Method for enhancing the immune function of bone marrow precursor cells by using 33-site monoubiquitin molecules

Abstract

The invention discloses a method for enhancing the immune function of bone marrow precursor cells by using a 33-site monoubiquitin molecule, and relates to the field of genetic engineering technology. Bone marrow precursor cells BMPCs (Bone Marrow Precursor Cells) are isolated from mouse bone marrow, and the bone marrow precursor cells are pretreated with a 33-site monoubiquitin molecule, and then adoptively immunized in vitro, and finally subjected to enzyme-linked immunosorbent assay and flow cytometry experiments. The experiment shows that the immune function of the treated bone marrow precursor cells is significantly enhanced, and their antigen-specific CTL induction ability is also enhanced, and their ability to kill tumor cells is enhanced; the 33-site monoubiquitin molecule can increase the ability of bone marrow precursor cells to transfer intracellular lymphocytes to produce perforin and granzyme B. Bone marrow precursor cells enhanced by the 33-site monoubiquitin molecule can be used in the preparation of tumor immunotherapy drugs.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to a method for enhancing the immune function of bone marrow precursor cells by using a 33-site monoubiquitin molecule. Background Art

[0002] Bone marrow (BM) is a spongy tissue that exists in the medullary cavity of long bones (such as humerus and femur) and the mesh between the loose bone of flat bones (such as ilium). It contains different types of stem cells, including hematopoietic stem cells and some less abundant non-hematopoietic mesenchymal stem cells. Hematopoietic stem cells can differentiate into three-lineage (white blood cells, red blood cells, platelets) progenitor cells in the hematopoietic microenvironment. Under the action of various hematopoietic factors, the three-lineage progenitor cells differentiate into various blood cell precursor cells. Under the conditions of a good environment and sufficient nutritional components, the precursor cells further differentiate into mature blood cells and enter the blood circulation. The bone marrow precursor cells mentioned in this article are partially differentiated dendritic cell precursor cells formed by hematopoietic stem cells under the stimulation of IL-4 (interleukin 4) and GM-CSF (granulocyte macrophage colony stimulating factor). Their differentiation potential is unipotent, which is less than that of progenitor cells. They can be regarded as the last step from stem cell differentiation to dendritic cells and are a relatively immature state of dendritic cells. (Dzierzak, E. & Speck, N.A. Of lineage and legacy: the development of mammalian hematopoietic stem cells. Nature Immunol. 9, 129–136 (2008).)

[0003] Ubiquitin (Ub) is composed of 76 amino acids and is widely expressed in eukaryotic cells. It contains 7 lysine residues (K6, K11, K27, K29, K33, K48 and K63). Usually, the C-terminal glycine of a ubiquitin molecule can combine with the ε-amino group of any lysine in another ubiquitin molecule to form polyubiquitination; therefore, 7 ubiquitin chains with different structures can be produced. The diverse ubiquitin chain connection methods can guide substrate proteins to different fates and cause different cellular reactions (Malynn BA, Ma A. Ubiquitin makes its mark on immune regulation Immunity, 2010, 33: 843-52.).

[0004] In addition, ubiquitin molecules can also be linked to substrate target proteins in a single molecule or linear manner to form monoubiquitination or linear ubiquitination to exert different regulatory functions.

[0005] In scientific research on ubiquitination, the ubiquitination of K48 and K63 is called classical ubiquitination because of its high content and relatively clear research, while other ubiquitination modes are called non-classical ubiquitination. In recent years, other types of ubiquitination chains assembled through K6, K11, K27, K29 and K33 residues have also gradually gained attention. For example, K6-linked ubiquitination has been shown to be associated with mitochondrial autophagy and DNA damage response; the presence of K27 and K29-linked chains promotes proteasome and autophagic degradation, as well as protein aggregation associated with Parkinson's disease (PD), etc. (Nucifora FC Jr, Nucifora LG, Ng CH, Arbez N, Guo Y, Roby E, et al. Ubiqutination via K27 and K29 ​​chains signals aggregation and neuronal protection of LRRK2 by WSB1. Nat Commun. 2016; 7: 11792.), while there are still few literature reports on K33.

