Patent Application
20210163891
The present invention relates to the field of biotechnology, and in particular, to engineered γδT cells, and uses and preparation methods for the cells.
Malignant tumors are one of the most dangerous diseases that endanger human health. Every year, about 8 million people die from cancer worldwide, and 6 people are diagnosed as having malignant tumors per 6 minutes in China. The development of new drugs and treatments for malignant tumors has become the global focus. Cancer immunotherapy was rated as the most important scientific breakthrough in 2013 by “Science”, among which adoptive cell immunotherapy is particularly interesting.
For adoptive cellular immunotherapy, the patient's own immune cells are expanded or modified in vitro, and then introduced back into the body, so as to achieve the purpose of treating tumors. This therapy has advantages, such as high specificity and few side effects, and is especially suitable for patients who cannot receive other treatments at the late stage. At present, T lymphocytes have become a new star of adoptive cellular immunotherapy.
T lymphocytes are divided into two major categories of αβT and γδT cells based on the structure of T cell receptor (TCR) on their surface. Human γδT cells are a unique subset of T lymphocytes that are conservatively evolved, their TCR receptors are composed of γ chains and δ chains; and they have anti-infection and anti-tumor effects in the immune monitoring of the body. There are many types and a variety of subtypes for γδT cells. Among them, the Vδ chain has 1-8 different subtypes, and the Vγ chain has 1-9 different subtypes. In human peripheral blood, γδT cells account for 1%-5% of the entire T lymphocytes, in which Vγ9Vδ2γδT cells account for 50%-95% of the entire γδT cells. Compared with αβT cells, γδT cells recognize more types of antigens, and are not restricted by major histocompatibility complex (MHC).
At present, the research on T cell adoptive immunotherapy mainly focuses on CAT-T therapy and TCR-T therapy. Among them, engineered TCR gene-modify T cells are mainly used in TCR-T therapy. Compared with the currently used αβTCR-transduced αβT cell protocol, αβTCR transduced γδT cells will naturaly possess advantages: its exogenous αβTCR hardly mismatches the endogenous γ chain and δ chain of γδT cells, thereby avoiding likely caused autoimmune reaction to the body.
In 2009, A Hiasa et al. [A Hiasal Gene Therapy (2009) 16, 620-6281 transduced αβTCR to γδT cells for the first time, and found that αβTCR-transduced γδT cells can transmit activation signals to cells through endogenous γδTCR, but also can respond to specific antigens through exogenous αβTCR. At the same time, from activation dynamics, αβTCR-transduced γδT cells respond quickly, compared with αβTCR-transduced αβT cells. However, unmodified wild-type αβTCR is used in Hiasa's solution. If only αβTCR is transduced into γδT cells, specific recognition of tumor antigens can not be achieved. Eventually, CD8 molecules have to be transduced to assist recognition, which undoubtedly increases the instability of the cell, and additional gene editing will bring more unpredictability and potential risk.
Therefore, there is an urgent need in the art for γδT cells that specifically recognize tumor antigens without co-transforming other CD molecules.
The object of the present invention is to provide a γδT cell that can specifically recognize a tumor antigen without co-transforming other CD molecules.
The object of the present invention is also to provide a preparation method and use of the γδT cells.
In the first aspect, the present invention provides a genetically modified γδT cell, into which a gene of high-affinity αβTCR has been transduced, and the affinity of the high-affinity αβTCR to its specific pMHC is at least two times that of the corresponding wild-type αβTCR.
In a preferred embodiment, the genetically modified γδT cells can specifically recognize a tumor antigen without exogenous CD8 molecules; preferably, the genetically modified γδT cells can specifically recognize a tumor antigen without any exogenous CD molecules; and most preferably, the genetically modified γδT cells can specifically recognize a tumor antigen without any exogenous auxiliary molecules.
In a specific embodiment, the equilibrium dissociation constant value KD of the affinity of the high-affinity αβ TCR to its specific pMHC is ≤10 μM.
In a specific embodiment, the equilibrium dissociation constant value KD of the affinity of the high-affinity αβTCR to its specific pMHC is ≤4 μM; for example, 5 nM≤KD≤4000 nM, 50 nM≤KD≤4000 nM, 100 nM≤KD≤4000 nM, 400 nM≤KD≤4000 nM, 450 nM≤KD≤4000 nM, 400 nM≤KD≤3000 nM, 500 nM≤KD≤3000 nM, 1000 nM≤KD≤3000 nM or 1500 nM≤KD≤3000 nM.
In a specific embodiment, the equilibrium dissociation constant value KD of the affinity of the high affinity αβ TCR to its specific pMHC is: 1 nM≤KD≤2000 nM; for example, 100 nM≤KD≤2000 nM, 400 nM≤KD≤2000 nM, 450 nM≤KD≤2000 nM, 500 nM≤KD≤2000 nM, 1000 nM≤KD≤2000 nM or 1500 nM≤KD≤2000 nM.
In a preferred embodiment, the gene of the high affinity αβ TCR is of full-length.
In a preferred embodiment, the nucleic acid sequence of the α-chain of the high-affinity αβTCR is SEQ ID NO: 5, and the nucleic acid sequence of the β-chain is SEQ ID NO: 7; or, the nucleic acid sequence of the α-chain is SEQ ID NO: 9, and the nucleic acid sequence of the β chain is SEQ ID NO: 11; or, the nucleic acid sequence of the α chain is SEQ ID NO: 13, and the nucleic acid sequence of the β chain is SEQ ID NO: 15; or, the nucleic acid sequence of the α chain is SEQ ID: NO: 17, and the nucleic acid sequence of the β chain is SEQ ID NO: 19; or, the nucleic acid sequence of the α chain is SEQ ID NO: 21, and the nucleic acid sequence of the β chain is SEQ ID NO: 23.
