CN111166867B - Function and application of PD-1 ubiquitination agonist - Google Patents

Abstract

The invention belongs to the technical field of life sciences, and specifically relates to the function and application of a PD-1 ubiquitination agonist. After extensive and in-depth research, the present invention finds for the first time that PD-1 will undergo ubiquitination and then be degraded, thereby enhancing the functions of T cell activation, proliferation and cytokine secretion, and improving the killing function of tumor-infiltrating T cells FBXO38 is a target that can promote the ubiquitination of PD-1, and promoting the expression of FBXO38 can significantly promote the degradation of PD-1. Inhibiting the expression of FBXO38 can inhibit the degradation of PD-1, which can be used to treat autoimmune diseases. Therefore, the present invention provides strong scientific evidence for the clinical immunotherapy of tumors and the treatment of autoimmune diseases from the level of clinical patient samples, the level of cell function and the level of molecules.

Description

Function and application of PD-1 ubiquitination agonist

technical field

The invention belongs to the technical field of life sciences, and specifically relates to the function and application of a PD-1 ubiquitination agonist.

Background technique

Malignant tumors are currently one of the most lethal diseases. Conventional treatment methods such as surgical resection, radiotherapy and chemotherapy are widely used in tumor treatment. However, these methods have limitations in the treatment of tumors, and it is difficult to completely cure them. Tumors, especially some metastatic malignant tumors.

As the most important line of defense for human health, the immune system is responsible for not only discovering and eliminating various pathogenic microorganisms (anti-infection) and harmful substances that may be produced in the body (anti-tumor), but also avoiding damage to the body or produce autoimmune disease. The immune system has a complex regulatory network to ensure that the immune response is in a balanced state. For example, the central tolerance mechanism ensures that the immune system clears self-reactive T cells, and the immunosuppressive cell-Treg suppresses the peripheral inflammatory response (Lohmann et al., 1996; van Noort et al., 1993). In addition, the immunosuppressive molecules expressed on the surface of immune cells are also an important part of ensuring immune balance, such as PD-1, CTLA-4, LAG-3 and TIM3 expressed on the surface of CD8+ T cells. After these immunosuppressive molecules are activated, they will start intracellular signal transduction pathways, transmit inhibitory signals to T cells, increase the threshold of T cell activation, and inhibit T cell activation, proliferation, cytokine secretion and other functions. The activation of these immunosuppressive molecules can inhibit the occurrence of autoimmune diseases. However, everything has two sides. In the tumor microenvironment, these immunosuppressive molecular pathways are usually continuously activated, resulting in the inhibition of the killing function and proliferation ability of tumor-infiltrating T cells, and tumor cells escape the killing of the immune system.

Recent studies have found that the continuous activation of the PD-1 pathway in the tumor microenvironment is an important mechanism of tumor immune escape. Tumor-infiltrating T cells highly express PD-1 molecules on the surface, and tumor cells highly express its ligand PD-L1 molecules, which results in the continuous activation of the PD-1 pathway in the tumor microenvironment, and tumor-infiltrating T cells become incompetent as a result. . Clinically, after blocking the PD-1 pathway with a PD-1 blocking antibody, the condition of about 20% of tumor patients will be alleviated. The emergence of PD-1 antibody is a milestone event in tumor treatment, allowing everyone to see the hope of treating tumors by activating the immune system, and opening up a new way of tumor treatment. However, many clinical data suggest that the effective rate of PD-1 antibodies against most solid tumors is only about 20%-40%, which means that most patients still cannot benefit from immunotherapy.

Contents of the invention

In order to overcome the problems existing in the prior art, the object of the present invention is the function and application of PD-1 ubiquitination agonists.

In order to achieve the above object and other related objects, the present invention adopts the following technical solutions:

The first aspect of the present invention provides the use of a PD-1 ubiquitination agonist in the preparation of a PD-1 degradation agent or in the preparation of a tumor immunotherapy drug.

In one embodiment, the tumor is selected from one of melanoma, non-small cell lung cancer, kidney cancer, head and neck squamous cell carcinoma, Hodgkin's lymphoma, gastric cancer, liver cancer, bladder cancer, endometrial cancer and colon cancer. one or more species.

Further, the PD-1 ubiquitination agonist refers to a molecule that can promote PD-1 ubiquitination.

Specifically, various chemical, physical, and biological methods can be used to promote PD-1 ubiquitination. including but not limited to:

(1) Regulating the PD-1 metabolic pathway to increase the ubiquitination level of PD-1;

(2) Directly link ubiquitin to PD-1.

Ubiquitin is a polypeptide consisting of 76 amino acids in eukaryotic cells. The PD-1 ubiquitination refers to linking ubiquitin to lysine of PD-1 for ubiquitination modification and affecting PD -1 signaling pathway or through the proteasome to degrade PD-1.

The PD-1 ubiquitination agonist can enhance functions such as activation, proliferation and cytokine secretion of T cells, improve the killing function and proliferation ability of tumor-infiltrating T cells, and cause tumor cells to be killed by the immune system.

The nucleotide sequence of the ubiquitin is shown in SEQ ID NO.1, specifically:

ATGCAGATCTTTGTGAAGACCCTCACTGGCAAAACCATCACCCTTGAGGTCGAGCCCAGTGACACCATTGAGAATGTCAAAGCCAAATTCAAGACAAGGAGGGTATCCCACCTGACCAGCAGCGTCTGATATTTGCCGGCAAACAGCTGGAGGATGGCCGCACTTCTCAGACTACAACATCCAGAAAGAGTCCACCCTGCACCTGGTGTTGCGCCTACCGCGGTGGATA

The Genbank accession number of the PD-1 is: AY238517.

Further, the PD-1 ubiquitination agonist can promote the degradation of PD-1.

In one embodiment, the PD-1 ubiquitination agonist may be a plasmid, carbohydrate, lipid, small molecular compound, RNA, polypeptide or protein packaged by a lentivirus or a retrovirus.

In one embodiment, the PD-1 ubiquitination agonist is a FBXO38 agonist.

Further, the FBXO38 agonist refers to a molecule that has a promoting effect on FBXO38.

The promoting effect on FBXO38 includes but not limited to: enhancing the activity of FBXO38, or promoting the transcription or expression of FBXO38 gene.

In one embodiment, the FBXO38 agonist is selected from molecules capable of increasing the expression level or activity of FBXO38.

The molecule capable of increasing the expression of FBXO38 or enhancing the activity may be a plasmid, carbohydrate, lipid, small molecular compound, RNA, polypeptide or protein packaged by a lentivirus or a retrovirus.

Optionally, the FBXO38 agonist may be a carrier that increases the expression of FBXO38. Specifically, it may be a vector containing FBXO38 gene and capable of expressing active FBXO38. The vector may be a plasmid vector, a lentiviral vector, or a retroviral vector. For example pHAGE, pMXs, MSCV.

The embodiment of the present invention specifically lists pHAGE-fEF1a-3Myc-FBXO38-IRES-ZsGreen and MSCV-3Myc-FBXO38-IRES-ZsGreen as carriers for increasing the expression of FBXO38. The specific pHAGE-fEF1a-3Myc-FBXO38-IRES-ZsGreen is as follows: the Fbxo38 sequence with 3 consecutive Myc tags at the N-terminal is inserted into the pHAGE vector, which is located before the IRES-ZsGreen. After transfecting cells, the FBXO38 molecule with Myc tag can be expressed in the cells, and ZsGreen indicates positive cells.

Specifically, MSCV-3Myc-FBXO38-IRES-ZsGreen is: insert the Fbxo38 sequence with 3 consecutive Myc tags at the N-terminal of the MSCV vector, before IRES-ZsGreen. After transfecting cells, the FBXO38 molecule with Myc tag can be expressed in the cells, and ZsGreen indicates positive cells.

The Genbank accession number of the human FBXO38 is: BC050424.

The Genbank accession number of the mouse FBXO38 is: AK031347.

The tumor immunotherapy drug has at least one of the following functions:

Enhance the killing function and proliferation ability of tumor-infiltrating T cells, inhibit cancer cell proliferation, reduce cancer cell viability, promote cancer cell apoptosis, and inhibit tumor growth.

The tumor immunotherapy drug necessarily includes a PD-1 ubiquitination agonist, and uses the PD-1 ubiquitination agonist as an active ingredient for the aforementioned functions.

In the tumor immunotherapy drug, the active ingredient that exerts the aforementioned functions may only be a PD-1 ubiquitination agonist, and may also contain other molecules that can perform similar functions.

The tumor immunotherapy drug can be a single-component substance or a multi-component substance.

The form of the tumor immunotherapy drug is not particularly limited, and can be in various forms such as solid, liquid, gel, semi-fluid, and aerosol.

Optionally, in the tumor immunotherapy drug, the PD-1 ubiquitination agonist is a FBXO38 agonist.

The tumor immunotherapy drugs are mainly targeted at mammals, such as rodents, artiodactyls, perissodactyls, lagomorphs, primates and the like. The primate is preferably a monkey, ape or Homo sapiens.

The second aspect of the present invention provides a tumor immunotherapy method, which is to administer a PD-1 ubiquitination agonist to a subject.

The subject may be a mammal. The mammal is preferably a rodent, an artiodactyla, a perissodactyla, a lagomorpha, a primate or the like. The primate is preferably a monkey, ape or human.

The subject may be a patient suffering from a tumor or an individual for whom treatment for a tumor is desired.

The PD-1 ubiquitination agonist can be administered to the subject before, during and after receiving tumor treatment.

In one embodiment, the tumor is selected from one of melanoma, non-small cell lung cancer, kidney cancer, head and neck squamous cell carcinoma, Hodgkin's lymphoma, gastric cancer, liver cancer, bladder cancer, endometrial cancer and colon cancer. one or more species.

The third aspect of the present invention provides a drug for tumor immunotherapy, including an effective amount of a PD-1 ubiquitination agonist.

Further, the tumor immunotherapy drug includes an effective amount of PD-1 ubiquitination agonist and a pharmaceutical carrier.

The tumor immunotherapy drug necessarily includes a PD-1 ubiquitination agonist, and uses the PD-1 ubiquitination agonist as an active ingredient for the aforementioned functions.

In the tumor immunotherapy drug, the active ingredient that exerts the aforementioned functions may only be a PD-1 ubiquitination agonist, and may also contain other molecules that can perform similar functions.

That is, the PD-1 ubiquitination agonist is the only active ingredient or one of the active ingredients of the tumor immunotherapy drug.

The tumor immunotherapy drug can be a single-component substance or a multi-component substance.

The form of the tumor immunotherapy drug is not particularly limited, and can be in various forms such as solid, liquid, gel, semi-fluid, and aerosol.

In one embodiment, the PD-1 ubiquitination agonist is a FBXO38 agonist.

Further, the FBXO38 agonist refers to a molecule that has a promoting effect on FBXO38.

The promoting effect on FBXO38 includes but not limited to: enhancing the activity of FBXO38, or promoting the transcription or expression of FBXO38 gene.

In one embodiment, the FBXO38 agonist is selected from molecules capable of increasing the expression level or activity of FBXO38.

The molecule capable of increasing the expression of FBXO38 or enhancing the activity may be a plasmid, carbohydrate, lipid, small molecular compound, RNA, polypeptide or protein packaged by a lentivirus or a retrovirus.

Optionally, the FBXO38 agonist may be a carrier that increases the expression of FBXO38. Specifically, it may be a vector containing FBXO38 gene and capable of expressing active FBXO38. The vector may be a plasmid vector, a lentiviral vector, or a retroviral vector. For example pHAGE, pMXs, MSCV.

The embodiment of the present invention specifically lists pHAGE-fEF1a-3Myc-FBXO38-IRES-ZsGreen and MSCV-3Myc-FBXO38-IRES-ZsGreen as carriers for increasing the expression of FBXO38. The specific pHAGE-fEF1a-3Myc-FBXO38-IRES-ZsGreen is as follows: the Fbxo38 sequence with 3 consecutive Myc tags at the N-terminal is inserted into the pHAGE vector, which is located before the IRES-ZsGreen. After transfecting cells, the FBXO38 molecule with Myc tag can be expressed in the cells, and ZsGreen indicates positive cells.

Specifically, MSCV-3Myc-FBXO38-IRES-ZsGreen is: insert the Fbxo38 sequence with 3 consecutive Myc tags at the N-terminal of the MSCV vector, before IRES-ZsGreen. After transfecting cells, the FBXO38 molecule with Myc tag can be expressed in the cells, and ZsGreen indicates positive cells.

Optionally, the pharmaceutical preparation also contains a pharmaceutically acceptable carrier.

The tumor immunotherapy drugs are mainly targeted at mammals, such as rodents, artiodactyls, perissodactyls, lagomorphs, primates and the like. The primate is preferably a monkey, ape or Homo sapiens.