[0006] The ubiquitination process is of great significance in BMPCs maturation, migration, adaptive immunity, and maintaining internal environment stability. You X et al. inhibited the ubiquitination of the NF-KB pathway with the ubiquitination inhibitor PYR-41 or Thalidomide, and observed that the recruitment of p97 and Sec61 to the endosome was reduced, resulting in a decrease in the cytoplasmic translocation of internalized antigens, which impaired the antigen presentation ability of BMPCs. When BMPCs are endocytosed, the ubiquitination of the B chain of MHC-II molecules can regulate the expression of MHC-II molecules. (Jancic C, Savina A, Wasmeier C, et al. Rab27a regulates phagosomal pH and NADPH oxidase recruitment to dendritic cell phagosomes [J]. Nat Cell Biol. 2007; 9 (4): 367-378). Both MARCH1 and MARCH8 can ubiquitinate MHC-II class molecules. MARCH1 negatively regulates CD86 ubiquitination, inhibiting IL-10 autocrine secretion and CTL induction. Hrd1 is a transmembrane protein in the endoplasmic reticulum that targets misfolded proteins for proteasome degradation. During inflammation, Hrd1 interacts with BIMP-1 (Blymphocyte-induced maturation protein 1) to promote BIMP-1 ubiquitination and self-degradation, mediating CD4+ T cell activation.

[0007] To date, K33-linked ubiquitination remains the least studied of all Ub linkage types, and there are few reports of its association with bone marrow precursor cells. This ubiquitination has been identified, but its role has not been further explored. A major breakthrough in K33 research was the structural analysis of TRABID and its K29 / K33 linkage specificity. However, reports on in-depth studies of its role in physiological processes are still very limited (Michal Tracz, Wojciech Bialek, et al. Beyond K48 and K63: non-canonical protein ubiqutination. Cell Mol Biol Lett. 2021; 26: 1.). This makes this type of ubiquitination connection even more mysterious, and researchers are urgently waiting to develop its potential functions and value. Summary of the invention

[0008] The purpose of the present invention is to provide a method for enhancing the immune function of bone marrow precursor cells by using a 33-site monoubiquitin molecule in view of the above-mentioned deficiencies in the prior art; the immune function of the treated bone marrow precursor cells is significantly enhanced, their antigen-specific CTL inducing ability is also enhanced, and their ability to kill tumor cells is enhanced; the 33-site monoubiquitin molecule can increase the ability of bone marrow precursor cells to transfer intracellular lymphocytes to produce perforin and granzyme B.

[0009] Another object of the present invention is to provide the use of bone marrow precursor cells enhanced by 33-site monoubiquitin molecules in the preparation of tumor immunotherapy drugs.

[0010] The method for enhancing the immune function of bone marrow precursor cells by using the 33-site monoubiquitin molecule comprises the following steps:

[0011] 1) Isolate bone marrow progenitor cells from mouse bone marrow;

[0012] 2) Bone marrow progenitor cells were pretreated with 33-site monoubiquitin molecules for in vitro adoptive immunization.

[0013] In step 1), the specific method for isolating bone marrow precursor cells from mouse bone marrow can be as follows: killing mice by cervical dislocation, disinfecting with 75% alcohol for 10 minutes, aseptically separating their humerus, femur and tibia, flushing out the bone marrow as completely as possible with a 5mL sterile syringe, and then fully grinding the bone marrow into a single cell suspension with the bottom of a 1mL sterile syringe; centrifuging at 1500rpm for 5min and discarding the supernatant, adding 2mL of red blood cell lysis buffer (ACK) to lyse red blood cells for 4-5min, and then adding 2mL of complete culture medium to terminate; centrifuging again, discarding the supernatant, adding cytokines IL4 and GM-CSF to make their final concentrations of 1ng / mL and 10ng / mL; culturing in a 37°C, 5% CO2 constant temperature incubator; on the 4th to 5th day, discarding the suspended cells in the supernatant, washing the bottom of the dish with PBS, and the cells attached to the bottom of the dish obtained are the bone marrow precursor cells (BMPCs) isolated from mouse bone marrow.

