Pharmaceutical composition for targeting myeloid cells so as to enhance efficacy of OX40 agonist antibody and application of pharmaceutical composition for targeting myeloid cells so as to enhance efficacy of OX40 agonist antibody

Abstract

The invention relates to the technical field of biological medicine, in particular to a pharmaceutical composition targeting myeloid cells to enhance the drug effect of an OX40 agonist antibody and application of the pharmaceutical composition. The pharmaceutical composition for targeting myeloid cells so as to enhance the drug effect of the OX40 agonist antibody comprises the OX40 agonist antibody, MPLA and IFN-gamma. The pharmaceutical composition acts on myeloid cells, reprogramming of tumor infiltration myeloid cells such as macrophages and neutrophils can be realized, and the drug effect of an OX40 agonist antibody can be significantly enhanced through combination treatment with the OX40 agonist antibody.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a pharmaceutical composition that targets myeloid cells and thereby enhances the efficacy of OX40 agonist antibodies and applications thereof. Background Art

[0002] OX40 (also known as CD134 or TNFRSF4) is a member of the tumor necrosis factor receptor superfamily. It is a type I transmembrane glycoprotein composed of 249 amino acids and is mainly expressed by T lymphocytes (constitutively expressed by regulatory T lymphocytes and expressed by effector T lymphocytes after activation, in CD8 + T and CD4 + Highly expressed in T lymphocytes). In multiple preclinical models, OX40 agonist antibodies induced T cell activation in in vitro and in vivo models, inhibited Treg-mediated immunosuppression, and showed tumor inhibitory efficacy in tumor-bearing mouse models. Currently, the effect of OX40 agonist antibodies combined with immune checkpoint antibodies or TLR9 agonist CpG is not good, and the effects of bispecific antibodies and fusion proteins based on OX40 agonist antibodies are also not ideal. Summary of the invention

[0003] The present invention aims to solve at least one of the technical problems existing in the related art. To this end, the present invention provides a pharmaceutical composition that targets myeloid cells and thereby enhances the efficacy of OX40 agonist antibodies, which targets myeloid cells and can achieve reprogramming of tumor-infiltrating myeloid cells such as macrophages and neutrophils to enhance the efficacy of OX40 agonist antibodies.

[0004] In one aspect of the present invention, the present invention provides a pharmaceutical composition for targeting myeloid cells and thereby enhancing the efficacy of OX40 agonist antibodies, the pharmaceutical composition comprising: OX40 agonist antibodies, MPLA, and IFN-γ.

[0005] Furthermore, the molar ratio of the OX40 agonist antibody, MPLA and IFN-γ is (32-34): (55-57): (32-34).

[0006] Furthermore, the molar ratio of the OX40 agonist antibody, MPLA and IFN-γ is 33:57:32.

[0007] Furthermore, the OX40 agonist antibody includes an OX40 humanized agonist antibody (2-2-1).

[0008] In another aspect of the present invention, the present invention provides a use of the pharmaceutical composition as described above for targeting myeloid cells and thereby enhancing the efficacy of OX40 agonist antibodies in the preparation of drugs for treating tumors.

[0009] Furthermore, the pharmaceutical composition for targeting myeloid cells and thereby enhancing the efficacy of OX40 agonist antibodies enhances the efficacy of OX40 agonist antibodies by reprogramming tumor-infiltrating myeloid cells.

[0010] Furthermore, the myeloid cells include monocytes, macrophages and / or neutrophils.

[0011] Further, reprogramming tumor-infiltrating myeloid cells comprises: The up-regulated genes in the mononuclear macrophages and / or neutrophils are enriched in at least one of the following innate immune signaling pathways: a cellular response to lipopolysaccharide, a cellular response to interferon gamma, and a cellular response to interferon beta.

[0012] In another aspect of the present invention, the present invention provides a drug for treating tumors, wherein the drug comprises the aforementioned drug composition that targets myeloid cells and thereby enhances the efficacy of OX40 agonist antibodies.

[0013] The above one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: The pharmaceutical composition of the present invention that targets myeloid cells and thus enhances the efficacy of OX40 agonist antibodies targets myeloid cells, can achieve reprogramming of tumor-infiltrating myeloid cells such as macrophages and neutrophils to enhance the efficacy of OX40 agonist antibodies, and can further enhance the body's anti-tumor immune response by reversing the polarization of macrophages and / or neutrophils. When the pharmaceutical composition of the present invention that targets myeloid cells and thus enhances the efficacy of OX40 agonist antibodies is used for mouse tumor treatment, the life span of the mice can be significantly extended. The combined use of MPLA and IFN-γ with OX40 agonist antibodies shows great clinical potential.