In one embodiment, the tumor is selected from one of melanoma, non-small cell lung cancer, kidney cancer, head and neck squamous cell carcinoma, Hodgkin's lymphoma, gastric cancer, liver cancer, bladder cancer, endometrial cancer and colon cancer. one or more species.

The fourth aspect of the present invention provides a drug combination for combined treatment of tumors, including an effective amount of a PD-1 ubiquitination agonist and at least one other drug for the treatment of tumors.

The other tumor treatment drugs refer to tumor treatment drugs other than PD-1 ubiquitination agonists.

The combination therapy drug combination can be any one of the following forms:

1) The PD-1 ubiquitination agonist and other tumor therapeutic drugs are prepared into independent preparations, the dosage forms of the preparations may be the same or different, and the routes of administration may also be the same or different.

When other tumor therapeutic drugs are anti-tumor antibodies, parenteral administration is generally used. When the other tumor treatment drug is a chemotherapeutic drug, the administration form can be relatively rich, and it can be gastrointestinal administration or parenteral administration. Dosing is generally recommended for the known route of administration of each chemotherapeutic agent.

2) The PD-1 ubiquitination agonist and other tumor treatment drugs are formulated into a compound preparation. When the PD-1 ubiquitination agonist and other tumor therapeutic drugs are administered through the same route of administration and applied simultaneously, the two can be formulated in the form of a compound preparation.

In one embodiment, the tumor is selected from one of melanoma, non-small cell lung cancer, kidney cancer, head and neck squamous cell carcinoma, Hodgkin's lymphoma, gastric cancer, liver cancer, bladder cancer, endometrial cancer and colon cancer. one or more species.

The fifth aspect of the present invention provides a tumor treatment method, which is to administer an effective amount of a PD-1 ubiquitination agonist to a subject, and administer an effective amount of other tumor therapeutic drugs to the subject and/or implement other tumor treatments to the subject means.

An effective amount of a PD-1 ubiquitination agonist and at least one effective amount of other tumor therapeutic drugs can be administered simultaneously or sequentially.

In one embodiment, the tumor is selected from one of melanoma, non-small cell lung cancer, kidney cancer, head and neck squamous cell carcinoma, Hodgkin's lymphoma, gastric cancer, liver cancer, bladder cancer, endometrial cancer and colon cancer. one or more species.

The other tumor treatment drugs include but are not limited to: anti-tumor antibodies, chemotherapy drugs or targeted drugs, etc.

The PD-1 ubiquitination agonist can be administered gastrointestinally or parenterally. The other drugs for treating tumors can be administered gastrointestinally or parenterally. For anti-tumor antibodies or chemotherapeutic drugs, parenteral administration is generally used.

The sixth aspect of the present invention provides the use of FBXO38 as a target in screening drugs for tumor immunotherapy or screening drugs for autoimmune diseases.

In the application of screening tumor immunotherapy drugs, the tumor is selected from melanoma, non-small cell lung cancer, kidney cancer, head and neck squamous cell carcinoma, Hodgkin's lymphoma, gastric cancer, liver cancer, bladder cancer, endometrial cancer and colon cancer one or more of.

In the application of screening tumor immunotherapy drugs, the application specifically refers to: using FBXO38 as an action object, screening candidate substances, and verifying whether the candidate substances can enhance the ubiquitination of PD-1 and/or whether they can have an effect on FBXO38. Facilitate the effect, if so, then identify it as a candidate drug for tumor immunotherapy.

In the application of screening tumor immunotherapy drugs, the candidate substance is selected from nucleic acid drugs, carbohydrate drugs, lipid drugs, small molecule drugs, polypeptide drugs or protein drugs.

In the application of screening drugs for the treatment of autoimmune diseases, the autoimmune diseases are selected from chronic lymphatic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, chronic ulcerative colitis, pernicious anemia with chronic atrophic gastritis , pulmonary hemorrhagic nephritic syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis, systemic lupus erythematosus, rheumatoid arthritis One or more of autoimmune diseases such as inflammation, systemic sclerosis, polyarteritis nodosa, systemic vasculitis, scleroderma, etc.

In the application of screening drugs for the treatment of autoimmune diseases, the application specifically refers to: using FBXO38 as an action object, screening candidate substances, and verifying whether the candidate substances can weaken PD-1 ubiquitination and/or whether they can inhibit FBXO38 If there is an inhibitory effect, it will be determined as a candidate drug for the treatment of autoimmune diseases.

In the application of screening drugs for treating autoimmune diseases, the candidate substance is selected from nucleic acid drugs, carbohydrate drugs, lipid drugs, small molecule drugs, polypeptide drugs or protein drugs.

The inhibitory effect on FBXO38 includes but not limited to: inhibiting the activity of FBXO38, or inhibiting the transcription or expression of FBXO38 gene.

The seventh aspect of the present invention provides a method for screening tumor immunotherapy drugs, including: verifying whether the drug to be screened can enhance PD-1 ubiquitination and/or whether it can promote FBXO38, and if so, determine it Candidate drug for tumor immunotherapy.

In one embodiment, the drug to be screened is acted on in vitro cells expressing PD-1, and it is determined whether the ubiquitination of PD-1 in the cells is enhanced.

For example, the screening method can be: culturing in vitro cells expressing PD-1 under conditions suitable for cell growth, setting up two groups of comparative experiments, one group adding the drug to be screened to the culture dish of the in vitro cells, and the other The same amount of saline was added to the group, and the other conditions were the same, and incubated to test whether the ubiquitination of PD-1 cells in vitro was enhanced.

The specific method for determining the enhanced ubiquitination can be: detection by western blot technology or detection by mass spectrometry technology.

In one embodiment, the drug to be screened is acted on in vitro cells expressing FBXO38, and it is determined whether the activity of FBXO38 in the cells is enhanced or the expression is upregulated.

For example, the screening method can be: culture in vitro cells expressing FBXO38 under conditions suitable for cell growth, and set up two groups of comparative experiments, one group adds the drug to be screened to the culture dish of the in vitro cells, and the other group adds Equal amount of saline, and other conditions are the same, incubate to test whether the activity of FBXO38 in the cells is enhanced or the expression is up-regulated.

The method for determining whether the activity of FBXO38 in the cells is enhanced or the expression is upregulated may be: real-time quantitative PCR, real-time quantitative PCR and Western Blot detection, or mass spectrometry detection.

In one embodiment, the drug for tumor immunotherapy is a nucleic acid drug, a carbohydrate drug, a lipid drug, a small molecule drug, a polypeptide drug or a protein drug.

The eighth aspect of the present invention provides a method for screening drugs for the treatment of autoimmune diseases, including: verifying whether the drug to be screened can weaken PD-1 ubiquitination and/or whether it has an inhibitory effect on FBXO38, and if so, determine it Drug candidates for the treatment of autoimmune diseases.

In one embodiment, the drug to be screened is acted on in vitro cells expressing PD-1 to determine whether the ubiquitination of PD-1 in the cells is weakened.

For example, the screening method can be: culturing in vitro cells expressing PD-1 under conditions suitable for cell growth, setting up two groups of comparative experiments, one group adding the drug to be screened to the culture dish of the in vitro cells, and the other The same amount of saline was added to the group, and the rest of the conditions were the same, and incubated to test whether the ubiquitination of PD-1 cells in vitro was weakened.

The specific method for determining the weakening of ubiquitination may be: detection by western blot technology or detection by mass spectrometry technology.

In one embodiment, the drug to be screened is acted on in vitro cells expressing FBXO38, and it is determined whether the activity or expression of FBXO38 in the cells is weakened.

For example, the screening method can be: culture in vitro cells expressing FBXO38 under conditions suitable for cell growth, and set up two groups of comparative experiments, one group adds the drug to be screened to the culture dish of the in vitro cells, and the other group adds Equal amount of normal saline, and other conditions are the same, incubate to test whether the activity of FBXO38 in the cells is weakened or the expression is reduced.

The method for determining whether the activity or expression of FBXO38 in cells has been weakened may be: real-time quantitative PCR, real-time quantitative PCR and Western Blot detection, or mass spectrometry detection.

In one embodiment, the drug for treating autoimmune diseases is nucleic acid drug, carbohydrate drug, lipid drug, small molecule drug, polypeptide drug or protein drug.

Compared with the prior art, the present invention has the following beneficial effects:

After extensive and in-depth research, the present invention found for the first time that PD-1 can undergo ubiquitination and then be degraded, thereby enhancing the functions of T cell activation, proliferation and cytokine secretion, and improving the killing function and proliferation of tumor-infiltrating T cells The ability to kill tumor cells by the immune system, FBXO38 is a target that can promote the ubiquitination of PD-1, and promoting the expression of FBXO38 can significantly promote the degradation of PD-1. Inhibiting the expression of FBXO38 can inhibit the degradation of PD-1 and can be used to treat autoimmune diseases. Therefore, the present invention provides strong scientific evidence for the clinical immunotherapy of tumors and the treatment of autoimmune diseases from the level of clinical patient samples, the level of cell function and the level of molecules.

Description of drawings

Figure 1. PD-1 undergoes ubiquitination modification and degradation after T cell activation

a, Co-immunoprecipitation and western blot were used to detect the ubiquitination of PD-1 in human peripheral blood mononuclear cells (PBMCs).

b, Co-immunoprecipitation and western blot were used to detect the dynamics of ubiquitination modification of PD-1 at different times in Jurkat cell lines.

c, Co-immunoprecipitation and western blot were used to detect the ubiquitination of PD-1 in Jurkat cell lines.

d, The effect of proteasome inhibitor MG132 on PD-1 was detected by flow cytometry.

e, The effect of proteasome inhibitor MG132 on ERK was detected by western blot.

f, The effect of lysosomal inhibitor NH4Cl on PD-1 was detected by flow cytometry.

g, The effect of lysosomal inhibitor NH4Cl on p62 was detected by western blot.

h, The effect of lysosomal inhibitor BFA on PD-1 was detected by flow cytometry.

i, The effect of lysosomal inhibitor BFA on p62 was detected by western blot.

Figure 2 FBXO38 specifically regulates the level of PD-1

a, The interaction between overtransformed HA-PD-1 and Myc-FBXO38 was detected by co-immunoprecipitation in 293FT cells.

b, The interaction between overtransformed Myc-FBXO38 and HA-PD-1 was detected by co-immunoprecipitation in 293FT cells.

c, The interaction between overtransformed Myc-FBXO38 and HA-PD-1 was detected by co-immunoprecipitation in Jurkat cells.

d, The interaction between overtransferred Myc-FBXO38 and endogenous PD-1 was detected by co-immunoprecipitation in Jurkat cells.

e, The interaction between overtransferred Myc-FBXO47 and HA-PD-1 was detected by co-immunoprecipitation in 293FT cells.

f, The overexpression of Myc-FBXO38 and Myc-FBXO47 in Jurkat cells was detected by western blot.

g, The surface level of CD3 in Jurkat cells overexpressing Myc-FBXO38 and Myc-FBXO47 was detected by flow cytometry.

h-i, The surface level of PD-1 in Jurkat cells overexpressing Myc-FBXO38 and Myc-FBXO47 was detected by flow cytometry.

j, The knock down efficiency of Fbxo38 was detected by real-time quantitative PCR.

k, Western blot method was used to detect the protein level of FBXO38 after Fbxo38knock down.

l, The surface level of CD3 in Jurkat cells with Fbxo38knock down was detected by flow cytometry.

m, The surface level of PD-1 in Jurkat cells with Fbxo38knock down was detected by flow cytometry.

n, The mRNA level of PD-1 in Jurkat cells with Fbxo38knock down was detected by real-time quantitative PCR.

o, Western blot method was used to detect the protein level of PD-1 in Jurkat cells with Fbxo38knock down.

Figure 3FBXO38 catalyzes the K-48 polyubiquitination modification of PD-1 and promotes its degradation

a, Co-immunoprecipitation and western blot techniques were used to detect the ubiquitination of PD-1 by FBXO38 in 293FT cells, and the effect of two conserved lysines in the intracellular region of PD-1 on the ubiquitination of PD-1.

b, Co-immunoprecipitation and western blot were used to detect the effect of FBXO38 overexpression on PD-1 ubiquitination in Jurkat cells.

c, Co-immunoprecipitation and western blot were used to detect the effect of FBXO38knock down on PD-1 ubiquitination in Jurkat cells.

d, Co-immunoprecipitation and western blot techniques were used to detect the effects of two conserved lysines K210 and K233 in the intracellular region of PD-1 on PD-1 ubiquitination in 293FT cells.

e, Co-immunoprecipitation and western blot techniques were used to detect the important role of F-box sequence in FBXO38-mediated PD-1 ubiquitination in 293FT cells.

f, Co-immunoprecipitation and western blot techniques were used to detect the type of PD-1 ubiquitination mediated by FBXO38 in 293FT cells. g, The role of FBXO38 in promoting PD-1 degradation was detected by western blot in 293FT cells.

h, The role of two conserved lysines in the intracellular region of PD-1 in the degradation of PD-1 was detected by western blot in 293FT cells.

i, The role of the F-box sequence of FBXO38 in mediating PD-1 degradation was detected by western blot in 293FT cells.

