CN116731193A - Recombinant proteins and uses thereof - Google Patents

Abstract

The application provides a recombinant protein and application thereof, wherein the recombinant protein comprises: at least one of the following: d4-2, C25, TPP-1, PPL-C and ERY2-4, the C-terminal of D4-2 with the N end of C25 links to each other, the C-terminal of C25 with the N end of TPP-1 links to each other, the C-terminal of TPP-1 with the N end of PPL-C links to each other, the C-terminal of PPL-C with the N end of ERY2-4. The recombinant protein prepared by the application can effectively solve the problem of immune response inhibition, has longer half-life in vivo compared with short peptide, can effectively treat or prevent cancers, and remarkably improves the anti-tumor effect.

Description

Recombinant proteins and uses thereof

Technical Field

The application belongs to the field of biological medicine, in particular to recombinant protein and application thereof, and more particularly relates to recombinant protein, nucleic acid molecules, expression vectors, recombinant cells, compositions, application of the recombinant protein, the nucleic acid molecules, the expression vectors, the recombinant cells or the compositions in preparation of medicines and the medicines.

Background

Immune checkpoint inhibitors (e.g., anti-CTLA-4, anti-PD-1/PD-L1, anti-LAG-3, anti-CD 47/sirpa) target the natural immune homeostatic pathway to drive a stronger immune response against tumors, have become a treatment strategy for advanced tumors, and blockade of immune checkpoints will overcome immune tolerance in the tumor microenvironment and enhance anti-tumor immunity, inducing continued tumor remission. Thus blocking inhibitory signals, reactivating immune function has become an effective means for treating cancer, and in 2011, the us FDA approved CTLA-4 antibodies for treatment of melanoma, and in 2014 approved PD-1 antibodies for treatment of melanoma and other cancers. Although immune blocking therapy has the advantages of strong specificity and high stability, long-term injection therapy is easy to induce the organism to generate anti-antibody and complement-mediated cytotoxicity: ADCC (antibody dependent cell mediated cytotoxicity) and CDC (complement dependent cytotoxicity) effects, whose discrete toxicities caused by nonspecific activation of the immune system cause the appearance of inflammatory symptoms in the corresponding organs, known as immune-related adverse reactions (immune-related adverse events, irAEs), which can involve multiple systems, multiple organs, occur at irregular times, during treatment or at any time after cessation of treatment, are important for the safe use of immune checkpoint inhibitors.

In addition, there are also a number of immunosuppressive mechanisms in the tumor microenvironment that even blocking the PD-L1, CTLA-4 pathways is insufficient to restore the immune system. Although PD-1 or PD-L1 antibodies alter the pattern of many advanced cancer patients, including melanoma, lung cancer, breast cancer, renal cancer, etc., good long-term results are achieved. However, is effective in only a fraction of patients and may produce toxic effects. Meanwhile, gene therapy is a potential effective method for treating genetic diseases, but factors such as the size, charge and stability of oligonucleotide and plasmid DNA for gene therapy cannot be effectively absorbed by cells, and the curative effect is seriously affected. Thus, there remains a need to further develop products directed against tumor immunosuppression.

Disclosure of Invention

The present application aims to solve, at least to some extent, one of the above technical problems or to at least provide a commercial choice.

In the application, the inventor designs a plurality of exogenous injection proteins aiming at tumor models, and obtains dominant recombinant proteins after a large number of experimental screening, the recombinant proteins can effectively solve the problems of immune response inhibition and the like, and have the characteristic of longer half-life in vivo compared with short peptides. Furthermore, the inventor loads the genome encoding the recombinant protein into a tumor specific T cell, and the T cell continuously expresses and secretes immune checkpoint inhibitory short peptide in tumor tissues, so that the problem of insufficient tissue penetration caused by short peptide injection is effectively avoided, the activation of the whole immune system is avoided, and the recombinant protein has very important benefit to tumor patients.

Thus, in a first aspect of the application, the application proposes a recombinant protein. According to an embodiment of the application, at least two of the following are included: d4-2, C25, TPP-1, PPL-C and ERY2-4. The inventor performs recombination and screening on various immune checkpoint inhibitory peptides through a large number of experiments to obtain various dominant recombinant proteins, and the recombinant proteins according to the embodiment of the application can effectively block the interaction between tumor cells expressing immune checkpoints and immune cells, can effectively solve the problem of immune response inhibition, and have longer in vivo half-life compared with short peptides, can effectively treat or prevent cancers, and can remarkably improve the anti-tumor effect.

In a second aspect of the application, the application provides a nucleic acid. According to an embodiment of the application, the nucleic acid encodes the recombinant protein of the first aspect. The recombinant protein coded by the nucleic acid can effectively block the interaction between tumor cells expressing immune check points PD-1/PD-L1, LAG-3, CTLA-4 or CD47/SIRP alpha and immune cells, can effectively solve the problem of immune response inhibition, has longer in vivo half-life compared with short peptides, can effectively treat or prevent cancers, and has obviously improved anti-tumor effect.

In a third aspect of the application, the application provides an expression vector. According to an embodiment of the application, the expression vector comprises: the nucleic acid molecule of the second aspect. The expression vector may include optional control sequences operably linked to the nucleic acid molecule. Wherein the control sequences are one or more control sequences that direct expression of the nucleic acid molecule in a host, which control sequences may be derived directly from the vector itself or may be exogenous, i.e., not derived from the vector itself. The expression vector provided by the embodiment of the application can efficiently express the recombinant protein in a proper host cell, the recombinant protein can effectively block the interaction between tumor cells expressing immune check points PD-1/PD-L1, LAG-3, CTLA-4 or CD47/SIRP alpha and immune cells, the problem of immune response inhibition can be effectively solved, and compared with a short peptide, the recombinant protein has longer half-life in vivo, can effectively treat or prevent cancers, and has obviously improved anti-tumor effect.

In a fourth aspect of the application, the application provides a method for preparing the recombinant protein according to the first aspect. According to an embodiment of the application, it comprises: introducing the expression vector of the third aspect into a cell; the cells are cultured under conditions suitable for protein expression and secretion to obtain the recombinant protein. The recombinant protein obtained by the method provided by the embodiment of the application can effectively block the interaction between tumor cells expressing immune check points PD-1/PD-L1, LAG-3, CTLA-4 or CD47/SIRP alpha and immune cells, can effectively solve the problem of immune response inhibition, has longer in vivo half-life compared with short peptides, can effectively treat or prevent cancers, and has obviously improved anti-tumor effect.

