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CN112011833B - Method for screening and isolating tumor neoantigens - Google Patents

Method for screening and isolating tumor neoantigens Download PDF

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CN112011833B
CN112011833B CN201910465670.3A CN201910465670A CN112011833B CN 112011833 B CN112011833 B CN 112011833B CN 201910465670 A CN201910465670 A CN 201910465670A CN 112011833 B CN112011833 B CN 112011833B
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黄跃进
焦顺昌
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Shanghai Jenomed Biotech Co ltd
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Abstract

The present invention relates to methods of screening and isolating tumor neoantigens. In particular, the invention relates to the establishment of a plurality of tumor neoantigen display (display) peptide libraries or tumor neoantigen peptide libraries capable of binding to HLA class I or II (HLA diagnostic) molecules of cancer patients and activating TCR (tumor-specific tumor-infiltrating lymphocytes) of the cancer patients, screening and separating the tumor-specific tumor neoantigens of the patients; and, in 3-4 weeks, personalized cancer vaccines are prepared, thereby developing a rapid, highly effective personalized solid tumor immunotherapy regimen. The invention can accurately, rapidly and efficiently capture the specific tumor neoantigen of a patient capable of exciting immune response under the conditions of no wound and no need of tumor tissues or cells of the patient, and can be prepared into an individual cancer vaccine, and has wide application prospect in the fields of tumor, autoimmune diseases and infectious diseases treatment.

Description

Method for screening and isolating tumor neoantigens
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a method for screening and separating tumor neoantigens. More particularly, the present invention relates to methods for preparing and screening tumor neoantigen display (display) peptide libraries or tumor neoantigen peptide libraries capable of binding to HLA class I or II (HLA diagnostic) molecules of cancer patients and activating TCR of tumor-specific tumor infiltrating lymphocytes of cancer patients.
Background
In recent years, the immunotherapy of tumors mainly comprises an immunity check point inhibitor, adoptive cell feedback, tumor vaccine and the like, and has become a new direction of anti-tumor drugs. Currently, various immunotherapies have been successfully achieved and have achieved good clinical efficacy, such as foreign anti-PD-1 and PD-L1 mabs have been approved by the FDA for sale, with Opdivo and Keytruda approved in china and two CAR-T cell therapy products marketed abroad, kymriah for nova and Yescarta for ket.
The so-called personalized cancer vaccine, i.e. a therapeutic anticancer vaccine tailored to the tumor neoantigens specific to the tumor cells of cancer patients, is a high-level stage of personalized accurate medical development. Some proteins in tumor are genetically mutated, and these abnormal proteins are key to the immune system to recognize cancer cells, if the vaccine containing tumor neoantigen is injected into human body, the T cells of immune system can find out cancer cells with the protein antigen and destroy them.
The journal 2017 of nature discloses an important achievement obtained in the field of cancer research on line, and the two personalized cancer vaccines are safe and effective in advanced melanoma clinical tests, so that the method has a milestone significance for developing a rapid and efficient personalized cancer immunotherapy scheme. Of course, its use also faces challenges, for example, many tumors contain less tumor neoantigens; the vaccine preparation time is long and needs 6-12 weeks; the cancerous tissue of the advanced patient needs to be excised, and the somatic mutation of the tumor is found and confirmed. In particular, the accuracy of predicting tumor neoantigens based on biological software is low, so that the effectiveness of personalized cancer vaccine treatment is reduced, and the huge clinical treatment requirements of cancer patients are difficult to meet.
Thus, there is a pressing need in the art to develop methods that can increase the effectiveness of personalized cancer vaccine immunotherapy.
Disclosure of Invention
It is an object of the present invention to provide a method for improving the effectiveness of an personalized cancer vaccine immunotherapy.
In a first aspect of the present invention, there is provided a method of isolating and screening tumor neoantigens, the method comprising the steps of:
(a) Preparing one or more tumor neoantigen display peptide libraries capable of binding to HLA class I or II molecules of a cancer patient by phage display (PHAGE DISPLAY) or other display techniques; wherein the display peptide library has one or more of the following features:
(i) The length of a plurality of polypeptide chains which form the peptide library and can be combined with HLA class I molecules of cancer patients is 8-11 amino acid residues, wherein certain sites contain amino acid residues with the same or similar properties, and other sites contain any amino acid residue, or are longer or shorter, but still can be combined with the HLA class I molecules effectively;
(ii) The plurality of polypeptide chains comprising the peptide library which are capable of binding to HLA class II molecules of a cancer patient may be from 10 to 25 amino acid residues, more preferably from 13 to 18 amino acid residues, wherein some of the sites comprise amino acid residues of the same or similar nature and others comprise any amino acid residue, or longer or shorter, but still be capable of efficiently binding to HLA class II molecules;
(iii) The polypeptide chain contains an added specific epitope coding sequence marker at the N end or the C end for downstream screening;
(iv) One or more tumor neoantigen display peptide libraries prepared that are capable of binding to HLA class I or II molecules of a cancer patient may be derived from synthetic, and/or cancer patient tumor tissue, and/or tumor cells including Circulating Tumor Cells (CTCs), and/or tumor cell lines;
(b) One or more tumor neoantigens display peptide libraries capable of binding to HLA class I or II molecules of a cancer patient are individually subjected to one or more rounds of screening with cells such as CD4 or CD8 lymphocytes or other killer T lymphocytes, macrophages, NK cells, etc. isolated from the immune system of the cancer patient, or with Dendritic Cells (DCs) or other Antigen Presenting Cells (APCs) derived from the patient that have been previously primed (primed) with a peptide library polypeptide, or with a mixture of peptide library polypeptides that have been directly mixed with Dendritic Cells (DCs) or other Antigen Presenting Cells (APCs) derived from the patient, with the addition of a peptide library polypeptide, thereby isolating potential tumor neoantigens that activate patient-specific CD4 or CD8 lymphocytes or other killer T lymphocytes, macrophages, NK cells, etc.;
Or alternatively
Using multiple polyclonal antibodies with magnetic beads or other labels separated from peripheral blood of cancer patients, carrying out one or more rounds of screening on one or more prepared tumor neoantigen display peptide libraries capable of binding with HLA I or II molecules of the cancer patients, thereby separating potential tumor neoantigens capable of specifically binding with one or more antibodies;
(c) Immune cell-antigen validation experiments or antigen-antibody binding validation experiments were performed on the isolated potential, patient-specific tumor neoantigens, thereby confirming the patient-specific tumor neoantigen components.