Figure 4 Effect of Fbxo38 knockout on T cell development

a, FBXO38 protein levels in CD8 ⁺ T cells of WT and FBXO38-CKO mice.

b, The ratio of CD4 ^- CD8 ^- (DN), CD4 ⁺ CD8 ⁺ (DP), CD4 ⁺ CD8 ^- (CD4SP) and CD4 ^- CD8 ⁺ (CD8SP) cells in thymocytes of WT and CKO mice detected by flow cytometry .

c, Detection of CD44 ⁺ CD25 ^- (DN1) in thymic CD4 ^- CD8 ^- cells of WT and CKO mice by flow cytometry

Proportions of CD44 ⁺ CD25 ⁺ (DN2), CD44 ^- CD25 ⁺ (DN3) and CD44 ^- CD25 ^- (DN4) cells.

d, The ratio of CD4 ⁺ and CD8 ⁺ T cells in the spleen of WT and CKO mice was detected by flow cytometry.

e, The ratio of CD4 ⁺ and CD8 ⁺ T cells in the lymph nodes of WT and CKO mice was detected by flow cytometry.

(CD44^loCD62L^hi),central memory(CD44^hiCD62L^hi；CM)和effector/effector memory(CD44^hi CD62L^lo,effector/EM)的比例。f, Detection of CD4 ⁺ T cells in the spleen of WT and CKO mice by flow cytometry

(CD44 ^lo CD62L ^hi ), the ratio of central memory (CD44 ^hi CD62L ^hi ; CM) and effector/effector memory (CD44 ^hi CD62L ^lo , effector/EM).

(CD44^loCD62L^hi),central memory(CD44^hiCD62L^hi；CM)和effector/effector memory(CD44^hi CD62L^lo,g, Detection of CD4 ⁺ T cells in lymph nodes of WT and CKO mice by flow cytometry

(CD44 ^lo CD62L ^hi ), central memory (CD44 ^hi CD62L ^hi ; CM) and effector/effector memory (CD44 ^hi CD62L ^lo ,

effector/EM) ratio.

(CD44^loCD62L^hi),central memory(CD44^hiCD62L^hi；CM)和effector/effector memory(CD44^hi CD62L^lo,effector/EM)的比例。h, CD8 ⁺ T cells in the spleen of WT and CKO mice detected by flow cytometry

(CD44 ^lo CD62L ^hi ), the ratio of central memory (CD44 ^hi CD62L ^hi ; CM) and effector/effector memory (CD44 ^hi CD62L ^lo , effector/EM).

(CD44^loCD62L^hi),central memory(CD44^hiCD62L^hi；CM)和effector/effector memory(CD44^hi CD62L^lo,i, Detection of CD8 ⁺ T cells in lymph nodes of WT and CKO mice by flow cytometry

(CD44 ^lo CD62L ^hi ), central memory (CD44 ^hi CD62L ^hi ; CM) and effector/effector memory (CD44 ^hi CD62L ^lo ,

effector/EM) ratio.

Figure 5 Effect of Fbxo38 knockout on T cell activation

a, Surface levels of TCR and CD28 in unactivated WT and CKO CD8 ⁺ T cells detected by flow cytometry.

b, The expression level of CD44 after activation of WT and CKO CD8 ⁺ T cells in vitro was detected by flow cytometry.

c, The expression level of LAG-3 after activation of WT and CKO CD8 ⁺ T cells in vitro was detected by flow cytometry.

d, The levels of IFN-γ, Granzyme B and TNF-α after activation of WT and CKO CD8 ⁺ T cells in vitro were detected by flow cytometry.

Figure 6 Effect of Fbxo38 knockout on T cell proliferation and apoptosis

a, Celltracker staining and flow cytometry were used to detect the proliferation of WT and CKO CD8 ⁺ T cells after in vitro activation.

b, Annexin V and propidium iodide (PI) staining and flow cytometry were used to detect the apoptosis of WT and CKO CD8 ⁺ T cells.

Figure 7 Effect of Fbxo38 knockout on CD8 ⁺ T cell transcriptome

和活化状态下的基因转录水平。图示为与T细胞功能相关的表面分子、功能分子以及重要转录因子的转录水平。a, RNA-seq analysis of WT and CKO CD8 ⁺ T cells in

and gene transcription levels in the activated state. The graph shows the transcription levels of surface molecules, functional molecules and important transcription factors related to T cell function.

Figure 8 Fbxo38 regulates the expression level of PD-1

a, The expression level of PD-1 after activation of WT and CKO CD8 ⁺ T cells in vitro was detected by flow cytometry.

b, The expression level of PD-1 after activation of WT and CKO CD4 ⁺ T cells in vitro was detected by flow cytometry.

c, The expression level of PD-1 in nTreg cells in WT and CKO spleens was detected by flow cytometry.

Figure 9Fbxo38 regulates the anti-tumor immunity of T cells

a, B16F10 subcutaneous tumor model was used to study the effect of FBXO38 knockout on tumor growth in mice.

b, B16F10 subcutaneous tumor model was used to study the effect of FBXO38 knockout on the survival of tumor-bearing mice.

c, The expression level of CD44 in tumor-infiltrating CD8 ⁺ T cells in the B16F10 subcutaneous tumor model was detected by flow cytometry.

d, The expression level of PD-1 in tumor-infiltrating CD8 ⁺ T cells in the B16F10 subcutaneous tumor model was detected by flow cytometry.

e, The expression levels of cytokines in tumor-infiltrating CD8 ⁺ T cells in the B16F10 subcutaneous tumor model were detected by flow cytometry.

f, The expression level of Ki-67 in tumor-infiltrating CD8 ⁺ T cells in the B16F10 subcutaneous tumor model was detected by flow cytometry.

g, Analysis of the number and ratio of tumor-infiltrating CD8 ⁺ T cells and CD4 ⁺ T cells in the B16F10 subcutaneous tumor model.

h, Analysis of the number of tumor-infiltrating Treg cells in the B16F10 subcutaneous tumor model.

i, The expression level of PD-1 in tumor-infiltrating CD4 ⁺ T cells in the B16F10 subcutaneous tumor model was detected by flow cytometry.

j, PD-1 expression levels in tumor-infiltrating Treg cells in the B16F10 subcutaneous tumor model were detected by flow cytometry.

Figure 10Fbxo38 knockout affects the anti-tumor immunity of T cells

a, The effect of FBXO38 knockdown on tumor growth in mice was investigated using the MC38 subcutaneous tumor model.

b, The expression level of PD-1 in tumor-infiltrating CD8 ⁺ T cells in the MC38 subcutaneous tumor model was detected by flow cytometry.

c, The expression level of PD-1 in tumor-infiltrating CD4 ⁺ T cells in the MC38 subcutaneous tumor model was detected by flow cytometry.

d, The expression level of PD-1 in tumor-infiltrating Treg cells in the MC38 subcutaneous tumor model was detected by flow cytometry.

e, Analysis of the number and proportion of tumor-infiltrating CD8 ⁺ T cells and CD4 ⁺ T cells in the MC38 subcutaneous tumor model.

f, Analysis of the number of tumor-infiltrating Treg cells in the MC38 subcutaneous tumor model.

Figure 11 Fbxo38 knockdown did not affect the activation of T cells

a, The knockdown efficiency of Fbxo38 was detected by real-time fluorescent quantitative PCR technique.

b, The knockdown efficiency of Fbxo38 was detected by western blot technique.

c, The expression levels of CD3 and CD28 on the cell surface after Fbxo38 knockdown were detected by flow cytometry

d, Western blot technique was used to detect the changes of TCR signaling pathway in activated CD8 ⁺ T cells in vitro after Fbxo38 knockdown.

e, The expression level of CD69 on the surface of activated CD8 ⁺ T cells in vitro after Fbxo38 knockdown was detected by flow cytometry.

f, The expression level of IFN-γ in CD8 ⁺ T cells activated in vitro for 24 hours after Fbxo38 knockdown was detected by flow cytometry.

g, The expression level of IFN-γ in CD8 ⁺ T cells activated in vitro for 48 hours after Fbxo38 knockdown was detected by flow cytometry.

Figure 12 Fbxo38 knockdown can upregulate the level of PD-1 and affect the tumor killing ability of CD8 ⁺ T cells

a, The PD-1 level on the surface of CD8 ⁺ T cells stimulated in vitro after Fbxo38 knockdown was detected by flow cytometry.

b, The expression of PD-L1 in EL-4 cells was detected by flow cytometry.

c, ELISA detection of CD8 ⁺ T cells killing EL-4 cells in vitro after Fbxo38 knockdown.

d, The overexpression efficiency of Fbxo38 in mouse CTL detected by flow cytometry.

e, The graph shows the level of PD-1 detected by flow cytometry after overexpression of Fbxo38 in mouse CTL.

f, The diagram shows the statistics of PD-1 levels after Fbxo38 overexpression in mouse CTL detected by flow cytometry.

Figure 13 FBXO38 endogenously regulates the anti-tumor ability of CD8 ⁺ T cells

a, Schematic illustration of the experimental approach for adoptive cellular immunotherapy in the B16F10 subcutaneous tumor model.

b, The proportion of reinfused CD8 ⁺ T cells (GFP ⁺ ) in tumor-infiltrating lymph nodes (dLN) at day 18 was detected by flow cytometry.

c, Flow cytometric detection of the proportion of reinfused CD8 ⁺ T cells (GFP ⁺ ) in non-tumor infiltrating lymph nodes (non-dLN) on day 18.

d, Flow cytometry detection of PD-1 levels in reinfused CD8 ⁺ T cells (GFP ⁺ ) in tumor-infiltrating lymph nodes (dLN) on day 18.

e, Ki-67 levels of reinfused CD8 ⁺ T cells (GFP ⁺ ) in tumor-infiltrating lymph nodes (dLN) at day 18 were detected by flow cytometry.

f, The tumor growth curve of mice after adoptive immunotherapy after knocking down Fbxo38.

g, Survival curve of mice after Fbxo38knock down and adoptive immunotherapy.

h, The growth curve of mouse tumors after Fbxo38knock down and adoptive immunotherapy combined with PD-1 antibody treatment.

i, Survival curve of mice after Fbxo38knock down and adoptive immunotherapy combined with PD-1 antibody treatment. In all figures, Error bars represent mean+/-SEM.

ns means no significant difference, *P<0.05, **P<0.01, ***P<0.001.

detailed description

So far, studies on the regulation of PD-1 expression have focused on the transcriptional level, and no studies on the post-transcriptional level of PD-1 have been published. At present, the regulation of PD-1 protein at the post-translational level is still blank. In the research of the present invention, it is found that PD-1 ubiquitination can degrade PD-1, thereby enhancing the functions of T cell activation, proliferation and cytokine secretion, and improving the killing function and proliferation ability of tumor-infiltrating T cells, so that Tumor cells are killed by the immune system, and FBXO38 is a target that can promote the ubiquitination of PD-1, and promoting the expression of FBXO38 can significantly promote the degradation of PD-1.

Ubiquitin is a very conserved polypeptide consisting of 76 amino acids in eukaryotic cells. It can be covalently activated by three enzymes (E1: Ub-activating enzyme; E2: Ub conjugating enzyme; E3: Ub protein ligase). Cross-linking to the lysine of the target protein forms ubiquitination modification of the target protein, thereby affecting the signaling pathway of the target protein or degrading the target protein through the proteasome. Ubiquitination regulates many important physiological processes in cells, such as controlling the cycle, down-regulating membrane proteins, and participating in signal transduction, etc. One ubiquitin can be attached to the lysine of the target protein to form a monoubiquitination modification, and many ubiquitins can also be attached to form a polyubiquitination modification. In the polyubiquitination modification, the polyubiquitination modification mediated by the 48th lysine on ubiquitin usually causes the target protein to be degraded through the proteasome pathway, and the polyubiquitination mediated by the 63rd lysine on the ubiquitin The induced polyubiquitination modification will play a role in many signal transduction pathways.

The PD-1 ubiquitination refers to linking ubiquitin to lysine of PD-1 for ubiquitination modification, affecting the signaling pathway of PD-1 or degrading PD-1 through proteasome.

PD-1 ubiquitination agonist

Refers to molecules that can promote the ubiquitination of PD-1.

The PD-1 ubiquitination agonist can enhance functions such as activation, proliferation and cytokine secretion of T cells, improve the killing function and proliferation ability of tumor-infiltrating T cells, and cause tumor cells to be killed by the immune system.

Further, the PD-1 ubiquitination agonist can promote the degradation of PD-1.

Further, the PD-1 ubiquitination agonist is a FBXO38 agonist.

