FIG. 1 is CD8 in tumor tissue microenvironment after attenuated Salmonella treatment ⁺ T cell analysis. Percentage of total CD8+ T cells within the tumor changed on day 3 after administration of B16F10 melanoma model, PBS and attenuated salmonella VNP20009. And (5) a histogram statistical comparison graph. 1: a PBS group; 2: VNP20009 group. (n =4,ns means no significant difference).

FIG. 2 is a graph of attenuated Salmonella treatment reversing depleted CD 8T cells in tumor tissues. B16F10 melanoma model, intratumoral depletion of CD8 on day 3 after administration of PBS and attenuated Salmonella VNP20009 ⁺ T cells (PD 1) ⁺ CD38 ^high ) In percentage (b)And (4) changing. 2A, representative streaming detection map; and 2B, a histogram statistical comparison graph. 1: a PBS group; 2: VNP20009 group. (n = 4. X.p)<0.001)。

FIG. 3 is a graph showing that attenuated Salmonella treatment increased PDL1 levels on the surface of tumor cells. Percentage of tumor cells in/highly expressing PDL1 in the tumor interior surface changed by day 3 after administration of B16F10 melanoma model, PBS and attenuated salmonella VNP20009. Representative streaming detection maps; and 3B, a histogram statistical comparison graph. 1: a PBS group; 2: VNP20009 group. (n =4, # P < 0.0001).

FIG. 4 is a graph showing that attenuated Salmonella treatment increased the level of monocyte surface PDL1 in tumor tissue. Percentage of monocytes that highly expressed PDL1 on the surface of tumors changed by day 3 after administration of B16F10 melanoma model, PBS and attenuated salmonella VNP20009. 4A. Representative streaming images; 4B, comparing the histogram statistics. 1: a PBS group; 2: VNP20009 group. (n =4, # P < 0.0001).

FIG. 5 shows VNP20009 (2X 10) of the present invention ⁴ ) Tumor growth profile in mice after combination with different doses of Nivolumab; the VNP20009 strain is administered at a dose of 2X10 ⁴ CFU, mice were treated for melanoma in combination with varying concentrations of Nivolumab. 0: a PBS group; 1: nivolumab =0mg/kg group 2: nivolumab =1.6mg/kg group; 3:3.2mg/kg group; 4:8mg/kg group.

FIG. 6 shows VNP20009 (2X 10) of the present invention ⁵ ) Tumor growth profile in mice after combination with different doses of Nivolumab; the VNP20009 strain is administered at a dose of 2X10 ⁵ CFU, mice were treated for melanoma in combination with varying concentrations of Nivolumab. 0: a PBS group; 1: nivolumab =0mg/kg group 2: nivolumab =1.6mg/kg group; 3:3.2mg/kg group; 4:8mg/kg group.

FIG. 7 shows VNP20009 (2X 10) of the present invention ⁶ ) Tumor growth profile in mice after combination with different doses of Nivolumab; the VNP20009 strain is administered at a dose of 2X10 ⁶ CFU, mice were treated for melanoma in combination with varying concentrations of Nivolumab. 0: a PBS group; 1: nivolumab =0mg/kg group 2: nivolumab =1.6mg/kg group; 3:3.2mg/kg group; 4:8mg/kg group.

FIG. 8 shows VNP20009 and Ni of the present inventionTumor growth curve of mouse melanoma after the combination of the volumab; selecting the best-effective dose combination group (VNP 20009= 2X 10) ⁶ CFU, nivolumab =8 mg/kg) mice were specifically analyzed for tumor growth. 1. A PBS group; 2. nivolumab group; 3. VNP20009 group; 4. VNP20009 and Nivolumab combination group.

FIG. 9 is a graph of tumor growth doubling time for mouse melanoma after the combination of VNP20009 and Nivolumab of the present invention. 1. A PBS group; 2. nivolumab group; 3. VNP20009 group; 4. VNP20009 and Nivolumab combination with P < 0.05 and P < 0.01.

FIG. 10 is a graph of the survival time of tumor-bearing mice after the combination of VNP20009 and Nivolumab of the present invention. 1. A PBS group; 2. nivolumab group; 3. VNP20009 group; 4. VNP20009 and Nivolumab combination group.

FIG. 11 is a graph showing the tumor growth delay time of melanoma in mice after the combination of VNP20009 and Nivolumab of the present invention; 1. a PBS group; 2. nivolumab group; 3. VNP20009 group; 4. VNP20009 and Nivolumab combination with P < 0.05.

FIG. 12 is a graph showing the tumor growth of mouse melanoma after the htrA-deficient strain of the present invention was used in combination with Nivolumab; when htrA deficient strain =2 × 10 ⁶ CFU, nivolumab =8mg/kg combined with tumor growth in mice of the group. 1. A PBS group; 2. nivolumab group; 3. htrA-deficient bacterium group; 4. htrA-deficient bacteria and Nivolumab. FIG. 9 is a graph showing the tumor growth doubling time of mouse melanoma after the combination of the htrA-deficient bacterium of the present invention and Nivolumab. 1. A PBS group; 2. nivolumab group; 3. htrA-deficient bacterium group; 4. the htrA-deficient bacteria and Nivolumab are combined, P is less than 0.05, and P is less than 0.01.

