Dendritic cell preparation and preparation method thereof
12440565 ยท 2025-10-14
Assignee
Inventors
Cpc classification
C12N5/0639
CHEMISTRY; METALLURGY
C12N2501/22
CHEMISTRY; METALLURGY
C12N2501/51
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
A61P35/00
HUMAN NECESSITIES
Abstract
The present application discloses a novel dendritic cell preparation and a preparation method thereof. The cell preparation comprises PD-L1 negative dendritic cells and/or dendritic cells with CD40 agonists bound to the cell surface. The preparation method comprises: blocking and/or activating dendritic cells with a treatment agent, wherein the treatment agent comprises PD-L1 antibodies and/or CD40 agonists. The specific method includes adding the treatment agent to block and/or activate dendritic cells either during the dendritic cell culture stage or after completion of the culture. The dendritic cell preparation provided by the present application has enhanced maturity, improved antigen presentation, and a superior capacity to activate and amplify antigen-specific T cells compared to conventional dendritic cells, and has broad application prospects in the field of cancer immunotherapy.
Claims
1. A novel dendritic cell preparation, comprising PD-L1 negative dendritic cells and dendritic cells with CD40 agonists bound to the cell surface, wherein the surface of PD-L1 negative dendritic cells in the preparation is bound to PD-L1 antibodies.
2. The dendritic cell preparation according to claim 1, wherein the proportion of PD-L1 negative dendritic cells is 80% of the total cells in the preparation, and the proportion of dendritic cells with CD40 agonists bound to the cell surface is 80% of the total cells in the preparation.
3. The novel dendritic cell preparation according to claim 1, wherein the proportion of dendritic cells with mature cell phenotype is 50% of the total cells in the preparation, or, the proportion is 80%.
4. The novel dendritic cell preparation according to claim 1, wherein each proportion of CD80, CD86, HLA-DR, and CD83 positive cells is 50% of the total cells in the preparation, or, the proportion is 80%.
5. The novel dendritic cell preparation according to claim 1, wherein the proportion of CD14 positive cells is 20% of the total cells in the preparation, or, the proportion is 10%, and/or wherein the sum of proportions of CD3, CD19, and CD56 positive cells is 20% of the total cells in the preparation; or, the sum of proportions is 10%; or, the sum of proportions is 5%.
6. The novel dendritic cell preparation according to claim 1, wherein the PD-L1 antibody is selected from a PD-L1 monoclonal antibody or a multispecific antibody targeting PD-L1; wherein, the PD-L1 antibody is optionally selected from a monoclonal antibody Atezolizumab, Adebrelimab, Durvalumab, or Avelumab.
7. The novel dendritic cell preparation according to claim 1, wherein the CD40 agonist is selected from a CD40 agonist antibody or a CD40 ligand, wherein the CD40 agonist antibody comprises a CD40 monoclonal antibody and a multispecific antibody targeting CD40, and the CD40 ligand comprises a CD40L recombinant protein and a fusion protein; optionally, the CD40 agonist is selected from the group consisting of: monoclonal antibodies Mitazalimab, Cifurtilimab, Sotigalimab, and bispecific antibody Tecaginlimab.
8. The novel dendritic cell preparation according to claim 1, wherein the dendritic cells are derived from human peripheral blood monocytes.
9. The novel dendritic cell preparation according to claim 1, wherein the dendritic cells in the preparation are further loaded with a tumor neoantigen or a tumor associated antigen, optionally, wherein the preparation further comprises a cell cryopreservation solution, a cell protectant, or a pharmaceutically acceptable carrier.
10. A method for preparing novel dendritic cells, comprising: blocking and activating dendritic cells with a treatment agent comprising PD-L1 antibodies and CD40 agonists.
11. The preparation method according to claim 10, wherein the PD-L1 antibody is selected from a PD-L1 monoclonal antibody or a multispecific antibody targeting PD-L1; optionally, the PD-L1 antibody is selected from monoclonal antibodies Atezolizumab, Adebrelimab, Durvalumab, or Avelumab; and wherein the CD40 agonist is selected from a CD40 agonist antibody or CD40 ligand, wherein the CD40 agonist antibody comprises a CD40 monoclonal antibody and a multispecific antibody targeting CD40, and the CD40 ligand comprises a CD40L recombinant protein or a fusion protein; optionally, the CD40 agonist is selected from the group consisting of: monoclonal antibodies Mitazalimab, Cifurtilimab, Sotigalimab, and bispecific antibody Tecaginlimab.
12. The preparation method according to claim 10, wherein a working concentration of the PD-L1 antibody is 1-50 g/mL, and a working concentration of the CD40 agonist is 0.1-10 g/mL, optionally, wherein a working concentration of the PD-L1 antibody is 10-20 g/mL, and a working concentration of the CD40 agonist is 1-5 g/mL.
13. The preparation method according to claim 10, wherein the blocking and activating comprises the step of adding the treatment agent to the dendritic cells and co-incubating them; optionally, a co-incubation condition is: a temperature of 35-38 C., and 5% CO.sub.2.
14. The preparation method according to claim 10, further comprising a step of promoting maturation culture of dendritic cells, optionally, wherein the dendritic cells are subjected to blocking and activation treatment during or after the maturation promoting culture process; optionally, the blocking and activation treatment is performed 1 to 6 hours before completion of the culture.
15. The preparation method according to claim 10, further comprising a step of loading a tumor neoantigen and/or a tumor associated antigen onto the dendritic cells, optionally wherein the dendritic cells are loaded with tumor neoantigen and/or tumor associated antigen before the blocking and activation treatment.
16. The preparation method according to claim 10, wherein the dendritic cells subjected to the blocking and activation treatment are PD-L1 negative dendritic cells and dendritic cells with CD40 agonists bound to the cell surface.
17. A dendritic cell preparation prepared by the preparation method according to claim 16, wherein the proportion of the PD-L1 negative dendritic cells is 80% of the total cells in the preparation, and the proportion of dendritic cells with CD40 agonists bound to the cell surface is 80% of the total cells in the preparation.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
(19) The following provides a detailed description and explanation of the implementation method of the present application through specific embodiments, but the following content should not be understood as any limitation on the present application. The technical terms mentioned in this specification have the same meaning as those commonly understood by those skilled in the art. In case of any conflict, the definition in this specification shall prevail. Under the inspiration of the present application, those ordinary skilled in the art can make various forms without departing from the protection scope of the purpose and claims of the present application, all of which fall within the scope of protection of the present application.
(20) Secondly, the examples or specific embodiments hereafter refer to specific features, structures, or characteristics that can be included in at least one embodiment of the present application. The term in one example or in one embodiment used in different parts of this specification does not necessarily refer to the same example or embodiment, nor is it a separate or selective example or embodiment that is mutually exclusive with other examples or embodiments. The preferred embodiment of the present application described in the following specification is intended for the general principles of the specification and is not intended to limit the scope of the present application. The protection scope of the present application shall be determined by the attached claims.
(21) The present application relates to a dendritic cell preparation comprising PD-L1 negative dendritic cells and/or dendritic cells with CD40 agonists bound to the cell surface.
(22) In specific embodiments, the proportion of PD-L1 negative dendritic cells in the cell preparation is 80% of the total cells. For example, the proportion of PD-L1 negative cells is 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%; and/or the proportion of dendritic cells with CD40 agonists bound to the cell surface is 80% of the total cells in the preparation. For example, the proportion of cells with CD40 agonists bound to the cell surface is 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
(23) In specific embodiments, the PD-L1 negative dendritic cells are dendritic cells with PD-L1 antibodies bound to the cell surface.
(24) In specific embodiments, the PD-L1 antibody is selected from PD-L1 monoclonal antibody or multispecific antibody targeting PD-L1.
