CN116554356A - Fusion protein of hyper IL-15, sCD4 and Fc and application thereof - Google Patents

Fusion protein of hyper IL-15, sCD4 and Fc and application thereof Download PDF

Info

Publication number
CN116554356A
CN116554356A CN202310547760.3A CN202310547760A CN116554356A CN 116554356 A CN116554356 A CN 116554356A CN 202310547760 A CN202310547760 A CN 202310547760A CN 116554356 A CN116554356 A CN 116554356A
Authority
CN
China
Prior art keywords
cells
hiv
fusion protein
scd4
hyperil
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202310547760.3A
Other languages
Chinese (zh)
Other versions
CN116554356B (en
Inventor
程亮
罗飞
王丽
侯炜
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wuhan University WHU
Original Assignee
Wuhan University WHU
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wuhan University WHU filed Critical Wuhan University WHU
Priority to CN202310547760.3A priority Critical patent/CN116554356B/en
Publication of CN116554356A publication Critical patent/CN116554356A/en
Application granted granted Critical
Publication of CN116554356B publication Critical patent/CN116554356B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2086—IL-13 to IL-16
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70514—CD4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a fusion protein of hyper IL-15, sCD4 and Fc and application thereof, and the fusion protein has the application of treating HIV or preparing medicines for treating HIV. The hyper IL-15 of the fusion protein is formed by fusing IL-15 and IL15-Rα sushi structural domain, sCD4 isThe extracellular region D1D2 domain of CD4 molecule and Fc is the Fc region of immunoglobulin. The fusion protein has the function of activating HIV latent virus library, and targets the virus library cells through sCD 4; the fusion protein can enhance HIV-specific CD8 + Effector functions of T cells and NK cells, thereby mediating killing and clearance of viral pool cells; the fusion protein can neutralize HIV free virus and further block new infection, so that the effect of treating AIDS is maximized.

