CN118403154A - Anticancer pharmaceutical composition and application thereof - Google Patents
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention belongs to the field of biotechnology, and discloses an anticancer pharmaceutical composition and application thereof, wherein the anticancer pharmaceutical composition is formed by combining an HSPC-96 tumor vaccine and a PD-1 antibody; the invention combines the PD-1 antibody and the HSPC-96 tumor vaccine for preparing the anticancer drug, and has the advantage that the HSPC-96 tumor vaccine can enable the PD-1 monoclonal antibody to be more effective for the immunotherapy of cancer.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an anticancer pharmaceutical composition and application thereof.
Background
Studies have shown that tumors have individual specificities, which are manifested not only between tumor types, but also between different individuals. Even for the same type of tumor, there is a significant difference in tumor biology and immunological properties among different individuals. This makes tumor vaccines designed against known tumor antigens ineffective in eliciting immune responses against tumor cells. The tumor autologous gp96 antigen complex (HSPC-96) carries complete tumor individual information, the immune treatment of the complex fully considers the individual difference, and the characteristics of gp96 natural T cell adjuvants are utilized. The gp96 polypeptide complex extracted from the allogeneic tumor tissue is used as an individual therapeutic vaccine for immunotherapy of tumors, not only fully considers the difference among individuals, but also considers the defect of insufficient new antigens of the tumor derived from the individual tissue, can achieve the best therapeutic effect, and is one of the important directions of the current medical and pharmaceutical development.
The heat shock protein glycoprotein 96-kDa (Gp 96) belongs to the family of heat shock proteins, and is mainly located in the Endoplasmic Reticulum (ER) and is a main partner. Gp96 has the ability to bind naturally to tumor-associated antigens (peptides), thereby forming Gp96 peptide complexes that can be taken up by antigen presenting cells (e.g., dendritic cells) and then elicit a natural and adaptive anti-tumor immune response. Thus, after purification of the complex from the patient's tumor, gp96 peptide complex can be developed into a personalized multivalent cancer therapeutic vaccine, commonly referred to as heat shock protein peptide complex 96 (hspc-96). Hspc-96 purified from tumor tissue is the antigen peptide pool most representative of tumor tissue, is a personalized multivalent anti-tumor vaccine, has unique immunological function, is the only natural adjuvant derived from mammalian cells discovered so far, and the immunological mechanism comprises two aspects: firstly, the antigen presentation of T cells is involved, and DC maturation is promoted; secondly, the natural immunity is effectively stimulated, and immune factors IL-6, IL-12, TNF and the like are generated, so that the nonspecific immune response of the organism is enhanced. Heat shock proteins exist in the cytoplasm and endoplasmic reticulum membrane and consist of multiple members such as HSP60, HSP70, HSP90, gp96, and the like. Heat shock proteins play an important role as chaperones in protein folding and transport, and another important function is to bind 5-25mer antigen polypeptides in cells, enter cells through Antigen Presenting Cell (APC) surface CD91 molecules and present the bound epitopes to MHC class I and II molecules to initiate specific T cell immune responses. PD1 (programmed CELL DEATH 1) is a member of the CD28 superfamily, expressed predominantly on the membrane surface of activated T cells, B cells, dendritic cells and mononuclear macrophages, and PD1 inhibits the innate or adaptive immune response of the body by interacting with its ligand molecule PDL1 (programmedcell DEATH LIGAND 1). Clinically, in the intratumoral and hepatoprotective cases of tumor patients, the killing effect of T cells is significantly inhibited due to the up-regulation of the expression of the activated PD1 molecules on the surface of T cells and the simultaneous up-regulation of the expression of PDL1 on the surface of target cell membranes by the interferon released by CTLs. At present, monoclonal antibodies against PD1 molecules have been developed for blocking the PD1/PDL1 interaction, restoring the suppressed T cell response, and thus killing tumor cells with activated T cells and corresponding cytokines, which becomes an important therapeutic tool for cancer immunotherapy.
