CN117624379A - Artificial protein and application thereof - Google Patents
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- CN117624379A CN117624379A CN202311540756.0A CN202311540756A CN117624379A CN 117624379 A CN117624379 A CN 117624379A CN 202311540756 A CN202311540756 A CN 202311540756A CN 117624379 A CN117624379 A CN 117624379A
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the technical field of artificial proteins, and particularly relates to an artificial protein and application thereof. The artificial protein has unique in-vivo and in-vitro immunocompetence and can meet the requirement of recovering the immune function of a patient; namely, the artificial protein structure can not only identify the exhausted immune cells, but also increase invasiveness and restore functions; and the clinical application of the composition can enhance the functions of inhibiting tumor growth and controlling virus infection, and has good clinical prospect and wide application range.
Description
Technical Field
The invention belongs to the technical field of artificial proteins, and particularly relates to an artificial protein and application thereof.
Background
Tumor occurs as a result of mutation of genes during division of cells in the body, and loss of control of growth of the mutated cells. If tumor cells cannot be cleared in a short period of time, continued antigen stimulation will gradually cause functional failure of tumor antigen specific T cells, forming tolerance to the tumor, the immune system is no longer sensitive to tumor cells, causing further growth and spread of the tumor, eventually forming cancer. The occurrence of chronic diseases caused by viral infection is also a consequence of antigen-specific cell failure, and numerous studies have demonstrated this conclusion.
Studies have also shown that due to sustained antigen stimulation, most Tumor Infiltrating Lymphocytes (TILs) and antiviral specific T cells highly express immunosuppressive receptors, in a state of functional failure, weakening or losing the function of specifically recognizing and killing antigen positive target cells. The depleting immune cells lose the ability to secrete functional cytokines, such as gamma interferon (ifnγ) and the like. Even if there are a large number of specific immune cells in the organism, the depleted immune cells cannot clear antigen positive target cells. Phenotypically, immunosuppressive checkpoint receptors that are ubiquitously highly expressed by depleting T cells include the programmed death receptor (PD-1), programmed death ligand (PD-L1/L2), T cell immunoglobulin 3 (Tim-3), cytotoxic T lymphocyte antigen 4 (CTLA-4), and lymphocyte activating gene 3 (Lag-3), among others. Activation of these signaling pathways can inhibit the phosphorylation reaction by inducing phosphatases, attenuate immune cell activation signals, inhibit the expansion of immune cells, reduce the function of effector immune cells, promote apoptosis of T cells, and cause immune tolerance. Therefore, the antigen specific immune cell population is activated, so that the antigen specific immune cell population can restore the function of killing antigen positive target cells, and then tumor cells or virus infected cells can be eliminated, and finally the aim of curing is achieved.
Antibodies specifically recognize protein antigens on the surface of target cells, checkpoint inhibitory antibodies suppress immunosuppressive signals on the surface of depleting cells, e.g., prevent the transduction and activation of inhibitory signals by binding of PD-1 antibodies (Pembrolizumab and Nivolumab) to PD-1, restore the function of depleting immune cells, and have been shown clinically to treat cancer, particularly in some patients, with complete tumor disappearance and longer duration of clinical effects. However, the clinical effect on patients lacking effector cells is not ideal, and the recovery time of such T cells is short, and specific immune cells in the patient can quickly return to the depleted state. At the same time, blocking T cell inhibition signals alone does not result in an increase in immune cell numbers, while patients lacking immune effector cells do not have substantially the clinical effect, thus further limiting the clinical therapeutic effect of checkpoint antibodies.
Cytokines have been approved for cancer treatment, and Interleukin-2 (IL-2) was approved in 1992 for colorectal cancer treatment. However, only a small proportion of patients benefit, while a large proportion of patients have serious side effects. High doses of IL-2 can cause shock and even death in patients, and finally, clinical trials of IL-15 treatment of tumors have not produced significant clinical effects.