Description

Fusion protein of hyper IL-15, sCD4 and Fc and application thereof
Technical Field
The invention belongs to the technical fields of genetic engineering and biomedical medical treatment, and particularly relates to fusion proteins of hyper IL-15, sCD4 and Fc, a pharmaceutical composition and a kit containing the fusion proteins, and application of the fusion proteins in preventing and treating HIV infection.
Background
HIV infection can cause CD4 in the body + In addition to the reduction of T, it also induces chronic inflammation and immune exhaustion, such as CD8 + T cell function depletion. In addition, HIV can form a latent infection in patients, resulting in the long-term presence of a pool of latent viruses. The existing clinical antiretroviral therapy (HAART or ART) can effectively inhibit HIV replication, and greatly promote the prevention and treatment effects of AIDS. However, HAART therapy has the following limitations: 1) HAART cannot thoroughly remove HIV virus in patients, and after stopping taking medicine, the virus can rebound rapidly, so that patients need to take medicine for life; 2) HAART treatment does not restore the patient's immune system to a completely normal state, and chronic inflammation and immune damage caused by HIV infection persist in a portion of the patient. Therefore, development of a novel therapeutic strategy is needed to cure AIDS.
How to efficiently activate the antiviral immunity of the organism and clear the virus library in the HIV patient is the key for curing HIV. Current studies indicate that the "activate" and "clear" regimen is a potential means of achieving functional cure for HIV. The protocol first uses a viral pool activating reagent to "activate" the latently infected cells, re-express HIV antigen, and promote exposure of the infected cells. By enhancing killing CD8 on the basis of "activating" the viral pool + T cell or NK cell function mediates clearance of HIV viral pool in hopes of achieving HIV functional cure. The development of drugs with dual effects (both "activation" and "clearance") has therefore become an important point of research.
Cytokine IL-15 vs CD8 + The function and dynamic balance of T cells and NK cells (natural killer cells) play a vital role. Animal experiment results show that IL-15 can enhance CD8 in vivo + T cells and NK cells have effector functions and have anti-tumor activity. More importantly, IL-15 does not cause obvious toxic and side effects in vivo, such as the death of T cell activation induced cells, the expansion of Treg cells and the like. However, because of the short in vivo half-life of IL-15, and because of its function, it often requires limitations such as cross presentation of IL-15Rα on antigen presenting cells, limiting its use. The inventor earlier work fuses IL-15 and IL-15 Ralpha chain to form hyper IL-15 (super IL 15), which can effectively prolong the half life of the super IL-15 in vivo and greatly enhance the activation of CD8 + T cell and NK cell function (Cheng, L., et al, hyper-IL-15suppresses metastatic and autochthonous liver cancer by promoting tumour-specific CD8+T cell responses. Journal of hepatology 61,1297-1303 (2014)). Studies show that hyper IL-15 can increase tumor specific CD8 in tumor-bearing mice + The number of T cells and the effect function of the T cells are enhanced, so that primary and metastatic liver cancer is effectively cleared. In HIV-infected humanized mice, IL-15 super-agonist (IL-15 binding IL15Rα and Fc fusion protein) treatment inhibited HIV viral replication (Seay, K., et al, in vivo activation of human NK cells by treatment with an interleukin-15superagonist potently inhibits acute in vivo HIV-1infection in humanized mice.Journal of virology 89,6264-6274 (2015)). Using SIV infection rhesus models, researchers have found that IL15 super agonist treatment can activate and drive SIV-specific CD8 + T cells enter B cell follicles, effectively reducing SIV replication in lymph nodes (Ayala, V.I., et al CXCR5-dependent entry of CD T cells into rhesus macaque B-cell follicles achieved through T-cell engineering. Journal of virology 91, e02507-02516 (2017); webb, G.M., et al human IL-15 super-magnet ALT-803direct SIV-specific CD8+T cells into B-cell follicles. Blood additives 2,76-84 (Webb, G.M., et al human IL-15 super-magnet N-803-promotes migration of virus-specific CD8+ T and NK cells to B cell follicles but does not reverse latency in ART-support, SHIV-fed magnets. PLoS pages 16, e1008339 (2020)). IL15 superagonists have, in addition to enhancing CD8 + The T cells and NK cells have the function of activating HIV latent virus library besides the antiviral effect function. Using human primary CD4 + T cell in vitro infection HIV latent infection model and treatment of patient peripheral blood derived cells with HAART, low concentration (1 nM) of IL-15 or IL15 super agonist is effective in activating HIV virus pool. More importantly, IL15 superagonist therapy also activates the HIV viral pool in humanized mice infected with HIV and treated with HAART (Jones, R.B., et al, A subset of latency-reversing agents expose HIV-infected resting CD4+T-cells to recognition by cytotoxic T-lymphocytes. PLoS pathens 12, e1005545 (2016)). In conclusion, the IL15 super-agonist has the dual functions of activating the HIV latent virus library and enhancing the immune response of the organism, and can be used as a potential drug for treating HIV.
The CD4 molecule acts as a receptor for HIV and can bind gp120 on the HIV envelope protein. The CD4 molecule consists of four domains, where D1D2 is the extracellular domain and D3D4 is the intracellular domain. Soluble CD4 molecules (sCD 4) consisting of the extracellular D1D2 domain of the CD4 molecule are able to bind to free HIV virus particles. The sCD4 protein is effective in preventing HIV and SHIV infection in humanized mice and rhesus monkeys. In addition to neutralizing HIV infection function, sCD4 or analogs of CD4 bind gp120 on the surface of infected cells, which can convert the HIV envelope protein from a "blocked" conformation to an "open" conformation, exposing it to more epitopes, promoting binding of endogenous antibodies to the HIV envelope protein to gp120, thereby mediating antibody-dependent cellular cytotoxicity (ADCC), clearing the infected cells.
Enhancing the body's humoral immune response against HIV is also a potential means to promote killing of the viral pool, however, broad spectrum neutralizing antibodies can only be produced in about 2% -5% of HIV patients. The broad-spectrum neutralizing antibody is obtained by separating the part of patients, and then expressed and returned to the patients by using a genetic engineering means, so that the method is a potential method for realizing the blocking of virus replication and the persistent control of viremia. Early studies showed that broad-spectrum neutralizing antibodies have good therapeutic effects in HIV-infected humanized mice and SHIV-infected monkey models. Clinical trials have also shown that the time required for viral rebound is significantly delayed after HIV patients have received broad-spectrum neutralizing antibody 3BNC117 treatment against the HIV envelope protein CD4 binding site. The antibody functional region Fc is required for the broad spectrum neutralizing antibodies to function in vivo, suggesting the importance of antibody-dependent cellular cytotoxicity for their function.
Latent infection of HIV virus and exhaustion of antiviral immune function of human body are main reasons that HIV can not be effectively cleared. How to effectively activate HIV latency library and simultaneously target and clear latency library cells is a difficult point of treating AIDS and is a problem to be solved in the field at present.
Disclosure of Invention
Although IL-15 super-agonists can activate HIV viral libraries, promote NK and CD8+ T cell function in HIV therapy, and inhibit HIV replication in humanized mice. However, in the course of application, the IL-15 superagonists, on the one hand, activate HIV viral libraries leading to new infections and the generation of new viral libraries; on the other hand, is unable to target exposed HIV + Cells, targeted to kill newly activated viral pool cells.
In order to overcome the above-mentioned shortcomings and drawbacks, the present invention aims to provide a fusion protein of HyperIL-15 with sCD4 and Fc and its application, which can neutralize free virus, target latent library cells and promote HIV-specific CD8 + T cells function to better inhibit HIV replication. The hyper IL-15 XSCD 4-Fc fusion protein of the invention can neutralize free viruses and activate NK and virus specific CD8 simultaneously with activating HIV virus library + T, targeted killing of virus pool activated cells. The hyper IL-15 xsCD 4-Fc fusion protein can be used as a new generation HIV immunotherapeutic medicine.
The aim of the invention is achieved by the following technical scheme:
the invention provides a fusion protein, which comprises three functional elements of cytokines hyper IL-15, sCD4 and an immunoglobulin Fc region (Fc), or comprises two functional elements of hyper IL-15 and sCD 4. The functional elements can be directly connected or connected through a linker.
The hyper IL-15 is formed by fusing IL-15 and IL-15-Rα sushi structural domain (SEQ ID NO. 13), or combining IL-15 and IL-15Rα (SEQ ID NO. 15). The IL-15 in the hyper IL-15 can be selected from human IL-15 (SEQ ID NO. 11) and human IL-15 mutant (N72D) (SEQ ID NO. 17). Preferably, a fusion protein of IL-15 (SEQ ID NO. 11) of human origin and an IL15-Rα sushi domain is used as the hyper IL-15 (SEQ ID NO. 9).
