T cell receptor and application thereof

Abstract

The present invention relates to the field of cancer immunotherapy. In particular, the present application relates, in some aspects, to binding molecules, such as complexes capable of specifically binding a major histocompatibility complex (MHC) molecule and an AFP peptide. The present invention relates to T cell receptors (TCRs), including antigen binding domains thereof, that bind to or recognize such peptide epitopes. The present application further relates to engineered cells comprising such binding molecules, such as TCR double antibody proteins or TCRs, and the like, and their use in cancer immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of cancer immunotherapy, and in particular relates to a T cell receptor capable of specifically binding to a complex of a major histocompatibility complex (MHC) molecule and an AFP peptide and an application thereof. Background Art

[0002] Immunotherapy achieves the effect of curing cancer by enhancing the function of the patient's immune system and using immune cells to eliminate cancer cells in the body. Cell therapy is an adoptive therapy that transfers T cells that are specifically reactive to target cell antigens into the patient's body so that they can act on target cells. The T cell receptor (TCR) is a membrane protein on the surface of T cells that can recognize antigenic short peptides on the surface of corresponding target cells. In the immune system, through the combination of antigenic short peptide-specific TCR and short peptide-major histocompatibility complex (pMHC complex), T cells and antigen presenting cells (APC) are in direct physical contact, and then other cell membrane surface molecules of both T cells and APCs interact, causing a series of subsequent signal transmission, so that T cells with different antigen specificities can exert immune effects on their target cells.

[0003] Engineered T cells containing tumor antigen-specific T cell receptors (TCRs) can overcome some of the challenges faced by current adoptive lymphocyte therapy approaches because it can rapidly generate tumor-reactive T lymphocytes with defined antigen specificity. There is a need for TCRs that target different tumor antigens and provide high antitumor efficacy in vivo and low off-target effects. Common problems with TCR-T cell therapy approaches include serious adverse events (such as central nervous system toxicity) in clinical trials, which may be related to inappropriate target selection (the so-called on-target off tumor effect) and biased expansion of T cell populations.

[0004] Alpha-fetoprotein (AFP) is a highly specific protein expressed by liver cancer cells. Many liver cancer patients (70-80%) have the characteristic of high expression of AFP gene during the onset of the disease. AFP is a germinal protein whose gene is turned on and expressed during fetal development, and is basically in a closed state two years after birth. However, when adults develop liver cancer or benign liver regeneration, the AFP gene is reactivated and expressed in large quantities. It is clinically considered to be a classic tumor marker for liver cancer and is therefore used as the gold standard for diagnosing liver cancer.

[0005] Human AFP belongs to α-globulin, and its electrophoretic movement is between albumin and globulin, with a molecular weight of about 64,000-72,000 and a sedimentation coefficient of 4.5. This protein is composed of about 18 kinds of amino acids, and carbohydrates account for about 4%. AFP from the fetus has a consistent electrophoretic movement, and AFP from tumors contains 4 variants or subcomponents, and their electrophoretic movements are slightly different. This difference is mainly in the carbohydrate content rather than the change of the peptide chain.

[0006] AFP-specific TCR and TCR-T therefore have the potential to become cancer therapies with high anti-tumor efficacy and low off-target effects. Summary of the invention

[0007] In order to achieve the above-mentioned object, the present invention discloses a method for identifying HLA-A restricted AFP. 158-166 T cell receptor for epitope peptide, consisting of α chain and β chain, The α chain and the β chain contain the following complementary determining regions: 1) the amino acid sequence of CDR1 on the α chain is shown in one of SEQ ID NOs: 7-12, the amino acid sequence of CDR2 on the α chain is shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 on the α chain is shown in SEQ ID NO: 3; The amino acid sequence of CDR1 on the β chain is shown in SEQ ID NO: 4, the amino acid sequence of CDR2 on the β chain is shown in SEQ ID NO: 5, and the amino acid sequence of CDR3 on the β chain is shown in SEQ ID NO: 6; or 2) the amino acid sequence of CDR1 on the α chain is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 on the α chain is shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 on the α chain is shown in SEQ ID NO: 3; The amino acid sequence of CDR1 on the β chain is shown in SEQ ID NO: 4, the amino acid sequence of CDR2 on the β chain is shown in SEQ ID NO: 14, and the amino acid sequence of CDR3 on the β chain is shown in SEQ ID NO: 6; or 3) the amino acid sequence of CDR1 on the α chain is shown in SEQ ID NO: 1 or 7, the amino acid sequence of CDR2 on the α chain is shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 on the α chain is shown in SEQ ID NO: 13; The amino acid sequence of CDR1 on the β chain is shown in one of SEQ ID NOs: 4, 15-17, the amino acid sequence of CDR2 on the β chain is shown in SEQ ID NO: 5, 14 or 18, and the amino acid sequence of CDR3 on the β chain is shown in SEQ ID NO: 6.