[0014] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 This is a schematic diagram of the drug composition for treating mice according to Example 1 of the present invention, which targets myeloid cells and thereby enhances the efficacy of OX40 agonist antibodies.

[0017] Figure 2 This is the tumor growth curve of the mice in the response group and the non-response group of MC38 tumor-bearing mice treated with the OX40 agonist antibody in Example 1 of the present invention.

[0018] Figure 3 This is a cell clustering diagram of single cell sequencing of the response group and the non-response group of MC38 tumor-bearing mice treated with OX40 agonist antibody in Example 1 of the present invention.

[0019] Figure 4 It is a bar graph showing the changes in the proportions of immune cell subtypes in the response group and the non-response group of MC38 tumor-bearing mice treated with the OX40 agonist antibody in Example 1 of the present invention.

[0020] Figures 5 to 11 They are CD8 T cells, CD4 T cells, macrophages, CD8T cells in Example 1 of the present invention. + INFγ + , the ratio of macrophage subtypes M1 / M2, neutrophils, and the release of granzyme B in neutrophils + neutrophil) changes.

[0021] Fig.12 and Fig.13 They are respectively the results of GO pathway enrichment analysis in biological process (BP) of up-regulated genes in monocytes, macrophages and neutrophils in the OX40 antibody treatment response group analyzed by single cell transcriptome sequencing in Example 1 of the present invention.

[0022] Figures 14 to 19 They are respectively the immune cell signaling pathways enriched in mononuclear macrophages in the response group analyzed by the GSEA method in Example 1 of the present invention.

[0023] Fig. 20 and Fig.21 They are respectively the tumor volume growth curve of MC38 tumor-bearing mice treated with the combined drug in Example 2 of the present invention and the corresponding mouse survival curve.

[0024] Fig. 22 and Fig.23 They are respectively the tumor volume growth curve of E.g7 tumor-bearing mice treated with the combined drug in Example 2 of the present invention and the corresponding mouse survival curve.

[0025] Fig.24 and Fig.25 They are respectively the tumor volume growth curve of B16 tumor-bearing mice treated with the combined drug in Example 2 of the present invention and the corresponding mouse survival curve.

[0026] Fig.26 This is a UMAP (Uniform Manifold Approximation and Projection) clustering diagram of single-cell transcriptome sequencing of tumor-infiltrating immune cells in MC38 tumor-bearing mice after combined drug treatment in Example 3 of the present invention.

[0027] Fig. 27 The single-cell transcriptome sequencing in Example 3 of the present invention detected the changes in the proportions of CD4 T cells and CD8 T cell subsets in different groups after combined drug treatment.

[0028] Fig.28 The single-cell sequencing in Example 3 of the present invention detects the proportional changes in the number of immune cells in different groups after combined drug use.

[0029] Fig.29 This is the change of M1 / M2 inside the tumor after using targeted myeloid combined drug treatment in Example 3 of the present invention.

[0030] Fig.30 The Granzyme B release of CD8 T cells in different groups after combined drug treatment in Example 3 of the present invention. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0032] In the description of the embodiments of the present invention, it should be noted that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the embodiments of the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore cannot be understood as limitations on the embodiments of the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.

[0033] In the description of the embodiments of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "connected" and "connection" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium. For ordinary technicians in this field, the specific meanings of the above terms in the embodiments of the present invention can be understood according to specific circumstances.

[0034] In the embodiments of the present invention, unless otherwise clearly specified and limited, the first feature being "above" or "below" the second feature may mean that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Moreover, the first feature being "above", "above" and "above" the second feature may mean that the first feature is directly above or obliquely above the second feature, or simply means that the first feature is higher in level than the second feature. The first feature being "below", "below" and "below" the second feature may mean that the first feature is directly below or obliquely below the second feature, or simply means that the first feature is lower in level than the second feature.

[0035] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0036] In one aspect of the present invention, the present invention provides a pharmaceutical composition for targeting myeloid cells and thereby enhancing the efficacy of OX40 agonist antibodies, the pharmaceutical composition comprising: OX40 agonist antibodies, MPLA (monophosphoryl lipid A) and IFN-γ (γ-interferon).

