CN116687970A - Application of autologous lymphocyte reinfusion preparation and PD-1/PD-L1 inhibitor in preparation of tumor treatment products - Google Patents
Application of autologous lymphocyte reinfusion preparation and PD-1/PD-L1 inhibitor in preparation of tumor treatment products Download PDFInfo
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- CN116687970A CN116687970A CN202310709134.XA CN202310709134A CN116687970A CN 116687970 A CN116687970 A CN 116687970A CN 202310709134 A CN202310709134 A CN 202310709134A CN 116687970 A CN116687970 A CN 116687970A
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- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
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Abstract
The invention discloses an application of autologous lymphocyte reinfusion preparation and PD-1/PD-L1 inhibitor in preparing a tumor treatment product. The invention provides autologous lymphocyte reinfusion preparation and application of PD-1/PD-L1 inhibitor in any of the following: 1) Preparing a tumor treatment product; 2) Preparing a product for inhibiting the growth of tumor cells; 3) Preparing a product for prolonging the life cycle of a tumor patient; 4) Preparing a product for promoting immune reconstitution of an organism; the experiments prove that the autologous lymphocyte reinfusion preparation and the PD-1/PD-L1 inhibitor have remarkable anti-tumor effects on a human liver cancer QGY7703 subcutaneous transplantation tumor model and a human liver cancer HepG2 in-situ transplantation tumor model, so that the survival time of mice is effectively prolonged, the autologous lymphocyte reinfusion preparation and the PD-1/PD-L1 inhibitor can be used for jointly treating liver cancer, and the PD-1/PD-L1 inhibitor assists the autologous lymphocyte reinfusion preparation to promote immune reconstruction of T lymphocytes activated by organisms.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to the field of immune cell treatment and antibody treatment, and in particular relates to application of an autologous lymphocyte reinfusion preparation and a PD-1/PD-L1 inhibitor in preparation of a tumor treatment product.
Background
Along with the annual rise of the incidence of tumors, the incidence and mortality of malignant tumors in China are the first worldwide. Discovery of mid-90 th century immune checkpoint therapy has opened a new era of tumor immunotherapy. Immune checkpoint inhibitors (immune checkpoint inhibitions, ICIs), especially inhibitors targeting the programmed death receptor-1 (programmed cell death 1, PD-1)/programmed death receptor ligand-1 (programmed cell death ligand1, PD-L1), treat tumors, and are of great importance in tumor immunotherapy. Compared with the traditional operation and chemoradiotherapy method, the immunotherapy has three advantages over the traditional therapy: (1) Broader spectrum of anti-cancer effects without distinguishing tumor origin (O drugs have been globally batched for treatment of 10 cancer species, K drugs batched for 15 cancer species); (2) It attacks tumors by activating the autoimmune system, with adverse effects less than chemotherapeutic drugs; (3) If the drug is effective on the patient, it may lead to long-term survival of the late-stage patient, even clinical cure, which is the greatest difference in immunotherapy from other therapies. At present, our country has accelerated the pace of the arrival of immunotherapy at the market, so far, 8 types of immune checkpoint inhibitors have been marketed in China, including 4 types of domestic PD-1 (terep Li Shan antibody, xindi Li Shan antibody, carilizumab, tirelizumab), 2 types of imported PD-1 (palbociclizumab Pembrolizumab (Keytruda), na Wu Liyou monoclonal antibody Nivolumab (Opdivo)) and 2 types of imported PD-L1 (atilizumab Atezolizumab (Tecentriq), duvalli You Shan anti Durvalumab (Imfinzi). ICI treatment has shown positive clinical results in various types of tumors, however, except for patients with higher response rate (40% -70%) to single PD-1 blockade in certain diseases (e.g. melanoma, merkel cell, hodgkin lymphoma, MSI-H tumor), the remission rate of most other cancer indications is only 10% -25%, furthermore, even in patients who initially respond to ICIs, diseases eventually may progress, therefore, other immunotherapy treatment strategies including adoptive cell therapy, including the recruitment of tumor cells by the transient cell type of the tumor, the tumor is a new tumor-infiltration strategy, and the tumor is restored to a tumor infiltration condition by the recruitment of new cell type of tumor cell type, and the tumor cell infiltration strategy is further developed.
Adoptive cellular immunotherapy (Adoptive cell transfer, ACT) is a highly personalized immunotherapy that achieves antitumor effects by activating and expanding tumor-specific or non-specific killer lymphocytes in vitro. ACT treatment is widely applied to various types of tumors. In the ACT process, a large number of antitumor lymphocytes are activated in vitro and proliferate rapidly, and the lymphocytes are infused into a patient body through adoptive feedback, and the normal antitumor immune response of the body is restored by activating body cells and humoral immune response, restarting and maintaining tumor-immune circulation, so as to achieve the purposes of controlling and eliminating tumors.