[0008] The present invention also protects polypeptides or protein molecules having functions similar to those of the T cell receptor of the present invention obtained by substitution and / or deletion and / or addition of one or more amino acid residues; or polypeptides or protein molecules having a sequence similarity of more than 90% and having the ability to recognize HLA-A restricted AFP. 158-166 Epitope peptides are functionally related to proteins.

[0009] Wherein, the T cell receptor recognizes HLA-A restricted AFP 158-166 The epitope peptide, the amino acid sequence of the epitope peptide is shown in SEQ ID NO: 19, and is specifically as follows: FMNKFIYEI (SEQ ID NO:19).

[0010] Preferably, the HLA typing includes: HLA-A*0203, HLA-A*0201, HLA-A*0206 or HLA-A*0207.

[0011] The second aspect of the present invention discloses a nucleic acid molecule encoding the above-mentioned T cell receptor.

[0012] In the present invention, it is well known to those skilled in the art that, due to the degeneracy of protein genetic codons, there are usually more than one codon that determines an amino acid, and the substitution of the third nucleotide in a triplet codon often does not change the composition of the amino acid, so the nucleotide sequences of genes encoding the same protein may be different. Based on the known codon table, those skilled in the art can completely deduce the nucleotide sequences of genes that can encode them from the amino acid sequences disclosed in the present invention, and obtain the nucleotide sequences by biological methods (such as PCR methods, mutation methods) or chemical synthesis methods. Therefore, the partial nucleotide sequences should be included in the protection scope of the present invention.

[0013] The third aspect of the present invention discloses an engineered immune cell expressing the above-mentioned T cell receptor.

[0014] The T cell receptor is introduced into immune cells by viral vector or non-viral vector transduction, CRISPR / Cas9 system, transposon system or mRNA transfection to obtain engineered immune cells.

[0015] The viral vector transduction can be carried out by retrovirus or lentivirus to transfer the TCR gene into T cells.

[0016] The non-viral vector transduction includes electroporation or nanoparticle technology to introduce TCR genes into T cells. The engineered immune cells are derived from NK cells or T cells.

[0017] The fourth aspect of the present invention discloses a TCR dual antibody protein, which is composed of an antibody targeting a receptor molecule on the cell membrane surface of an immune cell, and the extracellular region of the above-mentioned T cell receptor, and simultaneously binds to immune cells and tumor cells; the immune cells include T cells or NK cells; the antibodies to the receptor molecules on the cell membrane surface of the immune cell include scFv or Fab antibody fragments.

[0018] The immune cell cell membrane surface receptor molecules are preferably CD3, CD4, CD8, CD16a and / or NKG2D.

[0019] The extracellular region of the T cell receptor recognizes and binds to antigens on tumor cells.

[0020] The fifth aspect of the present invention discloses a TCR dual antibody mRNA preparation, including the above-mentioned TCR dual antibody protein, specifically using mRNA technology to express the TCR dual antibody protein in vivo.

[0021] The sixth aspect of the present invention discloses the use of the above-mentioned T cell receptor, nucleic acid molecule, engineered immune cell, TCR dual antibody protein or TCR dual antibody mRNA preparation in the preparation of a drug for preventing and / or treating a disease or condition expressing AFP.

[0022] Diseases or conditions in which AFP is expressed include liver cancer, germ cell tumors, hepatitis, gastric cancer, etc.

[0023] The drugs also include TCR protein drugs; The TCR protein drug is prepared from a multispecific T cell complex, and the multispecific T cell complex is prepared from the above-mentioned T cell receptor; The multispecific T cell complex comprises a TCR bispecific antibody, a TCR trispecific antibody, and a TCR tetraspecific antibody; And a multispecific T cell complex formed by T cell receptor, TCR bispecific antibody, TCR trispecific antibody or TCR tetraspecific antibody, connected with nuclear medicine, toxin, fluorescein and antibody fragment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The following examples are intended to be purely illustrative of the present application and therefore should not be considered to limit the invention in any way.The following examples and detailed description are provided by way of illustration and not by way of limitation.

[0025] Figure 1Shown is an HLA restriction assay of T cells expressing the AFP antigen-specific T cell receptor of AM14-M1 in the K562 cell line, and a HepG2 in vitro toxicity assay.