[0014] In step 2), the specific steps of pretreating bone marrow progenitor cells with 33-site monoubiquitin molecules for in vitro adoptive immunization can be as follows: first add 5-7.5 μM (5 μM to achieve the ideal effect) of ubiquitin mutant chain K33 Only (33-site monoubiquitin molecule) to the bone marrow progenitor cells separated in step 1), and after 30 minutes of action, load exogenous antigen (ovalbumin, OVA 50 μg / mL), and after 4 hours, collect the cells into a centrifuge tube, resuspend the cells with PBS and centrifuge to remove the influence of excess impurities; then resuspend the cells with 37°C preheated physiological saline or PBS to adjust the concentration, and intraperitoneally inoculate the same number (1×10 5Bone marrow precursor cells (100 cells / mL) were transferred into 4-6 week old C57BL / 6 female mice with the same genetic background; 5-7 days later, spleen and lymph nodes were obtained and ground to prepare cell suspension for later use.

[0015] The ubiquitin mutant chain K33 Only keeps lysine unchanged at the K33 Only site, and lysine at other sites is mutated to arginine. Considering that the ubiquitin connection may act at multiple points, relatively speaking, only retaining the target site and mutating other sites may be a better choice. Moreover, since it only keeps lysine unchanged at the K33 Only site, steps such as ubiquitin interference are omitted, and the treatment is more convenient and quick.

[0016] In order to verify the enhancing effect of 33-site monoubiquitin molecule on the immune function of bone marrow precursor cells, enzyme-linked immunosorbent assay and flow cytometry experiment can be performed.

[0017] The specific steps of the ELISpot assay are as follows: first, prepare the mouse spleen suspension concentration, dislocate and kill the adoptive immunization experimental mice and aseptically separate the spleen, lyse the red blood cells with red blood cell lysis solution, and adjust the suspension concentration to 1×10 7 / mL; secondly, the pre-coated IFN-γ antibody detection plate was activated with complete culture medium, and the suspension was added, i.e., 1×10 6 / well, add SIINFEKL 2-4μg / mL, and culture for 16-24h; after hypotonic lysis of cells with pre-cooled double distilled water, add biotin-IFN-γ and avidin-HRP antibody in sequence, and wash the plate with washing buffer during incubation; finally, prepare AEC color development solution for color development, rinse with clean water to terminate the experiment, and after the test plate is naturally dried, use ELISPOT image analysis system to take pictures and count the number of spots.

[0018] First, the mouse spleen suspension concentration was prepared. The adoptive immunization experimental mice were killed by dislocation and the spleen was aseptically separated. After the red blood cells were lysed with red blood cell lysis buffer, the suspension concentration was adjusted to 1×10 7 / mL; secondly, the pre-coated IFN-γ antibody detection plate was activated with complete culture medium, and the suspension was added, i.e., 1×10 6 / well, add SIINFEKL 2-4μg / mL, and culture for 16-24h; after hypotonic lysis of cells with pre-cooled double distilled water, add biotin IFN-γ and avidin-HRP antibody successively, and wash the plate with washing buffer during incubation; finally, prepare AEC color development solution for color development, and rinse with clean water to terminate the experiment. After the test plate is naturally dried, take pictures with ELISPOT image analysis system and count the number of spots.

[0019] The specific steps of the flow cytometry experiment can be:

[0020] Intracellular staining: To detect the expression of perforin and granzyme B in CD8+T lymphocytes in mouse spleen cells after the action of bone marrow precursor cells BMPCs, the mouse spleen cells were first ground to prepare a spleen cell suspension, and then the spleen cells were stained with CD8 antibody at the concentration recommended in the instructions at 4°C in the dark, and then the permeabilized cells were fixed with Fix / Permeabilization Solution at 4°C in the dark for 20 minutes; washed with PBS, and each antibody was combined in an appropriate volume of staining buffer according to the recommended amount in the instructions, stained at 4°C in the dark for 30 minutes, and washed with flow cytometer staining buffer; finally, the FACSCalibur flow cytometer was used for on-machine detection, and the obtained data were analyzed using Cytexpert software.

[0021] Experiments have shown that the immune function of the treated bone marrow precursor cells is significantly enhanced, their antigen-specific CTL induction ability is also enhanced, and their ability to kill tumor cells is also enhanced; the 33-site monoubiquitin molecule can increase the ability of bone marrow precursor cells to transfer intracellular lymphocytes to produce perforin and granzyme B.

[0022] Bone marrow precursor cells enhanced by the 33-site monoubiquitin molecule can be used in the preparation of tumor immunotherapy drugs.