CD137 expresses a TNFR receptor after T cells are stimulated, and the interaction of CD137-CD137L can cause immune cells to expand and survive, strengthen the functions of the immune cells and prevent the apoptosis of the immune cells. In antigen-stimulatory cells, CD137 can promote the function of antigen-presenting cells, enhancing activation of T cells. Experiments prove that the activation of CD137 molecule can increase the activity of antigen specific immune cells. Animal experiments show that only CD137 + Has the effect of controlling the growth of tumor. In recent years, CD137 + T cells controlling the exacerbation of tumor patients have further demonstrated that their expression in tumor microenvironment may be inhibited by PD-1 with reduced activity. Clinical studies have shown that CD137 expression in lymphocytes is positively correlated with survival in patients with colorectal cancer. Furthermore, in lung cancer patients, CD137 + Cell reduction is inversely related to patient survival. At the same time, CD8 + CD137 + The proportion of positive T cells has a clear positive correlation with survival rate of cancer patients, and a high proportion of CD3 + CD137 + PD1 + Immune cells significantly reduce the chance of patient deterioration.
In view of the above, up to now, there is no protein drug in the art that can specifically recognize depleting immune cells and increase immune cell function.
Disclosure of Invention
The invention aims to provide an artificial protein and application thereof, wherein the artificial protein can not only be used for depleting immune cells, but also can be used for enhancing the functions of the immune cells.
The invention provides an artificial protein, which comprises an identification functional region, an activation functional region and a nonfunctional amino acid fragment connected with the identification functional region and the activation functional region; the C end of the identification functional region is connected with the N end of the activation functional region through a nonfunctional amino acid fragment;
the recognition functional region comprises a PD-1 single-chain antibody, wherein the amino acid of the PD-1 single-chain antibody is shown as SEQ ID NO.1 or a mutant sequence with the same function as the amino acid shown as SEQ ID NO. 1;
the activating functional region includes cytokines IL-2, IL-7, IL-15, or IL-21.
Preferably, the amino acid sequence of the cytokine IL-2 is shown as SEQ ID NO.2, or a mutant sequence with the same function as the amino acid shown as SEQ ID NO. 2;
the amino acid sequence of the cytokine IL-7 is shown as SEQ ID NO.3, or a mutant sequence with the same function as the amino acid shown as SEQ ID NO. 3;
the amino acid sequence of the cytokine IL-15 is shown as SEQ ID NO.4, or a mutant sequence with the same function as the amino acid shown as SEQ ID NO. 4;
the amino acid sequence of the cytokine IL-21 is shown as SEQ ID NO.5, or a mutant sequence with the same function as the amino acid shown as SEQ ID NO. 5;
preferably, the amino acid sequence of the nonfunctional amino acid fragment is shown in SEQ ID NO. 6.
Preferably, the amino acid sequence of the artificial protein is shown as SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO. 10.
The invention also provides application of the artificial protein in preparation of medicines for recovering immune cell functions.
Preferably, the immune cells comprise T cells and/or NK cells.
Preferably, the medicament comprises a medicament that simultaneously recognizes depleting immune cells and/or enhances immune cell function.
Preferably, the medicament comprises a medicament for the treatment of cancer and/or chronic viral infection.
Preferably, the cancer comprises one or more of renal cell carcinoma, melanoma, lymphoma, colorectal cancer, liver cancer, head and neck squamous carcinoma, bladder cancer, and lung cancer.
The invention also provides a medicine for recovering the immune cell function, which comprises the artificial protein in the technical scheme.
The beneficial effects are that:
the invention provides an artificial protein, which comprises an identification functional region, an activation functional region and a nonfunctional amino acid fragment connected with the identification functional region and the activation functional region; the C end of the identification functional region is connected with the N end of the activation functional region through a nonfunctional amino acid fragment; the recognition functional region comprises a PD-1 single-chain antibody, and the amino acid of the PD-1 single-chain antibody is shown as SEQ ID NO. 1; the activating functional region includes cytokines IL-2, IL-7, IL-15, or IL-21. The artificial protein has unique in-vivo and in-vitro immunocompetence and can meet the requirement of recovering the immune function of a patient; namely, the artificial protein structure can not only identify the exhausted immune cells, but also increase invasiveness and restore functions; and the clinical application of the composition can enhance the functions of inhibiting tumor growth and controlling virus infection, and has good clinical prospect and wide application range.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is an electrophoresis detection diagram of the recombinant fusion artificial protein in examples 1 to 4;
FIG. 2 is a graph showing the results of in vitro culture activity and function assays of recombinant fusion artificial proteins on human T cells in example 1 and control;
FIG. 3 is a graph showing the results of in vitro culture activity and function measurement of human T cells by recombinant fusion artificial proteins in examples 2 to 4.
