WO2023226788A1 - Médicament pour immunisation combinée, composition de gel thermosensible, particule de chimiokine et leur utilisation - Google Patents

Médicament pour immunisation combinée, composition de gel thermosensible, particule de chimiokine et leur utilisation Download PDF

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WO2023226788A1
WO2023226788A1 PCT/CN2023/093856 CN2023093856W WO2023226788A1 WO 2023226788 A1 WO2023226788 A1 WO 2023226788A1 CN 2023093856 W CN2023093856 W CN 2023093856W WO 2023226788 A1 WO2023226788 A1 WO 2023226788A1
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chemokine
cells
tumor
gel
apd1
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郑毅然
韩文清
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苏州大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/195—Chemokines, e.g. RANTES
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1658—Proteins, e.g. albumin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells

Definitions

  • the invention relates to combined immunological drugs, thermosensitive gel compositions, chemokine particles and their applications.
  • Adoptive cell therapy represented by chimeric antigen receptor T cells (CAR-T)
  • CAR-T chimeric antigen receptor T cells
  • ACT adoptive cell therapy
  • solid tumors such as lung cancer, breast cancer, and liver cancer is still insufficient.
  • CAR-T therapy T cells extracted from the patient's peripheral blood are genetically modified so that they can specifically recognize tumor cells, activate and proliferate these cells in vitro, and then infuse them back into the patient's body.
  • adoptive T cells are immunosuppressed by cancer cells in the tumor microenvironment (TME), resulting in reduced function and reduced number.
  • CTL Cytotoxic CD8+ T lymphocytes
  • CXCR3 CXC-chemokine receptor 3
  • the main ligands of CXCR3 are CXCL9, CXCL10 and CXCL11.
  • CXCL9 also known as Monokine induced by gamma interferon (MIG)
  • MIG Monokine induced by gamma interferon
  • solid tumors express less chemokines and cannot effectively attract T cells to migrate to solid tumors.
  • the present invention provides combined immunological drugs, thermosensitive gel compositions, chemokine particles and their applications.
  • the present invention injects a mixed solution of CXCL9 chemokine particles, PD1 monoclonal antibodies and thermosensitive polymers next to solid tumors to form a mixture at body temperature.
  • the immune-modulating thermosensitive hydrogel loaded with CXCL9 chemokine particles and PD1 monoclonal antibodies enables the controlled release of the contained drugs near the tumor for a long time, improving the tumor targeting and anti-tumor killing power of immune cells.
  • the tumor-penetrating peptide iRGD can be loaded into thermo-sensitive gel or injected intravenously, and used in combination with immune-modulating thermo-sensitive gel to enhance the tumor infiltration ability of immune cells.
  • chemokine particles include amphiphilic polymers, chemokines, and albumin carriers, and the amphiphilic polymers include molecules that bind to albumin, affinity molecules, and albumin carriers. Water spacer groups and functional groups that react with chemokines;
  • the molecules that bind to albumin include lipophilic diacyl chains, lipophilic acyl chains, alkyl chains, fatty acids, vitamin E, and polypeptides containing the sequence AVGALEGPRNQDWLGVPRQL.
  • the structure of the hydrophilic spacer group is as follows:
  • X is a carbon chain containing multiple carbon atoms of ether bonds
  • the functional group that reacts with the chemokine includes a functional group that can react with the amino group, sulfhydryl group or disulfide bond of the chemokine.
  • the functional groups that react with the protein are as follows:
  • Y is oxygen, or a carbon chain of multiple carbon atoms containing ether bonds.
  • the functional groups that react with the protein are as follows:
  • Z is a carbon chain containing multiple carbon atoms.
  • the chemokine includes one or a combination of any two or more of CXCL9, CXCL10 and CXCL11.
  • the particle size of the albumin carrier is 1 to 1400 nm.
  • the preparation method of the above-mentioned chemokine particles includes the following steps:
  • BSA and DTSSP solution are mixed according to a molar ratio of 1: (10 to 300), and stirred for reaction. After the reaction is completed, centrifuge and wash with PBS to prepare NP;
  • the preparation method of the above-described chemokine particles includes the following steps:
  • thermosensitive gel composition includes the above-mentioned chemokine particles, PD-1 monoclonal antibody, and thermosensitive hydrogel.
  • thermosensitive gel composition includes the above-mentioned chemokine particles, PD-1 monoclonal antibody, iRGD polypeptide or polypeptide sequence including c(CRGDKGPDC), and thermosensitive hydrogel.
  • Combined immunological drugs include the above-mentioned thermosensitive gel composition administered near the tumor and iRGD or a polypeptide sequence containing c(CRGDKGPDC) administered intravenously.
  • the above-described chemokine particles, the above-described thermosensitive gel composition, and the above-described combined immunological drug in adoptive cell therapy and non-adopted cell immunotherapy; wherein the adoptive cells include CAR-T cells, CAR-NK cells, CAR-M cells, TCR-T cells, and neutrophils; non-adopted cells include endogenous CD8 + T cells, endogenous CD4 + T cells, endogenous NK cells, endogenous macrophages, endogenous Generate neutrophils.
  • iRGD can enhance the targeting and infiltration ability of drugs into tumors.
  • iRGD is a cyclic peptide composed of 9 amino acid residues c (CRGDKGPDC). During systemic circulation, it can specifically bind to integrin and neuropilin-1 (NRP-1) receptors overexpressed on tumor cells and activate NRP-1, increases the permeability of tumor blood vessels and tissues.
  • NRP-1 neuropilin-1
  • tumor cells In the TME, tumor cells often upregulate cell surface PD-L1 expression.
  • the immune checkpoint signaling pathway is activated, thereby inhibiting cell activity and reducing the anti-cancer immunity of T cells.