Disclosure of Invention
The invention aims to provide an application of autologous lymphocyte reinfusion preparation and PD-1/PD-L1 inhibitor in preparing a tumor treatment product.
In a first aspect, the invention provides an autologous lymphocyte reinfusion preparation and the use of a PD-1/PD-L1 inhibitor in any one of the following:
1) Preparing a tumor treatment product;
2) Preparing a product for inhibiting the growth of tumor cells;
3) Preparing a product for prolonging the life cycle of a tumor patient;
4) Preparing a product for promoting immune reconstitution of an organism;
the autologous lymphocyte reinfusion preparation comprises autologous lymphocytes, and specifically comprises autologous lymphocyte suspension liquid, which consists of autologous lymphocytes and buffer solution; in an embodiment of the invention the buffer is PBS.
The autologous lymphocytes are obtained by culturing peripheral blood mononuclear cells derived from a subject in an autologous lymphocyte culture solution;
in an embodiment of the invention, the autologous lymphocytes are peripheral blood derived from the subjectThe mononuclear cells are cultured in autologous lymphocyte culture solution until 15 days to obtain 2X10 concentration 7 Autologous lymphocyte suspension/mL.
In an embodiment of the invention, the autologous lymphocytes are cd3+cd56-, cd3+cd4+, cd3+cd8+, cd3+cd56+, cd3+cd45ro+, and the specific lymphocyte subpopulations are counted in absolute terms: cd3+cd56-:1.1X10 9 ，CD3+CD4+：0.75X10 9 ，CD3+CD8+：0.33X10 9 ，CD3+CD56+：0.1X10 9 ，CD3+CD45RO+：0.70X10 9 。
The subject is a human tumor patient;
the autologous lymphocyte culture solution comprises human gamma interferon IFN-gamma, human autologous plasma, anti-human CD3 monoclonal antibodies, IL-2, IL-7, IL-15, PHA-P and cell basal culture medium.
The autologous lymphocyte culture fluid described above is as follows: adding human gamma interferon IFN-gamma into a LONZA X-VIVO 15X culture medium to obtain a liquid which is a LONZA X-VIVO 15X basic culture medium containing IFN-gamma 1000U/ml, and then adding human autologous plasma, an anti-human CD3 monoclonal antibody, IL-2, IL-7, IL-15 and PHA-P into the liquid to obtain an autologous lymphocyte culture solution, wherein the concentration of the IFN-gamma in the autologous lymphocyte culture solution is 1000U/ml; the volume percentage of the human autologous plasma in the autologous lymphocyte culture solution is 10%; the concentration of the anti-human CD3 monoclonal antibody in the autologous lymphocyte culture solution is 100ng/ml; PHA-P concentration in autologous lymphocyte culture medium is 500ng/ml; the concentration of IL-2 in autologous lymphocyte culture solution is 500U/ml; IL-7 concentration in autologous lymphocyte culture medium is 25ng/ml; IL-15 was present in autologous lymphocyte culture medium at a concentration of 50ng/ml.
In the above, the tumor is of human or animal origin; the animal is a mammal, in particular a mouse in embodiments of the invention.
In such applications, autologous lymphocytes, whether they are human or animal, are derived from the subject.
In a second aspect, the invention provides the use of a PD-1/PD-L1 inhibitor in the preparation of an immunoreconstitution product for assisting an autologous lymphocyte reinfusion preparation in promoting activated T lymphocytes in the body.
In a third aspect, the invention provides a kit comprising the autologous lymphocyte reinfusion agent of the first aspect and the PD-1/PD-L1 inhibitor.
The kit has any one of the following functions:
1) Treating tumors;
2) Inhibiting tumor cell growth;
3) Prolonging the survival time of tumor patients;
4) Promoting immune reconstruction of organism.
Above, the autologous lymphocyte reinfusion preparation and the PD-1/PD-L1 inhibitor are packaged separately.
The above kit further comprises a readable carrier describing the following method of use: administering the autologous lymphocyte reinfusion agent and the PD-1/PD-L1 inhibitor of the first or second aspect at intervals; and the first administration is an autologous lymphocyte reinfusion preparation as described in the first or second aspects.
In the above, the dosage of the autologous lymphocyte reinfusion preparation is as follows: the dosage for human is not less than 2X10 9 Individual cells for mice of not less than 2X10 7 The method comprises the steps of carrying out a first treatment on the surface of the The PD-1/PD-L1 inhibitor is used according to the instruction or the known dosage, and in the embodiment of the invention, the PD-1/PD-L1 inhibitor is used for mice at a dosage of 5mg/kg, and the required injection volume is calculated according to the weight of the mice after the use and is configured into a solution with a concentration of 5 mg/mL.