[0026] Figure 2 Shown is an HLA restriction assay of T cells expressing the AFP antigen-specific T cell receptor of AM14-M2 in the K562 cell line, and a HepG2 in vitro toxicity assay.

[0027] Figure 3 Shown is the HLA restriction assay of T cells expressing the AFP antigen-specific T cell receptor of AM14-M3 in the K562 cell line, and the HepG2 in vitro toxicity assay.

[0028] Figure 4 Shown is the HLA restriction assay of T cells expressing the AFP antigen-specific T cell receptor of AM14-M4 in the K562 cell line, and the HepG2 in vitro toxicity assay.

[0029] Figure 5 Shown is the HLA restriction assay of T cells expressing the AFP antigen-specific T cell receptor of AM14-M5 in the K562 cell line, and the HepG2 in vitro toxicity assay.

[0030] Figure 6 HLA restriction assay of T cells expressing the AFP antigen-specific T cell receptor of AM14-M6 in the K562 cell line.

[0031] Figure 7 HLA restriction assay of T cells expressing the AFP antigen-specific T cell receptor of AM14-M7 in the K562 cell line.

[0032] Figure 8 Shown is the HLA restriction assay of T cells expressing the AFP antigen-specific T cell receptor of AM14-M8 in the K562 cell line, and the HepG2 in vitro toxicity assay.

[0033] Fig. 9 It showed that the AFP-TCR dual antibody protein AM14-M7 efficiently induced T cells to recognize the AFP-HLA-A*02:03 complex on the target cell membrane and activated T cells to secrete IFN-γ.

[0034] Fig.10 It shows that the AFP-TCR dual antibody protein AM14-M10-13 efficiently induces T cells to recognize the AFP-HLA-A*02:03 complex on the target cell membrane and activates T cells to express luciferase.

[0035] Fig.11It shows that the AFP-TCR dual antibody protein AM14-M14-18 efficiently induces T cells to recognize the AFP-HLA-A*02:03 complex on the target cell membrane and activates T cells to express luciferase. DETAILED DESCRIPTION

[0036] The following examples are intended to be purely illustrative of the present application and therefore should not be considered to limit the invention in any way.The following examples and detailed description are provided by way of illustration and not by way of limitation.

[0037] Example 1: Preparation of AFP-specific T cells TCR mRNA was prepared and activated CD3-T cells were electroporated to prepare TCR-T. On the first day, PBMCs were revived and incubated at 4°C for 5 minutes with pan-T sorting magnetic beads. Secondary antibodies were then added and incubated at 4°C for 10 minutes. The cell suspension was added to the sorting column and CD3-T cells were sorted by magnetic field. The sorted CD3-T cells were activated with T cell Transact (10µL / 1E6cell) and the cell density was adjusted to 1×10 using AIM-V medium. 6 Cells / mL. On the 7th day, activated CD3-T cells were collected, centrifuged at 350g for 5 minutes, the supernatant was removed and fresh culture medium was added, and samples were taken for counting. The cells were washed with PBS, the supernatant was removed, and the electroporation solution (1E6 cells / 20µL electroporation solution) was added for resuspending. 20µL of cell suspension was added to the EP tube, a total of 8 samples, and 2 µL of TCR mRNA (AM14-M1, AM14-M2, AM14-M3, AM14-M4, AM14-M5, AM14-M6, AM14-M7, and AM14-M8 mRNA) were added respectively, mixed, and added to the small slot of the 20µL system electroporation cup. The electroporation cup was gently tapped to ensure that the sample covered the bottom of the cup, and then placed in the electroporation instrument slot for electroporation under the set program.