The sCD4 consists of the D1D2 domain of the extracellular region of the CD4 of the HIV receptor protein, and the CD4 can be selected from the group consisting of CD4 molecules and mutants of various molecules of CD4, such as CD4 Q40A 、CD4 G38A 、CD4 K46A 、CD4 P48Q 、CD4 E85A Etc. The sCD4 is preferably sCD4 (SEQ ID NO. 21) or sCD4 Q40A (SEQ ID NO.23)。
The immunoglobulin Fc region may be selected from the group consisting of the constant region amino acid sequences of IgG1, igG2, igG3 or IgG4, preferably IgG1 (SEQ ID NO. 19). Wherein, igG1 has stronger capability of inducing ADCC and CDC effects and longer serum half-life, and is the most common antibody subtype of antibody drugs; igG2 and IgG4 have a weak ability to induce ADCC and CDC effects, but have a long serum half-life.
The fusion protein containing three functional elements is heterodimeric hyper IL-15 xsCD 4-Fc, and comprises a first polypeptide and a second polypeptide, wherein the first polypeptide is different from the second polypeptide; the first polypeptide comprises, in order from N-terminus to C-terminus, hyperIL-15, an immunoglobulin Fc region (HyperIL-15-Fc), and the second polypeptide comprises, in order from N-terminus to C-terminus, sCD4, an immunoglobulin Fc region (sCD 4-Fc). The immunoglobulin Fc region comprised by the first polypeptide is derived from an immunoglobulin of the same subtype as the immunoglobulin Fc region comprised by the second polypeptide. Preferably, the amino acid sequence of the first polypeptide is shown as SEQ ID NO.1, and the amino acid sequence of the second polypeptide is shown as SEQ ID NO. 3.
The fusion protein containing two functional elements is a monomer HyperIL-15-sCD4 or sCD4-HyperIL-15, and the connection combination mode of HyperIL-15-sCD4 or sCD4-HyperIL-15, preferably HyperIL-15-sCD4. The amino acid sequence of the super IL-15-sCD4 is preferably shown in SEQ ID NO.5, and the amino acid sequence of the sCD 4-super IL-15 is preferably shown in SEQ ID NO. 7.
The fusion protein of the invention has the following effects: the application of hyperIL15 xscd 4-Fc to human peripheral blood mononuclear cells can enhance CD8 + T and NK Activity and promote CD8 + T secretes effector cytokine function. The application of hyperIL15 xcd 4-Fc to activated HIV latent infected cell lines allows targeting of activated HIV latent infected cells in vitro. The application of hyperIL15 xscd 4-Fc to a mixed system of HIV-infected Tzmb-1 cell line and macrophage cell line can promote ADCC function in the macrophage line. The application of hyperIL15 xcd 4-Fc to free HIV neutralizes the free HIV virus in vitro, blocking its infection of target cells. Application of hyperIL15 xcd 4-Fc to a pool-activated latent infected cell line can promote binding of endogenous antibodies of HIV patients to the surface of infected cells, potentially promoting ADCC function. HyperIL15 XSCD 4-Fc was administered to human Peripheral Blood Mononuclear Cell (PBMCs) models of acute HIV infection in vitro to better inhibit HIV replication and kill p24 in vitro than does HyperIL15-Fc + T cells. Application of hyperIL15 xscd 4-Fc to HAART-treated PBMC can effectively enhance HIV-specific CD8 + T cell functions such as promoting secretion of TNF-a, IL-2 cytokines.
The present invention provides a nucleic acid molecule encoding the fusion protein HyperIL-15×sCD4-Fc, hyperIL-15-sCD4 or sCD4-HyperIL-15 of the present invention.
The nucleotide sequence of the nucleic acid molecule encoding the first polypeptide of the heterodimeric hyper IL-15 XSCD 4-Fc is preferably as shown in SEQ ID NO. 2; the nucleotide sequence of the nucleic acid molecule encoding the second polypeptide is preferably as shown in SEQ ID NO. 4.
The nucleotide sequence of the nucleic acid molecule encoding said monomeric HyperIL-15-sCD4 or sCD4-HyperIL-15 is preferably as shown in SEQ ID NO.6 or 8.
The present invention provides a vector comprising the nucleic acid molecule described above.
The present invention provides a cell comprising the fusion protein hyper IL-15 xsCD 4-Fc of the present invention, a nucleic acid molecule encoding the fusion protein or a vector expressing the fusion protein for use in the production of the fusion protein. The cells are selected from non-human mammalian cells, preferably CHO and HEK293 cells.
The invention provides application of the fusion protein, nucleic acid molecule, vector and/or cell in preparing a medicament for preventing or treating HIV infection; the HIV infection is preferably an HIV acute or chronic infected patient; more preferably HIV patients treated with clinical antiretroviral therapy (HAART).
Terms and definitions
Unless specifically stated otherwise, terms and definitions used in the present application are all meanings commonly used in the art and are known to those skilled in the art.
As used herein, the term "HIV viral pool" refers to cells that are latently infected or replicate at low levels by HIV, which cells integrate the HIV genome, but do not undergo or undergo at low levels the transcription of HIV and synthesis of related proteins, but under certain conditions re-initiate replication of HIV and produce HIV with replication and infection capabilities.
The 'HIV infection' described in the present invention is mainly HIV-1 infection; the virus of HIV-1 may be selected from JR-CSF, T278-50, 242-14, T250-4, HO86.8, DU422.01, X2088.c9, 6322.V4.C1, 6471.V1.C16, 6631.V3.C10, 620445.c1, etc.
The term "application" or "use" as used herein may be used for the purpose of disease prevention or treatment, or may be used for non-therapeutic purposes, such as scientific research.
The invention has the beneficial effects that:
(1) The hyper IL-15 xsCD 4-Fc/hyper IL-15-sCD4 fusion protein provided by the invention shows that the data can obviously activate CD8 + T and NK cells.
(2) The present invention provides a hyper IL-15 xsCD 4-Fc/hyper IL-15-sCD4 fusion protein, and the data show that the fusion protein can be targeted to bind activated HIV latent infection cells.
(3) The hyper IL-15 xsCD 4-Fc fusion protein provided by the invention can be combined with Fc receptor to perform ADCC function.
(4) The hyper IL-15 xsCD 4-Fc fusion protein provided by the invention, data show that free HIV virus can be effectively neutralized, and new infection can be blocked.
(5) The hyper IL-15 xsCD 4-Fc fusion protein provided by the invention, and the data show that the binding of endogenous antibodies to HIV can be effectively promoted + The cell surface has a function of potentially promoting ADCC.
(6) The hyper IL-15 xsCD 4-Fc fusion protein provided by the invention has the data that compared with hyper IL-15-Fc, the hyper IL-15-Fc can effectively inhibit HIV replication in vitro and better kill p24 + T cells.
(7) The hyper IL-15 xsCD 4-Fc fusion protein provided by the invention, and data show that the HIV specific CD8 of a patient treated by HAART can be effectively promoted + T cell function, promotes secretion of various cytokines.
(8) The hyper IL-15 xsCD 4-Fc fusion protein provided by the invention shows that the data can effectively activate the HAART source CD4 + HIV viral pool of cells.
Drawings
Fig. 1: schematic representation of HyperIL-15-sCD4/HyperIL-15 xsCD 4-Fc fusion protein and results of protein purification. (A) schematic structural diagram of HyperIL-15-sCD4 fusion protein. (B) schematic structural diagram of HyperIL-15×sCD4-Fc fusion protein. (C) SDS-PAGE identifies the purified hyper IL-15-sCD4 fusion protein. (D) SDS-PAGE identifies the purified hyper IL-15 XSCD 4-Fc fusion protein.
Fig. 2: hyperIL-15-sCD4/HyperIL-15 xsCD 4-Fc fusion protein significantly promotes CD8 + T and NK activation. (A) The fusion proteins HyperIL-15-sCD4 (10 nM) or HyperIL-15 xsCD 4-Fc (10 nM) stimulated PBMCs for 48 hours, and 1 xBFA was added to the culture system during the last 5 hours. (B-C) flow assay of CD8 after 48 hours of PBMC stimulation with HyperIL-15-sCD4 + Activation marker CD69 expression on T (B) and NK cells (C). (D-E) flow assay for CD8 after 48 hours of PBMC stimulation with HyperIL-15-Fc and HyperIL-15×sCD4-Fc + Activation marker CD69 expression on T (D) and NK cells (E). (F) Statistics CD69 + NK cell surface CD69 expression. (G) Streaming identification of CD8 + T cells secrete IFN- γ ability.
Fig. 3: efficient HIV targeting of HyperIL-15-sCD4/HyperIL-15 xsCD 4-Fc fusion proteins + And (3) cells. (A) Experimental procedure for in vitro detection of binding of hyperIL-15-sCD4/hyperIL-15×scd4-Fc to PMA stimulated ACH2 cells: ACH2 cells were stimulated with 10ng/mL PMA for 48h, incubated with fusion protein for 1h, stained with a-hIgG-APC antibody for 0.5h, and finally flow-on-machine detected. (B) q-PCR detects HIV gene transcription after ACH2 cells are activated by PMA. (C) Binding of HyperIL-15-sCD4 to PMA stimulated ACH2 cells was detected by flow. (D) Binding of HyperIL-15-Fc, hyperIL-15 XSCD 4-Fc to unstimulated or PMA-stimulated ACH2 cells was flow-tested.