[0037] The pharmaceutical composition of the present invention that targets myeloid cells and thus enhances the efficacy of OX40 agonist antibodies targets myeloid cells, can achieve reprogramming of tumor-infiltrating myeloid cells such as macrophages and neutrophils to enhance the efficacy of OX40 agonist antibodies, and can further enhance the body's anti-tumor immune response by reversing the polarization of macrophages and / or neutrophils. When the pharmaceutical composition of the present invention that targets myeloid cells and thus enhances the efficacy of OX40 agonist antibodies is used for mouse tumor treatment, the life span of the mice can be significantly extended. The combined use of MPLA and IFN-γ with OX40 agonist antibodies shows great clinical potential.

[0038] In some embodiments of the present invention, the molar ratio of the OX40 agonist antibody, MPLA and IFN-γ is (32-34): (55-57): (32-34).

[0039] In some preferred embodiments of the present invention, the molar ratio of the OX40 agonist antibody, MPLA and IFN-γ is 33:57:32.

[0040] In some embodiments of the present invention, the OX40 agonist antibody comprises an OX40 humanized agonist antibody (2-2-1).

[0041] In some specific embodiments of the present invention, the OX40 humanized agonist antibody (2-2-1) is prepared in the following manner: first, an antibody library binding to the OX40 target is screened by phage display technology, and then several active OX40 agonist antibodies are screened from the OX40 antibody binding library by high-throughput screening based on microfluidics technology, and the activities of these OX40 agonist antibodies are evaluated in vitro and in vivo, and finally the OX40 agonist antibody with the highest activity is screened and named 2-2-1.

[0042] The inventors of the present invention found that some mice showed primary or acquired drug resistance after treatment with OX40 agonist antibodies alone, and further found through single-cell sequencing and other technologies that the proportion of myeloid cells in non-responsive mice changed more significantly. Therefore, MPLA and IFN-γ were combined with OX40 agonist antibodies to achieve reprogramming of tumor-infiltrating myeloid cells such as macrophages and neutrophils to enhance the efficacy of OX40 agonist antibodies.

[0043] The inventors of the present invention also found that OX40 is a popular target for tumor immunotherapy. Current research basically focuses on the activation of T cells and the clearance of Treg by OX40 agonist antibodies, and no research has been found on the effects of OX40 agonist antibodies on myeloid cells. The present invention studies the effects of myeloid cells such as monocytes, macrophages and neutrophils on the efficacy of OX40 agonist antibodies, and further deepens the understanding of the combined use of OX40 agonist antibodies.

[0044] In another aspect of the present invention, the present invention provides a use of the pharmaceutical composition as described above for targeting myeloid cells and thereby enhancing the efficacy of OX40 agonist antibodies in the preparation of drugs for treating tumors.

[0045] In some embodiments of the present invention, the pharmaceutical composition for targeting myeloid cells and thereby enhancing the efficacy of OX40 agonist antibodies enhances the efficacy of OX40 agonist antibodies by reprogramming tumor-infiltrating myeloid cells.

[0046] In some embodiments of the present invention, the myeloid cells include monocytes, macrophages and / or neutrophils.

[0047] In some embodiments of the invention, reprogramming tumor-infiltrating myeloid cells comprises: The up-regulated genes in the mononuclear macrophages and / or neutrophils are enriched in at least one of the following innate immune signaling pathways: cellular response to lipopolysaccharide, cellular response to interferon gamma, and cellular response to interferon beta.

[0048] In some specific embodiments of the present invention, the tumor includes colon cancer, T lymphoma, melanoma and the like.

[0049] In another aspect of the present invention, the present invention provides a drug for treating tumors, wherein the drug comprises the aforementioned drug composition that targets myeloid cells and thereby enhances the efficacy of OX40 agonist antibodies.

