CN116159131B - Application of TRIM21 and promoter thereof in preparation of antitumor biotherapeutic drugs - Google Patents
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- CN116159131B CN116159131B CN202211514074.8A CN202211514074A CN116159131B CN 116159131 B CN116159131 B CN 116159131B CN 202211514074 A CN202211514074 A CN 202211514074A CN 116159131 B CN116159131 B CN 116159131B
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Abstract
Provides the application of TRIM21 and the promoter thereof in preparing antitumor biotherapeutic drugs. In particular, the use of TRIM21 and/or an enhancer thereof for the manufacture of an acquired immune enhancing agent for the treatment of melanoma in a subject, an acquired immune enhancing product comprising a therapeutically and/or prophylactically effective amount of TRIM21 and/or an enhancer thereof, and the use thereof in combination with other tumour therapeutic agents, in particular immunotherapeutic agents. The improvement of TRIM21 level or activity can effectively promote T cells to infiltrate the tumor part, thereby promoting the anti-tumor immunity of the organism, providing a new way for the treatment of tumors and having wide application prospect.
Description
Technical Field
The application belongs to the field of biotechnology and medicine, and particularly relates to an anti-tumor biological therapeutic drug, in particular to a drug for treating tumors by enhancing acquired immunity. More specifically, the application relates to the application of TRIM21 and an accelerant thereof in preparing anti-melanoma biological treatment medicaments (especially as anti-tumor acquired immunity enhancers), and corresponding tumor treatment methods and applications.
Background
The posttranslational modification of proteins is an important factor for regulating the functions and stability of proteins, while ubiquitination modification is one of the most important posttranslational modification forms and plays a very important role in protein localization, metabolism and functions. Meanwhile, ubiquitination modification is also involved in almost all life processes of cell proliferation, apoptosis, differentiation, migration, gene expression, transcriptional regulation, signal transduction, inflammatory immunity and the like, and is closely related to clinical tumor, infection and other diseases.
Ubiquitination refers to the process of specific binding modification of ubiquitin molecules under the action of special enzymes. The main enzymes involved in this process are four: e1 ubiquitin activating enzyme, E2 ubiquitin binding enzyme, E3 ubiquitin ligase and Deubiquitinase (DUB) mediating reverse deubiquitination. The E1 ubiquitin activating enzyme and the E2 ubiquitin ligase in organisms are few in types and high in universality; the E3 ubiquitin ligase is a key enzyme for determining the specificity of ubiquitination modification, so the variety is more, and about 600 or more. The triad protein (TRIM) family is a larger subfamily of E3 ubiquitin ligases. To date, nearly 80 TRIM family proteins have been found in the human genome, many of which have been demonstrated to have ubiquitin ligase activity to participate in various vital activities of the body.
TRIM21 is a TRIM family E3 ubiquitin enzyme with a molecular weight of 52kD and is therefore also known as Ro52. TRIM21 was originally found as an autoantigen in systemic lupus erythematosus (Systemic lupus erythematosus, SLE) and Sjogren's syndrome (Sjogren's syndrome), and was expressed in various cells, particularly immune cells such as T cells, macrophages, dendritic cells, and the like.
TRIM21 comprises, starting from the N-terminus, the RING-finger, B-box and Coiled-coil domains, respectively, and the PRYSPRY domain at the C-terminus. The RING-finger domain is TRIM21 as the basis of E3 ubiquitin ligase ubiquitination activity, the B-box domain is similar to the RING-finger domain, and participates in TRIM21 activity regulation in a competitive manner, the Coiled-coil domain mainly mediates the multimerization of the TRIM molecule, the C-terminal PRYSPRY domain participates in the recognition of specific substrates, and also contains an antibody binding site, is a determination domain of the specificity of TRIM21, and is also a key domain of antiviral immunity.
As a TRIM family E3 ubiquitin ligase, the ubiquitination function research of TRIM21 is mainly focused on the infection and inflammation fields, especially on the regulation of the antiviral immune response of organisms. Cell antivirus mainly recognizes viral nucleic acid RNA or DNA through Pattern Recognition Receptors (PRR) such as RIG-I and cGAS, and further mediates downstream MAVS/STING, and finally activates IRF3 and NFkB transcription factors to start the expression of interferon and inflammatory factors, thereby exerting antiviral effect. TRIM21 can regulate body antiviral effects at multiple levels:
(1) At the PRR level, TRIM21 can rapidly lyse the viral coat in an antibody-dependent manner, exposing the nucleic acids for recognition by RIG-I and cGAS (Watkinson RE et al, "TRIM21 Promotes cGAS and RIG-ISensing of Viral Genomes during Infection by Antibody-OPsonified viruses". PLoS Pathog.2015;11 (10): e 1005253);
(2) TRIM21 can also be directly combined with a DNA recognition receptor DDX41, promote the degradation of a proteasome pathway of the DDX41 through K48 ubiquitination, inhibit the generation of I-type interferon and negatively regulate the antiviral immunity of organisms;
(3) At the signal transduction linker protein level, TRIM21 can catalyze K27 ubiquitination of MAVS by binding to MAVS, promoting recruitment of TBK1 by MAVS, thereby enhancing activation of downstream signaling pathways and interferon production, enhancing antiviral effects (Xue BB et al, "TRIM21 promotes innate immune response to RNA viral infection through Lys-linked polyubiquitination of MAVS". J Virol,2018, 92 (14): e 00321);
(4) On the transcription factor level, TRIM21 has regulatory effects on IRF3, IRF5, IRF7, IRF8 and the like. TRIM21 can bind to the IAD domains of IRF3, IRF5 and IRF7 through the prypry domain, mediating degradation of IRFs through K48 ubiquitination, negatively regulating the production of type I interferons. The effect of IRF8 of TRIM21 differs from other IRFs in that ubiquitination does not mediate IRF8 degradation, but rather promotes expression of antiviral inflammatory factors by enhancing its transcriptional activity.
The specific function of the C-terminal PRYSPRY domain binding antibody of TRIM21 is the most important feature of TRIM21 in comparison to other TRIM family members. PRYSPRY contains two subdomains, PRY and SPRY, PRY binds to the CH2 domain of IgG and SPRY binds to the CH3 domain, monomer TRIM21 binds to only one heavy chain, and TRIM21 dimer binds to just two heavy chains of an antibody. TRIM21 confers unique antiviral immune functions through the ability of PRYSPRY to bind to the Fc segment of an antibody molecule (IgG, igM, igA).