In a preferred embodiment, the high affinity αβ TCR is transduced into the γδ T cells by viral vectors, including but not limited to lentiviral vectors.
In a preferred embodiment, the γδ pair of the γδT cell is Vγ1-9 and Vδ1-8.
In a preferred embodiment, the cells are Vγ9Vδ2γδT cells.
In a specific embodiment, the γδT cells are used to prepare medicaments for treating a tumor or an infectious disease.
In a specific embodiment, the infection is a viral infection or a bacterial infection.
In a preferred embodiment, the γδ T cells are derived from a treated subject itself or from a healthy donor.
In a second aspect, the present invention provides uses of the γδT cell of the first aspect for preparing a medicament for treating a tumor or infectious disease.
In a preferred embodiment, the infectious disease is a viral infection or a bacterial infection.
In a third aspect, the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier and the γδ T cell of the first aspect.
In a fourth aspect, the present invention provides a method for treating a disease, comprising administering to a subject in need thereof an appropriate amount of the γδ T cell of the first aspect or the pharmaceutical composition of the third aspect.
In a preferred embodiment, the disease is a tumor or an infectious disease.
In a preferred embodiment, the infectious disease is a viral infection or a bacterial infection.
In a fifth aspect, the present invention provides a method for preparing the γδT cell of the first aspect, the method comprising the following steps:
(i) transducing a gene of high-affinity αβTCR into a γδT cell;
(ii) culturing the γδT cell described in (i).
In a preferred embodiment, the affinity of the high-affinity αβTCR to its specific pMHC is at least two times that of the corresponding wild-type αβTCR.
It should be understood that, within the scope of the present invention, the above technical features of the present invention and the technical features specifically described in the following (e.g., examples) can be combined with each other, thereby forming a new or preferred technical solution, which is not necessary to be repeated one by one.
After extensive and in-depth research, the inventors unexpectedly discovered that γδT cells transduced with high-affinity αβTCR can specifically recognize tumor antigens without co-transforming other CD molecules. And such modified γδT cells showed unexpected and more excellent effects in experiment of activation function and killing function. Such modified cells are obviously more advantageous in terms of stability, since it is not necessary to co-transform other CD molecules, thereby greatly reducing the unpredictability and potential risks in the preparation process. On this basis, the present invention has been completed.
pMHC: major histocompatibility complex, i.e., MHC, is a generic name for all biocompatibility complex antigens and is a molecule encoded by the MHC gene family. Human MHC is often called HLA gene or HLA complex. A tumor antigen will be proteolytically processed into polypeptide fragments of 8-16 amino acid in length, i.e., CTL epitopes, in the cells. The polypeptide fragments combine with MHC molecules in the endoplasmic reticulum cavity to form a peptide-MHC complex, i.e., pMHC. pMHC is finally presented on the cell surface for recognition by the αβT cell receptor (αβTCR) on the surface of CD8+ T cells. Different αβTCR can recognize different pMHC molecules, and αβTCR is specific for pMHC recognition. Wild-type αβTCR exhibits a certain affinity for its specific pMHC.
High affinity αβTCR: refers to a αβTCR, whose affinity to its specific pMHC molecule is at least twice that of the corresponding wild-type αβTCR to the aforementioned pMHC molecule.
Tumor: refers to all types of cancer cell growth or carcinogenic processes, metastatic tissue or malignant transformed cells, tissues or organs, regardless of pathological type or stage of infection. Examples of tumors include, without limitation, solid tumors, soft tissue tumors, and metastatic lesions. Examples of solid tumors include: malignant tumors of different organs or systems, such as sarcoma, lung squamous cell carcinoma and cancer. For example: infected prostate, lung, breast, lymph, gastrointestinal (e.g., colon), and genitourinary tract (e.g., kidney, epithelial cells), pharynx. Lung squamous cell carcinoma includes malignant tumors, for example, most colon cancers, rectal cancer, renal cell cancer, liver cancer, non-small cell cancer of the lung, small intestine cancer, and esophageal cancer. Based on the teachings of the present invention, a skilled person in the art can understand that the high-affinity αβTCR-transduced γδT cells of the present invention can specifically recognize a tumor antigen without or without expressing exogenous auxiliary molecules, such as CD8 molecules, thereby achieving killing effects on tumor cells. Therefore, the various genetically modified γδT cells prepared by employing the idea of the present invention have potential therapeutic effects on various tumors.
Pharmaceutical carrier: also named as an excipient or stabilizer, the dosage and concentration of which are not toxic to the cells or individuals exposed to it. The physiologically acceptable carrier is generally a pH buffered aqueous solution. Examples of a physiologically acceptable carrier include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other sugars, including glucose, mannose or dextrin; complexing agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN′, polyethylene glycol (PEG) and PLURONICS′.
Genetically Modified γδT Cells
After extensive and in-depth research, the present inventors obtained a genetically modified γδT cell with excellent performance, into which a gene of high affinity αβTCR is transduced, and the affinity of the high affinity αβTCR to its specific pMHC is at least twice that of the corresponding wild-type αβTCR. Therefore, the γδ T cells of the present invention can specifically recognize a tumor antigen without or without expressing exogenous CD8 molecules; preferably, the γδ T cells of the present invention can specifically recognize a tumor antigen without or without expressing any exogenous CD molecules (e.g., but not limited to CD8, CD4 molecules); and most preferably, the γδT cells of the present invention can specifically recognize a tumor antigen without or without expressing any exogenous auxiliary molecules.