The PD-1/PD-L1 inhibitor is applied after the autologous lymphocyte reinfusion preparation and is used for assisting the autologous lymphocyte reinfusion preparation to be applied to lung cancer, liver cancer, colorectal cancer, gastric cancer, head and neck squamous carcinoma, urothelial carcinoma, melanoma, hodgkin lymphoma or renal carcinoma.
The above combination of an autologous lymphocyte reinfusion preparation and a PD-1/PD-L1 inhibitor may be administered several times at different time intervals according to a dosing regimen, i.e. within a specified period of time.
The dosing regimen of the compositions provided herein depends on the pharmacokinetic profile of the autologous lymphocyte reinfusion agent and the PD-1/PD-L1 inhibitor in the composition, i.e., the absorption, distribution, biochemical conversion (or metabolism) and excretion processes of the drug in the body, particularly the time-dependent regulation of blood concentration, and furthermore, the dosing strategy needs to be adjusted over time according to the individual needs of the patient.
In a fourth aspect, the invention provides the use of a kit according to the third aspect in any of the following:
1) Preparing a tumor treatment product;
2) Preparing a product for inhibiting the growth of tumor cells;
3) Preparing a product for prolonging the life cycle of a tumor patient;
4) And preparing the product for promoting the immune reconstruction of the organism.
Above, the PD-1/PD-L1 inhibitors include, but are not limited to, palbociclib mab Pembrolizumab (Keytruda), nal Wu Liyou mab Nivolumab (Opdivo), atilizumab Atezolizumab (Tecentriq) or dulcis You Shan-mab Durvalumab (Imfinzi).
In the above, the tumor is a metastatic tumor or a primary tumor, which is lung cancer, liver cancer, colorectal cancer, gastric cancer, head and neck squamous carcinoma, urothelial cancer, melanoma, hodgkin's lymphoma or renal cancer.
Further, the liver cancer is liver metastasis or primary liver cancer, specifically, liver cancer primary focus or liver cancer without metastasis, and in the embodiment of the invention, the in-situ cancer model of subcutaneous tumor of liver cancer xenograft or liver cancer xenograft is taken as an example.
The experiments prove that the autologous lymphocyte reinfusion preparation and the PD-1/PD-L1 inhibitor have remarkable anti-tumor effects on a human liver cancer QGY7703 subcutaneous transplantation tumor model and a human liver cancer HepG2 in-situ transplantation tumor model, so that the survival time of mice is effectively prolonged, the autologous lymphocyte reinfusion preparation and the PD-1/PD-L1 inhibitor can be used for jointly treating liver cancer, and the PD-1/PD-L1 inhibitor assists the autologous lymphocyte reinfusion preparation to promote immune reconstruction of T lymphocytes activated by organisms.
Drawings
FIG. 1 is a graph showing the results of tumor growth curves of various groups of the subcutaneous tumor QGY7703 model for treating the subcutaneous tumor QGY7703 model.
FIG. 2 shows the body weight results of mice in each treatment group in the subcutaneous tumor QGY7703 model.
FIG. 3 is a graph showing survival time of mice in each group in an in situ model of HepG2 human liver cancer xenograft.
FIG. 4 shows the body weight results of mice in each treatment group in the HepG2 human liver cancer xenograft in situ model.
Fig. 5 shows absolute counts of peripheral blood T lymphocyte subpopulations, P <0.05, P <0.01, P <0.001.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The human autologous plasma and human peripheral blood mononuclear cells (Peripheralbloo dmononuclear cell, PBMC) in the following examples were from tumor hospital tumor patients in the university of chinese medical science, beijing covarian academy of medical science, who have signed informed consent, and the study was approved by the ethical committee of tumor hospital in the university of chinese medical science, beijing covarian academy of medical science.
The LONZA X-VIVO 15X medium (product number: 04-418Q) in the following examples was a product of LONZA company, and the LONZA X-VIVO 15X medium contained human albumin, recombinant human insulin and pasteurized transferrin; the anti-human CD3 monoclonal antibody (clone number OKT 3) is a product of TAKARA company, and the product catalog number is T210; interleukin 2 (IL-2) is a product of Beijing Shuanglu pharmaceutical industry Co., ltd, and the product catalog number is national medicine standard S20020016; interleukin 15 (IL-15) is a product of Shanghai's Biotechnology Co., ltd, catalog number of GMP-101-15; interleukin 7 (IL-7) is a product of Beijing Bai Oy Libo Co., ltd, and the product catalog number is JN0349-KFR; IL-1 alpha is a product of Beijing Toari sea source biotechnology limited company, and the product catalog number is GMP-TL109; phytohemagglutinin P (PHA-P) is a sigma company product with a catalog number of L8754; CD3-PerCP-Cy5.5 is PeproTech (BioGems) product, catalog number: 05121-70-100; CD4-FITC is BD Pharmingen product, catalog number: 561005; CD8-APC was BD Pharmingen product, catalog number: 561952; CD56-PE is a BD Biosciences product, catalog number: 340685; CD45RO-APC is BD Pharmingen product, catalog number: 560899; human gamma interferon IFN-gamma is a product of Shanghai Prime biotechnology Co., ltd., product catalog number: GMP-106-06. Palbociclib was purchased from MSD Ireland (Carlow), catalog number S20180019. The PBS concentration in the examples described below was 0.01M and the pH was 7.4.