Further, the FBXO38 agonist refers to a molecule that has a promoting effect on FBXO38.

The promoting effect on FBXO38 includes but not limited to: enhancing the activity of FBXO38, or promoting the transcription or expression of FBXO38 gene.

Enhancing the activity of FBXO38 refers to increasing the activity of FBXO38. Preferably, the FBXO38 activity is increased by at least 10%, preferably by at least 30%, more preferably by at least 50%, more preferably by at least 70%, and most preferably by at least 90%, compared to before enhancement.

Promoting FBXO38 gene transcription or expression refers to: accelerating FBXO38 gene transcription, or increasing FBXO38 gene transcription activity, or accelerating FBXO38 gene expression, or increasing FBXO38 gene expression activity.

Those skilled in the art can use conventional methods to regulate the gene transcription or expression of FBXO38.

Preferably, FBXO38 gene transcription or expression is increased by at least 10%, preferably by at least 30%, more preferably by at least 50%, more preferably by at least 70%, and optimally by at least 90%, compared with wild type .

PD-1 ubiquitination agonist preparation of drugs

The drug is prepared by using the PD-1 ubiquitination agonist as the main active ingredient or one of the main active ingredients. Usually, in addition to the active ingredient, the medicine also includes one or more pharmaceutically acceptable carriers or excipients according to the requirements of different dosage forms.

"Pharmaceutically acceptable" means that the molecular entities and compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.

The "pharmaceutically acceptable carrier or excipient" should be compatible with the PD-1 ubiquitination agonist, that is, it can be blended with it without greatly reducing the effect of the pharmaceutical composition under normal circumstances. Specific examples of some substances that can be used as pharmaceutically acceptable carriers or excipients are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, Ethyl cellulose and methyl cellulose; Gum tragacanth powder; Malt; Gelatin; Talc; Solid lubricants such as stearic acid and magnesium stearate; Calcium sulfate; Vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive Oils, corn oil, and cocoa butter; polyols, such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; coloring flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer, etc. These substances are used as needed to aid the stability of the formulation or to help enhance the activity or its bioavailability or to produce an acceptable mouthfeel or odor in the case of oral administration.

In the present invention, unless otherwise specified, the pharmaceutical dosage form is not particularly limited, and can be made into dosage forms such as injections, oral liquids, tablets, capsules, dripping pills, sprays, etc., and can be prepared by conventional methods. The choice of drug dosage form should match the mode of administration.

Combination Therapy Combinations and Methods of Administration

The combination therapy drug combination can be any one of the following forms:

1) The PD-1 ubiquitination agonist and other anti-tumor drugs are made into independent preparations, the dosage forms of the preparations may be the same or different, and the routes of administration may also be the same or different. When in use, several medicines can be used at the same time, or several medicines can be used successively. In the case of sequential administration, other drugs should be administered to the body during the period when the first drug is still effective on the body.

2) The PD-1 ubiquitination agonist and other anti-tumor drugs are formulated into a compound preparation. When the PD-1 ubiquitination agonist and other anti-tumor drugs are administered by the same route of administration and applied simultaneously, the two can be formulated in the form of a compound preparation.

The commonly used methods of administration of antibodies are intravenous injection, intravenous drip or arterial infusion. Its usage and dosage can refer to the prior art.

Commonly used methods of administration of small molecule compounds can be gastrointestinal administration or parenteral administration. siRNA, shRNA, and antibodies are generally administered parenterally. Administration may be local or systemic.

Effective doses of PD-1 ubiquitination agonists and effective doses of other tumor therapeutic drugs can be administered synchronously or sequentially. When in use, effective doses of PD-1 ubiquitination agonists and effective doses of other tumor therapy drugs can be used simultaneously, or effective doses of PD-1 ubiquitination agonists and effective doses of other tumor therapy drugs can be used successively. use. In the case of sequential administration, other drugs should be administered to the living body during the period in which the first drug is still effective on the living body.

Chemotherapeutic drugs include alkylating agents (such as nimustine, carmustine, lomustine, cyclophosphamide, ifosfamide and glycosyl mustard, etc.), anti-metabolites (such as doxifluoroguanosine, Nucleotide analogues such as dosifuguanidine, fluorouracil, mercaptopurine, methotrexate), antitumor antibiotics (such as actinomycin D, doxorubicin and daunorubicin and other antibiotics), antitumor animal and plant Ingredient drugs (such as vinorelbine, paclitaxel, harringtonine, irinotecan, taxotere and vinblastine, etc.), anti-tumor hormone drugs (such as atamestane, anastrozole, aminoglutethimide, cisplatin, dacarbazine, oxaliplatin, lexatidine, coplatinoxa, mitoxantrone, and procarbazine.

Targeted drugs include EGFR blockers such as gefitinib (Gefitinib, Iressa, and Iressa) and erlotinib (Erlotinib, Tarceva), monoclonal antibodies to specific cell markers such as cetuximab (Cetuximab , Erbitux) and anti-HER-2 monoclonal antibodies (Herceptin, Trastuzumab, Herceptin), tyrosine kinase receptor inhibitors such as crizotinib (Crizotinib, Xalkori), anti-tumor angiogenesis drugs such as Bevacizumab, endostatin and Bevacizumab etc., Bcr-Abl tyrosine kinase inhibitors such as Imatinib and Dasatinib, anti-CD20 monoclonal antibodies such as Rituximab, IGFR-1 kinase inhibitors such as NVP-AEW541, mTOR kinase inhibitors such as CCI-779, ubiquitin-proteasome inhibitors Such as Bortezomib et al.

Other tumor treatment methods can be selected from surgical resection, radiofrequency ablation, argon-helium superconducting surgery, laser ablation, high-intensity focused ultrasound, and radiation therapy, including X-knife, R-knife, 3D-CRT and IMRT. Various.

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are to describe specific specific embodiments, It is not intended to limit the protection scope of the present invention. The test methods for which specific conditions are not indicated in the following examples are usually in accordance with conventional conditions, or in accordance with the conditions suggested by each manufacturer.

When the examples give numerical ranges, it should be understood that, unless otherwise stated in the present invention, the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to those skilled in the art's grasp of the prior art and the description of the present invention, the methods, equipment, and materials described in the embodiments of the present invention can also be used Any methods, apparatus and materials of the prior art similar or equivalent to the practice of the present invention.

Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, gene mutation, etc. technology and conventional techniques in related fields.

Example 1

1.1 Experimental materials

1.1.1 Buffer formulation

Lysis Buffer for Co-IP (for co-immunoprecipitation experiments in 293FT cells and Jurkat cells): 50mM Tris-HCl, pH 7.4, 155mM NaCl, 2mM EDTA, 2mM Na ₃ VO4, 20mM NaF, 10mM Iodoacetamide, 0.5% NP40, 1mM PMSF, 1mM DTT, Complete Protease Inhibitor Cocktail (Sigma);

Lysis Buffer for Ubiquitination (for mouse and human activated CD8 ⁺ T cell lysis, detection of ubiquitination of PD-1): 50mM Tris-HCl, pH 7.4, 155mM NaCl, 2mM EDTA, 2mM Na ₃ VO4, 20 mM NaF, 10 mM Iodoacetamide, 1% NP40, 0.1% SDS, 1 mM PMSF, 1 mM DTT, Complete Protease Inhibitor Cocktail (Sigma);

Lysis Buffer for Ubiquitination 1 (for ubiquitination assay in 293FT cells, containing 1% SDS): 50mM Tris-HCl, pH 7.4, 155mM NaCl, 2mM EDTA, 2mM Na ₃ VO4, 20mM NaF, 10mMIodoacetamide, 1% NP40 , 1% SDS, 1mM PMSF, 1mM DTT, Complete Protease Inhibitor Cocktail (Sigma);

Lysis Buffer for Ubiquitination 2 (for ubiquitination assay in 293FT cells, without SDS): 50mM Tris-HCl, pH 7.4, 155mM NaCl, 2mM EDTA, 2mM Na ₃ VO4, 20mM NaF, 10mMIodoacetamide, 1% NP40, 1mM PMSF, 1mM DTT, Complete Protease Inhibitor Cocktail (Sigma);

Tris buffer: 50mM Tris, 200mM NaCl, pH8.0;

PBS buffer solution: 8g NaCl, 0.2g KCl, 3.58g NaH ₂ PO ₄ 12H ₂ O, 0.27g K ₂ HPO ₄ , dilute to 1L, adjust the pH to 7.4, autoclave;

Protein electrophoresis buffer (Tris-glycine): 25mM Tris, 250mM glycine, 0.1% SDS;

Protein transfer buffer (Transfer Buffer): 39mM glycine, 48mM Tris, 0.037% SDS;

Stripping Buffer: 1.5% glycine, 0.1% SDS, 1% Tween 20, adjust the pH to 2.2;

TBST buffer: 150mM NaCl, 50mM Tris, 0.1% Tween 20, adjust the pH value to 7.4, used to prepare the primary antibody, secondary antibody and blocking solution in western blotting.

Western blot blocking solution: 10% BSA in TBST Buffer;

Magnetic bead sorting buffer (mouse/human CD8 ⁺ T cells): 2% FBS, 2mM EDTA dissolved in PBS, adjust pH to 7.2, 0.2μM membrane filter to sterilize;

Surface staining buffer: 0.1% BSA in PBS Buffer;

Intracellular staining buffer: 0.5% BSA in PBS Buffer, 0.1% Triton X-100;

1.1.2 Chemical reagents

1.1.3 Kits, cytokines, genetic tools and enzymes

1.1.4 Cell lines

Jurkat cell line: isolated from the peripheral blood of a 14-year-old boy with T-cell leukemia in the 1970s, this cell is a suspension cell and is a commonly used cell line for studying T cell signal transduction, which can be activated by CD3 antibody or PMA+Ionomycin . In this paper, the Fbxo38 gene was overexpressed or knocked down in the Jurkat cell line, and then the expression level and ubiquitination modification of PD-1 were detected.

HEK-293FT cell line: 293F cells are cell lines derived from human embryonic kidney cells, which are transfected with adenovirus type 5 DNA fragments. The cells can activate the promoters of certain viruses, promote protein expression, and have faster growth rate. 293FT cells are transfected with SV40 large T antigen on the basis of 293F cells, which can be expressed at a very high level from the vector containing the SV40 origin. The 293FT cell line grows fast and is very easy to transfect. It is a cell line commonly used in laboratories for overexpressing genes or for lentiviral packaging.

B16F10 melanoma cell line: isolated from C57BL/6 mouse skin tissue melanoma, morphologically spindle-shaped and epithelial cells. The cells are adherent cells, easy to transfect, and can be stably passaged. The cells are mainly used for the construction of mouse melanoma tumor models, as well as the research of cell proliferation, apoptosis and signal transduction

B16F10-OVA cell line: The OVA antigen is overexpressed in the B16F10 cell line and presented to the surface by the cells, as an antigen-presenting cell presenting the OVA antigen peptide to stimulate and activate T lymphocytes derived from OT-1. This cell line can successfully activate OT-1-derived T lymphocytes for use in ACT models.

EL4 cell line: The source of EL4 cell line is the C57 background lymphoma mouse, which is cultured in 1640 medium, presents a semi-suspension state, and will be slightly adhered to the wall. After PD-L1 detection, EL4 cells highly express PD-L1 protein, which can be used for killing experiments of CD8 ⁺ T cells.

1.1.5 Plasmid introduction

pHAGE-fEF1a-IRES-ZsGreen/mCherry (pHAGE vector): protein overexpression and lentiviral packaging vector, the promoter is fEF1a, and it also has the IRES sequence. IRES is a special DNA sequence that can independently initiate the translation process in the middle of mRNA. IRES is connected with fluorescent protein ZsGreen/mCherry, which can be used as an indicator of successful cell transfection. At the same time, the vector has LTRs sequence and can be used for lentivirus packaging to transfect difficult-to-transfect cell lines, such as Jurat cell lines and primary CD8 ⁺ T cells of mice.

pHAGE-fEF1a-HA-PD-1-IRES-mCherry: Insert the Pdcd1 sequence with HA tag at the N-terminus on the pHAGE vector, before IRES-mCherry. After transfecting cells, PD-1 molecules with HA tag can be expressed in cells, and mCherry indicates positive cells.

pHAGE-fEF1a-3Myc-FBXO38-IRES-ZsGreen: first made on the basis of pHAGE carrier

pHAGE-fEF1a-3Myc-IRES-ZsGreen plasmid. Combine 3 pairs of complementary fragments:

Complementary strand 1A (SEQ ID NO.2):

5'-CAGGTGTCGTGAAGCATGGAACAGAAATTGATAAGTGAGGAAGATTTAG-3';

Complementary strand 1B (SEQ ID NO.3):

5'-TTGCTCTAAATCTTCCTCACTTATCAATTTCTGTTCCATGCTTCACGACACCTG-3';

Complementary strand 2A (SEQ ID NO.4):

5'-AGCAAAAGCTCATTTCTGAAGAGGACTTGGAACAGAAATTGATAAGTGAGGAAGATT-3'

Complementary strand 2B ((SEQ ID NO.5):

5'-CCGCTAAATCTTCCTCACTTATCAATTTCTGTTCCAAGTCCTCTTCAGAAATGAGCTT-3';

Complementary strand 3A (SEQ ID NO.6):

5'-TAGCGGCCGCAGCAATGCATGCAGGCGCGCCAGAAACGCGTGGGCACCGGTCTGCAGC-3'; complementary strand 3B (SEQ ID NO.7):

5'-GCTGCAGACCGGTGCCCACGCGTTTCTGGCGCGCCTGCATGCATTGCTGCGG-3'.

