CN116875550A - FABP4 + C1q + Macrophage and its preparation method and use - Google Patents
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention relates to FABP4 + C1q + The macrophage and the preparation method and the application thereof belong to the field of tumor immunology and biotechnology, in particular discloses a preparation method of double-positive macrophage which simultaneously and highly expresses FABP4 and C1q and the application thereof in preparing tumor immunotherapy medicaments, wherein the FABP4 group is prepared by the double-positive macrophage + C1q + Macrophages have strong inflammatory cytokine production and phagocytic capacity, and have stronger anti-tumor function, FABP4 + C1q + Double-positive macrophages infiltrate more in tumor sites and regional drainage lymph node immune microenvironment, which suggests that the tumor immunotherapy has better curative effect. As can be seen, the in vitro culture induced FABP4 obtained by the invention + C1q + Macrophages, as an active ingredient of a tumor immunotherapeutic agent, against malignancyThe tumor has good therapeutic effect.
Description
Technical Field
The invention belongs to the field of tumor immunology and biotechnology, and in particular relates to FABP4 + C1q + Macrophages and methods of making and using the same.
Background
Malignant tumor is a disease with extremely bad prognosis, involves abnormal proliferation and differentiation of cells, and the main treatment ways include surgical excision, radiotherapy, chemotherapy, targeting, immunotherapy and other methods.
Immune checkpoint inhibitor (immune checkpointinhibitors, ICIs) drug-dominated immunotherapy significantly improved the prognosis of malignant patients in the last decade. However, the exact mechanism of action of immunotherapy, such as ICIs, and how to screen the benefited population becomes a great challenge. Currently, only a few studies have demonstrated that the efficacy of immunotherapy may be related to individual tumor immune microenvironment (tumor immunemicroenvironment, TME) immune infiltration heterogeneity. Therefore, it is important to explore potential predictive markers of tumor immunotherapy, and an individualized and accurate treatment strategy can be provided for patients.
Studies have shown that malignant TME will remodel following immunotherapy. Macrophages are taken as a group of cells with the most heterogeneity in TME, the gene expression profiles are diversified, the macrophages with different gene expression profiles also have different functions, and the macrophage subpopulations are required to be finely divided according to the gene expression profiles, so that the functional mechanism of novel macrophage subpopulations related to tumor immunotherapy is explored.
At present, no FABP4 is known + C1q + Macrophage studies are reported.
Disclosure of Invention
The invention aims to provide FABP4 + C1q + Macrophages and methods of making and using the same.
In order to solve the technical problems, the invention adopts the following technical scheme: FABP (Fabry-Perot Back propagation)4 + C1q + A method for preparing macrophages comprising the steps of:
s1, peripheral Blood Mononuclear Cells (PBMC) extraction: mononuclear cells were obtained from peripheral blood density gradient centrifugation and CD14 was sorted using magnetic beads + Monocytes;
S2、FABP4 + C1q + macrophage induction: CD14 selected in step S1 was resuspended and cultured with RPMI-1640 complete medium containing 10% FBS,1% diabody, 50ng/ml M-CSF + Monocytes, adjusted to a concentration of 1X 10 6 Inoculating the seeds into a cell culture dish for culture;
s3, induction culture conditions: semi-quantitatively changing liquid with 2-3 days as period, culturing for 7-9 days to obtain M0 macrophage, and separating FABP4 by flow fluorescence + C1q + Macrophages.
In the step S1, diluting healthy peripheral blood with an equal volume of PBS buffer solution to form a diluent, slowly adding the diluent into the cell layering solution along the pipe wall according to the ratio of the cell layering solution to the diluent of 1:1, and performing gradient centrifugation for 1800r/min for 18min; after centrifugation, slowly sucking and discarding the upper transparent PBS buffer solution by using a sterile suction tube, and inserting the sterile suction tube into a cloud and fog belt to suck peripheral blood mononuclear cells and washing for 2 times, wherein 1-2ml is left; CD14 microbeds magnetic bead sorting CD14 + Monocytes.
Another object of the present invention is to provide FABP4 obtained by the above-mentioned production method + C1q + Macrophages.
Another object of the present invention is to provide a FABP4 as described above + C1q + Use of macrophages.
The technical scheme for achieving the purpose is as follows:
FABP4 + C1q + application of macrophage in preparing tumor immunotherapy medicine is disclosed.
Further, the FABP4 + C1q + Macrophages simultaneously express FABP4 and C1q at high levels.