Normally, the antiviral ability of antibodies is to prevent virus invasion into cells by binding to neutralizing epitopes of the virus, which is commonly referred to as the virus neutralization reaction. However, the dominant epitopes of viral antigens are usually non-neutralizing epitopes, and therefore it appears that although antibodies bind to the virus, they do not prevent the virus from invading the host cell. In this case, invasion of the virus into the cell with non-neutralizing antibodies is a special scenario where TRIM21 exerts a unique effect. The virus and antibody complex entering the cytoplasm is recognized and combined by the PRYSPRY domain of TRIM21 in the cytoplasm, and after the TRIM21 enzyme activity is activated to mediate self ubiquitination, the virus and antibody complex is degraded through a proteasome pathway or an autophagy pathway, so that the virus is inactivated, and the organism is protected from infection. This unique effect of TRIM21 is also known as antibody-dependent intracellular neutralization (Antibody dependent intracellular neutralization, ADIN). The RNA, DNA and other mode molecules released after the virus particles are degraded can be further identified by RIG-I or cGAS and the like, so that the virus can be thoroughly cleared.
Viruses that are currently reported to be able to be cleared by TRIM21 are mainly a few encapsidated viruses, including Foot and Mouth Disease Virus (FMDV), human adenovirus type V, and the like. In addition, TRIM21 can also participate in the organism to resist the infection of intracellular bacteria such as salmonella (salmonella enterica) and the like by a similar mechanism. TRIM21 mediated intracellular neutralization effects, without the need for high concentrations of antibodies, can rapidly degrade viruses entering the cell, prevent their replication and reproduction in the cytoplasm, and trigger antiviral immunity, potentially playing an important role in early anti-infection. The characteristic of TRIM21 also derives the birth of TRIM-away technology, so that any intracellular protein can be targeted for degradation, and the method has wide application prospect in the pharmaceutical industry.
Melanoma is a tumor with high malignancy, and is easy to metastasize, and the incidence rate of melanoma tends to rise year by year. Clinical studies have found that the average 5-year survival rates of I, II, III, IV stage melanoma patients are about 90%, 70%, 40% and less than 10%, respectively, with median progression-free survival in stage IV patients of only 1.7 months, with metastasis being the leading cause of death. In recent years, along with the development of targeted therapy and immunotherapy, melanoma treatment has progressed rapidly, especially the development of immunotherapy, provides a wider choice and benefit for melanoma patients, and greatly prolongs the survival time of melanoma patients.
Immunotherapy of melanoma includes interferon therapy, adoptive immunotherapy, tumor vaccines, and in recent years, particularly popular immune checkpoint inhibitors. The median survival time of the patients treated by the Ipilimumab (Ipilimumab) combined with dacarbazine is obviously prolonged, and the survival rate of the patients in 5 years is improved to 18.2 percent and is higher than 8.8 percent of that of the control group. And another clinical test shows that the nivolumab treatment obviously improves the total survival rate of patients for one year, and the objective remission rate is 42 percent which is far higher than 14 percent of that of the dacarbazine group. Although monoclonal antibodies directed against CTLA4 and PD-1 are widely used as immune checkpoint inhibitors in the immunotherapy of melanoma, with excellent efficacy, due to the high heterogeneity of tumors and the complexity of tumor microenvironments, their effective rate or response rate is still less than 30%, there are still large numbers of melanoma patients who cannot benefit from it, and the responsiveness of different individuals to treatment cannot be predicted, even without any response in some "cold" tumors (i.e. tumors that are not recognized or do not elicit a strong immune system response for various reasons). Thus, a significant challenge facing current tumor immunotherapy is how to increase the patient response rate of immune checkpoint inhibitors, reduce drug resistance, and promote the application of immune checkpoint therapies in different tumors, benefiting more tumor patients.
Currently, one strategy to improve tumor immunotherapy is to develop biomarkers, such as PD-L1, tumor mutational burden TMB (tumor mutation burden), etc., that can be used to select potential responders or exclude potential non-responders. Another strategy is to combine drugs with different mechanisms of action, thereby exerting a synergistic effect and overcoming multiple mechanisms of drug resistance. Several combination therapies for different cancer types have been approved by the FDA to date, and combinations between radiotherapy, chemotherapy, targeted therapy and immunotherapy are being tested clinically. In addition, for cold tumors, the cold tumors are changed into hot tumors through combined medication, so that the curative effect of immunotherapy is improved, and the method is a hot spot for developing current medicines.
The TRIM family of molecules has been studied in the past in the development of tumorigenesis. TRIM11 promoted ovarian and gastric cancer progression, TRIM22 promoted microglioma progression, TRIM27 promoted non-small cell lung cancer progression, and TRIM35 and TRIM47 promoted breast cancer progression, both by modulating tumor cell proliferation apoptosis and invasion metastasis (YX Zhang et al, "The roles and targeting options of TRIM family proteins in tumor". Front Pharmacol.2022Sep30. DOI: 10.3389/fphar.2022.999380), suggesting that certain TRIM family molecules are involved in tumor biological behavior regulation. However, although TRIM family molecules have been studied in tumor prognosis, it is still a great difficulty to screen out specific TRIM molecules that are relevant to tumors and can be used as indicators of pathogenesis, treatment regimen selection and prognosis, given that TRIM functions are different and that the different tumors originate and pathogenesis are different.
At present, no research report on TRIM21 in the relevance of tumor (such as melanoma) treatment and immunotherapy is available at home and abroad.
There is a strong need in the art to find therapies that are effective for the treatment of tumors, particularly for the conversion of "cold" tumors that are not recognized or do not respond strongly by the immune system to "hot" tumors that are recognized by the immune system, in order to enhance the therapeutic efficacy of immunotherapy.
Disclosure of Invention
Provided herein are uses of TRIM21 and/or an enhancer thereof in tumor therapy, particularly tumor immunotherapy combination. The medicine, the medicine composition or the product of the invention can be used for enhancing the infiltration of tumor lymphocytes, converting cold tumor into hot tumor and improving the curative effect and the responsiveness of immunotherapy.