Put them into the tubes respectively, and then put the three tubes into boiling water, and let them cool down to room temperature naturally with the boiling water. Glue back the DNA fragments separately. Then these three DNA fragments were ligated together with T4 ligase, and the ligated product was glued back to obtain a 3Myc fragment, which was about 160 bp. The pHAGE vector was digested with Not I endonuclease and glued back. Finally, the cut pHAGE-fEF1a-IRES-ZsGreen vector and the 3Myc fragment were connected with the GBclonart seamless cloning kit to make the pHAGE-fEF1a-3Myc-IRES-ZsGreen plasmid. Using cDNA as a template,

Use Fbxo38 upstream primer (SEQ ID NO.8):

5'-TGAGGAAGATTTAGCGATGG GGCCACGAAA GAAAAGTG-3' and

Fbxo38 downstream primer (SEQ ID NO.9):

5'-TGCATGCATT GCTGCTTAAA TGTAGTCATC TTCAACTG-3';

Fbxo38 was amplified and the product was glued back. The pHAGE-fEF1a-3Myc-IRES-ZsGreen plasmid was cut with endonuclease Not I, and connected with GBclonart seamless cloning kit. The pHAGE-fEF1a-3Myc-FBXO38-IRES-ZsGreen plasmid was prepared. After transfecting cells, the FBXO38 molecule with Myc tag can be expressed in the cells, and ZsGreen indicates positive cells.

pHAGE-fEF1a-V5-Ub-IRES-ZsGreen: Insert the Ub sequence with the V5 tag at the N-terminal on the pHAGE vector, before IRES-ZsGreen, including Ub WT, UbK48R (change the 48th lysine of Ub into R), UbK63R (change the 63rd lysine of Ub to R) and UbKtoR (change both the 48th and 63rd lysine of Ub to R), which are used to detect the ubiquitous expression of PD-1 protein in 293FT cells prime modification.

MSCV-3Myc-FBXO38-IRES-ZsGreen: Use the following primers:

Fbxo38 upstream primer (SEQ ID NO.10):

5'-GCGCCGGAATTAGATCTCATGGAACAGAAATTGATAAGTGAGGAAG-3'

Fbxo38 downstream primer (SEQ ID NO.11):

5'-GGGCGGAATTCGTTAACCTTAAATGTAGTCATCTTCAAC-3'

The 3Myc-FBXO38 gene was amplified from the pHAGE-fEF1a-3Myc-FBXO38-IRES-ZsGreen plasmid. At the same time, the MSCV vector was cut with restriction endonuclease XhoI and glued back. And connected with GBclonart seamless cloning kit. The MSCV-3Myc-FBXO38-IRES-ZsGreen plasmid was made. After transfecting cells, the FBXO38 molecule with Myc tag can be expressed in the cells, and ZsGreen indicates positive cells.

pHAGE-fEF1a-3Myc-FBXO47-IRES-ZsGreen: Insert the Fbxo47 sequence with 3 consecutive Myc tags at the N-terminus on the pHAGE vector, before IRES-ZsGreen. After transfecting cells, the FBXO47 molecule with Myc tag can be expressed in the cells, and ZsGreen indicates positive cells.

PLKO.1G vector: used to generate gene-specific shRNA to knock down specific genes in cells. The vector can express GFP protein, which is used to indicate positive cells and can be screened. In this study, this vector was used to knock down the Fbxo38 gene in Jurkat and mouse primary CD8 ⁺ T cells.

psPAX2 and pMD2.G: Both belong to the second generation of lentiviral packaging plasmids, which are used together with the pHAGE vector as a three-plasmid system to produce lentiviruses. The main components of psPAX2 include the promoter and the GAG, POL, TAT, REV sequences behind it, and pMD2.G mainly include the promoter and the ENV sequence behind it. In the invention, psPAX2 and pMD2.G can be transfected with 293FT cell line with pHAGE vector and PLKO.1G vector for lentiviral packaging.

1.1.6 Antibody introduction

1.1.7 Introduction of Experimental Animals

Fbxo38 gene conditional knockout mice: Fbxo38 flox ^/ flox mice were purchased from Beijing CasGene Biotech Co., Ltd. (Beijing CasGene Biotech Company), and are called F0 generation. After the F0 generation mice grow up to 8 weeks, they can be mated with CD4 ^cre mice for multiple generations to obtain Fbxo38 ^CKO mice in which FBXO38 protein is specifically knocked out in CD4 and CD8 cells.

mice。OT-1 mice: C57BL/6-Tg(Tcrα, Tcrβ) 1100Mjb/J, Tcrα-V2 and Tcrβ-V5 genes were inserted into the mouse transgenic group, so the transgenic T cell receptor of this mouse can only recognize Ovalbumin 257 The -264 (OVA _257-264 ) residue peptide is used to study the response of CD8 ⁺ T cells to antigens. Like most TCR transgenic mice, these mice are somewhat immunodeficiency. The mice were of C57BL/6 background and were purchased from

mice.

C57BL/6 mice: directly purchased from Shanghai SLAC Company, SPF grade, 8-10 weeks for animal experiments.

All mice used in the present invention were raised in SPF environment, Fbxo38 conditional knockout mice and OT1 mice were identified by PCR before use. In the tumor experiment, the age difference between the mice in the control group and the experimental group was within 2 weeks, and the sexes were the same.

1.2 Experimental method

1.2.1 Cell experiments

1.2.1.1 Transfection of 293FT cells using liposomes

1. 1-2 days before cell transfection, subculture the cells at a ratio of 1:4-1:8. Use trypsin to digest 293FT cells (density 90%-100%) for 1 minute, remove trypsin, then quickly add DMEM medium, resuspend, blow and mix, pass into a new 10cm culture dish, and then use a pipette gun to blow for a few times Mix thoroughly once to avoid cell clumps.

2. Change the medium 4 hours before cell transfection. When the cell density reaches about 50%, replace the old medium with new DMEM medium. You can use a pipette to suck off 6ml of the old medium, slowly add 6ml of fresh DMEM medium preheated at 37 degrees, and continue to cultivate for 4h.

3. Transfection. According to different experimental purposes, different plasmid transfection amounts were selected, and the amount of Lipofectamine2000 transfection reagent was adjusted accordingly.

Generally, the amount of Lipofectamine 2000 (μl)/mass of plasmid (μg) is 2:1. The specific transfection steps are as follows: take two 1.5mL Ep tubes, add 250 μl OPTI-MEM medium respectively, add 10 μg plasmid to one tube, and add 20 μl Lipofectamine 2000 transfection reagent to the other tube, mix well and let stand for 5 minutes; Afterwards, the two tubes of solutions were aseptically mixed and allowed to stand at room temperature for another 20 minutes; then, the mixture was added dropwise to the cell culture medium, then mixed gently, and placed in a 37°C cell culture incubator to continue culturing. For the PD-1 ubiquitination experiment, the amount of plasmid transfection in a 10cm plate is: Myc-Fbxo38 15μg, HA-Pd1 3.5μg, V5-Ub 3μg, Lipofectamine 2000 30μl; for the PD-1 degradation experiment, the amount of plasmid transfection in a 6cm plate The amount of staining is: Myc-Fbxo38 4 μg, HA-Pd1 1 μg, Lipofectamine 2000 10 μl.

4. After 24-72 hours, observe the positive rate under a fluorescent microscope, and the successfully transfected cells will express fluorescent proteins.

This experimental method is suitable for gene overexpression or lentiviral packaging in 293FT cells.

1.2.1.2 Infection of Jurkat cell line with lentivirus system

1. Carry out lentiviral packaging according to the method described in 1.2.1.1, and use Lipofectamine 2000 to transfect the packaging plasmid and target gene plasmid into 293FT cell line. The quality of the plasmid is generally: 10 μg target plasmid + 7.5 μg psPAX2 + 3 μg pMD2.G.

2. After 24 hours of transfection, observe under a fluorescent microscope, the positive rate should reach more than 80%. If the target protein has a large molecular weight, the transfection efficiency may be low.

3. After 48-60 hours of transfection, collect the virus supernatant. Carefully put the virus supernatant into a 15ml centrifuge tube, centrifuge at 6000rpm/min for 5 minutes, then filter with a 0.45μm syringe filter to obtain the lentivirus supernatant, and then directly infect Jurkat cells with the lentivirus supernatant. Generally, 1-2×10 ⁶ Jurkat T cells can be added to 10 mL of virus liquid. For safety reasons, put the supernatant in a culture flask to infect cells. Visible fluorescence was visible 72 hours after infection. If the target gene is large, such as a 3500bp gene like Fbxo38, the positive rate is slightly lower, generally 30%; if it is a DNA sequence with only 20bp like shRNA, the general positive rate is 100%.

4. Generally, change the medium after 72-96 hours of infection. Centrifuge the virus supernatant at 500g, replace with fresh 1640 medium to resuspend the cells, and continue culturing for 72-96 hours to obtain relatively stable positive cells, which can be sorted by flow cytometry.

1.2.1.3 Infection of mouse primary cell lines with lentivirus system

(1) Virus packaging

1. Carry out lentivirus packaging according to the method described above, and use Lipofectamine 2000 to transfect 293FT cell line. The quality of the plasmid is: 10 μg PLKO.1G+7.5 μg psPAX2+3 μg pMD2.G. Each shRNA was transfected into three 10cm dishes for subsequent virus concentration.

2. Collect the virus supernatant according to the method described above, filter it with a 0.45 μm syringe filter, put all the 3 plates of virus supernatant into a 38ml ultracentrifuge tube, centrifuge at 27000rpm/min, 4 degrees for 2h. After centrifugation, quickly pour off the supernatant, add 500 μl 1640 medium without FBS along the tube wall, resuspend the virus, place it in 4 degrees, and use it overnight.

(2) CD8 ⁺ T cell culture and infection

CD8⁺ T细胞，培养在下面的培养基中：RPMI-1640+10％FBS+1％PS+IL-7(10ng/ml)+IL-15(100ng/ml)+0.05mM 2-mercaptoethanol，每孔培养200万细胞，培养48-72h，可以看到细胞体积变大，出现小的细胞团。感染前一天，在24孔板(货号351147,BD)中铺PBS稀释的RereoNectin(1mg/ml，T100B,Takara)，一般400μl/孔，4度过夜。1. Sorting Using Magnetic Beads

CD8 ⁺ T cells were cultured in the following medium: RPMI-1640+10%FBS+1%PS+IL-7(10ng/ml)+IL-15(100ng/ml)+0.05mM 2-mercaptoethanol, each 2 million cells were cultured in the well, and cultured for 48-72 hours, it can be seen that the cell volume becomes larger and small cell clusters appear. The day before infection, spread RereoNectin (1 mg/ml, T100B, Takara) diluted in PBS in a 24-well plate (Catalog No. 351147, BD), generally 400 μl/well, overnight at 4 hours.

2. Count the cells, then centrifuge the cultured CD8 ⁺ T cells, remove the supernatant according to the 2 million/15ml centrifuge tube, use 500ul concentrated virus supernatant to reselect 2 million CD8 ⁺ T cells, and then slowly add Centrifuge at 2000g for 60min at 37°C in wells lined with RereoNectin washed once with PBS.

3. After centrifugation, the cells will stick to the bottom of the 24-well plate, remove the supernatant directly, and continue to culture for 3 days with fresh medium containing IL-7 and IL-15.

4. Use a flow cytometer or a fluorescence microscope to detect the positive rate. Positive cells can be sorted according to the expression of GFP for subsequent functions or the cells can continue to be expanded and cultured for use in the ACT model.

During the entire infection process, cells were cultured with only IL-7 and IL-15 in a memerylike state, but TCR signaling was not activated, so these cells did not express PD-1 protein.

1.2.1.4 Overexpression of FBXO38 in primary mouse cell lines by retroviral system

Overexpression of FBXO38 protein in mouse CD8 ⁺ T cells by retrovirus MSCV.

(1) Virus packaging

1. Perform retrovirus packaging according to the method described in 1.2.1.1, and use Lipofectamine 2000 to transfect the MSCV-3Myc-FBXO38-IRES-ZsGreen plasmid into plat E cells.