(25) In some specific embodiments, the PD-L1 monoclonal antibody is selected from monoclonal antibodies Atezolizumab, Adebrelimab, Durvalumab, or Avelumab.
(26) In specific embodiments, the CD40 agonist is selected from CD40 agonist antibodies or CD40 ligands, wherein the CD40 agonist antibodies include CD40 monoclonal antibodies and multispecific antibodies targeting CD40, and the CD40 ligands include CD40L recombinant proteins or fusion proteins.
(27) In some specific embodiments, the CD40 agonist is selected from CD40 monoclonal antibodies Mitazalimab, Cifurtilimab, Sotigalimab, or bispecific antibody Tecaginlimab.
(28) In specific embodiments, the dendritic cells are dendritic cells that bind PD-L1 antibody and CD40 agonist on the cell surface, or dendritic cells that bind PD-L1 and CD40 bispecific antibody, or dendritic cells that bind multispecific antibody targeting PD-L1 and CD40.
(29) In specific embodiments, the dendritic cells are derived from human peripheral blood monocytes.
(30) In a specific embodiment, in the preparation provided by the present application, the proportion of CD80, CD86, HLA-DR, or CD83 positive cells to the total cells in the preparation is 50%, preferably 80%.
(31) In a specific embodiment, in the preparation provided by the present application, the proportion of CD14 positive cells to the total cells in the preparation is 20%, preferably 10%.
(32) In a specific embodiment, in the preparation provided by the present application, the sum proportion of CD3, CD19, and CD56 positive cells to the total cells in the preparation is 20%, preferably 10%, and further preferably 5%.
(33) In specific embodiments, the dendritic cells in the preparation may also be loaded with a tumor neoantigen and/or a tumor associated antigen.
(34) In a specific embodiment, tumor antigens can be loaded during the maturation promoting phase of monocyte culture. The methods of loading antigens include but are not limited to adding antigen polypeptides or electroporation of mRNAs encoding antigens.
(35) In specific embodiments, the preparation of the present application further comprises a cell cryopreservation solution, a cell protectant, or a pharmaceutically acceptable carrier.
(36) In the present application, those skilled in the art can fully understand that any cell cryopreservation solution can be used to preserve the preparation of the present application. Those skilled in the art can choose based on existing commercially available cell cryopreservation solutions, or choose based on literature reports, or prepare using commercially available cell cryopreservation solutions themselves. Those skilled in the art can fully understand that any cell protectant can be used to preserve the preparation of the present application. Those skilled in the art can choose based on existing commercially available cell protectants, choose based on literature reports, or prepare using commercially available cell protectants themselves.
(37) In some specific embodiments, the preparation of the present application comprises a cell cryopreservation solution and a cell protectant, wherein the composition of the cell cryopreservation solution and cell protectant is 40-90% CryoStor CS10 cryopreservation solution+530% human serum albumin solution+530% NaCl injection.
(38) The present application also relates to a method for preparing dendritic cells, comprising: blocking and/or activating dendritic cells with a treatment agent comprising PD-L1 antibody and/or CD40 agonist.
(39) In specific embodiments, the PD-L1 antibody is selected from PD-L1 monoclonal antibodies or multispecific antibodies targeting PD-L1.
(40) In specific embodiments, the CD40 agonist is selected from CD40 agonist antibodies or CD40 ligands, wherein the CD40 agonist antibodies include CD40 monoclonal antibodies and multispecific antibodies targeting CD40, and the CD40 ligands include CD40L recombinant proteins or fusion proteins.
(41) In specific embodiments, the treatment agent includes PD-L1 antibodies and CD40 agonists. For example, the treatment agent can consist of one of the following combinations: PD-L1 monoclonal antibodies with CD40 agonist monoclonal antibodies, bispecific antibodies or multispecific antibodies targeting CD40 or CD40L recombinant proteins; or PD-L1 and CD40 bispecific antibodies; multispecific antibodies targeting PD-L1, CD40, and other sites; or a combination of bispecific antibodies or multispecific antibodies targeting PD-L1 with CD40 monoclonal antibodies or CD40L recombinant proteins.
(42) In some specific embodiments, the PD-L1 monoclonal antibody is selected from monoclonal antibodies Atezolizumab, Adebrelimab, Durvalumab, or Avelumab.
(43) In some specific embodiments, the CD40 agonists are selected from CD40 monoclonal antibodies Mitazalimab, Cifurtilimab, Sotigalimab, or bispecific antibody Tecaginlimab.
(44) The PD-L1 antibody, CD40 agonist antibody, CD40 ligand, and other agents described in the present application can be obtained by purchasing commercially available products (e.g., monoclonal antibodies Atezolizumab and Mitazalimab), or can be synthesized by those skilled in the art according to conventional methods.
(45) In specific embodiments, the working concentration of the PD-L1 antibody is 1 g/mL, preferably 1-50 g/mL, further preferably 10-20 g/mL, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 g/mL.
(46) In specific embodiments, the working concentration of the CD40 agonist is 0.1 g/mL, preferably 0.1-10 g/mL, further preferably 1-5 g/mL, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 g/mL.
(47) In specific embodiments of the present application, the blocking and/or activation treatment comprises the step of adding a treatment agent such as PD-L1 antibodies and/or CD40 agonists to dendritic cells and co-incubating them.
(48) In some specific embodiments, the co-incubation condition is at the temperature of 35-38 C. and 5% CO.sub.2. In a specific embodiment, the co-incubation condition is at the temperature of 37 C. and 5% CO.sub.2.
(49) In the embodiments of the present application, the preparation method further comprises a step of maturation promoting culture of the dendritic cells. Further preferably, the blocking and/or activation treatment is performed on mature dendritic cells prior to completion of the culture.
(50) In specific embodiments, mature dendritic cells for use in the dendritic cell preparation of the present application or for use in the preparation method of the present application can be obtained through the following methods:
(51) Mature dendritic cells were obtained by culturing monocytes from human peripheral blood or CD34+ progenitor cells from human bone marrow or umbilical cord blood, using serum-free medium in combination with a differentiation promoting cytokine composition and a maturation promoting cytokine composition.
(52) The serum-free culture medium used in the present application has no specific brand or ingredient restrictions, and any common serum-free culture medium in the art can be used. Serum comprises various plasma proteins, polypeptides, carbohydrates, growth factors, hormones, etc. Due to the complexity and variability of serum components between batches, consistency of ingredients cannot be guaranteed. In addition, while serum contains many beneficial ingredients for cell growth, it also includes potentially harmful ingredients, such as complement, antibodies, endotoxins, etc. Consequently, cells cultured with high concentration serum are not suitable for clinical use, as this increases the risk of clinical allergies. In contrast serum-free culture medium has no adverse effects on the growth, differentiation, morphology, and function of dendritic cells.
(53) In a specific embodiment, the serum-free culture medium used is a culture medium with explicit chemical ingredients and without animal derived ingredients. The manufacturing process, ingredient testing, and release of the product comply with GMP guidelines and can be used for in vitro preparation of clinical grade dendritic cell drugs.