Glioblastoma multiforme (GBM) is one of the most fatal brain cancers, accounting for 48.6% of all primary brain malignancies, and constitutes a great threat to human health because current therapies have the lowest efficacy. Primary surgical resection, adjuvant radiation therapy and temozolomide chemotherapy are the standard of treatment for GBM; however, patient survival was only slightly increased, with median Overall Survival (OS) reported to be 14.6 months, with 5 year survival below 10%. Thus, new treatments are urgently needed to improve the prognosis of such fatal brain cancer patients. Recently, the advent of Immune Checkpoint Blockade (ICB) using anti-apoptotic protein-1 (anti-PD-1) antibodies as a novel therapeutic strategy for immunotherapy has shown promise in many solid tumors including melanoma and non-malignant tumors.
Several new immunization methods are being developed in the treatment of GBMs and encouraging results have been achieved in preclinical studies and early trials. However, the recent stage III CHECKMATE 143 clinical trial evaluating anti-PD-1 in GBM patients failed to improve mOS compared to current standards of care. The lack of significant clinical efficacy of anti-PD-1 therapies is believed to be due in part to poor antigen presentation/initiation, low quality neoantigen and relatively low mutation leading to intrinsic resistance of the immune system, as well as extrinsic resistance from the immunosuppressive Tumor Microenvironment (TME). Furthermore, regulatory T cells (Tregs), a population of immunosuppressive T cells that are not normally seen in the brain, are significantly increased in the representation of advanced gliomas such as GBM.
The existing anticancer drugs have the following defects: the prior art only can carry out single antibody treatment, and has poor cancer inhibiting effect. In the context of GBM, immunosuppression in TME is likely to occur, we have attempted to evaluate the efficacy of hspc-96 tumor vaccine therapy with anti-PD-1 survival in the mouse GBM model, and to evaluate the immunological effects of this dual immunotherapy. There has been no report on the combined use of PD-1 antibodies and HSPC-96 for the preparation of anticancer drugs. The combined use of PD-1 antibodies and HSPC-96 for the treatment of cancer is of profound clinical importance.
Disclosure of Invention
The invention aims to provide an anticancer pharmaceutical composition and application thereof.
In order to achieve the aim of the invention, the technical scheme adopted is as follows:
In a first aspect, the invention discloses an anticancer pharmaceutical composition consisting of a combination of a hspc-96 tumor vaccine and a PD-1 antibody.
The invention is further provided with: the mass ratio of the PD-1 antibody to the HSPC-96 tumor vaccine is 20% -50%.
The invention is further provided with: the content of the PD-1 antibody in 0.1ml of the anticancer pharmaceutical composition is 250 mg/kg/dose.
The invention is further provided with: the content of the HSPC-96 tumor vaccine in 0.1ml of the anticancer pharmaceutical composition is 2 ug/10 ug/only.
The invention is further provided with: the HSPC-96 tumor vaccine is prepared by fully grinding tumor tissues and sequentially carrying out ConA affinity chromatography and strong anion exchange chromatography.
The invention is further provided with: the tumor tissue is mouse tumor tissue.
In a second aspect, the invention discloses the use of an anti-cancer pharmaceutical composition according to any one of claims 1 to 6 for the treatment of tumors.
The invention is further provided with: the tumor is glioma.
The invention is further provided with: the tumor is esophageal tumor, lung tumor, pancreatic tumor, colon tumor and rectal tumor.
The invention is further provided with: the tumor is liver tumor, kidney tumor and stomach tumor.
In summary, compared with the prior art, the invention has the advantage that the PD-1 monoclonal antibody can be more effective in treating cancers by combining the PD-1 antibody and the HSPC-96 tumor vaccine for preparing anticancer drugs.
Drawings
FIG. 1 is a bioluminescence imaging diagram provided in this embodiment;
Fig. 2 is a life cycle graph provided in this embodiment.
Detailed Description
In order to better understand the technical solutions of the present invention, those skilled in the art will clearly and completely describe the technical solutions of the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by a person skilled in the art without any inventive effort, are intended to be within the scope of the present invention based on the embodiments of the present invention.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
In a first aspect of the present embodiments, an anti-cancer pharmaceutical composition is provided, the anti-cancer pharmaceutical composition consisting of a combination of a hspc-96 tumor vaccine and a PD-1 antibody.