  • Immune checkpoint inhibitors such as PD-1 antibodies (aPD1) and PD-L1 antibodies can effectively block the PD-1 signaling pathway, thereby restoring T cell activity.
  • the injectable hydrogel system can locally and targetedly deliver drugs and controllable sustained-release drugs. It has great potential in improving drug efficacy and reducing systemic toxicity. It has high biocompatibility and can be widely used in the biomedical field.
  • Thermoresponsive gels are a broad class of supramolecular hydrogels that gel through hydrophobic interactions. It is a liquid within a certain temperature range and can transform into a gel as the temperature changes.
  • Distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) block copolymer has good biocompatibility, and carboxyl, amino, maleimide or N-hydroxysulfo groups are usually used in the application process.
  • Groups such as succinimide modify the ends of DSPE-PEG to link other functional molecules at one end.
  • the present invention has the following beneficial effects:
  • This invention uses an injectable temperature-sensitive polymer PLGA-PEG-PLGA, which forms next to solid tumors at body temperature.
  • Hydrogel long-term controlled release of chemokine particles and immune checkpoint inhibitor PD-1 monoclonal antibody (aPD1), enhances the targeting of adoptive T cells to solid tumors and immune suppression in the tumor microenvironment (TME) resistance, and by combining the tumor-penetrating peptide iRGD to enhance the infiltration of adoptive T cells into solid tumors, thereby effectively treating solid tumors.
  • Chemokine particles are composed of distearoylphosphoethanolamine-polyethylene glycol-N-hydroxysuccinimide (DSPE-PEG-NHS, DP-NHS) modified CXCL9 (CXCL9-DP) and albumin submicron carrier (SMP) was constructed (CXCL9-DP@SMP).
  • thermosensitive gel CMP/aPD1@Gel
  • CMP/aPD1@Gel thermosensitive gel
  • CMP/aPD1@Gel co-loaded with CXCL9-DP@SMP
  • aPD1 CMP/aPD1@Gel
  • iRGD increased the adoptive T in the tumor by 21 times compared with the same amount of free drugs.
  • the number of cells, and these adoptive T cells highly express functional proteins such as IFN- ⁇ and Granzyme B, and have tumor cell killing ability.
  • This technology can be applied to a variety of adoptive T cell therapies such as chimeric antigen receptor T cells (CAR-T) and T cell receptor T cells (TCR-T) therapy, providing a new platform and method for enhancing adoptive T cell therapy. ideas.
  • Figure 1 is (A) the preparation flow chart of the immunomodulatory thermosensitive hydrogel and (B) the schematic diagram of enhancing the anti-tumor efficacy of adoptive T cells in vivo;
  • Figure 2 shows the preparation and characterization of BSA carrier and determination of drug loading capacity.
  • A Particle size and PDI of nanoparticles (NP) obtained from BSA:DTSSP with different reaction molar ratios.
  • B Particle size distribution of BSA NP (1:200).
  • C Schematic diagram of different preparation methods of submicron particles (SMP).
  • E Particle size distribution of BSA SMP.
  • E Particle size distribution of BSA SMP.
  • G TEM image of NP.
  • H TEM image of SMP.
  • I Combination of DP-FITC and SMP under fluorescence microscope.
  • J Drug loading capacity of DP-FITC on BSA carrier.
  • K Drug loading capacity of DP-Cytochrome C-Cy5 on BSA NP/SMP;
  • Figure 3 shows the preparation of thermosensitive hydrogel and the determination of phase transition temperature and drug loading.
  • A Illustration of phase transition of hydrogel.
  • B Phase transition temperatures of thermosensitive copolymers with different mass ratios.
  • C Cytochrome C can be loaded per mg of PLGA-PEG-PLGA hydrogel.
  • D Loading efficiency of hydrogels for different drugs and carriers;
  • Figure 4 shows the in vitro degradation and drug release kinetics of thermosensitive gel, and the in vitro migration experiment of chemokine carriers.
  • A Degradation of thermosensitive hydrogel in the physiological environment of 37°C in vitro.
  • B Release kinetics of CC-Cy5 in each carrier system.
  • C Release kinetics of aPD1-FITC in hydrogels.
  • D Effect of different reaction molar ratios of DSPE-PEG-NHS on the activity of CXCL9
  • E Schematic diagram of Transwell migration experiment.
  • Figure 5 shows the biodegradation of thermosensitive gel in mice.
  • A Balb/c mice without tumor.
  • B C57Bl/6 B16-OVA subcutaneous melanoma margin in mice;
  • Figure 6 shows the in vivo release of thermosensitive gel.
  • A IVIS fluorescence imaging of mice at different time points.
  • Figure 7 shows the expression of CD8 and CXCR3 in spleen cells of OT-1 mice during the activation process.
  • A Flow cytometry histogram of the proportion of CD8+ T cells.
  • B Quantitative analysis of the proportion of CD8+ T cells.
  • C Flow cytometry histogram of cells expressing CXCR3.
  • Figure 8 shows that the chemokine biogel synergizes with free aPD1 and iRGD to significantly inhibit the growth of subcutaneous melanoma.
  • A Experimental schedule.
  • B Tumor volume changes during treatment in tumor-bearing mice.
  • C Relative changes in body weight.
  • Figure 9 shows the in vivo evaluation of the anti-tumor effect of the co-loaded drug combination biogel.
  • A Experimental schedule.
  • B Tumor volume changes during treatment in tumor-bearing mice.
  • C Relative changes in body weight.
  • Figure 10 shows the in vivo immunological evaluation of co-loaded drug combination biogels.
  • A Experimental schedule.
  • B Tumor volume changes during treatment in tumor-bearing mice.
  • Figure 11 shows flow cytometry analysis of immune cells in tumors.
  • A Representative flow chart of each group showing the proportion of CD8+ T cells among lymphocytes.