(54) After obtaining the progenitor cells, the cells are suspended in serum-free medium at a concentration of 1-510.sup.6 cells/mL, and the differentiation promoting cytokine composition of rhGM-CSF (recombinant human granulocyte-macrophage colony stimulating factor) and rhIL-4 (recombinant human interleukin 4) are added. The specific composition can be referred to reference 1 [Laura Ridolfi, Francesco de Rosa, Laura Fiammenghi et al. Complementary vaccination protocol with dendritic cells pulsed with autologous tumour lysate in patients with resected stage III or IV melanoma: protocol for a phase II randomised trial (ACDC Adjuvant Trial). [J]. BMJ Open, 2018, 8(8)]. The cell suspension is transferred into a culture flask and cultured in a 37 C., 5% CO.sub.2 incubator for 2-3 days before being supplemented with culture medium. The culture is continued for 2-3 days to obtain immature dendritic cells (imDCs). Then, the imDCs are induced into mature DC cells using a maturation promoting cytokine composition (conventional maturation promoting cytokine compositions are used, including but not limited to rhGM-CSF, rhIL-4, rhTNF-, etc.). In specific embodiment, the maturation promoting cytokine composition can adopt the composition described in the above literature.
(55) In specific embodiments, the dendritic cells are subjected to the blocking and/or activation treatment during or after the maturation promoting culture process. In a specific embodiment, the treatment is performed 1 to 6 hours prior to completion of the culture.
(56) In the embodiments of the present application, the preparation method further comprises the step of loading a tumor neoantigen or a tumor associated antigen onto the dendritic cells.
(57) In a specific embodiment, the antigen is loaded before the blocking and/or activation treatment (immature dendritic cell stage). The methods of loading antigens include but are not limited to adding antigen polypeptides to the cell culture system or electroporation mRNA encoding an antigen into the dendritic cells.
(58) In a specific embodiment, the tumor neoantigen polypeptides are dissolved according to their respective solubility properties and added to the imDC cell suspension for co-incubation and culture.
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(60) In specific embodiments of the present application, the dendritic cells subjected to the blocking and/or activation treatment are PD-L1 negative and/or with CD40 agonists bound to the cell surface.
(61) In a specific embodiment, the dendritic cell preparation provided by the present application is prepared by the following method:
(62) Monocytes from humans are cultured in serum-free medium in combination with the differentiation promoting cytokine composition and the maturation promoting cytokine composition to obtain dendritic cells with mature phenotypes;
(63) Optionally, during the maturation promoting stage, tumor neoantigens or tumor associated antigens can be loaded, ways for antigen loading include but are not limited to adding antigen polypeptides or electroporation of mRNA encoding an antigen;
(64) Dendritic cells with mature phenotypes and loaded with antigens are co-cultured with CD40 agonist antibody and/or PD-L1 antibody (or multispecific antibody targeting at least one of PD-L1 and CD40);
(65) Optionally, the treated cells are washed, purified and suspended in a cell cryoprotectant to obtain dendritic cell preparations.
(66) The positive/negative expression levels of molecular markers such as PD-L1, CD80, CD86, HLA-DR, CD83, CD14, CD3, CD19, CD56, and the binding rate of CD40 agonists in the present application can be determined by conventional cellular and molecular biology detection methods in the art. For example, the expression levels of molecular markers such as PD-L1, CD80, CD86, HLA-DR, CD83, CD14, CD3, CD19, and CD56 on the surface of the dendritic cells, as well as binding rate of the CD40 agonists, can be determined by detection methods such as flow cytometry (FACS), immunofluorescence (IF), immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), and Western blot. Those skilled in the art can also use other appropriate detection methods to determine. There are no restrictions in the present application. Furthermore, the above detection methods can also be used to determine various functional indicators of the dendritic cells.
(67) For example, in the present application, flow cytometry (FACS) can be used to detect the proportion of PD-L1 positive cells in the dendritic cell preparation of the present application, and then subtract the number of PD-L1 positive cells from the total number of cells to calculate the proportion of PD-L1 negative cells. Of course, those skilled in the art can also understand that based on the different methods used, the proportion of PD-L1 negative cells in the cell preparations can be directly detected.
(68) In the present application, flow cytometry (FACS) can be used to detect the number of cells in the dendritic cell preparation that have surface-bound CD40 agonists, and further calculate their proportion in the cell preparation.
(69) In a specific embodiment, immunofluorescence (IF) can be used to detect the number of PD-L1 positive cells in the preparation, as well as the number of cells that have surface-bound CD40 agonists, and further calculate the proportion of PD-L1 negative cells to the total cells in the preparation and the proportion of cells that have surface-bound CD40 agonists to the total cells in the preparation.
(70) In a specific embodiment, cell surface enzyme-linked immunosorbent assay (ELISA) can be used to detect the number of PD-L1 positive cells in the preparation, as well as the number of cells that have surface-bound CD40 agonists, and further calculate the proportion of PD-L1 negative cells to the total cells in the preparation and the proportion of cells that have surface-bound CD40 agonists to the total cells in the preparation.
(71) In specific embodiments of the present application, flow cytometry is used to detect the proportion of PD-L1 and CD40 molecules on the surface of dendritic cells bound to their respective antibodies. For CD40, the binding level of antibody is directly detected by fluorescently labeled anti-human IgG antibodies; while for PD-L1, the binding level of PD-L1 antibody is indirectly detected by detecting the unblocked PD-L1 sites on the cell surface using fluorescently labeled PD-L1 antibodies.
(72) In a specific embodiment of the present application, the flow cytometry detection method used is as follows:
(73) The sample cells are added to PBS, washed and centrifuged, after which the supernatant is discarded. The cells are then resuspended in PBS to the appropriate concentration. A dead/live cell stain is added, and mixed well, incubated at room temperature in dark for 10 minutes. After incubation, PBS is added, followed by centrifugation, washing and discarding the supernatant. Cells are resuspended in Cell Staining Buffer, and the appropriate flow cytometry antibodies are added to the tubes of each group (as described in the method section of reference 2 [Sara Nava, Marta Dossena, Simona Pogliani, et al. An optimized method for manufacturing a clinical scale dendritic cell-based vaccine for the treatment of glioblastoma. [J]. PLoS One, 2012, 7:12.]). The mixture is vortexed and incubated in the dark at room temperature for 30 minutes. After incubation, PBS is added to each tube and mixed, centrifuged, washed and the cells are resuspended. The samples are analyzed by flow cytometry to determine the positive rates of various cell surface markers such as CD80, CD86, HLA-DR, CD83, CD3/CD56/CD19, CD14, PD-L1, and the binding rate of cells bound with CD40 agonists. In the present application, there are no specific limitations on the flow cytometer used.
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(75) For the dendritic cell preparation provided by the present application, the expression of PD-L1 molecular markers on the surface of dendritic cells is detected by flow cytometry. Preferably, the proportion of PD-L1 positive dendritic cells is reduced to 20% (i.e., the PD-L1 antibody blocking efficiency is 80%). In a specific embodiment, the dendritic cell PD-L1 positivity rate blocked with PD-L1 monoclonal antibody decreases from 99.99% before treatment to 5.27% after treatment (as shown in
(76) In addition, the ability of antigen-specific T cells activated by dendritic cells (T cells secreting IFN- cytokines are considered antigen-specific T cells activated by dendritic cells) is detected using the classic Elispot method (enzyme-linked immunospot). The number of activated T cells stimulated by dendritic cells activated by CD40 monoclonal antibody is 35.5% higher than that in the untreated group (as shown in
(77) Compared with conventional dendritic cells, the innovation of the present application lies in co-culturing dendritic cells with mature cell phenotypes and loaded with antigens with CD40 agonists and/or PD-L1 antibodies (or multispecific antibody comprising at least one of CD40 and PD-L1 antibodies), followed by washing, purification, and cryopreservation to obtain a dendritic cell injection with strong anti-cancer/cancer prevention effects. Preferably, the ingredients of the cryoprotectant for dendritic cells are 40-90% CryoStor CS10 cryopreservation solution+530% human serum albumin solution+530% NaCl injection. Preferably, the proportion of PD-L1 negative dendritic cells is 80%, and the proportion of dendritic cells bound with CD40 agonists is 80%.