2. After 48 hours of transfection, collect the virus supernatant, filter it with a 0.45 μm syringe filter, and then concentrate the virus supernatant 50-70 times at room temperature with an ultrafiltration tube (Millipore, 100KD) at a speed of 3000 g.

3. The spleen of OT1 mice was taken out, ground up, red blood cells were lysed, and activated in 1640 medium containing 10nM OVA and 10ng/ml IL-2 for 24 hours. Activated cells were resuspended with 1ml virus medium (including 500ul concentrated virus, 500ul fresh complete RPMI-1640 medium and 10μg/ml polybree) into a 24-well plate treated with RetroNectin (1mg/ml, T100B, Takara) middle. Next, the cells were centrifuged at 2500 rpm at 37 degrees for 2 hours, and then cultured in an incubator for 10 hours. Then transfect again as above. After the two transfections, the cells were resuspended into new complete RPMI-1640 medium for culture. GFP positive cells are FBXO38 overexpressed cells. During the above process, there was always 10 ng/ml IL-2 in the culture medium.

1.2.2 Animal experiments

1.2.2.1 Isolation of CD8+ cells from mouse spleen

1. Fbxo38 ^CKO or C57BL6 mice around 8-10 weeks old were killed by cervical dislocation;

2. Sterilize with 70% ethanol, take out the spleen, place it in PBS, crush the whole spleen with tweezers and grind it, pass through a 40 μm nylon filter to obtain a single cell suspension;

3. Centrifuge at 500g for 5min, resuspend to 10 ⁸ /mL with MACS buffer, and transfer to a 5mL flow tube;

5. Add 15 μL Biotin-labeled negative selection antibody Cocktail (Stem Cell, mouse CD8 ⁺ T Cell Isolation Kit, 19852) to 1 mL of cells, and incubate at room temperature for 15 minutes;

6. Add 30 μL Streptavidin cross-linked magnetic beads and continue to incubate at room temperature for 5 minutes;

7. Add MACS buffer to 3mL volume, then insert the flow tube into the magnetic pole, and let stand at room temperature for 2 minutes;

8. Flip the magnetic pole, pour the liquid into a new 15mL centrifuge tube, and centrifuge at 400g for 10min at 4°C;

9. Resuspend in PBS or RPMI-1640 medium and count, the cells are negatively selected CD4 ⁺ or CD8 ⁺ T cells;

10. Take a small part of the cells, stain with anti-CD4 or anti-CD8, and check the purity of the cells by flow cytometry, generally the purity is greater than 90%.

1.2.2.2 Anti-CD3/CD28 antibody activates mouse CD8+ cells

1. One day in advance, dilute the antibody with PBS with pH = 9.0, the final concentration is anti-CD3 (2 μg/ml) + anti-CD28 (2 μg/ml). In a 48-well plate, generally add 140 μl of PBS.

2. Isolate the primary CD8 ⁺ T cells of the mouse, count them, resuspend them in RPMI 1640 medium containing 10% FBS + 10ng/ml IL-2 to 4×10 ⁵ /mL, and add them to a 48-well plate, each well 400,000 cells.

3. Between 24 and 96 hours, the cells were harvested for surface staining to detect PD-1 expression or internal staining to detect cytokine secretion.

4. Surface staining to detect the expression of cell surface molecules. After harvesting the cells, centrifuge at 400 g for 5 minutes at room temperature, remove the supernatant, then add 500 μl 4°C pre-cooled PBS, and centrifuge again to remove the supernatant. Then resuspend the cells with PBS + 0.1% BSA + antibody staining solution, and place them in the dark at 4 degrees for 45 minutes. Then add 500 μl PBS, centrifuge at 400 g for 5 minutes at 4 degrees, remove the supernatant, then add 500 μl PBS to resuspend the cells, and perform flow cytometric detection.

5. Intracellular staining to detect cytokine secretion. After the cells are harvested, surface staining is usually carried out first, such as using anti-CD8 to mark the CD8 molecules on the cell surface, and the specific method is the same as above. After washing once with PBS, add 300 μL of 4% PFA, fix at room temperature for 10 minutes, then centrifuge at 1500g for 5 minutes, remove the supernatant; add 1ml of PBS, centrifuge at 1500g, wash again; then add PBS+0.5%BSA+0.1%Triton X -100+ antibodies (such as IFN-γ, TNF-α, Granzyme B), stained at 4 degrees for 2 hours or overnight; add 500μl PBS, centrifuge at 1500g, remove the supernatant; add 500μl PBS to resuspend cells, and perform flow cytometry.

6. Detection of cell proliferation. Before stimulating CD8 ⁺ cells, first incubate with 0.5 μm living cell dye CellTracker ^TM Deep Red fluorescent dye (C34565, Thermo Fisher Scientific) at 37 degrees for 20 minutes, then wash three times with PBS, wash off the dye and then stimulate. Cells were harvested at different times, and then the proliferation was detected by flow cytometry.

7. Cell apoptosis detection. After the CD8 ⁺ cells, the cells were harvested at different times, and the apoptosis was detected with a cell apoptosis kit (eBioscience).

1.2.2.3 Induction of OT1 mouse CTL cells

1. Mice were sacrificed by cervical dislocation, sterilized with 70% ethanol, and the spleen was removed.

2. Add 5mL PBS to a 6cm Petri dish, and place the cell strainer in the Petri dish. The spleen was torn up with forceps, placed on a strainer, and ground with a syringe plunger until all cells diffused through the strainer into PBS.

3. Add the obtained cell suspension into a 50mL centrifuge tube, add 45mL erythrocyte lysate, invert and mix well and let stand for 2-3min. Centrifuge at 500g for 5min and discard the supernatant.

4. Resuspend with 50mL PBS, filter with a cell strainer (optional), centrifuge at 500g for 5min, and discard the supernatant.

5. Resuspend cells with 30mL1640 medium, add 30μL 10μg/mL IL-2 (final concentration 10ng/mL), 3uL 100μM OVA _257-264 (final concentration 10nM), mix well and add to a medium-sized culture flask, place in cultured in a cell culture incubator.

6. Generally, the medium turns yellow after three days, and then change the medium every day, and passage according to the ratio of 1:3. The cells on the fifth day are in better condition. At the same time, cells can also be taken every day for surface staining to detect the expression level of PD-1.

1.2.2.4 Killing test of CD8+ cells

1. Induction of CTL. According to the method described in 1.2.2.3, CTL cells are induced from the spleen of OT1 mice. Generally, the CTL cells on the fifth to sixth day are used for the test, and the cell state is better at this time.

2. Incubate target cells EL-4 cells with 10nM OVA _257-264 at 37°C for 1 hour; wash effector CTL cells and target cells twice with killing buffer (phenolfree-RPMI, 2% FBS), and resuspend to 1 million/ mL and 0.1million/mL concentration. In a U-bottom 96-well plate, add 100 μL of target cells to each space; add CTL cells diluted in a gradient according to the ratio of fixed-effect CTL cells to target cells (10:1, 5:1, 2.5:1, 1.25:1), 250 g Centrifuge for 4 minutes, incubate together in an incubator for 4 hours, collect 50 μL of supernatant, and use Promega kit to detect LDH released by target cell death, and calculate the killing efficiency of effector CTL cells. The control wells were set according to the requirements of the kit.

1.2.2.5 B16F10 and MC38 tumor models

1. B16F10 and MC38 cell culture. The cells were digested with trypsin, washed three times with PBS, and then filtered with a 40 μm filter membrane to obtain a single cell suspension, and the cell density was adjusted to 3.2 million/ml. When culturing B16F10 cells, pay attention to changing the medium in time to avoid excessive cell density affecting the cell state. In the process of digestion with trypsin, the digestion time of B16F10 was slightly longer than that of 293FT, about 1.5 minutes, and the digestion time of MC38 cells was shorter, about 45 seconds. Before inoculating mice, control the density of B16F10 and MC38 cells at about 80%. At this time, the cells are in the best state and the tumor formation rate is the highest.

2. B16F10 and MC38 cell transplantation: 8-10 week-old mice were used for tumor cell transplantation experiments. The specific operation was as follows: firstly inject 100 μL of 3% pentobarbital sodium into the anesthetized mouse, and subcutaneously inject 0.4 One million B16F10 or 0.5 million MC38 single-cell suspensions were injected into the epidermis slightly lower on the back of the mouse body near the abdomen, with an injection volume of 125 μl. In C57BL/6 background mice, tumor formation was visually observed 6-8 days after subcutaneous injection of tumor cells.

3. Tumor Measurement and Sacrifice. About 8-10 days, the tumor size was recorded with a vernier caliper, and the long diameter (mm) and wide diameter (mm) of the tumor were measured, and the tumor size was expressed as the long diameter multiplied by the wide diameter. Generally speaking, the tumor reaches 10mm×10mm in about 13 days, and 15mm×15mm in about 18-20 days. Mice whose long diameter or wide diameter of the tumor is greater than 15mm, or mice with tumor ulceration greater than 10mm, or mice with skin ulceration at the tumor lesion and secondary infection during the experiment and feeding process are euthanized .

4. Phenotype analysis of tumor infiltrating lymphocytes. Generally on day 14-16, when the tumor size was about 12mm X 12mm, the mice were sacrificed by cervical dislocation, sterilized with 70% ethanol, the spleen was taken out and cut into small pieces (3mm X 3mm) and resuspended with 10ml of digestive solution ( 5% FBS, 20mM Glutamine, 50μM β-mercaptoethanol, 1.6mg/ml Collagenase IV, 1.6mg/ml Collagenase I, 0.02% DNase I), placed in a 37-degree turntable for 1.5 hours, you can see that most of the tumor tissue Digested into a cell suspension, leaving only a small amount of fat or unknotted hoof tissue, then filtered with a 70 μm filter to obtain a single cell suspension, and then used 40% and 70% Percoll for density gradient centrifugation, tumor infiltrating lymphocytes Will be enriched at the interface of 40% and 70% Percoll. Carefully aspirate the lymphocytes with a pipette or a pipette gun, and wash them twice with PBS to obtain enriched tumor-infiltrating lymphocytes, which can be directly stained on the surface to detect the expression of CD8, CD44, PD-1 and other molecules. If it is necessary to detect cytokine secretion function, it needs to be stimulated with 1640 medium containing 1 μM Ionomycin + 50 ng PMA + 5 μg/ml BFA at 37 degrees for 4 hours, and then perform intracellular staining.

1.2.2.6 Adoptive cell therapy (ACT) model

1. Subcutaneously inoculate 0.4 million B16F10-OVA cells into C57 mice according to the method described in 1.2.2.5, with at least 15 mice in each experimental group.

2. According to the method of 1.2.1.3 lentiviral system infection of primary mouse cell lines, knock down Fbxo38 gene in CD8 cells with OT1 background, sort GFP positive cells and continue to culture with IL-7 and IL-15 Medium culture for three to four days, generally the cells can be expanded three times.

3. On the fourth day after B16F10-OVA cell injection, 1.25 million/only Fbxo38 knockdown cells were injected into the tail vein, and the control group was injected with PBS.

4. Count the tumor size of the control group and the experimental group at about 8-10 days, once every two days.

5. If it is necessary to analyze the phenotype of the transferred OT1CD8T cells, kill the mice at about 18 days; if anti-PD-1 combined with ACT treatment is required, inject PD-1 antibody at about 10 days, and clone the PD-1 antibody The number is J43, 100μg/monkey, injected once every three days, a total of four injections.

1.2.2.7RNA-seq, library construction and bioinformatics analysis

CD8⁺ T细胞，一部分直接用TRIZOL裂解，另一部分按照1.2.2.2所述方法在48孔板中刺激，抗体浓度是anti-CD3(2μg/ml)+anti-CD28(2μg/ml)，刺激时间96小时，收获细胞之后也用TRIZOL裂解。提取总RNA并且通过Agilent Bioanalyzer 2100检测RNA的质量，并且通过RNAClean XP Kit(Cat A63987,Beckman Coulter)和RNase-Free DNase Set(Cat#79254,QIAGEN)两个试剂盒进一步纯化RNA。1. RNA-seq sample preparation. Spleens isolated from three pairs of female Fbxo38 ^CKO and corresponding littermate wild-type mice

A part of CD8 ⁺ T cells was directly lysed with TRIZOL, and the other part was stimulated in a 48-well plate according to the method described in 1.2.2.2. The antibody concentration was anti-CD3 (2 μg/ml)+anti-CD28 (2 μg/ml), and the stimulation time At 96 hours, cells were also lysed with TRIZOL after harvest. Total RNA was extracted and the quality of RNA was detected by Agilent Bioanalyzer 2100, and RNA was further purified by two kits, RNAClean XP Kit (Cat A63987, Beckman Coulter) and RNase-Free DNase Set (Cat #79254, QIAGEN).