Fig. 4: the binding of the HyperIL-15×sCD4-Fc fusion protein to Fc receptors and the mediation of ADCC. (a) fusion protein binding Fc receptor protocol: the mouse macrophage line RAW264.7 or the human monocyte line Thp1 is incubated with the fusion protein for 20min, then incubated with the corresponding fluorescent secondary antibody for 0.5h, and finally the detection is carried out on the machine in a flow mode. (B) Flow assay binding of HyperIL-15×sCD4-Fc to Raw264.7. (C) Flow assay binding of HyperIL-15-sCD4, hyperIL-15×sCD4-Fc to Thp-1 cells. (D) ADCC experimental design: PBMCs were used as Effector cells, and HIV-infected TZM-bl cells were used as Target cells, according to E: t=10:1 or 30:1, and adding the fusion protein, co-culturing for 10 hours, and detecting the luciferase activity of the cells. (E) The level of luciferase activity in the co-culture system was measured to reflect the relative number of TZM-bl cells.
Fig. 5: the hyper IL-15 XSCD 4-Fc fusion protein effectively neutralizes free HIV. Experimental procedure for neutralization of free HIV by fusion proteins: will be 200TCID 50 And (3) incubating the virus and the fusion protein for 1h, then infecting TZM-bl cells by the incubated virus, and carrying out luciferase activity measurement after 44h of infection. (B) HIV-1 virus was co-incubated with various concentrations of HyperIL-15 XSCD 4-Fc at 37℃for 1h before re-infection of TZM-bl cells (1X 10) 4 ) Luciferase activity was detected in TZM-bl cells after 44 hours.
Fig. 6: hyperIL-15×sCD4-Fc fusion proteins promote binding of endogenous antibodies to HIV-infected patients by HIV + Cell surface. (A) Fusion proteins promote binding of endogenous antibodies of a patient to HIV + Cell surface protocol: after PMA stimulation of ACH2 cells for 48 hours, serum from healthy persons (3 samples) or serum from HIV patients (3 samples) was added, followed by PBS, hyperIL-15-Fc or HyperIL-15×sCD4-Fc fusion protein, and ACH2 cells hIgG was flow tested + Cell ratio. (B) a streaming results presentation graph. (C) Statistics of hIgG + Proportion of cells. (D) Statistics of hIgG + MFI of cell surface hIgG.
Fig. 7: hyperIL-15×sCD4-Fc fusion proteins inhibit HIV replication more efficiently than do HyperIL-15-Fc. The experimental design flow of the HIV replication inhibition of fusion protein: PHA blast cells and HIV + PHA blast cells and autologous spleen cells were prepared according to 1:1:10 and adding the same concentration of hyper IL-15-Fc or hyper IL-15 XSCD 4-Fc, carrying out p24 ELISA assay on day 6, and carrying out flow identification on day 7. (B) ELISA detects p24 content in the supernatant of the cells at day 6 of co-culture. (C) Flow assay p24 in cells after 7 days of co-culture + T cell ratio. (D) Streaming detection of p24 + hIgG in T cells + Proportion of cells. (E-F) flow assay after 7 days of co-cultivation, CD8 + Number of T (E) and NK cells (F). (G-H) flow assay Co-cultivation of CD8 after 7 days + T (G) and NK (H) ability to secrete multiple cytokines (Gzmb and IFN-. Gamma.).
Fig. 8: hyperIL-15×sCD4-Fc fusion proteins significantly promote HIV-specific CD8 + T cell function. (A) Fusion proteins promote HIV-specific CD8 + Experimental design flow of T cell function: stimulation of HAART-treated HIV-infected person-derived PBMCs with HyperIL-15×sCD4-Fc for 60 hours followed by addition of HIV gag and pol polypeptide pool, stimulation for 8 hours, flow assay for CD8 + T cells secrete cytokines. Streaming identification of CD8 + T cells secrete TNF-alpha (B) and IL-2 (C) and IFN-gamma (D).
Fig. 9: hyperIL-15×sCD4-Fc fusion protein capable of activating CD4 + HIV viral pool in cells. (A) fusion protein activation HIV viral library experimental design: purification of CD4 from PBMC of HAART treated patient origin + Cells were treated with the HyperIL-15 XSCD 4-Fc fusion protein for 3 days, and viral-associated RNA was quantified in the cells after harvesting the cells. (B) Statistical HyperIL-1Fold increase in viral RNA in 5 XSCD 4-Fc treated versus PBS group cells.
Detailed Description
The present invention will be described in further detail by the following examples, but it should be understood that the present invention is not limited by the following.
The sequences involved in the present invention are shown in Table 1 below:
TABLE 1
The following examples relate to materials and methods:
(1) Cells and reagents:
HEK293T cells were supplied by the professor Wang Li (university of armed university medical institute) and cultured in DMEM medium (Sigma, D6429-500 mL). ACH2 cells were supplied by the professor Zhang Liguo (institute of bioscience of chinese academy of sciences) and cultured in 1640 medium (Sigma, R8758-500 mL). RAW264.7 cells were supplied by the professor Cai Cheguo (university of Wuhan medical institute) and cultured in DMEM medium (Sigma, D6429-500 mL). TZM-bl cells were supplied by Zhang Liguo professor (Proc. Natl. Acad. Biol.Acad. Sci. China) and cultured in DMEM medium (Sigma, D6429-500 mL). HIV patient PBMCs and plasma were provided by the teaching of the south-middle-chinese university hospital Xiong Yong.
(2) Construction of the fusion protein of HyperIL-15 and sCD 4:
construction of HyperIL-15: IL-15-Rα sushi domain and linker SGGSGGGGSGGGSGGGGSLQ are connected to IL-15 to obtain hyper IL-15, the amino acid sequence of which is shown in SEQ ID NO.9, and the encoding nucleotide sequence of which is shown in SEQ ID NO. 10.
Monopolymer (HyperIL-15-sCD 4): the super IL-15 is connected with the sCD4N end through a connector GGGGSGGGGSGGS and then connected with 3 xflag to obtain the super IL-15-sCD4, the amino acid sequence of the super IL-15-sCD4 is shown as SEQ ID NO.5, and the encoding nucleotide sequence is shown as SEQ ID NO. 6.
Heterodimer (HyperIL-15×sCD 4-Fc): the first polypeptide is hyper IL-15 through linker
GGGGSGGGGSGGGGS is connected to hyper IL-15-Fc of IgG1-Fc N end, its amino acid sequence is shown in SEQ ID NO.1, and its coding nucleotide sequence is shown in SEQ ID NO. 2. The second polypeptide is sCD4 through linker
GGGGSGGGGSGGGGS is connected to the N end of IgGFc and then connected with 3×flag to obtain sCD4-Fc, the amino acid sequence of which is shown as SEQ ID NO.3, and the encoding nucleotide sequence of which is shown as SEQ ID NO. 4. The nucleotide sequences encoding the first and second polypeptides were cloned into the pee6.4 vector (Lonza), respectively. heterodimerization of HyperIL-15 and sCD4 was generated using the previously reported knob-to-eyes technique (Ridgway, J.B., presta, L.G. & Carter, P. 'Knobs-into-eyes'
Engineering of antibody CH3, domains for heavy chain, protein Engineering, design and Selection, 617-621 (1996). Plasmid ratio 1:1 into 293T cells. Following transfection, the supernatant was collected at 48h for protein purification.
(3) Flow cytometry:
binding of the fusion protein was detected using APC-anti-human IgG Fc (bioleged, 410711). Specific antibodies: anti-CD 3 antibodies (Biolegend, 300411), anti-CD 56 antibodies (Biolegend, MHCD 5617), anti-CD 8 antibodies (Biolegend, 344712), anti-CD 69 antibodies (Biolegend, 310904), anti-IFN-gamma antibodies (Biolegend, 502524), anti-Flag antibodies (Biolegend, 637309). Cells were suspended in FACS buffer (1% bovine serum albumin), incubated on ice for 10 min with Fc receptor blocking solution (Biolegend, 422302) and on ice for 30 min with the corresponding specific antibodies. Samples were analyzed on Fortessa flowcytometer (BD bioscience). Data was analyzed using FlowJo software (TreeStar).
(4) HyperIL-15-sCD4/HyperIL-15 xsCD 4-Fc stimulated CD8 + T cell and NK cell activity:
in human PBMC (2X 10) 5 ) HyperIL-15-sCD4/HyperIL-15 xsCD 4-Fc protein (10 nM) was added to the culture system. After 24 hours, the cells were collected,streaming CD8 + T(CD3 + CD8 + ) And NK (CD 3) - CD56 + ) Expression of cell surface activating molecule CD69 and intracellular detection of IFN- γ secretion.
(5) hyper IL-15 XSCD 4-Fc neutralization study:
different concentrations of HyperIL-15 XSCD 4-Fc were compared with HIV-1 virus (200 TCID 50 ) After 1 hour of co-incubation at 37℃in a total volume of 150. Mu.L, 10 was added 4 TZM-bl (total volume 100. Mu.L) cells. The cells were lysed by removing the cell supernatant and adding 50. Mu.L of lysate after culturing for 48 hours. The supernatant of the cell lysate was collected, centrifuged at 12000rpm for 5min, 40. Mu.L of the supernatant was taken, 100. Mu.L of the luciferase assay reagent was added, and after mixing with a pipette, measurement was performed by a fluorescent microplate reader.
(6) Super IL-15-sCD 4/super IL-15 xsCD 4-Fc targeting HIV latent infected cells study:
taking 5×10 5 ACH2 was stimulated in 48-well plates with PMA at a final concentration of 10ng/mL for 48h. Control cells and PMA stimulated cells were collected, washed with 1% FPBS, incubated on ice for 1h with 0.1. Mu.g of the HyperIL-15-sCD4/HyperIL-15 xsCD 4-Fc fusion protein, washed with 1% FPBS, incubated on ice for 0.5h with anti-Flag-PE/anti-hIgG-APC secondary antibody, and on-stream.
(7) HyperIL-15×sCD4-Fc promotes endogenous antibody recognition of HIV + Cell study:
ACH cells were stimulated with 10ng/mL PMA for 48h to give activated ACH2 cells. Taking 5×10 4 Activated ACH cells, plasma (1:500) of healthy human or HIV patient was added to each, PBS was added to the system, 5. Mu.M HyperIL-15-Fc or 5. Mu.M HyperIL-15 XSCD 4-Fc was incubated at room temperature for 20min, 500. Mu.L of 1% FPBS was added to wash through, anti-hIgG-APC secondary antibody was added to incubate for 0.5h, and the flow-on machine was started.
(8) HyperIL-15×sCD4-Fc binding studies to FcR:
cell lines RAW264.7 derived from mouse macrophages and Thp1 cells derived from human monocytes, both of which express mouse FcR and human FcR, respectively, were used as subjects.