(78) Compared with the conventional culture method of dendritic cells, the present application innovatively adds PD-L1 antibody (or multispecific antibody targeting PD-L1) and CD40 agonists (including CD40 agonist monoclonal antibody or multispecific antibody targeting CD40 or CD40L recombinant proteins, etc.) to enhance the function of dendritic cells during the preparation stage of dendritic cells. On one hand, it blocks the PD-L1 molecules on the surface of mature dendritic cells, enhancing the ability of dendritic cells to activate T cells; on the other hand, it activates CD40 receptors on the surface of dendritic cells, increases the expression of co-stimulatory molecules and antigen presentation efficiency, and enhances their ability to stimulate and activate T cells.
(79) In order to further demonstrate the ability of dendritic cells in the present application to activate naive antigen-specific T cells (CTL cells), as well as the ability of activated CTL cells to specifically recognize and kill tumor target cells, the following experiments are designed for verification:
(80) KLE, a human endometrial carcinoma cell line expressing p53.sup.R175H neoantigen (HMTEVVRHC), is used as the target cell, and CFPAC-1, a human pancreatic cancer cell line not expressing the neoantigen, is used as the negative control cell (both cell lines are HLA-A*02:01 subtype). ImDC cells cultured from healthy donor monocytes of the same HLA subtype are used to prepare Neo-DC cells (mature dendritic cells loaded with p53.sup.R175H neoantigen polypeptide during the maturation promoting stage and treated with CD40 and PD-L1 antibodies) and Mock DC cells (mature dendritic cells loaded with the same but not treated with PD-L1 and CD40 monoclonal antibodies as a control), respectively. CD8+T cells from the same healthy donor are stimulated in vitro for 10 days using Neo-DC and Mock DC cells to obtain p53.sup.R175HT T cells and control T cells. The composition of the CD8.sup.+ T cells obtained from in vitro stimulation is analyzed using flow cytometry, and the killing effect of these cells on target cells and negative control tumor cells is evaluated. The experimental results confirm that, Neo-DC cells exhibit superior induction of CD8+T cell activation compared to Mock DC cells, generating antigen-specific T cells (CTL cells) with a higher proportion of effector memory T cell subpopulations. These CTL cells have a strong ability to kill tumor cells.
(81) In order to evaluate the anti-tumor effect of dendritic cells in the tumor model of humanized huPBMC-NCG mice subcutaneously transplanted with human pancreatic cancer cell CFPAC-1, the following experiment is designed: CFPAC-1 cells in logarithmic growth phase is collected and subcutaneously inoculated into huPBMC-NCG mice. When the average volume of tumors in mice reaches a predefined volume, the mice are randomly divided into 3 groups (G1-G3) based on their body weight and tumor volume, with 6 mice in each group. Each mouse is injected intraperitoneally with 100 L PBMC of HLA-A*02:01 subtype for humanized immune system reconstruction. The day of injection is set as day DO. At the same time, monocytes from the same donor source are used to prepare Neo-DC cells (mature dendritic cells loaded with neoantigen polypeptide generated by human pancreatic cancer cell KRAS.sup.G12V mutation and treated with CD40 antibody and PD-L1 antibody) and Mock DC cells (mature dendritic cells loaded with the same neoantigen polypeptide but not treated with CD40 antibody and PD-L1 antibody) according to the cell preparation process of the present application. The vehicle is injected subcutaneously into the mice in G1 group that have completed immune reconstitution as a control; Mock DC cells are injected subcutaneously into the mice in G2 group; the same dose of Neo-DC cells are injected subcutaneously into the mice in G3 group. The administration method is subcutaneous two-point injection in the groin area of the hind legs of mice, with an administration frequency on D14, D18, D22, and D30 after completing immune reconstitution, for a total of 4 doses. The pharmacodynamic evaluation of the test substance in the tumor model of humanized huPBMC-NCG mice with subcutaneous transplantation of human pancreatic cancer cell CFPAC-1 is comprehensively assessed based on the observation indicators such as changes in tumor volume, body weight, as well as CBA analysis, GvHD scoring, and peripheral blood immune cell detection results during and after administration. The experimental results finally show that repeated subcutaneous injections of Neo-DC cells and Mock DC cells in human PBMC reconstituted immunodeficiency mice (at the same time, human pancreatic cancer cells are subcutaneously tumorigenic) both have an effect on tumor lesion growth inhibition, and there are significant statistical differences compared with the vehicle control, and the effect of Neo-DC cells is more significant than that of Mock DC cells on tumor growth inhibition. This indicates that subcutaneous injection of Neo-DC cells has a stronger effect on inhibiting and treating human pancreatic cancer lesions in humanized huPBMC-NCG mice.
(82) Human clinical trials are carried out using one or more of the several injection single drugs of Neo-DC cell injection in the present application, or combined with PD-1 antibodies, PD-L1 antibodies, temozolomide, etc., and the results show that Neo-DC cell injection has good clinical safety, and whether used alone or in combination with other drugs, it has certain clinical efficacy for patients with pancreatic cancer, liver cancer, and glioblastoma.
(83) The above indicates that the Neo-DC cell injection of the present application has good anti-cancer and cancer prevention activity, as well as clinical accessibility, and has broad application prospects in the field of immunotherapy for cancer and other diseases; and it can be used alone or in combination with PD-1 antibodies, PD-L1 antibodies, temozolomide and other radiochemotherapy drugs for clinical treatment of cancer or postoperative recurrence prevention. The cell drugs and/or combination drugs of the present application can be applied to solid tumors such as breast cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, nasopharyngeal cancer, gastric cancer, colorectal cancer, renal cancer, bladder cancer, prostate cancer, sarcoma, esophageal cancer, cervical cancer, gallbladder cancer, glioblastoma or melanoma, as well as blood cancers such as leukemia, lymphoma and multiple myeloma. Compared with the existing traditional dendritic cell culture methods, the present application significantly improves the maturity, antigen presentation ability, and the ability to activate specific T cells, by adding PD-L1 antibodies and/or CD40 agonists during the dendritic cell culture process, thereby enhancing their anti-cancer and cancer prevention effects in clinical practice.
Examples
(84) The present application provides a general and/or specific description of the materials and testing methods used in the experiments. In the following examples, unless otherwise specified, % represents volume percentage. The reagents or instruments used without specifying the manufacturer are all conventional reagent products that can be commercially obtained. The imDC cells and mDC cells are respectively immature dendritic cells and mature dendritic cells cultured using the methods described in Example 1 and Example 2 of the present application. The Neo-DC cells described in the present application are mature dendritic cells loaded with corresponding antigens cultured using the method described in Example 6 of the present application.
Example 1: Preparation of Monocytes and Culture Method of imDC Cells (Immature Dendritic Cells)
(85) The culture method of imDC cells includes four steps: apheresis or peripheral blood acquisition, PBMC separation and purification, monocyte sorting and purification, and imDC cell culture.
(86) 1. Apheresis or Peripheral Blood Acquisition
(87) On the day of collection or the day before, donors were screened for routine blood count, coagulation and infectious disease to confirm that the donors had no active infectious diseases such as hepatitis B, hepatitis C, syphilis and HIV, etc. Select the white blood cell mode on the blood cell separator for apheresis or direct intravenous collection of peripheral blood. After transporting the collected blood samples to the laboratory, the blood samples that had been preliminarily confirmed to be qualified would proceed to the next step of cell sorting, strictly following aseptic procedures.