[0038] Wild-type AM14 includes: α chain, CDR1: VSGLRG (SEQ ID NO: 1) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSNSGYALNF (SEQ ID NO: 3) Beta chain, CDR1:SEHNR (SEQ ID NO: 4) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0039] AM14-M1 includes: α chain, CDR1: SRMLRG (SEQ ID NO: 7) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSNSGYALNF (SEQ ID NO: 3) β chain, CDR1: SEHNR (SEQ ID NO: 4) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0040] AM14-M2 includes: α chain, CDR1: SSMLRG (SEQ ID NO: 8) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSNSGYALNF (SEQ ID NO: 3) β chain, CDR1: SEHNR (SEQ ID NO: 4) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0041] AM14-M3 includes: α chain, CDR1: STLLRG (SEQ ID NO: 9) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSNSGYALNF (SEQ ID NO: 3) β chain, CDR1: SEHNR (SEQ ID NO: 4) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0042] AM14-M4 includes: α chain, CDR1: SSLLRG (SEQ ID NO: 10) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSNSGYALNF (SEQ ID NO: 3) Beta chain, CDR1:SEHNR (SEQ ID NO: 4) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0043] AM14-M5 includes: α chain, CDR1: HSLLRG (SEQ ID NO: 11) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSNSGYALNF (SEQ ID NO: 3) Beta chain, CDR1:SEHNR (SEQ ID NO: 4) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0044] AM14-M6 includes: α chain, CDR1: VSLLRG (SEQ ID NO: 12) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSNSGYALNF (SEQ ID NO: 3) Beta chain, CDR1:SEHNR (SEQ ID NO: 4) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0045] AM14-M7 includes: α chain, CDR1: VSGLRG (SEQ ID NO: 1) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSWPSYALNF (SEQ ID NO: 13) Beta chain, CDR1:SEHNR (SEQ ID NO: 4) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0046] AM14-M8 includes: α chain, CDR1: VSGLRG (SEQ ID NO: 1) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSNSGYALNF (SEQ ID NO: 3) Beta chain, CDR1:SEHNR (SEQ ID NO: 4) CDR2:FIFGAQ (SEQ ID NO: 14) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0047] Example 2: Validation of AFP-specific T cell receptor AM14-M1 TCR expression and peptide recognition assay K562 cells with HLA-A*02:03 / HLA-A*02:01 / HLA-A*02:06 / HLA-A*02:07 genotypes were loaded with AFP 158-166 The peptides were used to stimulate T cell receptor transfected T cells in HLA restriction assays, measured by flow cytometry and human IFN-γ ELISA.

[0048] TCR-transduced T cells were collected and washed by adding 2 mL of PBS, centrifuged at 350 g for 5 minutes, and the supernatant was completely aspirated. Then, TCR-transduced T cells were resuspended by adding AIM-V medium (containing 5% AB serum) and adjusted to 1×10 6 cells / mL, 100µL was added to a 96-well plate. 158-166 Peptide target cells were mixed with tumor-specific T cells, brefeldin A (final concentration 3 µg / mL) was added to the wells, and cultured for 4 hours. After 4 hours, the cells were collected and stained for intracellular IFN-γ, surface CD3, and TCR expression, and detected by flow cytometry.

[0049] For IFN-γ ELISA, 100 µL / well of AFP 158-166 K562 cells (1×10 6 cells / mL) and 100 µL / well of TCR-transduced T cells (1×10 6Cells / mL) were mixed into 96-well plates and cultured in an incubator for 24 hours. After 24 hours, 175 µL of supernatant was collected per well in the 96-well plate and used for ELISA detection of IFN-γ using the IFN-γ ELISA HRP kit. On day 1, high protein binding ELISA plates were coated with antibody 1-D1K (IFN-γ ELISA HRP kit) diluted to 2 µg / mL in PBS (pH 7.4) (by adding 100 µL / well) and incubated overnight at 4°C. On day 2, the plates were washed twice with PBS (200 µL / well). The plates were blocked by adding 200 µL / well of culture medium and incubated for 1 hour at room temperature (RT). Human IFN-γ standard (IFN-γ ELISA HRP kit) was prepared in 2 mL PBS (with 1% BSA) to a concentration of 0.5 µg / mL and placed at RT for 15 minutes, then the tubes were vortexed. 100 µL / well of sample or standard was diluted in culture medium and incubated at RT for 2 hours. Samples and standard probes were tested in duplicate. The plate was washed five times with PBS containing 0.05% Tween 20. 100 µL / well of antibody 7-B6-1-biotin (IFN-γ ELISA HRP kit) was added at 1 µg / mL (in PBS), incubated at RT for 1 hour, and washed. 100 µL / well of streptavidin-HRP (IFN-γ ELISA HRP kit) diluted 1:1000 in PBS was added, incubated at RT for 1 hour, and washed. Add 100 µL / well of TMB substrate solution and incubate in the dark at RT for 15 to 30 minutes until the solution in the well turns a visible blue color. Add 100 µL / well of stop solution to stop the enzyme reaction. The solution color changes from blue to yellow. Measure the light intensity at 450 nm with an ELISA reader.

[0050] like Figure 1 As shown in A and B, T cells transfected with AM14-M1 can recognize AFP presented by HLA-A*02:03 / HLA-A*02:01 / HLA-A*02:06 / HLA-A*02:07 158-166 Antigenic peptide.