In a fifth aspect of the application, the application provides a recombinant cell. According to an embodiment of the application, the recombinant cell carries a nucleic acid according to the second aspect, an expression vector according to the third aspect or expresses a recombinant protein according to the first aspect. The recombinant protein obtained by expressing and secreting the recombinant cells provided by the embodiment of the application under a certain condition can effectively block the interaction between tumor cells expressing immune check points PD-1/PD-L1, LAG-3, CTLA-4 or CD47/SIRP alpha and immune cells, can effectively solve the problem of immune response inhibition, has longer half-life in vivo compared with immune check point inhibition short peptides, can effectively treat or prevent cancers, and has obviously improved anti-tumor effect.

In a sixth aspect of the application, the application provides a composition. According to an embodiment of the application, it comprises: the recombinant protein of the first aspect, the nucleic acid of the second aspect, the expression vector of the third aspect, or the recombinant cell of the fifth aspect. As described above, the recombinant protein according to the embodiment of the application can effectively block the interaction between tumor cells expressing immune checkpoints PD-1/PD-L1, LAG-3, CTLA-4 or CD47/SIRP alpha and immune cells, can effectively solve the problem of immune response inhibition, and has longer in vivo half-life compared with immune checkpoint inhibition short peptides, so that the composition containing the recombinant protein, such as food composition, pharmaceutical composition and the like, can also effectively solve the problem of immune response inhibition, has longer effective time in vivo, can effectively treat or prevent cancers, has remarkable effect of treating or preventing tumors, and has higher safety and smaller side effect.

In a seventh aspect of the application, the application provides the use of a recombinant protein according to the first aspect, a nucleic acid according to the second aspect, an expression vector according to the third aspect, a recombinant cell according to the fifth aspect or a composition according to the sixth aspect for the preparation of a medicament. According to an embodiment of the application, the medicament is for the treatment or prevention of cancer. According to the recombinant protein, the recombinant protein obtained by using nucleic acid, an expression vector and recombinant cells or part of protein in the composition, provided by the embodiment of the application, the interaction between tumor cells expressing immune check points PD-1/PD-L1, LAG-3, CTLA-4 or CD47/SIRP alpha and immune cells can be effectively blocked under a certain condition, the problem of immune response inhibition can be effectively solved, and compared with short peptides, the recombinant protein has a longer in vivo half-life, can effectively treat or prevent cancers, and has a significantly improved anti-tumor effect.

In an eighth aspect of the application, the application provides a medicament. According to an embodiment of the application, the medicament comprises: the recombinant protein of the first aspect, the nucleic acid of the second aspect, the expression vector of the third aspect, the recombinant cell of the fifth aspect, or the composition of the sixth aspect. As described above, the recombinant protein of the embodiment of the application can effectively block the interaction between tumor cells expressing immune checkpoints PD-1/PD-L1, LAG-3, CTLA-4 or CD47/SIRP alpha and immune cells, so that the problem of immune response inhibition can be solved, and compared with a short peptide, the recombinant protein has longer in vivo half-life, so that the medicament containing the recombinant protein and a series of related substances has obvious effect of treating or preventing cancers.

In a ninth aspect of the application, the application provides an immune effector cell. According to an embodiment of the application, the immune effector cell carries the nucleic acid molecule, expression vector or expresses the recombinant protein as described above. According to the specific embodiment of the application, the effector cells have tumor specificity, the T cells can continuously express and secrete immune checkpoint inhibitory peptide in tumor tissues, the problem of insufficient tissue penetration caused by peptide injection is effectively avoided, the effector cells are only expressed on the surfaces of the tumor cells, the activation of the whole immune system is avoided, the anti-tumor effect is obvious, the safety is high, and the side effect is small.

In a tenth aspect of the application, the application provides a method of treating or preventing cancer. According to an embodiment of the application, the method comprises administering to the subject a recombinant protein, expression vector, recombinant cell, immune effector cell, composition or medicament as previously described. As described above, the recombinant protein of the embodiment of the application can effectively block the interaction between tumor cells expressing immune checkpoints PD-1/PD-L1, LAG-3, CTLA-4 or CD47/SIRP alpha and immune cells, so that the problem of immune response inhibition can be solved, and compared with a short peptide, the recombinant protein has longer in vivo half-life, therefore, the recombinant protein and a series of substances related to the recombinant protein are given to a subject, and the recombinant protein has obvious effect of treating or preventing cancers.

Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application.

Drawings

The foregoing and/or additional aspects and advantages of the application will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 is a schematic diagram of recombinant protein Blockine and Fusion-Blockine structures according to an embodiment of the application, wherein n represents an nth immune checkpoint inhibitory peptide, and n is a positive integer;

FIG. 2 is a graph of the efficiency results of identifying Blockine, fusion-Blockine to OT-1T cell engineering by flow through EGFP expression in accordance with an embodiment of the present application;

FIG. 3 is a graph of the results of identifying secreted and expressed Block-4 of OT-1T cells by Dot-blot against an HA tag according to an embodiment of the present application;

FIG. 4 is a graph showing the detection results of the proteins Block 1-6 and Fusion-Block on tumor growth in a tumor mouse model according to an embodiment of the present application, wherein Control represents a Control group; and

FIG. 5 is a graph showing the results of detection of tumor growth in a tumor mouse model by recombinant proteins Fusion-block-S, fusion-block-1-4 according to an embodiment of the present application, wherein Control represents a Control group.

Detailed Description

Reference will now be made in detail to embodiments of the present application, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the drawings are illustrative and intended to explain the present application and should not be construed as limiting the application.

Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present application, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise.

"operably linked" herein refers to the linkage of a foreign gene to a vector such that control elements within the vector, such as transcription control sequences and translation control sequences, and the like, are capable of performing their intended functions of regulating transcription and translation of the foreign gene.

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

In order that the application may be more readily understood, certain technical and scientific terms are defined below. Unless clearly defined otherwise herein in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

In a first aspect of the application, the application provides a recombinant protein comprising at least two of the following: d4-2, C25, TPP-1, PPL-C and ERY2-4. The inventor performs recombination and screening on various immune checkpoint inhibitory peptides through a large number of experiments to obtain various dominant recombinant proteins, the recombinant proteins can effectively solve the problem of immune response inhibition, and compared with short peptides, the recombinant proteins have longer half-life in vivo, can effectively treat or prevent cancers, and remarkably improve the anti-tumor effect.

According to some embodiments of the application, the recombinant protein may further include at least one of the following additional technical features:

according to some embodiments of the application, the recombinant proteins include D4-2, C25, TPP-1, PPL-C and ERY2-4.