In some aspects herein, there is provided the use of the tri-motif E3 ubiquitin ligase 21 and/or an enhancer thereof in the manufacture of an acquired immune enhancer for the treatment of melanoma in a subject.
In some embodiments, TRIM21 is a substance selected from the group consisting of: trim21 gene, trim21 mRNA, cDNA, TRIM protein or an active fragment of any of the foregoing.
In some embodiments, TRIM21 includes genes and proteins thereof. The TRIM21 gene is transcribed and translated into a TRIM21 protein product in a subject.
In some embodiments, TRIM21 is from a mammal, such as a human, a non-human primate (e.g., gorilla, ape), a rodent (e.g., rat, mouse, guinea pig), a pet (e.g., cat, dog), a livestock (e.g., horse, cow, sheep, pig, rabbit).
In some embodiments, TRIM21 has a sequence known in the art or is a derivative thereof.
In some embodiments, TRIM21 is a molecule comprising the sequence:
(a) TRIM21 molecules having sequences shown in Gene ID 6737 (human), gene ID 20821 (mouse), gene ID 308901 (rat), and the like;
(b) A molecule which hybridizes under stringent conditions to the sequence defined in (a);
(c) A TRIM21 molecule having 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5% or more, or any number or range of numbers therebetween) sequence homology to the sequence of (a) or (b) and encoding a polypeptide or protein having acquired immune enhancing activity, e.g., a TRIM21 molecule obtained via codon optimization;
(d) Any of the polypeptides encoded by the TRIM21 molecules, or derivative proteins obtained by substituting, deleting or adding one or more amino acids in the polypeptides.
In some embodiments, the promoter of TRIM21 is selected from: substances that increase the level of TRIM21, substances that increase TRIM21 activity, and/or substances that delay the metabolism of TRIM 21.
In some embodiments, the promoter of TRIM21 is selected from: natural purification substances, modified natural purification substances, semisynthetic substances, and chemically synthesized substances.
In some embodiments, the promoter of TRIM21 is derived from a mammal, e.g., from: humans, non-human primates (e.g., gorillas, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), livestock (e.g., horses, cattle, sheep, pigs, rabbits).
In some embodiments, the promoter of TRIM21 is selected from: the over-expression vector of TRIM21, the nanoparticle carrying gene, the viral vector carrying the target gene or vector, the PEG modified protein, the protein microsphere, the liposome wrapping the target gene or protein, and the extracellular vesicle carrying the target gene or protein.
In some embodiments, the tumor is a melanoma or portion thereof that has a poor immune response or is in need of improvement of an immunotherapeutic response.
In some embodiments, a subject for which an adaptive immune enhancer is useful is selected from the group consisting of: a tumor patient with acquired immunodeficiency or injury; tumor patients who are not effective or have poor effects or expected to have poor effects in tumor immunotherapy; a patient undergoing, to be undergoing, or having undergone tumor immunotherapy; or a tumor patient having two or more of the foregoing.
In some embodiments, the subject is a mammal, e.g., a human, a non-human primate (e.g., gorilla, ape), a rodent (e.g., rat, mouse, guinea pig), a pet (e.g., cat, dog), a livestock (e.g., horse, cow, sheep, pig, rabbit).
In some embodiments, the acquired immune potentiators herein are contained in a pharmaceutical composition or kit.
In some embodiments, the pharmaceutical composition or kit is a pharmaceutical composition or kit suitable for administration by a means selected from the group consisting of: oral administration, injection (e.g., direct naked DNA or protein injection, liposome-encapsulated DNA, RNA or protein injection), gold-coated gene gun bombardment, plasmid DNA carrying by replication-defective bacteria, DNA carrying by replication-defective adenovirus or protein encoded by the target gene, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, transdermal administration.
In some embodiments, the pharmaceutical composition or kit further comprises or is used in combination with other anti-tumor active ingredients, e.g., selected from the group consisting of: tumor immunotherapeutic agents (e.g., PD-1 antibodies, PD-L1 antibodies, adoptive cell therapeutic agents, antigen chimeric receptor T cell therapeutic agents), chemotherapeutic agents.
In some embodiments, the pharmaceutical composition or kit optionally further comprises: pharmaceutically or immunologically acceptable carriers, adjuvants, containers, packages, administration devices, and indications (e.g., instructions or instructions for administration of TRIM21 and/or its promoters to treat tumors in a subject, etc.).
In some embodiments, the acquired immune enhancer herein is a tumor immunotherapy enhancer, e.g., an enhancer of tumor immunotherapy selected from the group consisting of: PD-1 antibody therapy, PD-L1 antibody therapy, adoptive cell therapy, and antigen chimeric receptor T cell therapy.
In some aspects herein, there is provided an obtained immune enhancing product comprising:
(A) A therapeutically and/or prophylactically effective amount of TRIM21 and/or a promoter thereof;
(B) Pharmaceutically or immunologically acceptable carriers or excipients;
(C) Optionally, one or more other antitumor active ingredients.
In some embodiments, the obtained immune enhancing product is an anti-tumor pharmaceutical composition or kit, such as a tumor immunotherapy enhancing product.
In some embodiments, the TRIM21 and its promoters are as described in detail herein. In some embodiments, the tumor is as described in detail herein. In some embodiments, the product is used in the treatment of a tumor patient as described in detail herein. In some embodiments, the additional anti-tumor chemical component is as detailed in itself. In some embodiments, the form of the product is as described in detail herein.
In some aspects herein, a method of treating a tumor is provided, the method comprising administering TRIM21 and/or an enhancer thereof described herein to a subject in need of tumor treatment.
In some embodiments, the method further comprises administering additional tumor therapy before, during, and/or after the administration of TRIM21 and/or an enhancer thereof.
In some embodiments, the other tumor treatments include, but are not limited to: surgery, radiation therapy, chemotherapy, immunotherapy, preferably immunotherapy. In some embodiments, immunotherapy includes, but is not limited to: PD-1 antibody therapy, PD-L1 antibody therapy, adoptive cell therapy, and antigen chimeric receptor T cell therapy.
In some aspects herein, TRIM21 and/or promoters thereof are provided for use in enhancing acquired immunity in tumor therapy.