[0050] The present invention is further described below in conjunction with specific examples. It should be noted that the following examples are only used to explain the present invention and are not to be construed as limiting the present invention. Example

[0051] The specific steps used in the following examples for the division of the response group and the non-response group, the single-cell sequencing analysis of mouse tumor-infiltrating immune cells, the combined use of MPLA, IFN-γ and OX40 agonist antibodies to treat various mouse tumor models, and the multicolor flow cytometry analysis of changes in mouse tumor-infiltrating immune cells are as follows: 1. Construction of bilateral subcutaneous tumor-bearing model in mice and division into response group and non-response group The specific operation steps are as follows: MC38 (mouse colon cancer) tumor cells were revived in vitro and expanded with DMEM complete medium (88% DMEM basal medium + 10% FBS + 1% PS + 1% Hepes); when the tumor cells expanded to a certain number, they were digested and counted with 0.25% trypsin, and the number of cells was 1×10 6 The number of cells per side was used to carry out bilateral subcutaneous tumor implantation in mice; it took about a week for the tumor volume of the mice to grow to 50-100mm 3 The mice were randomly divided into a control group and a treatment group. The mice in the treatment group were intraperitoneally administered with OX40 agonist antibodies every two days. On the seventh day after treatment, the tumor on one side was removed for single-cell sequencing analysis of the changes in tumor-infiltrating immune cells, and the wound was sutured. At the same time, the size of the tumor on the other side was monitored. The tumor volume and weight of the mice were measured every three days, and GraphPad Prism was used to draw the mouse tumor growth curve and mouse survival curve (tumor volume grew to 1500mm 3 The mice were killed by default) to determine the responding and non-responding groups to the OX40 agonist antibody treatment. 2. Single-cell sequencing analysis of tumor-infiltrating immune cells in mice in the response and non-response groups

[0052] The specific operation steps are as follows: MC38 tumor-bearing mice were grown to a tumor volume of 50-100mm 3 The OX40 agonist antibody was used to treat the tumor every two days. After three treatments, one side of the tumor was removed in a sterile environment and digested and ground into a single cell suspension. CD45 positive magnetic beads were used to detect the CD45 in the tumor tissue. +Immune cells were isolated, and their cell number and cell activity were measured, and the cell viability was ensured to be above 90%. After labeling the immune cells of each mouse with hashtag antibodies, single-cell transcriptome sequencing samples were prepared using the 10×Genomics single-cell transcriptome sequencing platform. Master Mix (mainly including poly-dT primers and enzymes for related reactions, etc.), cell suspension, gel beads containing barcodes, and oil droplets were added to different chambers of Chromium Chip B, and gel beads (Gel Bead-In-EMulsion, GEM) wrapped in oil droplets were formed through the microfluidic "double cross" cross system. GEMs were collected and mixed evenly. The gel beads automatically dissolved in each oil droplet to release a large number of primers carrying barcodes, and the cells were lysed to release mRNA. The mRNA was then reversely transcribed under the action of reverse transcriptase to produce cDNA carrying barcode and UMI (Unique Molecular Identifier) ​​information. After preparing the cDNA library according to the above standard process, it was sent to Novogene for sequencing using an Illumina sequencer. After sequencing is completed, data analysis is performed to determine the differences in immune cell changes and related signaling pathways in the tumor tissues of mice in the response group and the non-response group. 3. Combination of MPLA, IFN-γ and OX40 agonist antibody for the treatment of various mouse tumor models

[0053] The specific steps are as follows: After analyzing the signal pathways in the tumors of mice that respond to and do not respond to OX40 agonist antibody treatment using a bilateral subcutaneous tumor-bearing model and single-cell sequencing technology, the drug combination strategy of MPLA, IFN-γ and OX40 agonist antibody was determined based on this. The mice were then inoculated with different tumor cells, including mouse colon cancer (MC38), mouse T lymphoma (E.g7) and mouse melanoma (B16). When the tumor volume grew to 50-100 mm 3 The drug combination treatment was carried out every two days. The OX40 agonist antibody was intraperitoneally administered at a dose of 5 mg / kg, and MPLA and IFN-γ were administered intratumorally at doses of 1 μg and 5 μg, respectively. The tumor volume growth curve and mouse survival curve were drawn by measuring the tumor volume. The tumor volume grew to 1500mm 3 The mouse is killed by default. 4. Analysis of changes in mouse tumor-infiltrating immune cells using multicolor flow cytometry

[0054] The specific operation steps are as follows: After three drug treatments, the tumor-bearing mice were anesthetized and killed, and their tumor tissues were removed and digested and ground in vitro to prepare single-cell suspensions. Then a series of flow analysis schemes (panels) were designed to group and functionally analyze the immune cells in the tumor (including T cells, macrophages, NK cells, DC cells, neutrophils, etc.). The single-cell suspension was added to a V-bottom 96-well plate and blocked. After the blocking, flow staining was performed in the order of dead and live cell staining, surface antigen staining, and intracellular antigen staining after cell fixation and membrane permeation. The samples were analyzed using a BD LSRFortessa flow cytometer, and the flow data were analyzed using FlowJo software to compare the changes in immune cells and their functions in the tumor after combined treatment. Example 1