In particular, the equilibrium dissociation constant value KD of affinity of the high-affinity αβTCR to its specific pMHC≤10 μM. More particularly, the equilibrium dissociation constant value KD of the affinity of the high-affinity αβTCR to its specific pMHC is ≤4 μM; for example, 5 nM≤KD≤4000 nM, 50 nM≤KD≤4000 nM, 100 nM≤KD≤4000 nM, 400 nM≤KD≤4000 nM, 450 nM≤KD≤4000 nM, 400 nM≤KD≤3000 nM, 500 nM≤KD≤3000 nM, 1000 nM≤KD≤3000 nM or 1500 nM≤KD≤3000 nM.
In another preferred example, the equilibrium dissociation constant value KD of the affinity of the high affinity αβ TCR to its specific pMHC is: 1 nM≤KD≤2000 nM; for example, 100 nM≤KD≤2000 nM, 400 nM≤KD≤2000 nM, 450 nM≤KD≤2000 nM, 500 nM≤KD≤2000 nM, 1000 nM≤KD≤2000 nM or 1500 nM≤KD≤2000 nM.
Manners for determining the affinity between αβ TCR and its specific pMHC are well-known to a skilled person in the art. The binding affinity can be determined by any suitable method and using any suitable instrument, including but not limited to Biacore SPR or ForteBio, etc. For example, the binding affinity can be determined according to the instructions of the relevant instrument. It should be noted that when determining the binding affinity, both of αβTCR and pMHC are in a form of soluble protein, that is, both of αβTCR and pMHC are soluble in water. Methods for obtaining αβTCR and pMHC complexes in a soluble form are well-known to a skilled person, such as literature (Jonathan M. Boulter et al, 2003, Protein Engineering, 16 (9): 707-711) or patent literature PCT/CN2016/077680.
It is well known that the affinity is inversely proportional to the equilibrium dissociation constant KD. Therefore, if the affinity of TCR is doubled, the KD value will be reduced by 50%. The conversion relationship between KD value units are well-known to a skilled person in the art, that is, 1 M=1000 μM, 1 μM=1000 nM, and 1 nM=1000 μM. The binding constant is expressed as ka, and the dissociation constant is expressed as kd, KD=kd/ka. T1/2 (half-life) is calculated as In2 divided by the dissociation constant (kd). Therefore, if T1/2 is doubled, kd will be reduced by 50%. The same test protocol is advantageously employed to detect the binding affinity or binding half-life of a given TCR for several times, for example 3 or more times, and the average of the results are taken.
In a preferred embodiment, such detection is performed using Biacore SPR method. Biacore SPR: Surface plasmon resonance measurement method, that is, the resonance principle that the surface plasmon is sensitive to changes in the refractive index of a biosensor at 150 nm is empolyed to respond to changes in the concentration of the molecule on the surface of a biosensor in a real-time, non-invasive manner, in a real-time, non-invasive way, thereby detecting the interaction between the ligand and the analyte on the biosensor chip.
T lymphocytes are divided into two major categories of αβT and γδT cells based on the structure of T cell receptor (TCR) on their surface. The TCR on the surface of αβT cells is composed of α chain and β chain, which is called as αβTCR; the TCR on the surface of γδT cell is composed of γ chain and δ chain, which is called as γδTCR. Among them, the Vδ chain has 1-8 different subtypes, and the Vγ chain has 1-9 different subtypes. Among them, Vγ9Vδ2γδT cells are also called as Vδ2T cells, which can be used interchangeably in the present invention. In a preferred embodiment of the present invention, the gene of a high-affinity αβ TCR transduced into a γδ T cell is of full-length, including an extracellular region, transmembrane region and cytoplasmic region.
Preferably, the high affinity αβ TCR is transduced into a γδ T cell by viral vectors. Viral vectors include but are not limited to adenovirus vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, retrovirus vectors, lentivirus vectors, and baculovirus vectors. In a preferred embodiment of the present invention, the used vector is a lentiviral vector. Preferably, the gene of a high affinity αβ TCR is integrated into the chromosome of a γδ T cell.
The γδT cells of the present invention can be used to treat a tumor or infectious disease. The tumor includes but not limited to liver cancer, lung cancer, gastric cancer, malignant melanoma, prostate cancer, bladder cancer, breast cancer, multiple myeloma, hepatocellular carcinoma, oral squamous cell carcinoma and esophageal cancer, and the like. The infections include viral infections, bacterial infections, fungal infections and protozoan infections. The γδT cells of the present invention can be applied to adoptive immunotherapy, and many suitable methods for adoptive therapy are known to a skilled person in the art (e.g., Rosenberg et al., (2008) Nat Rev Cancer 8 (4): 299-308).
The recognition of an antigen by a γδT cell is not limited by the complex formed by major histocompatibility complex (MHC) and specific antigen short peptides, the γδT cell has extensive anti-tumor and anti-infective properties, and after being stimulated by the antigen, it can respond quickly and expand in great quantity, so as to meet the clinical dosage. Therefore, when treating the above-mentioned diseases, the γδT cells can be extracted not only from the patient but also from other healthy donors. The T cells may be derived from T cells isolated from a subject, or may be a part of a mixed cell population isolated from the subject, such as a part of a peripheral blood lymphocyte (PBL) population. For example, the cells can be isolated from peripheral blood mononuclear cells (PBMC). Alternatively, the cells of the invention may also be or derived from stem cells, such as hematopoietic stem cells (HSC).