Detailed Description
The invention provides an artificial protein, which comprises an identification functional region, an activation functional region and a nonfunctional amino acid fragment connected with the identification functional region and the activation functional region; the C end of the identification functional region is connected with the N end of the activation functional region through a nonfunctional amino acid fragment;
the recognition functional region comprises a PD-1 single-chain antibody, wherein the amino acid of the PD-1 single-chain antibody is shown as SEQ ID NO.1 or a mutant sequence with the same function as the amino acid shown as SEQ ID NO. 1;
the activating functional region includes cytokines IL-2, IL-7, IL-15, or IL-21.
In the invention, the amino acid sequence shown in SEQ ID NO.1 specifically comprises the following steps: EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKGGGGSGGGGSGGGGSGGGSGGGSQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS. The recognition functional region of the invention recognizes a phenotype receptor of an depleting immune cell, and the phenotype receptor of the depleting immune cell is an immune checkpoint PD-1 with a co-inhibitory function. Such receptor genes are overexpressed on the surface of immune cells, and activation of this signaling pathway leads to failure and loss of function of immune cells.
In the invention, the amino acid sequence of the cytokine IL-2 is preferably shown as SEQ ID NO.2 or a mutant sequence with the same function as the amino acid shown as SEQ ID NO. 2; the amino acid sequence shown in SEQ ID NO.2 specifically comprises the following steps: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP KLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLIS NINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT.
The amino acid sequence of the cytokine IL-7 is preferably shown as SEQ ID NO.3 or a mutant sequence with the same function as the amino acid shown as SEQ ID NO. 3; the amino acid sequence shown in SEQ ID NO.3 specifically comprises the following steps: DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNN EFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTIL LNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTC WNKILMGTKEH.
The amino acid sequence of the cytokine IL-15 is preferably shown as SEQ ID NO.4 or a mutant sequence with the same function as the amino acid shown as SEQ ID NO. 4; the amino acid sequence shown in SEQ ID NO.4 specifically comprises the following steps: GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQS MHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANN SLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS.
The amino acid sequence of the cytokine IL-21 is preferably shown as SEQ ID NO.5 or a mutant sequence with the same function as the amino acid shown as SEQ ID NO. 5; the amino acid sequence shown in SEQ ID NO.5 specifically comprises the following steps: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPE DVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQK HRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS.
The cytokine moiety of the present invention binds to its receptor expressed on the surface of T cells and NK cells, thereby exerting activating and invasive functions. Cytokines are either present in mutant form, which means that their effect on non-target cells is enhanced or reduced by mutation of the amino acid sequence or by mere use of a functional region polypeptide, without altering their essential function. For example, by altering the amino acid sequence of a cytokine, the mutant may reduce or increase activation of T regulatory cells, reduce effects on non-target cells and tissue organs, and thereby reduce side effects for clinical use.
In the present invention, the amino acid sequence of the non-functional amino acid fragment is preferably as shown in SEQ ID NO.6, specifically: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. The nonfunctional amino acid fragment plays a role in connecting and identifying the functional region protein and activating the functional region protein.