According to some embodiments of the application, the C-terminal of D4-2 is connected to the N-terminal of C25, the C-terminal of C25 is connected to the N-terminal of TPP-1, the C-terminal of TPP-1 is connected to the N-terminal of PPL-C, and the C-terminal of PPL-C is connected to the N-terminal of ERY2-4. The inventor carries out recombination and screening on various immune checkpoint inhibitory peptides through a large number of experiments to obtain various dominant recombinant proteins, wherein when the C end of D4-2 is connected with the N end of C25, the C end of C25 is connected with the N end of TPP-1, the C end of TPP-1 is connected with the N end of PPL-C, and the C end of PPL-C is connected with the N end of ERY2-4, the obtained recombinant proteins can effectively block the interaction between tumor cells expressing immune checkpoints PD-1/PD-L1, LAG-3, CTLA-4 or CD47/SIRP alpha and immune cells, can effectively solve the problem of immune response inhibition, and compared with the single short peptides D4-2, C25, TPP-1, PPL-C or ERY2-4, the obtained recombinant proteins have longer half-life in vivo, can effectively treat or prevent cancers, and can obtain extremely remarkable anti-tumor effects.

According to some embodiments of the application, the amino acid sequence shown as SEQ ID NO. 30, or an amino acid sequence having at least 95%, 90%, 85% identity to the amino acid sequence shown as SEQ ID NO. 25, is included.

Fusion-block S has the amino acid sequence shown below:

YASYHCWCWRDPGRSAAWGQAILEGELAWLEGGGGGAGQLADLKRQLAWWKQAAKDWSISARYSAVYSIHPSWAVPMTYRAAVSVSHFQKVWVV(SEQ ID NO：30)。

in a second aspect of the application, the application provides a nucleic acid encoding the recombinant protein of the first aspect. According to the nucleic acid-encoded recombinant protein provided by some specific embodiments of the application, the interaction between tumor cells expressing immune checkpoints PD-1/PD-L1, LAG-3, CTLA-4 or CD47/SIRP alpha and immune cells can be effectively blocked, the problem of immune response inhibition can be effectively solved, compared with a short peptide, the nucleic acid-encoded recombinant protein has a longer in vivo half-life period, can effectively treat or prevent cancers, and the anti-tumor effect is remarkably improved.

According to some embodiments of the application, the nucleic acid may further comprise at least one of the following additional technical features:

according to some embodiments of the application, the nucleic acid has the sequence of SEQ ID NO:8, and a nucleotide sequence shown in SEQ ID NO.

The gene encoding Fusion-block S has the nucleotide sequence shown below:

AAGGACTGGAGTATAAGCGCCCGATACTCCGCAGTATACAGTATCCATCCCTCATGGCAGTTCCAATGACCTATCGCGCCGCATACGCCAGCTACCACTGCTGGTGCTGGAGGGACCCCGGCAGGAGCGTCTCCGTGAGCCACTTCCAGAAGGTGTGGGGCAGCCTGGGGGCAGGCCATATTGGAAGGCGAGCTTGCATGGCTGGAGGGCGGGGGCGGCGGTGCTGGCCAACTGGCCGACCTGAAACGGCAGCTTGCTTGGTGGAAGCAAGCTGCA(SEQ ID NO：8)。

it should be noted that, for the nucleic acids mentioned in the present specification and claims, one skilled in the art will understand that either one or both of the complementary double strands are actually included. For convenience, in the present description and claims, although only one strand is shown in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid sequences of the present application include DNA forms or RNA forms, one of which is disclosed, meaning the other is also disclosed.

In a third aspect of the present application, the present application provides an expression vector comprising: the nucleic acid molecule of the second aspect. The expression vector may include optional control sequences operably linked to the nucleic acid molecule. Wherein the control sequences are one or more control sequences that direct expression of the nucleic acid molecule in a host, which control sequences may be derived directly from the vector itself or may be exogenous, i.e., not derived from the vector itself. The expression vector provided by some specific embodiments of the application can efficiently express the recombinant protein in a proper host cell, the recombinant protein can effectively block the interaction between tumor cells expressing immune check points PD-1/PD-L1, LAG-3, CTLA-4 or CD47/SIRP alpha and immune cells, the problem of immune response inhibition can be effectively solved, and compared with short peptides, the recombinant protein has longer in vivo half-life, can effectively treat or prevent cancers, and has obviously improved anti-tumor effect.

In a fourth aspect of the application, the application provides a method for preparing a recombinant protein according to the first aspect, comprising: introducing the expression vector of the third aspect into a cell; the cells are cultured under conditions suitable for protein expression and secretion to obtain the recombinant protein. The recombinant protein obtained by the method according to some specific embodiments of the application can effectively block the interaction between tumor cells expressing immune checkpoints PD-1/PD-L1, LAG-3, CTLA-4 or CD47/SIRP alpha and immune cells, can effectively solve the problem of immune response inhibition, has longer half-life in vivo compared with short peptides, can effectively treat or prevent cancers, and has obviously improved anti-tumor effect.

According to some embodiments of the application, the method may further comprise at least one of the following additional technical features:

according to some embodiments of the application, the cell is a eukaryotic cell.

According to some embodiments of the application, the eukaryotic cell is a mammalian cell.

According to some embodiments of the application, the mammalian cell comprises at least one selected from CHO-K1, CHOs, 293F, 293T, CAR-T, TCR-T, TIL, CAR-NK, CAR-M and CAR-DC.

According to some embodiments of the application, the mammalian cells do not include animal germ cells, fertilized eggs, or embryonic stem cells.

In a fifth aspect of the application, the application provides a recombinant cell carrying a nucleic acid according to the second aspect, or an expression vector according to the third aspect. The recombinant protein secreted by the recombinant cells according to some specific embodiments of the application can effectively block the interaction between tumor cells expressing immune checkpoints PD-1/PD-L1, LAG-3, CTLA-4 or CD47/SIRP alpha and immune cells, can effectively solve the problem of immune response inhibition, has longer in vivo half-life compared with short peptides, can effectively treat or prevent cancers, and has obviously improved anti-tumor effect.

It should be noted that the recombinant cells of the present application are not particularly limited, and may be prokaryotic cells, eukaryotic cells, or phage. The prokaryotic cell can be escherichia coli, bacillus subtilis, streptomycete or proteus mirabilis and the like. The eukaryotic cells can be fungi such as pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces, trichoderma and the like, insect cells such as armyworm and the like, plant cells such as tobacco and the like, and mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells and the like. In some embodiments, the recombinant cells of the application are preferably mammalian cells, including T cells, BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

The term "suitable conditions" as used herein refers to conditions suitable for expression of the recombinant protein of the present application. Those skilled in the art will readily appreciate that conditions suitable for expression of the recombinant protein include, but are not limited to, suitable transformation or transfection means, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, suitable cell culture time. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the conditions for optimal expression of the recombinant protein according to the specific environment of the laboratory.

In a sixth aspect of the application, the application provides a composition comprising: the recombinant protein of the first aspect, the nucleic acid of the second aspect, the expression vector of the third aspect, or the recombinant cell of the fifth aspect. As described above, the recombinant protein according to some embodiments of the present application can effectively block the interaction between tumor cells expressing immune checkpoints PD-1/PD-L1, LAG-3, CTLA-4 or CD47/SIRP alpha and immune cells, and thus, the composition comprising the recombinant protein, such as food composition, pharmaceutical composition, etc., can effectively solve the problem of immune response inhibition as well, and has longer effective duration in vivo, can effectively treat or prevent cancer, has remarkable tumor treatment or prevention effect, and has higher safety and less side effects.