In another preferred embodiment, in step (c), an in vitro (ex vivo) validation experiment of patient-specific CD4 or CD8 lymphocytes or other killer T lymphocytes, macrophages, NK cells, etc., or an antigen-antibody binding in vitro validation experiment is performed, thereby confirming the patient-specific tumor neoantigen component.
In another preferred embodiment, in step (c), a validation experiment of in vivo (in vivo) activation of patient-specific CD4 or CD8 lymphocytes or other killer T lymphocytes, macrophages, NK cells, etc. is performed to confirm the patient-specific tumor neoantigen component.
In another preferred embodiment, the peptide library may also be used to pre-warn the patient treated with PD-1 or a PD-L1 inhibitor or an autoimmune response caused by PD-1 or a PD-L1 inhibitor in a patient treated with PD-1 or a PD-1 and/or PD-L1 expression-deficient patient, thereby discriminating whether the patient is suitable for PD-1 or PD-L1 inhibitor immunotherapy and avoiding serious cardiotoxic side effects.
In another preferred embodiment, the display peptide library has features (i), (ii), (iii) and (iv); or (i), (iii) and (iv): or (ii), (iii) and (iv).
In another preferred embodiment, the phage display or other display technique includes, but is not limited to: phage display technology, yeast display technology, mRNA display technology, cell display technology, and the like.
In another preferred embodiment, the one or more tumor neoantigen display peptide libraries prepared are capable of binding to HLA class I or II molecules of a cancer patient, preferably 1-100 tumor neoantigen display peptide libraries, more preferably 1-50, such as 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 20, 30, 40, 50 tumor neoantigen display peptide libraries.
In another preferred embodiment, one or more of the tumor neoantigen display peptide libraries prepared for binding to HLA class I or II molecules of a cancer patient are subjected to one or more rounds of screening, preferably 1-20 rounds of screening, more preferably 1-10 rounds of screening, 1-5 rounds of screening, such as 1, 2, 3, 4, 5 rounds of screening, respectively.
In another preferred embodiment, other antigen presenting cells include, but are not limited to: mononuclear/macrophages, B lymphocytes, endothelial cells, fibroblasts, epithelial and mesenchymal cells, eosinophils, and the like.
In another preferred embodiment, the method comprises the steps of:
(a) Preparing one or more tumor neoantigen peptide libraries capable of binding to HLA class I or II molecules of cancer patients by bacterial expression or cell expression technology; wherein the peptide library has one or more of the following features:
(i) The plurality of polypeptide chains comprising the peptide library that are capable of binding to HLA class I molecules of a cancer patient can be 8 to 11 amino acid residues in length, wherein some of the sites comprise amino acid residues of the same or similar nature and other sites comprise any amino acid residue, or are longer or shorter, but still capable of efficiently binding to HLA class I molecules;
(ii) The plurality of polypeptide chains comprising the peptide library which are capable of binding to HLA class II molecules of a cancer patient may be from 10 to 25 amino acid residues, more preferably from 13 to 18 amino acid residues, wherein some of the sites comprise amino acid residues of the same or similar nature and others comprise any amino acid residue, or longer or shorter, but still be capable of efficiently binding to HLA class II molecules;
(iii) The polypeptide chain contains an added specific epitope coding sequence marker at the N end or the C end for downstream screening;
(iv) One or more tumor neoantigen peptide libraries prepared that bind to HLA class I or II molecules of a cancer patient may be derived from synthetic, and/or cancer patient tumor tissue, and/or tumor cells including CTCs, and/or tumor cell lines;
(b0) Sensitizing (priming) patient-specific dendritic cells or other antigen presenting cells in vitro with one or more tumor neoantigen polypeptides expressed in a tumor neoantigen peptide pool capable of binding to HLA class I or class II molecules of a cancer patient, thereby obtaining sensitized (primed) dendritic cells or other antigen presenting cells;
(b) One or more rounds of screening of the primed (primed) patient-specific dendritic cells or other antigen presenting cells, respectively, with magnetic beads or other labeled CD4 or CD8 lymphocytes or other killer T lymphocytes, macrophages, NK cells, etc., isolated from the immune system of a cancer patient, thereby isolating potential tumor neoantigens capable of activating patient-specific CD4 or CD8 lymphocytes or other killer T lymphocytes, macrophages, NK cells, etc.;
Or alternatively
One or more rounds of screening are respectively carried out on one or more prepared tumor neoantigen peptide libraries capable of combining with HLA I or II molecules of cancer patients by using a plurality of polyclonal antibodies with magnetic beads or other labels, which are separated from peripheral blood of the cancer patients, so that potential tumor neoantigens capable of specifically combining with one or more antibodies are separated;
(c) Immune cell-antigen validation experiments or antigen-antibody binding validation experiments were performed on the isolated potential, patient-specific tumor neoantigens, thereby confirming the patient-specific tumor neoantigen components.