[0051] In vitro toxicity assay T cells transfected with T cell receptor AM14-M1 were used as effector cells and mixed with target cells at a ratio of 5:1 as the experimental group. The target cells used in this experiment were HepG2 cells that naturally expressed HLA-A*02:01 and AFP proteins. T cells that were not transfected with T cell receptors were mixed with target cells at the same ratio as the control group. The killing ability of the effector cells of the experimental group and the control group on target cells was continuously measured for 20 hours. After 20h, the killing plate was removed, centrifuged at 500g, and 175 µL of supernatant was collected from each well and used for ELISA detection of IFN-γ using the IFN-γ ELISA HRP kit.

[0052] like Figure 1 As shown in C and D, the T cells transfected with AM14-M1 had a significant killing effect on HepG2 target cells that naturally expressed AFP protein, and secreted IFN-γ, killing the target cells, with an effector-target ratio (E: T) of 5: 1. The untransfected T cells in the control group had no killing effect on the target cells.

[0053] Example 3: Validation of AFP-specific T cell receptor AM14-M2-8 The AFP-specific T cell receptor AM14-M2-8 was tested in the same manner as in Example 2 in terms of TCR expression and peptide recognition, HLA restriction, in vitro toxicity, and IFN-γ ELISA. Figure 2-8 shown.

[0054] Figure 2 A and B show that T cells transfected with AM14-M2 can recognize AFP presented by HLA-A*02:03 / HLA-A*02:01 / HLA-A*02:06 / HLA-A*02:07 158-166 Antigenic peptides; Figure 2 C and D in the middle show that T cells transfected with AM14-M2 have a significant killing effect on HepG2 target cells that naturally express AFP protein, and the target cells are killed; the effector-target ratio (E:T) is 5:1.

[0055] Figure 3 A and B show that T cells transfected with AM14-M3 can recognize AFP presented by HLA-A*02:03 / HLA-A*02:01 / HLA-A*02:06 / HLA-A*02:07 158-166 Antigenic peptides; Figure 3 C and D in the middle show that T cells transfected with AM14-M3 have a significant killing effect on HepG2 target cells that naturally express AFP protein, and the target cells are killed; the effector-target ratio (E:T) is 5:1.

[0056] Figure 4A and B show that T cells transfected with AM14-M4 can recognize AFP presented by HLA-A*02:03 / HLA-A*02:01 / HLA-A*02:06 / HLA-A*02:07 158-166 Antigenic peptides; Figure 4 C and D in the middle show that T cells transfected with AM14-M4 have a significant killing effect on HepG2 target cells that naturally express AFP protein, and the target cells are killed; the effector-target ratio (E:T) is 5:1.

[0057] Figure 5 A and B show that T cells transfected with AM14-M5 can recognize AFP presented by HLA-A*02:03 / HLA-A*02:01 / HLA-A*02:06 / HLA-A*02:07 158-166 Antigenic peptides; Figure 5 C and D in the middle show that T cells transfected with AM14-M5 have a significant killing effect on HepG2 target cells that naturally express AFP protein, and the target cells are killed; the effector-target ratio (E:T) is 5:1.

[0058] Figure 6 A and B show that T cells transfected with AM14-M6 can recognize AFP presented by HLA-A*02:03 / HLA-A*02:01 / HLA-A*02:06 / HLA-A*02:07 158-166 Antigenic peptide.

[0059] Figure 7 A and B show that T cells transfected with AM14-M7 can recognize AFP presented by HLA-A*02:03 / HLA-A*02:01 / HLA-A*02:06 / HLA-A*02:07 158-166 Antigenic peptide.

[0060] Figure 8 A and B show that T cells transfected with AM14-M8 can recognize AFP presented by HLA-A*02:03 / HLA-A*02:01 / HLA-A*02:06 / HLA-A*02:07 158-166 Antigenic peptides; Figure 8 C and D in the middle show that T cells transfected with AM14-M8 have a significant killing effect on HepG2 target cells that naturally express AFP protein, and the target cells are killed; the effector-target ratio (E:T) is 5:1.

[0061] Example 4: Validation of AFP-TCR dual antibody protein The purpose of this example is to verify the recognition of target cells and epitopes by AFP-TCR dual antibody protein and the functional detection of inducing T cells in vitro.

[0062] Ten AFP antigen-specific T cell receptors AM14-M7, AM14-M10, AM14-M11, AM14-M12, AM14-M13, AM14-M14, AM14-M15, AM14-M16, AM14-M17 and AM14-M18 were prepared for in vitro functional validation experiments.

