CN117778330A - Novel coronavirus specific T cells and uses thereof - Google Patents

Abstract

The present invention provides novel coronavirus-specific T cells and their applications. Specifically, a specific T cell targeting the novel coronavirus (SARS-CoV-2) is provided, which is induced by the novel coronavirus-restricted epitope polypeptide or its sensitized dendritic cells. Preparations and products containing the specific T cells are also provided, which can be used to treat and prevent the new coronavirus SARS-CoV-2 to produce specific immune effects.

Description

Novel coronavirus-specific T cells and their applications

Technical Field

The present invention relates to the fields of immunology and medicine. More specifically, the present invention relates to novel coronavirus (especially its M protein) specific T cells (e.g., effector T cells or helper T cells) and their applications, which can be stimulated and produced by dendritic cells sensitized by novel coronavirus SARS-CoV-2 HLA-A2 restricted epitope polypeptides (e.g., polypeptides of SEQ ID NO: 5). The present invention also relates to compositions comprising the specific T cells and their applications, such as for the preparation of products for the detection, diagnosis, prevention and/or treatment of SARS-CoV-2 related diseases.

Background Art

Since Doherty and Zinkernagel discovered that cytotoxic T lymphocytes (CTL) can kill cells infected by exogenous microorganisms, and this killing effect depends on the dual recognition of CTL by exogenous peptides and self molecules (MHC), more and more studies have shown that virus-specific CTL-mediated cellular immunity has the function of clearing viruses and is one of the main mechanisms for host defense against viral infections. Doherty and Zinkernagel also won the Nobel Prize in Physiology and Medicine in 1996 for their discovery. Because of this, the research on CTL epitopes and CTL-mediated cellular immune responses has received more and more attention, and the concept of T cell vaccine was subsequently proposed.

Cellular immunity plays a key role in anti-tumor and anti-viral immunity. The antigens recognized by T cells are peptides that bind to MHC class I or class II molecules on the cell surface, and their length is about 8 to 12 amino acids. Cytotoxic T cells (CTLs), the main effector cells that kill tumor cells, recognize endogenous peptides that bind to MHC class I molecules. There is evidence that synthetic peptides can directly bind to MHC class I molecules, and they have the same effectiveness as natural endogenous peptides in activating the immune system.

T cells expressing CD4 (CD4+T cells) are an important immune cell in the human immune system and the most important hub cell for regulating immune responses. CD4 molecules are mainly expressed by helper T cells (Th). They are a leukocyte differentiation antigen and a receptor for Th cell TCR antigen recognition. They bind to the non-peptide region of MHC class II molecules and participate in the process of Th cell TCR antigen recognition. CD8 molecules are a leukocyte differentiation antigen and a glycoprotein on the surface of some T cells. They are used to assist T cell receptors (TCR) in recognizing antigens and participate in the transduction of T cell activation signals. They are also called TCR co-receptors. T cells expressing CD8 (CD8+T cells) usually differentiate into cytotoxic T cells (CTLs) after activation and can specifically kill target cells. Cytotoxic T cells are direct killer cells in immune responses.

Peptide vaccines have become a new strategy for combating malignant tumors and are also the most studied tumor therapeutic vaccines. The most studied ones are (1) gp100: The peptides G9154 (KTWGQYWQV) and G9209 (ITDQVPPFSV) derived from gp100 can both induce antigen-specific CTLs. When used to sensitize peripheral blood lymphocytes (PBL) from HLA-A*0201-positive melanoma patients, the ability to induce specific CTLs was significantly enhanced; (2) CEA: Using a method similar to the above, the CEA peptide CAP1-6D used by Zaremba et al. can not only sensitize CEA-specific CTLs in vitro, with a much higher efficacy than CAPI, but can also induce CEA-specific CTLs in vivo (but CAP1 cannot); and the sensitized CTLs can also recognize CAP1. More importantly, these CTLs can lyse homologous human tumors expressing CEA; (3) MAGE-2: MAGE-2 is widely present in many tumors such as melanoma, laryngeal cancer, lung cancer, sarcoma, etc. Three peptides have been found in MAGE-2 that can form stable complexes with MHC class I molecules at 37°C, at least two of which can be processed and presented by HLA-A*0201, becoming new candidates for peptide vaccine research; (4) P21WAFI: Fusion of P21WAFI peptide with endogenous peptide can inhibit tumor growth; (5) HER2/neu: Peptides derived from HER2/neu can sensitize specific CTLs in vitro and can also induce specific CTLs in mice, and can inhibit tumor growth. In addition, vaccines using HPV peptides are also on the market.

Dendritic cells (DC) are a type of immune cell discovered by Professor Ralph M. Steinman, winner of the 2011 Nobel Prize in Physiology or Medicine. They are named for their mature morphology with many dendritic or pseudopodia-like protrusions. DC plays a key role in acquired immune response and is the strongest professional antigen presenting cell (APC) known so far. It can efficiently ingest, process, handle and present antigens, activate CD4+ helper T cells (T helper lymphocyte, Th) and cytotoxic T cells (cytotoxic T cell, CTL), and is at the center of initiating, regulating, and maintaining immune response.