In another preferred embodiment, the peptide library may also be used to pre-warn the patient treated with PD-1 or a PD-L1 inhibitor or an autoimmune response caused by PD-1 or a PD-L1 inhibitor in a patient treated with PD-1 or a PD-1 and/or PD-L1 expression-deficient patient, thereby discriminating whether the patient is suitable for PD-1 or PD-L1 inhibitor immunotherapy and avoiding serious cardiotoxic side effects.
In another preferred embodiment, the bacterial expression or cellular expression techniques include, but are not limited to: bacterial expression systems, viral expression systems, yeast expression systems, insect cell expression systems, mammalian cell expression systems, animal and plant bioreactor expression systems, cell-free expression systems, and the like.
In another preferred embodiment, the one or more tumor neoantigen peptide libraries prepared or prepared are capable of binding to HLA class I or II molecules of a cancer patient, preferably 1-100 tumor neoantigen peptide libraries, more preferably 1-50, such as 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 20, 30, 40, 50 tumor neoantigen peptide libraries.
In another preferred embodiment, one or more of the tumor neoantigen peptide libraries prepared to bind to HLA class I or II molecules of a cancer patient is subjected to one or more rounds of screening, preferably 1-20 rounds of screening, more preferably 1-10 rounds of screening, 1-5 rounds of screening, such as 1,2,3,4, 5 rounds of screening, respectively.
In another preferred embodiment, other antigen presenting cells include, but are not limited to, monocytes/macrophages, B lymphocytes, endothelial cells, fibroblasts, epithelial and mesenchymal cells, eosinophils, and the like.
In another preferred embodiment, the tumor neoantigen encoding sequence elements are selected from the group consisting of: DNA sequence elements, RNA sequence elements, and/or polypeptide sequence elements.
In another preferred embodiment, the DNA sequence element comprises 1 to 12 DNA variants, more preferably 1 to 5, each comprising at least 1 polypeptide chain coding sequence; and/or
The RNA sequence elements comprise 1-12 RNA variants, more preferably 1-5 RNA variants, each comprising at least 1 polypeptide chain coding sequence; and/or
The peptide chain sequence element contains 3-100 amino acids.
In another preferred embodiment, the peptide chain sequence element is preferably 5-80 amino acids, more preferably 5-,40, such as 5, 10, 15, 20, 30, 40 amino acids.
In another preferred embodiment, the method further comprises the steps of: screening a single chain antibody (scFV) display peptide library for single chain antibodies (scFV) that specifically bind to the tumor neoantigen encoding sequences based on the one or more tumor neoantigen encoding sequences screened, and constructing and/or expanding T cells (CAR-T) expressing a Chimeric Antigen Receptor (CAR), wherein the CAR contains the scFV as an extracellular antigen binding domain.
In another preferred embodiment, based on the one or more tumor neoantigen encoding sequences selected, it is preferably 1-10 tumor neoantigen encoding sequences, more preferably 1-5, 1-2 tumor neoantigen encoding sequences.
In another preferred embodiment, the single-chain antibody is obtained by a single-chain antibody phage display technique, a yeast display technique, an mRNA display technique, a cell display technique, or the like.
In another preferred embodiment, the method further comprises the steps of: there is provided a personalized cell product (CAR-T cell) made by the method of the invention.
In another preferred embodiment, the Chimeric Antigen Receptor (CAR) expressing T cells are for return to the subject.
In another preferred embodiment, the reinfusion further comprises additional administration of CAR-T cells, TCR-T cells and/or co-stimulatory factors against the universal tumor antigen.
In another preferred embodiment, the method further comprises the steps of: based on the one or more tumor neoantigen encoding sequences screened, T Cell Receptors (TCRs) that specifically bind to the tumor neoantigen encoding sequences are screened, and T cells (TCR-T) expressing the TCRs are constructed and/or expanded.
In another preferred embodiment, based on the one or more tumor neoantigen encoding sequences selected, it is preferably 1-10 tumor neoantigen encoding sequences, more preferably 1-5, 1-2 tumor neoantigen encoding sequences.
In another preferred embodiment, the method further comprises providing a personalized cell product (TCR-T cells) produced by the method of the invention.
In another preferred embodiment, the T cells of the TCR-are used for feedback to the subject.
In another preferred embodiment, the reinfusion further comprises additional administration of CAR-T cells, TCR-T cells and/or co-stimulatory factors against the universal tumor antigen.
In another preferred embodiment, the method further comprises the steps of: based on the one or more tumor neoantigen encoding sequences screened, the patient's dendritic cells or other antigen presenting cells are primed (primed) in vitro to obtain primed (primed) dendritic cells or other antigen presenting cells. And co-culturing the sensitized dendritic cells or other antigen presenting cells with T cells of the subject in vitro to produce DC-CTL cells or APC-CTL cells.
In another preferred embodiment, based on the one or more tumor neoantigen encoding sequences selected, it is preferably 1-10 tumor neoantigen encoding sequences, more preferably 1-5, 1-2 tumor neoantigen encoding sequences.
In another preferred embodiment, there is provided an individualized cell product (DC-CTL cell or APC-CTL cell) produced by the method of the present invention.
In another preferred embodiment, the primed dendritic cells or other antigen presenting cells and/or DC-CTL cells are used for feedback to the subject.