Further, the FABP4 + C1q + Inflammatory cytokines produced by macrophages include, but are not limited to, TNF- α, IL-6, and IL-1β.
Further, the types of tumors include solid tumors and non-solid tumors.
Further, the types of tumors include lung cancer, ovarian cancer, liver cancer, pancreatic cancer, colon cancer, rectal cancer, renal cancer, prostate cancer, thyroid cancer, tongue cancer, melanoma, lymphoma, thyroid cancer, breast cancer, head and neck squamous carcinoma, and gastric cancer.
Further, the tumor is lung cancer.
Fatty acid binding protein FABP4 belongs to one of the important members of the fatty acid binding protein family, involved in lipid metabolism processes and inflammatory diseases. FABP4 has an important influence on cellular and systemic lipid signaling mediators and inflammatory processes, and macrophages expressing FABP4 often induce macrophage inflammation and play an important role in atherosclerosis, novel coronavirus infection, and the development of a variety of cancers.
Complement protein C1q is a recognition molecule of the classical pathway of complement, has complement and non-complement functions, and plays an important role in TME. C1q can bind to various ligands, both self-derived and non-self, and regulate immune and non-immune cell functions to perform complement functions; the non-complement function of C1q is manifested in inducing angiogenesis, synaptic pruning, participation in inflammatory infection of necrotic and tumor cells, and clearance of other apoptotic cells.
The beneficial effects of the invention are as follows:
the invention discloses a preparation method of double-positive macrophages with high expression of FABP4 and C1q simultaneously and application thereof in preparing tumor immunotherapy medicaments, wherein the FABP4 group + C1q + Macrophages have strong inflammatory cytokine production and phagocytic capacity, and have stronger anti-tumor function, FABP4 + C1q + Double-positive macrophages infiltrate more in tumor sites and regional drainage lymph node immune microenvironment, which suggests that the tumor immunotherapy has better curative effect.
As can be seen, the in vitro culture induced FABP4 obtained by the invention + C1q + Macrophages, which are the effective components of tumor immunotherapeutic drugs, have good therapeutic effects on malignant tumors. Special purposeHas good curative effect on lung cancer.
Furthermore, FABP4 of the present invention + C1q + Macrophages can be used as biological markers for evaluating tumor immunotherapy effect and the like, and the FABP4 group + C1q + Macrophages provide a new way for tumor immunotherapy and have clinical transformation prospects.
Drawings
The advantages and the manner of carrying out the invention will become more apparent from the following detailed description, taken in conjunction with the accompanying drawings, in which the content shown is meant to illustrate, but not to limit, the invention in any sense, and wherein:
FIG. 1 shows CD68 in tumor tissue and regional draining lymph nodes + FABP4 + C1q + Multicolor immunohistochemical staining patterns of macrophages.
FIG. 2 shows CD68 in lymph nodes of the different patients of FIG. 1 + FABP4 + C1q + Comparison of macrophage infiltration number.
FIG. 3 shows CD68 in tumor tissue of different patients of FIG. 1 + FABP4 + C1q + Comparison of macrophage infiltration number.
FIG. 4 is FABP4 + C1q + Macrophages and non-FABP 4 + C1q + Normalized mode fluorescence front for inflammatory cytokine production by macrophages.
FIG. 5 is FABP4 + C1q + Macrophages and non-FABP 4 + C1q + Mean fluorescence intensity of macrophages producing inflammatory cytokines versus graph.
FIG. 6 is FABP4 + C1q + Macrophages and non-FABP 4 + C1q + Normalized mode fluorescence peak profile of macrophage phagocytosis.
FIG. 7 is FABP4 + C1q + Macrophages and non-FABP 4 + C1q + Average fluorescence intensity for macrophage phagocytosis.
FIG. 8 is FABP4 + Comparison of inflammatory cytokine expression levels after siRNA transfection by macrophagesA drawing.
FIG. 9 is FABP4 + Comparison of macrophage fatty acid degradation pathway enzyme and macrophage fatty acid synthetase expression level after siRNA transfection.
FIG. 10 is FABP4 + Macrophage apoptosis assay following siRNA transfection.
FIG. 11 is FABP4 + Macrophage apoptosis rate comparison graph after siRNA transfection.
FIG. 12 is C1q + Normalized mode fluorescence peak pattern of inflammatory cytokines after siRNA transfection by macrophages.
FIG. 13 is C1q + Mean fluorescence intensity contrast of inflammatory cytokines after siRNA transfection by macrophages.