[0023] Compared with the prior art, the present invention has the following outstanding advantages:

[0024] The present invention treats bone marrow precursor cells BMPCs with 33-site monoubiquitin molecules. Experiments have shown that 33-site monoubiquitin molecules can increase the ability of bone marrow precursor cells BMPCs to produce perforin and granzyme B in recipient lymphocytes, as well as the ability to kill tumor cells. Its antigen-specific CTL induction ability is also enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a diagram of 33-site monoubiquitin molecules enhancing the transfer of recipient spleen antigen-specific CTL.

[0026] Figure 2 Figure 33 shows the enhanced translocation of perforin production by 33-site monoubiquitin molecules into recipient spleen cells.

[0027] Figure 3 Figure 33 shows the enhanced translocation of monoubiquitin molecules into recipient spleen cells to produce granzyme B.

[0028] Figure 4 This is a diagram showing the tumor-killing effect of 33-site monoubiquitin molecules on transfected recipient spleen cells. DETAILED DESCRIPTION

[0029] The following embodiments will further illustrate the present invention in conjunction with the accompanying drawings.

[0030] In the present embodiment, BMPCs were treated with 33-site monoubiquitin molecules, and the ELISpot assay and flow cytometry were used to detect the ability of BMPCs to induce antigen-specific CTLs, the ability of BMPCs to produce perforin and granzyme B in the recipient lymphocytes, and the ability of the treated lymphocytes to kill tumor cells.

[0031] The embodiment of the present invention includes the following steps:

[0032] 1) Induction and isolation of bone marrow progenitor cells (BMPCs);

[0033] The specific method for isolating bone marrow precursor cells from mouse bone marrow can be as follows: kill the mouse by cervical dislocation, disinfect with 75% alcohol for 10 minutes, aseptically separate the humerus, femur and tibia, flush out the bone marrow as completely as possible with a 5mL sterile syringe, and then fully grind the bone marrow into a single cell suspension with the bottom of a 1mL sterile syringe; centrifuge at 1500rpm for 5min and discard the supernatant, add 2mL red blood cell lysis solution (ACK) to lyse the red blood cells for 4-5min, and then add 2mL complete culture medium to terminate; centrifuge again, discard the supernatant, add cytokines IL4 and GM-CSF to make the final concentrations of 1ng / mL and 10ng / mL; place in a 37°C, 5% CO2 constant temperature incubator for culture; on the 4th to 5th day, discard the suspended cells in the supernatant, rinse the bottom of the dish with PBS, and the cells attached to the bottom of the dish are the bone marrow precursor cells (BMPCs) isolated from the mouse bone marrow.

[0034] 2) Pretreatment of bone marrow precursor cells:

[0035] In vitro adoptive immunization experiments:

[0036] The isolated bone marrow progenitor cells were first added with ubiquitin mutant chain K33 Only (33-site monoubiquitin molecule) 5-7.5 μM (5 μM to achieve the ideal effect), and after 30 minutes of action, exogenous antigen (ovalbumin, OVA 50 μg / mL) was loaded. After 4 hours, the cells were collected into a centrifuge tube, resuspended with PBS and centrifuged to remove excess impurities; the cells were resuspended with 37°C pre-warmed physiological saline or PBS and the concentration was adjusted. The same number (1×10 5 Bone marrow precursor cells (100 cells / mL) were transferred into 4-6 week old C57BL / 6 female mice with the same genetic background; 5-7 days later, spleen and lymph nodes were obtained and ground to prepare cell suspension for later use.

[0037] Enzyme-linked immunospot assay (Elispot)

[0038] To prepare the concentration of mouse spleen suspension, the adoptive immunization experimental mice were killed by dislocation and the spleen was aseptically isolated. After lysing the red blood cells with red blood cell lysis buffer, the suspension concentration was adjusted to 1×10 7 / mL; secondly, the pre-coated IFN-γ antibody detection plate was activated with complete culture medium (1640 + 10% FBS, FBS: fetal bovine serum) for 20 min, and 100 μL of the prepared cell suspension was added, i.e., 1×10 6 / well, add GF257 (2-4μg / mL), and culture in a 37℃, 5% CO2 constant temperature incubator for 16-24h; then, after hypotonic lysis of the cells with pre-cooled double distilled water, add biotin-IFN-γ first, incubate on a shaker at 37℃ for 1h, wash the plate with washing buffer and dry it, incubate it with avidin-HRP antibody under the same conditions for 1h, wash the plate with washing buffer again, and dry it for the last time; finally, prepare AEC color development solution for color development at room temperature in the dark, and terminate the experiment by rinsing with plenty of clean water after the color development is completed. After the test plate is naturally dried, take pictures with ELISPOT image analysis system and count the number of spots.