In another preferred embodiment, other antigen presenting cells include, but are not limited to, monocytes/macrophages, B lymphocytes, endothelial cells, fibroblasts, epithelial and mesenchymal cells, eosinophils, and the like.
In a second aspect of the invention there is provided a personalized cancer vaccine made by the method of any one of the first aspects of the invention. The vaccine can be used for treating tumors, autoimmune diseases and infectious diseases.
In another preferred embodiment, the vaccine optionally further comprises an adjuvant.
In another preferred embodiment, the adjuvant is selected from the group consisting of: poly-ICLC, TLR,1018ISS, aluminum salts, amplivax, AS15, BCG, CP-870, 893, cpg7909, cyaa, dslim, gm-CSF, IC30, IC31, imiquimod, imuFact IMP321, IS Patch, ISS, ISCOMATRIX, juvlmmune, lipoVac, MF59, monophosphoryl lipid a, meng Dani de IMS 1312, meng Dani de ISA 206, meng Dani de ISA 50V, meng Dani de ISA-51, ok-432, om-174, om-197-MP-EC, ONTAK, PLGA microparticles, requimod, SRL172, viral microsomes and other virus-like particles, YF-17d, vegf trap, R848, β -glucan, pam3Cys, ajuira QS21 piercer, vadimezan or AsA404 (DMXAA), and the like.
In another preferred embodiment, the personalized cancer vaccine may also be administered in combination with other drugs and/or therapies.
In another preferred embodiment, the additional agent or therapy comprises an anti-immunosuppressive agent, chemotherapy, radiation therapy, or other targeted agent.
In another preferred embodiment, the anti-immunosuppressive drug includes, but is not limited to, an anti-CTLA-4 antibody, an anti-PD 1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TIGI T antibody, an anti-VISTA antibody, an anti-CD 25 antibody, an anti-CD 47 antibody, an IDO inhibitor, or the like.
In a third aspect of the invention there is provided a personalized cell product made by the method of any one of the first aspects of the invention. The personalized cell product can be used for treating tumors, autoimmune diseases and infectious diseases.
In another preferred embodiment, the cell product is selected from the group consisting of: CAR-T cells, TCR-T cells, DC-CTL cells, APC-CTL cells, or a combination thereof.
In a fourth aspect of the invention there is provided a method of inducing a tumour-specific immune response in a subject suffering from cancer comprising administering to a subject in need thereof a personalized cancer vaccine according to the second aspect of the invention, or a personalized cell product according to the third aspect of the invention.
In another preferred embodiment, the personalized cancer vaccine may also be used to prepare a pharmaceutical composition for the combined administration of a therapeutic agent for cancer.
In another preferred embodiment, the personalized cancer vaccine and adjuvant may also be administered in combination with other drugs and/or therapies.
In another preferred embodiment, the additional agent or therapy comprises an anti-immunosuppressive agent, chemotherapy, radiation therapy, or other targeted agent.
In another preferred example, the anti-immunosuppressive drug includes the anti-immunosuppressive drug including, but not limited to, an anti-CTLA-4 antibody, an anti-PD 1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TIGI T antibody, an anti-VISTA antibody, an anti-CD 25 antibody, an anti-CD 47 antibody, an IDO inhibitor, or the like.
In a fifth aspect of the invention there is provided a method of individualizing a subject suffering from cancer, or from an autoimmune disease, or from an infectious disease, comprising administering to a subject in need thereof an individualised cancer vaccine according to the second aspect of the invention, or an individualised cell product according to the third aspect of the invention.
In another preferred embodiment, the method comprises administering a pharmaceutical composition for treating cancer, including an antibody drug, a cellular immunotherapeutic drug (e.g., CAR-T cells, TCR-T cells, DC-CTL cells, etc.), or a combination thereof.
Drawings
FIG. 1 shows a procedure for establishing a tumor neoantigen display (display) peptide library capable of binding to HLA class I or II (HLA diagnostic) molecules of a cancer patient and activating TCR of specific tumor infiltrating lymphocytes of the cancer patient, and screening for potential tumor neoantigen components.
Detailed Description
The present inventors have conducted extensive and intensive studies and have for the first time developed a method for effectively improving the effectiveness of immunotherapy with an individualized cancer vaccine. The invention screens and separates out the specific tumor neoantigen of the patient by establishing a tumor neoantigen display (display) peptide library or a tumor neoantigen peptide library which can be combined with HLA I or II molecules of the cancer patient and activate TCR of the specific tumor infiltrating lymphocyte of the cancer patient; and in a short time (e.g., within 3-4 weeks), personalized cancer vaccines are prepared, thereby developing a rapid, highly effective personalized solid tumor immunotherapy regimen. The invention can accurately, rapidly and efficiently capture the specific tumor neoantigen of a patient capable of exciting immune response under the conditions of no wound and no need of tumor tissues or cells of the patient, and can be prepared into an individual cancer vaccine, and has wide application prospect in the fields of tumor, autoimmune diseases and infectious diseases treatment. On this basis, the present invention has been completed.
Definition of the definition
"Tumor neoantigen (neoantigen)" refers to a novel antigen that is expressed on the surface of a tumor cell but is not present on normal cells, and is also called a unique tumor antigen. Such antigens may be present in tumors of the same tissue type in different individuals, e.g., melanoma specific antigens encoded by human malignant melanoma genes may be present in melanoma cells of different individuals, but normal melanoma cells are not expressed. Such antigens can also be shared by tumors of different histological types, such as mutated ras oncogene products found in the digestive tract, lung cancer, etc., but because of their amino acid sequence differences from the normal protooncogene ras expression products, they can be recognized by the immune system of the body, stimulating the immune system of the body to attack and eliminate tumor cells. Tumor neoantigens mainly induce T cell immune responses.