Statistical analysis in the following examples: the tumor volumes and tumor weights were statistically analyzed between groups using One-Way ANOVA test using IBM SPSS Statistics 22.0.0 statistical software, with p <0.05 considered significant differences.
For a better understanding of the present invention, the application of the autologous lymphocyte reinfusion preparation provided by the present invention, the autologous lymphocyte reinfusion preparation and the combined preparation with the PD-1/PD-L1 inhibitor are described by the following specific examples.
Example 1 preparation of autologous lymphocytes
1. Autologous lymphocyte culture solution
Experimental group-autologous lymphocyte culture fluid: adding human gamma interferon IFN-gamma into LONZA X-VIVO 15X culture medium to obtain LONZA X-VIVO 15X basic culture medium containing 1000U/ml IFN-gamma, and then adding human autologous plasma, anti-human CD3 monoclonal antibody, IL-2, IL-7, IL-15 and PHA-P into the basic culture medium to obtain autologous lymphocyte culture solution; wherein, the concentration of IFN-gamma in autologous lymphocyte culture solution is 1000U/ml; the volume percentage of the human autologous plasma in the autologous lymphocyte culture solution is 10%; the concentration of the anti-human CD3 monoclonal antibody in the autologous lymphocyte culture solution is 100ng/ml; PHA-P concentration in autologous lymphocyte culture medium is 500ng/ml; the concentration of IL-2 in autologous lymphocyte culture solution is 500U/ml; IL-7 concentration in autologous lymphocyte culture medium is 25ng/ml; IL-15 was present in autologous lymphocyte culture medium at a concentration of 50ng/ml.
Control group-autologous lymphocyte culture broth: adding human gamma interferon IFN-gamma into a LONZA X-VIVO 15X culture medium to obtain a LONZA X-VIVO 15X basic culture medium containing 1000U/ml IFN-gamma, and then adding human autologous plasma, an anti-human CD3 monoclonal antibody, IL-2 and IL-1 alpha into the basic culture medium to obtain autologous lymphocyte culture solution of a control group, wherein the concentration of IFN-gamma in the autologous lymphocyte culture solution is 1000U/ml; the volume percentage of the human autologous plasma in the autologous lymphocyte culture solution is 10%; the concentration of the anti-human CD3 monoclonal antibody in the autologous lymphocyte culture solution is 100ng/ml; the concentration of IL-2 in autologous lymphocyte culture solution is 500U/ml; IL-1 alpha was present in autologous lymphocyte culture medium at a concentration of 1ng/ml.
The human autologous plasma is prepared according to the following method: transferring 50mL of peripheral blood of a subject (tumor patient) into 2 centrifuge tubes with the volume of 50mL according to 25 mL/tube, centrifuging at 2000rpm for 10 minutes, and transferring the supernatant into a new 50mL centrifuge tube to obtain autologous plasma; placing the autologous plasma of the collected subject in a 56 ℃ water bath for 30 minutes, cooling in a refrigerator for 10 minutes at 2-8 ℃, centrifuging at 2000rpm for 10 minutes at low temperature, discarding precipitated proteins to obtain the inactivated autologous plasma, and storing at 2-8 ℃ for later use.
2. Induction culture of autologous lymphocytes
The peripheral blood collection amount was calculated as follows: when the absolute value of donor lymphocyte is 0.8-1.6X10 9 Per liter, blood collection amount (mL) =80 ml×10 9 Absolute value of (/ L)/lymphocyte; when the absolute value of donor lymphocytes is > 1.6X10 9 The blood collection amount (mL) =50ml, and the amount of blood may be adjusted appropriately according to the procedure.
The invention takes 50mL of peripheral blood as an example, the experiment is repeated three times, and each experimental method is as follows:
(1) Peripheral blood 50mL was withdrawn from the tumor patient in an empty stomach state, anticoagulated with heparin tube, separated with lymphocyte separation liquid (ficoll) (purchased from beijing solebao technologies, ltd., product catalog No. P4350) and human Peripheral Blood Mononuclear Cells (PBMCs) were collected and cultured in T75 culture flasks.