[0055] The specific steps of the drug composition design are as follows: 1. Using the mouse subcutaneous bilateral tumor-bearing (MC38) model, the tumor on one side of the mouse was monitored to determine whether the mouse responded to the treatment with the OX40 agonist antibody. The tumor-bearing mice were then divided into a control IgG group (isotype control irrelevant antibody IgG group or isotype control irrelevant antibody group), an OX40 agonist antibody response group (or simply referred to as a response group) and an OX40 agonist antibody non-response group (or simply referred to as a non-response group). The tumor on the other side was removed at the early stage of treatment for multi-color flow cytometry analysis and single-cell transcriptome sequencing analysis. Based on various bioinformatics analyses, the precise composition, quantity and functional status of tumor-infiltrating immune cells in mice in the OX40 agonist antibody treatment response and non-response groups were compared. The results are as follows: Figures 1 to 11 As shown (where Figure 2 The left and right sides of each group correspond to the left and right tumors in each group of mice. Several cell subpopulations and signaling pathways related to OX40 agonist antibody resistance were discovered (mainly including innate immune signaling pathways such as cellular response to lipopolysaccharide, cellular response to interferon amma and cellular response to interferon beta), and related drug targets were further sought through related signaling pathways for combined drug treatment.

[0056] 2. Through the analysis of differential gene enrichment signal pathways of macrophages and neutrophils in the single-cell transcriptome sequencing results, it was found that the up-regulated genes of mononuclear macrophages and neutrophils in the response group were enriched in the innate immune signal pathways cellular response to lipopolysaccharide, cellular response to interferon gamma and cellular response to interferon beta. GSEA analysis (Gene Set Enrichment Analysis) showed that the innate immune-related pathways of NOD like receptor signaling pathway, C-type lectin receptor signaling pathway, RIG-I-like receptor signaling pathway and Toll-like receptor signaling pathway were significantly up-regulated in the response group, suggesting that the innate immune signaling pathway may be related to the response to OX40 agonist antibodies. For specific results, please refer to Figures 12 to 19 ( Fig.14 In the middle, NES = 2.08, P < 0.001, q < 0.001, the ranking value of the data set when the gene ranking amount is 0 is 9245; Fig.15 In the middle, NES = 1.95, P < 0.001, q < 0.001, and the ranking value of the data set when the gene ranking amount is 0 is 9245; Fig.16 In the middle, NES = 1.91, P < 0.001, q < 0.001, and the ranking value of the data set when the gene ranking amount is 0 is 9245; Fig.17 In the middle, NES=1.72, P<0.005, q=0.004, the ranking value of the data set when the gene ranking amount is 0 is 9245; Fig.18 In the middle, NES=1.70, P<0.001, q=0.005, the ranking value of the data set when the gene ranking amount is 0 is 9245; Fig.19 In the middle, NES=1.68, P<0.001, q=0.006, the ranking value of the data set when the gene ranking amount is 0 is 9245; Figures 14 to 19In the figure, the name of the horizontal axis is Rank in OrderedDataSet, which represents the ranking value of the data set; there are two vertical axes, one is Enrichment score, which represents the enrichment score, and the other is Ranked list metric, which represents the gene ranking amount). The GSEA enrichment analysis of monocytes and macrophages in the response group showed that multiple innate immune signaling pathways were upregulated in the response group.

[0057] Drugs that can modulate the above differential signaling pathways are co-administered with OX40 agonist antibodies, and it is determined that MPLA and IFN-γ are combined with OX40 agonist antibodies. Example 2 Evaluation of therapeutic effects of pharmaceutical compositions targeting myeloid cells to enhance the efficacy of OX40 agonist antibodies