To date, three highly pathogenic human coronaviruses (CoVs) have been identified, including the Middle East respiratory syndrome coronavirus (MERS-CoV), the severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), and a 2019 novel coronavirus (SARS-CoV-2, referred to as the new coronavirus).

The human-to-human transmission rate of the new coronavirus has exceeded that of SARS-CoV and MERS-CoV. The main clinical symptoms of patients infected with SARS-CoV-2 are fever, cough, and shortness of breath, and laboratory tests often show multiple ground-glass shadows in the lungs. The condition of some patients can rapidly deteriorate and develop serious complications, including acute respiratory distress syndrome, acute kidney injury, secondary infection, inflammatory cytokine storm, etc. Some patients eventually die of respiratory failure, multiple organ dysfunction or shock.

The pathogen SARS-CoV-2, which causes the disease, has been identified in the laboratory as a newly discovered beta coronavirus. It belongs to the same virus genus as SARS-CoV, the pathogen of SARS in 2003, and has 79% genetic similarity. Its diameter is 60-140nm, with an envelope and a single-stranded, positive-sense RNA genome. The SARS-CoV-2 genome has 5' and 3' untranslated regions on both sides. The 5' end includes two long open reading frames (ORFs) encoding 16 non-structural proteins; the remaining genome near the 3' end mainly encodes structural proteins and other auxiliary proteins. The structural proteins of the virus mainly include spike protein (S protein), membrane glycoprotein (M protein), small envelope protein (E protein), and nucleocapsid protein (N protein).

The S protein plays the most important role in the attachment, fusion and entry of the virus, and is also the main target of antibodies, entry inhibitors and vaccines. The S protein mediates the entry of the virus into the host cell, first binding to the host receptor through the receptor binding domain (RBD) of the S1 subunit, and then fusing the virus and host cell membrane through the S2 subunit. The S protein can bind to host cells and mediate viral infection. It is the main antigen protein for reference in the development of antibodies and vaccines. However, SARS-CoV-2 continues to accumulate mutations in the host, especially mutations in structural proteins, resulting in the generation of several SARS-CoV-2 mutants with adaptive advantages, such as Omicron. These mutants usually have stronger infectious or pathogenic potential, and mutations may lead to changes in antigenic characteristics, thereby affecting the prevention and treatment effects of preventive vaccines and therapeutic antibodies on mutants, resulting in immune escape of the virus in the body.

The M protein is essentially a type of transmembrane protein. Its structural characteristics are that it has three domains, namely the N-terminal extracellular domain, the three transmembrane domains, and the internal C-terminal domain. It plays an important role in the morphogenesis and maintenance of the virus and is the most abundant glycoprotein in the virus particle. More importantly, the M gene is relatively conservative, and the frequency of mutation of the M protein is much lower than that of the S protein. Vaccines or antibodies developed based on the M protein are not likely to have reduced prevention and treatment effects due to viral mutations. Therefore, if the M protein is used as a target for immunization of the body, on the one hand, it will cause the body to produce a specific immune response to the viral M protein, so that the body can effectively eliminate the virus, providing measures for the prevention and treatment of SARS-CoV-2. On the other hand, its relatively conservative low mutation characteristics can further avoid the immune escape of the virus caused by mutations, and has a broader spectrum of effects.

There is still an urgent need in the art to develop effective drugs and methods that can effectively prevent and treat the new coronavirus SARS-CoV-2 and its related diseases.

Summary of the invention

The present application provides specific T cells targeting the new coronavirus (SARS-CoV-2) and related preparations and products, which can be directly or indirectly used in the detection, diagnosis, prevention and/or treatment of diseases related to the new coronavirus.

In some aspects of the present invention, a specific T cell targeting the new coronavirus (SARS-CoV-2) is provided.

In some embodiments, the specific T cells are cytotoxic T cells, helper T cells, or a combination thereof.

In some embodiments, the specific T cells herein are induced using SARS-CoV-2M protein epitope polypeptides or dendritic cells sensitized thereto.

In some embodiments, the sequence of the epitope polypeptide used to directly or indirectly stimulate the production of specific T cells is as follows: FLWLLWPVT (SEQ ID NO: 5).

In some embodiments, the epitope polypeptide used to directly or indirectly stimulate the production of specific T cells is an HLA-A2 restricted epitope peptide.

In some embodiments, the affinity coefficient of the epitope polypeptide used to directly or indirectly stimulate the production of specific T cells to the cell surface HLA-A*0201 molecule is at least 2.0, for example, at least 2.2, or at least 2.5.

In some embodiments, the dendritic cells used to stimulate the production of specific T cells are mature dendritic cells, for example, cells that express characteristic surface molecules of mature dendritic cells (eg, CD80, CD83, CD86).

In some embodiments, the dendritic cells used to stimulate the production of specific T cells present SARS-CoV-2 M protein antigens on their surface.

In some embodiments, the primed dendritic cells used to stimulate the production of specific T cells have enhanced antigen presentation capabilities compared to unprimed reference dendritic cells.

In some embodiments, the dendritic cells used to stimulate the production of specific T cells are derived from bone marrow cells, umbilical cord blood cells, and peripheral blood mononuclear cells.