"MHC" is a generic term for all biocompatible complex antigens, meaning molecules encoded by the MHC gene family (MHC class I, class II, class III), which are located on the cell surface and whose primary function is to bind peptide chains derived from pathogens, which are displayed on the cell surface to facilitate T-cell recognition and to perform a range of immune functions. MHC CLASS I are located on the surface of a typical cell and provide conditions within the typical cell, such as when the cell is infected with a virus, short peptide chains of associated viral outer membrane fragments are presented outside the cell by MHC, and can be recognized by cd8+ T cells, etc. for killing. MHC class II is located only on Antigen Presenting Cells (APCs), such as macrophages, CD4+ T helper cells, and the like. Such provision is the case outside the cell, such as when bacteria invade the tissue, and after ingestion by macrophages, bacterial debris is presented to helper T cells using MHC to initiate an immune response. MHC class III encodes mainly complement components, tumor Necrosis Factor (TNF), etc. Human MHC is commonly referred to as HLA (human leucocyte antigen), a human humoral cellular antigen. MHC genes, located on the short arm of human chromosome six, are highly polymorphic.
"CD8+ T cells" generally refer to T cells that express CD8 on the cell surface. While CD8 (cluster of differentiation) is a transmembrane glycoprotein, used as a co-receptor for TCR. Similar to TCR, CD8 binds to MHC CLASS I molecules for recognition of killing by cd8+ T cells and the like.
"CD4+ T helper cells" generally refer to T helper cells that express CD4 on the cell surface, and belong to a class of humoral cells. While CD4 (cluster of differentiation 4) is a glycoprotein, which acts as a co-receptor for TCR and assists TCR in recognizing APC. CD4 binds to MHC CLASS II molecules for identification of, for example, cd8+ T cells.
"Immunoadjuvant", also known as a nonspecific immunoproliferative agent. Does not itself have antigenicity, but can enhance immunogenicity or alter the type of immune response together with the antigen or by pre-injection into the body.
The term "DNA, RNA, peptide chain" refers to DNA, RNA, and/or peptide chains.
"CAR-T", which is collectively referred to as chimeric antigen receptor T cell immunotherapy, is one of the more effective methods of immunotherapy of malignant tumors at present. Chimeric Antigen Receptors (CARs) are the core component of CAR-T, conferring to T cells the ability to recognize tumor antigens in an HLA-independent manner, which enables CAR engineered T cells to recognize a broader range of targets than native T cell surface receptor TCRs. Has better curative effect on acute leukemia and non-Hodgkin lymphoma.
"TCR-T", collectively known as T Cell Receptor (TCR) chimeric T cells (TCR-T), destroy tumor cells by increasing the "affinity" of these TCRs for the corresponding tumor neoantigens by means of partial genetic engineering. The genetically engineered TCR technology is also known as affinity-enhanced TCR technology. Two latest immune cell technologies, which are currently used as adoptive cell feedback therapy (ACT) technology, are widely focused and studied due to their ability to express cells, such as tumor cells, that specifically recognize specific receptor targets.
The DC-CTL can specifically present a certain tumor antigen by the impact of autologous or same tumor cell lysate, so that cytotoxic lymphocyte (CTL) aiming at a certain specific tumor cell is induced, and the anti-tumor effect is improved. A large amount of clinical data at home and abroad show that the DC-CTL immunotherapy combines all advantages of DC and CTL, has obvious curative effect on a plurality of tumors, and has positive effects on controlling the recurrence and metastasis of the tumors, improving the immunity of a patient body and improving the quality of life. DC-CTL has become one of the main therapeutic methods of current biological therapy, and is also one of the most promising tumor therapeutic means in the future radical cure of tumors.
Accelerating the treatment process of individual cancer vaccine
At present, the development and preparation of personalized cancer vaccines takes about 6 to 12 weeks from the excision of cancerous tissues of a patient, and is expensive, which is a lengthy process especially for metastatic cancer patients; the cancerous tissue of the advanced patient needs to be excised, and the somatic mutation of the tumor is found and confirmed. Especially, the accuracy of predicting tumor neoantigens based on the biological software is low, so that the effectiveness of the personalized cancer vaccine treatment is reduced, and the huge clinical treatment requirements of cancer patients are difficult to meet. Through extensive and intensive studies, the inventor firstly proposes to establish a tumor neoantigen display peptide library or a tumor neoantigen peptide library which can be combined with HLA I or II molecules of a cancer patient and can activate TCR of specific tumor-infiltrating lymphocytes of the cancer patient, and then to screen and isolate the specific tumor neoantigens of the patient; and, in 3-4 weeks, personalized cancer vaccines are prepared, thereby developing a rapid, highly effective personalized solid tumor immunotherapy regimen.
The invention can accurately, rapidly and efficiently capture the specific tumor neoantigen of the patient which can excite the anti-cancer immune response under the conditions of no wound and no need of tumor tissues or cells of the patient, and can be prepared into individual cancer vaccine, and has wide application prospect in the field of tumor treatment.