  • B Quantitative analysis of the proportion of CD8+T cells in lymphocytes.
  • C Representative flow chart of each group showing the proportion of adoptive T cells (CD8+Thy1.1+) among CD8+ T cells.
  • D Quantitative analysis of the proportion of adoptive T cells in lymphocytes.
  • E Quantitative analysis of the proportion of adoptive T cells (CD8+Thy1.1+) among CD8+ T cells.
  • F Quantification of adoptive T cells (CD8+Thy1.1+) per mg of tumor.
  • Figure 12 shows the expression of functional proteins of immune cells in tumors.
  • A Representative flow chart of the proportion of IFN- ⁇ expression in CD8+ T cells.
  • B Quantitative analysis of IFN- ⁇ expression in CD8+ T cells.
  • C Representative flow chart showing the proportion of Granzyme B expression in CD8+ T cells.
  • Figure 13 shows the analysis of immune cells in lymphoid organs and peripheral blood.
  • A ACT(CD8+) in lymph node cells Representative flow chart of the proportion of Thy1.1+).
  • B Quantitative analysis of the proportion of ACT (CD8+Thy1.1+) in lymph node cells.
  • C Representative flow chart of the proportion of ACT (CD8+Thy1.1+) in spleen lymphocytes.
  • D Quantitative analysis of the proportion of ACT (CD8+Thy1.1+) in spleen lymphocytes.
  • E Representative flow chart of the proportion of ACT (CD8+Thy1.1+) in peripheral blood CD8+ T cells.
  • Figure 14 shows the evaluation of the systemic anti-tumor effect of the co-loaded drug combination biogel.
  • A Experimental schedule.
  • B Primary tumor volume changes during treatment in tumor-bearing mice.
  • C Distal tumor volume changes.
  • D Relative changes in body weight.
  • Figure 15 shows the tumor re-challenge experiment in cured mice.
  • A Experimental schedule.
  • B Changes in tumor volume in tumor-bearing mice.
  • chemokine particles including amphiphilic polymers, chemokines, and albumin carriers.
  • the amphiphilic polymers include molecules that bind to albumin, hydrophilic spacers, and react with chemokines. functional groups;
  • Molecules that bind to albumin include lipophilic diacyl chains, lipophilic acyl chains, alkyl chains, fatty acids, vitamin E, and polypeptides containing the sequence AVGALEGPRNQDWLGVPRQL.
  • the chemokine includes one or a combination of any two or more of CXCL9, CXCL10 and CXCL11.
  • the particle size of the albumin carrier is 1 to 1400 nm.
  • the structure of the hydrophilic spacer group is as follows:
  • X is a carbon chain containing multiple carbon atoms of ether bonds; among them, n is 1 to 200.
  • hydrophilic spacer group is as follows:
  • functional groups reactive with chemokines include functional groups capable of reacting with amino, sulfhydryl, or disulfide bonds of the chemokine.
  • the functional groups that react with the protein are as follows:
  • Y is oxygen, or a carbon chain of multiple carbon atoms containing ether bonds.
  • the functional groups that react with the protein are as follows:
  • Z is a carbon chain containing multiple carbon atoms.
  • amphiphilic polymers include DP-NHS and DP-MAL.
  • the preparation method of the above-mentioned chemokine particles includes the following steps:
  • BSA solution and DTSSP solution Use PBS to prepare BSA solution and DTSSP solution.
  • the preparation method of the above-described chemokine particles includes the following steps:
  • the preparation of SMP includes 3 different methods, as follows:
  • Two-step method Mix 0.5 parts by weight of BSA and 1 part by weight of DTSSP and react for 1.5 hours; then add 0.5 parts by weight of BSA and 1 part by weight of DTSSP into the reaction system and continue the reaction for 1.5 hours;
  • the reacted sample solution was centrifuged and resuspended in PBS to prepare SMP.
  • thermosensitive gel composition includes the above-mentioned chemokine particles, PD-1 monoclonal antibody, and thermosensitive hydrogel.
  • Chemokine particles, PD-1 monoclonal antibodies and thermosensitive polymers are mixed in a PBS solution, and the mixed solution is injected next to the tumor.
  • An immunomodulatory thermosensitive hydrogel loaded with chemokine particles and PD1 monoclonal antibodies is formed next to the tumor at body temperature. .
  • thermosensitive gel composition includes the above-mentioned chemokine particles, PD-1 monoclonal antibody, iRGD polypeptide or polypeptide containing c(CRGDKGPDC) sequence, and thermosensitive hydrogel.
  • Chemokine particles, PD-1 monoclonal antibodies, iRGD polypeptides or polypeptide sequences containing c(CRGDKGPDC), and temperature-sensitive polymers are mixed in PBS solution, and the mixed solution is injected next to the tumor, and a loaded chemoattractant is formed next to the tumor at body temperature.
  • Combined immunological drugs include the above-mentioned thermosensitive gel composition administered near the tumor and iRGD polypeptide or polypeptide sequence containing c(CRGDKGPDC) administered intravenously.
  • the above-described chemokine particles, the above-described thermosensitive gel composition, and the above-described combined immunological drugs in adoptive cell therapy and non-adopted cell therapy; wherein the adoptive cells include CAR-T cells, CAR -NK cells, CAR-M cells, TCR-T cells, neutrophils.
  • adoptive cells include CAR-T cells, CAR -NK cells, CAR-M cells, TCR-T cells, neutrophils.
  • Non-adopted cells include endogenous CD8 + T cells, endogenous CD4 + T cells, endogenous NK cells, endogenous macrophages, and endogenous neutrophils.
  • This technology uses hydrogel to cleverly combine chemokine CXCL9, immune checkpoint inhibitor PD-1 monoclonal antibody (aPD1) and tumor-penetrating peptide iRGD to enhance the targeting and infiltration of adoptive T cells into solid tumors. ability and intratumoral immune effects, thereby effectively treating solid tumors.