It is noted that the compositions include combinations that are separated in time and/or space, so long as they are capable of co-acting to achieve the objects of the present application. For example, the ingredients contained in the composition may be administered to the subject in whole or separately. When the components contained in the composition are separately administered to a subject, the individual components may be administered to the subject simultaneously or sequentially.

In a seventh aspect, the application provides the use of a recombinant protein according to the first aspect, a nucleic acid according to the second aspect, an expression vector according to the third aspect, a recombinant cell according to the fifth aspect or a composition according to the sixth aspect for the preparation of a medicament for the treatment or prevention of cancer. According to the recombinant protein, the recombinant protein obtained by using nucleic acid, an expression vector and recombinant cells or part of protein in a composition of some specific embodiments of the application, the interaction between tumor cells expressing immune checkpoints PD-1/PD-L1, LAG-3, CTLA-4 or CD47/SIRP alpha and immune cells can be effectively blocked under certain conditions, so that the problem of immune response inhibition is effectively solved, and compared with a short peptide, the recombinant protein has a longer in vivo half-life, can effectively treat or prevent cancers, and has a remarkably improved anti-tumor effect.

According to some embodiments of the present application, the above-mentioned medicament may further comprise at least one of the following additional technical features:

according to some embodiments of the application, the cancer comprises at least one of: gastric cancer, intestinal cancer, head and neck cancer, melanoma.

In an eighth aspect of the application, the application provides a medicament comprising: the recombinant protein of the first aspect, the nucleic acid of the second aspect, the expression vector of the third aspect, the recombinant cell of the fifth aspect, or the composition of the sixth aspect. As described above, the recombinant protein of the embodiment of the application can effectively block the interaction between tumor cells expressing immune checkpoints PD-1/PD-L1, LAG-3, CTLA-4 or CD47/SIRP alpha and immune cells, so that the problem of immune response inhibition can be solved, and compared with a short peptide, the recombinant protein has longer in vivo half-life, so that the medicament containing a series of substances related to the recombinant protein has obvious effect of treating or preventing cancers.

According to some embodiments of the present application, the above-mentioned medicament may further comprise at least one of the following additional technical features:

according to some embodiments of the application, the cancer comprises at least one of: gastric cancer, intestinal cancer, head and neck cancer, melanoma.

According to some embodiments of the present application, there is provided a medicament comprising a pharmaceutically acceptable carrier and an effective amount of an active ingredient of the recombinant protein.

As used herein, the term "effective amount" or "effective dose" refers to an amount that is functional or active in and acceptable to a human and/or animal.

As used herein, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

The medicament of the application contains safe and effective amount of the active ingredients of the application and pharmaceutically acceptable carriers. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation is matched with the administration mode, and the dosage forms of the medicine are injection, oral preparation (tablet, capsule and oral liquid), transdermal agent and sustained release agent. For example, by using physiological saline or an aqueous solution containing glucose and other auxiliary agents by conventional methods. The medicament is preferably manufactured under aseptic conditions.

The effective amount of the active ingredient described herein may vary depending upon the mode of administration, the severity of the condition being treated, and the like. The selection of the preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, separate doses may be administered several times per day, or the dose may be proportionally reduced, as dictated by the urgent need for the treatment of the condition.

Pharmaceutically acceptable carriers described herein include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be compatible with the mode of administration and will be well known to those of ordinary skill in the art.

In a ninth aspect of the application, the application provides an immune effector cell carrying a nucleic acid molecule, expression vector or recombinant protein as described hereinbefore. According to the specific embodiment of the application, the immune effector cells have tumor specificity, the T cells can continuously express and secrete immune checkpoint inhibitory peptide in tumor tissues, the problem of insufficient tissue penetration caused by peptide injection is effectively avoided, the expression is only carried out on the surfaces of the tumor cells, the activation of the whole immune system is avoided, the anti-tumor effect is obvious, the safety is high, and the side effect is small.

In a tenth aspect of the application, the application provides a method of treating or preventing cancer comprising administering to a subject a recombinant protein, expression vector, recombinant cell, immune effector cell, composition or medicament as described previously. As described above, the recombinant protein according to some embodiments of the present application can effectively block the interaction between tumor cells expressing immune checkpoints PD-1/PD-L1, LAG-3, CTLA-4 or CD47/SIRP alpha and immune cells, thereby solving the problem of immune response inhibition, and has a longer in vivo half-life than a short peptide, so that the recombinant protein and a series of substances related thereto are administered to a subject, and also have a remarkable effect of treating or preventing cancer.

The embodiments will be described in detail below. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

EXAMPLE 1 construction of expression vectors

In this embodiment, expression vectors of Blockine and Fusion-Blockine are constructed, and specific experimental operation steps are as follows:

construction of expression vectors

1.1 obtaining immune checkpoint inhibitory peptides

The inventor obtains nucleotide sequences encoding immune checkpoint inhibitory peptides D4-2 (Block 1), C25 (Block 2), TPP-1 (Block 3), PPL-C (Block 4) and ERY2-4 (Block 5) by a chemical synthesis method, and constructs nucleotide fragments encoding recombinant protein Fusion Block by using the nucleotide sequences, wherein the nucleotide sequences are respectively as follows: fusion Blockine S (D4-2) - (C25) - (TPP-1) - (PPL-C) - (ERY 2-4); fusion Block 1 (TPP-1) - (ERY-2) - (D4-2) - (C25) - (PPL-C); fusion Block 2 (PPL-C) - (D4-2) - (ERY 2-4) - (C25); fusion Block ine3 (TPP-1) - (D4-2) - (ERY 2-4) - (PPL-C) - (C25); fusion Block 4 (C25) - (TPP-1) - (ERY 2-4) - (D4-2) - (PPL-C), the specific nucleotide sequences of which are shown in Table 1.

Table 1:

in addition, the inventors obtained nucleotide sequences encoding human or mouse IL-2 secretion signal peptide (SEQ ID NO: 6) and HA (hemagglutinin) (SEQ ID NO: 7) tags by a chemical synthesis method, and linked the nucleotide sequences encoding the signal peptide and the tag, respectively, as shown in Table 2:

table 2:

the nucleotide sequence codes shown in Table 2 obtain the structure of a block and Fusion block precursor protein shown in figure 1, wherein the N end of the block and Fusion block precursor protein is connected with an IL-2 signal peptide, the C end of the block and Fusion block precursor protein is connected with an HA tag, the signal peptide helps to locate the block and Fusion block protein, and the signal peptide is hydrolyzed and separated after the protein is mature, so that the finally obtained block and Fusion block protein does not contain the IL-2 signal peptide.