Taking 5×10 4 RAW264.7 cells, one group of themMouse serum (as Fc block) was added for 10min incubation, PBS or 0.1. Mu.g of HyperIL-15 XSCD 4-Fc was added to the system, incubated at room temperature for 20min, 500. Mu.L of 1% FPBS was added for one wash, anti-hIgG-APC secondary antibody was added for 0.5h incubation on ice, and the system was run on stream.
For Thp1 cells, the experiments were divided into three groups (PBS, hyperIL-15-sCD4, hyperIL-15×sCD 4-Fc). Taking 5×10 4 Thp1 cells were incubated at room temperature for 20min with PBS, hyperIL-15-sCD4 or HyperIL-15 xsCD 4-Fc, washed once with 500. Mu.L of 1% FPBS, incubated on anti-Flag-PE secondary antibody for 0.5h, and on-stream.
(9) HyperIL-15×sCD4-Fc antibody dependent cell-mediated cytotoxicity:
3×10 5 TZM-bl cells were seeded in 12-well plates 12h later with HIV-1 (3X 10) 5 pfu) (total volume 700 μl) of infected cells. After 24h, 1.3mL of DMEM complete medium was added and the culture was continued for 48h. Removing cell culture supernatant, digesting the cells with 5mM EDTA for 5min, transferring the cells into 1.5mL EP tube, and resuspending the cells with complete medium to a cell density of 2X 10 5 cells/mL were used as Target cells. RAW264.7 cells served as Effector cells. 100 mu LTarget cells were seeded in 96-well plates, incubated with 10. Mu.g/mL of the HyperIL-15×sCD4-Fc fusion protein for 1h, and 2×10 5 (E:T＝10:1)、6×10 5 (E: t=30:1) RAW264.7 cells. After 8h incubation, cells were collected for luciferase activity assay. mu.L of the cell lysate was added to 100. Mu.L of the luciferase assay reagent, and the mixture was mixed with a gun and then measured by a fluorescent microplate reader.
(10) In vitro inhibition of HIV replication studies by HyperIL-15×sCD4-Fc fusion protein:
PHA-blast cells were obtained by activating PBMC with PHA-P (4. Mu.g/mL) and IL-2 (100U/mL) for 48 h. 300TCID 50 /10 6 PHA-blast cells/100. Mu.L were statically infected in an incubator for 3h, and washed once with 10mL of complete medium to obtain HIV + PHA-blast cells. 5X 10 of its own origin 6 Cells and 5X 10 5 PHA-blast cells and 5X 10 5 HIV + PHA-blast cells were co-cultured in 24-well plates and PBS, hyperIL-15-Fc (10 nM) or HyperIL-1 was added5 XSCD 4-Fc (10 nM), total medium volume 1 mL/well. After 4 days of culture, half-volume liquid change was performed. After 6 days of culture, cell supernatants were collected for p24 ELISA assays. After 7 days of culture, the cells were collected and analyzed by flow-through staining.
(11) HyperIL-15×sCD4-Fc fusion proteins promote HIV-specific CD8 + T function study:
5X 10 derived from HAART patient treatment 5 PBMC were plated into 96-well plates, 10nM of HyperIL-15 XSCD 4-Fc was added, incubated for 60h, stimulated with HIV gag (2. Mu.g/mL) and pol (2. Mu.g/mL) polypeptide pools for 3h, and incubated for a further 5h with 2 XSFA. Cell collection and flow-through identification of CD8 + T ability to secrete TNF-a, IL 2.
(12) Functional study of the activation of HIV viral pool by the HyperIL-15×sCD4-Fc fusion protein:
purification of CD4 from PBMCs (volunteers negative for plasma disease detection) of HAART-treated patients Using a botin-CD4 antibody + Cells, CD4 obtained + Cells were packed in 4.5X10 cells 6 Cells/well were plated into 12 well plates, 3mL1640 complete medium was added to each well plate, 1. Mu.M HIV integrase inhibitor (Ralterravir) was added to the final concentration, PBS or 10nM HyperIL-15×sCD4-Fc fusion protein was added, and cells were harvested after 3 days of culture. Total cellular RNA was extracted using the RNA extraction kit (TIANGEN, DP 451). RNA was inverted to cDNA using the reverse transcription kit (HiScript II Q RT SuperMix for qPCR, R222-01).
The expression level of CD4 mRNA was detected by primers 5'-GGTGCGAGAGCGTCAGTATTAAG-3' and 5'-AGCTCCCTGCTTGCCCATA-3'; the expression level of HIV gag was detected by primers 5'-GGTGCGAGAGCGTCAGTATTAAG-3' and 5'-AGCTCCCTGCTTGCCCATA-3' and probe primer FAM-AAAATTCGGTTAAGGCCAGGGGGAAAGAA-QSY7 (TAMRA), and finally the expression level of HIV RNA was normalized according to the expression level of CD4 mRNA.
(13) Statistical analysis:
the data are shown as average. Statistical analysis was compared using unpaired Student's two-tailed t-test. Analysis was performed using GraphPad Prism version 9.0 (GraphPad Software). Statistically significant differences of p <0.05, p <0.0l, and p <0.001 are expressed by x, and x, respectively.
EXAMPLE 1HyperIL-15-sCD4/HyperIL-15 XSCD 4-Fc fusion protein design and expression purification
The existing high-efficiency antiretroviral therapy developed clinically can inhibit HIV replication, but cannot clear the virus latent bank, and patients need to take the drug for life. How to target and clear the cells of the latent library while activating the HIV latent library is a difficult point for treating AIDS. Cytokine IL-15 has the potential to activate HIV latency repertoire and enhance CD8 + T cell killing function. The extracellular region sCD4 of the HIV receptor molecule CD4 can be combined with HIV envelope proteins, and can play a role in targeting and neutralizing HIV viruses. Based on this we designed the fusion protein HyperIL-15-sCD4. The immunoglobulin Fc region is a necessary structure for the development of antibody-dependent cell-mediated cytotoxicity (ADCC) and can effectively prolong the half-life of fusion proteins. We therefore fused the Fc region to the HyperIL-15-sCD4 end to further enhance the function of HyperIL-15-sCD4. The invention designs a single-polymer fusion protein composed of two functional elements of hyper IL-15 and sCD4 (figure 1A) and a fusion protein heterodimer hyper IL-15 xsCD 4-Fc composed of three functional elements of hyper IL-15, sCD4 and IgG1-Fc (figure 1B). HEK 293T cells were co-transfected with the plasmids and purified using Flag agarose gel beads as in FIGS. 1C-D, high purity HyperIL-15-sCD4 and heterodimeric HyperIL-15 xsCD 4-Fc fusion proteins were obtained. The novel fusion protein drug can exert various therapeutic effects of activating HIV latency library (hyper IL-15), targeting activated latency library cells, neutralizing newly generated free virus (sCD 4), enhancing CD 8T cell killing function (hyper IL-15), mediating antibody dependent cellular cytotoxicity (Fc) and the like.
+ EXAMPLE 2 activation of CD8T cells in vitro by HyperIL-15-sCD4/HyperIL-15×sCD4-Fc fusion protein NK cells
In HIV infection, CD8 + The T cells are continuously exposed to viral antigen stimulation, undergo functional depletion, and have reduced effector function and re-responsiveness. Patient CD8 even with HAART therapy to control viral replication + T still has difficulty in thoroughly recovering its effector function,it is difficult to exert an effective killing effect. Thus recovering or enhancing CD8 + T cell function is critical for the treatment of HIV. Beta receptor of IL-15 in CD8 + High expression on T and NK cells, but IL-15 function often requires IL-15 ra cross-presentation on antigen presenting cells limiting their use. The hyper IL-15-sCD4 constructed by the invention
The fusion protein of the hyper IL-15 xsCD 4-Fc fuses IL-15 and IL-15Rα sushi domain together, overcomes the limitation that the IL-15 needs cross presentation for functioning and further enhances the function of the IL-15.
To determine whether the HyperIL-15-sCD4/HyperIL-15 xsCD 4-Fc fusion protein has the function of HyperIL-15. Human PBMCs were stimulated in vitro with the HyperIL-15-sCD4, hyperIL-15×sCD4-Fc fusion protein (FIG. 2A). The result shows that the hyper IL-15-sCD4/hyper IL-15 xsCD 4-Fc (10 nM) can significantly improve the activation marker protein CD69 in human PBMCs-derived CD8 + T cells and NK cells (FIG. 2B-E). At the same concentration, the super IL-15 XSCD 4-Fc can better promote CD69 compared with the super IL-15-Fc + CD8 + T ratio (fig. 2D) and promotes expression of NK cell surface CD69 molecules (fig. 2F). Meanwhile, hyperIL-15×sCD4-Fc promotes CD8 + T cells secrete the effector cytokine IFN- γ (fig. 2G). The above experiments prove that the super IL-15-sCD 4/super IL-15 xsCD 4-Fc retains the function of super IL-15 for CD8 + T cells and NK cells have good activation.
EXAMPLE 3HyperIL-15-sCD4/HyperIL-15 xsCD 4-Fc targeting activated HIV latent infected cells
HIV-latently infected cells will under specific conditions be activated by transcription of HIV, expressing HIV-associated proteins such as gp160 and the like. Wherein gp160 is processed and matured into gp120 and gp41 proteins in the golgi apparatus, gp120 and gp41 form complexes which are transported to the cell membrane, and thus the activated HIV latent infection cell surface presents HIV envelope proteins. The CD4 molecule acts as a receptor for HIV and its extracellular domain (sCD 4) can bind gp120 on the HIV envelope protein. In the present invention, the sCD4 domain of fusion proteins is used to target HIV + And (3) cells.
This example uses a T cell line ACH2 cell that integrates the HIV genome and mimics HIV latent infection. ACH2 cells were stimulated with PMA to initiate HIV transcription and protein expression, and this system was used to examine the function of HIV-latently infected cells activated by binding to HyperIL-15-sCD4, hyperIL-15 xsCD 4-Fc (FIG. 3A). ACH2 cells stimulated with PMA (10 ng/mL) up-regulated 300-fold HIV-associated gene expression (FIG. 3B). PMA-activated ACH2 was incubated with HyperIL-15-sCD4, and showed about 58% of cells bound to the fusion protein (FIG. 3C). Further experimental investigation revealed that the HyperIL-15-Fc and HyperIL-15 XSCD 4-Fc only weakly bound to unstimulated ACH2 cells (4%). HyperIL-15-Fc bound weakly to activated ACH2 cells (4%), whereas up to 50% of activated ACH2 cells were bound by HyperIL-15×sCD4-Fc (FIG. 3D). The overall results show that hyper IL-15-sCD4/hyper IL-15 xsCD 4-Fc can effectively target activated HIV virus pool cells.