In some embodiments, the dendritic cells used to stimulate the production of specific T cells are derived from mammals, such as humans, non-human primates, and mice.

In some aspects, a preparation is provided comprising the specific T cells herein.

In some embodiments, the preparation is a cell preparation.

In some embodiments, the form of dendritic cells in the preparation can be selected from the following group: specific T cells alone, or complexes thereof with viral therapeutic drugs, cytotoxic drugs, radionuclides, toxins, detectable markers, and prodrug activating enzymes.

In some aspects, provided are uses of the specific T cells and/or preparations described herein for the preparation of products for the detection, diagnosis, prevention and/or treatment of SARS-CoV-2 related diseases or disorders.

In some embodiments, the product is an adoptive T cell therapy drug or pharmaceutical composition.

In some aspects, methods for detecting, diagnosing, preventing and/or treating SARS-CoV-2-related diseases or conditions are provided, comprising using the specific T cells or preparations described herein to directly or indirectly detect, diagnose, prevent and/or treat SARS-CoV-2-related diseases or conditions.

In some embodiments, a method for preventing and/or treating a SARS-CoV-2-related disease or condition is provided, the method comprising administering to a subject in need thereof a specific T cell or preparation described herein.

In some aspects, a method of preparing a specific T cell as described herein is provided, the method comprising:

(a) providing SARS-CoV-2 epitope polypeptides or dendritic cells sensitized thereto;

(b) inducing T cells or their precursors using the epitope polypeptide or sensitized dendritic cells.

In some embodiments, the SARS-CoV-2 epitope polypeptide used in the method for preparing specific T cells comprises consecutive amino acid residues of the SARS-CoV-2 M protein, and the sequence of the consecutive amino acid residues is FLWLLWPVT. In some embodiments, the epitope polypeptide used to prepare specific T cells is an HLA-A2 restricted epitope peptide.

Those skilled in the art may arbitrarily combine the above technical solutions and technical features without departing from the inventive concept and protection scope of the present invention. Other aspects of the present invention are obvious to those skilled in the art due to the disclosure of this article.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further described below in conjunction with the accompanying drawings, wherein these drawings are only for illustrating the embodiments of the present invention rather than for limiting the scope of the present invention.

Figure 1: SMp-11 epitope peptide has high affinity with HLA-A*0201 molecule. Table 1 shows the flow cytometry results for predicting the binding force between epitope peptide and HLA-A*0201 molecule. The higher the fluorescence coefficient, the higher the affinity between the epitope peptide and HLA-A*0201 (generally, a fluorescence coefficient greater than 1 indicates that the epitope peptide has high affinity with HLA-A*0201).

Figure 2: Epitope-specific immune response induced by immunization of transgenic mice with mouse DCs sensitized with epitope peptides. This figure shows the results of Elispot analysis ("****", P<0.0001, "ns" = no significant difference).

Figure 3: Successful induction and culture of mature human dendritic cells (DCs) sensitized with SMP-11 epitope peptides in vitro. This figure shows the expression of mature marker molecules on the surface of DC cells after SMP-11 induction and culture by flow cytometry.

Figure 4: Effector T lymphocytes induced by SMp-11-sensitized human DCs have SMp-11 epitope-specific killing effects. This figure shows the killing effect of SMp-11-specific CTLs induced by CFSE/7-AAD flow cytometry ("*", P<0.05; "****", P<0.0001, "ns" = no significant difference).

Figure 5: SMp-11-specific human CTLs have a high proportion in effector T cells induced by sensitized dendritic cells (DCs). This figure shows the results of Tetramer flow cytometry (OVA-loaded Tetramer was used as the flow cytometry control group).

Figure 6: SMp-11-specific human CTL can specifically secrete the cytokine IFN-γ under target cell stimulation. This figure is the Elispot result analysis ("****", P<0.0001, "ns" = no significant difference) (target cells loaded with irrelevant peptide OVA were used as the control group).

DETAILED DESCRIPTION

The present application provides specific T cells targeting the novel coronavirus (SARS-CoV-2), and their preparations and products. The specific T cells may be cytotoxic T cells, helper T cells or a combination thereof, which have a significant killing effect on HLA-A2 expressing cells expressing SARS-CoV-2 proteins (e.g., M proteins), especially containing the epitope polypeptides identified herein, and thus have great practical value in the detection, diagnosis, prevention and/or treatment of novel coronavirus-related diseases.

Specifically, we used the HLA-A2 restricted epitope polypeptide of the SARS-CoV-2 novel coronavirus protein obtained by screening to sensitize dendritic cell precursors of various sources (such as peripheral blood mononuclear cells) to obtain mature sensitized dendritic cells. Epitope peptides that can effectively induce an immune response in vivo were obtained by screening, and dendritic cells sensitized by them were used to further efficiently induce specific, HLA-A*0201 restricted cytotoxic T lymphocytes and helper T cells. Therefore, this application is of great significance for the development of SARS-CoV-2 prevention and/or treatment vaccines and preparations.