T-cell and B-cell responses to potential tumor neoantigens in peptide libraries
In the present invention, one or more rounds of screening of the prepared tumor neoantigen display peptide library, or libraries, respectively, capable of binding to the HLAI or class II molecules of a cancer patient is performed with magnetic beads or other labeled patient CD4 or CD8 cells, or a plurality of polyclonal antibodies, isolated from the peripheral blood of the cancer patient, thereby isolating potential tumor neoantigens capable of specifically binding to one or more antibodies. Since the patient's immune system is self-tolerized by the fact that T and B lymphocytes, which recognize self-antigens, are cleared or in a non-responsive state prior to their maturation in the central immune system (thymus and bone marrow), the vast majority of short peptide chains within the synthetic peptide pool that are identical or extremely similar to the patient's self-antigen sequences will not be screened through one or more rounds of screening of patient mature T cells or B cells. In addition, mature T and B lymphocytes may develop tolerance against self or foreign antigens due to the presence of peripheral tolerance in the patient's immune system, i.e., in peripheral immune organs.
One or more tumor neoantigen peptide libraries which can be combined with HLA I or II molecules of a cancer patient can be obtained from artificial synthesis and/or tumor tissues of the cancer patient and/or tumor cells including CTC and/or tumor cell lines, and can separate the tumor neoantigens specific to the patient and exclude most of non-tumor antigens through one or more rounds of screening by using high-titer IgG antibodies existing in serum of the patient through an ELISA method.
Method for separating and screening tumor neoantigens
The present invention provides a novel method for isolating and screening tumor neoantigens.
Referring to fig. 1, the method of the present invention typically comprises the steps of:
(a) Preparing one or more tumor neoantigen display peptide libraries capable of binding to HLA class I or II molecules of a cancer patient by phage display (PHAGE DISPLAY) or other display techniques;
(b) One or more tumor neoantigens display peptide libraries capable of binding to HLA class I or II molecules of a cancer patient are individually subjected to one or more rounds of screening with cells such as CD4 or CD8 lymphocytes or other killer T lymphocytes, macrophages, NK cells, etc. isolated from the immune system of the cancer patient, or with Dendritic Cells (DCs) or other Antigen Presenting Cells (APCs) derived from the patient that have been previously primed (primed) with a peptide library polypeptide, or with a mixture of peptide library polypeptides that have been directly mixed with Dendritic Cells (DCs) or other Antigen Presenting Cells (APCs) derived from the patient, with the addition of a peptide library polypeptide, thereby isolating potential tumor neoantigens that activate patient-specific CD4 or CD8 lymphocytes or other killer T lymphocytes, macrophages, NK cells, etc.;
or using multiple polyclonal antibodies with magnetic beads or other markers separated from peripheral blood of cancer patients to respectively perform one or more rounds of screening on one or more prepared tumor neoantigen display peptide libraries capable of binding with HLA I or II molecules of the cancer patients, thereby separating potential tumor neoantigens capable of specifically binding with one or more antibodies;
(c) Immune cell (e.g., CD4 and/or CD 8) -antigen validation experiments or antigen-antibody binding validation experiments were performed on isolated potential, patient-specific tumor neoantigens, thereby validating the patient-specific tumor neoantigen components.
In the invention, the peptide library can also early warn autoimmune response caused by PD-1 or PD-L1 inhibitor treatment in patients treated by PD-1 or PD-L1 inhibitor or patients with PD-1 and/or PD-L1 low expression, so as to distinguish whether the patients are suitable for PD-1 or PD-L1 inhibitor immunotherapy, and serious cardiotoxic side effects are avoided.
The main advantages of the invention include:
(a) The invention can rapidly and efficiently prepare the personalized cancer vaccine, thereby providing a personalized solid tumor immunotherapy scheme.
(B) The invention can accurately, rapidly and efficiently capture the specific tumor neoantigen of a patient capable of exciting immune response under the conditions of no wound and no need of tumor tissues or cells of the patient, and can be prepared into an individual cancer vaccine, and has wide application prospect in the fields of tumor, autoimmune diseases and infectious diseases treatment.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
Unless otherwise indicated, the materials or reagents used in the examples were all commercially available products.
Example 1 preparation of multiple tumor neoantigen phage display (PHAGE DISPLAY) peptide libraries capable of binding to HLA class I or II molecules of cancer patients and screening for tumor neoantigens
By a DNA synthesis method, different DNA fragments are respectively synthesized according to the conserved amino acid coding sequence of a short peptide chain combined with human HLA I or II molecules, the length of the conserved amino acid coding sequence, the different marker amino acid coding sequences positioned at the C end of the conserved amino acid coding sequence and the length of the marker amino acid coding sequences, and then PCR amplification is carried out, and the DNA fragments are respectively subcloned into a plasmid of a phage display system, T7Select 10-3b (Novagen).
According to the above method, each of the prepared tumor neoantigen phage display peptide libraries capable of binding to HLA class I or II molecules of cancer patients contains at least 10 8 unique polypeptides and has the following characteristics:
(i) The plurality of polypeptide chains comprising the peptide library that are capable of binding to HLA class I molecules of a cancer patient can be 8 to 11 amino acid residues in length, wherein some of the sites comprise amino acid residues of the same or similar nature and other sites comprise any amino acid residue, or are longer or shorter, but still capable of efficiently binding to HLA class I molecules;
(ii) The plurality of polypeptide chains comprising the peptide library which are capable of binding to HLA class II molecules of a cancer patient may be from 10 to 25 amino acid residues, more preferably from 13 to 18 amino acid residues, wherein some of the sites comprise amino acid residues of the same or similar nature and others comprise any amino acid residue, or longer or shorter, but still be capable of efficiently binding to HLA class II molecules;
(iii) The polypeptide chain contains a specific epitope coding sequence marker at the N end or the C end and is used for downstream screening;
(iv) One or more tumor neoantigen display peptide libraries that are capable of binding to HLA class I or II molecules of a cancer patient can be prepared from synthetic, and/or cancer patient tumor tissue, and/or tumor cells including CTCs, and/or tumor cell lines.