1.2 construction of expression vectors

The nucleotide sequences of block and Fusion-block obtained in Table 2 of step 1.1, carrying the IL-2 signal peptide and the detection tag HA, were ligated into the expression retroviral vector by homologous recombination using BamH-1 cleavage, respectively (Addgene, # 12282). The obtained cloning vector was sequenced and the sequencing results were consistent with expectations.

Example 2 preparation of recombinant proteins

Isolation and purification of CD8 from spleen and lymph node tissues of OT-I transgenic mice ⁺ T lymphocytes and activated, and viral transduction was performed 24 hours after activation; after 6 hours, the culture medium containing 20U/mL IL-2 needs to be replaced for continuous culture, and after 12 hours, transduction is performed again, 4 times of virus transduction are performed in total, and 3 times of culture medium containing 20U/mL IL-2 needs to be replaced in the period of time, so that the transduction efficiency is improved. After a further 24 hours of culture with a new medium, the engineered T cells were isolated using flow sorting. The specific operation steps of the T cell separation, activation and virus transduction flow are as follows:

(1) Packaging the retrovirus: the plasmid obtained in example 1 was transfected into Plat-E cells of a retrovirus packaging cell line using PEI, cultured using RPMI-1640 medium (350-000-CL), and virus supernatants were collected after 48 and 72 hours;

(2) Isolation and transduction of T cells: tumor-specific T cells were cultured by pre-plating 3-5 μg/mL anti-mouse-CD3 epsilon antibody in 6 well plates, diluting with 1 x dpbs, adding 1mL per well, incubating in 37 ℃ cell incubator for 1-3h or overnight at 4 ℃, and gently washing with 1 x dpbs once after completion. Isolation of CD8 from spleen and lymph node tissues of OT-I mice using MojoSort CD8 negative selection (Biolegend) magnetic bead sorting kit ⁺ T cells, plated in 6 well plates, 2 mL/well T cell medium, 1 x 10 ⁶ -2*10 ⁷ Per well OT-I T cells, 2. Mu.g/mL anti-mouse-CD28 antibody and 20U/mL IL-2 cytokine were added for 24h activation.

(3) After activation of anti-tumor OT-I T cells, the virus liquid supernatants (6 mL/well) packed with individual short peptides and fusion short peptides were transduced into 1mL OT-I T cells (1 x 10) ⁶ -2*10 ⁷ cells), 10mM HEPES, 500uL FBS, 8. Mu.g/mL Polybrene were added, and after centrifugation at 1200g at 32℃for 90min, the culture was continued overnight, and after 6 hours the medium containing 20U/mL IL-2 cytokine was replaced, and after 12 hours secondary viral transduction was performed. A total of 4 viral transduction were performed.

(4) EGFP detection by Flow cytometry after 24h ⁺ The positive rate, i.e. the transduction efficiency was measured, and the specific experimental results are shown in fig. 2.

(5) Detecting the secretory expression of the target short peptide outside the T cells: t cells successfully transduced are sorted by using a flow sorting technology (transduction efficiency is more than 63.3% and is optimal, EGFP is obtained ⁺ Shown in fig. 2); culturing T cells selected by nutrients, collecting culture supernatant after 24 hours, and verifying secretion effect of the Blockine by using Dot-blot experiment, wherein the Dot-blot experiment result of the Blockine-4 protein is taken as an example, and the result is shown in figure 3; wherein the amino acid sequence of each protein obtained is as follows:

immune checkpoint inhibitory peptide Blockine1 (D4-2) comprises the following amino acid sequence:

KDWSISARYSAVYSIHPSW(SEQ ID NO:23)。

immune checkpoint inhibitory peptide Blockine2 (C25) comprises the amino acid sequence:

AVPMTYRAA(SEQ ID NO:24)。

immune checkpoint inhibitory peptide Blockine3 (TPP-1) comprises the following amino acid sequence:

YASYHCWCWRDPGRS(SEQ ID NO:25)。

immune checkpoint inhibitory peptide Blockine4 (PPL-C) includes the following amino acid sequences:

VSVSHFQKVWVV(SEQ ID NO:26)。

immune checkpoint inhibitory peptide Blockine5 (ERY 2-4) comprises the following amino acid sequence:

AAWGQAILEGELAWLEGGGGGAGQLADLKRQLAWWKQAA(SEQ ID NO:27)。

the human or mouse IL-2 secretion signal peptide comprises the following amino acid sequence:

MYRMQLLSCIALSLALVTNS(SEQ ID NO:28)。

HA comprises the following amino acid sequence:

YPYDVPDYA(SEQ ID NO:29)。

fusion Blockine S comprises the amino acid sequence shown below:

YASYHCWCWRDPGRSAAWGQAILEGELAWLEGGGGGAGQLADLKRQLAWWKQAAKDWSISARYSAVYSIHPSWAVPMTYRAAVSVSHFQKVWVV(SEQ ID NO:30)。

fusion block 1 comprises the amino acid sequence shown below:

VSVSHFQKVWVVKDWSISARYSAVYSIHPSWYASYHCWCWRDPGRSAAWGQAILEGELAWLEGGGGGAGQLADLKRQLAWWKQAAAVPMTYRAA(SEQ ID NO:31)。

fusion block 2 comprises the amino acid sequence shown below:

YASYHCWCWRDPGRSKDWSISARYSAVYSIHPSWAAWGQAILEGELAWLEGGGGGAGQLADLKRQLAWWKQAAVSVSHFQKVWVVAVPMTYRAA(SEQ ID NO:32)。

fusion block 3 comprises the amino acid sequence shown below:

AVPMTYRAAYASYHCWCWRDPGRSAAWGQAILEGELAWLEGGGGGAGQLADLKRQLAWWKQAAKDWSISARYSAVYSIHPSWVSVSHFQKVWVV(SEQ ID NO:33)。

fusion block 4 comprises the amino acid sequence shown below:

KDWSISARYSAVYSIHPSWAVPMTYRAAYASYHCWCWRDPGRSVSVSHFQKVWVVAAWGQAILEGELAWLEGGGGGAGQLADLKRQLAWWKQAA(SEQ ID NO:34)。

example 3 evaluation of anti-tumor Effect of recombinant proteins

In this example, the recombinant proteins Block-1, 2, 3, 4, 5, fusion-Block-S and Fusion-Block-1-4 obtained in example 2 were tested for their antitumor ability, and specific experimental procedures were as follows:

(1) Construction of tumor-bearing mouse model

The inventors constructed a melanoma tumor model by inoculating 50, 6-week-old male C57BL/6 mice with melanoma (B16F 10-Ova) cells (2X 10) ⁵ /v), tumor formation after 12 d.