FIG. 14 is C1q + Comparison of inflammatory cytokine expression levels after siRNA transfection by macrophages.
FIG. 15 is C1q + Normalized mode fluorescence peak pattern of macrophage phagocytosis after siRNA transfection by macrophages.
FIG. 16 is C1q + Mean fluorescence intensity contrast for macrophage phagocytosis following siRNA transfection.
Detailed Description
The present invention will be described in further detail below with reference to the drawings and preferred embodiments, so that those skilled in the art can better understand the technical solutions of the present invention.
Multicolor immunohistochemical staining:
s1, baking slices: placing paraffin-embedded pathological tissue slices in a rack, and roasting slices 2 h at 65 ℃;
s2, can moving: sequentially dewaxing in gradient in xylene-I, xylene-II and xylene-III; and (3) rehydrating: absolute ethanol-I, absolute ethanol-II, 95% ethanol, 75% ethanol;
s3, fixing with 10% neutral formalin for 10 min;
s4, antigen retrieval: high-temperature antigen retrieval using (ph=9.0) an antigen retrieval liquid microwave oven;
s5, developing: after the sections are cooled, drawing tissue boundaries by an immunohistochemical hydrophobic pen, and sealing for 10 min by using primary anti-dilution (sealing liquid) in an Opal-7-color-Manual IHC kit to reduce non-specificity;
s6, incubating primary antibody: removing the surface sealing liquid of the slice, dripping the primary antibody, covering the tissue, and placing the wet box at 4 ℃ overnight;
s7, incubating a secondary antibody: the next day, the wet box is taken out from the refrigerator, the wet box is put at room temperature for half an hour, and horseradish peroxidase secondary antibody (common to mice and rabbits) in the Opal-7-color-Manual IHC kit is dripped on the tissue and incubated for 10 min at room temperature;
s8, incubating fluorescent dye: after the secondary antibody incubation is completed, opal fluorescent dye of a specific channel is selected and dripped into the slice tissue, the tissue is covered, and the incubation is carried out for 10 min at room temperature;
s9, repairing the second round of antigen: after the fluorescent dye is incubated, 3 times of washing with 1 XTBST are carried out for 3 min each time; performing antigen retrieval for the second round, microwave oven high Wen Xiufu antigen, and staining for multiple rounds: repeating the steps S6-S9;
s10, cell nucleus staining: after cooling, adding DAPI dye to dye cell nuclei, and incubating for 10 min at room temperature;
s11, sealing piece: after incubation of DAPI is completed, an anti-fluorescence attenuation tablet sealing agent is applied for sealing;
the results show that:
as shown in fig. 1 to 3: staining by polychromatic immunohistochemistry, CD68 was found in tumor tissue and locally draining lymph node tissue of MPR (major pathological remission) patients + FABP4 + C1q + Macrophages infiltrate more than non-MPR (non-major pathological remission) patients.
This result suggests FABP4 + C1q + Macrophage infiltration affects the effective rate of immunotherapy, especially anti-PD-1 therapy.
The invention evaluates the curative effect of immunotherapy by applying MPR, and the curative effect is good when the active tumor component in the tumor tissue excised by surgery is less than 10 percent.
In vitro CD68 + FABP4 + C1q + Macrophage function exploration:
peripheral blood mononuclear cellPBMC) extraction and FABP4 + C1q + Macrophage induction:
s1, diluting healthy peripheral blood with an equal volume of PBS buffer solution to form a diluent, and obtaining a cell layering solution (Ficoll): diluent = 1:1, diluent is slowly added into Ficoll along the tube wall, gradient centrifuged 1800r/min,18min (9-0 rise); after centrifugation, the upper layer of transparent PBS buffer is slowly sucked and discarded by a sterile suction tube, about 1-2ml remains, and the suction tube is inserted into a cloud and fog belt to suck PBMCs and washed for 2 times; magnetic beads (CD 14 microblads) sorting CD14 + Monocytes;
s2, culturing and inducing macrophages: the CD14 selected above was resuspended and cultured with RPMI-1640 complete medium containing 10% FBS,1% diabody (mixture of Streptomyces lividans), 50ng/ml M-CSF + Monocytes, adjusted to a concentration of 1X 10 6 Inoculating the seeds into a cell culture dish for culture;
s3, semi-quantitative liquid exchange is carried out with 2-3 days as a period during culture; culturing for 7-9 days to obtain M0 macrophage, and separating FABP4 by flow fluorescence + C1q + Macrophages.