  • This technology loads CXCL9 and aPD1 with hydrogel, and injects the temperature-sensitive hydrogel into solid tumors, so that the contained drugs can be released in a controlled and controlled manner next to the tumor for a long time. Due to the small molecular weight of the chemokine CXCL9 (only 12kDa), it will be rapidly released from the hydrogel.
  • CXCL9-DP DSPE-PEG
  • SMP submicron particles
  • aPD1 released from the hydrogel helps intratumoral adoptive T cells resist the immunosuppression of cancer cells and improves efficacy (Figure 1B).
  • This method can be applied to a variety of adoptive T cell therapies such as CAR-T and TCR-T, providing new ideas for the treatment of solid tumors.
  • the preparation method of this system is simple, the raw materials are biodegradable, and it has high clinical translation potential.
  • Mouse CXCL9, IL-2 and IL-7 were purchased from Peprotech (Rocky Hill, NJ, USA).
  • Anti-PD1 (clone number RMP1-14), anti-CD3 (clone number 145-2C11) and anti-CD28 (clone number 37.51) antibodies were purchased from Bioxcell (Lebanon, NH, USA).
  • Ovalbumin peptide (257-264) was purchased from InvivoGen (San Diego, CA, USA).
  • EasySep TM mouse CD8+T cell isolation kit was purchased from StemCell Technologies (Vancouver, BC, Canada).
  • Ficoll-Paque Plus was purchased from Cytiva (Pharmacia, Uppsala, Sweden).
  • LIVE/DEAD Fixable Dead Cell Stain Kit was purchased from Invitrogen (Carlsbad, CA, USA).
  • Anti-mouse CD183(CXCR3)APC, CD8a FITC/PE, Thy1.1APC/FITC, CD45FITC, CD4APC-Cy7, CD69BV421, IFN- ⁇ APC, Ki67BV421, Granzyme B PE were purchased from Biolegend (San Diego, CA, USA).
  • 3,3'-Dithiobis(sulfosuccinimidylpropionate) (DTSSP) was purchased from Abcam (Cambridge, UK).
  • mice C57BL/6 female mice (6 weeks old) and BALB/C female mice (6 weeks old) were purchased from Cavens (Changzhou, China).
  • OT-1 mice were purchased from Shanghai Model Organisms Center (Shanghai, China) and bred by our.
  • Pmel-1 mice were purchased from Jackson Laboratory (Maine, USA) and breed on their own. All mice were kept in SPF-grade rooms, 5 mice per cage, with sufficient feed and drinking water. The light and dark cycle of the breeding room is 12 hours each (7:00 am-7:00 pm), and the room temperature is 25 ⁇ 1°C. All animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Soochow University. The animal experimental protocol was conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23 Rev. 1985).
  • the B16-OVA cell line was purchased from ATCC (Rockville, MD, USA) and cultured in DMEM medium containing 10% fetal bovine serum (FBS).
  • FBS fetal
  • BSA (1mg/mL) and DTSSP (10mg/mL) solutions.
  • BSA and DTSSP were mixed according to the molar ratio of 1:10, 1:20, 1:50, 1:100, 1:200, and 1:300 respectively.
  • the reaction was carried out at 600 rpm for 1 hour.
  • the reacted solution was placed in a 50 kDa ultrafiltration tube, centrifuged (4000 rpm, 4 min), and washed twice with PBS to prepare NP.
  • a laser nanoparticle size and potentiometer (90Plus Particle Size Analyzer, Brookhaven) was used to measure the hydrated particle size and Zeta potential of the prepared NP and SMP, as shown in Table 1 and Table 2 below. 20 ⁇ L of NP and SMP with appropriate concentrations were dropped on the copper mesh, dried at room temperature, and observed using a transmission electron microscope (HT7700, Hitachi) to observe their morphological characteristics.
  • CXCL9 and DP-NHS (reaction molar ratios 1:1, 1:2, 1:5, 1:10) were mixed in PBS solution and reacted on a four-dimensional rotator at room temperature overnight to prepare CXCL9-DP.
  • Cytochrome C a protein with a similar molecular weight and close isoelectric point to CXCL9, was used as the model protein.
  • CC-Cy5 and DP-NHS molar ratio 1:5 connected with Cy5 fluorescent molecules were mixed in PBS solution and reacted on a four-dimensional rotator overnight to prepare CC-Cy5-DP.
  • thermosensitive hydrogel (Gel)
  • CC-Cy5-DP was synthesized by reacting CC-Cy5 and DP-NHS at a molar ratio of 1:5.
  • Different carriers CC-Cy5-DP@NP, CC-Cy5-DP@SMP, CC-Cy5@Gel, CC-Cy5-DP@NP@Gel and CC-Cy5-DP@
  • SMP@Gel set free CC-Cy5 as the control group.
  • Ern is the cumulative release percentage of CC-Cy5 at a certain time point
  • Ve is the displacement volume of the release medium, which is 200 ⁇ L
  • V0 is the total volume of the release medium, which is 20 mL
  • FCC-Cy5 is the initial concentration of CC-Cy5 in the dialysis bag The total amount of fluorescence
  • fn is the sample fluorescence reading of the nth replacement sampling.
  • aPD1-FITC and 8mg PLGA-PEG-PLGA were mixed in PBS solution and gelled at 37°C.
  • the loaded aPD1-FITC hydrogel was released for 7 days using the same method.
  • 200 ⁇ L solution was drawn from the centrifuge tube to detect FITC fluorescence, and 200 ⁇ L of the same temperature solution was added.
  • PBS Calculate the cumulative release percentage of aPD1-FITC.