The features mentioned in the present invention or the features mentioned in the embodiments may be combined. All the features disclosed in this specification may be used in any combination form, and each feature disclosed in the specification may be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

All numerical ranges provided herein are intended to clearly include all values falling between the range endpoints and the numerical ranges between them. For example, 1 to 3 includes endpoints 1 and 3, specific integer numerical points 2 and non-integer numerical points therein (for example, but not limited to: 1.2, 1.5, 1.8, 2.1, 2.3, 2.4, 2.8, etc.), and sub-ranges thereof (for example, but not limited to: 1 to 2, 2 to 3, 1 to 1.2, 1.5 to 1.8, etc.).

As used herein, “containing,” “having,” or “including” encompasses “comprising,” “consisting mainly of,” “consisting essentially of,” and “consisting of;” “consisting mainly of,” “consisting essentially of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

As used herein, "epitope polypeptide", "specific (poly)peptide", "HLA-A2 restricted epitope (poly)peptide" and "SARS-CoV-2 HLA-A2 restricted epitope (poly)peptide" are used interchangeably and refer to epitope peptides having the following characteristics: they are derived from various proteins of SARS-CoV-2 (such as M protein, N protein, S protein and E protein), have high affinity to HLA-A2, can be used to sensitize dendritic cells or induce specific T cells, and the sensitized dendritic cells and/or specific T cells can effectively induce a specific immune response against SARS-CoV-2 in vivo.

Through extensive screening, the inventors screened candidate epitope peptides with high affinity to HLA-A*0201 molecules from various proteins of SARS-CoV-2, and used these candidate epitope peptides to further sensitize DC and prepare specific T cells, unexpectedly obtaining highly effective epitope peptides, corresponding sensitized DC and specific T cells that can significantly induce specific immune response in mice. On this basis, the present application provides an excellent restricted epitope (poly)peptide SMp-11, whose amino acid sequence is shown in SEQ ID NO: 5, corresponding to amino acids 53-61 of SARS-CoV-2 M protein.

The protein or polypeptide of the present invention may be a naturally purified product, or a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (eg, bacteria, yeast, higher animal, insect and mammalian cells) using recombinant technology.

As used herein, the terms "dendritic cells (DC) of the present invention" and "sensitized dendritic cells" are used interchangeably, and both refer to dendritic cells sensitized after stimulation with SARS-CoV-2 M protein epitope polypeptides. Dendritic cells can be derived from mammals, preferably obtained from rats, mice or humans.

The dendritic cells used to prepare the sensitized dendritic cells of the present invention may be immature dendritic cells or dendritic cell precursors. For example, dendritic cells can be induced from bone marrow cells, umbilical cord blood cells, and peripheral blood mononuclear cells using methods known in the art (e.g., GM-CSF, IL-4, etc.). In some embodiments, promoting the maturation of immature dendritic cells or their precursors includes further adding a maturation stimulant, such as TNF-α, when the epitope polypeptide is added.

Sensitized dendritic cells can promote the phagocytic function of immature and mature dendritic cells, enhance the antigen presentation function of dendritic cells, and promote the production of effector cells, and can thus be used to detect, diagnose, prevent and/or treat new coronavirus infectious diseases or conditions.

"Specific T cells" or "effector T cells" or "induced T cells" are used interchangeably and refer to T cells that specifically target the new coronavirus SARS-CoV-2 (especially its M protein) in this application. In some embodiments, the specific T cells herein can efficiently produce killer cytokines, such as IFN-γ, under target cell stimulation. In some embodiments, the specific T cells herein have a specific killing effect on SARS-CoV-2M protein epitope polypeptides.

The specific T cells and/or products herein can be used to prepare a variety of products. The products may include, but are not limited to, one or more selected from the group consisting of: a drug, a pharmaceutical composition, or a kit containing an effective amount of the dendritic cells of the present invention and a pharmaceutically or immunologically acceptable carrier. In some embodiments, the product is, for example, a T cell vaccine or drug against novel coronavirus infection.

In a preferred embodiment, the pharmaceutical composition can be used to detect, diagnose, prevent and/or treat diseases associated with SARS-CoV-2, chronic diseases caused by it, and/or its symptoms. For example, the pharmaceutical composition of the present invention can be used to prevent or treat infectious diseases or symptoms caused by the new coronavirus, such as lung or other tissue damage, complications, multiple organ failure, etc. caused by it.

In some embodiments, the products herein can be used to prevent, eliminate or alleviate novel coronavirus infection or at least one symptom thereof in a subject, such as respiratory symptoms (such as nasal congestion, sore throat, hoarseness), headache, cough, sputum, fever, rales, wheezing, dyspnea, pneumonia caused by infection, severe acute respiratory syndrome, renal failure, etc.

The specific T cells of the present invention can be directly administered to a subject in need of prevention or treatment by injection or the like. T cells or their precursors (preferably autologous T cells or their precursors) can be induced in vitro, for example, by induction with sensitized dendritic cells loaded with epitope polypeptides, and the generated specific T cells are infused into a subject in need (e.g., a supplier of T cells or their precursors). The amount of the cells is preferably 10 ⁴ -10 ⁸ specific T cells/time, more preferably 10 ⁵ -10 ⁷ specific T cells/time, and most preferably 10 ⁶ -10 ⁷ specific T cells/time.