(2) Tumor model mouse treatment

Control and experimental groups were set up and the OT-1EGFP obtained in example 2 was purified using flow cytometry ⁺ After positive cells are sorted, the positive cells are infused back into a melanin tumor model body through orbital intravenous injection, and the infusion quantity is 2 x 10 ⁵ -5*10 ⁵ /only. Wherein, the control group was given T cells not transduced and expressing short peptide (i.e., T cells transduced with empty plasmid used in example 1) to the tumor mice respectively for adoptive feedback, the experimental group was given T cells transduced and expressing short peptide or fusion short peptide to the tumor mice respectively for adoptive feedback, and the mice after adoptive feedback were fed for 14 days, and tumor size and body weight of the mice were measured every three days after treatment, thereby evaluating the antitumor effect of the secretory immune checkpoint inhibitory peptide. When the tumor size is not more than 1000mm ³ At this time, tumor-infiltrating lymphocytes were isolated and analyzed for changes in tumor microenvironment.

(3) Treatment effect monitoring

Specific experimental results are shown in fig. 4 and 5, wherein fig. 4 shows that at day 14, all of block-1, 2, 3, 4, 5 and Fusion-block-1 can effectively slow down the growth of mouse tumor, and the effect of Fusion-block-1 is most remarkable, and block-5 (block-PPLC) times. FIG. 5 shows that Fusion-Block-1, 2, 3, 4, S was effective in slowing down tumor growth in mice at day 14, wherein Fusion-Block-S showed more excellent anti-tumor effects.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present application have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the application, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the application.