[0039] The statistical methods are as follows:

[0040] The measurement data of each group were expressed as mean ± standard deviation. GraphPad Prism 5 was used for statistical analysis, and the differences were analyzed using the Student's t test. P < 0.05 was considered statistically significant.

[0041] The 33-site monoubiquitin molecule enhances the transfer of spleen antigen-specific CTL to the recipient. Figure 1 BMPCs isolated by induction were treated with 33-site monoubiquitin molecule K33 Only (5-7.5μM) and exogenous OVA (50μg / mL), and equal numbers of BMPCs were transferred into 4-6 week-old C57BL / 6 female mice of the same genotype via the peritoneal cavity. 5-7 days after BMPCs were transferred into mice, spleen cells (SP) were taken from mice for Elispot experiment. Data are shown as mean +- standard deviation, and the student's t test was used between groups, *P<0.05, **P<0.01, ***P<0.001, n=3. OVA: ovalbumin.

[0042] Experimental results: Elispot test results showed that bone marrow progenitor cells (BMPCs) treated with K33-only ubiquitin molecules loaded with exogenous antigens and transfused into mice increased the ability to induce spleen cell-specific CTL production by 140%.

[0043] Conclusion: The immune function of BMPCs treated with 33-site monoubiquitin molecules combined with OVA antigen is enhanced, and the ability to induce the production of spleen antigen-specific CTL is further enhanced.

[0044] The 33-site monoubiquitin molecule enhances the expression of perforin in transfected spleen cells. Figure 2 , the induced and isolated BMPCs were treated with 33-site monoubiquitin molecule K33 Only (5-7.5μM) and exogenous OVA (50μg / mL), and the same number of BMPCs were transferred into the same genotype 4-6 week old C57BL / 6 female mice via the peritoneal cavity. 5-7 days after the BMPCs were transferred into the mice, the small spleen was taken and ground to obtain a cell suspension, and the expression of perforin was detected by flow cytometry. The results were expressed in a bar graph obtained by Prism analysis. OVA: ovalbumin.

[0045] Results: The results of flow cytometry showed that the RID value of the control group was set to 1, and the production of perforin by CTL mediated by bone marrow progenitor cells BMPCs treated with 33-site monoubiquitin molecules (K33Only) increased by 70%.

[0046] Conclusion: BMPCs treated with 33-site monoubiquitin molecules combined with OVA antigens can increase the secretion of perforin by spleen CTLs.

[0047] The monoubiquitin molecule at site 33 enhances the expression of granzyme B in transfected spleen cells. Figure 3 . The induced and isolated BMPCs were treated with the 33-site monoubiquitin molecule K33 Only (5-7.5μM) and exogenous OVA (50μg / mL), and the same number of BMPCs were transferred into the same genotype 4-6 week old C57BL / 6 female mice via the peritoneal cavity. After 5-7 days, the spleen of the mice was taken and ground to obtain a cell suspension, and the expression of granzyme B was detected by flow cytometry, which was represented by the bar graph obtained by Prism analysis. OVA: ovalbumin.

[0048] Experimental results: Flow cytometry results showed that if the control group value was set to 1, the production of granzyme B by BMPCs-mediated CTL increased by 60% after K33 Only treatment.

[0049] Conclusion: BMPCs treated with 33-site monoubiquitin molecules combined with OVA antigens increase the secretion of granzyme B by spleen CTLs.

[0050] The monoubiquitin molecule at site 33 enhances the tumor-killing effect of transfected spleen cells Figure 4,The induced and isolated BMPCs were treated with 33-site monoubiquitin molecule K33 Only (5-7.5μM) and exogenous OVA (50μg / mL), and the same number of BMPCs were transferred into the same genotype 4-6 week old C57BL / 6 female mice via the peritoneal cavity. 5-7 days after BMPCs were transferred into mice, small spleen cells were taken and mixed with EG7 cells at a ratio of 0.5:1, 1:1, 5:1 (number of spleen cells: number of EG7 cells) for 8 hours, and the tumor killing efficiency was detected by flow cytometry. The results were expressed in line graphs and bar graphs using Prism analysis. OVA: ovalbumin.