(88) 2. PBMC Separation and Purification
(89) The day on which the blood samples were processed was designated as Day 0 of the culture. Plasma was separated from the blood sample, and an appropriate amount of PBS solution was pipetted to the blood cell sample. The diluted blood was slowly loaded onto the Ficoll solution surface and centrifuged for 30 minutes. After centrifugation, the white membrane layer was carefully transferred into a new centrifuge tube using a pipette. PBS was added to dilute and mix the sample, followed by centrifugation and washing. The cell pellet was then resuspended in sorting buffer to obtain PBMC suspension.
(90) 3. Monocyte Sorting and Purification
(91) Using MACS technology for sorting the monocytes, the specific purification process was as follows:
(92) Based on the PBMC cell count obtained in the previous step, the cells were adjusted to the appropriate concentration using a sorting buffer. CD14 magnetic beads were added, mixed thoroughly, and incubated in a 2-8 C. for 15 minutes. Following incubation, 10-20 mL of buffer per 110.sup.8 cells was added, mixed well and centrifuged. The supernatant was discarded, buffer was added to resuspend the cells, and the volume was adjusted before centrifuging again. After discarding the supernatant, the cells were resuspended in the buffer and mixed thoroughly. The cell sorting column was placed in the sorting magnetic rack, rinsed with buffer, and the cell suspension was applied to the sorting column at a uniform rate. Once all the cell suspensions had passed through, the column was washed with buffer and removed from the magnetic rack. CD14+cells were collected from the column effluent, centrifuged, and resuspended in a serum-free culture medium to obtain the CD14+monocyte suspension. The cell concentration and viability were then measured.
(93) 4. Obtaining imDC Through Monocyte Differentiation Promoting Culture
(94) The monocyte suspension obtained from the above steps was adjusted to a concentration of 1-510.sup.6 cells/mL using the serum-free culture medium. Recombinant human GM-CSF (rhGM-CSF) and recombinant human IL-4 (rhIL-4) were added as differentiation-promoting cytokines. The cell suspension was transferred to a culture flask and incubated at 37 C. in a 5% CO.sub.2 incubator. After 2-3 days of incubation, additional medium was supplemented, and culturing continued for another 2-3 days to obtain immature dendritic cells (imDCs).
Example 2: Culture, Antigen Loading, and Treatment Method of mDC Cells (Mature Dendritic Cells)
(95) imDC cells were collected and subjected to a maturation promoting culture. The required amount of cytokines was calculated based on the volume of the maturation promoting medium, and each maturation promoting cytokine was added to the serum-free medium (the maturation promoting cytokine composition included, but was not limited to rhGM-CSF, rhIL-4, rhTNF-, etc., for example, as described in reference 1 above). Antigen polypeptides (specific selection can be found in the following examples) were dissolved according to their respective solubility properties, filtered through a membrane, and added to the imDC cell suspension. The imDC cells were then transferred to a culture flask and incubated at 37 C., in a 5% CO.sub.2 incubator for further maturation.
(96) On day 6 or 7 of the dendritic cell (DC) maturation culture, approximately one day prior to completion of the maturation process, PD-L1 monoclonal antibody and a DC cell agonist (specific examples are provided below) were added to the culture medium at the specified working concentration. The culture was incubated at 37 C. in a 5% CO.sub.2 incubator for 1-6 hours. After incubation, the cells were examined under an inverted microscope to observe and record the morphological characteristics of the mature cells. The cells were then collected into centrifuge tubes, centrifuged, and the pellets were retained for quality control. The cell pellets were resuspended with a washing solution, and the total cell count was determined. Based on the count, an aliquot was taken for the assessment of live cell concentration, viability, and the expression of surface markers, including CD80, CD86, HLA-DR, CD83, and PD-L1. After sampling, the cells were centrifuged again, and the supernatant was discarded to prepare the cells for subsequent experiments.
(97) Furthermore, Cryoprotectants were prepared according to clinical concentration standards, using the following composition: 40-90% CryoStor CS10 cryopreservation solution, 5-30% human serum albumin solution, and 5-30% NaCl injection. Based on the results of cell counting, the cell pellets were resuspended in the cryoprotectant, and an appropriate sample was taken for safety testing. The cell suspension in the cryoprotectant was then accurately pipetted, aliquoted, and cryopreserved according to established protocols.
Example 3: Determination of PD-L1 Monoclonal Antibody Treatment on Enhancing mDC Cell Function
(98) In this example, two commercially available humanized monoclonal antibodies, Adebrelimab and Atezolizumab, were utilized. The experiment first confirmed whether co-incubation of PD-L1 monoclonal antibodies with mature dendritic cells (mDCs) effectively reduced the proportion of PD-L1-positive mDCs. Additionally, it was assessed whether this co-incubation enhanced the ability of mDCs to activate T cells.
(99) Experimental method: using the methods described in Examples 1 and 2, monocytes from HLA-A*02:01 subtype healthy donors were cultured in vitro to obtain imDC cells. These imDCs were then loaded with cytomegalovirus CMV polypeptides and subjected maturation promoting culture. The cells were divided into the following experimental groups for PD-L1 antibody treatment: A. control (without PD-L1 antibody in the culture system); B. Adebrelimab treatment; C. Atezolizumab treatment. For both treatment, the PD-L1 antibodies were added to the culture system one day prior to completion of mDC cell culture and incubated at 37 C. for 3 hours. After incubation, the mature dendritic cells (mDCs) were collected, washed, and subjected to cell counting and viability testing, along with flow cytometric analysis of cell phenotypes. The blocking effect of PD-L1 molecules on the surface of DC was analyzed, and the ability of mDC cells to activate T cells was assessed using Elispot. The results of the three batches of experiments are shown in Table 1 and
(100) TABLE-US-00001 TABLE 1 statistics of mDC cell detection data in the control group and PD-L1 monoclonal antibody treatment group Cell PD-L1 monoclonal Number of cells culture antibody PD-L1.sup.+ secreting IFN- batch (2 g/mL) (%) (per million) 1.sup.st control group 99.94 1095 Adebrelimab group 2.39 1780 Atezolizumab group 2.56 1825 2.sup.nd control group 99.97 2640 Adebrelimab group 0.13 3080 Atezolizumab group 0.18 2985 3.sup.rd control group 99.88 1715 Adebrelimab group 1.52 1925 Atezolizumab group 1.38 1950
(101) The experimental results show that, at a PD-L1 monoclonal antibody concentration of 2 g/mL, a significant difference exists in the T cell activation capacity of mDC cells between the control, the Adebrelimab treatment, and the Atezolizumab treatment. However, there is no significant difference between the two antibody treatments. The data on the number of T cells that secrete IFN-, activated by mDC cells analyzed using Elispot (
(102) Subsequently, the working concentration of the Atezolizumab monoclonal antibody was explored and optimized, and the blocking effect of monoclonal antibodies of different gradient concentrations for PD-L1 molecules on the surface of DC was tested. The results are shown in Table 2 and
(103) TABLE-US-00002 TABLE 2 detection results of the proportion of PD-L1.sup.+ mDC cells treated with different concentrations of PD-L1 monoclonal antibody Concentration of Atezolizumab proportion of PD-L1.sup.+ mDC cell monoclonal antibody (ug/mL) (%) 50 0.87 15 0.95 1 1.74 0.1 18.96 0.01 87.52 0.001 95.63
(104) The results show that as the concentration of PD-L1 monoclonal antibody treatment increases, the proportion of PD-L1.sup.+ mDC cells decreases, showing an S-shaped curve; when the concentration of Atezolizumab monoclonal antibody is 1 g/mL, it can block over 98% of PD-L1 molecules on the surface of mDC cells.