According to 1X10 6 The human peripheral blood mononuclear cells were inoculated into the autologous lymphocyte culture medium of each group prepared above (on day 0 of culture) at 37deg.C in 5% CO2Culturing under the culture medium (corresponding autologous lymphocyte culture solution prepared in the first step) at intervals of day (day 2), and standing for 72 hr (day 5) to obtain culture solution (I);
(2) And (3) adding the prepared corresponding autologous lymphocyte culture solution to 250mL into the culture solution (I), standing and culturing in an incubator for 96 hours (9 days of culture), and then sucking 20mL of liquid for microorganism culture detection. After the detection of the microbial culture is negative, continuing to culture to prepare a culture solution (II);
table 1 shows items of microorganism detection
(3) And (3) adding the prepared corresponding autologous lymphocyte culture solution to 900mL of the culture solution (II), standing for 72h (12 days of culture), and then sucking 20mL of the culture solution for microorganism culture detection. After the detection of microorganisms is negative, continuing to culture to prepare a culture solution (III);
(4) Adding the prepared corresponding autologous lymphocyte culture solution to 1800mL into the culture solution (III), standing for 72h (15 days), and then sucking 10-20 mL of liquid for microorganism culture detection. And after the detection of microorganisms is negative, continuing to culture to obtain an experimental group autologous lymphocyte solution and a control group autologous lymphocyte solution.
Autologous lymphocyte concentration in autologous lymphocyte solution of experimental group is 2X10 7 The solute is autologous lymphocytes, and the solvent is experimental group-autologous lymphocyte culture solution;
the concentration of autologous lymphocytes in the control autologous lymphocyte solution is 2X10 7 and/mL, wherein the solute is autologous lymphocytes, and the solvent is control group-autologous lymphocyte culture solution.
3. Verification
Respectively taking the experimental groups obtained by the two stepsSomatic lymphocyte solution and control group autologous lymphocyte solution, 1000g centrifugate for 10 min, collect precipitate, wash with PBS, make cell suspension with PBS, concentration is 1×10 6 The following antibodies were added to each cell per 100 uL: after incubation for 30min at a dark place, CD3-PerCP-Cy5.5, CD4-FITC, CD8-APC, CD56-PE, CD45RO-APC, centrifugation at 1500rpm for 5 min, PBS washing for 2 times, finally adding 100 μl of 2% paraformaldehyde, shaking and mixing, and standing for 20 min. Flow cytometry detects the immunophenotype of the surface of immune cells, i.e., the content of T lymphocyte subpopulations.
The data were processed using SPSS 20.0 statistical software, the experimental results were expressed as mean.+ -. Standard deviation, and the t-test was used for differential significance typing.
Experimental results show that by absolute counting of peripheral blood T lymphocyte subpopulations, compared with control autologous lymphocytes, the experimental autologous lymphocytes CD3+CD56-, CD3+CD4+, CD3+CD8+, CD3+CD56+, CD3+CD45RO+ are significantly increased, wherein CD3+CD56-is increased by 30.4%
P < 0.001), cd3+cd4+ increased by 31.9% (< P < 0.001), cd3+cd8+ increased by 22.8% (< P < 0.01), cd3+cd56+ increased by 28.9% (< P < 0.05), cd3+cd45ro+ increased by 31.6% (< P < 0.01) (fig. 5).
Therefore, the purity of the monocyte-induced autologous lymphocytes is remarkably improved by culturing the autologous lymphocyte culture solution of an experimental group. The following experiments employed autologous lymphocytes from the experimental group.
Example 2 autologous lymphocyte reinfusion preparation and use of the combination with PD-1/PD-L1 inhibitor in the treatment of liver cancer
In the study, autologous lymphocytes from human prepared in example 1 are cultured in an autologous lymphocyte culture solution of an experimental group to prepare an autologous lymphocyte reinfusion preparation, and the autologous lymphocyte reinfusion preparation is reinfusion into a mouse tumor model to treat liver cancer in combination with a PD-1/PD-L1 inhibitor (the experimental example is palbociclizumab).
Since the human intended autologous lymphocyte reinfusion dose is 2X10 9 Cell/human, approximately 1-fold the number of human PBMC, and mice with approximately 10 PBMC per mouse 6 Each mouse thus deduced corresponds toThe clinical feedback dose of the human is about 10 6 Only, i.e. 5X10 7 Cells/kg (mouse body weight calculated as 20 g). The study used 20 times the converted dose as the dosing: that is, the autologous lymphocyte dose returned from the mice is 2×10 7 /only.
The autologous lymphocyte reinfusion preparation of the experimental group is prepared by centrifuging 1000g of autologous lymphocyte solution of the experimental group prepared in example 1 for 10 minutes, collecting precipitate, washing with PBS, and preparing into 5X10 solution with PBS 7 The experimental group autologous lymphocyte suspension liquid of/mL is recorded as an experimental group autologous lymphocyte reinfusion preparation;
the control autologous lymphocyte reinfusion preparation is prepared by centrifuging 1000g of the control autologous lymphocyte solution prepared in example 1 for 10 min, collecting precipitate, washing with PBS, and preparing into 5X10 solution with PBS 7 The control autologous lymphocyte suspension/mL was used as a control autologous lymphocyte reinfusion preparation.