[0063] IFN-γ ELISA Activated T cells with HLA-A*02:03 genotype and AFP were used in a 1:1 ratio. 158-166 After 48 h of co-culture of K562 cells transduced with HLA-A*02:03 and the dual-antibody protein to be tested, human IFN-γ ELISA was performed.

[0064] On the first day, HLA-A*02:03 transduced K562 cells were collected, centrifuged at 350 g for 5 minutes, and the supernatant was completely aspirated. The cell pellet was resuspended and adjusted to approximately 1×10 by adding complete 1640 medium (1640 medium + 10% fetal bovine serum + 1% double antibody). 6 cells / mL, AFP was added at 1 µg / ml 158-166 Incubate with antigen peptide for 2 hours. Collect loaded AFP 158-166 The HLA-A*02:03 transduced K562 cells after antigen peptide and HLA-A*02:03 transduced K562 cells (negative control group) were centrifuged at 350g for 5 minutes, and the supernatant was completely aspirated. Wash with 10 mL PBS (containing 2% fetal bovine serum), centrifuge at 350g for 5 minutes, and aspirate the supernatant completely. Resuspend the cell pellet and adjust to 1×10 by adding complete AIM-V medium (AIM-V medium + 2% human serum + 1% double antibody). 6 Cells / mL, 100 µL / well were added to a 96-well round-bottom plate. Activated T cells were collected, centrifuged at 350 g for 5 minutes, and the supernatant was completely aspirated. The cell pellet was resuspended and adjusted to 1×10 by adding complete AIM-V medium (AIM-V medium + 2% human serum + 1% double antibody). 6 cells / mL, add 20nM protein concentration of dual antibody protein (final concentration after dilution is 10nM), mix well and add AFP loading at 100 µL / well 158-166Antigenic peptide followed by HLA-A*02:03 transduction of K562 cells and HLA-A*02:03 transduction of K562 cells were mixed thoroughly and cultured for 48 hours. On the second day, high protein binding ELISA plates were coated (by adding 100 µL / well) with antibody 1-D1K (IFN-γ ELISA HRP kit) diluted to 2 µg / mL in PBS (pH 7.4) and incubated overnight at 4°C. On the third day, the ELISA plates were washed twice with PBS (200 µL / well). The ELISA plates were blocked by adding 200 µL / well of culture medium and incubated for 1 hour at room temperature (RT). Human IFN-γ standard (IFN-γ ELISA HRP kit) was prepared in 2 mL PBS (with 1% BSA) to a concentration of 0.5 µg / mL and placed at RT for 15 minutes, and the tubes were then vortexed. 100 µL / well of sample or standard diluted in culture medium and incubated at RT for 2 hours. Wash the ELISA plate five times with PBS containing 0.05% Tween 20. Add 100 µL / well of antibody 7-B6-1-biotin (IFN-γ ELISA HRP kit) at 1 µg / mL (in PBS), incubate at RT for 1 hour, and wash. Add 100 µL / well of streptavidin-HRP (IFN-γ ELISA HRP kit) diluted 1:1000 in PBS, incubate at RT for 1 hour, and wash. Add 100 µL / well of TMB substrate solution and incubate at RT in the dark for 15 to 30 minutes until the solution in the well turns a visible blue color. Add 100 µL / well of stop solution to stop the enzyme reaction. The solution color changes from blue to yellow. Measure the light intensity at 450 nm with an ELISA reader. The final measured value is the experimental group measured value minus the negative control group measured value.

[0065] like Fig. 9 As shown, compared with wild-type AM14, AM14-M7 has a stronger and more obvious function of inducing T cell activation and IFN-γ secretion at a TCR dual antibody concentration of 10 nM.

[0066] Jurkat Luciferase Assay At a 1:1 ratio, use load AFP 158-166 The HLA-A*02:03 transduced K562 cells after antigen peptide and the HLA-A*02:03 transduced K562 cells were co-cultured with Jurkat Luciferase cells and the double antibody of the protein to be detected for 24 hours, and then the luciferase expression was detected.