In some embodiments, the TRIM21 and its promoters are as described in detail herein. In some embodiments, the tumor is as described in detail herein. In some embodiments, the TRIM21 and its promoters are used in the treatment of tumor patients as described in detail herein. In some embodiments, the additional anti-tumor chemical component is as detailed in itself.
Any combination of technical solutions and features described herein may be implemented by those skilled in the art without departing from the inventive concept and scope of the present invention. Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.
Drawings
The present invention will be further described with reference to the accompanying drawings, wherein these drawings are provided only for illustrating embodiments of the present invention and are not intended to limit the scope of the present invention.
Fig. 1: qRT-PCR detection of expression of TRIM21 in melanoma tissues and paracancerous tissues; p values were calculated using T test, P <0.01 representing the most significant difference.
Fig. 2: knocking out TRIM21 expression of melanoma cells B16 by using CRISPR-cas9 gene editing technology, wherein:
Trim21 -/- and (3) cells: cells from which TRIM21 was successfully knocked out;
Trim21 +/+ control cells: cells of TRIM21 were not knocked out.
Fig. 3: comparison of tumor growth curves and mouse survival curves after TRIM21 knockout melanoma and control vaccinated mice. "ns": no significant differences; * *: p <0.01, very significant difference.
Fig. 4: detection of TRIM21 knockout melanoma by tissue immunofluorescence (TRIM 21 -/- ) And control tumors (CTR, trim21 +/+ ) Medium T cell infiltration, wherein:
bright fluorescent spot (red fluorescence in color photographs): CD3, CD4 or CD8;
dark fluorescent spots (blue fluorescent in color photographs): and (3) cell nucleus.
Fig. 5: correlation of TRIM21 expression level with PD-L1 antibody therapeutic responsiveness:
fig. 5A: response of TRIM21 knockout melanoma B16 and control tumor to PD-L1 antibody treatment;
fig. 5B: TRIM21 overexpressed melanoma YUMM1.7 and control tumors were responsive to PD-L1 antibody treatment.
Fig. 6: total survival time Kaplan-Meier survival curves of TRIM21 high and low expressing melanoma patients were compared.
Fig. 7: comparison of Kaplan-Meier survival curves after immune checkpoint inhibitor anti-PD-L1 treatment in TRIM21 high and low expressing patients, i.e. comparison of efficacy of anti-PD-L1 immunotherapy in TRIM21 high and low expressing patients.
Detailed Description
Provided herein are uses of TRIM21 (including its coding molecules and proteins and active fragments thereof) and/or its promoters in tumor therapy, particularly tumor immunotherapy combination. The medicine, the medicine composition or the product of the invention can be used for enhancing the infiltration of tumor lymphocytes, converting cold tumor into hot tumor and improving the curative effect and the responsiveness of immunotherapy.
The inventors have found through long-term and intensive studies that TRIM21 molecules are low-expressed in tumor tissue cells of tumor patients, and that the expression level thereof is correlated with tumor prognosis. Tumor T cell infiltration of TRIM21 is reduced, the survival time of a subject is shortened, and the curative effect of PD-1 immunotherapy is weak. Whereas increasing TRIM21 levels or activity (e.g., over-expressing TRIM 21) in tumor tissues or cells can improve tumor T cell infiltration and effectively improve the efficacy of immunotherapy (e.g., anti-PD-L1 therapy). The use of TRIM21 and/or an enhancer thereof as an acquired immune enhancer in the treatment of a tumor in a subject, particularly in combination with a tumor immunotherapy, is disclosed for the first time herein.
All numerical ranges provided herein are intended to expressly include all values and ranges of values between the endpoints of the range. The features mentioned in the description or the features mentioned in the examples can be combined. All of the features disclosed in this specification may be combined with any combination of the features disclosed in this specification, and the various features disclosed in this specification may be substituted for any alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the disclosed features are merely general examples of equivalent or similar features.
As used herein, "comprising," having, "or" including "includes" including, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …" and "consisting of … …" are under the notion of "containing", "having" or "including".
Numerical ranges herein include the endpoints thereof and each specific numerical point and subrange within the numerical range. For example, 1-3 includes endpoints 1 and 3, specific integer number points 2 and non-integer number points therein (e.g., without limitation, 1.2, 1.5, 1.8, 2.1, 2.3, 2.4, 2.8, etc.), and sub-ranges thereof (e.g., without limitation, 1-2, 2-3, 1-1.2, 1.5-1.8, etc.).
TRIM21 molecules
As used herein, the term "TRIM21" or "TRIM21 molecule" has its broad meaning, including TRIM21 gene, mRNA, cDNA, TRIM precursor of TRIM21, TRIM21 protein, modified or cleaved products thereof, or active fragments. According to the teachings of the present application, TRIM21 molecules can play an important role in enhancing the acquired immune response in tumor therapy, particularly tumor therapy, and can be used to effectively increase the sensitivity and effect of anti-tumor therapy. In a preferred embodiment of the present application, the tumor is a melanoma, e.g. a melanoma with a poor immune response.
The TRIM21 molecules described herein can be derived from humans, non-human primates (e.g., gorillas, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), livestock (e.g., horses, cattle, sheep, pigs, rabbits), and the like.
The TRIM21 molecules described herein can be, for example:
(a) TRIM21 molecules having sequences shown in Gene ID 6737 (human), gene ID 20821 (mouse), gene ID 308901 (rat), and the like;
(b) A molecule which hybridizes under stringent conditions to the sequence defined in (a);
(c) Has 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 98% >, and/or more than 70% of the sequence of (a) or (b),
99%, 99.5%, or any number or range of values therebetween) and the encoded polypeptide or protein has acquired immune enhancing activity, such as a TRIM21 molecule obtained via codon optimization;
(d) A polypeptide encoded by any one of the TRIM21 molecules described above (e.g., a polypeptide as described in np_ 003132), or a derivative protein of the polypeptide in which one or more amino acids have been substituted, deleted or added.
As used herein, the term "stringent conditions" refers to: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturing agents such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42℃and the like during hybridization; or (3) hybridization only occurs when the identity between the two sequences is at least 50%, preferably 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, more preferably 95% or more. For example, the sequence may be the complement of the sequence defined in (a).