1. Autologous lymphocyte reinfusion preparation and PD-1/PD-L1 inhibitor are combined to inhibit QGY7703 liver cancer cells
1. Subcutaneous tumor QGY7703 model for xenograft of human liver cancer and administration
QGY-7703 human hepatoma tumor cell line (available from Australian Rui Siemens Biotechnology (Shanghai) Inc. under the product catalog number ORC 0402) in logarithmic growth phase was used for in vivo tumor inoculation as follows:
washing tumor cells QGY-7703 with PBS, and resuspending to a concentration of 2×10 7 Per ml of tumor cell suspension, 100 μl/mouse (when the inoculation is marked as D0) was inoculated subcutaneously in the right flank of male NCG mice. After the tumor grows until the average tumor volume reaches 20-30mm 3 And obtaining a subcutaneous tumor QGY7703 model, namely a mouse liver cancer xenograft subcutaneous tumor model.
The subcutaneous tumor QGY7703 model was dosed according to tumor volume (group-wise, PG-D0), for a total of 4 groups of 6 animals, with the specific dosing schedule shown in Table 2 below.
Table 2 shows the dosage regimen
Note that: the control group in the table above is group1, vehicle is PBS; purchased from marsupuno life technologies, inc., catalog number: PB180327; groups 2-6 are treatment groups; * Dosing volume: the autologous lymphocyte reinfusion preparation is injected at a concentration of 400 mu L/injection, and the cells are ensured not to precipitate or agglomerate before injection, and each group of autologous lymphocyte suspensions is slowly injected during injection; v. the following: (tail) intravenous injection; i.p.: intraperitoneal injection. Qw×5 represents once weekly administration for 5 times, tiw× 4wks,from the 2nd week represents twice weekly administration for 4 weeks, and administration starts from week 2.
The palbociclib is dissolved in normal saline before injection to obtain a solution with the concentration of 5mg/mL, and the required injection volume is calculated according to the weight of the mice to achieve the therapeutic dose of 5mg/kg shown in Table 2.
2. Detection of
1) Measurement and experimental index of mouse weight
The tumor volume was measured 2 times per week using a vernier caliper, the body weight of the mice was weighed with an electronic balance, and the long and short diameters of the tumor were measured, and the volume RTV calculation formula was: volume rtv=0.5×long diameter×short diameter 2 。
The treatment/control tumor volume ratio T/C values were calculated from tumor volumes, treatment/control tumor volume ratio T/C (%) = treatment RTV mean/control RTV mean x 100%.
Tumor growth inhibition rate TGI TV (％)＝(1－T/C)×100％。
Wherein the average RTV of tumor volumes of treatment group (T) or control group (C) is the ratio of tumor volume after administration to that before administration. The tumor growth inhibition rate is more than or equal to 60 percent, and p <0.05 is effective after statistical treatment.
At the end of the experiment, animals are euthanized, peeled tumors are weighed and placed in order for photographing, and the tumor weight inhibition rate TGI is calculated TW (％)，TGI TW (%) = (1-mean tumor weight in treated group/mean tumor weight in control group) ×100%. Tumor regrowth inhibition rate is more than or equal to 60 percent, and p is treated by statistics<0.05 is effective.
During the course of the experiment, animals should be euthanized if any of the following conditions occur:
1) The animals are abnormal or paralyzed and can not ingest drinking water by themselves;
2) The weight of the animals was reduced by more than 20% of the weight at the beginning of the drug treatment;
3) Veterinary believes that animal welfare and/or other abnormalities that are normally experienced are severely affected
The results of the tumor growth curves of the subcutaneous tumor QGY7703 model treated by each Group in Table 2 are shown in FIG. 1, groups 1-6 correspond to groups 1-6 in Table 2, respectively; as can be seen, the tumor volume of the experimental group cell product + palbociclib combined group (group 6) was significantly lower than that of the Vehicle control group (p < 0.001); the tumor volume of the experimental group cell product + palbociclizumab combination group (group 6) was significantly lower than its corresponding experimental group cell product single drug treatment group (p < 0.05).
The tumor growth inhibition ratio (TGI) of the single drug group (group 2), the single drug group (group 3), the Pabo Li Zhushan antibody group (group 4), the combination of the cell product of the control group and the Pabolizumab (group 5) and the combination of the cell product of the test group and the Pabolizumab (group 6) is calculated from the results of the figure 1 to obtain the day of the experiment (PG-D35) TV % by weight) were 20%, 23%, 31%, 29% and 49%, respectively.