Example 4: Determination of DC Cell Agonist Treatment on Enhancing mDC Cell Function
(105) Five DC cell agonists were selected for this example: agonist A (OK432), agonist B (PolyI: C), agonist C (CD40 monoclonal antibody Cifurtilimab), agonist D (CD40 monoclonal antibody Mitazalimab), and agonist E (CD40L recombinant protein). The objective was to verify the effects of these different agonists on the quality and function of mature dendritic cells (mDCs).
(106) Experimental method: Utilizing the methods described in Examples 1 and 2, monocytes from HLA-A*02:01 subtype healthy donors were cultured in vitro to obtain imDC cells, which were then loaded with cytomegalovirus CMV polypeptides and subjected to imDC cell maturation promoting culture. On the last day of dendritic cell maturation promoting culture, different agonists (working concentration of 2 g/mL) were added to the culture medium according to the settings in the five experimental treatment groups mentioned above, alongside a control treatment (conventional method for maturation promoting culture) was set up. After culture, mDC cells were harvested and washed, followed by counting and survival rate determined, flow cytometry for phenotypic analysis, and Elispot assays to evaluate the ability of mDCs to activate T cells. Differences among groups were analyzed, comparing yield, survival rate, cytokine secretion and maturity, as well as antigen presentation to determine the impact of various agonists on the quality and function of mDC. The experimental results are shown in Table 3 and
(107) TABLE-US-00003 TABLE 3 statistics of mDC cell detection data in different agonist treatment groups Number of survival cells mDC rate of secreting yield mDC CD83+ IL-12p70 IFN- Agonist (%) (%) (%) (pg/mL) (per million) Control group 20.7 85.8 81.00 50.0 1370 Agonist A 20.7 84.6 83.40 409.8 1630 OK432 Agonist B 18.0 81.3 82.64 396.9 1675 PolyI: C Agonist C 20.6 89.1 93.24 1491.7 2205 CD40 monoclonal antibody Cifurtilimab Agonist D 18.7 88.1 95.73 1468.3 2105 CD40 monoclonal antibody Mitazalimab Agonist E CD40L 19.4 83.7 88.20 874.4 1970 recombinant protein
(108) The experimental results show that, compared to the control group, all five agonists can enhance the maturity, cytokine secretion, and T cell activation ability of DC cells to varying degrees. However, the treatment effect of CD40 monoclonal antibody and CD40L recombinant protein is significantly better than that of OK432 and PolyI: C. The Cifurtilimab group and Mitazalimab group show the most significant improvement in the ability of mDC cells to activate T cells, and there is no significant difference between the two antibody groups, followed by the improvement effect of CD40L. Subsequently, Cifurtilimab monoclonal antibodies were selected for exploration and optimization of working concentrations. The effects of different gradient concentrations of mAb on the maturation phenotype of DC cells (CD83 molecule), IL-12 cytokine secretion, and the enhancement of activated T cells were detected. The results are shown in Table 4 and
(109) TABLE-US-00004 TABLE 4 statistical analysis of data from mDC cells treated with CD40 mAb at various concentrations Concentration mDC mDC Number of cells of Cifurtilimab yield survival CD83+ IL-12p70 secreting IFN- mAb (g/mL) (%) rate (%) (%) (pg/mL) (per million) 0.001 24.6 91.4% 83.37 10.2 1075 0.01 24.1 90.6% 84.64 10.0 1110 0.1 23.3 89.2% 93.29 309.3 1370 1 24.9 91.4% 94.09 312.3 1465 3 24.7 89.9% 95.65 397.1 1575 10 22.7 88.0% 95.73 401.5 1625
(110) The results show that increasing concentrations of CD40 monoclonal antibody treatment, result in increased the secretion of CD83 and IL-12 cytokine, as well as enhanced T cell activation by mDCs, following an S-shaped curve; when the concentration of Cifurtilimab mAb is 0.1 g/mL, it can significantly improve various functional and activity indices of the mDC cells.
Example 5: Combination Treatment of PD-L1 Monoclonal Antibody and DC Cell Agonist for Enhancing mDC Cell Function
(111) This example used PD-L1 monoclonal antibody (Atezolizumab), a combination of PD-L1 monoclonal antibody (Atezolizumab) and CD40 monoclonal antibody (Cifurtilimab), and PD-L1 and CD40 bispecific antibody for experiments (CD40-PD-L1-IgG4 bispecific antibody is a bispecific antibody comprising Fab and Fc synthesized by Sino Biological Inc. commissioned by the applicant, which comprises four polypeptide chains (i.e., two light chains and two heavy chains), and the two branches of the Fab bind to different antigens (i.e., CD40 and PD-L1) respectively. A CrossMab design was adopted, the constant region remained unchanged; the anti-PD-L1 light chain variable region was swapped with the anti-PD-L1 heavy chain variable region, so that the light chain of the anti-CD40 paired with the heavy chain of the anti-CD40, and the light chain of the anti-PD-L1 paired with the heavy chain of the anti-PD-L1. Among them, the sequences of CD40_L, CD40_H, PD-L1_L and PD-L1_H are shown as SEQ ID Nos. 1-4, respectively).
(112) SEQ ID No.1 (CD40_L):
(113) DIQMTQSPSSLSASVGDRVTIKCQASQSISSRLAWYQQKPGKPPKLLIYR ASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQCTGYGISWPIGGGTKVEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(114) SEQ ID No.2 (CD40_H):
(115) QVQLVESGGGVVQPGRSLRLSCAASGFSFSSTYVCWVRQAPGKGLEWI ACIYTGDGTNYSASWAKGRFTISKDSSKNTVYLQMNSLRAEDTAVYFCARPDITYGFAI NFWGPGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(116) SEQ ID No.3 (PD-L1_L):
(117) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLE WMGRIGPNSGFTSYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYD YFDYWGQGTTVTVSSASVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC
(118) SEQ ID No.4 (PD-L1_H):
(119) DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKL LIYAASNLESGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEIK ASTKGPSVFPLAPCSRSTSESTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDEKVESKYGPPCPPCPAPEFEGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEM TKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(120) Experimental method: using the methods described in Examples 1 and 2, monocytes from HLA-A*02:01 subtype healthy donors were cultured in vitro to obtain imDC cells, which were then loaded with cytomegalovirus CMV polypeptide and subjected to imDC cell maturation promoting culture, and the following experimental groups were set: A. control group (conventional DC culture maturation promoting cytokine combination plus PD-L1 monoclonal antibody treatment); B. combination of monoclonal antibodies (conventional DC culture maturation promoting cytokine combination plus PD-L1 monoclonal antibody and CD40 monoclonal antibody treatment); C. bispecific antibody group (conventional DC culture maturation promoting cytokine combination plus PD-L1CD40 bispecific antibody). The antibody treatment method was: the corresponding antibody was added to the culture system and incubated at 37 C. for 3 hours before completion of the mDC cell culture. After incubation, the mDC cells were harvested and washed, followed by counting and survival rate detection, flow cytometry phenotype analysis, and Elispot assay for DC's ability to activate T cells. Differences among groups were analyzed, and yield, survival rate, cytokine secretion and maturity, as well as antigen presentation were compared to determine the impact of monoclonal and/or bispecific antibodies on the quality and function of the mDCs. The experimental results are shown in Table 5 and
(121) TABLE-US-00005 TABLE 5 statistical analysis of mDC cell detection data in each treatment group batch of survival rate Number of cells Cell Antibody mDC yield of mDC CD83.sup.+ IL-12p70 secreting IFN- culture combination (%) (%) (%) (pg/mL) (per million) 1.sup.st control 27.1 88.1 92.69 4.1 2965 monoclonal 27.4 90.1 96.88 450.2 2985 antibody combination bispecific 28.2 88.4 98.77 447.1 3080 antibody 2.sup.nd control 28.8 87.8 64.27 4.8 1535 monoclonal 25.7 88.1 92.29 1215.9 2515 antibody combination bispecific 23.4 89.2 91.62 922.8 2350 antibody 3.sup.rd control 35.2 90.6 89.18 5.7 640 monoclonal 36.1 89.2 97.63 1108.8 1045 antibody combination bispecific 34.9 88.7 97.83 992.3 1065 antibody 4.sup.th control 24.0 83.4 75.24 5.0 2605 monoclonal 24.9 82.5 96.09 453.5 2890 antibody combination bispecific 22.5 79.0 94.29 455.1 3155 antibody