A patient can be treated by isolating γδ T cells from the patient with a relevent disease, introducing high-affinity αβ TCR into the T cells, and then reintroducing these genetically modified cells into the patient. It is also possible to isolate γδT cells from a healthy people, and introduce high-affinity αβTCR into the γδT cells, so as to obtain modified γδT cells for future use and reintroduce to a people in need thereof. The amount of reinfusion can be determined by a physician.
Therefore, the γδ T cells of the present invention can be obtained from various sources, including but not limited to commercial sources.
It should be understood that an internationally accepted single letter is used herein to represent the name of an amino acid, and the corresponding three-letters abbreviation of amino acids are: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), Val (V);
1. The γδT cells of the present invention, into which a high-affinity αβTCR is transduced can specifically recognize a tumor antigen without being co-transformed with other CD molecules, and the preparation method and process thereof are relatively convenient.
2. The γδT cells of the present invention, into which a high-affinity αβTCR is transduced exhibit unexpected and excellent activation functions and killing effects on target cells.
The following specific examples further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. Experimental methods without specific conditions in the following examples are generally performed in accordance with conventional conditions, for example (Sambrook and Russell et al. Molecular cloning: Molecular Cloning-A Laboratory Manual (Third Edition) (2001) CSHL The conditions described in the company) or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercially available channels.
Before describing the present invention, it should be understood that the present invention is not limited to the described specific methods and experimental conditions, since such methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments and is not intended to be limiting, and the scope of the present invention will be limited only by the appended claims.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled person in the art to which this invention belongs.
Although any methods and materials similar or equivalent to those described in the present invention can be used in practicing or testing of the present invention, the preferred methods and materials are exemplified herein.
The full-length genes of α and β chains of a TCR were connected to a lentivirus expression vector.
Sources of Main Materials
Three lentivirus packaging plasmids pMDLg/pRRE (Cat. No. 12251), pRSV-REV (Cat. No. 12253), pMD2.G (Cat. No. 12259) were purchased from addgene; and the plasmid PEI (Cat. No. 23966) was purchased from polyscience.
Liposome transfection was used by the inventors to co-transfect the 293T packaging cells with the third-generation of lentiviral system 4 plasmid (3 plasmids using pLenti (addgene) as the lentiviral vector, and containing other components necessary for constructing infectious but non-replicating lentiviral particles) to prepare lentivirus containing the target T cell receptor gene.
Methods
Cell culture: In a 15 cm dish, a suspension of 1.5-2*107 293 T cells (DMEM medium containing 10% FBS) was evenly plated, placed at 37° C., under 5% CO2 overnight; and when about 80% confluence was achieved, lentivirus packaging was conducted.
Lentivirus packaging: the expression plasmid PLenti-TCRα-P2A-TCRβ (15 ug/dish) and packaging plasmids pMDLg/pRRE (5 ug/dish), pRSV-REV (5 ug/dish) and pMD2.G (5 ug/dish) and 1800 microliters of opti-MEM medium was mixed thoroughly, and placed at room temperature for 5 minutes to form a DNA mixture; PEI (60 ug/dish) and 1800 microliters of opti-MEM medium were taken and mixed thoroughly, and placed at room temperature for 5 minutes to form a PEI mixture solution. The DNA mixture and the PEI mixture were mixed together, placed for 30 minutes at room temperature, then 3150 microliters of opti-MEM medium was added, mixed well and added to a 293T cell culture dish, in which DMEM medium had been changed to 11.25 ml of opti-MEM medium, mixed gently and incubated for 7-8 hours at 37° C. under 5% CO2, and then the transfection medium was changed to DMEM medium containing 10% FBS. The supernatant virus liquid was collected at 48 hours and 72 hours after transfection, respectively. The collected virus liquid was centrifuged at 3000 g for 10-15 minutes to remove cell debris; after filteration through a 0.45 micron filter, a concentration tube with 50 KD cut-off value was used to conduct centrifugation at low-temperature. The virus liquid obtained from 1 dish was finally concentrated to 1 ml of virus concentrate, and then divided and stored at −80° C.
γδT cells were transduced with the lentiviral expression vector containing the gene of interest constructed in Example 1.
Sources of Main Reagents and Material
Human peripheral blood mononuclear cells (PBMC) were from healthy donors. Zoledronete was purchased from NOVARTIS, batch number: 50425, imported drug registration account: H20140218; Recombinant Human Interleukin-2 (IL-2) for Injection: Shandong Quangang Pharmaceutical Co., Ltd., batch number: 201407004; IL-7: PEPROTECH, Cat. No.: AF-200-07; IL-15: PEPROTECH, Cat. No.: AF-200-15; IL-21: PEPROTECH, Cat. No.: AF-200-21; medium: 10% FBS (Australian fetal bovine serum, inactivated): purchased from Gibco, Cat. No. 1828728; PRMI1640 (Gibco, Life Technologies) purchased from Gibco, Cat. No. 8117107; flow cytometer: BD FACSAriaIII.
Methods
(a) Vγ9Vδ2γδT cell stimulation: PBMCs from a healthy donor were resuspended in a complete medium, RPMI1640+10% Au+1% PS and mixed homogeneously. The cells were divided into a 24-well plate at a concentration of 2*106/ml. The initial stimulation is recorded as DO, and zoledronete (working concentration was 5 uM) is added to the PBMCs in each wells, and the cytokine IL-2 (working concentration was 100 IU/ml) was also supplemented; and in such system, directed induction and activation of Vγ9Vδ2γδT cells present in PBMC was initiated. The plate was placed in a 37° C., 5% CO2 incubator. In the follow-up culture, half of the medium was exchanged by adding fresh medium and IL-2 every 2-3 days.