In the invention, the amino acid sequence of the artificial protein is shown as SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO. 10. The amino acid sequence shown in SEQ ID NO.7 of the invention specifically comprises the following steps: EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQK PGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKGGGGSGGGGSGGGGSGGGSGGGSQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT; the amino acid sequence shown in SEQ ID NO.8 specifically comprises the following steps: EIVLTQSPATLSLSPGERATLSCRASKGVSTS GYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKGGGGSGGGGSGGGGSGGGSGGGSQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH; the amino acid sequence shown in SEQ ID NO.9 specifically comprises the following steps: EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAP RLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKGGGGSGGGGSGGGGSGGGSGGGSQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS; the amino acid sequence shown in SEQ ID NO.10 specifically comprises the following steps: EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSY LHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKGGGGSGGGGSGGGGSGGGSGGGSQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS. In the invention, the SEQ ID NO.7 is formed by sequentially connecting a PD-1 single-chain antibody shown as SEQ ID NO.1, a nonfunctional amino acid fragment shown as SEQ ID NO.6, a linker 'GGGS' and a cytokine IL-2 shown as SEQ ID NO. 2; the SEQ ID NO.8 is formed by sequentially connecting a PD-1 single-chain antibody shown as SEQ ID NO.1, a nonfunctional amino acid fragment shown as SEQ ID NO.6, a linker 'GGGS' and a cytokine IL-7 shown as SEQ ID NO. 3; the SEQ ID NO.9 is formed by sequentially connecting a PD-1 single-chain antibody shown as SEQ ID NO.1, a nonfunctional amino acid fragment shown as SEQ ID NO.6, a linker 'GGGS' and a cytokine IL-15 shown as SEQ ID NO. 4; the SEQ ID NO.10 is formed by sequentially connecting a PD-1 single-chain antibody shown as SEQ ID NO.1, a nonfunctional amino acid fragment shown as SEQ ID NO.6, a linker 'GGGS' and a cytokine IL-21 shown as SEQ ID NO. 5.
The recognition functional region in the artificial protein shown in SEQ ID NO. 7-10 recognizes the immune cells with failure and is a receptor PD-1 for recognizing the phenotype of the immune cells with failure by using a PD-1 single-chain antibody; the activating functional region adopts IL-2, IL-7, IL-15 or IL-21 to activate immune cells, thereby achieving the functions of identifying the failure immune cells and enhancing the functions of the immune cells.
The invention also provides application of the artificial protein in preparation of medicines for recovering immune cell functions. In the present invention the immune cells preferably comprise T cells and/or NK cells, more preferably T cells or NK cells; the T cells preferably comprise specific T cells.
The medicament of the invention preferably comprises a medicament for simultaneously recognizing an depleting immune cell and/or enhancing immune cell function, further comprises a medicament for simultaneously recognizing an depleting immune cell and enhancing immune cell function, and more preferably comprises a medicament for simultaneously blocking immune cell PD-1 expression and increasing CD137 expression. The enhancing cellular functions of the present invention preferably includes increasing invasive functions of immune cells.
The medicament of the invention preferably comprises a medicament for the treatment of cancer and/or chronic viral infections, more preferably a medicament for the treatment of cancer and chronic viral infections. The cancer of the present invention preferably comprises one or more of renal cell carcinoma, melanoma, lymphoma, colorectal cancer, liver cancer, head and neck squamous carcinoma, bladder cancer and lung cancer.
The invention also provides a medicine for recovering the immune cell function, which comprises the artificial protein in the technical scheme. The artificial protein of the present invention is preferably an active ingredient in the drug. The medicaments of the invention are preferably used alone or in combination with other medicaments or methods of treatment; the other drugs preferably include chemotherapeutic drugs, targeted drugs or antibody drugs; the method of treatment preferably comprises cytotherapeutic live radiation therapy.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
The construction, production and purification of the gene of the artificial protein structure comprises the following steps:
1) According to the C-terminal amino acid sequence of the human PD-1 single-chain antibody (SEQ ID NO. 1) and the N-terminal amino acid sequence of interleukin 2 (SEQ ID NO. 2), through corresponding base synthesis, enzyme digestion and further cloning, the C-terminal amino acid sequence of the human PD-1 single-chain antibody (SEQ ID NO. 1) and the N-terminal amino acid sequence of interleukin 2 (SEQ ID NO. 2) are connected through nonfunctional artificial construction amino acid (SEQ ID NO. 6) and linker 'GGGS', an artificial protein structure amino acid sequence (SEQ ID NO. 7) is formed, and a nucleotide sequence encoding the artificial protein structure amino acid sequence (SEQ ID NO. 7) is cloned into a pcDNA3.1 (-) vector.
2) The expression vector of the protein structure was transfected into chinese hamster ovary Cells (CHO). Transfected cells were placed at 37℃in 5% CO 2 Culturing in incubator, taking supernatant after one week, purifying by protein A affinity chromatography, and finally purifying protein to obtain designed recombinant fusion artificial protein.