The composition of the present invention may also contain T lymphocytes or natural killer cells induced from spleen or peripheral blood. Dendritic cells and natural killer cells may also be administered before, during or after administration of the specific T cells of the present invention.

As used herein, the term "comprising" or "including" includes "comprising", "consisting essentially of", and "consisting of". As used herein, the term "acceptable" ingredients are substances that are suitable for use in humans and/or animals and/or other subjects (such as cells) without excessive adverse reactions (such as toxicity, irritation, and allergic reactions), that is, substances with a reasonable benefit/risk ratio. As used herein, the term "effective amount" refers to an amount that can produce a function or activity on humans and/or animals and/or other subjects (such as cells) and can be accepted by the subject.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent, which may include various excipients and diluents. The term refers to such pharmaceutical carriers: they are not essential active ingredients themselves and are not overly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art, and a full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Acceptable carriers 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 agents or emulsifiers, flavoring agents, pH buffer substances, etc. may also be present in these carriers. Typically, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably, the pH is about 6-8.

The active substance in the composition of the present invention accounts for 0.001-99.9wt% of the total weight of the composition, preferably 1-95wt%, more preferably 5-90wt%, and more preferably 10-80wt%, and the remainder is pharmaceutically acceptable carriers and other additives.

As used herein, the term "unit dosage form" means that for the convenience of taking, the composition of the present invention is prepared into a dosage form required for a single dose, including but not limited to various solid doses (such as tablets), liquid doses, capsules, and sustained-release preparations.

In another preferred embodiment of the present invention, the composition is in unit dosage form or multiple dosage form. In another preferred embodiment of the present invention, 1 to 6 doses of the composition of the present invention are administered daily, preferably 1 to 3 doses are administered; most preferably, the daily dose is 1 dose.

It should be understood that the effective dose of the active substance used may vary with the severity of the subject to be administered or treated. The specific situation is determined according to the individual situation of the subject (e.g., the subject's weight, age, physical condition, the desired effect), which is within the scope of the skilled physician's judgment.

The composition of the present invention may be in solid form (such as granules, tablets, lyophilized powder, suppositories, capsules, sublingual tablets) or liquid form (such as oral liquid) or other suitable shapes. In some embodiments, the composition of the present invention may be administered by injection, such as subcutaneous injection, intravenous injection or infusion. In some embodiments, the administration route may be: (1) direct naked protein injection; (2) connecting the active substance to a transferrin/poly-L-lysine complex to enhance its biological effect; (3) forming a complex between the active substance and a positively charged lipid to overcome the difficulty of crossing the cell membrane caused by the negative charge of the phosphate backbone; (4) liposome encapsulation administration; (5) combining with cholesterol to increase its cytoplasmic retention time by 10 times; (6) using immunoliposomes for transport to enable its specific transport to target tissues and target cells; (7) in vitro transfection; (8) electroporation for introduction into target cells.

The specific T cells and related preparations and products of the present invention can be used in combination with antiviral infection drugs, and can also be combined with other drugs and treatment methods for the prevention and treatment of new coronavirus infectious diseases or symptoms.

Example

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Those skilled in the art may make appropriate modifications and changes to the present invention, and these modifications and changes are within the scope of the present invention.

The experimental methods for which the specific conditions are not specified in the following examples can be conventional methods in the art, for example, refer to "Molecular Cloning Laboratory Manual" (3rd edition, New York: Cold Spring Harbor Laboratory Press, 1989) or follow the conditions recommended by the supplier. The DNA sequencing method is a conventional method in the art and can also be tested by commercial companies.

Unless otherwise indicated, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

Example 1: Affinity Identification of Epitope Peptides and HLA-A*0201 Molecule

Peptide binding experiments were used to screen epitope peptides with high affinity to HLA-A*0201. First, T2 cells (Fuheng Biotechnology, FH0150) were collected, washed three times with serum-free 1640 medium, and the cell concentration was adjusted to 2×10 ⁶ /ml, and plated in a 24-well plate, 1ml/well. Then, 50μM candidate peptides and 3μg/ml β2 microglobulin were co-incubated for 18h at 37°C and 5% CO ₂ in an incubator. The incubated cells were washed three times with ice PBS, PE-labeled HLA-A2-specific flow cytometry antibodies (BiolegendInc) were added, incubated at 4°C for 30min, and the mean fluorescence intensity was detected by flow cytometry after PBS washing. The HLA-A2-restricted influenza virus epitope peptide GILGFVFTL was used as a positive control, and simple T2 cells without peptide stimulation were used as background controls.

Results: Flow cytometry was used to detect the binding of peptides to HLA-A*0201 molecules. This is based on the fact that the binding of exogenous peptides to MHC class I molecules on the surface of T2 cells can increase the expression of MHC class I molecules on their surface. The more stable the binding, the more MHC class I molecules can be detected. The average fluorescence intensity is used as the detection index. The results are measured using the fluorescence coefficient (FI). Peptide FI>1 is considered to be a high-affinity epitope.