(b) Lentiviral transduction of Vγ9Vδ2γδT cells: At D2/D3 after activation, the concentrated lentivirus with the target gene was added into a 200 μl of infection system in a 48-well plate at a proportion of MOI=5. 10 ug/ml of protamine was also supplemented at the same time. At D4, Vγ9Vδ2γδT cells transduced with lentivirus were transferred from a 200-microliter system in the 48-well plate to 1-ml system in a 24-well plate for cultivation.
The binding affinity can be determined by any suitable method using any suitable instrument, including but not limited to Biacore SPR or ForteBio, etc. For example, it can be determined according to the instruction manual of the relevant instrument. It should be noted that both αβTCR and pMHC can exist in a form of soluble protein when determining binding affinity. Methods for obtaining αβTCR and pMHC complexes in a soluble form are well-know to a skilled person in the art, such as described in a literature (Jonathan M. Bouter et al, 2003, Protein Engineering, 16 (9): 707-711) or patent literature PCT/CN2016/077680.
The soluble pMHC complex can be prepared according to the following method: MHC heavy and light chains are expressed by using inclusion bodies, then short peptides are synthesized, and the dissolved MHC heavy chain and light chain as well as short peptides were added into a renaturation buffer and purified. The biotinylated pMHC molecules can be aliquoted and stored at −80° C.
In this example, the affinity between the αβTCR protein and its specific pMHC was determined by using a Biacore instrument from GE. The streptavidin was fixed to the surface of a CMS chip by amino-coupling method, and the fixing amount was greater than several hundred RU. After the biotinylated non-specific pMHC and specific pMHC were captured in the reference channel and the detection channel, respectively, biotin was used to block remaining biotin binding sites of streptavidin. The TCR was diluted into several suitable concentrations in a running buffer and injected sequentially. The dissociation time will depend on the TCR affinity. The analysis software of Biacore was usd to analyze the measured data, the 1:1 combining model was used to fit the obtained kinetic parameters, and the equilibrium dissociation constant KD=kd/ka.
This experiment was conducted to verify the activation of γδ T cells transduced with high affinity 1G4 TCR that specifically respond to target cells T2. The effects were significantly better than that of γδT cells transduced with wild-type 1G4TCR. The IFN-γ production was detected by ELISPOT test as the readout value of TCR-transduced γδT cell activation.
Sources of Main Reagents and Material
10% FBS (Gibco, Catalog number 16000-044), RPMI 1640 (Gibco, Catalog number C11875500bt), PVDF ELISPOT 96-well plate (Merck Millipore, Catalog number MSIPS4510), human IFN-γ ELISPOT PVDF-enzyme kit (BD) containing all other required reagents (capture and detection antibody, streptavidin-alkaline phosphatase and BCIP/NBT solution), Elispot's Kit (BD™ ELISPOT ELISPOT Set Instruction Manual Catalog number: 551282), short peptide: P1B specific for the antigen NY-ESO-1 157-165, sequence of which is SLLMWITQC, HLA genotype of which is A0201, P72A for the control antigen GP100, sequence of which is YLEPGPVTA, and corresponding HLA genotype of which is A0201.
Methods
Preparation of Target Cells
The target cell in this example is a lymphoblastoma T2 cell. The HLA type of the cell is A0201, but it does not express the antigen NY-ESO-1 specific for 1G4TCR; T2 cells loaded with antigen NY-ESO-1 short peptide P1B (loading concentration was 0.1 μM\0.01 μM\0.0001 μM) and control peptide P72A (loading concentration was 0.1 μM), and T2 cells without short peptides were used as the three types of target cells in this experiment.
Preparation of Effector Cells
Different groups of effector cells were prepared: Vδ2T cells without being transduced with 1G4TCR and Vδ2T cells transduced with 1G4TCR of different affinities. The type of γδT cells is Vγ9Vδ2γδT cells (Vδ2T cells). The 1G4TCR (Yangbing Zhao, * The Journal of Immunology, 2007, 179: 5845-5854) is a high-affinity αβ TCR, and the affinity of the TCR used in this example is 32 μM (wild type) (the nucleic acid sequence of α chain is SEQ ID NO: 1, the amino acid sequence of α chain is SEQ ID NO: 2; and the nucleic acid sequence of β chain is SEQ ID NO: 3, the amino acid sequence of β chain is SEQ ID NO: 4), 4 μM (the nucleic acid sequence of α chain is SEQ ID NO: 5, the amino acid sequence of α chain is SEQ ID NO: 6; and the nucleic acid sequence of β chain is SEQ ID NO: 7, the amino acid sequence of β chain is SEQ ID NO: 8), 1.07 μM (the nucleic acid sequence of α chain is SEQ ID NO: 9, the amino acid sequence of α chain is SEQ ID NO: 10; the nucleic acid sequence of β chain is SEQ ID NO: 11, the amino acid sequence of β chain is SEQ ID NO: 12), 450 nM (the nucleic acid sequence of α chain is SEQ ID NO: 13, the amino acid sequence of α chain SEQ ID NO: 14; and the nucleic acid sequence of of β chain is SEQ ID NO: 15, the amino acid sequence of β chain is SEQ ID NO: 16), 84 nM (the nucleic acid sequence of α chain is SEQ ID NO: 17, the amino acid sequence of α chain is SEQ ID NO: 18; the nucleic acid sequence of β chain is SEQ ID NO: 19, the amino acid sequence of β chain is SEQ ID NO: 20) and 5 nM (the nucleic acid sequence of α chain is SEQ ID NO: 21, the amino acid sequence of α chain is SEQ ID NO: 22; and the nucleic acid sequence of β chain is SEQ ID NO: 23, and the amino acid sequence of β chain is SEQ ID NO: 24).