Example 2
1) According to the N-terminal amino acid sequence of human PD-1 single-chain antibody (SEQ ID NO. 1) and interleukin 7 (SEQ ID NO. 3), through gene corresponding base synthesis, enzyme digestion and further cloning, the N-terminal amino acid sequence of human PD-1 single-chain antibody (SEQ ID NO. 1) and interleukin 7 (SEQ ID NO. 3) is connected through nonfunctional artificial construction amino acid (SEQ ID NO. 6) and linker 'GGGS', an artificial protein structure amino acid sequence (SEQ ID NO. 8) is formed, and a nucleotide sequence encoding the artificial protein structure amino acid sequence (SEQ ID NO. 8) is cloned into pcDNA3.1 (-) vector.
2) As in example 1.
Example 3
1) According to the C-terminal amino acid sequence of the human PD-1 single-chain antibody (SEQ ID NO. 1) and the N-terminal amino acid sequence of interleukin 15 (SEQ ID NO. 4), through gene corresponding base synthesis, enzyme digestion and further cloning, the C-terminal amino acid sequence of the human PD-1 single-chain antibody (SEQ ID NO. 1) and the N-terminal amino acid sequence of interleukin 15 (SEQ ID NO. 4) are connected through nonfunctional artificial construction amino acid (SEQ ID NO. 6) and linker 'GGGS', an artificial protein structure amino acid sequence (SEQ ID NO. 9) is formed, and a nucleotide sequence encoding the artificial protein structure amino acid sequence (SEQ ID NO. 8) is cloned into a pcDNA3.1 (-) vector.
2) As in example 1.
Example 4
1) According to the C-terminal amino acid sequence of the human PD-1 single-chain antibody (SEQ ID NO. 1) and the N-terminal amino acid sequence of interleukin 21 (SEQ ID NO. 5), through corresponding base synthesis, enzyme digestion and further cloning, the C-terminal amino acid sequence of the human PD-1 single-chain antibody (SEQ ID NO. 1) and the N-terminal amino acid sequence of interleukin 21 (SEQ ID NO. 5) are connected through nonfunctional artificial construction amino acid (SEQ ID NO. 6) and linker 'GGGS', an artificial protein structure amino acid sequence (SEQ ID NO. 10) is formed, and a nucleotide sequence encoding the artificial protein structure amino acid sequence (SEQ ID NO. 8) is cloned into a pcDNA3.1 (-) vector.
2) As in example 1.
The results of the electrophoresis test of the recombinant fusion artificial proteins prepared in examples 1 to 4 are shown in FIG. 1, wherein lanes 1 to 4 in FIG. 1 sequentially show the results of the display of the recombinant fusion artificial proteins in examples 1 to 4. The molecular weight of the purified protein structure was confirmed to be about 67kD by the electrophoresis detection in fig. 1, demonstrating that the recombinant artificial protein structure designed according to the present invention can be produced by CHO cells. Finally, the protein concentration was measured with a spectrophotometer and diluted in PBS for further in vivo and in vitro activity testing and functional studies.
Application example 1
The activity and function of the recombinant fusion artificial proteins in examples 1 to 4 on human T cells cultured in vitro were determined as follows:
human peripheral blood was isolated and purified into PBMC fraction by lymphocyte density gradient centrifugation (Ficoll) and then diluted to a cell density of 5X 10 in 24-well plates with X-Vivo15 medium 6 Per mL, adding test protein to a final concentration of 100ng/mL; wherein the test proteins are recombinant fusion artificial proteins in examples 1 to 4, respectively. Then placed at 37 ℃ and 5% CO 2 Culturing in incubator for 72h, staining cells after collection with corresponding flow antibody (Biolegend) (corresponding PD-1 antibody is APC and CD137 antibody is PE), performing phenotype measurement and data analysis by a flow cytometer after washing, and the results are shown in fig. 2-3, wherein positive rate of PD-1 is shown in the vertical direction in fig. 2-3, positive rate of CD137 is shown in the horizontal direction, and the Control group and the recombinant fusion artificial protein in example 1 are shown in the left-to-right direction in fig. 2, and the recombinant fusion artificial proteins in examples 2-4 are shown in the left-to-right direction in fig. 3.