The calculation formula of fluorescence coefficient (FI) is as follows:

According to this method, epitope polypeptides with high affinity to HLA-A2 were screened from the membrane glycoprotein M protein (SEQ ID NO: 10), the spike glycoprotein S protein (SEQ ID NO: 11), and the envelope protein E protein (SEQ ID NO: 12) of the SARS-CoV-2 coronavirus. The average fluorescence intensity and fluorescence coefficient of the exemplary epitope peptides involved in the screening can be found in Table 1 and Figure 1:

Table 1. Fluorescence coefficient FI of different epitope peptides binding to HLA-A*0201 molecules on the surface of T2 cells detected by flow cytometry

Results: The affinity results of the exemplary candidate epitope polypeptides to the HLA-A*0201 molecule are shown in Table 1. The results show that polypeptide epitopes No. 1, 3, 5, 7, and 8 (named epitope peptide 1, epitope peptide 3, epitope peptide 5 (also known as SMp-11), epitope peptide 7, and epitope peptide 8, respectively) ranked at the top, with their average fluorescence intensities greater than 10,000, and fluorescence coefficients FI greater than 1, and even reaching above 2.0; among them, the FI of the SMp-11 epitope FLWLLWPVT was as high as 2.73.

Conclusion: Through screening, peptide epitopes with high affinity to HLA-A*0201 molecules were obtained, and the top five peptide epitopes (1, 3, 5, 7, 8) were selected for further screening and identification.

Example 2: Detection of IFN-γ secretion from splenocytes of HLA-A2 transgenic mice immunized with epitope peptide-sensitized DC

The five potential polypeptide epitopes (epitope peptides 1, 3, 5, 7, and 8) obtained by screening according to Example 1 were further tested for their immunogenicity in HLA-A2 transgenic mice. Dendritic cells (DC) derived from bone marrow of HLA-A2.1/K ^b transgenic mice (Jackson Laboratory, 003475) were prepared according to conventional methods. The mice were killed by cervical dislocation and the femur and tibia were removed. PBS was extracted with a 1 ml syringe and the needle was inserted into the bone marrow cavity to flush out the bone marrow. Then, the bone marrow red blood cells were lysed with Tris-NH ₄ Cl solution, the supernatant was discarded by centrifugation, and DC was induced and cultured in 1640 medium containing 10% FBS, 1 ng/ml mouse IL-4 (PeproTech, 214-14-5UG), and 10 ng/ml mouse GM-CSF (PeproTech, 315-03-50UG). DCs cultured to day 5 were collected, the cell concentration was adjusted to 1×10 ⁶ cells/ml, epitope peptides (final concentration 20 μg/ml) were added, and culture was continued in a 37°C, 5% CO ₂ incubator until day 6. TNF-α (30 ng/ml) was added to stimulate maturation, and the culture was continued until day 8 to obtain epitope peptide-sensitized DC cells.

Peptide-sensitized DCs were collected, washed three times with PBS, and the cell concentration was adjusted to 1×10 ⁷ cells/ml. Each male transgenic mouse was subcutaneously injected with 0.1 ml in the abdomen, and immunized three times in total, with an interval of one week. Mice that were incubated with DC without epitope peptide or subcutaneously injected with PBS at the same time were used as negative control mice. Seven days after the last immunization, the spleens of mice in each group (sensitized DC group, empty DC group, PBS group) were removed aseptically, and the red blood cells were lysed to prepare a single cell suspension. The spleen cell suspension (1×10 ⁶ cells/ml) was added to the ELISPOT pre-coated plate (MabTech Inc), 200 μl per well, and the corresponding epitope peptide (final concentration 20 μg/ml) was added to stimulate the culture for 24 hours. Splenocytes stimulated with PMA (Dayou, 2030421) were used as positive stimulation control wells. After the culture was completed, the cells were emptied, washed with PBS 5 times, and the color developing solution was added for color development. After sufficient drying, the plate reader was used for counting and statistical analysis.

Results: The results of Elispot detection are shown in Figure 2. The results showed that compared with the negative control group (non-sensitized DC or PBS-immunized mice), the spleen cells of mice immunized with SMp-11 (epitope peptide 5) could secrete a significantly increased number of IFN-γ spots under the stimulation of SMp-11 epitope peptide. However, there was no significant difference in the secretion of the other four epitope peptides (epitope peptides 1, 3, 7, and 8) compared with the control.

Moreover, although epitope peptide 8 and epitope peptide 5 contain 7 identical consecutive amino acids and have high similarity in sequence, their effects in inducing specific immune responses are completely opposite: the results show that epitope peptide 8 cannot induce specific immune responses and is not a specific epitope; while epitope peptide 5 can not only efficiently induce immune responses, but also the immune responses have excellent specificity.

Conclusion: In view of the complexity of the in vivo functions of organisms, it is unpredictable whether the polypeptide epitope with high affinity to the HLA-A*0201 molecule can sensitize DC and the ability of sensitized DC to induce immune response in vivo, and its specific immune induction effect needs to be verified by in vivo testing. The in vivo test of this example verifies that the SMp-11 epitope polypeptide of the present invention can effectively sensitize DC, and the sensitized DC can significantly induce a specific IFN-γ immune response against the SMp-11 epitope in mice.