ELISPOT Operation
Plates were repared according to the instruction provided by the manufacturer as follows: anti-human IFN-γ capture antibody was diluted at 1:200 by using 10 ml of sterile PBS per plate, and then 100 μl of diluted capture antibody was added to each well. The plate was incubated at 4° C. overnight. After incubation, the plate was washed to remove excess capture antibody. 100 microliters of RPMI1640 medium containing 10% FBS was added to each well and the plate was incubated at room temperature for 2 hours to block the plate. The medium was then washed away from the plate, and any residual washing buffer was removed from the ELISPOT plate using paper. The effector cells and target cells prepared above were added to the ELISPOT well plate through centrifugation and counting: for 100 μl of target cells, 2*105 cells per ml (20,000 cells per well), and for 100 μl of effector cells, 2*104 cells per ml (2000 cells per well). Note: The short peptide dilution produced by the gradient dilution was directly used to resuspend the T2 cells. In all wells, additions in triplicate were conducted.
Then the plate was incubated at 4° C. overnight (37° C., 5% CO2). On the next day, the culture medium was discarded, the plate was washed twice with double-distilled water, and then washed with the washing buffer for three times. The residual washing buffer was removed on a paper tissue. Then the primary antibody was diluted with PBS containing 10% FBS and added to each well at 100 μl per well. The plate was incubated at room temperature for 2 hours, the washed with a washing buffer for three times, and the excess washing buffer was removed on a paper tissue.
Streptavidin-alkaline phosphatase was diluted at 1:100 with PBS containing 10% FBS, 100 μl of diluted streptavidin-alkaline phosphatase was added to each well, and the plate was incubated at room temperature for 1 hour. Then the plate was washed for 3 times with a washing buffer and 2 times with PBS, and pat the excess washing buffer and PBS were removed on a paper tissue. After washing, 100 μl/well of BCIP/NBT solution provided by the kit was added for development. The plate was covered with a tin foil during development to avoid light, and incubated for 5-15 minutes. During this period, spots on the developed plate were routinely checked to determine the best time to quench the reaction. The BCIP/NBT solution was removed and the plate was rinsed with double distilled water to stop quench the development reaction, and spin-dried. And then the bottom of the plate was removed. The plate was dried at room temperature until each well is completely dry, and then the immunospot plate counter (CTL, Cellular Technology Limited) was used to count the number of spots formed on the bottom membrane in the plate.
Results
The release of IFN-γ from the γδ T cells transduced with the TCR of the present invention in response to T2 cells loaded with NY-ESO-1 157-165 P1B SLLMWITQC short peptide and T2 cells loaded with the control peptide P72A was detected by ELISPOT experiment (as described above). The number of detected spots was analyzed and plotted using the software GraphPad Prism7.
The experimental results are shown in
This experiment was conducted to verify the activation of high affinity 1G4 TCR-transduced γδT cells that specifically respond to tumor cell lines. The achieved effects were significantly better than thoes of γδT cells transduced with wild-type 1G4TCR. The IFN-γ production detected by the ELISPOT test was used as the readout value of TCR-transduced γδT cell activation. The specific operation of ELISPOT experiment is as described in Example 4.
Sources of Main Reagents and Material
10% FBS (Gibco, Catalog number 16000-044), RPMI 1640 (Gibco, Catalog number C11875500bt), PVDF ELISPOT 96-well plate (Merck Millipore, Catalog number MSIPS4510), human IFN-γ ELISPOT PVDF-enzyme kit (BD) containing all other required reagents (capture and detection antibody, streptavidin-alkaline phosphatase and BCIP/NBT solution), Elispot's Kit (BD™ ELISPOT ELISPOT Set Instruction Manual Catalog number: 551282), tumor cell line: IM9 is multiple myeloma, HLA genotype of which is A0201, also express NY-ESO-1 antigen; U266B1 is myeloma, plasma cells, HLA genotype of which is A0201, also expresses NY-ESO-1 antigen; NCI-H1299 is a lung cancer cell line that expresses NY-ESO-1 antigen, but HLA genotype of which is not A0201, as a control; LCLs are immortalized lymphoblastoid cell lines, which does not express NY-ESO-1 antigen, and the HLA genotype of which is not A0201, as a control. The above tumor cell lines were purchased from ATCC.
Methods
Preparation of Target Cells
The target cells of this experimental example were IM9 and U266B1 as the positive tumor cell line of this experiment. The HLA genotype of these two cell lines is A0201, and they also express the antigen NY-ESO-1; NCI-H1299 expresses NY-ESO-1 antigen, but the HLA genotype is not A0201; LCLs neither express NY-ESO-1 antigen, nor the HLA genotype is A0201, these two cells were used as negative tumor cell lines in this experiment.
Preparation of Effector Cells
Preparation of effector cells was described in the examples. Different groups of effector cells were prepared: Vδ2T cells without being transduced with 1G4TCR and Vδ2T cells transduced with 1G4TCRs of different affinities of 32 μM (wild type), 4 μM, 1.07 μM, 450 nM, 84 nM and 5 nM.