At the end of the experiment (PG-D35), tumor weight inhibition ratio (TGI) of control cell product single drug group (group 2), experimental cell product single drug group (group 3), pabo Li Zhushan anti-group (group 4), control cell product+Pabolizumab combination group (group 5), experimental cell product+Pabolizumab combination group (group 6) TW The% of the total tumor weight was 21%, 23%, 39%, 40% and 53%, respectively, and the results of the comparison were consistent with the results of the comparison by tumor volume, and the tumor weight of the experimental group cell product + palbociclib combined group (group 6) was significantly lower than that of the Vehicle control group (group 1) (p)<0.001 A) is provided; the tumor weight of the experimental group cell product + palbociclib combined group (group 6) was significantly lower than that of the corresponding experimental group cell product single drug treatment group (p<0.05)。
3. Post-dosing response and weight change in experimental animals
During the treatment period, the test substance TA cell products and the palbociclib are well tolerated after administration, each group of mice normally ingests drinking water without obvious acute abnormal symptoms, and the weight of each treatment group of mice is relatively stable without stopping drug.
The weight measurement results of the mice in each treatment Group are shown in FIG. 2, and groups 1 to 6 correspond to groups 1 to 6 in Table 2, respectively; at the end of the experiment (PG-D35), there was no significant difference (p > 0.05) in the recombination between mice in each group.
In conclusion, the experimental group autologous lymphocyte and palbociclib monoclonal antibody combined group (group 6) has remarkable anti-tumor effect (inhibiting tumor volume or quality, improving tumor growth inhibition rate or improving tumor weight inhibition rate) on a human liver cancer QGY7703 subcutaneous transplantation tumor model, and compared with the experimental group autologous lymphocyte single drug treatment (group 3) and palbociclib monoclonal antibody single drug treatment (group 4), the tumor inhibition effect is improved to a certain extent, and has obvious treatment advantages; the comparison among the mouse recombinations of the groups shows that the TA cell products and the palbociclib are combined to have no obvious toxicity.
2. Autologous lymphocyte reinfusion preparation and combination of autologous lymphocyte reinfusion preparation and PD-1/PD-L1 inhibitor for inhibiting HepG2 liver cancer cells
1. HepG2 human liver cancer xenograft in-situ model and drug administration
Human hepatoma cells HepG2 with luciferase markers were resuspended in PBS: matrigel (1:1) was adjusted to a concentration of 5.0X10 6 +M/ml tumor cell suspension, inoculated in liver parenchyma of 6-8 week old NCG breed mice that open the abdomen after anesthesia, 30 μL/mouse; tumors grew 5 days after HepG2 cell inoculation (D5, inoculation when the day is marked as D0) until the average tumor volume reached 20-30mm 3 And obtaining the HepG2 human liver cancer xenograft in-situ model, namely the mouse liver cancer xenograft in-situ model.
HepG2 human liver cancer xenograft in situ model was randomly grouped (group-wise astronomical PG-D0) for a total of 4 groups of 6, and dosing treatment was started on the day of grouping. The specific dosing regimen is shown in table 3 below.
Table 3 shows the dosage regimen
Note that: the control group in the table is group1, and groups 2-6 are all treatment groups; * Dosing volume: dosing volume: autologous lymphocyte reinfusion preparation is 400 mu L/mouse, and the cells are ensured not to precipitate or agglomerate before injection, and each group of autologous lymphocyte suspension is slowly injected during injection; v. the following: (tail) intravenous injection; i.p.: intraperitoneal injection. Qw×5 represents once weekly administration for 5 times, tiw× 4wks,from the 2nd week represents twice weekly administration for 4 weeks, and administration starts from week 2.
The palbociclib is dissolved in normal saline before injection to obtain a solution with the concentration of 5mg/mL, and the required injection volume is calculated according to the weight of the mice to achieve the therapeutic dose of 5mg/kg shown in Table 3.
2. Detection of
Tumor-bearing mice survival: the health state of the mice is observed, the survival time of euthanasia is recorded when each mouse dies or reaches the euthanasia end point, the Median Survival (MST) of the tumor-bearing mice in each group and the prolongation rate (ILS%) of the survival of the tumor-bearing mice in the treatment group are calculated, and the calculation formula is as follows: (treatment group MST/control group MST-1). Times.100%. It was observed that the end of 43 days after grouping.
During the course of the experiment, animals should be euthanized if any of the following conditions occur:
1) The animals are abnormal or paralyzed and can not ingest drinking water by themselves;
2) The weight of the animals was reduced by more than 20% of the weight at the beginning of the drug treatment;
3) Veterinarians consider it to seriously affect animal welfare and/or other abnormalities where experiments are normally conducted.