[0067] On the first day, HLA-A*02:03 transduced K562 cells were collected, centrifuged at 350 g for 5 minutes, and the supernatant was completely aspirated. The cell pellet was resuspended and adjusted to approximately 1×10 by adding complete 1640 medium (1640 medium + 10% fetal bovine serum + 1% double antibody). 6 cells / mL, AFP was added at 1 µg / ml 158-166 Incubate with antigen peptide for 2 hours. Collect loaded AFP 158-166 The HLA-A*02:03 transduced K562 cells after antigen peptide and HLA-A*02:03 transduced K562 cells (negative control group) were centrifuged at 350g for 5 minutes, and the supernatant was completely aspirated. Wash with 10 mL PBS (containing 2% fetal bovine serum), centrifuge at 350g for 5 minutes, and aspirate the supernatant completely. Resuspend the cell pellet and adjust to approximately 4×10 by adding complete 1640 medium (1640 medium + 10% fetal bovine serum + 1% double antibody). 5 cells / mL, 50 µL / well was added to a 96-well round-bottom plate. Jurkat Luciferase cells were collected, centrifuged at 350 g for 5 minutes, and the supernatant was completely aspirated. The cell pellet was resuspended and adjusted to 4×10 by adding complete 1640 medium (1640 medium + 10% fetal bovine serum + 1% double antibody). 5 cells / mL, add 2nM protein concentration of dual antibody protein (final concentration after dilution is 1nM), mix well and add AFP loading at 50 µL / well 158-166 The HLA-A*02:03 transduced K562 cells after antigen peptide and the HLA-A*02:03 transduced K562 cells were fully mixed and cultured for 24 hours. On the second day, the detection reagent in the firefly luciferase reporter gene detection kit was restored to room temperature in advance, and the cell mixture after co-culture was blown to take 60μl of cell suspension into the white ELISA plate, and then 60μl of detection reagent was mixed with the cell suspension, and the reaction was fully reacted for 10-20min, and the fluorescence value was detected by chemiluminescence using an enzyme reader. The final measured value is the measured value of the experimental group minus the measured value of the negative control group.

[0068] like Fig.10 As shown, compared with the wild-type AM14, at a TCR dual antibody concentration of 1 nM, AM14-M10, AM14-M11, AM14-M12, and AM14-M13 have stronger and more obvious functions of inducing T cell activation and luciferase expression.

[0069] like Fig.11As shown, compared with AM14-M10, at a TCR dual antibody concentration of 1 nM, AM14-M14, AM14-M15, AM14-M16, AM14-M17 and AM14-M18 have stronger and more obvious functions of inducing T cell activation and luciferase expression.

[0070] AM14-M7 includes: α chain, CDR1: VSGLRG (SEQ ID NO: 1) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSWPSYALNF (SEQ ID NO: 13) Beta chain, CDR1:SEHNR (SEQ ID NO: 4) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0071] AM14-M10 includes: Alpha chain, CDR1: SRMLRG (SEQ ID NO: 7) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSWPSYALNF (SEQ ID NO: 13) Beta chain, CDR1:SEHNR (SEQ ID NO: 4) CDR2: FQNEAQ (SEQ ID NO: 14) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0072] AM14-M11 includes: α chain, CDR1: VSGLRG (SEQ ID NO: 1) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSWPSYALNF (SEQ ID NO: 13) Beta chain, CDR1:LGMNR (SEQ ID NO: 15) CDR2:FIFGAQ (SEQ ID NO: 14) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0073] AM14-M12 includes: Alpha chain, CDR1: VSGLRG (SEQ ID NO: 1) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSWPSYALNF (SEQ ID NO: 13) Beta chain, CDR1: YDHNR (SEQ ID NO: 16) CDR2: FQNDYL (SEQ ID NO: 18) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0074] AM14 - M13 includes: Alpha chain, CDR1: SRMLRG (SEQ ID NO: 7) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSWPSYALNF (SEQ ID NO: 13) Beta chain, CDR1: LGMNR (SEQ ID NO: 15) CDR2: FIFGAQ (SEQ ID NO: 14) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0075] AM14 - M14 includes: Alpha chain, CDR1: SRMLRG (SEQ ID NO: 7) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSWPSYALNF (SEQ ID NO: 13) Beta chain, CDR1: LGMNR (SEQ ID NO: 15) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0076] AM14 - M15 includes: Alpha chain, CDR1: SRMLRG (SEQ ID NO: 7) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSWPSYALNF (SEQ ID NO: 13) β chain, CDR1: YDHNR (SEQ ID NO: 16) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0077] AM14 - M16 includes: α chain, CDR1: SRMLRG (SEQ ID NO: 7) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSWPSYALNF (SEQ ID NO: 13) β chain, CDR1: SEYRW (SEQ ID NO: 17) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0078] AM14 - M17 includes: α chain, CDR1: SRMLRG (SEQ ID NO: 7) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSWPSYALNF (SEQ ID NO: 13) β chain, CDR1: SEHNR (SEQ ID NO: 4) CDR2: FIFGAQ (SEQ ID NO: 14) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0079] AM14 - M18 includes: α chain, CDR1: SRMLRG (SEQ ID NO: 7) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSWPSYALNF (SEQ ID NO: 13) β chain, CDR1: SEHNR (SEQ ID NO: 4) CDR2: FQNDYL (SEQ ID NO: 18) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