Detection of FABP4 by flow cytometry + C1q + Macrophages and non-FABP 4 + C1q + Macrophage inflammatory cytokine production level and phagocytic capacity.
The results show that:
as shown in fig. 4 and 5: FABP4 + C1q + Macrophage (FABP 4) high C1q + Macrophages), IL-6 (interleukin-6), IL-1 beta (interleukin-1 beta) inflammatory cytokine production capacity is greater than that of non-FABP 4 + C1q + Macrophage (non FABP 4) high C1q + Macrophages).
As shown in fig. 6 and 7: FABP4 + C1q + Macrophage (FABP 4) high C1q + Macrophages) phagocytic capacity greater than non-FABP 4 + C1q + Macrophage (non FABP 4) high C1q + Macrophages).
Knocking down FABP4 expression in macrophages can reduce the production level of inflammatory cytokines IL-6 and IL-1 beta of the macrophages, increase apoptosis rate of the macrophages and promote decomposition of fatty acids of the macrophages:
PBMC extraction and CD14 separation + Monocytes, after 7 days of incubation with M-CSF, were subjected to siRNA transfection by induced macrophages.
The results show that:
as shown in fig. 8: macrophage inflammatory cytokines IL-6 and IL-1. Beta. Expression levels were reduced in the siFABP4 group (interfering FABP4 group). This result suggests that FABP4 may enhance the inflammatory cytokine production capacity of macrophages.
As shown in fig. 9: the expression of macrophage fatty acid degradation pathway enzyme (CPT 1, ACADM, HADHA) of siFABP4 group (interference FABP4 group) is increased, and the expression of fatty acid synthase (FASN, ACACACA) is decreased. This result suggests that FABP4 promotes macrophage fatty acid synthesis, regulates macrophage energy metabolism and storage.
As shown in fig. 10 and 11: the rate of macrophage apoptosis was higher in the siFABP4 group (interfering FABP4 group). This result suggests that FABP4 may play an important role in macrophage survival.
Knocking down C1q expression in macrophages will reduce their inflammatory cytokine production and phagocytic function:
PBMC extraction and CD14 separation + Monocytes, after 7 days of incubation with M-CSF, were subjected to siRNA transfection by induced macrophages.
The results show that:
as shown in fig. 12 to 14: the inflammatory cytokines IL-6, IL-1. Beta. And TNF-. Alpha.produced by macrophages of group siC1q (interfering with group C1 q) were reduced in levels. This result suggests that C1q may enhance the inflammatory cytokine production capacity of macrophages.
As shown in fig. 15 and 16: siC1 group 1q (interfering with the phagocytic capacity of macrophages in group C1 q) decreased. This result suggests that C1q plays an important role in phagocytic function of macrophages.
The p-value in fig. 2, 3, 5, 7, 8, 9, 11, 13, 14 and 16 is a parameter for determining the hypothesis test result, where p-value <0.05, p-value <0.01, p-value <0.001, p-value <0.0001, and ns-p-value >0.05.
In the experimental process, the invention analyzes the tumor and the macrophage landscape difference in the related tissues of non-small cell lung cancer (non-small cell lung cancer, NSCLC) patients operated after ICIS combined chemotherapy by using a single cell sequencing technology, and identifies a group of FABP4 related to the curative effect of the immune combined chemotherapy + C1q + Macrophages.
Tumor tissue and regional drainage lymph node tissue of MPR patient after ICIS combined chemotherapy are infiltrated with more FABP4 + C1q + Macrophages. And FABP4 + C1q + Macrophages have powerful pro-inflammatory cytokine production and phagocytic capacity. The above results suggest FABP4 + C1q + Macrophages can be used as a predictive target for immunotherapy of malignant tumors, and have a powerful antitumor effect.
The foregoing describes the embodiments of the present invention in detail, but the description is only a preferred embodiment of the present invention and should not be construed as limiting the scope of the invention. All equivalent changes and modifications within the scope of the present invention are intended to be covered by this patent.