The experimental results are shown in
Different from the traditional 51Cr release method, this experiment uses a colorimetric-based detection method-CytoTox 96® non-radioactive cytotoxicity test to quantitatively measure the lactate dehydrogenase (LDH) released during cell lysis. The reaction principle is: cells will release LDH into the supernatant of the medium due to lysis, which can be detected by a 30-minute coupled enzyme reaction. In the enzyme reaction, LDH can convert a tetrazolium salt (INT) into red formazan. The amount of red product produced is directly proportional to the number of lysed cells. A standard 96-well plate reader was used to collect the visible light absorbance data at 490 nm. This method is used to reflect the lethality to target cells when effector cells and target cells are incubated together.
Materials
CytoTox 96® non-radioactive cytotoxicity test (Promega; Catalog No.: G1780) kit and supporting substrate mixture, test buffer, lysis solution and stop buffer. Medium: 10% FBS (Gibco, Catalog number: 16000-044), added to RPMI1640 (Gibco, Catalog number: C11875500bt) medium without phenol red. Note: FBS has been inactivated before use. Round bottom 96-well tissue culture plate (Corning, Catalog number: 3799); 96-well immunoplate (Thermo, Catalog number: 167008). Tumor cell lines: A375 melanoma (HLA type of which is A0201, and which expresses antigen NY-ESO-1) and NCI-H1650 lung cancer cell line (HLA type of which is A0201 and which does not express antigen NY-ESO-1) were purchased from ATCC; NCI-H1299 lung cancer cells (HLA type of which is A0201 and which expresses antigen NY-ESO-1), which is in-house modified from the wild type purchased from the ATCC.
Methods
Preparation of Target Cella
The positive tumor cell lines (both expressing HLA-A2 and antigen NY-ESO-1) used in the experiment were: A375 and NCI-H1299, and the negative tumor cell lines (expressing only one of HLA-A2 and NY-ESO-1) is: NCI-H1650. The adjusted target cell density was 2*105 cells/ml, and 2*104 cells/100 μl was added in each well.
Preparation of Effector Cells
The effector cells were prepared as described in Example 2. The effector cells in this experiment were:
Vδ2γδT cells (control) without being transduced with lentivirus (control), Vδ2γδT cells transduced with GFP (control) and Vδ2γδT cells transduced with wild-type (KD=32 μM) 1G4TCR, as well as Vδ2γδT cells transduced with 1G4TCRs of different affinities. The KD values of the high-affinity 1G4 TCR were 4 μM, 1.07 μM, 450 nM, 84 nM, and 5 nM, respectively, and all of the above cells were obtained by expanding a polyclonal cell population obtained by BD Aria flow sorting. The ratio of effector cells to target cells (E:T ratio) was: 10:1, 5:1, 1:1 and 1:5, respectively.
Experiment Groups were Set as Follows:
The experimental group, in which Vδ2γδT cells transduced with NY-ESO-1 specific 1G4TCR mutants of different affinities specifically kill tumor cell lines, was set as follows: 100 μl of target cells was firstly added to a round bottom 96-well plate; and effector cells were also added to the round-bottom 96-well plate in a volume of 100 microliters at 4 effector-to-target ratios.
All of control groups were set as follows: spontaneous LDH experimental well of effector cell: 100 μl of effector cell suspension was added; spontaneous LDH experimental group of target cell: 100 μl of target cell suspension was added; maximum LDH release group of target cell: 100 μl of target cell suspension was added; background control group of medium: only 200 μl of medium; volume-corrected control group: only 200 μl of medium. All of experimental wells and control wells were prepared in triplicate, and the final volume of all of wells was 200 μl (supplemented with phenol red-free complete medium).
The plate was placed in a 37° C., 5% CO2 incubator and incubated for 24 hours. 10 μl of lysis solution (10×) per 100 μl of medium was added to maximum LDH release well of target cell and volume correction well to lyse the cells; and the plate was incubated in 37° C., 5% humidified CO2 incubator for 45 minutes, centrifuged at 250×g for 4 minutes. 50 μl of supernatant was taken from each well and transferred to a new 96-well flat-bottom (enzyme analysis) plate. 50 μl of a prepared substrate mixture (12 ml of Assay Buffer was added to a bottle of Substrate Mix and dissolve it at room temperature) was added to each well, and the plate was covered with a tin foil or an opaque box to protect the plate from light. The plate was incubated at room temperature for 30 minutes. 50 μl of stop solution was added to each well to quench the reaction. Absorbance at 490 nm was measured within one hour after adding the stop solution, the data were exported and saved in EXCEL format for analyzing results. The counting formula is as follows:
% Cytotoxicity=[100*(experiment−spontaneous effector cell−spontaneous target cell)]/(maximum target cell−spontaneous target cell). Note: for the experimental well, different effect target ratios (mean)−medium background (mean); for the target cell spontaneous well, target cell spontaneous well (mean)−medium background (mean); for effector cell spontaneous well, spontaneous effector cells of different proportions (mean)−medium background (mean); and for the highest well of target cell, the highgest target cell (mean)−volume correction control (mean). The final drawing software is GraphPad Prism7.
Results
The experimental results are shown in
According to a rough estimate, compared with the killing percentage of the wild type αβTCR on target cells, when the KD value of αβTCR affinity was between 4 μM-5 nM, the killing percentage of transduced γδT cells on target cells at different effector-target ratios was increased at least 1 times, preferably, at least a 2-3 times. Generally, when the affinity of αβTCR was 1.07 μM, the killing efficiency of γδT cells was more excellent.