Claims

1. A T cell receptor comprising an α chain and a β chain, characterized in that The α chain and the β chain contain the following complementary determining regions: 1) the amino acid sequence of CDR1 on the α chain is shown in one of SEQ ID NOs: 7-12, the amino acid sequence of CDR2 on the α chain is shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 on the α chain is shown in SEQ ID NO: 3; The amino acid sequence of CDR1 on the β chain is shown in SEQ ID NO: 4, the amino acid sequence of CDR2 on the β chain is shown in SEQ ID NO: 5, and the amino acid sequence of CDR3 on the β chain is shown in SEQ ID NO: 6; or 2) The amino acid sequence of CDR1 on the α chain is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 on the α chain is shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 on the α chain is shown in SEQ ID NO: 3; The amino acid sequence of CDR1 on the β chain is shown in SEQ ID NO: 4, the amino acid sequence of CDR2 on the β chain is shown in SEQ ID NO: 14, and the amino acid sequence of CDR3 on the β chain is shown in SEQ ID NO: 6; or 3) the amino acid sequence of CDR1 on the α chain is shown in SEQ ID NO: 1 or 7, the amino acid sequence of CDR2 on the α chain is shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 on the α chain is shown in SEQ ID NO: 13; The amino acid sequence of CDR1 on the β chain is shown in one of SEQ ID NOs: 4, 15-17, the amino acid sequence of CDR2 on the β chain is shown in SEQ ID NO: 5, 14 or 18, and the amino acid sequence of CDR3 on the β chain is shown in SEQ ID NO:

6.

2. The T cell receptor according to claim 1, characterized in that The T cell receptor recognizes HLA-A restricted AFP 158-166 The epitope peptide has an amino acid sequence as shown in SEQ ID NO:

19.

3. The T cell receptor according to claim 2, characterized in that The HLA typing is HLA-A, and HLA-A is HLA-A*0203, HLA-A*0201, HLA-A*0206 or HLA-A*0207.

4. A nucleic acid molecule encoding the T cell receptor according to any one of claims 1 to 3.

5. An engineered immune cell, characterized in that: Expressing the T cell receptor according to any one of claims 1 to 3.

6. An engineered immune cell according to claim 5, characterized in that: The T cell receptor is introduced into immune cells by viral vector or non-viral vector transduction, CRISPR / Cas9 system, transposon system or mRNA transfection to obtain engineered immune cells; the engineered immune cells include T cells or NK cells.

7. A TCR dual antibody protein, characterized in that: The TCR dual antibody protein is composed of an antibody targeting a receptor molecule on the cell membrane surface of an immune cell and the extracellular region of the T cell receptor as claimed in any one of claims 1 to 3, and binds to both immune cells and tumor cells at the same time; The immune cells include T cells or NK cells; The antibodies to the receptor molecules on the cell membrane surface of the immune cells include scFv or Fab antibody fragments.

8. A TCR dual antibody protein according to claim 7, characterized in that: The immune cell cell membrane surface receptor molecules are CD3, CD4, CD8, CD16a and / or NKG2D.

9. A TCR dual antibody protein according to claim 7, characterized in that: The extracellular region of the T cell receptor recognizes and binds to antigens on tumor cells.

10. A TCR dual-antibody mRNA preparation, characterized in that: The invention comprises the TCR dual antibody protein according to any one of claims 7 to 9, and the TCR dual antibody protein is expressed in vivo using mRNA technology.

11. Use of the T cell receptor according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the engineered immune cell according to any one of claims 5 to 6, the TCR dual antibody protein according to any one of claims 7 to 9, or the TCR dual antibody mRNA preparation according to claim 10 in the preparation of a drug for preventing and / or treating a disease or condition expressing AFP.

12. The use according to claim 11, characterized in that The drugs also include TCR protein drugs; The TCR protein drug is prepared from a multispecific T cell complex, and the multispecific T cell complex is prepared from the T cell receptor according to any one of claims 1 to 3; The multispecific T cell complex comprises a TCR bispecific antibody, a TCR trispecific antibody, and a TCR tetraspecific antibody; And a multispecific T cell complex formed by T cell receptor, TCR bispecific antibody, TCR trispecific antibody or TCR tetraspecific antibody, connected with nuclear medicine, toxin, fluorescein and antibody fragment.

Images