The full-length TRIM sequences herein, or fragments thereof, can be obtained generally by PCR amplification, recombinant methods, or synthetic methods. For PCR amplification, primers can be designed according to the nucleotide sequences disclosed in the present invention, and the related sequences can be obtained by amplification. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
It is to be understood that the TRIM21 molecules herein are preferably obtained from humans, and that other TRIM21 molecules obtained from other animals that are highly homologous (e.g., have a sequence identity of 70% or more, 75% or more, 80% or more, more preferably 85% or more, such as 85%, 90%, 95%, 98% or even 99% or more) to human TRIM21 are also within the scope of the preferred equivalents of the present invention. Methods and tools for aligning sequence identity are also well known in the art, such as BLAST.
TRIM21 promoter
The invention also relates to an "accelerator" of TRIM 21. The term "accelerator" refers to a substance that can increase the level and/or activity of TRIM21 or delay its degradation. Accelerators useful in the present invention include, but are not limited to: the over-expression vector of TRIM21, the nanoparticle carrying gene, the viral vector carrying the target gene or vector, the PEG modified protein, the protein microsphere, the liposome wrapping the target gene or protein, and the extracellular vesicle carrying the target gene or protein.
The TRIM21 and the promoter thereof can play a role in promoting the sensitivity and effect of anti-tumor treatment, and can be further used for anti-tumor treatment by improving T cell infiltration, improving tumor cell immune response and the like. In addition, by improving the sensitivity of an anti-tumor drug (such as a tumor immunotherapeutic agent), the TRIM21 and/or the accelerator thereof can be used in combination in tumor treatment, so that the dosage or the frequency of the anti-tumor drug can be reduced, the toxic and side effects can be reduced, and the compliance of patients can be improved.
The promoters of the present application may be obtained and tested for their effectiveness using methods and platforms known in the art.
Medicament or pharmaceutical composition and therapeutic application thereof
The invention also provides a medicament or a pharmaceutical composition, which contains an effective amount of TRIM21 and/or an accelerator thereof and a pharmaceutically or immunologically acceptable carrier. As used herein, the term "active" or "active herein" are used interchangeably to refer to a TRIM21 molecule and/or an enhancer thereof.
As used herein, the term "pharmaceutically/immunologically acceptable" ingredients are substances that are suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., commensurate with a reasonable benefit/risk ratio. As used herein, the term "effective amount" refers to an amount that is functional or active in and acceptable to a human and/or animal.
As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. The term refers to such agent carriers: they are not per se essential active ingredients and are not overly toxic after administration. Suitable vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington pharmaceutical sciences (Remington's Pharmaceutical Sciences, mack Pub.Co., N.J.1991).
The pharmaceutically acceptable carrier in the composition may contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting or emulsifying agents, flavoring agents, pH buffering substances, etc. may also be present in these carriers. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8.
The active substances in the composition account for 0.001 to 99.9 weight percent of the total weight of the composition; preferably 1 to 95wt%, more preferably 5 to 90wt%, and even more preferably 10 to 80wt% of the total weight of the composition. The rest is pharmaceutically acceptable carrier and other additives.
As used herein, the term "unit dosage form" refers to a dosage form that is required to prepare a composition of the present invention for administration in a single administration, including but not limited to various solid (e.g., tablet), liquid, capsule, sustained release formulations.
In some embodiments herein, the composition is in unit dosage form or multiple dosage form. As used herein, the term "unit dosage form" refers to a dosage form that is prepared from the products of the present disclosure into a dosage form required for a single administration for ease of administration, including, but not limited to, various solid agents (e.g., tablets), liquid agents, capsules, sustained release agents. In some embodiments herein, the compositions herein are administered at an appropriate frequency, e.g., daily, every other day, weekly, every other or two weeks, monthly, every other month, or two months, e.g., 1 to 6 doses.
It will be appreciated that the effective dose of the active agent used may vary with the severity of the subject to be administered or treated. The specific conditions are determined according to the individual condition of the subject (e.g., the subject's weight, age, physical condition, effect to be achieved), which is within the scope of judgment of a skilled physician.
The composition of the invention can be solid (such as granules, tablets, freeze-dried powder, suppositories, capsules, sublingual tablets) or liquid (such as oral liquid) or other suitable shapes. The administration route can be as follows: (1) direct naked DNA/RNA injection; (2) Ligating the TRIM21 molecule-related cDNA, mRNA with the transferrin/poly L-lysine complex to enhance its biological effect; (3) cDNA, mRNA and proteins form complexes with positively charged lipids to overcome the difficulties of crossing cell membranes due to negative charge of the phosphate backbone; (4) The liposome is used for wrapping cDNA, mRNA and protein and then mediating to enter cells, which is beneficial to the smooth entry of macromolecules and is free from the hydrolysis of various extracellular enzymes; (5) Binding of cDNA, mRNA and protein to cholesterol increases its cytoplasmic retention time by a factor of 10; (6) The immunoliposome is used for transporting cDNA, mRNA and protein, so that the cDNA, mRNA and protein can be specifically transported to target tissues and target cells; (7) The cDNA, mRNA and protein are transfected into the transfer cell (such as fibroblast) in an external way, so that the related medicine can be loaded into the target cell well; (8) Electroporation (electric corporation), i.e., the introduction of RNA, cDNA, mRNA, etc., into target cells by means of electric current.
In some embodiments, the invention also provides an anti-tumor method comprising administering to a subject in need thereof a therapeutically effective amount of TRIM21 and/or an enhancer thereof. Preferably, other anti-tumor agents, especially anti-tumor immunotherapeutic agents, are administered before, during or after the administration of TRIM21 and/or its accelerator(s) of the present application.
Examples
The present application is further described below with reference to specific embodiments and figures. It should be understood that these examples are illustrative only of the present application and are not intended to limit the scope of the present application. Appropriate modifications and variations of the invention may be made by those skilled in the art, and are within the scope of the invention.
The reagents and starting materials used in the present invention are commercially available or may be prepared by literature procedures. The following examples are not drawn to specific conditions, and are generally performed according to conventional conditions such as Michael R.Green et al, fourth edition, new York, cold spring harbor laboratory Press, molecular cloning: the conditions described in the laboratory guidelines (New York: cold Spring Harbor Laboratory Press, 2017) are either conventional or recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
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, any methods and materials similar or equivalent to those described herein can be used in the present application. The preferred methods and materials described herein are presented for illustrative purposes only.