  • the 0.1% gelatin solution was spread on an inverted Transwell chamber (3415, Costar), and MS1 endothelial cells (1 ⁇ 105) were inoculated and cultured on the front of the chamber. Two days later, TNF- ⁇ (20ng/mL) was added to activate the MS1 cells in the chamber and upregulate the expression of adhesion molecules. After 4 hours, 200 ⁇ L of RPMI 1640 complete medium containing IL-2 (10ng/mL) and IL-7 (10ng/mL) containing 2.5 ⁇ 105 CD8+ T cells was added to each well in the upper chamber.
  • the lower chamber is divided into 7 groups: 1No CXCL9; 2Free CXCL9; 3CXCL9-DP; 4CXCL9-DP@NP; 5CXCL9-DP@NP@Gel; 6CXCL9-DP@SMP; 7CXCL9-DP@SMP@Gel (except 1 group without CXCL9, The remaining groups all contained an equal amount of 0.25 ⁇ g CXCL9.
  • RPMI 1640 complete medium for CD8+ T cells, aspirate half of the liquid in the lower chamber, replace it with fresh above-mentioned RPMI 1640 complete medium, and continue sampling on the 5th day.
  • thermosensitive hydrogel 1.12 Degradation of thermosensitive hydrogel in in vitro physiological environment
  • thermosensitive gel 1.13 In vivo biodegradation of thermosensitive gel
  • thermosensitive gel 1.14 In vivo release of thermosensitive gel
  • mice were randomly divided into the following 6 groups: 1CC-Cy5; 2CC-Cy5@Gel; 3CC-Cy5@NP; 4CC-Cy5@NP@Gel; 5CC-Cy5@SMP; 6CC-Cy5@SMP@Gel. 50 ⁇ L of different sample solutions (containing an equal amount of 44 ⁇ g CC-Cy5) were subcutaneously injected into the right back of mice.
  • mice After the OT-1 (or Pmel-1) mice were euthanized, the spleens were removed, ground on a 70 ⁇ m cell strainer, rinsed, and centrifuged (700 g, 4 min). The cell pellet was treated with ACK red blood cell lysis buffer, centrifuged again, and resuspended in RPMI 1640 containing OVA257-264peptide (or gp100) (1 ⁇ g/mL), IL-2 (10ng/mL) and IL-7 (1ng/mL). In the culture medium, adjust the cell density to 2 ⁇ 106 cells/mL. After incubation at 37°C for 3 days, lymphocytes were purified by Ficoll-paque plus gradient centrifugation.
  • the collected cells were further cultured in RPMI 1640 medium containing IL-2 (10ng/mL) and IL-7 (10ng/mL). Change the medium every 2 days. On days 0, 3, 4 and 5 of cell activation, 1 ⁇ 106 cells were taken for flow cytometric antibody staining to detect the proportion of CD8+ T cells and the expression of CXCR3+ in OT-1 mice.
  • B16-OVA cells were dissolved in sterile PBS (4 ⁇ 106 cells/mL), Matrigel was added at a volume of 1:1, and 2 ⁇ 105 cells were inoculated subcutaneously in C57BL/6 mice.
  • tumor-bearing mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg/kg to eliminate lymphocytes in the mice.
  • the tumor-bearing mice were randomly divided into 6 groups, and the injection doses of relevant drugs in each group were OT-1CD8+T (5 ⁇ 106), aPD1 (30 ⁇ g), iRGD (200 ⁇ g), and CXCL9 (15 ⁇ g) activated for 5 days.
  • each mouse in group 1 was injected with 200 ⁇ L sterile PBS into the tail vein, and each mouse in the remaining groups was injected with 5 ⁇ 106 activated OT-1CD8+ T cells into the tail vein for 5 days.
  • mice The survival status of the mice was observed every 2 days, and changes in body weight and tumor size of mice in each group during treatment were recorded. A tumor volume exceeding 1000 mm3 was used as the end point of the experiment for each mouse.
  • B16-OVA cells were dissolved in sterile PBS (1 ⁇ 107 cells/mL), Matrigel was added in a volume of 4:1, and 8 ⁇ 105 cells were inoculated subcutaneously in C57BL/6 mice.
  • tumor-bearing mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg/kg to eliminate lymphocytes in the mice.
  • the tumor-bearing mice were randomly divided into 6 groups, and the injection doses of relevant drugs in each group were OT-1CD8+T (5 ⁇ 106), aPD1 (30 ⁇ g), iRGD (200 ⁇ g), and CXCL9 (15 ⁇ g) activated for 5 days.
  • the first drug was grouped as follows. Four days after administration, each mouse in group 1 was injected with 200 ⁇ L sterile PBS into the tail vein, and each mouse in the remaining groups was injected with 5 ⁇ 106 activated OT-1CD8+ T cells into the tail vein for 5 days.
  • mice The survival status of the mice was observed every 2 days, and changes in body weight and tumor size of mice in each group during treatment were recorded. A tumor volume exceeding 1000 mm3 was used as the end point of the experiment for each mouse.
  • mice Dissolve B16-OVA cells in sterile PBS (1.25 ⁇ 107 cells/mL), add Matrigel in a volume of 4:1, and inoculate 1 ⁇ 106 cells subcutaneously in C57BL/6 mice.
  • tumor-bearing mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg/kg to eliminate lymphocytes in the mice.
  • Tumor-bearing mice were randomly divided into 4 group, the injection doses of relevant drugs in each group were Pmel-1CD8+T (5 ⁇ 106), aPD1 (30 ⁇ g), iRGD (200 ⁇ g), and CXCL9 (15 ⁇ g) activated for 5 days.
  • the first drug was grouped as follows. Four days after administration, each mouse in group 1 was injected with 200 ⁇ L sterile PBS into the tail vein, and each mouse in the remaining groups was injected with 5 ⁇ 106 Pmel-1 CD8 + T cells into the tail vein to activate Pmel-1 CD8 + T cells for 5 days.
  • mice The changes in body weight and tumor volume of mice in each group were recorded every 2 days. Two days after the second administration, mice were euthanized, and tumors, spleens, inguinal lymph nodes, and peripheral blood were collected. After red lysis, peripheral blood, spleen cells, and lymph node cells were stained with flow cytometry antibodies to detect the expression of Thy1.1, CD8a, CD4, and CD45.
  • the cells obtained by grinding the tumors were incubated with eBioscienceTM Cell Stimulation Cocktail (Invitrogen) for 6 hours at 37°C, and the expression levels of Thy1.1, CD8a, CD25, IFN- ⁇ , Granzyme B, etc. in the cells were detected by flow cytometry.
  • thermosensitive gel co-loaded with CXCL9 and aPD1
  • B16-OVA cells were dissolved in sterile PBS (1 ⁇ 107 cells/mL), Matrigel was added in a volume of 4:1, and inoculated subcutaneously in C57BL/6 mice at both ends, with an inoculation number of 8 ⁇ 105 cells.
  • tumor-bearing mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg/kg to eliminate lymphocytes in the mice.
  • the tumor-bearing mice were randomly divided into 4 groups, and the injection doses of relevant drugs in each group were OT-1CD8+T (5 ⁇ 106), aPD1 (30 ⁇ g), iRGD (200 ⁇ g), and CXCL9 (15 ⁇ g) activated for 5 days.
  • the first drug was grouped as follows. Four days after administration, each mouse in group 1 was injected with 200 ⁇ L sterile PBS into the tail vein, and each mouse in the remaining groups was injected with 5 ⁇ 106 activated OT-1CD8+ T cells into the tail vein for 5 days.
  • mice The survival status of the mice was observed every 2 days, and changes in body weight and tumor size of mice in each group during treatment were recorded. A tumor volume exceeding 1000 mm3 was used as the end point of the experiment for each mouse.
  • B16-OVA cells were dissolved in sterile PBS (1.25 ⁇ 107 cells/mL), Matrigel was added in a volume of 4:1, and inoculated subcutaneously in C57BL/6 mice, with an inoculation number of 1 ⁇ 106 cells.
  • Cured C57BL/6 mice and healthy C57BL/6 mice of the same age were used to construct the Rechallenge tumor-bearing model.
  • Free aPD1 (30 ⁇ g) was injected into the tail vein on days 11, 14, 18, 21, 24, and 27 days after tumor grafting. Starting from the 8th day after tumor grafting, the survival status of the mice was observed every 2 days, and changes in body weight and tumor size of mice in each group during treatment were recorded. A tumor volume exceeding 1000 mm3 was used as the end point of the experiment for each mouse.
  • n-hydroxysuccinimide (NHS) functional groups at both ends of 3,3'-dithiobis(sulfosuccinimide propionate) can react with the amino groups in the protein to form an amide reaction.
  • Albumin (BSA) is cross-linked to form carrier particles.
  • NP nanoparticles
  • SMP submicron particles
  • DP-FITC FITC fluorescent molecules
  • BSA SMP BSA SMP
  • DP-FITC FITC fluorescent molecules
  • Figure 2I DSPE
  • the amount of DP-FITC that can be bound to NP/SMP is dose-dependent on the added DP-FITC.
  • the amount of DP-FITC added exceeds 40 ⁇ g, the combination of DP-FITC and BSA tends to be saturated.
  • Each mg NP can bind up to approximately 36 ⁇ g DP-FITC
  • each mg SMP can bind up to approximately 34 ⁇ g DP-FITC ( Figure 2J).
  • Cytochrome C (CC) consists of 108 amino acid residues, the protein molecular weight is 12.3kDa, the isoelectric point is 8.7, and the number of lysine residues is 16.
  • CXCL9 consists of 105 amino acid residues, the protein molecular weight is 12.2kDa, the isoelectric point is 9.8, and the number of lysine residues is 21. Therefore, CC is suitable as a model protein of CXCL9 to conduct research on physical and chemical properties.
  • CC-Cy5-DP can be obtained by reacting CC with fluorescent molecule Cy5 with DP.
  • CC-Cy5-DP@NP represents the combination of CC-Cy5-DP and NP
  • CC-Cy5@SMP represents the combination of CC-Cy5 and SMP.
  • CC-Cy5-DP passed DSPE specifically binds to NP/SMP.
  • NP can load 32 ⁇ g CC-Cy5/mg NP
  • SMP can load 39 ⁇ g CC-Cy5/mg SMP, which is more than 3 times the amount of CC loaded by electrostatic adsorption (CC-Cy5@SMP). ( Figure 2K).
  • the PLGA-PEG-PLGA polymer aqueous solution can gel at 37°C at a concentration of 14% (mass/volume, m/v) to 22% (m/v). The higher the w/v% ratio, the better the gelation. The lower the temperature. In order to ensure that the hydrogel does not gel at room temperature but gels quickly at body temperature, the w/v% of the hydrogel used in subsequent experiments was 16% ( Figure 3B).
  • thermosensitive hydrogel can contain up to ⁇ 0.35 mg CC/mg PLGA-PEG-PLGA ( Figure 3C).
  • the encapsulation efficiency of the hydrogel for 15 ⁇ g of free CC-Cy5 and the same amount of CC-Cy5 loaded on BSA NP and SMP was close to 100%, indicating that the BSA carrier did not affect the loading of chemokines.
  • the loading efficiency of aPD1 in the hydrogel was also close to 100%, indicating that the molecular weight and type of protein drugs have no significant impact on the drug loading efficiency of the hydrogel (Figure 3D).
  • thermosensitive gel 2.3 Investigation of in vitro degradation and drug release kinetics of thermosensitive gel
  • thermosensitive copolymer solution containing Ponceau (10%) was laid out in the shape of CAR-T on the surface of the culture dish, and gelled at 37°C. Then PBS was added to submerge the hydrogel and placed on a shaker (100 rpm, 37°C), observe every day. During the 8-day observation period, the thermosensitive gel showed no obvious changes in the first 4 days. After 4 days, the hydrogel gradually began to degrade as time went by (Figure 4A).
  • thermosensitive gel CC-Cy5@Gel, CC-Cy5-DP@NP@Gel and CC-Cy5-DP@SMP@Gel.
  • Free CC-Cy5 was released rapidly and almost completely diffused outside the dialysis bag within 24 hours.
  • the hydrogel in the CC-Cy5@Gel group can delay the release of CC-Cy5, but due to the large pore size of the hydrogel, CC-Cy5 with a molecular weight of only 12kDa will flow out of the hydrogel, and nearly 80% of CC will be released within 3 days. -Cy5 release.
  • the CC-Cy5-DP@NP and CC-Cy5-DP@SMP groups can delay the release of CC-Cy5, reaching 80% release on 3d and 4d respectively. This is mainly due to the NP and SMP carriers themselves.
  • CC-Cy5-DP@NP and CC-Cy5-DP@SMP were loaded into hydrogels (CC-Cy5-DP@NP@Gel, CC-Cy5-DP@SMP@Gel) can further sustain the release of CC-Cy5.
  • the sustained release effect of CC-Cy5-DP@NP@Gel is not obvious, mainly because the particle size of NP is smaller and cannot stay in the hydrogel for a long time.
  • the sustained release effect of the CC-Cy5-DP@SMP@Gel group was significantly improved compared to CC-Cy5-DP@SMP, and the release time was nearly five times longer than that of the free CC-Cy5 group ( Figure 4B).
  • the total amount of CC-Cy5 cumulatively released by each carrier is basically the same.
  • the release of free aPD1 in the hydrogel takes about 7 days. This is due to its larger molecular weight (150kDa), so the release period in the in vitro hydrogel system is longer than that of free CC ( Figure 4C).
  • MS1 cells were first plated in the upper chamber, and 2 days later, equal amounts of CXCL9 free and loaded in BSA particles/hydrogel were added to the lower chamber, and an equal volume of T cell culture medium containing cytokines was added.
  • Add an equal amount of activated CD8+ T cells and the same T cell culture medium to the upper chamber keep the liquid levels in the upper and lower chambers equal, and count the number of cells in the lower chamber every day.
  • the number of CD8+ T cells in the lower chamber increased significantly in the Free CXCL9 (Free CXCL9) group, approximately 2.3 times after 8 days, proving that the chemokine CXCL9 can induce the migration of CXCR3+ T cells.
  • the number of CD8+ T cells recruited by DP-modified CXCL9 (CXCL9-DP) and unmodified CXCL9 was basically the same, indicating that DP modification did not affect the chemotactic function of CXCL9.
  • the number of CD8+T cells recruited in the lower chamber after 8 days in the CXCL9-DP@NP group and the CXCL9-DP@SMP group was about 2.3 times and 3.3 times in the CXCL9 group, indicating that the combination of CXCL9-DP and NP/SMP can sustain the release of CXCL9 and prolong the Its action time.
  • thermosensitive hydrogel encapsulated CXCL9 The number of CD8+T cells collected in the lower chamber of -DP@SMP after 8 days was nearly 7 times that of No CXCL9 and nearly 2 times that of the CXCL9-DP@SMP group ( Figure 4F).
  • thermosensitive gel 2.5 In vivo biodegradation and drug release of thermosensitive gel
  • a mixed solution of CC-Cy5-DP@SMP and PLGA-PEG-PLGA polymer was injected subcutaneously into normal BALB/c mice and C57BL/6 mice bearing B16-OVA transplanted tumors. It can be observed that after injection of the mixed solution, both normal BALB/c mice ( Figure 5A) and tumor-bearing C57BL/6 mice ( Figure 5B) Can form hydrogel. As time goes by, the hydrogel degrades in the body and releases CC-Cy5. The blue color under the skin of the mice becomes lighter and lighter, and basically disappears after 8 days. We are also using small animal in vivo imaging equipment to detect the release of drugs contained in temperature-sensitive gels in the body.
  • mice BALB/C female mice were subcutaneously injected with free CC-Cy5 or CC-Cy5-loaded particles and thermosensitive gel, and the Cy5 fluorescence at the injection site of the mice was detected at different time points ( Figure 6A-B).
  • the fluorescence intensity of the free CC-Cy5 group and CC-Cy5@Gel group decreased rapidly in vivo, and 65% of the fluorescence had disappeared after 1 day. This may be due to the small molecular weight of CC-Cy5, which easily leaks out of the hydrogel, so the release kinetics of CC-Cy5@Gel are similar to those of free CC-Cy5.
  • the subcutaneous fluorescence intensity of the CC-Cy5-DP@NP and CC-Cy5-DP@NP@Gel groups was still ⁇ 50% after 2 days, which was approximately twice that of the free CC-Cy5 group. This is probably due to the retention of BSA nanoparticles under the skin, but because the NP particle size is not large enough, it is easy to leak out of the gel, resulting in little difference between the CC-Cy5-DP@NP@Gel group and the CC-Cy5-DP@NP group. .
  • the release curves of the CC-Cy5-DP@SMP group are similar to those of the CC-Cy5-DP@SMP group, but the hydrogel group loaded with CC-Cy5-DP@SMP can significantly improve the drug retention effect.
  • the CC-Cy5-DP@SMP@Gel group maintained 80% of the fluorescence signal after 2 days, and still had 20% of the signal after 8 days, which was consistent with the in vitro release kinetics results.
  • CD8+T cells obtained from OT-1 transgenic mice can specifically recognize the MHC class I molecule-OVA257-264 antigen peptide complex, and thus can recognize and kill B16-OVA tumor cells.
  • CD8+T cells obtained from OT-1 transgenic mice can specifically recognize the MHC class I molecule-OVA257-264 antigen peptide complex, and thus can recognize and kill B16-OVA tumor cells.
  • we crushed and split the spleen of OT-1 mice and combined it with OVA257-264 antigen Peptides and cytokines were co-incubated to activate Ficoll-purified lymphocytes for 3 days and then cultured and proliferated. After spleen cells of OT-1 mice were lysed, only 25% of CD8+ T cells were present.
  • aPD1+iRGD group T+aPD1+iRGD
  • tail vein injection of CD8+T+aPD1+subcutaneous injection of free CXCL9 group T+aPD1+CXCL9
  • tail vein injection of CD8+T+aPD1+iRGD+subcutaneous injection of free CXCL9 group T +aPD1+CXCL9+iRGD
  • tail vein injection of CD8+T+aPD1+subcutaneous injection of CMP@Gel group T+aPD1+CMP@Gel
  • tail vein injection of CD8+T+aPD1+iRGD+subcutaneous injection of CMP@Gel group T +aPD1+CMP@Gel+iRGD
  • the T+aPD1+CXCL9 group showed better anti-tumor effect than T+aPD1, proving that the chemotactic ability of CXCL9 to recruit CD8+ T cells helps to enhance the anti-tumor effect of adoptive T cells.
  • the results of the T+aPD1+iRGD group compared with the T+aPD1 group, and the T+aPD1+CMP@Gel+iRGD group compared with the T+aPD1+CMP@Gel group proved that the tumor blood vessel penetration performance of iRGD peptide improves the effect of adoptive T cells on entities. tumor efficacy.
  • the results of the T+iRGD+aPD1+CMP@Gel group and the T+iRGD+CMP@Gel group prove that immune checkpoint inhibition of aPD1 is necessary to improve the efficacy of solid tumors.
  • the results of the T+iRGD+CMP/aPD1@Gel group and the T+iRGD+aPD1+CMP@Gel group prove that compared with tail vein injection of free aPD1, aPD1 loaded in hydrogel is more conducive to enhancing the treatment of combination therapy Effect.
  • thermosensitive gel co-loaded with CMP and aPD1 combined with iRGD we constructed a B16-OVA tumor model subcutaneously in C57BL/6 female mice, extracted splenocytes from Pmel-1 mice, and activated them with gp100 peptide. After the tumor volume reached 85 mm3, activated Pmel-1Thy1.1+CD8+ T cells were injected for adoptive T cell therapy.
  • the drug co-loaded immunogel can also significantly increase the number of endogenous CD8+T cells per mg of solid tumors.
  • the T+iRGD+CMP/aPD1@Gel group is 6 times higher than the free drug T+iRGD+aPD1+CXCL9 group.
  • Figure 11G The gel group not only increased the number of adoptive T cells compared with the free drug group, but also the expression levels of IFN- ⁇ ( Figure 12A-B) and Granzyme B (Figure 12C-D) of adoptive T cells in the T+iRGD+CMP/aPD1@Gel group.
  • thermosensitive gel also increased the proportion of adoptive T cells in inguinal lymph nodes (Figure 13A-B), spleen (Figure 13C-D) and blood (Figure 13E-F), which were 2.4% of the free drug group respectively. , 2.6 and 1.8 times.
  • thermosensitive gel therapy can stimulate systemic anti-cancer immune responses to treat non-gel-near tumors and achieve the effects of systemic treatment and treatment of metastatic tumors.
  • thermosensitive gel can trigger systemic immune responses and treat systemic or metastatic tumors (Figure 14), rather than being limited to tumors near the gel injection site.
  • thermosensitive gel promotes the generation of anti-tumor immune memory
  • C57BL/6 mice that were cured by the gel group in the previous experiment were subcutaneously inoculated (90 days later) with B16-OVA tumor cells to perform a tumor re-challenge experiment.
  • Healthy C57BL/6 mice of the same age were subcutaneously inoculated with tumor cells as a control group.
  • aPD1 was injected into the tail vein on days 11, 14, 18, 21, 24 and 27 respectively after tumor grafting (Fig. 15A). 12.5% of the mice in the cured group did not develop tumors after tumor re-challenge, and another 50% of the mice in the cured group had tumors that were completely eliminated by aPD1 after growth, while every mouse in the control group had tumors.

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Abstract

L'invention concerne un médicament pour l'immunisation combinée, une composition de gel thermosensible, une particule de chimiokine et leur utilisation. La présente invention utilise une solution mixte d'un polymère thermosensible injectable PLGA-PEG-PLGA, d'une particule de chimiokine, et d'un anticorps monoclonal d'inhibiteur de point de contrôle immunitaire PD-1 pour former un hydrogel thermosensible près d'une tumeur solide à la température corporelle, et la chimiokine et l'aPD1 sont libérés de manière contrôlée pendant une longue période, de sorte que la propriété de ciblage d'un lymphocyte T adoptif sur la tumeur solide et la résistance à l'immunosuppression dans un micro-environnement tumoral sont améliorées. Au moyen de la combinaison de gel thermosensible immunomodulateur et d'un peptide de pénétration tumorale iRGD, l'infiltration du lymphocyte T adoptif dans la tumeur solide est améliorée, ce qui permet de traiter efficacement la tumeur solide. La particule de chimiokine est construite par une chimiokine modifiée par distéaroyl phosphoéthanolamine-polyéthylène glycol-N-hydroxysuccinimide et un support d'albumine.
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