In the specific implementation process, the preparation flow of the HSPC-96 tumor vaccine is as follows:
1. The cells, instruments and medicines are prepared.
A) 10 ten thousand GL261 cells (namely, mouse glioblastoma cells) with the concentration of 5 ul/mouse glioblastoma cells are prepared, digested by cytopancreatin, 180g is measured and centrifuged for 5 minutes, and then resuspended by using sterile PBS (BL 302A, biosharp) with the concentration of 1% matrigel and pH of 7.2-7.4;
b) Sucking the cell suspension by a pipette, pulling out a needle of a 25uL microinjector (80465, hamilton), holding the microinjector horizontally, and injecting the cell suspension from the microinjector by the pipette;
c) Analgesic (PBS diluted 10 times, 100uL each, 2mL each) was taken.
Ic injection procedure.
A) Isoflurane (RWD) gas anesthetized 6 week old C57BL/6N mice;
b) Subcutaneous injection of 100uL meloxicam between the ears;
c) Fixing the mouse into the three-dimensional positioning device, sterilizing, and pulling the legs of the mouse down to ensure complete anesthesia;
d) Cutting the scalp between the two ears of the mouse to find a pre-chimney;
e) Injecting 5uL working cell liquid for 2min at a depth of 3mm and a depth of 2mm at the right side of the pre-chimney, and slowly withdrawing the needle after waiting for 30-60 seconds;
f) Sealing the needle hole with bone wax, dripping brucine onto the wound and skull, and closing the incision with adhesive glue or suture;
3. Observations (body weight measured daily, behavior state of mice observed);
the end time stamp of this process is:
i) The mice lost more than 20% of their original weight;
ii) mice develop hemiplegia symptoms;
iii) The tumors of the mice stand out from their cranium.
Hspc-96 vaccine preparation.
A) Neck-pulling and killing the tumor-forming mice at the end point time, and aseptically taking brain tumor tissues;
b) Grinding and crushing mouse tumor tissue, and subjecting supernatant fluid obtained by centrifugation to ConA affinity chromatography and strong anion exchange chromatography in sequence to obtain HSPC-96 vaccine.
The tumor tissue to be surgically resected includes, but is not limited to, glioma tissue, esophageal cancer tissue, lung cancer tissue, pancreatic cancer tissue, colon cancer tissue, rectal cancer tissue, liver cancer tissue, kidney cancer tissue, stomach cancer tissue, and the like. It should be further noted that the above purification steps include the following specific steps:
i) Regulating the pH value of the tumor tissue lysate to pH7.5; slowly loading the sample to a ConA affinity chromatographic column at a runoff rate of 0.15 cm/min; washing ConA chromatographic column at a radial flow rate of 0.15cm/min, wherein the volume of the ConA chromatographic column is 30 times that of the column, and the solution is 200mM PBS solution; eluting ConA chromatographic column affinity component at a radial flow rate of 0.15cm/min, wherein the elution buffer is a 200mM PBS solution of alpha-D-glucopyranose with a concentration of 100g/L, the elution volume is 3 times of the volume of the chromatographic column, and the elution process comprises eluting 2 volumes of the eluent, stopping for 40 minutes, eluting 1 volume of the eluent and eluting 1 volume of the 200mM PBS solution;
ii) loading the 4 volumes of eluent from step i onto a strong anion chromatographic column at a radial flow rate of 1.2 cm/min; washing the hybrid protein at a runoff speed of 1.2cm/min, wherein the buffer solution comprises PB 2 chromatographic column volumes, 200mM PBS 10 chromatographic column volumes and 300mM PBS 30 chromatographic column volumes in sequence; eluting the target component at a runoff rate of 1.2cm/min, eluting the volumes of 4 chromatographic columns by using 600mM PBS as a buffer solution, and concentrating and changing the obtained eluent to obtain the autologous gp 96-antigen complex vaccine for treatment.
Preferably, the mass ratio of the PD-1 antibody to the HSPC-96 tumor vaccine is 20% -50%.
Preferably, the PD-1 antibody content in 0.1ml of the anticancer pharmaceutical composition is 250 mg/kg/dose.