Example 1: preparation of TRIM21 detection kit
A TRIM21 detection kit is prepared which is suitable for detecting the expression of TRIM21 in a biological sample by a real-time quantitative reverse transcription PCR (qRT-PCR) method, according to the following composition:
(a) A container (one of the products of Takara Corp., cat# RR 037) containing a reverse transcriptase and an RNase inhibitor;
(b) Is filled with reverse transcription 5 Xbuffer (75mM KCl,500mM Tris-Cl, pH 8.3, 25 ℃,3mM MgCl) 2 10mM DTT);
(c) A vessel containing a target gene reverse transcription primer (10. Mu.M);
TRIM21 reverse transcription primer:
Oligo(dT)：5'-TTTTTTTTTTTTTTTTTT-3'(SEQ ID NO:7)；
(d) Vessel containing the primers upstream and downstream of the gene of interest PCR (10. Mu.M each):
quantitative PCR primers:
5'-TGCCTGGACCCCTTCGT-3' (upstream, SEQ ID NO: 1); and
5'-GCCTCCTGGCTGATTTCTTTA-3' (downstream, SEQ ID NO: 2);
(e) A vessel containing internal reference reverse transcription primer and PCR upstream and downstream primers (10. Mu.M):
reverse transcription reaction primer of internal reference beta-actin:
Oligo(dT)：5'-TTTTTTTTTTTTTTTTTT-3'(SEQ ID NO:7)；
Quantitative PCR primers:
5'-CCATCGTCCACCGCAAAT-3' (upstream, SEQ ID NO: 3); and
5'-GCTGTCACCTTCACCGTTCC-3' (downstream, SEQ ID NO: 4);
(f) A container (Takara Co., ltd., product No. RR 430) containing dNTP, fluorescent dye TB Green and 2 XPCR buffer; and
(g) Instructions for use.
Example 2: relation between TRIM21 expression level and melanoma
The expression of TRIM21mRNA levels in tumor tissues and paracancerous tissues of 88 melanoma patients collected from the long-sea hospital in 2016 to 2020 were analyzed using the detection kit of example 1 using a real-time quantitative reverse transcription PCR (qRT-PCR) method.
Tissue total RNA was extracted using TRIzol (Invitrogen). qRT-PCR was performed on a lightcycler1.5 (Roche) real-time quantitative PCR instrument using the test kit of example 1.
Relative quantitative use of TRIM21 2 -ΔΔCt Calculations (beta-actin is an internal reference) (Livak, KJ. et al, "Analysis of relative gene expression data using real-time quantitative PCR and the 2- ΔΔCt method". Methods.2001; 25:402-408).
The qRT-PCR analysis found that TRIM21 expression was very significantly reduced in tumor tissue compared to paracancerous non-tumor tissue (FIG. 1, p < 0.01).
Example 3: comparison of melanoma cell tumor volumes after TRIM21 knockout
According to the literature (Genome engineering using the CRISPR-Cas9 system. Ran FA, hsu PD, wright J, agarwala V, scott DA, zhang F.Nat Protoc.2013Nov;8 (11): 2281-308.), TRIM21 expression of melanoma cells B16 was knocked out using CRISPR-Cas9 gene editing technique to obtain Trim21 -/- Cells and control cells without Trim21 knockdown. The specific method comprises the following steps:
trim21 gRNA upstream and downstream sequences were mixed and inserted into PX458 (from Addgene, plasmid # 48138) as Trim21 gRNA vector, and B16 cells were transfected with JetPEI transfection reagent (from Polyplus Co., ltd., product No. 101000053) at a final transfection concentration of 1. Mu.g/ml.
Trim21 gRNA upstream primer:
5'-caccGCATTGACACCCAGAAGTCG-3'(SEQ ID NO:5)
trim21 gRNA downstream primer:
5'-aaacCGACTTCTGGGTGTCAATGC-3'(SEQ ID NO:6)
after transfection, single cells were sorted to the well plate by a Cell flow Sorter (Sony SH800 Cell Sorter), and after 3 weeks, monoclonal cells were collected and protein was extracted for Western Blot detection.
Western Blot was performed according to literature (Tao Y et al, UFL1 promotes antiviral immune response by maintaining STING stability independent of UFMylation. Cell Death Differ (2022): https:// doi. Org/10.1038/s 41418-022-01041-9), and TRIM21 antibody (available from CST company under the trade designation # 92043) was diluted 1:1000. Selection of TRIM21 successful knockout cell clone as TRIM21 -/- Cells, cells not knocked out TRIM21 as TRIM21 +/+ Control cells (fig. 2).
Rag2 is a recombinase necessary for the development process of T cells and B cells, and the T cells and the B cells of the mice cannot develop and mature after the deletion, so that an acquired immunodeficiency model is formed. Equal amount of 2×10 5 Trim21 -/- The cells and the control B16 cells were inoculated to Rag2, respectively -/- Mice (purchased from Jackson Lab, accession number 008449) and immunized female C57BL/6 mice (purchased from Shanghai Bikea)Ste), tumor volume was measured subcutaneously at regular intervals, and the two tumor growth rates were compared. Tumor volumes were calculated as per literature (Qi, r. Et al, "Notch1 signaling inhibits growth of human hepatocellular carcinoma through induction of cell cycle arrest and apoptosis", cancer res.2003; 63:8323-8329), specific formulas: tumor volume= (length x width 2 )/2。
The results showed that there was no difference in the size of the two tumor volumes in immunodeficient mice (FIG. 3, left panel), indicating Trim21 -/- There was no significant difference in growth between the cells and the control B16 cells themselves. In immunized normal C57BL/6 mice, trim21 -/- The tumor cells grew significantly faster than the control cells (FIG. 3, right panel), indicating that the presence of the acquired immune system inhibited Trim21 +/+ Tumor growth, thus showing Trim21 -/- Is faster.
The above results suggest that the acquired immune system is more likely to kill Trim21 +/+ Tumor cells, but against Trim21 -/- The tumor killing ability is weak.
Example 4: relation between TRIM21 expression level and T cell infiltration of melanoma
Subcutaneous tumors of C57BL/6 mice in example 3 were stained for immunofluorescence after tumor tissue was harvested on day 12 and fixed.