Experiments end the survival observations 48 days after cell inoculation (PG-D43), the survival curves of the mice in each group and comparison are shown in FIG. 3, groups 1-6 correspond to groups 1-6 in Table 2, respectively; it can be seen that median survival times of the experimental group cell product single drug group (group 3), the pamphlet Li Zhushan antibody group (group 4), the control group cell product+pamphlet combined group (group 5), the experimental group cell product+pamphlet combined group (group 6) were 11 days, 12 days, 16 days, 14 days, 22 days and 28 days, respectively. Compared with the Vehicle control group (group 1), the survival time of the cell product of the experimental group and the palbociclib combined group (group 6) is obviously prolonged (p < 0.05), and the survival time of the cell product of the experimental group and the palbociclib combined group (group 6) is obviously longer than that of the corresponding single drug treatment group (p < 0.05).
As shown in FIG. 4, the results of comparison between the mouse recombinations of the groups are shown in FIG. 4, the ordinate is the weight of the mice per day of detection/weight of the mice in the Group x100%, and groups 1 to 6 correspond to groups 1 to 6 in Table 2, respectively, and no significant difference (p > 0.05) exists between the mouse recombinations of the groups.
In conclusion, the experimental group autologous lymphocyte and palbociclib monoclonal antibody combined treatment (group 6) has remarkable anti-tumor effect on the human liver cancer HepG2 in-situ transplantation tumor model, effectively prolongs the survival time of mice, and has obvious tumor inhibition effect which is obviously improved compared with the experimental group autologous lymphocyte single drug group (group 3) and palbociclib monoclonal antibody single drug (group 4) treatment, and has obvious treatment advantages; the comparison among the mouse recombinations of the groups shows that the cell products of the experimental groups and the palbociclib are combined and have no obvious toxicity.
Claims (8)
1. Use of an autologous lymphocyte reinfusion preparation and a PD-1/PD-L1 inhibitor in any one of the following:
1) Preparing a tumor treatment product;
2) Preparing a product for inhibiting the growth of tumor cells;
3) Preparing a product for prolonging the life cycle of a tumor patient;
4) Preparing a product for promoting immune reconstitution of an organism;
the autologous lymphocyte feedback preparation comprises autologous lymphocytes;
the autologous lymphocytes are obtained by culturing peripheral blood mononuclear cells derived from a subject in an autologous lymphocyte culture solution;
the autologous lymphocyte culture solution comprises human gamma interferon IFN-gamma, human autologous plasma, anti-human CD3 monoclonal antibodies, IL-2, IL-7, IL-15, PHA-P and cell basal culture medium.
Use of a PD-1/PD-L1 inhibitor for the preparation of an immunoreconstitution product for assisting an autologous lymphocyte reinfusion preparation in promoting activated T lymphocytes in the body.
3. A kit comprising the autologous lymphocyte reinfusion preparation of claim 1 or 2 and the PD-1/PD-L1 inhibitor.
4. A kit according to claim 3, wherein:
the kit has any one of the following functions:
1) Treating tumors;
2) Inhibiting tumor cell growth;
3) Prolonging the survival time of tumor patients;
4) Promoting immune reconstruction of organism.
5. The kit of claim 3 or 4, wherein: the kit further comprises a readable carrier describing the following method of use: administering the autologous lymphocyte reinfusion preparation of claim 1 or 2 and the PD-1/PD-L1 inhibitor at intervals; and the first administration is an autologous lymphocyte reinfusion preparation according to claim 1 or 2.
6. Use of a kit according to any one of claims 3 to 5 in any one of the following:
1) Preparing a tumor treatment product;
2) Preparing a product for inhibiting the growth of tumor cells;
3) Preparing a product for prolonging the life cycle of a tumor patient;
4) And preparing the product for promoting the immune reconstruction of the organism.
7. The use according to claim 1 or 2 or 6 or the kit according to any one of claims 3 to 5, characterized in that: the PD-1/PD-L1 inhibitor is palbociclib monoclonal antibody, sodium Wu Liyou monoclonal antibody, actigb monoclonal antibody or dulcis You Shan antibody.
8. The use according to claim 1 or 2 or 6 or the kit according to any one of claims 3 to 5, characterized in that: the tumor is lung cancer, liver cancer, colorectal cancer, gastric cancer, head and neck squamous carcinoma, urothelial cancer, melanoma, hodgkin's lymphoma or renal cancer.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118161619A (en) * | 2024-05-16 | 2024-06-11 | 哈尔滨医科大学附属肿瘤医院(哈尔滨医科大学附属第三医院、哈尔滨医科大学第三临床医学院、黑龙江省肿瘤医院) | Pharmaceutical combination of adoptive cell therapy product and immune checkpoint blocker and application |
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- 2023-06-15 CN CN202310709134.XA patent/CN116687970A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118161619A (en) * | 2024-05-16 | 2024-06-11 | 哈尔滨医科大学附属肿瘤医院(哈尔滨医科大学附属第三医院、哈尔滨医科大学第三临床医学院、黑龙江省肿瘤医院) | Pharmaceutical combination of adoptive cell therapy product and immune checkpoint blocker and application |
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