(122) The experimental results show that there is no significant difference in the yield and survival rate of mDC cells among groups of the monoclonal antibody combination, the bispecific antibody, and the control (the mean yield of the control group, the monoclonal antibody combination group, and the bispecific antibody group are 28.8%, 28.5%, and 27.3%, respectively; and the mean survival rate are 87.5%, 87.5%, and 86.3%, respectively). For the DC maturity index (represented by the proportion of CD83.sup.+ cells), the proportions of the mean CD83.sup.+ cells of the three groups are 80.35%, 95.72%, and 95.63%, respectively; indicating that adding combination of two types of monoclonal antibodies or adding a bispecific antibody can significantly improve the cell maturity of DC cells, with the increase rate of CD83 positive ratio being 19.1% and 19.0%, respectively. For the IL-12 cytokine secretion index, the mean values of the three groups are 4.9 pg/mL, 746.3 pg/mL, and 704.1 pg/mL, respectively, indicating that adding two types of monoclonal antibodies or adding a bispecific antibody can both significantly enhance the ability of mDC cells to secrete cytokines. There are also significant differences in the number of IFN- secreting T cells (reflecting the mDC antigen presentation ability) among the three groups, with mean values of 1936, 2359, and 2413, respectively. Compared with the control group, the monoclonal antibody combination group and the bispecific antibody group increase by 21.8% and 24.6%, respectively, indicating that addition of the monoclonal antibody combination or the bispecific antibody can both significantly enhance the ability of DC cells to activate T cells, leading to a significant increase in the number of IFN- secreting T cells. Based on the analysis of the above indexes, it is determined that co-culturing with PD-L1 mAb and CD40 mAb or PD-L1CD40 bispecific antibody before DC harvesting can promote DC maturation, cytokine secretion, and T cell activation without significantly affecting the yield and survival rate of mDC cells.
Example 6: Dendritic Cells Activate Cytotoxic T Lymphocyte (CTL) and Induce their Tumor Cell Killing In Vitro
(123) In order to further demonstrate the ability of dendritic cells presenting tumor neoantigen to activate initial antigen-specific T cells (CTL cells), as well as the ability of activated CTL cells to specifically recognize and kill tumor target cells, the following experiments were designed:
(124) KLE (a human endometrial carcinoma cell line expressing p53.sup.R175H neoantigen (HMTEVVRHC)) was used as the target cell, and CFPAC-1 (a human pancreatic cancer cell line not expressing the neoantigen) was used as the negative control cell (both cell lines are HLA-A*02:01 subtype). Monocytes from healthy donor of the same HLA subtype were cultured to obtain imDC cells, the imDC cells were then loaded with antigen (in this example, the antigen was p53.sup.R175H neoantigen polypeptide), and treated with CD40 antibody (Cifurtilimab, 2 g/mL) and PD-L1 antibody (Atezolizumab, 2 g/mL) during the maturation promoting stage to obtain mature dendritic cells (in the following examples, mature dendritic cells loaded with corresponding antigens and treated by this method are referred to as Neo-DC cells); and mature DC cells from the same source but without PD-L1 monoclonal antibody and CD40 monoclonal antibody treatment were taken as control cells (hereinafter referred to as Mock DC). The specific culture, loading, and antibody treatment methods are as described in Examples 1 and 2. CD8.sup.+ T cells from the same healthy donor were stimulated in vitro for 10 days by either Neo-DC or Mock DC cells to obtain p53.sup.R175HT T cells and control T cells. The composition of the CD8+T cells obtained from such in vitro stimulation was analyzed using flow cytometry, and the killing effect of these cells on target cells and negative control tumor cells was detected. The experimental results are shown in
(125) Results and analysis: the cell surface glycoprotein CD69 and chemokine receptor CXCR3 are important markers of effector T cell activation. Memory is one of the important biological characteristics of effector T cells, and the memory of T cells is formed during the proliferation and differentiation of antigen-specific T cell clones. After activation, Naive T cells (CCR7.sup.+CD45RA.sup.+) can differentiate into central memory T cells (CCR7.sup.+CD45RA.sup., T.sub.CM), effector memory T cells (CCR7.sup.CD45RA.sup., T.sub.EM), and terminally differentiated T effector memory cells re-expressing CD45RA (T.sub.eff, CCR7.sup.CD45RA.sup.+). The reactive memory of T cells is mediated by T.sub.CM, which can respond to antigen stimulation to proliferate and differentiate into effector cells. Protective memory is mediated by T.sub.EM and can migrate to peripheral tissues for immediate effects when needed. Flow cytometry analysis of CD8.sup.+ T cells derived from the same healthy donor and stimulated with either Neo-DCs or Mock DCs for 10 days, with pre-stimulation CD8.sup.+ T cells serving as a baseline sample. The results show that the CXCR3 expression level on the surface of CD8.sup.+ T cells before stimulation is 0.55%; after stimulation, the CXCR3 expression levels on the surface of the p53.sup.R175H T cells and control T cells reach 78.2% and 73.9%, both significantly higher than that before culture; indicating both Neo-DCs and Mock DCs can effectively activate CD8.sup.+ T cells. The experiment also found that the proportions of T.sub.CM cell subpopulation in p53.sup.R175H T and control T cells are 7.88% and 5.59%, respectively, both of which are significantly higher than 1.84% (before culture); the proportions of T.sub.EM cell subpopulation are 54.8% and 28.9%, both significantly higher than 7.19% (before culture), indicating that Neo-DC cells and Mock DCs can significantly induce CT8.sup.+ T cells to differentiate into central memory T cells and effector memory T cell subpopulations. However, Neo-DC cells have a stronger induction function, and the proportions of induced central memory T cells and effector memory T cell subpopulations are respectively 1.4 times and 1.9 times that of the control T cells.
(126) In order to test the ability of effector T cells in p53.sup.R175H T and control T cells to recognize and kill tumor cells, KLE tumor cells expressing p53 neoantigen (abbreviated as KLE.sup.mut) were used as target cells, while CFPAC-1 tumor cells expressing wild-type p53 were used as negative control cells (abbreviated as CFPAC-1.sup.wt). The p53.sup.R175H T or control T cells obtained in the previous experiment were co-cultured with these two types of cells at different effector-to-target ratios of 2.5:1, 5:1, and 10:1, respectively. The death of the target cells was monitored using RTCA cell analyzer, and the killing activity of the T cells against tumor cells was calculated. The killing effect of CTL cells on tumor cells was detected using impedance method of the analyzer. Tumor cells (KLE.sup.mut or CFPAC-1.sup.wt) with a density of 110.sup.4 cells per 100 L were placed into a 96 well resistance detection microplate. After 24 hours, different proportions of effector cells (p53.sup.R175H T or control T) were added to detect resistance changes in the system. The cytotoxicity of effector cells within 6 hours was calculated according to Cytolysis=(CI well without T-cellsCI experimental well)/CI well without T-cells100%. The experimental results are shown in
(127) Results and analysis: under three different effector-to-target ratios, p53.sup.R175H T can significantly kill tumor target cells KLE.sup.mut that express p53.sup.R175H antigen compared to control T, but the killing effect on CFPAC-1.sup.wt cells not expressing p53.sup.R175H antigen is not significant. Statistical analysis was conducted on the killing efficiency at the 4-hour time point of co-culture of effector cells and target cells. Under three different effector-to-target ratios (2.5:1, 5:1, 10:1), the killing efficiency of p53.sup.R175H T on target cells is respectively about 3 times, 2 times, and 1.5 times that of the control T, the differences are significant; the difference of killing efficiency between p53.sup.R175H T on the KLE.sup.mut target cells and the negative control CFPAC-1 cells is greater, with the former being about 12 times, 3 times, and 2.5 times higher than the latter, respectively. This indicates that p53.sup.R175H T has a significant ability to specifically recognize and kill target cells expressing p53.sup.R175H T antigen.