[0058] The specific operation steps are as follows: Different groups of mice were inoculated with mouse colon cancer (MC38), mouse T lymphoma (E.g7) and mouse melanoma (B16). The MC38 model (i.e., mice inoculated with MC38) was divided into: Control IgG group, MPLA+OX40 agonist antibody (2-2-1) group, IFN-γ+OX40 agonist antibody (2-2-1) group, OX40 agonist antibody (2-2-1) group, MPLA+IFN-γ+control IgG group and MPLA+IFN-γ+OX40 agonist antibody (2-2-1) group. The E.g7 model (i.e., mice inoculated with E.g7) and the B16 model (i.e., mice inoculated with B16) were divided into: Control IgG group, OX40 agonist antibody (2-2-1) group, MPLA+IFN-γ+control IgG group and MPLA+IFN-γ+OX40 agonist antibody (2-2-1) group. There were 6 mice in each group in the three models. When the tumor volume of the mice grew to 50-100mm 3 The drug combination treatment was carried out every two days. The OX40 agonist antibody was intraperitoneally administered at a dose of 5 mg / kg, and MPLA and IFN-γ were administered intratumorally at doses of 1 μg and 5 μg, respectively. The tumor volume growth curve and mouse survival curve were drawn by measuring the tumor volume. The tumor volume grew to 1500mm 3 The mouse is killed by default.

[0059] The therapeutic effects of MPLA, IFNγ and OX40 agonist antibody combination were evaluated in MC38, B16 and E.g7 mouse tumor models. The results showed that the drug combination enhanced the anti-tumor efficacy of OX40 agonist antibody in all three tumor models and significantly prolonged the survival of mice. OX40 humanized mice were inoculated with MC38, B16 or E.g7 mouse tumor cells and the tumor volume grew to 50-100 mm 3 When the mice were divided into groups (MC38 model: control IgG group, MPLA+OX40 agonist antibody (2-2-1) group, IFN-γ+OX40 agonist antibody (2-2-1) group, OX40 agonist antibody (2-2-1) group, MPLA+IFN-γ+control IgG group and MPLA+IFN-γ+OX40 agonist antibody (2-2-1) group. E.g7 and B16 models were grouped as control IgG group, OX40 agonist antibody (2-2-1) group, MPLA+IFN-γ+control The mice were divided into IgG group and MPLA+IFN-γ+OX40 agonist antibody (2-2-1) group (6 mice in each group in the three models). The drugs MPLA (1 μg) and IFNγ (5 μg) were intratumorally administered every three days, and the OX40 agonist antibody was intraperitoneally administered at a dose of 5 mg / kg for a total of four times. The tumor volume of the mice was monitored and the tumor volume was greater than or equal to 1500 mm. 3 Finally, Graphpad was used to plot the mouse tumor growth curve and mouse survival curve. The results are as follows Figure 20 to Figure 25 As shown ( Figure 20 to Figure 21 In the assay, the isotype control irrelevant antibody IgG group corresponds to the Control IgG group, the MPLA+OX40 agonist antibody group corresponds to the MPLA+OX40 agonist antibody (2-2-1) group, the IFN-γ+OX40 agonist antibody group corresponds to the IFN-γ+OX40 agonist antibody (2-2-1) group, the OX40 agonist antibody group corresponds to the OX40 agonist antibody (2-2-1) group, the MPLA+IFN-γ+isotype control irrelevant antibody IgG group corresponds to the MPLA+IFN-γ+control IgG group, the MPLA+IFN-γ+OX40 agonist antibody group corresponds to the MPLA+IFN-γ+OX40 agonist antibody (2-2-1) group, Figure 22 to Figure 25In the figure, the isotype-irrelevant antibody IgG group corresponds to the Control IgG group, the OX40 agonist antibody group corresponds to the OX40 agonist antibody (2-2-1) group, the MPLA+IFN-γ+isotype control irrelevant antibody IgG group corresponds to the MPLA+IFN-γ+Control IgG group (M-Control IgG), and the MPLA+IFN-γ+OX40 agonist antibody group corresponds to the MPLA+IFN-γ+OX40 agonist antibody (2-2-1) group). Example 3 Exploration of the mechanism of action of the anti-tumor efficacy of drug combinations

[0060] OX40 humanized mice were inoculated with MC38 tumor cells and the tumors were grown to 50-100 mm 3 The mice were divided into groups (specific groups included: isotype control irrelevant antibody IgG group (labeled as control IgG), OX40 agonist antibody (2-2-1) group, MPLA+IFN-γ+control IgG group (labeled as M-control IgG) and MPLA+IFN-γ+OX40 agonist antibody (2-2-1) group (labeled as M-OX40), each group had 6 mice / group, and were treated according to different groups. The combined drugs MPLA (1 μg) and IFNγ (5 μg) were intratumorally administered, and the OX40 agonist antibody (2.5 mg / kg) was intraperitoneally administered. The treatment was once every three days. After two treatments, the tumor-infiltrating immune cells were isolated for single-cell transcriptome sequencing (the results are shown in Figure 2). Fig.26 The results showed that the proportion of CD8-TEM cells was upregulated in the combined treatment group, while the proportion of CD4-Treg cells was significantly downregulated (the results are shown in Fig. 27 ), while the number of monocytes and macrophages was more significantly downregulated in the combined treatment group (results shown in Fig.28 as shown).