One or more tumor neoantigen phage display peptide libraries prepared as described above are individually screened one or more rounds of selection for potential tumor neoantigens capable of activating patient-specific CD4 or CD8 lymphocytes or other killer T lymphocytes, macrophages, NK cells, etc., using CD4 or CD8 lymphocytes or other killer T lymphocytes specifically isolated from the immune system of a cancer patient, or Dendritic Cells (DCs) or other Antigen Presenting Cells (APCs) from the patient, or a mixture of peptide library polypeptides directly mixed with Dendritic Cells (DCs) or other Antigen Presenting Cells (APCs) from the patient, with the addition of a prior peptide library polypeptide-primed (primed) with magnetic beads or other markers.
EXAMPLE 2 preparation of fresh tumor specimen cDNA library from cancer patients and screening for tumor neoantigens
In this example, cDNA libraries were made from fresh tumor specimens from patients, packaged into lambda phage vectors and expressed recombinantly in E.coli. Transferring the recombinant protein to a nitrocellulose membrane, and separating the tumor neoantigens specific to the patient by one or more rounds of screening by using high-titer IgG antibodies existing in the serum of the patient through an enzyme-linked immunosorbent assay.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims (22)

1. A method of isolating and screening tumor neoantigens, said method comprising the steps of:
(a) Preparing one or more tumor neoantigen display peptide libraries capable of binding to a cancer patient HLAI or class II molecule by phage display (PHAGE DISPLAY) or other display techniques; wherein the display peptide library has features (i), (ii), (iii) and (iv); or (i), (iii) and (iv): or (ii), (iii) and (iv):
(i) The length of a plurality of polypeptide chains which form the peptide library and can be combined with HLA class I molecules of cancer patients is 8-11 amino acid residues, wherein certain sites contain amino acid residues with the same or similar properties, and other sites contain any amino acid residue, or are longer or shorter, but still can be combined with HLAI class molecules effectively;
(ii) The length of a plurality of polypeptide chains which form the peptide library and can be combined with HLA class II molecules of cancer patients is 10-25 amino acid residues, wherein certain sites contain amino acid residues with the same or similar properties, and other sites contain any amino acid residue, or are longer or shorter, but still can be combined with HLAII class molecules effectively;
(iii) The polypeptide chain contains an added specific epitope coding sequence marker at the N end or the C end for downstream screening;
(iv) One or more tumor neoantigen display peptide libraries prepared capable of binding to HLA class I or II molecules of a cancer patient may be derived from synthetic, and/or cancer patient tumor tissue, and/or tumor cells, including circulating tumor cells, and/or tumor cell lines;
(b) One or more tumor neogenesis antigen display peptide libraries capable of binding to HLA class I or II molecules of a cancer patient are individually subjected to one or more rounds of screening with magnetic beads or other labeled CD4 or CD8 lymphocytes or other killer T lymphocytes, macrophages, NK cells isolated from the immune system of the cancer patient, or Dendritic Cells (DCs) or other Antigen Presenting Cells (APCs) derived from the patient that have been primed with a peptide library polypeptide in advance of the magnetic beads or other labels, or a mixture of peptide library polypeptides directly mixed with Dendritic Cells (DCs) or other Antigen Presenting Cells (APCs) derived from the patient, thereby isolating potential tumor neogenesis antigens capable of activating patient-specific CD4 or CD8 lymphocytes or other killer T lymphocytes, macrophages, NK cells;
Or alternatively
Using multiple polyclonal antibodies with magnetic beads or other markers separated from peripheral blood of cancer patients, carrying out one or more rounds of screening on one or more prepared tumor neoantigen display peptide libraries capable of combining with HLAI or II molecules of the cancer patients, thereby separating potential tumor neoantigens capable of specifically combining with one or more antibodies;
(c) Immune cell-antigen validation experiments or antigen-antibody binding validation experiments were performed on the isolated potential, patient-specific tumor neoantigens, thereby confirming the patient-specific tumor neoantigen components.
2. The method of claim 1, wherein in step (ii) the plurality of polypeptide chains comprising the peptide library capable of binding to a cancer patient class HLAII molecule are 13-18 amino acid residues in length.
3. The method of claim 1, wherein in step (c) an in vitro (ex vivo) validation experiment of patient specific CD4 or CD8 lymphocytes or other killer T lymphocytes, macrophages, NK cells, or antigen-antibody binding in vitro validation experiment is performed to confirm patient specific tumor neoantigen components.
4. The method of claim 1, wherein in step (c) an in vivo (in vivo) activation of patient-specific CD4 or CD8 lymphocytes or other killer T lymphocytes, macrophages, NK cells is performed to confirm the patient-specific tumor neoantigen component.
5. The method of claim 1, wherein the peptide library is further capable of alerting the patient to autoimmune response caused by PD-1 or PD-L1 inhibitor therapy in a patient treated with PD-1 or PD-L1 inhibitor therapy or a patient with low expression of PD-1 and/or PD-L1 inhibitor therapy, thereby identifying whether the patient is suitable for PD-1 or PD-L1 inhibitor immunotherapy and avoiding serious cardiotoxic side effects.
6. The method of claim 1, wherein the one or more tumor neoantigen display peptide libraries prepared are 1-100 tumor neoantigen display peptide libraries capable of binding to HLA class I or II molecules of cancer patients.
7. The method of claim 1, wherein one or more rounds of screening are performed on one or more tumor neoantigen display peptide libraries prepared to bind to a cancer patient HLAI or class II molecule, respectively.