RNA Sample Preparation Kit(Illumina,USA)试剂盒构建文库，并且进一步通过

2.0Fluorometer(Life Technologies)和validated byAgilent 2100bioanalyzer(Agilent Technologies)对文库进行纯化、富集和定量，。2. Library construction. use

The RNA Sample Preparation Kit (Illumina, USA) kit was used to construct the library, and further passed

2.0 Fluorometer (Life Technologies) and validated by Agilent 2100bioanalyzer (Agilent Technologies) were used to purify, enrich and quantify the library.

3. Sequencing. Clusters were generated using cBot and sequenced using Illumina HiSeq 2500 (Illumina, USA).

4. Gene expression analysis. The sequencing read value of each gene will be converted into FPKM value, which is calculated according to the following formula:

1.2.3 Biochemical experiments

1.2.3.1 Co-immunoprecipitation

1. Take at least 10 ⁷ cells (applicable to Jurkat and 293FT cells), centrifuge at 500g for 5 minutes, discard the supernatant, lyse with LysisBuffer for Co-IP, and lyse with a rotary shaker at 4°C at more than 20rpm for half an hour until the cell suspension becomes clear , a flocculent precipitate appeared. Centrifuge at the maximum speed at 4°C for 5 minutes, and pick out the flocculent precipitate with a 200 μL pipette tip. Take 30 μL of the supernatant, add 4 times concentrated SDS loading buffer and mix well, cook the sample in a heater at 100°C for 5 minutes for three times, and keep it as the whole cell lysate sample.

2. Add 40 μL Protein G Sepharose beads to the remaining supernatant, and incubate on a rotary shaker at 4°C for 2 hours to remove non-specific binding proteins. Centrifuge at 400g at 4°C for 5min, remove the supernatant into a new EP tube, add 1.5μg-3μg antibody, incubate on a rotary shaker at 4°C for 12h, then add 60μL Protein G Sepharose beads, and incubate for 4h.

3. Centrifuge at 400 g for 5 min at 4°C, remove the supernatant, and wash 3 times with 1 mL of 0.5% NP-40 IP lysate. Aspirate the supernatant with the tip of a flat pipette, add 30 μL SDS loading buffer and mix well, cook the sample in a heater at 100°C, shake once every 5 minutes, a total of three times. Centrifuge at the maximum speed for 5 minutes, and use a flat pipette tip to draw the lysate into a new EP tube for Western Blot experiments.

1.2.3.2 Detection of PD-1 ubiquitination in 293FT cells

1. In 293FT cells, transfect the plasmids of PD-1, FBXO38, and Ub, collect the cells after 36 hours, directly blow off the 293FT cells from the 10cm dish with PBS, centrifuge at 500g, remove the supernatant, and wash once with PBS .

2. Use 100ul of Lysis Buffer for Ubiquitination 1 to lyse the cells, pipette until viscous, vortex 3-5 times, then incubate on a 100-degree cooker for 10 minutes, then put it on ice to cool down, about 10 minute.

3. Add 900 μl of Lysis Buffer for Ubiquitination 2, the purpose is to dilute the concentration of SDS to 0.1%, which is convenient for subsequent immunoprecipitation experiments. After these treatments, the sample will form a compact agglomerate, and then use an ultrasonic breaker to break the agglomerate to form a homogeneous emulsion, which is placed on a 4-degree rotary bed for 1 hour.

4. Centrifuge at the highest speed at 4°C for 10 minutes, remove the precipitate, keep the supernatant, perform IP with anti-HA antibody at a concentration of 4 μg/ml, and take part of the supernatant as a whole cell lysis sample.

5. Refer to the co-immunoprecipitation method in 1.2.3.1 for the remaining steps.

1.3 Test statistical analysis method

All experiments were performed in at least 3 independent repetitions (some mouse tumor experiments were performed in duplicate). All data are expressed as mean and standard deviation or standard error (mean±SD or mean±SEM). Define a P value less than 0.05 as a significant difference, specifically: *p<0.05; **p<0.01; ***p<0.001; n.s.p>=0.05, no significant difference.

For the experimental data of n=3: if there is no significant difference in the variance of the two groups of samples, use the two-tailed unpaired t-test for difference detection; if there is a significant difference in the variance of the two groups of samples, use the two-tailed unpaired t- test with Welch's correction for difference detection.

For the experimental data with n>3, first use the Kolmogorov-Smirnov test to detect whether the data conforms to the normal distribution: if it conforms to the normal distribution, and there is no significant difference in the variance, use the two-tailed unpaired t-test for difference detection; if it conforms to the normal distribution, and there is a significant difference in variance, use two-tailed unpaired t-test with Welch's correction for difference detection; if it does not meet the normal distribution, use Mann-Whitney test for difference detection.

For the human samples in a and e in Figure 3.4.1, first use the Kolmogorov-Smirnov test to detect whether the data conforms to the normal distribution: if it conforms to the normal distribution, use the paired t-test for difference detection; if it does not conform to the normal distribution , using the Wilcoxon matched-pairs signed rank test for difference detection.

The mouse tumor growth curve was analyzed using two-way ANOVA test; the survival curve was analyzed using log-rank (Mantel-Cox) test.

1.4 Experimental results

1.4.1 Ubiquitination of PD-1 after T cell activation

The present invention directly uses PD-1 antibody (EH12.1, BD) to immunoprecipitate PD-1 protein in activated human peripheral blood mononuclear cells (PBMC) and Jurkat cell lines, and then uses ubiquitin-specific antibody for immunoblotting It was found that PD-1 has obvious ubiquitination modification in human PBMC and Jurkat cell line, and there is also a dynamic change of ubiquitination modification in Jurkat cell line (Fig. 1. ac). Wherein, the specific method for isolating PBMC cells from human blood refers to Lymphoprep ^TM Density Gradient Medium for the Isolation of Mononuclear Cells Separation Kit (Catalog #07801) of STEMCELL Company.

In the present invention, proteasome degradation pathway inhibitor MG132 or lysosomal degradation pathway inhibitor NH4Cl and BFA were added to the medium of mouse CTL cells expressing PD-1, and it was found that only MG132 could lead to the accumulation of PD-1, while NH4Cl and BFA There was no effect on the expression of PD-1 (Fig. 1.di). In order to ensure that the concentrations of MG132, NH4Cl and BFA are sufficient to inhibit proteasome and lysosomal degradation pathways, the present invention detects ERK1/2 protein and p62 protein respectively as positive controls for proteasome degradation pathway and lysosomal degradation pathway. The present invention finds that in the same cells, ERK1/2 protein and p62 protein are accumulated. Therefore, the above data suggest that PD-1 protein has obvious dynamic changes after CD8 ⁺ induced expression, and the ubiquitination modification and proteasome-dependent degradation of PD-1 may be the important mechanism.

1.4.2 FBXO38 specifically regulates the expression of PD-1 in Jurkat cell lines

In the present invention, lipo2000 is used to overexpress Fbxo38 in 293FT cells, a lentiviral packaging system is used to infect Jurkat cells, and pHAGE-fEF1a-3Myc-FBXO38-IRES-ZsGreen is used to overexpress Fbxo38 in Jurkat cells. In 293FT cells and Jurkat cell lines, immunization Co-precipitation experiments proved that Myc-FBXO38 can interact with HA-PD-1, and exogenously overexpressed Myc-FBXO38 can also interact with endogenously induced PD-1 (Figure 2.a-d). In 293FT cells, Myc-FBXO47 could also interact with HA-PD-1 (Fig. 2.e). In order to detect the influence of FBXO38 and FBXO47 on the stability of PD-1, the present invention uses pHAGE-fEF1a-3Myc-FBXO38-IRES-ZsGreen and pHAGE-fEF1a-3Myc-FBXO47-IRES-ZsGreen to overexpress Fbxo38 and Fbxo47 gene, it was found that overexpression did not affect the expression of CD3 on the surface of Jurkat cell lines, but after anti-CD3 stimulation, the expression level of PD-1 in Jurkat cells with FBXO38 overexpression was significantly lower than that in the control group, while Jurkat cells with FBXO47 overexpression PD-1 in cells was not affected (Figure 2.f-i), these data suggest that although both FBXO38 and FBXO47 can interact with PD-1, the role of FBXO38 may be more important. Further, the present invention successfully knocked down the Fbxo38 gene with shRNA in the Jurkat cell line, its transcription and protein levels were significantly reduced, while the expression of CD3 on the cell surface was not affected (Fig. 2.j-l). After anti-CD3 stimulation, the expression of PD-1 in the Fbxo38 knockdown group was significantly higher than that in the control group, while there was no significant difference in the transcript level between the two groups (Fig. 2.m-n). Further, the antibody IP:PD-1 of the co-immunoprecipitation of PD-1 in the present invention and the detection of Fbxo38 knockdown by western blot can significantly increase the level of PD-1 protein (Fig. 2.o). Combined with co-immunoprecipitation experiments and overexpression and knockdown experiments in Jurkat cells, the present invention believes that FBXO38 is a key protein that regulates the stability of PD-1.

The DNA nucleotide sequence encoding the shRNA is 5'GACTTCCTTTGTATCAGCTTA 3' (SEQ ID NO.12).

1.4.3 FBXO38 catalyzes the K-48 polyubiquitination modification of PD-1 and promotes its degradation

The present invention first overexpresses Myc-Fbxo38, HA-Pd1 and V5-Ub in 293FT cells, and finds that FBXO38 can promote the ubiquitination of PD-1, and all the two lysines in the intracellular segment of PD-1 are mutated After changing to R, the ubiquitination signal of PD-1 completely disappeared, which indicated that FBXO38 may catalyze the ubiquitination of PD-1 at these two sites (Fig. 3.a). In the Jurkat cell lines overexpressing and knocking down Fbxo38, the present invention found that FBXO38 can significantly affect the ubiquitination modification of endogenous PD-1 (Fig. 3.b-c). The present invention compared the ubiquitination signals of wild-type PD-1 and K210R, K233R, and KtoR with both K mutations, and found that the K210R mutation did not affect the ubiquitination signal of PD-1, while the K233R and KtoR mutations almost No ubiquitination modification of PD-1 was detected, which indicated that FBXO38 catalyzed ubiquitination modification at the K233 site of PD-1 (Fig. 3.d). FBXO38 belongs to the F-box E3 ligase family. This family has an important F-box functional domain. After the F-box is deleted in the present invention, it is found that the ability of FBXO38 to catalyze the ubiquitination of PD-1 has been significantly affected (Fig. 3.e). In the present invention, different V5-Ub mutations were transfected into 293FT cells, and it was found that after the K-48R mutation, the ubiquitination signal of PD-1 was significantly affected, while the signal of K63R was hardly affected, suggesting that FBXO38 is also catalyzed PD-1 produces K-48-type polyubiquitination modifications (Fig. 3.f). Finally, the present invention uses the protein synthesis inhibitor CHX to block protein synthesis, detects the protein stability of PD-1, and finds that FBXO38 can significantly promote the degradation of PD-1. If the two lysines of PD-1 are mutated into R or the F-box domain of FBXO38 is deleted, PD-1 will become more stable (Figure 3.g-i). Therefore, the above data indicate that FBXO38 can catalyze the K-48 type polyubiquitination modification at the K233 site of PD-1 and promote the degradation of PD-1.

1.4.4 Effect of Fbxo38 knockout on T cell development

The present invention uses a C57 background Fbxo38 conditional knockout mouse (Fbxo38 ^CKO ) ( FIG. 4 a ).

状态，central memory(CD44^hiCD62L^hi)和effector/effector memory(CD44^hiCD62L^lo)细胞也没有显著的差别(图4.d-i)。所以，这些数据说明，Fbxo38基因敲除不影响T细胞的发育和外周的免疫稳态。In the present invention, the influence of Fbxo38 gene knockout on mouse T cell development is first detected by flow cytometry. In the thymus, the development of CD4 and CD8 cells was not significantly different in Fbxo38 ^CKO mice compared with controls, including DN (CD4 ^- CD8 ^- ), DP (CD4 ⁺ CD8 ⁺ ), CD4SP (CD4 ⁺ CD8 ^- ) and CD8SP (CD4 ^- CD8 ⁺ ), there was no obvious difference in DN1, DN2, DN3 and DN4 stages of DN cells (Fig. 4.bc). In the peripheral spleen and lymph nodes, the ratio of CD4 and CD8 cells in Fbxo38 ^CKO mice was not significantly different from that in the control group, and most of the CD4 and CD8 cells remained in

State, central memory (CD44 ^hi CD62L ^hi ) and effector/effector memory (CD44 ^hi CD62L ^lo ) cells also had no significant difference (Figure 4.di). Therefore, these data suggest that Fbxo38 gene knockout does not affect T cell development and peripheral immune homeostasis.