[0061] In addition, in order to further study the changes in immune cell subtypes in tumors before and after combined treatment, we used 1×10 6 / mouse inoculated with MC38 (mouse colon cancer) tumor cells and waited until the tumor volume grew to 50-100mm 3The mice were randomly divided into Control IgG group, OX40 agonist antibody (2-2-1) group, MPLA+IFN-γ+Control IgG group (M-Control IgG) and MPLA+IFN-γ+OX40 agonist antibody (2-2-1) group (M-OX40). Treatment was once every three days, OX40 agonist antibody was intraperitoneally administered at a dose of 5 mg / kg, and MPLA and IFN-γ were administered intratumorally at doses of 1 μg and 5 μg, respectively. After the third treatment, the mouse tumors were removed, digested and ground to prepare single-cell suspensions, and multi-color flow cytometry was used to further detect changes in the proportions of immune cell subsets inside the tumor. The results of multi-color flow cytometry showed that the proportions of CD4 T, CD8 T, macrophages and neutrophils changed significantly after combined use, especially the ratio of M1 / M2 in CD4 T cells and macrophages and the release of GranzymeB in CD8 T cells (results are shown in Figure 2). Fig.29 and Fig.30 As shown, Fig.29 and Fig.30 In the data, the isotype-irrelevant antibody IgG group corresponded to the Control IgG group, the OX40 agonist antibody (2-2-1) group corresponded to the OX40 agonist antibody group, the M_isotype control irrelevant antibody group corresponded to the MPLA+IFN-γ+Control IgG group (M-Control IgG), and the M_OX40 agonist antibody group corresponded to the MPLA+IFN-γ+OX40 agonist antibody (2-2-1) group (M-OX40)), indicating that the combined treatment may further enhance the body's anti-tumor immune response mainly by reversing the polarization of macrophages and enhancing the killing function of T and NK.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pharmaceutical composition for targeting myeloid cells and thereby enhancing the efficacy of an OX40 agonist antibody, characterized in that: include: OX40 agonist antibodies, MPLA, and IFN-γ.

2. The pharmaceutical composition for targeting myeloid cells and thereby enhancing the efficacy of OX40 agonist antibodies according to claim 1, characterized in that: The molar ratio of the OX40 agonist antibody, MPLA and IFN-γ is (32-34): (55-57): (32-34).

3. The pharmaceutical composition for targeting myeloid cells and thereby enhancing the efficacy of OX40 agonist antibodies according to claim 2, characterized in that: The molar ratio of the OX40 agonist antibody, MPLA and IFN-γ is 33:57:

32.

4. The pharmaceutical composition for targeting myeloid cells and enhancing the efficacy of OX40 agonist antibodies according to any one of claims 1 to 3, characterized in that: The OX40 agonist antibody includes an OX40 humanized agonist antibody (2-2-1).

5. Use of the pharmaceutical composition for targeting myeloid cells and thereby enhancing the efficacy of OX40 agonist antibodies as claimed in any one of claims 1 to 4 in the preparation of drugs for treating tumors.

6. The use according to claim 5, characterized in that: The pharmaceutical composition that targets myeloid cells and thereby enhances the efficacy of OX40 agonist antibodies enhances the efficacy of OX40 agonist antibodies by reprogramming tumor-infiltrating myeloid cells.

7. The use according to claim 6, characterized in that: The myeloid cells include monocytes, macrophages and / or neutrophils.

8. The use according to claim 7, characterized in that: Reprogramming tumor-infiltrating myeloid cells includes: The up-regulated genes in the mononuclear macrophages and / or neutrophils are enriched in the following innate immune signaling pathways: at least one of the cell response to lipopolysaccharide, the cell response to gamma-interferon, and the cell response to beta-interferon.

9. A drug for treating tumors, characterized in that: The drug comprises a pharmaceutical composition of any one of claims 1 to 4 that targets myeloid cells and thereby enhances the efficacy of OX40 agonist antibodies.

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