EXAMPLE 4HyperIL-15 XSCD 4-Fc targeted killing of HIV-infected cells
The fusion protein HyperIL-15×sCD4-Fc is added with an immunoglobulin Fc region to lead the immunoglobulin Fc region to mediate the function of ADCC to kill HIV targeted by the fusion protein + And (3) cells.
To verify the ADCC function of the fusion protein, the ability of the HyperIL-15×sCD4-Fc to bind FcR on Thp-1 (human monocyte line) and Raw264.7 (mouse macrophage line) was tested in vitro (FIG. 4A). The results demonstrate that HyperIL-15×sCD4-Fc binds to Raw264.7 in an FcR-dependent manner (FIG. 4B). Further studies showed that, in contrast to HyperIL-15-sCD4, hyperIL-15 xsCD 4-Fc could bind to Thp1 cells, whereas HyperIL-15-sCD4 was unable to bind to Thp1 cells (FIG. 4C), eliminating the possibility of binding other portions of the HyperIL-15 xsCD 4-Fc fusion protein to Thp 1.
In a further experiment, raw264.7 cells were co-cultured with HIV-infected TZM-bl cells, while HyperIL-15×sCD4-Fc protein or PBS control was added, and after 10 hours, the relative numbers of TZMb1 cells were reflected by measuring the level of luciferase activity (FIG. 4D). The result shows that the hyper IL-15×sCD4-Fc fusion protein can promote the Raw264.7 to kill the TZMb1 cells infected by HIV, so that the TZM in a co-culture system b1 decreased cell number (fig. 4E). The result suggests that: hyperIL-15 XSCD 4-Fc binding HIV + After the target cells, it is possible to achieve killing of the target cells by ADCC.
EXAMPLE 5HyperIL-15 XSCD 4-Fc neutralization of free HIV virus
In the treatment of chronic infection with HIV using the "activation" and "clearing" strategies, the activated HIV viral pool cells will produce new free viruses which may lead to new infections. sCD4 in the fusion proteins of the invention is able to neutralize free virus, blocking new infections.
To verify the function of fusion proteins to neutralize HIV, the effect of hyper IL-15×sCD4-Fc protein neutralization of free HIV virus was detected by measuring luciferase activity in TZM-bl cells (a HeLa cell line that had been engineered to highly express CD4, CCR5 and CXCR4 molecules and integrated firefly luciferase under control of the long repeat ends of HIV and E.coli β -galactosidase reporter) after pre-incubation of varying concentrations of hyper IL-15×sCD4-Fc protein with free HIV virus particles (FIG. 5A). As shown in FIG. 5B, 100. Mu.g/mL of the fusion protein can effectively neutralize free virus to TZM-bl cells with a neutralization efficiency of about 80%, and IC is obtained by calculating the fusion protein 50 A concentration of 7.89. Mu.g/mL, i.e., the HyperIL-15 XSCD 4-Fc fusion protein, was effective in blocking half of HIV infection at a concentration of 7.89. Mu.g/mL.
+ EXAMPLE 6HyperIL-15 XSCD 4-Fc promotes endogenous antibody recognition of HIV cells
There are a large number of endogenous antibodies to HIV envelope proteins in HIV patients, which have the effect of recognizing HIV-infected cell surface viral envelope proteins. However, in the normal case the HIV envelope proteins on the surface of the infected cells are in a "closed" conformation, and the internal antigens of the envelope proteins are not exposed, so that many antibodies directed against the HIV envelope proteins cannot recognize the infected cells. Early studies showed that sCD4 molecules bind to the envelope protein on the surface of infected cells, promoting the conversion of the envelope protein from a "closed" conformation to an "excess" conformation, promoting the "opening" of the envelope protein, thereby promoting the binding of endogenous antibody phase to the envelope protein in the patient and mediating ADCC, clearing the infected cells. However, HIV infection results in a significant decrease in target cell CD4 molecule expression, and HIV envelope proteins on the surface of the infected cells tend to be in a "closed" conformation, with some endogenous antibodies not binding, thereby evading ADCC-mediated killing.
To verify that fusion proteins promote endogenous antibody recognition of HIV + Cells, hyperIL-15-Fc, hyperIL-15×scd4-Fc fusion protein were incubated with PMA-activated ACH2 cells and plasma from healthy or HIV patient was added by assaying hIgG + The proportion of cells and the binding strength were used to determine whether the fusion protein HyperIL-15×sCD4-Fc promoted endogenous antibody binding to the cell surface (FIG. 6A). The results show that there are some endogenous antibodies to HIV in the plasma of HIV patients + Background binding of cells, but hardly present in healthy human plasma (fig. 6B). The 5. Mu.M HyperIL-15 XSCD 4-Fc fusion protein promotes the binding of endogenous antibodies of the patient to HIV compared to the control (PBS or 5. Mu.M HyperIL-15-Fc) + Cells, particularly exhibiting a significant increase in hIgG + Cell ratio (FIGS. 6B-C) and binding strength (FIG. 6D). The above results show that the HyperIL-15 XSCD 4-Fc fusion protein has the effect of promoting the binding of endogenous antibodies of patients to HIV + Cells, and enhance ADCC to mediate killing of potential functions of target cells.
EXAMPLE 7HyperIL-15×sCD4-Fc inhibits HIV replication better than HyperIL-15-Fc
Through the research, the novel fusion protein of the HyperIL-15×sCD4-Fc can effectively promote CD8 + Activation of T and NK cells (FIGS. 2B-C) and promotion of CD8 + T cells secrete cytokines (fig. 2D). Compared with the HyperIL-15-Fc, the HyperIL-15×sCD4-Fc has the function of targeted killing of HIV + The effect of the cells (FIG. 3, FIG. 4), neutralising the free HIV virus, blocks the new infection (FIG. 5). These results suggest that the constructed hyper IL-15 XSCD 4-Fc fusion proteins of the present invention may be able to inhibit HIV replication better than hyper IL-15-Fc.
This practice isExamples an in vitro acute infection model was constructed to investigate whether the HyperIL-15 XSCD 4-Fc fusion protein was able to better inhibit HIV replication. As shown in FIG. 7A, HIV-infected PHA-blast cells were combined with autologous spleen immune cells and uninfected PHA-blast cells according to 1:10:1, co-cultivation was performed in the same ratio. After 6 days of co-culture, p24 in the cell supernatants was assayed and both the HyperIL-15-Fc and HyperIL-15×sCD4-Fc were found to have an inhibitory effect on HIV replication, but the effect of HyperIL-15×sCD4-Fc was 2.6 times that of HyperIL-15-Fc (FIG. 7B). After 7 days of incubation, the results showed that both HyperIL-15-Fc and HyperIL-15×sCD4-Fc significantly reduced p24 + T cell ratio (FIG. 7C), promotion of CD8 + Proliferation of T (FIG. 7D) and NK cells (FIG. 7E) and promotion of CD8 + T (fig. 7F) and NK cells (fig. 7G) ability to secrete cytokines. Furthermore, the super IL-15 XSCD 4-Fc is capable of targeting p24 compared to super IL-15-Fc + T cells (fig. 7H). The results in summary show that: hyperIL-15 XSCD 4-Fc is better able to inhibit HIV replication in vitro than does HyperIL-15-Fc.
+ EXAMPLE 8HyperIL-15 XSCD 4-Fc promotes HIV-specific CD8T function
HIV specific CD8 + T cells play an important role in the clearance of HIV-infected cells. However, in HIV-1 chronically infected patients, CD8 is specific for it + T cell function is depleted, its effector function and re-response capacity are reduced, and it cannot be completely restored by HAART treatment. Thus restoring HIV-specific CD8 + T cells are of great importance for the cure of HIV.
To further determine whether the HyperIL-15×sCD4-Fc fusion protein was able to enhance HIV-specific CD8 + T cell function, treatment of HAART with HyperIL-15×sCD4-Fc patient derived PBMCs for 60h followed by stimulation with HIV gag and pol polypeptide pool (FIG. 8A). The results showed that the addition of the super IL-15 XSCD 4-Fc treated with the polypeptide pool stimulated group significantly enhanced the secretion of TNF- α, IL-2 and IFN- γ by CD8+ T cells compared to the PBS treated group. The results in summary show that: the super IL-15×sCD4-Fc fusion protein can significantly enhance the HIV specific CD8 of a patient treated by HAART + T function.
+ EXAMPLE 9 activation of the latent CD4 cell Virus pool by HyperIL-15×sCD4-Fc in vitro
The low level or non-expression of HIV-associated genes in latently infected cells makes it difficult for latently infected cells to be found by immune cells, resulting in failure of the immune system to recognize and clear latently infected cells. Therefore, the activation of HIV virus library activator to activate HIV related gene expression and antigen expression in latent infected cell has important significance in eliminating HIV virus library.
To further determine whether the HyperIL-15 XSCD 4-Fc fusion protein was effective in activating the HIV viral pool. Purification of CD4 from long-term HAART-treated patients with PBMCs (volunteers negative for plasma disease detection) + Cells, with the addition of HIV integrase inhibitor (Ralterravir) PBS or 10nM HyperIL-15×sCD4-Fc (FIG. 9A). The results showed that the HyperIL-15 XSCD 4-Fc fusion protein treated CD4 compared to the PBS group + The cell group can significantly increase the expression of HIV RNA associated with the cell. This result shows that the HyperIL-15 XSCD 4-Fc fusion protein has the function of activating HIV virus library.