1.4.5 Effect of Fbxo38 knockout on T cell activation, proliferation and apoptosis

The present invention uses flow cytometry to detect the expression levels of CD3 and CD28 on the surface of CD8 ⁺ T cells in Fbxo38 ^CKO and control mice, and finds that knockout of Fbxo38 does not affect the expression of CD3 and CD28 (Fig. 5.a). After anti-CD3 and anti-CD28 stimulation, the expression of activation molecule CD44 on the surface of Fbxo38 ^CKO and CD8 ⁺ T cells in the control group was comparable (Fig. 5.b). At the same time, the present invention also used flow cytometry to detect the ability of CD8 ⁺ T cells to secrete cytokines after activation, and found that the secretion of IFN-γ, TNF-α and GranzymB were not significantly affected at the two time points of 72h and 96h. effect (Fig. 5.d).

1.4.6 Effect of Fbxo38 knockout on T cell proliferation and apoptosis

The present invention further uses Celltracker staining and flow cytometry to detect the proliferation of WT and CKO CD8 ⁺ T cells after in vitro activation, and uses Annexin V and propidium iodide (PI) staining and flow cytometry to detect the proliferation of WT and CKO CD8 ⁺ T cells. In terms of apoptosis, it was found that knockout of Fbxo38 did not affect the proliferation and apoptosis of CD8 ⁺ T cells ( FIG. 6 ).

1.4.7 Effect of Fbxo38 knockout on CD8 ⁺ T cell transcriptome

和活化状态下的基因转录水平，选择了三对Fbxo38^CKO和对照组小鼠，分离CD8⁺ T细胞，经过anti-CD3和anti-CD28刺激之后，通过RNA-Seq检测了多个细胞表面分子、细胞因子和活化分子以及转录因子的表达变化，发现Fbxo38敲除也不影响CD8⁺ T细胞中这些功能分子的表达。但是，CD8⁺ T细胞活化之后，PD-1的蛋白水平在Fbxo38敲除的CD8⁺ T细胞中有了明显的升高。相反，另外一个免疫抑制分子LAG-3的表达水平并没有差异。在这些实验说明，在CD8+ T细胞中，FBXO38的主要靶点是PD-1，在PD-1通路没有激活的情况下，Fbxo38敲除的小鼠具有正常的活化能力(图7a)。The present invention uses RNA-seq to analyze WT and CKO CD8 ⁺ T cells in

and the gene transcription level in the activated state, selected three pairs of Fbxo38 ^CKO and control mice, isolated CD8 ⁺ T cells, after anti-CD3 and anti-CD28 stimulation, detected multiple cell surface molecules, Expression changes of cytokines and activating molecules, as well as transcription factors, found that Fbxo38 knockdown also did not affect the expression of these functional molecules in CD8 ⁺ T cells. However, after CD8 ⁺ T cell activation, the protein level of PD-1 was significantly increased in Fbxo38 knockout CD8 ⁺ T cells. In contrast, there was no difference in the expression levels of another immunosuppressive molecule, LAG-3. These experiments demonstrated that in CD8+ T cells, the main target of FBXO38 is PD-1, and in the absence of activation of the PD-1 pathway, Fbxo38 knockout mice had normal activation ability (Fig. 7a).

1.4.8 The effect of Fbxo38 knockout on PD-1

The present invention detects the expression levels of PD-1 on the surface of CD8 ⁺ , CD4 ⁺ T cells and Treg cells in WT and Fbxo38 ^CKO mice by flow cytometry. After anti-CD3 and anti-CD28 stimulation, the PD-1 levels on the surface of CD8 ⁺ and CD4 ⁺ T cells were significantly higher in Fbxo38 ^CKO compared with WT (Fig. 8.ab). At the same time, the present invention also used flow cytometry to detect the PD-1 level of Treg (CD4 ⁺ CD25 ⁺ ) cells in the mouse spleen, and found that the PD-1 level in Treg was significantly increased in Fbxo38 ^CKO compared with WT (Fig. 8.c ). In summary, the knockout of Fbxo38 significantly increases the expression level of PD-1 in T cells.

1.4.9 Fbxo38 knockout affects T cell anti-tumor immunity

The invention uses a tumor model to study the effect of FBXO38 on the anti-tumor immunity of CD8 ⁺ T cells under physiological conditions. B16F10 cells highly express PD-L1 molecules, which means that the PD-1-PD-L1 pathway is continuously activated in the B16F10 model, and the PD-1 pathway plays an important role in the tumor immune escape of this model. B16F10 is a very malignant tumor model, and the tumor growth rate is very fast. However, tumor growth was faster and survival was shorter in Fbxo38 ^CKO mice compared with control mice (Fig. 9.ab). Further analysis of tumor-infiltrating T cells found (Fig. 9.c), more than 90% of tumor-infiltrating CD8 ⁺ T cells were CD44-positive, which meant that these cells were activated by antigen, while Fbxo38 ^CKO mice CD8 ⁺ T cells have higher expression levels of PD-1, lower proliferative capacity (Ki67 expression) and defects in the function of secreting cytokines (IFN-γ, TNF-α and GranzymB), CD4 ⁺ , CD8 ⁺ and Treg cells The ratio of was not affected (Fig. 9.dj).

1.4.10 Fbxo38 knockout affects T cell anti-tumor immunity

The present invention uses the MC38 model, and it is also found that CD8 ⁺ T cells in Fbxo38 ^CKO mice have higher expression levels of PD-1 and faster tumor growth (Fig. 10.af). These data indicate that FBXO38 protein can affect the function of CD8 ⁺ T cells by regulating the expression of PD-1.

1.4.11 Fbxo38 knockdown did not affect the activation of T cells

The present invention knocks down the Fbxo38 gene in OT1CD8 ⁺ T cells by shRNA in vitro, and observes the PD-1 level of CD8 ⁺ T cells in the Fbxo38 knockdown group and the control group. After screening, the present invention selects two Fbxo38-specific shRNAs, which can effectively knock down endogenous Fbxo38 after verification by qPCR and immunoblotting (Fig. 11.ab). Like in Fbxo38-knockout CD8 ⁺ T cells, Fbxo38 knockdown did not affect the expression of CD3 and CD28 molecules on the cell surface (Fig. 11.c). Furthermore, the present invention detected the effect of Fbxo38 knockdown on the TCR signaling pathway, and found that CD3 phosphorylation, PLC-phosphorylation and ERK phosphorylation were not affected by Fbxo38 knockdown, T cell activation molecule CD69 and cytokine IFN- The secretion of was also not affected (Fig. 11.dg).

The DNA nucleotide sequence of encoding described shRNA is:

5'GTGGATGCGACTGGTTGATAT 3' (SEQ ID NO.13)

5'CGTAATCGTAACGGAGCATTT 3' (SEQ ID NO.14)

1.4.12 Fbxo38 knockdown up-regulates the expression of PD-1 and affects the killing ability of CD8 ⁺ T cells

After activating T cells with anti-CD3 and anti-CD28 antibodies, the level of PD-1 was detected by flow cytometry, and it was found that the expression level of PD-1 in the cells of the Fbxo38 knockdown group was significantly increased (Fig. ⁺ T cell phenotypes were consistent. In order to study whether the high expression of PD-1 will affect the function of CD8 ⁺ T cells, the present invention selects the OT1 CD8 ⁺ T cell killing system with EL-4 cells as target cells. EL-4 cells highly express PD-L1, which can effectively activate the PD-1 pathway when OT1CD8 ⁺ T cells are killed (Figure 12.b). Compared with the control group, the killing ability of the cells in the Fbxo38 knockdown group was significantly affected; at the same time, anti-PD-1 could completely restore this functional defect (Fig. 12.c). To further verify that FBXO38 regulates the PD-1 level of CD8 ⁺ T cells, Fbxo38 was overexpressed with retrovirus MSCV in OVA-activated CTL, and the level of PD-1 was found to be significantly reduced (Figure 12.df). These data suggest that PD-1 is the main target of FBXO38.

1.4.13 FBXO38 endogenously regulates the anti-tumor ability of CD8+ T cells

The present invention uses adoptive T cell therapy (Adoptive Transfer Model) to treat the B16F10 tumor model for research. In this model, the present invention knocks down the Fbxo38 gene in OT1CD8 ⁺ T cells in vitro by shRNA (the shRNA used is the same as that used in 1.4.11), sorts and expands these cells, and then injects them into tumor-bearing mice (B16F10-OVA), to observe the ability of CD8 ⁺ T cells in the Fbxo38 knockdown group and control group to control the tumor (Fig. 13.a). In the present invention, the unstimulated Fbxo38 knockdown group and control group cells were reinfused into B16F10-OVA tumor-bearing mice, and it was found that in the inguinal lymph node (draining lymph node) of the mice, the proportion of cells in the Fbxo38 knockdown group and the level of Ki67 The expression level was significantly lower than that of the control group, while the expression level of PD-1 was significantly higher than that of the control group; and in the non-draining lymphnode, the proportion of cells in the two groups was equivalent, indicating that Fbxo38 knockdown did not affect T cells Migrate to secondary lymphoid organs (Fig. 13.be). Therefore, the ability of cells in the Fbxo38 knockdown group to control the tumor was significantly lower than that in the control group, and the survival period of the mice was also shorter (Figure 13.fg). This data is consistent with the data of the Fbxo38 conditional knockout mice. Finally, the present invention studies the combined treatment of anti-PD-1 and ACT on the control of tumors, and finds that in the Fbxo38 knockdown and control groups, the combination treatment is significantly better than the ACT treatment alone, and after the combination treatment, the two groups are less There was no significant difference in tumor size and survival in the mice (Fig. 13.hi), which indicated that anti-PD-1 could restore the functional defect caused by Fbxo38 knockdown. These data, combined with the Fbxo38 knockdown conditional knockout data, indicate that FBXO38 can regulate the anti-tumor immune function of CD8 ⁺ T cells by specifically regulating the expression level of PD-1.

The above examples are intended to illustrate the disclosed embodiments of the present invention, and should not be construed as limiting the present invention. In addition, various modifications set forth herein, as well as changes in the method and composition of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in connection with various specific preferred embodiments of the invention, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as mentioned above which are obvious to those skilled in the art to obtain the invention should be included in the scope of the present invention.

sequence listing

<110> Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

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Claims (12)

1. Use of a PD-1 ubiquitinated agonist in the preparation of a PD-1 degradant or in the preparation of a medicament for the immunotherapy of cancer, said PD-1 ubiquitinated agonist being an FBXO38 agonist.
2. 2. Use according to claim 1, further comprising one or more of the following features:

   a. the PD-1 ubiquitination agonist is a molecule having a promoting effect on PD-1 ubiquitination;

   b. the PD-1 ubiquitination agonist is selected from lentivirus or retrovirus packaged plasmid, carbohydrate, lipid, small molecule compound, RNA, polypeptide or protein.
3. 3. Use according to claim 1, further comprising one or more of the following features:

   c. the FBXO38 agonist is a molecule having a promoting effect on FBXO 38;

   d. the FBXO38 agonist is selected from a molecule capable of increasing the expression or activity of FBXO 38.
4. 4. Use according to claim 3, characterized in that in characteristic d the molecule capable of increasing the expression or activity of FBXO38 is selected from the group consisting of lentivirus or retrovirus packaged plasmids, carbohydrates, lipids, small molecule compounds, RNA, polypeptides or proteins.
5. 5. An immunotherapeutic drug for tumors, comprising an effective amount of a PD-1 ubiquitination agonist, said PD-1 ubiquitination agonist being an FBXO38 agonist.
6. Use of FBXO38 as an action target in screening a tumor immunotherapy medicament or screening an autoimmune disease treatment medicament.
7. 7. A method for screening a tumor immunotherapy drug, comprising: and (4) verifying whether the medicament to be screened can have a promoting effect on the FBXO38, and if so, determining the medicament to be screened as a candidate medicament for tumor immunotherapy.
8. 8. The method for screening a tumor immunotherapeutic drug according to claim 7, which comprises: the drug to be screened is acted on the in vitro cell expressing FBXO38 to determine whether the FBXO38 in the cell has activity enhancement or expression up-regulation.
9. 9. The method for screening a tumor immunotherapeutic drug according to claim 8,

   the method for determining whether the FBXO38 has increased viability or up-regulated expression in the cell is: real-time quantitative PCR and/or Western Blot detection, or mass spectrometric detection.
10. 10.A method for screening a therapeutic agent for an autoimmune disease, comprising: and (4) verifying whether the medicament to be screened can inhibit the FBXO38, and if so, determining the medicament to be screened as a candidate medicament for treating the autoimmune disease.
11. 11. The method for screening for an autoimmune disease therapeutic agent according to claim 10, comprising:

    the drug to be screened is applied to the in vitro cells expressing FBXO38 to determine whether the FBXO38 in the cells has weakened activity or reduced expression.
12. 12. The method of screening for an autoimmune disease therapeutic agent according to claim 11,

    the method for determining whether the FBXO38 has decreased viability or expression in a cell is: real-time quantitative PCR and/or Western Blot detection, or mass spectrometric detection.

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