[0051] Results: Flow cytometry results showed that the efficiency of BMPCs-mediated CTL killing of tumor cells was improved after K33 Only treatment.

[0052] Conclusion: The immune function of BMPCs treated with 33-site monoubiquitin molecules combined with OVA antigen is enhanced, and the ability of spleen antigen-specific CTL to kill tumors is further enhanced.

Claims

1. 33-site monoubiquitin molecule method for enhancing the immune function of bone marrow precursor cells, characterized in that The following steps are involved: 1) Isolate bone marrow progenitor cells from mouse bone marrow; 2) Bone marrow progenitor cells were pretreated with 33-site monoubiquitin molecules for in vitro adoptive immunization.

2. The method for enhancing the immune function of bone marrow precursor cells by using a 33-site monoubiquitin molecule as claimed in claim 1, characterized in that In step 1), the specific method for isolating bone marrow precursor cells from mouse bone marrow is as follows: kill the mouse by cervical dislocation, disinfect with 75% alcohol for 10 minutes, aseptically separate the humerus, femur and tibia, flush out the bone marrow as completely as possible with a 5mL sterile syringe, and then fully grind the bone marrow into a single cell suspension with the bottom of a 1mL sterile syringe; centrifuge at 1500rpm for 5min and discard the supernatant, add 2mL of red blood cell lysis solution ACK to lyse the red blood cells for 4-5min, and then add 2mL of complete culture medium to terminate; centrifuge again, discard the supernatant, add cytokines IL4 and GM-CSF to make the final concentrations of 1ng / mL and 10ng / mL; place in a 37°C, 5% CO2 constant temperature incubator for culture; on the 4th to 5th day, discard the suspended cells in the supernatant, rinse the bottom of the dish with PBS, and the cells attached to the bottom of the dish are the bone marrow precursor cells BMPCs isolated from the mouse bone marrow.

3. The method for enhancing the immune function of bone marrow precursor cells by using a 33-site monoubiquitin molecule as claimed in claim 1, characterized in that In step 2), the specific steps of pretreating bone marrow precursor cells with 33-site monoubiquitin molecules for in vitro adoptive immunization are: first adding ubiquitin mutant chain K33 Only to the bone marrow precursor cells separated in step 1), after the action, loading exogenous antigen OVA, collecting cells into a centrifuge tube, resuspending the cells with PBS and centrifuging to remove the influence of excess impurities; then resuspending the cells with pre-warmed saline or PBS to adjust the concentration, and transferring the same number of bone marrow precursor cells into 4-6 week old C57BL / 6 female mice with the same genetic background through the peritoneal cavity; after 5-7 days, the spleen and lymph nodes are taken and ground to prepare a cell suspension for use.

4. The method for enhancing the immune function of bone marrow precursor cells by using a 33-site monoubiquitin molecule as claimed in claim 3, characterized in that Add 5-7.5 μM ubiquitin mutant chain K33 Only and act for 30 min. Load the exogenous antigen OVA 50 μg / mL. After 4 h, collect the cells into a centrifuge tube.

5. The method for enhancing the immune function of bone marrow precursor cells by using a 33-site monoubiquitin molecule as claimed in claim 4, characterized in that Add 5 μM of ubiquitin mutant chain K33 Only.

6. The method for enhancing the immune function of bone marrow precursor cells by using a 33-site monoubiquitin molecule as claimed in claim 3, characterized in that The temperature of the pre-warmed saline solution is 37°C.

7. The method for enhancing the immune function of bone marrow precursor cells by using a 33-site monoubiquitin molecule as claimed in claim 3, characterized in that The ubiquitin mutant chain K33 Only keeps lysine unchanged at the K33 site, and lysine at other sites is mutated to arginine.

8. Use of bone marrow progenitor cells treated with the method for enhancing the immune function of bone marrow progenitor cells with the 33-site monoubiquitin molecule as claimed in claim 1 in the preparation of tumor immunotherapy drugs.

Images