Claims (10)

1. A fusion protein, characterized in that: the fusion protein comprises three functional elements of the hyper IL-15, sCD4 and an immunoglobulin Fc region, or comprises two functional elements of hyper IL-15 and sCD 4;
The hyper IL-15 is formed by fusing IL-15 and IL-15-Rα sushi structural domain, or combining IL-15 and IL-15Rα; the sCD4 consists of a D1D2 domain of the extracellular region of CD 4; the immunoglobulin Fc region is selected from a constant region amino acid sequence of IgG1, igG2, igG3 or IgG 4;
the fusion protein containing three functional elements is heterodimeric hyper IL-15 xsCD 4-Fc, and comprises a first polypeptide and a second polypeptide, wherein the first polypeptide is different from the second polypeptide; the first polypeptide comprises hyper IL-15 and an immunoglobulin Fc region from the N end to the C end, and the second polypeptide comprises sCD4 and an immunoglobulin Fc region from the N end to the C end;
the fusion protein containing two functional elements is a monomer hyper IL-15-sCD4 or sCD4-hyper IL-15.
2. The fusion protein of claim 1, wherein:
the IL-15 is selected from human IL-15 and human IL-15 mutant N72D;
the CD4 is selected from CD4 molecule and CD4 mutant.
3. The fusion protein of claim 1, wherein:
the amino acid sequence of the hyper IL-15 is shown as SEQ ID NO. 9;
the amino acid sequence of sCD4 is shown as SEQ ID NO.21 or SEQ ID NO. 23;
the amino acid sequence of the immunoglobulin Fc region is shown as SEQ ID NO. 19.
4. The fusion protein of claim 1, wherein:
the amino acid sequence of the first polypeptide of the heterodimer hyper IL-15 xsCD 4-Fc is shown as SEQ ID NO.1, and the amino acid sequence of the second polypeptide is shown as SEQ ID NO. 3;
the amino acid sequence of the monomer super IL-15-sCD4 or sCD 4-super IL-15 is shown in SEQ ID NO.5 or 7.
5. A nucleic acid molecule encoding the fusion protein of any one of claims 1-4.
6. The nucleic acid molecule of claim 5, wherein:
1) The nucleotide sequence of the nucleic acid molecule encoding the first polypeptide of the heterodimer hyper IL-15 xsCD 4-Fc is shown in SEQ ID NO. 2; the nucleotide sequence of the nucleic acid molecule encoding the second polypeptide of the heterodimer is shown in SEQ ID NO. 4;
2) The nucleotide sequence of the nucleic acid molecule encoding the monomer super IL-15-sCD4 or sCD 4-super IL-15 is shown in SEQ ID NO.6 or 8.
7. A carrier, characterized in that: the vector comprises the nucleic acid molecule of claim 5 or claim 6.
8. A medicament, characterized in that: the medicament comprising the fusion protein of any one of claims 1-4.
9. A cell, characterized in that: the cell comprises the fusion protein of any one of claims 1-4, the nucleic acid molecule of claim 5 or 6, or the vector of claim 7.
10. Use of the fusion protein of any one of claims 1-4, the nucleic acid molecule of claim 5 or 6 and/or the vector of claim 7 for the preparation of a medicament for the prevention or treatment of HIV infection.
CN202310547760.3A 2023-05-16 2023-05-16 Fusion protein of hyper IL-15, sCD4 and Fc and application thereof Active CN116554356B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310547760.3A CN116554356B (en) 2023-05-16 2023-05-16 Fusion protein of hyper IL-15, sCD4 and Fc and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310547760.3A CN116554356B (en) 2023-05-16 2023-05-16 Fusion protein of hyper IL-15, sCD4 and Fc and application thereof