γδT cells transduced with αβ1G4TCRs of different high affinities exhibited basically no killing effects on negative tumor cell lines. γδT cells that have not been transduced with exogenous αβTCR or γδT cells that have only been transfected with GFP exhibited basically no response to the relevant tumor cell lines.
Luciferase reporter gene refers to a reporter system that uses luciferin as a substrate to detect fireflyluciferase activity. Luciferase can catalyze the conversion of luciferin to oxyluciferin through oxidation, and bioluminescence will be emitted during the oxidation of luciferin. The bioluminescence released during the oxidation of luciferin is measured by a fluorescence analyzer. The energy for the redox reaction is necessarily provided by cells, therefore, we incubated effector cells with specific target cells, which will kill the target cells, thereby reducing the energy of the target cells. And the luminescence when catalyzing the fluorescein substrate will be reduced, which can indirectly reflect the killing ability of effector cells on target cells and can be used as a verification to check the function of lactate dehydrogenase LDH. This method is used to reflect the killing effects on target cells when effector cells and target cells are incubated together.
Main Reagents and Materials
The substrate catalyzed by luciferase is D-luciferin (a potassium ion salt, 150 μg/ml, Cayman Chemical, USA); 96-well plate for experiment: purchased from Corning, Catalog number: 3792; tumor cell line: A549 with luciferase was purchased from ATCC. In this experiment, a cell line which expresses NY-ESO-1 antigen and is of HLA-A0201 was constructed.
Preparation of Target Cells
As target cells, A549 expressing Luciferase, NY-ESO-1 and A0201 were used as positive tumor cells, and wild-type A549 (Luciferase) was used as negative control tumor cells and a control group with only corresponding target cells.
Preparation of Effector Cells
The effector cells were prepared as described in Example 2, including Vδ2T cell group without being transduced with lentivirus, Vδ2T cells transduced with GFP, and Vδ2T cell group transduced with wild-type αβ1G4TCR (KD=32 uM) and Vδ2T cell group transduced with high affinity αβ1G4TCR with KD=1.07 uM.
Methods
The effector cells and target cells were resuspended in RPMI1640+10% FBS+1% PS complete medium. The effector cells and target cells were innoculated into a 96-well plate at E:T=5:1 and 2*104 target cells/well; incubated at 37° C., 5% CO2 for 24 hours; 100 ul of Luciferase substrate was added to each well of the experimental group and the control group in darkness, respectively; the target cell viability in the experimental group and the control group was detected by exciting light at 450 nM using a microplate reader; excel data was derived and the killing efficiency of effector cells was calculated according to the formula. The formula is as follows: killing %=(1-signal of experimental group/maximum signal of target cell) %. The final drawing software is GraphPad Prism7.
Results
The detection result is shown in
Real-time dynamic live cell imaging analyzer—IncuCyte Zoom, a non-invasive method is used to record the real-time growth status of cells. The killing effects of γδT transduced with TCRs of different affinities can be dynamically compared.
Main Reagents and Materials
96-well plate: purchased from Corning, Catalog number: 3603;
YO-YO-3 staining: NCI-H1299 lung cancer cells, purchased from ATCC is a wild-type expressing antigen NY-ESO-1, but the HLA type thereof is not A0201;
Real-time dynamic live cell imaging analyzer—IncuCyte Zoom: American Essen Company.
Preparation of Target Cells
NCI-H1299: The wild type purchased from ATCC was modified in the laboratory of the inventors, and the wild type NCI-H1299 cell line was directly transduced with the HLA-A0201 lentivirus to construct the a positive tumor cell line which expresses NY-ESO-1 antigen and is also of HLA-A0201.
Preparation of Effector Cells
Effector cells was prepaed as described in Example 2, different groups of effector cells were prepared, including Vδ2T cells without being transduced with 1G4TCR and Vδ2T cells transduced with GFP and
Vδ2T cells transduced with 1G4TCRs of different affinities of 32 μM (wild type), 4 μM, 1.07 μM, 450 nM, 84 nM and 5 nM.
Methods
The target cells were digested and centrifuged; resuspended in phenol red-free RPMI1640+10% FBS complete medium, and counted, 7*103 cells/well/100 ul. The target cells were evenly spread in a 96-well plate; placed in a 37° C., 5% CO2 incubator and incubated overnight; effector cells were prepared at a E:T=5:1, and centrifuged; the old culture medium was discarded and replaced with new phenol red-free RPMI1640+10% FBS medium (the medium was mixed with YOYO-3 dye of a final concentration of 10000*). The effector cells were incubated with the target cells in the experimental group at 3.5*104 cells/well/100 ul. 100 ul of complete medium was added to each well in the control group of target cells or effector cells to ensure that the final concentration of YOYO-3 dye in each well is 10000*; the plate was placed in the Real-time dynamic live cell imaging analyzer—IncuCyte Zoom dedicated to Incucyte detection; real-time observation was started and pictures were taken; and the test results were processed and the data were exported and analyzed by using IncuCyte ZooM 2016A. The final drawing software is GraphPad Prism7.
Results
Detection results are shown in the
The sequences involved in the present invention are shown in the following table:
All documents mentioned in the present invention are cited as references in this application, as if each document is individually cited as a reference. In addition, it should be understood that after reading the above teachings of the present invention, a skilled person in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201711124057.2 | Nov 2017 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2018/115235 | 11/13/2018 | WO | 00 |