SEQUENCE LISTING

<110> Bozhen Biotechnology (Hangzhou) Co., ltd

<120> recombinant protein and use thereof

<130> PDI210493

<160> 34

<170> PatentIn version 3.5

<210> 1

<211> 57

<212> DNA

<213> Artificial Sequence

<220>

<223> 1

<400> 1

aaggactgga gtataagcgc ccgatactcc gcagtataca gtatccatcc ctcatgg 57

<210> 2

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> 2

<400> 2

gcagttccaa tgacctatcg cgccgca 27

<210> 3

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> 3

<400> 3

tacgccagct accactgctg gtgctggagg gaccccggca ggagc 45

<210> 4

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<223> 4

<400> 4

gtctccgtga gccacttcca gaaggtgtgg g 31

<210> 5

<211> 117

<212> DNA

<213> Artificial Sequence

<220>

<223> 5

<400> 5

gcagcctggg ggcaggccat attggaaggc gagcttgcat ggctggaggg cgggggcggc 60

ggtgctggcc aactggccga cctgaaacgg cagcttgctt ggtggaagca agctgca 117

<210> 6

<211> 60

<212> DNA

<213> Artificial Sequence

<220>

<223> 6

<400> 6

atgtacagga tgcagctcct gtcttgcatt gcactgagtc tggcactcgt cacaaacagc 60

<210> 7

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> 7

<400> 7

tatccatacg acgtgccaga ttatgct 27

<210> 8

<211> 277

<212> DNA

<213> Artificial Sequence

<220>

<223> 8

<400> 8

aaggactgga gtataagcgc ccgatactcc gcagtataca gtatccatcc ctcatgggca 60

gttccaatga cctatcgcgc cgcatacgcc agctaccact gctggtgctg gagggacccc 120

ggcaggagcg tctccgtgag ccacttccag aaggtgtggg gcagcctggg ggcaggccat 180

attggaaggc gagcttgcat ggctggaggg cgggggcggc ggtgctggcc aactggccga 240

cctgaaacgg cagcttgctt ggtggaagca agctgca 277

<210> 9

<211> 277

<212> DNA

<213> Artificial Sequence

<220>

<223> 9

<400> 9

tacgccagct accactgctg gtgctggagg gaccccggca ggagcgcagc ctgggggcag 60

gccatattgg aaggcgagct tgcatggctg gagggcgggg gcggcggtgc tggccaactg 120

gccgacctga aacggcagct tgcttggtgg aagcaagctg caaaggactg gagtataagc 180

gcccgatact ccgcagtata cagtatccat ccctcatggg cagttccaat gacctatcgc 240

gccgcagtct ccgtgagcca cttccagaag gtgtggg 277

<210> 10

<211> 232

<212> DNA

<213> Artificial Sequence

<220>

<223> 10

<400> 10

gtctccgtga gccacttcca gaaggtgtgg gaaggactgg agtataagcg cccgatactc 60

cgcagtatac agtatccatc cctcatgggc agcctggggg caggccatat tggaaggcga 120

gcttgcatgg ctggagggcg ggggcggcgg tgctggccaa ctggccgacc tgaaacggca 180

gcttgcttgg tggaagcaag ctgcagcagt tccaatgacc tatcgcgccg ca 232

<210> 11

<211> 277

<212> DNA

<213> Artificial Sequence

<220>

<223> 11

<400> 11

tacgccagct accactgctg gtgctggagg gaccccggca ggagcaagga ctggagtata 60

agcgcccgat actccgcagt atacagtatc catccctcat gggcagcctg ggggcaggcc 120

atattggaag gcgagcttgc atggctggag ggcgggggcg gcggtgctgg ccaactggcc 180

gacctgaaac ggcagcttgc ttggtggaag caagctgcag tctccgtgag ccacttccag 240

aaggtgtggg gcagttccaa tgacctatcg cgccgca 277

<210> 12

<211> 277

<212> DNA

<213> Artificial Sequence

<220>

<223> 12

<400> 12

gcagttccaa tgacctatcg cgccgcatac gccagctacc actgctggtg ctggagggac 60

cccggcagga gcgcagcctg ggggcaggcc atattggaag gcgagcttgc atggctggag 120

ggcgggggcg gcggtgctgg ccaactggcc gacctgaaac ggcagcttgc ttggtggaag 180

caagctgcaa aggactggag tataagcgcc cgatactccg cagtatacag tatccatccc 240

tcatgggtct ccgtgagcca cttccagaag gtgtggg 277

<210> 13

<211> 144

<212> DNA

<213> Artificial Sequence

<220>

<223> 13

<400> 13

atgtacagga tgcagctcct gtcttgcatt gcactgagtc tggcactcgt cacaaacagc 60

aaggactgga gtataagcgc ccgatactcc gcagtataca gtatccatcc ctcatggtat 120

ccatacgacg tgccagatta tgct 144

<210> 14

<211> 114

<212> DNA

<213> Artificial Sequence

<220>

<223> 14

<400> 14

atgtacagga tgcagctcct gtcttgcatt gcactgagtc tggcactcgt cacaaacagc 60

gcagttccaa tgacctatcg cgccgcatat ccatacgacg tgccagatta tgct 114

<210> 15

<211> 132

<212> DNA

<213> Artificial Sequence

<220>

<223> 15

<400> 15

atgtacagga tgcagctcct gtcttgcatt gcactgagtc tggcactcgt cacaaacagc 60

tacgccagct accactgctg gtgctggagg gaccccggca ggagctatcc atacgacgtg 120

ccagattatg ct 132

<210> 16

<211> 118

<212> DNA

<213> Artificial Sequence

<220>

<223> 16

<400> 16

atgtacagga tgcagctcct gtcttgcatt gcactgagtc tggcactcgt cacaaacagc 60

gtctccgtga gccacttcca gaaggtgtgg gtatccatac gacgtgccag attatgct 118

<210> 17

<211> 204

<212> DNA

<213> Artificial Sequence

<220>

<223> 17

<400> 17

atgtacagga tgcagctcct gtcttgcatt gcactgagtc tggcactcgt cacaaacagc 60

gcagcctggg ggcaggccat attggaaggc gagcttgcat ggctggaggg cgggggcggc 120

ggtgctggcc aactggccga cctgaaacgg cagcttgctt ggtggaagca agctgcatat 180

ccatacgacg tgccagatta tgct 204

<210> 18

<211> 364

<212> DNA

<213> Artificial Sequence

<220>

<223> 18

<400> 18

atgtacagga tgcagctcct gtcttgcatt gcactgagtc tggcactcgt cacaaacagc 60

aaggactgga gtataagcgc ccgatactcc gcagtataca gtatccatcc ctcatgggca 120

gttccaatga cctatcgcgc cgcatacgcc agctaccact gctggtgctg gagggacccc 180

ggcaggagcg tctccgtgag ccacttccag aaggtgtggg gcagcctggg ggcaggccat 240

attggaaggc gagcttgcat ggctggaggg cgggggcggc ggtgctggcc aactggccga 300

cctgaaacgg cagcttgctt ggtggaagca agctgcatat ccatacgacg tgccagatta 360

tgct 364

<210> 19

<211> 364

<212> DNA

<213> Artificial Sequence

<220>

<223> 19

<400> 19

atgtacagga tgcagctcct gtcttgcatt gcactgagtc tggcactcgt cacaaacagc 60

tacgccagct accactgctg gtgctggagg gaccccggca ggagcgcagc ctgggggcag 120

gccatattgg aaggcgagct tgcatggctg gagggcgggg gcggcggtgc tggccaactg 180

gccgacctga aacggcagct tgcttggtgg aagcaagctg caaaggactg gagtataagc 240

gcccgatact ccgcagtata cagtatccat ccctcatggg cagttccaat gacctatcgc 300

gccgcagtct ccgtgagcca cttccagaag gtgtgggtat ccatacgacg tgccagatta 360

tgct 364

<210> 20

<211> 319

<212> DNA

<213> Artificial Sequence

<220>

<223> 20

<400> 20

atgtacagga tgcagctcct gtcttgcatt gcactgagtc tggcactcgt cacaaacagc 60

gtctccgtga gccacttcca gaaggtgtgg gaaggactgg agtataagcg cccgatactc 120

cgcagtatac agtatccatc cctcatgggc agcctggggg caggccatat tggaaggcga 180

gcttgcatgg ctggagggcg ggggcggcgg tgctggccaa ctggccgacc tgaaacggca 240

gcttgcttgg tggaagcaag ctgcagcagt tccaatgacc tatcgcgccg catatccata 300

cgacgtgcca gattatgct 319

<210> 21

<211> 364

<212> DNA

<213> Artificial Sequence

<220>

<223> 21

<400> 21

atgtacagga tgcagctcct gtcttgcatt gcactgagtc tggcactcgt cacaaacagc 60

tacgccagct accactgctg gtgctggagg gaccccggca ggagcaagga ctggagtata 120

agcgcccgat actccgcagt atacagtatc catccctcat gggcagcctg ggggcaggcc 180

atattggaag gcgagcttgc atggctggag ggcgggggcg gcggtgctgg ccaactggcc 240

gacctgaaac ggcagcttgc ttggtggaag caagctgcag tctccgtgag ccacttccag 300

aaggtgtggg gcagttccaa tgacctatcg cgccgcatat ccatacgacg tgccagatta 360

tgct 364

<210> 22

<211> 364

<212> DNA

<213> Artificial Sequence

<220>

<223> 22

<400> 22

atgtacagga tgcagctcct gtcttgcatt gcactgagtc tggcactcgt cacaaacagc 60

gcagttccaa tgacctatcg cgccgcatac gccagctacc actgctggtg ctggagggac 120

cccggcagga gcgcagcctg ggggcaggcc atattggaag gcgagcttgc atggctggag 180

ggcgggggcg gcggtgctgg ccaactggcc gacctgaaac ggcagcttgc ttggtggaag 240

caagctgcaa aggactggag tataagcgcc cgatactccg cagtatacag tatccatccc 300

tcatgggtct ccgtgagcca cttccagaag gtgtgggtat ccatacgacg tgccagatta 360