Tissue immunofluorescence method: taking mouse tumor tissue, embedding the mouse tumor tissue in a plastic small box by using OCT glue, and freezing and preserving the mouse tumor tissue at-80 ℃. Frozen microtomes 6 micron sections were left at room temperature for 20 minutes. Serum from primary species was selected for blocking for 1 hour at room temperature. The primary antibody was incubated for 12 hours. PBS was washed 3 times. Secondary antibody Anti-Rabbit IgG H & L (AF 594) (from Abcam, cat. No. ab 150080) was incubated for 2 hours at room temperature. PBS was washed 3 times. DAPI staining for 15 min. PBS was washed 3 times. Cover glass was covered and observed under a laser confocal microscope. The primary antibody used is described below.
Research on T cell infiltration in tumor tissue by immunofluorescence method, and labeling tumor tissue sections with CD3, CD4 and CD8 (CD 3 antibody, catalog No. ab5690; CD4 antibody, catalog No. ab183685; CD8 antibody, catalog No. a) b217344, all available from abcam, usa), T cells were observed in Trim21 +/+ (i.e., control) and Trim21 -/- Tumor tissue infiltration; the lighter (corresponding to red fluorescence) is CD3/CD4/CD8 and the darker (corresponding to blue DAPI fluorescence) is stained nuclei.
The results show that: trim21 +/+ Tumor tissue local is no matter total CD3 + T cells, or CD4 + T cells and CD8 + T cells are all significantly more than Trim21 -/- Tumor tissue (fig. 4), suggesting that Trim21 expression promotes T cell infiltration.
The above results suggest that in Trim21 -/- In tumor cells, T cells are difficult to infiltrate, forming a microenvironment resembling a "cold" tumor; trim21 expression promotes T cell infiltration, which converts "cold" tumors to "hot" tumors.
Example 5: relation of TRIM21 expression to PD-1 antibody immunotherapy
5.1 relation of TRIM21 expression in melanoma cell B16-derived tumors with PD-1 immunotherapy
Trim21 in example 3 +/+ And Trim21 -/- Melanoma cells were individually placed in 100. Mu.l PBS and inoculated subcutaneously (2X 10) in immunized normal C57BL/6 mice 5 Starting on day 10, 100 μg of PD-L1 antibody (PBS in 100 μl of final volume) was administered for intraperitoneal injection immunotherapy (inoviumab anti-mouse PD-L1, bioXcell, clone No. 10f9g2), control group was administered with an equivalent amount of homologous IgG (InVivoMAb rat IgG2b isotype control, bioXcell, BE 0090) once for 3 days, and tumor size was detected until the end of the experiment.
As a result, it was found that in Trim21 -/- Among tumors, PD-L1 immunotherapy can slow down tumor growth slightly, but is not statistically significant; and in Trim21 +/+ Tumor, PD-L1 immunotherapy was able to inhibit tumor growth very significantly (fig. 5A).
The results suggest that the immunotherapy of PD-L1 and the like is performed on Trim21 +/+ The tumor is more responsive, and exerts larger anti-tumor effect, but in Trim21 -/- In cold tumor, soundThe response is weaker.
Relation of TRIM21 expression in 5.2YUMM1.7 melanoma cells with PD-1 immunotherapy
A YUMM1.7 cell line that overexpresses TRIM21 was constructed using a YUMM1.7 melanoma cell line (available from Sier, inc., cat# M5-0701) and a control cell line. The specific method comprises the following steps:
the TRIM21 gene sequence was inserted into the pCDH-CMV-MCS-EF1-copGFP vector (available from vast, inc., cat# P0376) according to the method described in literature (Liu, X., chen, L., fan, Y.et al., IFITM3 promotes bone metastasis of prostate cancer cells by mediating activation of the TGF-. Beta.signaling pathway.cell Death Dis 10,517 (2019): https:// doi.org/10.1038/s 41419-019-1750-7), and after packaging as a lentivirus, YUMM1.7 cells were infected, and control cells were infected with lentivirus containing no load. The flow cytometer separately sorts CopGFP positive cells as Trim21 over-expressing cells and Ctr control cells.
Equal amounts of these two melanoma cells were inoculated subcutaneously (2X 10) in immunized normal C57BL/6 mice with 100. Mu.l volume PBS 5 Starting on day 10, 100 μg PD-L1 antibody (formulated in PBS, 100 μl final volume) was given intraperitoneally for immunotherapy (inovivomab anti-mouse PD-L1, bioXcell, clone No. 10f.9g2), control group was given an equivalent amount of homologous IgG (InVivoMAb rat IgG2b isotype control, bioXcell, BE 0090) and once for 3 days, tumor size was monitored until the end of the experiment.
As a result, it was found that PD-L1 immunotherapy was able to slightly slow down tumor growth in tumors of the control group, but was not statistically significant; whereas in Trim21 overexpressed tumors, PD-L1 immunotherapy was able to significantly inhibit tumor growth (fig. 5B).
The results indicate that the immunotherapy such as PD-L1 is easier to respond in Trim21 high-expression tumors, exerts larger anti-tumor effects, and has weaker responsiveness in Trim21 low-expression 'cold' tumors. By increasing the Trim21 expression level in tumor cells, for example, by using Trim21, its expression precursors, biological or chemical substances that promote Trim21 expression by tumor cells, which are targeted for delivery to tumors, the response of tumor-derived immunity can be increased, thereby achieving a more efficient treatment of tumors.
Example 6: relation between TRIM21 expression level and survival curve of melanoma patient
The tumor tissues of 88 melanoma patients collected from the Hospital in the sea were subjected to a real-time quantitative PCR method, and the expression level of TRIM21 in the tumor tissues was measured according to the kit method in example 1, and divided into two groups, namely, high and low, according to the expression median of TRIM21, 44 individuals each. No significant differences were seen between the age, sex, clinical stage, tumor site, and immunotherapy for the two groups of patients (table 1):
TABLE 1 analysis of TRIM21 expression in melanoma tissues in relation to patient clinical factors
The correlation of TRIM21 expression with patient survival was analyzed and the P-value was calculated using log-rank test, the results are shown in FIG. 6.