In FIGS. 2-3, it is shown that the 4 artificial protein structure treatments significantly increased CD137 expression in T cells compared to the Control (Control) without the addition of the protein group, and that the CD137 positive rate in the specific Control group was only 4.06%, whereas the CD137 positive rates in the test groups with the recombinant fusion artificial proteins of examples 1-4 were 21.2%, 19.8%, 11.3% and 25.7% in this order. Meanwhile, compared with a Control (Control) without protein (7.64%), after the culture of 4 protein structures, the expression of PD-1 is obviously reduced, the positive rate of PD-1 in a specific Control group is as high as 7.64%, and the positive rate of PD-1 in a test group added with the recombinant fusion artificial protein in examples 1-4 is only 0.14%, 0.05%, 0.03% and 0.02% in sequence. Experiments prove that the recombinant protein structure designed according to the invention not only can activate human T cells and increase the expression of CD137 molecules, but also can shield the signal of PD-1+ on the cell surface so as to achieve the activity generated by designing the target protein structure.
As can be seen from the above examples, the artificial protein structure of the present invention can recognize the depleting immune cells, activate and increase the invasiveness of the immune cells, and restore the function of the immune cells to kill antigen-positive cells. The protein structure has the function of a non-protein structure, not only can activate immune cells, but also can shield the signal of PD-1, and induces the parent wetting of immune cell tumors. Because the occurrence and diffusion of tumors and chronic viral infection are the results of immune cell failure and immune system tolerance, the recovery of the functions of the failed immune cells and the number of amplified immune cells can enhance the anti-tumor and anti-chronic viral infection capability of organisms, and by analogy, the clinical application of the protein structure can inhibit the growth of tumors and control the viral infection, and has good clinical prospect and wide application range.
The artificial protein is a double-function recombinant protein structure, can be singly applied or combined with chemotherapy, targeted drugs, antibody drugs, cell therapy and radiotherapy, and is used for preparing drugs for treating diseases caused by immune cell failure, such as drugs for treating cancers, drugs for treating chronic viral infection and the like.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. An artificial protein comprising a recognition domain, an activation domain, and a non-functional amino acid fragment linking the recognition domain and the activation domain; the C end of the identification functional region is connected with the N end of the activation functional region through a nonfunctional amino acid fragment;
the recognition functional region comprises a PD-1 single-chain antibody, wherein the amino acid of the PD-1 single-chain antibody is shown as SEQ ID NO.1 or a mutant sequence with the same function as the amino acid shown as SEQ ID NO. 1;
the activating functional region includes cytokines IL-2, IL-7, IL-15, or IL-21.
2. The artificial protein according to claim 1, wherein the amino acid sequence of the cytokine IL-2 is shown as SEQ ID No.2 or a mutant sequence having the same function as the amino acid shown as SEQ ID No. 2;
the amino acid sequence of the cytokine IL-7 is shown as SEQ ID NO.3, or a mutant sequence with the same function as the amino acid shown as SEQ ID NO. 3;
the amino acid sequence of the cytokine IL-15 is shown as SEQ ID NO.4, or a mutant sequence with the same function as the amino acid shown as SEQ ID NO. 4;
the amino acid sequence of the cytokine IL-21 is shown as SEQ ID NO.5 or a mutant sequence with the same function as the amino acid shown as SEQ ID NO. 5.
3. The artificial protein according to claim 1 or 2, wherein the amino acid sequence of the nonfunctional amino acid fragment is shown in SEQ ID No. 6.
4. The artificial protein of claim 1, wherein the amino acid sequence of the artificial protein is as shown in SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 or SEQ ID No. 10.
5. Use of an artificial protein according to any one of claims 1 to 4 for the preparation of a medicament for restoring immune cell function.
6. The use according to claim 5, wherein the immune cells comprise T cells and/or NK cells.
7. The use according to claim 5 or 6, wherein the medicament comprises a medicament which simultaneously recognizes depleting immune cells and/or enhances immune cell function.
8. The use according to claim 5, wherein the medicament comprises a medicament for the treatment of cancer and/or chronic viral infection.
9. The use of claim 8, wherein the cancer comprises one or more of renal cell carcinoma, melanoma, lymphoma, colorectal cancer, liver cancer, head and neck squamous carcinoma, bladder cancer, and lung cancer.
10. A medicament for restoring immune cell function, comprising the artificial protein of any one of claims 1 to 4.
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