8. The method of claim 1, wherein the one or more tumor neoantigen display peptide libraries prepared to bind to a cancer patient HLAI or class II molecule are each subjected to 1-20 rounds of screening.
9. The method of claim 1, wherein the one or more tumor neoantigen display peptide libraries prepared to bind to a cancer patient HLAI or class II molecule are screened for 1-10 rounds of selection, respectively.
10. The method of claim 1, wherein the one or more tumor neoantigen display peptide libraries prepared to bind to a cancer patient HLAI or class II molecule are subjected to 1-5 rounds of screening, respectively.
11. The method of claim 1, wherein the additional antigen presenting cells comprise: mononuclear/macrophages, B lymphocytes, endothelial cells, fibroblasts, epithelial and mesenchymal cells, eosinophils.
12. The method of claim 1, comprising the steps of:
(a) Preparing one or more tumor neoantigen peptide libraries capable of binding to HLA class I or II molecules of cancer patients by bacterial expression or cell expression technology; wherein the peptide library has one or more of the following features:
(i) The plurality of polypeptide chains comprising the peptide library that are capable of binding to HLA class I molecules of a cancer patient may be 8-11 amino acid residues in length, wherein some of the sites comprise amino acid residues that are identical or of similar nature, and others comprise any amino acid residue, or are longer or shorter, but still capable of efficiently binding to HLAI class molecules;
(ii) The length of a plurality of polypeptide chains which form the peptide library and can be combined with HLA class II molecules of cancer patients is 10-25 amino acid residues, wherein certain sites contain amino acid residues with the same or similar properties, and other sites contain any amino acid residue, or are longer or shorter, but still can be combined with HLAII class molecules effectively;
(iii) The polypeptide chain contains an added specific epitope coding sequence marker at the N end or the C end for downstream screening;
(iv) One or more tumor neoantigen peptide libraries prepared that bind to HLA class I or II molecules of a cancer patient may be derived from synthetic, and/or cancer patient tumor tissue, and/or tumor cells, including circulating tumor cells, and/or tumor cell lines;
(b0) Sensitizing (priming) patient-specific dendritic cells or other antigen presenting cells in vitro with one or more tumor neoantigen polypeptides expressed in a tumor neoantigen peptide pool capable of binding to HLA class I or class II molecules of a cancer patient, thereby obtaining sensitized (primed) dendritic cells or other antigen presenting cells;
(b) One or more rounds of screening of the above primed (primed) patient-specific dendritic cells or other antigen presenting cells with magnetic beads or other labeled CD4 or CD8 lymphocytes or other killer T lymphocytes, macrophages, NK cells, respectively, isolated from the immune system of a cancer patient, thereby isolating potential tumor neoantigens that activate the patient-specific CD4 or CD8 lymphocytes or other killer T lymphocytes, macrophages, NK cells;
Or alternatively
One or more rounds of screening are respectively carried out on one or more prepared tumor neoantigen peptide libraries capable of combining with HLA I or II molecules of cancer patients by using a plurality of polyclonal antibodies with magnetic beads or other labels, which are separated from peripheral blood of the cancer patients, so that potential tumor neoantigens capable of specifically combining with one or more antibodies are separated;
(c) Immune cell-antigen validation experiments or antigen-antibody binding validation experiments were performed on the isolated potential, patient-specific tumor neoantigens, thereby confirming the patient-specific tumor neoantigen components.
13. A method according to claim 12, wherein in step (ii) the plurality of polypeptide chains comprising the peptide library capable of binding to the HLAII class of molecules of cancer patients are 13-18 amino acid residues in length.
14. The method of claim 1 or 12, wherein the tumor neoantigen encoding sequence elements are selected from the group consisting of: DNA sequence elements, RNA sequence elements, and/or polypeptide chain sequence elements.
15. The method of claim 14, wherein the DNA sequence element comprises one or more DNA variants, each comprising one or more polypeptide chain coding sequences; and/or said RNA sequence elements comprise one or more RNA variants, each comprising one or more polypeptide chain coding sequences; and/or said peptide chain sequence element comprises 3-100 amino acids.
16. The method according to claim 1 or 12, further comprising the steps of: screening a single chain antibody (scFV) display peptide library for single chain antibodies (scFV) that specifically bind to the tumor neoantigen encoding sequences based on the one or more tumor neoantigen encoding sequences screened, and constructing and/or expanding T cells (CAR-T) expressing a Chimeric Antigen Receptor (CAR), wherein the CAR contains the scFV as an extracellular antigen binding domain.
17. The method according to claim 1 or 12, further comprising the steps of: based on the one or more tumor neoantigen encoding sequences screened, T Cell Receptors (TCRs) that specifically bind to the tumor neoantigen encoding sequences are screened, and T cells (TCR-T) expressing the TCRs are constructed and/or expanded.
18. The method of claim 1, further comprising the step of: based on the one or more tumor neoantigen encoding sequences screened, the patient's dendritic cells or other antigen presenting cells are primed (primed) in vitro to obtain primed (primed) dendritic cells or other antigen presenting cells.
19. The method according to claim 12 or 18, further comprising the step of: in vitro, the sensitized dendritic cells or other antigen presenting cells are co-cultured with T cells of the patient to produce DC-CTL cells or APC-CTL cells.
20. A personalized cancer vaccine, characterized in that said vaccine is made by the method of any one of claims 1-19.
21. An individualized cell product produced by the method of claim 16, 17 or 18.
22. The personalized cell product of claim 21, wherein said cell product is selected from the group consisting of: CAR-T cells, TCR-T cells, DC-CTL cells, APC-CTL cells, or a combination thereof.
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