Example 3: Characterization of DC cells sensitized by SMp-11 epitope peptide

Peripheral blood mononuclear cells from healthy people were isolated, and PBMCs were resuspended in RPMI1640 serum-free medium. Samples were counted, and the cell density was adjusted to 5×10 ⁶ cells/ml. 2 ml was spread in each well of a 6-well culture plate, and incubated overnight at 37°C and 5% CO _2. The next day, the non-adherent cells (mainly lymphocytes) were shaken and blown off, and the cells were collected and frozen; the adherent cells were monocytes, and 2 ml of complete medium containing human recombinant GM-CSF (50 ng/ml) and human recombinant IL-4 (10 ng/ml) were added to each well of the six-well plate, and 2 ml of the same medium was added every other day. On the fifth day, immature dendritic cells (DC) induced by monocyte differentiation were collected, and SMp-11 epitope peptide was added to 20 μg/ml. On the sixth day, human TNF-α was added to 10 ng/ml to stimulate maturation. Mature DC cells sensitized by SMp-11 epitope peptide were obtained by culture on the eighth day.

Mature DC cells were taken and resuspended to 1×10 ⁶ /100 μl with PBS. 1 μl of FITC-CD80, PE-CD83, and APC-CD86 flow cytometry antibodies (Biolegend Inc) were added. After mixing, the cells were incubated at 4°C in the dark for 30 min. After washing once with PBS, the expression of CD80, CD83, and CD86 molecules, characteristic surface molecules of mature DCs, was detected by flow cytometry (Figure 3).

Results: The expression of characteristic surface molecules of DC cells sensitized by SMp-11 epitope peptide is shown in Figure 3. The results showed that the characteristic molecules CD80, CD83, and CD86 on the surface of DC cells sensitized by SMp-11 epitope peptide after stimulation and maturation were all highly expressed.

Conclusion: Mature SMp-11 peptide-sensitized DC cells were successfully induced in vitro.

Example 4: Induction and culture of SMp-11-specific human CTL and detection of specific killing effect

Autologous T cells and dendritic cells were isolated from purchased human PBMC cells (Sai Li Biotechnology, s2001002) using conventional methods. The cultured T cells were stimulated once a week with mature SMp-11-sensitized autologous dendritic cells for a total of three times. After three weeks of culture and expansion, effector cells were collected and their specific killing effects were detected using the CFSE/7-AAD method.

T2 cells loaded with SMp-11, T2 cells loaded with OVA polypeptide (SEQ ID NO: 13, SIINFEKL, i.e., OVA _257-264 ) and empty T2 cells without polypeptide loading were used as target cells, incubated with CFSE working solution at 37°C for 15 minutes (200 μl CFSE/2×10 ⁶ cells), washed once with complete medium, and the cell concentration was adjusted to 1×10 ⁵ cells/ml with complete medium, and added to a 96-well round-bottom plate with 100 μl per well.

Effector cells were added at three different effector-target ratios of 10:1, 5:1, and 2.5:1. SMp-11, OVA _257-264 , or empty T2 cells without effector cells were used as background control groups. The cells were incubated at 37°C for 4 hours, centrifuged and collected, and labeled with 7-AAD working solution at 4°C for 15 minutes. The cells were washed twice with PBS and resuspended, and the CFSE and 7-AAD fluorescence signals were detected by flow cytometry. The killing rate was calculated as follows:

Killing rate (%) = CFSE-7AAD double positive cells in experimental group (%) - CFSE-7AAD double positive cells in background group (%)

Results: The results of the CFSE/7-AAD killing assay are shown in Figure 4, which show that SMp-11-sensitized DCs can effectively induce T lymphocytes, causing them to show a significantly higher killing efficiency against target cells loaded with SMp-11 epitope peptides than the control group (simple T2 cells or T2 cells loaded with irrelevant peptides).

Conclusion: SMp-11-sensitized DCs induced the generation of effector T lymphocytes with excellent SMp-11 epitope-specific killing activity.

Example 5: Detection of the proportion of SMp-11-specific human CTLs in effector T cells induced in vitro

20 μl of peptide FLEX-T ^TM Tetramer monomer (Biolegend Inc; 280003) was mixed with 20 μl of SMp-11 epitope peptide (400 μM), placed on ice and irradiated with ultraviolet light (366 nm) for 30 minutes, then incubated at 37°C in the dark for 30 minutes, then 3.3 μl of PE-streptavidin (Biolegend Inc) was added and mixed, and then placed on ice in the dark for 30 minutes, and finally 2.4 μl of blocking solution was added and mixed (blocking solution: 1.6 μl 50 mM D-biotin + 6 μl 10% (w/v) NaN ₃ + 192.4 μl PBS). Tetramer loaded with OVA polypeptide (SIINFEKL, OVA _257-264 , SEQ ID NO: 13) prepared in the same way was used as an irrelevant peptide control. Tetramer loaded with OVA polypeptide (SIINFEKL, OVA _257-264 ) served as an irrelevant peptide control.

The effector T cells induced in vitro by the same method as in Example 4 were resuspended in PBS to 2×10 ⁶ cells/200 μl, and 2 μl of the prepared SMp-11-Tetramer or OVA-Tetramer was added. After mixing, the cells were incubated at 4°C in the dark for 30 minutes. After washing once with PBS, the proportion of SMp-11-specific CTLs binding to SMp-11-Tetramer was detected by flow cytometry.