tgct 364

<210> 23

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> 23

<400> 23

Lys Asp Trp Ser Ile Ser Ala Arg Tyr Ser Ala Val Tyr Ser Ile His

1 5 10 15

Pro Ser Trp

<210> 24

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> 24

<400> 24

Ala Val Pro Met Thr Tyr Arg Ala Ala

1 5

<210> 25

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> 25

<400> 25

Tyr Ala Ser Tyr His Cys Trp Cys Trp Arg Asp Pro Gly Arg Ser

1 5 10 15

<210> 26

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> 26

<400> 26

Val Ser Val Ser His Phe Gln Lys Val Trp Val Val

1 5 10

<210> 27

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> 27

<400> 27

Ala Ala Trp Gly Gln Ala Ile Leu Glu Gly Glu Leu Ala Trp Leu Glu

1 5 10 15

Gly Gly Gly Gly Gly Ala Gly Gln Leu Ala Asp Leu Lys Arg Gln Leu

20 25 30

Ala Trp Trp Lys Gln Ala Ala

35

<210> 28

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> 28

<400> 28

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser

20

<210> 29

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> 29

<400> 29

Tyr Pro Tyr Asp Val Pro Asp Tyr Ala

1 5

<210> 30

<211> 94

<212> PRT

<213> Artificial Sequence

<220>

<223> 30

<400> 30

Tyr Ala Ser Tyr His Cys Trp Cys Trp Arg Asp Pro Gly Arg Ser Ala

1 5 10 15

Ala Trp Gly Gln Ala Ile Leu Glu Gly Glu Leu Ala Trp Leu Glu Gly

20 25 30

Gly Gly Gly Gly Ala Gly Gln Leu Ala Asp Leu Lys Arg Gln Leu Ala

35 40 45

Trp Trp Lys Gln Ala Ala Lys Asp Trp Ser Ile Ser Ala Arg Tyr Ser

50 55 60

Ala Val Tyr Ser Ile His Pro Ser Trp Ala Val Pro Met Thr Tyr Arg

65 70 75 80

Ala Ala Val Ser Val Ser His Phe Gln Lys Val Trp Val Val

85 90

<210> 31

<211> 94

<212> PRT

<213> Artificial Sequence

<220>

<223> 31

<400> 31

Val Ser Val Ser His Phe Gln Lys Val Trp Val Val Lys Asp Trp Ser

1 5 10 15

Ile Ser Ala Arg Tyr Ser Ala Val Tyr Ser Ile His Pro Ser Trp Tyr

20 25 30

Ala Ser Tyr His Cys Trp Cys Trp Arg Asp Pro Gly Arg Ser Ala Ala

35 40 45

Trp Gly Gln Ala Ile Leu Glu Gly Glu Leu Ala Trp Leu Glu Gly Gly

50 55 60

Gly Gly Gly Ala Gly Gln Leu Ala Asp Leu Lys Arg Gln Leu Ala Trp

65 70 75 80

Trp Lys Gln Ala Ala Ala Val Pro Met Thr Tyr Arg Ala Ala

85 90

<210> 32

<211> 94

<212> PRT

<213> Artificial Sequence

<220>

<223> 32

<400> 32

Tyr Ala Ser Tyr His Cys Trp Cys Trp Arg Asp Pro Gly Arg Ser Lys

1 5 10 15

Asp Trp Ser Ile Ser Ala Arg Tyr Ser Ala Val Tyr Ser Ile His Pro

20 25 30

Ser Trp Ala Ala Trp Gly Gln Ala Ile Leu Glu Gly Glu Leu Ala Trp

35 40 45

Leu Glu Gly Gly Gly Gly Gly Ala Gly Gln Leu Ala Asp Leu Lys Arg

50 55 60

Gln Leu Ala Trp Trp Lys Gln Ala Ala Val Ser Val Ser His Phe Gln

65 70 75 80

Lys Val Trp Val Val Ala Val Pro Met Thr Tyr Arg Ala Ala

85 90

<210> 33

<211> 94

<212> PRT

<213> Artificial Sequence

<220>

<223> 33

<400> 33

Ala Val Pro Met Thr Tyr Arg Ala Ala Tyr Ala Ser Tyr His Cys Trp

1 5 10 15

Cys Trp Arg Asp Pro Gly Arg Ser Ala Ala Trp Gly Gln Ala Ile Leu

20 25 30

Glu Gly Glu Leu Ala Trp Leu Glu Gly Gly Gly Gly Gly Ala Gly Gln

35 40 45

Leu Ala Asp Leu Lys Arg Gln Leu Ala Trp Trp Lys Gln Ala Ala Lys

50 55 60

Asp Trp Ser Ile Ser Ala Arg Tyr Ser Ala Val Tyr Ser Ile His Pro

65 70 75 80

Ser Trp Val Ser Val Ser His Phe Gln Lys Val Trp Val Val

85 90

<210> 34

<211> 94

<212> PRT

<213> Artificial Sequence

<220>

<223> 34

<400> 34

Lys Asp Trp Ser Ile Ser Ala Arg Tyr Ser Ala Val Tyr Ser Ile His

1 5 10 15

Pro Ser Trp Ala Val Pro Met Thr Tyr Arg Ala Ala Tyr Ala Ser Tyr

20 25 30

His Cys Trp Cys Trp Arg Asp Pro Gly Arg Ser Val Ser Val Ser His

35 40 45

Phe Gln Lys Val Trp Val Val Ala Ala Trp Gly Gln Ala Ile Leu Glu

50 55 60

Gly Glu Leu Ala Trp Leu Glu Gly Gly Gly Gly Gly Ala Gly Gln Leu

65 70 75 80

Ala Asp Leu Lys Arg Gln Leu Ala Trp Trp Lys Gln Ala Ala

85 90

Claims (14)

1. A recombinant protein comprising at least two of the following:

d4-2, C25, TPP-1, PPL-C and ERY2-4.

2. The recombinant protein according to claim 1, including D4-2, C25, TPP-1, PPL-C and ERY2-4.

3. The recombinant protein according to claim 2, wherein said D4-2C-terminus is linked to said C25N-terminus, said C25C-terminus is linked to said TPP-1N-terminus, said TPP-1C-terminus is linked to said PPL-C N-terminus, and said PPL-C-terminus is linked to said ERY 2-4N-terminus.

4. The recombinant protein according to claim 1, wherein said recombinant protein has an amino acid sequence as shown in SEQ ID No. 30, or an amino acid sequence having at least 95%, 90%, 85% identity with the amino acid sequence as shown in SEQ ID No. 25.

5. A nucleic acid encoding the recombinant protein of any one of claims 1-4.

6. The nucleic acid of claim 5, wherein the nucleic acid has the sequence of SEQ ID NO:8, and a nucleotide sequence shown in SEQ ID NO.

7. An expression vector comprising the nucleic acid of claim 5 or 6.

8. A method for preparing the recombinant protein according to any one of claims 1 to 4, comprising:

introducing the expression vector of claim 7 into a recipient cell;

the recipient cells carrying the expression vector are cultured under conditions suitable for the expression and secretion of the protein, so as to obtain the recombinant protein.

9. The method of claim 8, wherein the recipient cell comprises a eukaryotic cell;

optionally, the eukaryotic cell comprises a mammalian cell,

optionally, the mammalian cells comprise at least one selected from the group consisting of T cells, CHO-K1, CHOs, 293F, 293T, CAR-T, TCR-T, TIL, CAR-NK, CAR-M, and CAR-DC.

10. A recombinant cell carrying the nucleic acid of claim 5 or 6, the expression vector of claim 7 or the recombinant protein of any one of claims 1-4.

11. An immune effector cell carrying the nucleic acid of claim 5 or 6, the expression vector of claim 7, or the recombinant protein of any one of claims 1-4;

optionally, the immune effector cell is a T cell, preferably CD8 ⁺ T cells.

12. A composition, comprising:

the recombinant protein of any one of claims 1-4, the nucleic acid of claim 5 or 6, the expression vector of claim 7, the recombinant cell of claim 10, or the immune effector cell of claim 11.

13. Use of the recombinant protein of any one of claims 1-4, the nucleic acid of claim 5 or 6, the expression vector of claim 7, the recombinant cell of claim 10, the immune effector cell of claim 11, or the composition of claim 12 in the manufacture of a medicament for treating or preventing cancer;

optionally, the cancer comprises at least one selected from the group consisting of: gastric cancer, intestinal cancer, head and neck cancer and melanoma.

14. A medicament, comprising: the recombinant protein of any one of claims 1-4, the nucleic acid of claim 5 or 6, the expression vector of claim 7, the recombinant cell of claim 10, the immune effector cell of claim 11, or the composition of claim 12, for use in the treatment or prevention of cancer;

optionally, the cancer comprises at least one selected from the group consisting of: gastric cancer, intestinal cancer, head and neck cancer and melanoma.