(128) The above experimental results confirm that Neo-DC cells have a better ability to activate CD8.sup.+ T cells than Mock DC cells, obtaining antigen-specific T cells (CTL cells) with a higher proportion of effector memory T cell subpopulation, wherein the CTL cells have a strong ability to kill tumor cells.
Example 7. Pharmacodynamic Evaluation of Neo-DC Cells in the Subcutaneous Transplantation Model of CFPAC-1 Tumor in huPBMC-NCG Mice
(129) The CFPAC-1 human pancreatic cancer cells in logarithmic growth phase were collected and resuspended to a suspension of 110.sup.7 cells/mL with DPBS, then subcutaneously inoculated 18 huPBMC-NCG mice (200 L/mouse). When the average volume of tumors in mice reached the preset volume, the mice were randomly divided into 3 groups (G1-G3) based on their body weight and tumor volume, with 6 mice in each group. Each mouse was injected intraperitoneally with 100 L PBMC of HLA-A*02:01 subtype for humanized immune system reconstruction. The day of injection was set as DO. At the same time, monocytes cells from the same donor source were used to prepare Neo-DC cells (in this example, the cells were mature dendritic cells loaded with neoantigen polypeptide generated by human pancreatic cancer cell KRAS.sup.G12V mutation and treated with CD40 antibody and PD-L1 antibody) and Mock DC cells (mature dendritic cells loaded with the same neoantigen polypeptide but not treated with CD40 antibody and PD-L1 antibody) according to the cell preparation process of Example 6. The mice in G1 group, which had undergone immune reconstitution, were injected subcutaneously with vehicle as a control; the G2 group mice were injected subcutaneously with Mock DC cells (100 L/mouse); the G3 group mice were injected subcutaneously with the same dose of Neo-DC cells. The administration method was subcutaneous two-point injection in the groin area of the hind legs of mice, with a frequency of administration on D14, D18, D22, and D30 after completing immune reconstitution, for a total of 4 doses. The pharmacodynamic evaluation of the test substance in the humanized huPBMC-NCG mouse model with subcutaneous transplantation of human pancreatic cancer cells CFPAC-1 was carried out based on the changes in tumor volume, body weight and other observation indexes during and after drug administration, as well as CBA analysis, GvHD scoring, and peripheral blood immune cell detection results.
(130) Results and analysis: during the experiment, flow cytometry of peripheral blood of the mice shows that more than half of the mice in each group have a proportion of hCD45.sup.+ immune cells greater than 10% in their peripheral blood; in hCD45.sup.+ immune cells, the proportion of T cells is greater than 99%, indicating the successful construction of the human PBMC immune reconstruction model. The data in
(131) In summary, the results of this experiment show that repeated subcutaneous injections of Neo-DC cells and Mock DC cells in immunodeficiency mice reconstituted by human PBMC (at the same time, human pancreatic cancer cells formed subcutaneous tumorigenesis) both have an effect on tumor lesion growth inhibition, and there are significant statistical differences compared with vehicle control group, and the tumor growth inhibition effect of Neo-DC cells is more significant than that of Mock DC cells. This indicates that subcutaneous injection of Neo-DC cells at this dose has a stronger effect on inhibiting and treating human pancreatic cancer lesions in humanized huPBMC-NCG mice.
Example 8: Clinical Study on the Safety and Efficacy of Neo-DC Cell Therapy for Advanced Malignant Solid Tumors
(132) In a single-center, single-arm, prospective phase I clinical study, the subject population was patients with advanced malignant common solid tumors. Neo-DC cells (loaded with the patient's tumor antigen) were administered as a monotherapy or in combination with PD-1 monoclonal antibody following the patient's surgery. The dosage regimen was: the subjects received 5 subcutaneous injections of 1.0 mL each after surgery, with a two-week interval between the first three injections and a three weeks interval between the last two injections; if combined with PD-1 monoclonal antibody, PD-1 monoclonal antibody was subcutaneously injected every three weeks after surgery at the clinically recommended dose. There was a total of 3 subjects with advanced malignant solid tumors who completed treatment evaluation. Safety evaluation results: the only adverse reaction related to the drug was transient fever after cell injection, which disappeared within 1-2 days without treatment; all subjects did not experience any other unexpected adverse events, no serious adverse events, and no dose-limiting toxicity. Effectiveness evaluation results: all 3 subjects showed strong anti-tumor T cell immune response in their bodies. Among them, for one subject with advanced pancreatic cancer and systemic metastasis (case P1), after 3 months of treatment with Neo-DC cell injection combined with PD-1 monoclonal antibody, the lesion shrank by 64%, RECIST rating PR, followed up for 28 months; for a subject with lung metastasis from laryngeal cancer (case P2), after 3 months of treatment with Neo-DC cell injection monotherapy, the lesion shrank by 46%, RECIST rating PR, followed up for 36 months; for a subject with advanced multifocal hepatocellular carcinoma (Case P3), after 8 months of treatment with Neo-DC cell injection monotherapy combined with PD-1 monoclonal antibody, the lesion shrank by 13%, RECIST rating SD, followed up for 32 months. The experimental results are shown in
Example 9: Assessment of Safety and Preliminary Clinical Efficacy of Postoperative Adjuvant TMZ and Neo-DC Cell Injection in Patients with Glioblastoma
(133) In a single-center, single-arm, prospective phase I clinical study, several subjects aged 18-75 years with newly diagnosed primary glioblastoma multiforme (GBM) confirmed by histopathology were included. The newly diagnosed glioblastoma was WHO grade IV confirmed by histopathology examination, and wild-type IDH1 and IDH2 genes confirmed by molecular pathological diagnosis, with the tumor enhancement lesion resection exceeding 90%. After completing surgical resection, the subjects received standard treatment (SoC) and underwent 5-6 subcutaneous injections of Neo-DC cell (loaded with patient tumor antigen) injection during routine adjuvant TMZ chemotherapy. If progress is confirmed upon completion of injection treatment, continuation with PD-1 monoclonal antibody or bevacizumab treatment may be considered. A total of 10 subjects completed the full cycle treatment. Safety evaluation results: the only adverse reaction related to the drug was transient fever after cell injection, which disappeared within 1-2 days without treatment; all subjects did not experience any other unexpected adverse events, no serious adverse events, and no dose limiting toxicity. The clinical efficacy evaluation results as of Apr. 1, 2024: 8 subjects achieved sustained stability of the lesion (SD), with no change in the target lesion during the evaluation period, and a DCR of 80%; a subject maintained a long-term recurrence free survival status for 23 months (as shown in
(134) The above are only preferred examples of the present application and do not impose any form of limitation on the present application. Any technical skilled in the art may use the disclosed technical content to modify or change it into equivalent examples. However, any simple modifications, equivalent changes, or modifications made to the above examples based on the technical essence of the present application, as long as they do not depart from the content of the technical solution of the present application, still fall within the protection scope of the technical solution of the present application.