Results: The Tetramer detection results of SMp-11-specific CTL are shown in Figure 5, which show that the proportion of SMp-11-specific CTL in effector T cells induced by SMp-11-sensitized DCs reached 2.02%, while that in the irrelevant peptide control group was only 0.37%.

Conclusion: The proportion of SMp-11-specific human CTLs in effector T cells induced by SMp-11-sensitized DCs was significantly increased.

Example 6: Detection of IFN-γ cytokine secretion by SMp-11-specific human CTL

The effector T cells induced in vitro by the same method as above were resuspended in complete medium to 2×10 ⁶ cells/ml and added to the ELISPOT pre-coated plate at 100 μl per well. T2 cells loaded with SMp-11, T2 cells loaded with OVA polypeptide (SIINFEKL, OVA _257-264 ) and empty T2 cells without polypeptide were used as stimulator cells, and the cell concentration was adjusted to 1×10 ⁶ cells/ml with complete medium. They were added to the ELISPOT pre-coated plate (MabTech Inc) at different effector-target ratios (1:0.1, 1:0.25, 1:0.5). T2 cells loaded with SMp-11, OVA _257-264 or empty without effector cells were used as background control groups, incubated at 37°C for 24 hours, emptied the cells, washed 5 times with PBS, and then added with colorimetric solution for color development. After sufficient drying, the number of IFN-γ spots displayed was counted with a plate reader and statistically analyzed. The proportion of CTL cells secreting IFN-γ was calculated based on the proportion of CD8-positive T cells and the number of IFN-γ spots in effector T cells.

The calculation formula of the proportion of CTL cells secreting IFN-γ is as follows:

Results: The results of Elispot detection are shown in Figure 6. The results showed that the induced SMp-11-specific CTLs had significantly higher IFN-γ secretion than the control group (simple T2 cells or T2 cells loaded with irrelevant peptides) under the stimulation of target cells loaded with SMp-11 epitope peptides, and the proportion of CTLs secreting IFN-γ was as high as 0.87% under the stimulation of the lowest proportion of stimulating cells.

Conclusion: SMp-11-specific CTL induced by SMp-11-sensitized DCs can specifically and efficiently secrete IFN-γ under the stimulation of target cells.

The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Appendix. Sequence information

Claims (10)

1. A specific T cell targeting the new coronavirus (SARS-CoV-2), the specific T cell is induced and produced by using SARS-CoV-2M protein epitope polypeptide or its sensitized dendritic cells. 2. The specific T cell according to claim 1, wherein the sequence of the epitope polypeptide is as shown in FLWLLWPVT (SEQ ID NO: 5), or the sensitized dendritic cells are composed of the sequence as FLWLLWPVT (SEQ ID NO: 5). Sensitization to the epitope polypeptide shown in ID NO:5). 3. The specific T cell of claim 1, wherein the specific T cell is a cytotoxic T cell, a helper T cell, or a combination thereof. 4. The specific T cell according to claim 3, wherein The epitope polypeptide is an HLA-A2 restricted epitope peptide; and/or The affinity coefficient between the epitope polypeptide and the cell surface HLA-A*0201 molecule is at least 2.0, for example, at least 2.2, or at least 2.5. 5. The specific T cell according to claim 3, wherein the dendritic cells are mature dendritic cells, for example, expressing characteristic surface molecules of mature dendritic cells (such as CD80, CD83, CD86); and / or The dendritic cells present SARS-CoV-2M protein antigen on their surface; and/or The dendritic cells have enhanced antigen presentation ability compared with unsensitized reference dendritic cells; and/or The dendritic cells enhance the secretion of cytokines (e.g., interferons, such as IFN-γ) or chemokines; and/or The dendritic cells can effectively induce effector cells targeting the M protein of the new coronavirus (SARS-CoV-2); and/or The dendritic cells are derived from bone marrow cells, umbilical cord blood cells, and peripheral blood mononuclear cells; and/or The dendritic cells are derived from mammals, such as humans, non-human primates, and mice. 6. A product comprising the specific T cells according to any one of claims 1 to 5. 7. The product of claim 6, wherein the product is a cell product; and/or The form of the specific T cells is selected from the group consisting of cells alone or in complexes with viral therapeutic drugs, cytotoxic drugs, radionuclides, toxins, detectable markers, and prodrug-activating enzymes. 8. The specific T cells according to any one of claims 1 to 5, and the product according to any one of claims 6 to 7 are prepared for use in the detection, diagnosis, and diagnosis of SARS-CoV-2 related diseases. Applications in preventive and/or therapeutic products. 9. The use according to claim 8, wherein the product is an adoptive T cell therapy drug or a pharmaceutical composition. 10. A method for preparing specific T cells according to any one of claims 1 to 5, said method comprising: (a) Provide the SARS-CoV-2M protein epitope polypeptide or its sensitized dendritic cells as mentioned in any one of claims 1, 2 or 4; (b) Inducing T cells or their precursors using the epitope polypeptide or sensitized dendritic cells.