The result shows that: lower expression of TRIM21 was significantly correlated with lower overall survival of the patient (fig. 6).
Example 7: relation between TRIM21 expression level and PD-1 antibody immunotherapeutic effect
Tumor tissue samples of patients with advanced melanoma, which received immune checkpoint inhibitor anti-PD-1 antibody therapy (Pembrolizumab, treatment dose of 2mg/kg Q3W), were collected, the relative expression levels of TRIM21 in tumor tissue were detected using the kit of example 1, and the correlation between TRIM21 expression and patient survival was analyzed according to the median high and low expression groups, and the P value was calculated using log-rank test.
The result shows that: high expression of TRIM21 was significantly correlated with longer overall survival of patients after PD-1 immunotherapy (fig. 7).
The above results indicate that: the expression of TRIM21 is obviously reduced in the tumor tissue of a melanoma patient, and the high expression of TRIM21 can promote T cells to infiltrate the tumor tissue and strengthen tumor immunity, so that the growth of the tumor is inhibited, and the immunotherapy such as PD-1 can exert better curative effect. Clinically, low TRIM21 expression is significantly associated with poor prognosis in patients, and TRIM 21-low expressing patients are more refractory to PD-1 antibody immunotherapy. The result suggests that there is a close correlation between TRIM21 and the occurrence and development of melanoma, and TRIM21 or its promoter can improve the responsiveness and efficacy of antibody immunotherapy such as PD-1, PD-L1, etc. by promoting T cell infiltration.
While the preferred embodiments of the present invention have been illustrated and described, the present invention is not limited to the embodiments, and various equivalent modifications and substitutions can be made by one skilled in the art without departing from the spirit of the present invention, and these equivalent modifications and substitutions are intended to be included in the scope of the present invention as defined in the appended claims.
Sequence information
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Claims (12)
1. Use of the tri-motif E3 ubiquitin ligase 21 (TRIM 21) and/or a promoter thereof for the manufacture of an adaptive immunopotentiator for the treatment of melanoma in a subject, the TRIM21 promoter increasing the level and/or activity or delaying the degradation of TRIM21, wherein the TRIM21 promoter is selected from the group consisting of: an over-expression vector of TRIM21, a nanoparticle carrying TRIM21 gene, a viral vector carrying TRIM21 gene, a PEG modified TRIM21 protein, a TRIM21 protein microsphere, a liposome-encapsulated TRIM21 gene or protein, and a TRIM21 gene or protein carried by extracellular vesicles.
2. The use according to claim 1, wherein the TRIM21 is a substance selected from the group consisting of:Trim21a gene, a Trim21 mRNA, cDNA, TRIM protein, or an active fragment of any of the foregoing.
3. The use of claim 1, wherein the subject has a poor or in need of improvement of an immunotherapeutic response.
4. The use of claim 1, wherein the subject is selected from the group consisting of: a tumor patient with acquired immunodeficiency or injury; tumor patients who are not effective or have poor effects or expected to have poor effects in tumor immunotherapy; a patient undergoing, to be undergoing, or having undergone tumor immunotherapy; or a tumor patient having two or more of the foregoing.
5. The use of claim 1, wherein the subject is a mammal.
6. The use of claim 1, wherein the enhancer is contained in a pharmaceutical composition or kit.
7. The use according to claim 6, wherein the pharmaceutical composition is adapted to be administered by a means selected from the group consisting of: oral, injectable, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, transdermal.
8. The use according to claim 6, wherein the pharmaceutical composition or kit further comprises or is used in combination with other anti-tumor active ingredients.
9. The use according to claim 8, wherein the other anti-tumor active ingredient is selected from the group consisting of: tumor immunotherapeutic agents or chemotherapeutic agents.
10. The use according to claim 8, wherein the other anti-tumor active ingredient is selected from the group consisting of: ipilimumab or nivolumab.
11. The use of claim 9, wherein the tumor immunotherapeutic agent is selected from the group consisting of: PD-1 antibodies, PD-L1 antibodies, adoptive cell therapeutics, and antigen chimeric receptor T cell therapeutics.
12. The use of any one of claims 1-11, wherein the acquired immune enhancer is a tumor immunotherapy enhancer.
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|---|---|---|---|---|
| CN111337678A (en) * | 2020-02-21 | 2020-06-26 | 杭州凯保罗生物科技有限公司 | Biomarker related to tumor immunotherapy effect and application thereof |
| CN111569065A (en) * | 2020-05-18 | 2020-08-25 | 华东理工大学 | Composition for degrading endogenous Survivin of cells through targeted ubiquitination of nano antibody and application of composition |
| CN112870359A (en) * | 2021-01-22 | 2021-06-01 | 苏州系统医学研究所 | TRIM41 inhibitor, anti-inflammatory effect and application thereof |
| CN115028738A (en) * | 2022-06-14 | 2022-09-09 | 天津医科大学 | Fc fusion protein dual-targeting degradation agent and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111337678A (en) * | 2020-02-21 | 2020-06-26 | 杭州凯保罗生物科技有限公司 | Biomarker related to tumor immunotherapy effect and application thereof |
| CN111569065A (en) * | 2020-05-18 | 2020-08-25 | 华东理工大学 | Composition for degrading endogenous Survivin of cells through targeted ubiquitination of nano antibody and application of composition |
| CN112870359A (en) * | 2021-01-22 | 2021-06-01 | 苏州系统医学研究所 | TRIM41 inhibitor, anti-inflammatory effect and application thereof |
| CN115028738A (en) * | 2022-06-14 | 2022-09-09 | 天津医科大学 | Fc fusion protein dual-targeting degradation agent and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| Jiangyan Chen et al..TRIM14 regulates melanoma malignancy via PTEN/PI3K/AKT and STAT3 pathways.《AGING》.2021,第13卷(第9期),13225-13238. * |
| L. Yang et al..Upregulated E3 ligase tripartite motif-containing protein 21 in psoriatic epidermis ubiquitylates nuclear factor-kB p65 subunit and promotes inflammation in keratinocytes.《British Journal of Der matolog y》.2020,1-12. * |
| 李玉琴等.TＲIM 家族蛋白质在自噬调控中的作用.《中国生物化学与分子生物学报》.2021,第37卷(第10期),1305-1313. * |
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