Preferably, the HSPC-96 tumor vaccine content in 0.1ml of the anticancer pharmaceutical composition is 2 ug/10 ug/min.
Preferably, the HSPC-96 tumor vaccine is prepared by sufficiently grinding tumor tissue, and sequentially performing ConA affinity chromatography and strong anion exchange chromatography.
Preferably, the tumor tissue is murine tumor tissue.
In a second aspect of this embodiment, there is provided the use of an anticancer pharmaceutical composition as described above for the treatment of a tumor.
Preferably, the tumor is a glioma.
Preferably, the tumor is an esophageal tumor, a lung tumor, a pancreatic tumor, a colon tumor, and a rectal tumor.
Preferably, the tumor is a liver tumor, a kidney tumor, or a stomach tumor.
In a third aspect of this embodiment, a method for constructing an animal model of C57BL/6N mouse supratentorial glioma is provided.
1. Preparing cells, instruments and medicines.
A) Cell number 10 ten thousand GL261 cells/5 ul/cell transfected with luciferase gene, cell pancreatin digestion, 180g centrifugation for 5min followed by resuspension at above concentration with sterile pH7.2-7.4PBS (BL 302A, biosharp) containing 1% Matrigel (356234 Corning Matrigel);
b) Sucking the cell suspension by a pipette, pulling out a needle of a 25uL microinjector (80465, hamilton), holding the microinjector horizontally, and injecting the cell suspension from the microinjector by the pipette;
c) Analgesic (meloxicam, diluted 10-fold in PBS, 100uL each, 2mL each) was taken.
Ic injection procedure.
A) Isoflurane (RWD) gas anesthetized 6 week old C57BL/6N mice;
b) Subcutaneous injection of 100uL meloxicam between the ears;
c) Fixing the mouse into the three-dimensional positioning device, sterilizing, and pulling the legs of the mouse down to ensure that the mouse is completely anesthetized;
d) Cutting the scalp between the two ears to find the anterior chimney;
e) Injecting 5uL working cell liquid for 2min at a depth of 3mm and a depth of 2mm at the right side of the pre-chimney, and slowly withdrawing the needle after waiting for 30-60 s;
6) The pinholes were sealed with bone wax, brucine was added dropwise to the wound and skull, and the incision was closed with adhesive glue or sutures.
3. Observations (body weight measured daily, behavior state of mice observed).
The end time stamp of this process is:
i) The mice lost more than 20% of their original weight;
ii) mice develop hemiplegia symptoms;
iii) The tumors of the mice stand out from their cranium.
In a fourth aspect of this example, there is provided an assay for the combined use of a hspc-96 tumor vaccine and a PD-1 antibody for the preparation of an anti-cancer drug, the assay being performed in time (days), the specific steps of:
day-1, cyclophosphamide CTX (HY-17420, MCE) in PBS, 2 mg/min, subcutaneously;
constructing a Day0 and C57BL/6N mouse supratentorial glioma animal model;
day1, four packets: 6 HSPC-96 vaccines combined with PD-1 mab (BE 0146, inVivoMab) in group, 6 PD-1 mab in group, 5 HSPC-96 vaccines in group, 5 PBS in control group; HSPC-96 vaccine in combination with PD-1 mab group and HSPC-96 vaccine group: injecting HSPC-96 vaccine into abdominal cavity, 2ug/100 ul/patient; PD-1 mab group and PBS control group: injecting 100ul of PBS into the abdominal cavity;
day4, hspc-96 vaccine in combination with PD-1 mab group and hspc-96 vaccine group: injecting HSPC-96 vaccine into abdominal cavity, 2ug/100 ul/patient; PD-1 mab group and PBS control group: injecting 100ul of PBS into the abdominal cavity;
day7, hspc-96 vaccine in combination with PD-1 mab group: injecting HSPC-96 vaccine into abdominal cavity, 2ug/100 ul/PD-1 250 mg/kg/PD; hspc-96 vaccine group: injecting HSPC-96 vaccine into abdominal cavity, 2ug/100 ul/patient; PD-1 mab group: PD-1 250 mg/kg/person; PBS control group: injecting 100ul of PBS into the abdominal cavity; mice were intraperitoneally injected with 150ng/g D-fluorescein (Perkinelmer, 122796) and imaged with a bioluminescence imaging system (In Vivo IMAGING SYSTEM, IVIS) 10 minutes after injection. Analyzing the IVIS data using in vivo imaging software (CALIPER LIFE SCIENCES);