Claims (8)
1. FABP4 + C1q + The preparation method of the macrophage is characterized by comprising the following steps: the method comprises the following steps:
s1, peripheral blood mononuclear cell extraction: mononuclear cells were obtained from peripheral blood density gradient centrifugation and CD14 was sorted using magnetic beads + Monocytes;
S2、FABP4 + C1q + macrophage induction: CD14 selected in step S1 was resuspended and cultured with RPMI-1640 complete medium containing 10% FBS,1% diabody, 50ng/ml M-CSF + Monocytes, adjusted to a concentration of 1X 10 6 Inoculating the seeds into a cell culture dish for culture;
s3, induction culture conditions: semi-quantitatively changing liquid with 2-3 days as period, culturing for 7-9 days to obtain M0 macrophage, and separating FABP4 by flow fluorescence + C1q + Macrophages with a function of promoting the growth of human body。
2. The method of manufacturing according to claim 1, characterized in that: in the step S1, diluting healthy peripheral blood with an equal volume of PBS buffer solution to form a diluent, slowly adding the diluent into the cell layering solution along the pipe wall according to the ratio of the cell layering solution to the diluent of 1:1, and performing gradient centrifugation for 1800r/min for 18min; after centrifugation, slowly sucking and discarding the upper transparent PBS buffer solution by using a sterile suction tube, and inserting the sterile suction tube into a cloud and fog belt to suck peripheral blood mononuclear cells and washing for 2 times, wherein 1-2ml is left; CD14 sorting Using CD14 microbeds magnetic beads + Monocytes.
3. FABP4 obtained by the production method according to claim 1 or 2 + C1q + Macrophages.
4. A FABP4 as in claim 3 + C1q + Application of macrophage in preparing tumor immunotherapy medicine is disclosed.
5. Use according to claim 4, characterized in that: the FABP4 + C1q + Macrophages simultaneously express FABP4 and C1q at high levels.
6. Use according to claim 4, characterized in that: the FABP4 + C1q + Inflammatory cytokines produced by macrophages include TNF- α, IL-6 and IL-1β.
7. Use according to claim 4, characterized in that: types of tumors include solid tumors and non-solid tumors.
8. Use according to claim 4, characterized in that: the tumor types include lung cancer, ovarian cancer, liver cancer, pancreatic cancer, colon cancer, rectal cancer, renal cancer, prostate cancer, thyroid cancer, tongue cancer, melanoma, lymphoma, thyroid cancer, breast cancer, head and neck squamous carcinoma, and gastric cancer.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104302322A (en) * | 2012-03-26 | 2015-01-21 | 日本化学药品株式会社 | Agent for preventing or treating giant cell tumor or chondrosarcoma arising in bone/soft tissue |
| US20180355037A1 (en) * | 2017-03-24 | 2018-12-13 | Meng ZHENG | Anti-human adrb3 monoclonal antibody and application thereof in disease diagnosis and treatment |
| CN109071663A (en) * | 2016-02-05 | 2018-12-21 | 奥里尼斯生物科学公司 | Bispecific signal transduction agent and application thereof |
| US20200354424A1 (en) * | 2018-01-26 | 2020-11-12 | Orionis Biosciences, Inc. | Xcr1 binding agents and uses thereof |
| CN112805004A (en) * | 2018-07-02 | 2021-05-14 | 艾珀特玛治疗公司 | Targeted delivery of therapeutic agents to human adipocytes |
| US20230159932A1 (en) * | 2020-04-22 | 2023-05-25 | Direct Biologics Llc | Methods and compositions for treating inflammatory conditions associated with infectious disease |
-
2023
- 2023-09-08 CN CN202311155081.8A patent/CN116875550B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104302322A (en) * | 2012-03-26 | 2015-01-21 | 日本化学药品株式会社 | Agent for preventing or treating giant cell tumor or chondrosarcoma arising in bone/soft tissue |
| CN109071663A (en) * | 2016-02-05 | 2018-12-21 | 奥里尼斯生物科学公司 | Bispecific signal transduction agent and application thereof |
| US20180355037A1 (en) * | 2017-03-24 | 2018-12-13 | Meng ZHENG | Anti-human adrb3 monoclonal antibody and application thereof in disease diagnosis and treatment |
| US20200354424A1 (en) * | 2018-01-26 | 2020-11-12 | Orionis Biosciences, Inc. | Xcr1 binding agents and uses thereof |
| CN112805004A (en) * | 2018-07-02 | 2021-05-14 | 艾珀特玛治疗公司 | Targeted delivery of therapeutic agents to human adipocytes |
| US20230159932A1 (en) * | 2020-04-22 | 2023-05-25 | Direct Biologics Llc | Methods and compositions for treating inflammatory conditions associated with infectious disease |
Non-Patent Citations (1)
| Title |
|---|
| JIHAO TU: "Single-cell RNA datasets and bulk RNA datasets analysis demonstrated C1Q+ tumor-associated macrophage as a major and antitumor immune cell population in osteosarcoma", FRONT. IMMUNOL, vol. 14, pages 1 - 11 * |
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