Specific t cell receptors against epitopes of mutant myd88l265p protein for adoptive t cell therapy
A specific and variable technology, applied in the field of immunotherapy, which can solve the problems of limited practicality of TCR engineering and small number of patients
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Embodiment 1
[0132] Example 1: Generation of Mutation-Specific T Cells
[0133] PBMCs were isolated from the blood of HLA-B7 positive healthy donors. Monocytes were isolated by plastic adhesion to generate dendritic cells (DC), and then cultured in RPMI containing 800 IU / ml GM-CSF and 10 ng / ml IL-4 in 1% human serum for 3 days, followed by adding 10 ng / ml Immature dendritic cells (imDC) were cultured overnight in the presence of ml LPS and 50 ng / ml interferon gamma (IFN gamma) to allow maturation. Mature dendritic cells (mDC) were then loaded with a mutant peptide (RPIPIKYKAM, SEQ ID NO:2) and used to prime autologous CD8-positive naive T cells (5×10 5 T cells / well, varying with donor-dependent DC-T cell ratio). After 10 days, cells in each well were stained with a specific streptamer (HLA*B07:02-RPIPIKYKAM), or after short (~6 hr) peptide restimulation for T cell activation markers such as CD137 (4-1BB)) was stained. Re-stimulate positively stained wells with peptide-loaded autologous...
Embodiment 2
[0136] Example 2: Identification of Mutation-Specific T Cell Receptors (TCRs)
[0137] After final detection with IFNγ ELISA, viable CD8 and streptamer positive cells were isolated separately by FACS from each reactive T cell clone. Perform total RNA isolation. TCRα and β genes were amplified by 5'-RACEPCR and cloned. Multiple bacterial clones from each TCR chain were sequenced to analyze T cell clonality. Table 1 shows the CDR3 of MyD88-L265P mutation-specific TCR, its sequence number and gene subtype, and Table 2 shows the list of amino acid sequences of CDR1 and CDR2.
[0138] Table 1
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[0142] Table 2
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[0145] The identified variable domains were combined with the sequences of the murine constant domains for experimental characterization of the TCR and synthesized by codon optimization for expression in human cells. A kit of TCR genes encoding TRBV-binding murine TRBC and TRAV-binding TRAC isolated from the p2...
Embodiment 3
[0146] Embodiment 3: TCR affinity analysis
[0147] Peripheral CD8+ T cells from HLA-B7-positive healthy donors were successfully transduced to express mutation-specific TCR without signs of fratricide, and compared with HLA*B07:02-transduced cells loaded with different concentrations of mutant peptides K562 cells were co-cultured. IFNγ response was determined by ELISA. image 3 A / C shows nonlinear curve analysis of TCR-engineered T cells to IFNγ response to mutant peptide titration. Responses to mutant peptides were detected at concentrations as low as 10 -4 μg / ml, K D Values are in the nanomolar (high affinity) range. image 3 B / D shows a non-linear curve analysis of the IFNγ response to the corresponding wild-type peptide titration. Mutation-specific TCRs showed 10,000-fold greater affinity for the mutant peptide. Table 3 shows the affinities of the different TCRs analyzed.
[0148] Table 3: TCR affinity to SEQ ID NO:2 in the context of HLA-B*07:02 (shown as K D ). ...
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