Day10, hspc-96 vaccine in combination with PD-1 mab group: injecting HSPC-96 vaccine into abdominal cavity, 2ug/100 ul/PD-1 250 mg/kg/PD; hspc-96 vaccine group: injecting HSPC-96 vaccine into abdominal cavity, 2ug/100 ul/patient; PD-1 mab group: PD-1 250 mg/kg/person; PBS control group: injecting 100ul of PBS into the abdominal cavity;
Day12, mice were intraperitoneally injected with 150ng/g D-fluorescein (PerkinElmer, 122796), and imaged with a bioluminescence imaging system (In Vivo IMAGING SYSTEM, IVIS) 10 minutes after injection; analyzing the IVIS data using in vivo imaging software (CALIPER LIFE SCIENCES);
Day13, hspc-96 vaccine in combination with PD-1 mab group: injecting HSPC-96 vaccine into abdominal cavity, 2ug/100 ul/PD-1 250 mg/kg/PD; hspc-96 vaccine group: injecting HSPC-96 vaccine into abdominal cavity, 2ug/100 ul/patient; PD-1 mab group: PD-1 250 mg/kg/person; PBS control group: injecting 100ul of PBS into the abdominal cavity;
Day14, mice were intraperitoneally injected with 150ng/g D-fluorescein (PerkinElmer, 122796), and imaged with a bioluminescence imaging system (In Vivo IMAGING SYSTEM, IVIS) 10 minutes after injection; analyzing the IVIS data using in vivo imaging software (CALIPER LIFE SCIENCES);
Day35, mice were intraperitoneally injected with 150ng/g D-fluorescein (PerkinElmer, 122796), and imaged with a bioluminescence imaging system (In Vivo IMAGING SYSTEM, IVIS) 10 minutes after injection; analyzing the IVIS data using in vivo imaging software (CALIPER LIFE SCIENCES);
The test results are shown in fig. 1 and 2. From the figure, the mice of the HSPC-96 vaccine combined PD-1 monoclonal antibody group have a significantly longer survival period than other groups.
In this test, from Day1, weight measurement was performed every three days, and the condition of the mice was observed.
In conclusion, the invention has the advantage that the PD-1 monoclonal antibody can be more effective in treating cancers by combining the PD-1 antibody and the HSPC-96 tumor vaccine for preparing the anticancer drug.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (10)
1. An anticancer pharmaceutical composition, characterized in that: the anticancer pharmaceutical composition is formed by combining an HSPC-96 tumor vaccine and a PD-1 antibody.
2. The anticancer pharmaceutical composition of claim 1, wherein the mass ratio of the PD-1 antibody to the hspc-96 tumor vaccine is 20% -50%.
3. The anticancer pharmaceutical composition of claim 1, wherein the PD-1 antibody is present in an amount of 250 mg/kg/serving as a basis for 0.1ml of the anticancer pharmaceutical composition.
4. The anticancer pharmaceutical composition according to claim 3, wherein the hspc-96 tumor vaccine is present in an amount of 2 ug/10 ug/v in 0.1ml of the anticancer pharmaceutical composition.
5. The anticancer pharmaceutical composition of claim 1, wherein the hspc-96 tumor vaccine is prepared by subjecting tumor tissue to ConA affinity chromatography and strong anion exchange chromatography in that order.
6. The anti-cancer pharmaceutical composition of claim 5, wherein the tumor tissue is murine tumor tissue.
7. Use of an anticancer pharmaceutical composition as defined in any one of claims 1 to 6 for the treatment of tumors.
8. The use of claim 7, wherein the tumor is a glioma.
9. The use of claim 7, wherein the tumor is a liver cancer, an esophageal tumor, a lung tumor, a pancreatic tumor, a colon tumor, or a rectal tumor.
10. The use of claim 9, wherein the neoplasm is a liver tumor, a kidney tumor, or a stomach tumor.
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