FIG. 13 is a graph showing the survival time of tumor-bearing mice after the htrA-deficient bacterium of the present invention and Nivolumab are used in combination. 1. A PBS group; 2. nivolumab group; 3. htrA-deficient bacterium group; 4. htrA-deficient bacteria and Nivolumab.

FIG. 14 is a graph showing the tumor growth delay time of mouse melanoma after the htrA-deficient bacterium of the present invention and Nivolumab are used together. 1. A PBS group; 2. nivolumab group; 3. htrA-deficient bacterial group; 4. the htrA-deficient bacteria and Nivolumab are combined, P is less than 0.05, and P is less than 0.01.

FIG. 15 is a graph showing the tumor growth of melanoma in mice following the combination of VNP20009 and Carrilizumab (Suzhou Shengdiya biomedical corporation) of the present invention; 1. a PBS group; 2. a group of Cayleigh mabs; 3. VNP20009 group; 4. VNP20009 and charizumab combination.

FIG. 16 is a graph showing the analysis of bacterial titer in tumor-bearing mouse tissues after the combined use of VNP20009 and Nivolumab according to the present invention; attenuated salmonella typhimurium tumor targeting assay bacterial titers (in Log10 (CFU /) g) in liver, spleen and tumor tissues were examined since liver and spleen are the most distributed normal tissues. 1. VNP20009 group; 2. VNP20009 and Nivolumab combination group. Wherein: the bacterial titer in the liver was VNP20009 group, VNP20009 and Nivolumab combination group; the bacterial titer in the spleen is VNP20009 group, and VNP20009 and Nivolumab combination group; the bacterial titers in the tumors were VNP20009 group, VNP20009 and Nivolumab combination group.

FIG. 17 is a graph of the analysis of the bacterial titer in tumor-bearing mouse tissues after the combined use of htrA-deficient bacteria and Nivolumab of the present invention; attenuated salmonella typhimurium tumor targeting assay since liver and spleen are the most bacterially distributed normal tissues, the titers of bacteria in liver, spleen and tumor tissues were examined (in units of Log10 (CFU /) g): 1. htrA-deficient bacterial group; 2. htrA-deficient bacteria and Nivolumab. Wherein: the bacterial titer in the liver is htrA-deficient bacterium group, and the htrA-deficient bacterium and Nivolumab combined group; the bacterial titer in the spleen is htrA-deficient bacterium group, and the htrA-deficient bacterium and Nivolumab combined group; the bacterial titer in the tumor is htrA-deficient bacterium group, and the htrA-deficient bacterium and Nivolumab combined group; ns, P > 0.05.

FIG. 18 is an SDS-PAGE analysis diagram of the separation and purification process of the anti-PD 1 nm antibody according to the present invention; m: a protein Marker;1: bacteria lysis supernatant; 2: bacteria cracking and precipitation; 3: elution with 10mM imidazole; 4:20mM imidazole; 5: elution with 40mM imidazole; 6:60mM imidazole elution; 7:80mM imidazole; 8: elution with 100mM imidazole; 9:150mM imidazole.

FIG. 19 is a tumor growth profile of anti-PD 1 nanobody proteins of the present invention in combination with attenuated Salmonella VNP 20009; 1: a PBS group; 2: anti-PD 1 nanobody PD1nb group; 3: attenuated salmonella VNP20009 control group (VNP-NC); 4: anti-PD 1 Nanobody proteins were combined with attenuated Salmonella VNP20009 (VNP-NC + PD1 nb). i.p.: and (4) carrying out intraperitoneal injection.

FIG. 20 is a graph showing the tumor growth doubling time of the anti-PD 1 nano-antibody protein of the present invention in combination with attenuated Salmonella VNP20009, i.e., tumors ranging from 1000mm ³ Up to 2000mm in length ³ The number of days required; 1: a PBS group; 2: anti-PD 1 nanobody PD1nb group; 3: an attenuated salmonella VNP20009 control group (VNP-NC); 4: anti-PD 1 Nanobody proteins were combined with attenuated Salmonella VNP20009 (VNP-NC + PD1 nb).

FIG. 21 is a graph of tumor growth delay time for the anti-PD 1 nanobody protein of the present invention in combination with attenuated Salmonella VNP20009. 1: a PBS group; 2: anti-PD 1 nanobody PD1nb group; 3: an attenuated salmonella VNP20009 control group (VNP-NC); 4: anti-PD 1 nano-antibody protein was combined with Salmonella attenuated VNP20009 (VNP-NC + PD1 nb).

FIG. 22 is a graph showing the effect of anti-melanoma lung metastasis by the combination of anti-PD 1 nanobody protein of the present invention and Salmonella attenuated VNP20009. Lung metastasis status in mice; b. quantitatively analyzing the area of the lung tumor metastasis. The scale is 5 mm. 1: a PBS group; 2: anti-PD 1 nanobody PD1nb group; 3: an attenuated salmonella VNP20009 control group (VNP-NC); 4: anti-PD 1 Nanobody proteins were combined with attenuated Salmonella VNP20009 (VNP-NC + PD1 nb).

FIG. 23 is a graph of lung metastasis and ImageJ analyzed lung tumor metastasis area for groups of mice of the present invention; fig. 23a is a photograph of lung metastasis of each group of mice recorded, and fig. 23 is a graph of lung tumor metastasis area analyzed by bmagej of the present invention.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. However, it is easily understood by those skilled in the art that the descriptions of the embodiments are only for illustrating the present invention and should not be construed as limiting the present invention as detailed in the claims. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

Example 1

Analysis of immune checkpoint protein expression in tumor tissue microenvironment following attenuated salmonella treatment

(1) Establishment of mouse melanoma model

B16F10 mouse melanoma is a highly invasive and metastatic malignancy. B16F10 mouse melanoma cells are digested with 0.5% pancreatin after growing to exponential growth phase in DMEM cell culture medium, then centrifuged for 3min at 1000rpm/min, the supernatant culture solution is removed, washed for 2 times with PBS and then counted, and finally the cells are resuspended with PBS to adjust the final concentration of the cells to 2x10 ⁶ one/mL. Each C57BL/6 mouse was inoculated with 100. Mu.L of the vaccine at the mouse axillary fat pad, i.e., 2X10 ⁵ One/only. After inoculation, mice were housed in clean-grade animal houses until the tumor volume of the mice had grown to approximately 100mm ³ The subsequent experiments were performed.

C57/B6 mice bearing B16F10 mice melanoma were randomized into PBS and VNP20009 groups. The PBS group was injected intraperitoneally with 100. Mu.L PBS; VNP20009 intraperitoneal injection of 5X 10 ⁵ CFU bacteria (in exponential growth phase, dissolved in 100. Mu.L PBS). Tumor bearing mice were sacrificed on day 3 post-dosing and tumors were dissected and incubated at 37 degrees in digestion medium (10U/mL collagenase I, 400U/mL collagenase IV, 30U/mL DNase I, all diluted in HBSS) with gentle inversion shaking for 30-50 minutes. The cell pellet was removed through a 40- μm cell filter to obtain a single cell suspension. Cells were stained with a specific dead cell dye (BD, 564407), incubated for 10-15 minutes at room temperature in the dark, and then subjected to combinatorial staining using the following anti-mouse antibodies: CD45-PE-Cy7 (BD, clone No. 30-F11), CD11b-APC (BD, clone No. 561690), F4/80-BV421 (BD, clone No. T45-2342), CD86-PE (BD, clone No. GL 1), CD3e-FITC (BD, clone No. 145-2C 11), CD8-APC (BD, clone No. 53-6.7), PD1-APC (BD, clone No. J43), PDL1-BV421 (BD, clone No. MIH 5) and CD38-BV421 (BD, clone No. 90/CD 38). After incubation for 20 min at room temperature in the dark, PBS was washed 1-2 times, the collected cells were carefully resuspended and analyzed by flow cytometry (BD Canto II).

Treatment with attenuated salmonella VNP20009 does not substantially alter CD8 in tumor tissue ⁺ Content of T cells (FIG. 1), but PD1 ⁺ CD38 ^High CD8 ⁺ The proportion of T cells did not follow CD8 ⁺ T cells remained unchanged and their proportion was significantly reduced (by about 60%) (fig. 2). PD1 ⁺ CD38 ^High CD8 ⁺ T cells are depleted CD8 ⁺ T cells, the invention discovers for the first time that the attenuated salmonella VNP20009 can reduce exhausted PD1 ⁺ CD38 ^High CD8 ⁺ T cells, which are one of the important mechanisms for the antitumor efficacy of the attenuated salmonella VNP20009, are disclosed for the first time.

The present invention also found that treatment with attenuated salmonella VNP20009 significantly increased the expression of PDL1 on the surface of tumor cells (by about 2.47-fold) (fig. 3). On one hand, the improvement of PDL-1 expression on the surface of the tumor cell indicates that the immunosuppressive activity of the tumor cell is improved after the attenuated salmonella VNP20009 is treated; on the other hand, it is predicted that tumor cells may be more sensitive to treatment with PDL-1 inhibitors.

The present invention also found that treatment with attenuated salmonella VNP20009 significantly increased monocyte surface PDL-1 expression in tumor tissue (increased by about 2.52-fold) (fig. 4). An increase in the expression of PDL-1 on the surface of monocytes would also indicate an increase in immunosuppressive activity in the tumor microenvironment following treatment with attenuated salmonella VNP20009.

Transcriptomics studies confirmed not only the changes in PD-1, PDL-1 expression, but also showed similar trends for other immune checkpoints, such as CTLA-4, TIM-3, LAG3, etc.

The above findings indicate that attenuated salmonella VNP20009 treatment can produce both anti-tumor beneficial changes in the tumor microenvironment and immune checkpoint changes that are not beneficial for tumor treatment. The change of the immune check points of the tumor microenvironment unfavorable for tumor treatment indicates that the combined application of the attenuated salmonella VNP20009 and the immune check point inhibitor is expected to improve the change which is unfavorable for tumor treatment and is caused by the attenuated salmonella VNP20009 treatment, solve the problem of the current immune check point inhibitor treatment and generate the synergistic curative effect.

Example 2

Antitumor Effect of a combination regimen of attenuated Salmonella and PD-1/PD-L1 immune checkpoint antibody blockers

The concentration settings for the screening optimized Nivolumab in combination with attenuated salmonella of the present invention are shown in table 1:

TABLE 1

(2) Administration mode

After tumor-bearing mice are randomly grouped, the bacteria are injected into the abdominal cavity, the PD-1 immune checkpoint antibody is injected into the abdominal cavity on the third day after the bacteria injection, and then the drug is administered once every 2 days for three times.

Different doses of attenuated salmonella VNP20009 and its mutant strains and PD-1 immune checkpoint antibodies, VNP20009: 2X10 ⁴ ，5×10 ⁴ ，1×10 ⁵ ，2×10 ⁵ ，5×10 ⁵ ，1×10 ⁶ ，2×10 ⁶ (unit: CFU/only); nivolumab or carprilizumab, administered at the respective doses: 1.0,1.6,3.2,8 (unit: mg/kg); tumor growth was observed in 10 mice per group. The attenuated salmonella and the Nivolumab or the garelizumab ozogamicin have better dose-dependent anti-tumor curative effect; the curative effect of Nivolumab is slightly better than that of the carprilizumab; and performing optimal combination screening.

In order to better and finely analyze the combined anti-tumor curative effect of the attenuated salmonella and the Nivolumab or the Carrilizumab, the PBS group is respectively arranged after the optimal combined dose combination is screened out; VNP20009 group; a delta htrA mutant strain group; nivolumab group; VNP20009+ Nivolumab combination group; delta htrA mutant strain + Nivolumab combined group; wherein the Salmonella dose group is selected from 0,2 × 10 ⁴ ，2×10 ⁵ ，2×10 ⁶ (unit: CFU/only); dose group selection for Nivolumab: 0,1.6,3.2,8 (unit: mg/kg), 50 mice per group. 10 mice were sacrificed at 11, 14 and 17 days, and blood, spleen, liver and tumor tissues were collected and analyzed (FIGS. 5 to 7).

In the mouse melanoma model, the tumor growth doubling time of the VNP20009 and Nivolumab combination group increased from 1.86 days in the PBS control group, 2.46 days in the Nivolumab group, and 6.62 days in the VNP20009 group to 9.73 days (fig. 8, 9), the tumor growth doubling time of the combination group was 295.5% longer than that of the Nivolumab group, 47.0% longer than that of the VNP20009 group, and even the tumor growth doubling time of the VNP20009 group was 169.1% longer than that of the Nivolumab group; compared with the PBS control group, the doubling time of the tumor growth of the Nivolumab group after treatment is prolonged by 32.3 percent, the doubling time of the tumor growth of the VNP20009 group after treatment is prolonged by 255.9 percent, and the doubling time of the tumor growth of the VNP20009 and Nivolumab combined group is prolonged by 423.1 percent. Compared with the PBS control group, the tumor growth doubling time of the VNP20009 and Nivolumab combination group is increased by 423.1 percent, which is far greater than the sum (288.3 percent) of the tumor growth doubling time after the Nivolumab group is treated by 32.3 percent and the tumor growth doubling time after the VNP20009 group is treated by 255.9 percent, and the sum of the tumor growth doubling time increase amplitude caused by the curative effect of the VNP20009 and Nivolumab combination group and the sum of the tumor growth doubling time increase amplitude caused by the curative effect of the Nivolumab group and the VNP20009 group is 1.47 times of the sum of the theoretical values of the two groups of the curative effects, so that the VNP20009 and Nivolumab combination group really generate the synergistic curative effect.

The tumor growth delay time of mouse melanoma was extended from 9.83 days in PBS group, 11.83 days in Nivolumab group, and 17.16 days in VNP20009 group to 24.5 days after the combination of VNP20009 and Nivolumab (fig. 10, 11), 107.1% longer than that in Nivolumab group, 42.8% longer than that in VNP20009 group, and 45.1% longer than that in Nivolumab group even though the tumor growth delay time in VNP20009 group was extended. Compared with the PBS control group, the growth delay time of the tumor after the treatment of the Nivolumab group is prolonged by 20.3 percent, the growth delay time of the tumor after the treatment of the VNP20009 group is prolonged by 74.6 percent, and the growth delay time of the tumor of the VNP20009 and Nivolumab combination group is prolonged by 149.2 percent. Compared with the PBS control group, the tumor growth delay time of the VNP20009 and Nivolumab combination group is prolonged by 149.2 percent, which is far greater than the sum (94.9 percent) of the tumor growth delay time prolonged by 20.3 percent after the Nivolumab group is treated and the tumor growth delay time prolonged by 74.6 percent after the VNP20009 group is treated, and the extent of the tumor growth delay time prolonged by the curative effect of the VNP20009 and Nivolumab combination group is 1.57 times of the theoretical value of the sum of the two groups of curative effects compared with the theoretical value of the extent of the tumor growth delay time prolonged by the curative effect of the Nivolumab group and VNP20009 group after the Nivolumab group and VNP20009 group are treated independently, so that the VNP20009 and Nivolumab combination group really produce the synergistic curative effect.

In contrast, in the same mouse melanoma model, when the same antibody was used in combination with htrA-deficient VNP20009 mutant strain, the tumor growth doubling times of the htrA-deficient VNP20009 mutant strain and Nivolumab combination group were extended from 1.86 days of PBS group, 2.46 days of Nivolumab group, and 2.17 days of htrA-deficient group to 2.67 days (fig. 12, 13), and the tumor growth doubling times of the combination group were respectively extended by 43.5% compared to PBS control group, 8.5% compared to Nivolumab group, 18.7% compared to htrA-deficient group, and 13.4% compared to htrA-deficient group. Compared with a PBS control group, the doubling time of the tumor growth of the Nivolumab group after treatment is prolonged by 32.3%, the delay time of the tumor growth of the htrA-deficient bacterium group after treatment is prolonged by 16.7%, and the doubling time of the tumor growth of the htrA-deficient bacterium and Nivolumab combined group is prolonged by 30.3%. Compared with the PBS control group, the doubling time of the tumor growth of the htrA-deficient bacterium and Nivolumab combination group is prolonged by 30.3%, which is less than the doubling time of the tumor growth of the Nivolumab group after treatment by 32.3%, which is more less than the sum of the doubling time of the tumor growth of the Nivolumab group after treatment by 32.3% and the doubling time of the tumor growth of the htrA-deficient bacterium group after treatment by 16.7% (49%). In contrast, the doubling time of tumor growth after treatment in the VNP20009 group was 255.9%, and the doubling time of tumor growth in the VNP20009 and Nivolumab combination group was 423.1%. The defect in the htrA gene resulted in a substantial reduction in the anti-tumor efficacy of VNP20009 strain alone or in combination with immune checkpoint antibody inhibitors.

The tumor growth delay time of the htrA-deficient bacteria and Nivolumab combination group was increased from 9.83 days in the PBS control group, 11.83 days in the Nivolumab group, and 10.66 days in the htrA-deficient bacteria group to 12.5 days (fig. 14, 15); the tumor growth delay time of the htrA-deficient bacterium and Nivolumab combination group is respectively prolonged by 27.2 percent compared with that of a PBS control group, 5.7 percent compared with that of a Nivolumab group, 17.3 percent compared with that of the htrA-deficient bacterium group, and the tumor growth delay time of the Nivolumab group is also prolonged by 11.0 percent compared with that of the htrA-deficient bacterium group. Compared with a PBS control group, the growth delay time of the tumor after the treatment of the Nivolumab group is prolonged by 20.3 percent, the growth delay time of the tumor after the treatment of the htrA-deficient bacterium group is prolonged by 8.4 percent, and the growth delay time of the tumor of the htrA-deficient bacterium and Nivolumab combination group is prolonged by 27.2 percent; compared with the PBS control group, the tumor growth delay time of the htrA-deficient bacterium and Nivolumab combination group is prolonged by 27.2 percent, which is less than the sum (28.7 percent) of 20.3 percent of the tumor growth delay time after the Nivolumab group is treated and 8.4 percent of the tumor growth delay time after the htrA-deficient bacterium group is treated. In contrast, the tumor growth delay time after treatment in VNP20009 group was increased by 74.6%, and that in the combination of VNP20009 and Nivolumab was increased by 149.2%. Therefore, defects in the htrA gene resulted in a greatly reduced antitumor efficacy of VNP20009 strain alone or in combination with immune checkpoint inhibitors.

The calrayleigh mab produced in Suzhou Shengdiya biomedical corporation, when used in combination with VNP20009 and its mutant, showed similar enhanced antitumor efficacy, and was also closely related to the presence or absence of htrA (FIG. 16).

It follows that the htrA gene is a crucial key gene for the antitumor activity of VNP20009, especially its enhanced antitumor activity when used in combination with immune checkpoint antibodies.

Example 3

Tissue distribution of bacteria on tumor-bearing mice in a combination regimen

Fifth day of bacterial treatment of tumor-bearing mice, the mice were randomly sacrificed and tumors, livers and spleens of the tumor-bearing mice were taken under sterile conditions, weighed separately and homogenized in 2mL PBS using a tissue homogenizer (frequency: 60Hz; time: 100 s). Different tissues are diluted according to different gradients, coated in an LB plate and inverted in a bacterial incubator at 37 ℃ for 12 hours, colony counting is carried out, and the quantity difference of VNP20009 and mutant strains thereof in mouse tissues is analyzed in a comparative way.

Liver and spleen are the most distributed normal tissues for the bacteria, therefore, in the present invention, we tested the titers of bacteria in liver, spleen and tumor tissues. In the VNP20009 group, the bacterial titer in the liver was 5.766, the bacterial titer in the spleen was 8.477, and the bacterial titer in the tumor was 12.172; in the VNP20009 and Nivolumab combination group, the bacterial titer in liver was 5.784, the bacterial titer in spleen was 8.484, and the bacterial titer in tumor was 12.927 (FIG. 17). It can be seen that the bacterial titers in the liver, spleen and tumor tissues of the VNP20009 and Nivolumab combination group were not significantly changed compared to the VNP20009 treatment group. In the htrA-deficient bacterium group, the bacterial titer in the liver is 5.686, the bacterial titer in the spleen is 8.117, and the bacterial titer in the tumor is 10.712; in the htrA-deficient strain and Nivolumab combination group, the bacterial titer in liver was 5.674, the bacterial titer in spleen was 7.984, and the bacterial titer in tumor was 10.987 (fig. 18). It can be seen that the htrA-deficient bacteria and Nivolumab combination group had unchanged bacterial titers in liver, spleen and tumor tissues compared to the htrA-deficient bacteria treatment group.

The bacterial titer in the liver of the htrA-deficient bacterial group is 0.99 times that of the VNP20009 group, the bacterial titer in the spleen of the htrA-deficient bacterial group is 0.94 times that of the VNP20009 group, and the bacterial titer in the tumor of the htrA-deficient bacterial group is 0.88 times that of the VNP20009 group; the bacterial titer in the liver of the htrA-deficient strain and Nivolumab combination group was 0.98 times that of the VNP20009 group, the bacterial titer in the spleen of the htrA-deficient strain group was 0.94 times that of the VNP20009 group, and the bacterial titer in the tumor of the htrA-deficient strain group was 0.85 times that of the VNP20009 group. It can be seen that the toxicity of htrA-deficient bacteria and their combination with Nivolumab to the liver and spleen is slightly lower than VNP20009.

The invention also utilizes PD-1 nano antibody (Chinese invention application number: 202210011915.7), PDL-1 nano antibody (ZL 201811065587.9) and attenuated salmonella VNP20009 and mutant strains thereof to carry out the experiment, and obtains similar results. The following mainly takes PD-1 nano antibody as an example.

Example 4

Expression and purification of anti-PD 1 nano antibody protein

Carrying out double enzyme digestion on a nucleotide sequence (Chinese invention application number: 202210011915.7) of the anti-PD 1 nano antibody by using EcoR I and Hind III, carrying out agarose gel electrophoresis gel cutting to recover a digestion product, inserting the digestion product into pET32a, and constructing an expression vector of the anti-PD 1 nano antibody; transformed into BL21 (DE 3) and IPTG induced expression. Centrifuging the bacterial liquid to obtain bacterial sediment, resuspending the bacterial sediment by using a lysis buffer solution, crushing the bacterial sediment by using ultrasound or high pressure, and centrifuging and collecting a supernatant. Obtaining the purified anti-PD 1 nano antibody by a Ni-IDA affinity chromatography method. Then, the detection was carried out by SDS-PAGE (FIG. 19).

Example 5

Anti-tumor effect of anti-PD 1 nano antibody protein and attenuated salmonella VNP20009

The experimental animal mice are 5-7 weeks old female BALB/C mice or C57BL/6 mice (Changzhou animal center). The mice were randomly assigned to different groups, 7-8 per group. C57BL/6 mice were inoculated with 100. Mu.l of 2X10 axilla in the right forelimb ⁵ B16F10 cells for tumorigenesis. The tumor of the mouse grows to about 100-150mm ³ Then the treatment is carried out. Wherein the anti-PD 1 nano antibody PD1nb group is obtained by intraperitoneal or intravenous injection of the purified PD1nb antibody (5 mg kg-1), and the medicine is injected for 1 time every other day and is continuously injected for 4 times or more; VNP20009 group is intraperitoneal injection 1X10 ⁶ 100 μ l VNP20009 strain, only inoculated once initially; the combined group of VNP20009 and anti-PD 1 nano antibody PD1nb is a mixed mode of the two groups. The PBS group was intraperitoneally injected with 100. Mu.l sterile PBS alone. Tumor volume was calculated according to the formula V = length × width ² X 0.52. Tumor size measurements were taken at intervals for a particular group of mice and calculated for mapping tumor growth curves. Values are expressed as SD ± SEM (fig. 20).

The fold growth time of tumor was 2.168 days in PBS group, 2.355 days in anti-PD 1 nanobody PD1nb group, 2.790 days in VNP20009 group, and 3.678 days in VNP20009 and anti-PD 1 nanobody PD1nb combined group (fig. 21, table 2). Compared with the PBS group, the tumor fold growth time of the PD1nb group is prolonged by 8.6 percent, the tumor fold growth time of the VNP20009 group is prolonged by 28.7 percent, and the tumor fold growth time of the VNP20009 and anti-PD 1 nano antibody PD1nb combined group is prolonged by 69.6 percent; compared with the PD1nb group, the tumor fold growth time of the VNP20009 group is prolonged by 18.5 percent, and the tumor fold growth time of the VNP20009 and anti-PD 1 nano antibody PD1nb combined group is prolonged by 56.2 percent; compared with the VNP20009 group, the fold growth time of tumors in the VNP20009 and anti-PD 1 nanobody PD1nb combined group was extended by 31.8%. Compared with the PBS control group, the tumor fold growth time of the VNP20009 and anti-PD 1 nano antibody PD1nb combination group is prolonged by 69.6 percent, which is far greater than the sum (37.3 percent) of the tumor fold growth time of the PD1nb group prolonged by 8.6 percent and the tumor fold growth time of the VNP20009 group prolonged by 28.7 percent, and the extent of the tumor growth fold time prolongation caused by the curative effect of the VNP20009 and anti-PD 1 nano antibody combination group is 1.87 times of the theoretical value of the addition of the two groups of curative effects compared with the theoretical value of the addition of the extent of the tumor growth fold time of each group caused by the curative effect after the single treatment of the PD1 nano antibody group and the VNP20009 group, so that the VNP20009 and anti-PD 1 nano antibody PD1nb combination indeed produces the synergistic therapeutic effect. The comparison of the tumor fold growth time and the tumor delay time between different groups of the anti-PD 1 nano antibody protein and the attenuated salmonella VNP20009 of the invention is shown in Table 2:

TABLE 2

1: a PBS group; 2: anti-PD 1 nanobody PD1nb group; 3: an attenuated salmonella VNP20009 control group; 4: anti-PD 1 nano-antibody protein and attenuated salmonella VNP20009 combination group.

The tumor delay time was 2.170 days for the PBS group, 2.317 days for the anti-PD 1 nanobody PD1nb group, 2.850 days for the VNP20009 group, and 4.094 days for the VNP20009 and anti-PD 1 nanobody PD1nb combined group (VNP-NC + PD1 nb) (fig. 22, table 2). Compared with the PBS group, the tumor delay time of the PD1nb group is prolonged by 6.8 percent, the tumor delay time of the VNP20009 group is prolonged by 31.3 percent, and the tumor delay time of the VNP20009 and anti-PD 1 nano antibody PD1nb combined group is prolonged by 88.7 percent; compared with the PD1nb group, the tumor delay time of the VNP20009 group is prolonged by 23.0 percent, and the tumor delay time of the VNP20009 and anti-PD 1 nano antibody PD1nb combined group is prolonged by 76.7 percent; compared with the VNP20009 group, the tumor delay time was prolonged by 43.6% in the group combining VNP20009 and anti-PD 1 nanobody PD1 nb. Compared with the PBS control group, the tumor delay time of the combined group of VNP20009 and anti-PD 1 nano antibody PD1nb is prolonged by 88.7 percent, which is far greater than the sum (38.1 percent) of the tumor delay time of the PD1nb group prolonged by 6.8 percent and the tumor delay time of the VNP20009 group prolonged by 31.3 percent, and the sum of the tumor delay time prolonged amplitude caused by the curative effect of the combined group of VNP20009 and anti-PD 1 nano antibody and the theoretical value of the sum of the tumor delay time prolonged amplitude caused by the curative effect of the anti-PD 1 nano antibody group and the VNP20009 group is 2.33 times of the theoretical value of the sum of the two groups of curative effects, therefore, the combined use of VNP20009 and anti-PD 1 nano antibody PD1nb can indeed produce synergistic curative effect.

The anti-PD 1 nanobody in combination with VNP20009 produced a more significant synergistic therapeutic effect compared to PD-1 antibody and VNP20009.

Example 6

anti-PD 1 nano antibody protein and attenuated salmonella VNP20009 combined anti-tumor metastasis effect

For the lung metastasis model of B16F10, 100. Mu.l of a solution containing 1X10 was injected via the tail vein ⁶ B16F10 cell suspension (C57 BL/6), treatment started on day 6 post-injection, treatment groups were as above, and mice were sacrificed at day 18 post-injection, lung metastases were recorded in each group of mice photographed (fig. 23A), and ImageJ analyzed lung tumor metastasis area (fig. 23B).

The tumor area of lung metastasis in PBS group was 133.2. + -. 14.27mm ² The tumor area of the lung metastasis of the anti-PD 1 nano antibody PD1nb group is 71.52 +/-5.31 mm ² The lung metastasis tumor area of VNP20009 group was 32.97. + -. 3.606mm ² The lung metastasis tumor area of the VNP20009 and + anti-PD 1 nano antibody PD1nb combined group is 18.04 +/-1.528 mm ² . Compared with the PBS group, the lung tumor metastasis of the anti-PD 1 nano antibody PD1nb group is reduced by 46.3%, the lung tumor metastasis of the VNP20009 group is reduced by 75.2%, VNP20009 and anti-PD 1 nano antibody PDThe lung tumor metastasis in the 1nb combination group was reduced by 86.5%. Compared with the anti-PD 1 nano antibody PD1nb group, the lung tumor metastasis of the VNP20009 group is reduced by 53.9 percent, and the lung tumor metastasis of the VNP20009 and anti-PD 1 nano antibody PD1nb combined group is reduced by 74.8 percent; compared with the VNP20009 group, the lung tumor metastasis was reduced by 45.3% in the VNP20009 and anti-PD 1 nanobody PD1nb combination group. The result shows that the combined group of VNP20009 and anti-PD 1 nano antibody PD1nb has a significantly increased tumor metastasis inhibition effect compared with any single treatment mode.

The same treatment as the anti-PD 1 nano antibody is carried out by combining the anti-PD-L1 nano antibody (ZL 201811065587.9) with the recombinant salmonella VNP20009 and a mutant strain thereof. In the melanoma model, the tumor growth doubling time of the PBS group was 2.48 days, the tumor fold growth time of the anti-PD-L1 nanobody was 2.98 days, the tumor fold growth time of the VNP20009 group was 3.25 days, and the tumor fold growth time of the VNP20009 and + anti-PD-L1 nanobody combination group was 3.82 days.

The immune checkpoint antibody inhibitors represented by the PD1, PD-L1 antibodies and the nano antibodies all aim at a plurality of different antigen epitopes, but all show enhanced anti-tumor curative effects when combined with the recombinant salmonella VNP20009, so that the enhanced combined effect is proved to be unrelated to the immune checkpoint antigen epitopes, and therefore, the immune checkpoint antibody inhibitors have universality.

The invention also utilizes CTLA-4 antibody, nano antibody, TIM-3 antibody and LAG3 antibody to carry out the research of the main contents in the research contents, obtains similar results and shows enhanced anti-tumor curative effect when being combined with the recombinant salmonella VNP20009.

The foregoing description shows and describes several preferred embodiments of the present invention, but as aforementioned, it is to be understood that the invention is not limited to the forms disclosed herein, and is not to be construed as excluding other embodiments, but is capable of various other combinations, modifications, and variations within the scope of the inventive concept as expressed above, or as known in the art to which this invention pertains. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. An attenuated salmonella and immune checkpoint inhibitor combination, characterized in that: the attenuated salmonella typhimurium and the immune checkpoint inhibitor combined drug are combined by attenuated salmonella typhimurium and an immune checkpoint inhibitor, and the attenuated salmonella typhimurium is the bacterium which is attenuated salmonella typhimurium VNP20009 and a genetically modified strain thereof; the blocking agent of the immune checkpoint comprises but is not limited to antibodies and nanobodies, and the immune checkpoint inhibitor is CTLA-4, TIM-3, LAG3, nivolumab or Ceriluzumab and PD-1/PDL-1 nanobody.

2. The attenuated salmonella and immune checkpoint inhibitor combination of claim 1, wherein: the htrA of the attenuated salmonella typhimurium VNP20009 is a key gene for producing synergistic effect by combined treatment.

3. The attenuated salmonella and immune checkpoint inhibitor combination of claim 1, wherein: the attenuated salmonella typhimurium VNP20009 reduces PD1 ⁺ CD38 ^High CD8 ⁺ T cells remarkably improve the expression of PDL1 on the surface of tumor cells and remarkably improve the expression level of PDL1 on the surface of mononuclear cells in tumor tissues.

4. The method of use of the combination of attenuated salmonella and an immune checkpoint inhibitor according to claim 1, characterized in that: the administration time interval and times of the salmonella typhimurium and the PD-1/PD-L1 immune checkpoint antibody/nano antibody blocker are specifically as follows: the Nivolumab or the Carriluzumab or the PD-1/PD-L1 nano antibody is administrated at the fourth day after the bacteria administration, and the administration is carried out three times or more at intervals of two days each time; the administration mode of the PD-1/PD-L1 immune checkpoint antibody/nano antibody blocking agent is intraperitoneal or intravenous administration.

5. The method of claim 4, wherein the attenuated Salmonella is administered in combination with an immune checkpoint inhibitor,the method is characterized in that: different doses of attenuated salmonella VNP20009 and PD-1/PD-L1 immune checkpoint antibody/nanobody blockers, VNP20009: 2X10 ⁴ ，5×10 ⁴ ，1×10 ⁵ ，2×10 ⁵ ，5×10 ⁵ ，1×10 ⁶ ，2×10 ⁶ The unit is CFU/only; nivolumab or carprilizumab or PD-1/PD-L1 nano antibody, the administration dose is respectively: 1.0,1.6,3.2,8, said units being mg/kg.

6. The method of claim 5, wherein the combination of the attenuated salmonella and the immune checkpoint inhibitor comprises: the attenuated salmonella is subjected to combined treatment by intravenous injection, intraperitoneal injection, oral administration, subcutaneous administration and intratumoral administration.

7. The method of claim 6, wherein the combination of the attenuated salmonella and the immune checkpoint inhibitor comprises: after the attenuated salmonella and a PD-1/PD-L1 immune checkpoint antibody blocker Nivolumab or a Carrilizumab or a PD-1/PD-L1 nano antibody are jointly applied, the growth of malignant tumors of mice is remarkably inhibited, the survival time is remarkably prolonged, and the synergistic effect is remarkable.

8. Use of the attenuated salmonella of any one of claims 1 to 7 in the preparation of an anti-tumor medicament for the combined treatment of a malignancy with an immune checkpoint inhibitor.

9. Use of the attenuated salmonella of any one of claims 1 to 7 in combination with an immune checkpoint inhibitor for the treatment of a malignant tumor in the preparation of a medicament against tumor metastasis.