Publications (2)

Publication Number Publication Date
CN116554356A true CN116554356A (en) 2023-08-08
CN116554356B CN116554356B (en) 2024-01-23

Family

ID=87501495

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310547760.3A Active CN116554356B (en) 2023-05-16 2023-05-16 Fusion protein of hyper IL-15, sCD4 and Fc and application thereof

Country Status (1)

Country Link
CN (1) CN116554356B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117645673A (en) * 2023-11-22 2024-03-05 武汉大学 Fusion protein of IL-21, sCD4 and Fc and application thereof
CN117645673B (en) * 2023-11-22 2024-05-10 武汉大学 Fusion protein of IL-21, sCD4 and Fc and application thereof

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040265306A1 (en) * 2001-10-25 2004-12-30 James Arthos Efficient inhibition of hiv-1 viral entry through a novel fusion protein including of cd4
CN102085367A (en) * 2011-01-19 2011-06-08 烟台荣昌生物工程有限公司 Application of optimized TACI (Transmembrane Activator and CAML Interactor)-Fc fusion protein in preparation of medicaments for treating rheumatoid arthritis
CN103059103A (en) * 2012-12-03 2013-04-24 中国人民解放军第四军医大学 Specific CD4+T cell epitope peptide of Hantaan virus nucleoprotein
US20130236456A1 (en) * 2012-03-08 2013-09-12 Georgia Health Sciences University Research Institute, Inc. IMMUNOGLOBULIN Fc FRAGMENT TAGGING ACTIVATION OF ENDOGENOUS CD4 AND CD8 T CELLS AND ENHANCEMENT OF ANTITUMOR EFFECTS OF LENTIVECTOR IMMUNIZATION
CN105980410A (en) * 2014-02-06 2016-09-28 豪夫迈·罗氏有限公司 Interleukin-2 fusion proteins and uses thereof
CN108699131A (en) * 2015-11-21 2018-10-23 艾滋基金会研究所 With dual antiviral and immunoregulatory activity HIV antibody derivative
WO2019204665A1 (en) * 2018-04-18 2019-10-24 Xencor, Inc. Pd-1 targeted heterodimeric fusion proteins containing il-15/il-15ra fc-fusion proteins and pd-1 antigen binding domains and uses thereof
CN111527109A (en) * 2017-12-26 2020-08-11 南京金斯瑞生物科技有限公司 Fusion protein dimer with antibody Fc region as framework and application thereof
CN112105645A (en) * 2018-04-18 2020-12-18 Xencor股份有限公司 IL-15/IL-15RA heterodimer FC fusion protein and application thereof
CN112584870A (en) * 2018-06-11 2021-03-30 威斯塔解剖学和生物学研究所 Compositions and methods for in vivo post-translational modification
CN112585161A (en) * 2018-05-04 2021-03-30 免疫靶向有限公司 Interleukin 15 fusion proteins and compositions and methods of treatment thereof
WO2022216993A2 (en) * 2021-04-08 2022-10-13 Marengo Therapeutics, Inc. Multifuntional molecules binding to tcr and uses thereof

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040265306A1 (en) * 2001-10-25 2004-12-30 James Arthos Efficient inhibition of hiv-1 viral entry through a novel fusion protein including of cd4
CN102085367A (en) * 2011-01-19 2011-06-08 烟台荣昌生物工程有限公司 Application of optimized TACI (Transmembrane Activator and CAML Interactor)-Fc fusion protein in preparation of medicaments for treating rheumatoid arthritis
US20130236456A1 (en) * 2012-03-08 2013-09-12 Georgia Health Sciences University Research Institute, Inc. IMMUNOGLOBULIN Fc FRAGMENT TAGGING ACTIVATION OF ENDOGENOUS CD4 AND CD8 T CELLS AND ENHANCEMENT OF ANTITUMOR EFFECTS OF LENTIVECTOR IMMUNIZATION
CN103059103A (en) * 2012-12-03 2013-04-24 中国人民解放军第四军医大学 Specific CD4+T cell epitope peptide of Hantaan virus nucleoprotein
CN105980410A (en) * 2014-02-06 2016-09-28 豪夫迈·罗氏有限公司 Interleukin-2 fusion proteins and uses thereof
CN108699131A (en) * 2015-11-21 2018-10-23 艾滋基金会研究所 With dual antiviral and immunoregulatory activity HIV antibody derivative
CN111527109A (en) * 2017-12-26 2020-08-11 南京金斯瑞生物科技有限公司 Fusion protein dimer with antibody Fc region as framework and application thereof
WO2019204665A1 (en) * 2018-04-18 2019-10-24 Xencor, Inc. Pd-1 targeted heterodimeric fusion proteins containing il-15/il-15ra fc-fusion proteins and pd-1 antigen binding domains and uses thereof
CN112105645A (en) * 2018-04-18 2020-12-18 Xencor股份有限公司 IL-15/IL-15RA heterodimer FC fusion protein and application thereof
CN112867734A (en) * 2018-04-18 2021-05-28 Xencor股份有限公司 PD-1 targeting heterodimeric fusion proteins comprising an IL-15/IL-15Ra Fc fusion protein and a PD-1 antigen binding domain and uses thereof
CN112585161A (en) * 2018-05-04 2021-03-30 免疫靶向有限公司 Interleukin 15 fusion proteins and compositions and methods of treatment thereof
CN112584870A (en) * 2018-06-11 2021-03-30 威斯塔解剖学和生物学研究所 Compositions and methods for in vivo post-translational modification
WO2022216993A2 (en) * 2021-04-08 2022-10-13 Marengo Therapeutics, Inc. Multifuntional molecules binding to tcr and uses thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CAROLINE JOCHEMS等: "The multi-functionality of N-809, a novel fusion protein encompassing anti-PD-L1 and the IL-15 superagonist fusion complex.", 《ONCOIMMUNOLOGY》, vol. 8, no. 2, XP093036897, DOI: 10.1080/2162402X.2018.1532764 *
CHUNXIAO YU等: "Activatable Fc-IL-15 and anti-PD1-IL-15 fusion molecules with extended half-life.", 《CANCER RESEARCH》, vol. 81 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117645673A (en) * 2023-11-22 2024-03-05 武汉大学 Fusion protein of IL-21, sCD4 and Fc and application thereof
CN117645673B (en) * 2023-11-22 2024-05-10 武汉大学 Fusion protein of IL-21, sCD4 and Fc and application thereof

Also Published As

Publication number Publication date
CN116554356B (en) 2024-01-23

Similar Documents

Publication Publication Date Title
Arunachalam et al. T cell-inducing vaccine durably prevents mucosal SHIV infection even with lower neutralizing antibody titers
Lagenaur et al. Prevention of vaginal SHIV transmission in macaques by a live recombinant Lactobacillus
Lusso et al. Cryptic nature of a conserved, CD4-inducible V3 loop neutralization epitope in the native envelope glycoprotein oligomer of CCR5-restricted, but not CXCR4-using, primary human immunodeficiency virus type 1 strains
Vangelista et al. Engineering of Lactobacillus jensenii to secrete RANTES and a CCR5 antagonist analogue as live HIV-1 blockers
Lee et al. Antibody-dependent cellular cytotoxicity against reactivated HIV-1-infected cells
CN115710311A (en) Antibodies or antigen-binding fragments thereof to coronaviruses
Maréchal et al. Opposite effects of SDF-1 on human immunodeficiency virus type 1 replication
Melchers et al. Targeting HIV-1 envelope glycoprotein trimers to B cells by using APRIL improves antibody responses
Dashti et al. SMAC mimetic plus triple-combination bispecific HIVxCD3 retargeting molecules in SHIV. C. CH505-infected, antiretroviral therapy-suppressed rhesus macaques
Chen et al. The early antibody-dependent cell-mediated cytotoxicity response is associated with lower viral set point in individuals with primary HIV infection
Veillette et al. Role of HIV-1 envelope glycoproteins conformation and accessory proteins on ADCC responses
CN111944054B (en) anti-BCMA CAR and expression vector and application thereof
Kutzler et al. Chemokine-adjuvanted electroporated DNA vaccine induces substantial protection from simian immunodeficiency virus vaginal challenge
IL287311B1 (en) Antibodies and processes for preparing the same
Adalid-Peralta et al. Stimulation of the primary anti-HIV antibody response by IFN-α in patients with acute HIV-1 infection
Münch et al. Hemofiltrate CC chemokine 1 [9-74] causes effective internalization of CCR5 and is a potent inhibitor of R5-tropic human immunodeficiency virus type 1 strains in primary T cells and macrophages
WO2020236753A1 (en) Methods of identifying hiv patients sensitive to therapy with gp120 v3 glycan-directed antibodies
Yan et al. CCL19 and CCL28 assist herpes simplex virus 2 glycoprotein D to induce protective systemic immunity against genital viral challenge
Nacsa et al. Contrasting effects of low-dose IL-2 on vaccine-boosted simian immunodeficiency virus (SIV)-specific CD4+ and CD8+ T cells in macaques chronically infected with SIVmac251
Mselle et al. Human uterine natural killer cells but not blood natural killer cells inhibit human immunodeficiency virus type 1 infection by secretion of CXCL12
CN116333166B (en) Recombinant interferon targeting HIV gp120 protein and application thereof
CN116554356B (en) Fusion protein of hyper IL-15, sCD4 and Fc and application thereof
Bovolenta et al. Human T-cell leukemia virus type 2 induces survival and proliferation of CD34+ TF-1 cells through activation of STAT1 and STAT5 by secretion of interferon-γ and granulocyte macrophage–colony-stimulating factor
CN117645673B (en) Fusion protein of IL-21, sCD4 and Fc and application thereof
CN112175092B (en) Dual-target chimeric antigen receptor simultaneously targeting LMP1 and GP350 and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant