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T-CELL RECEPTORS WHICH RECOGNISE FRAMESHIFT MUTANTS OF TGFbetaRII

A technology of cell receptors and immune effector cells, applied to genetically modified cells, animal cells, receptors/cell surface antigens/cell surface determinants, etc., can solve problems such as the impossibility of predicting the functional sequence of the TCR chain

Active Publication Date: 2019-02-12
UNIV OSLO HF
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, even when having a CDR3 sequence, it is still impossible to predict the functional sequence of the entire TCR chain, since a functional TCR requires two additional CDR sequences in each chain

Method used

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  • T-CELL RECEPTORS WHICH RECOGNISE FRAMESHIFT MUTANTS OF TGFbetaRII
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  • T-CELL RECEPTORS WHICH RECOGNISE FRAMESHIFT MUTANTS OF TGFbetaRII

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0241] Materials and methods:

[0242] Cell Lines, Media and Reagents

[0243] TGFβRII frameshift mutation-responsive, HLA-A2-restricted CTL (cytotoxic T lymphocyte) clones were isolated from the blood of MSI+ colon cancer patients and cloned by limiting dilution. The patient was vaccinated with the 23-mer TGFβRII(-1A) frameshift peptide (peptide having SEQ ID NO: 49). The clinical trial was approved by the Norwegian Medicines Agency, the Southern Medical Research Ethics Committee and the Hospital Review Board. Treatment was carried out in accordance with the Declaration of Helsinki of the World Medical Association. Informed consent was obtained from the patient. An autologous Epstein Barr virus transformed lymphoblastoid cell line (EBV-LCL) was generated by transformation of B cells from a donor. Antigen processing-deficient T2 cell lines are used as T cell targets in flow cytometry and cytotoxicity assays. Colon cancer cell lines HCT116, SW480 and LS174T and human emb...

Embodiment 2

[0282] To study CD4 transduced with Radium-1 TCR + and CD8 + T cells kill target cells, which are stably transduced to express luciferase. Two sets of target cells were used: the HCT116 cell line and the Granta cell line. HCT116 cells are described above; the Granta cell line is a human B-cell lymphoma cell line. Changes in bioluminescence were used to measure changes in the number of target cells during culture of Radium-1-transduced T cells, representing killing of target cells by T cells.

[0283] Luciferase-transduced target cells were co-cultured with effector T cells on effectors to a target (E:T) ratio of 30:1 and bioluminescence was measured. Cells were co-cultured for 24 hours and bioluminescence was measured at 1, 2, 3, 4, 5, 8, 11, 20, 21, 22, 23 and 24 hours. Effector T cells were co-cultured with Granta cells with and without exogenous peptide p621 (SEQ ID NO:55) comprising the sequence of SEQ ID NO:1.

[0284] purified CD4 + T cells and purified CD8 transdu...

Embodiment 3

[0291] The soluble His-tagged Radium-1 TCR encoded by the scTCR of SEQ ID NO: 69 was expressed in HEK cells. Supernatants of cells expressing HEK were isolated. SupT1 cells (HLA-A2 negative cell line) were transduced to express HLA-A2 fused to a non-specific irrelevant peptide not recognized by Radium-1 or a TGFβRII frameshift peptide. Transduced cells were incubated with soluble Radium-1 TCR for 30 minutes at room temperature; non-transduced cells were also incubated with soluble TCR as an additional negative control. After incubation, cells were washed and then stained with allophycocyanin (APC) to identify soluble TCR binding. Staining was performed using a primary mouse anti-His antibody followed by a secondary APC-conjugated anti-mouse IgG antibody. The stained cells were then analyzed by flow cytometry ( Figure 12 ).

[0292] like Figure 12 As shown in A and 12B, essentially no staining of the negative control was observed, demonstrating that soluble Radium-1 TCR ...

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Abstract

The present invention relates to TCR molecules which recognise neopeptides produced as a result of the cancer-associated '-1A' frameshift mutation in human TGFbetaRII. The TCR molecules are capable ofbinding a peptide of SEQ ID NO: 1 when said peptide is presented by a Class I MHC, and comprise an alpha-chain domain and a beta-chain domain, each chain domain comprising three CDR sequences, wherein a) CDRs 1, 2 and 3 of the alpha-chain domain have the sequences of SEQ ID NOs: 2, 3 and 4 respectively; and b) CDRs 1, 2 and 3 of the beta-chain domain have the sequences of SEQ ID NOs: 5, 6 and 7 respectively, and wherein one or more of said CDR sequences may optionally be modified by substitution, addition or deletion of 1 or 2 amino acids. Nucleic acid molecules encoding such TCRs are provided, as are soluble TCR molecules with these CDR sequences. The nucleic acid molecules of the invention can be used to modify immune effector cells to express a TCR as defined herein, and such modifiedimmune effector cells are useful in therapy for cancer, as are soluble TCRs as defined above.

Description

technical field [0001] The present invention relates to T cell receptors (TCRs) that recognize frameshift mutants of transforming growth factor-beta receptor II (TGFβRII). These TCRs can be used to treat cancers, especially cancers that contain specific frameshift mutations of TGFβRII. The present invention provides TCR molecules, nucleic acid molecules encoding such TCRs and vectors containing these nucleic acid molecules. The provided nucleic acid molecules and vectors can be used to modify immune effector cells, including T cells in particular, to express TCRs. Nucleic acid molecules and vectors can also be used to modify production host cells to produce TCRs. Such modified immune effector cells can be used in adoptive cell transfer therapy. In particular, the TCRs of the invention are based on or derived from a specific TCR, identified herein as Radium-1, which was identified in cytotoxic T lymphocyte (CTL) clones isolated from clinically responding patients immunized w...

Claims

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Application Information

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IPC IPC(8): C07K14/725C07K14/495
CPCC07K14/495C07K14/7051A61P1/00A61P35/00A61P35/02A61P37/04A61K39/464434C12N5/0636A61K2239/50A61K39/4632A61K39/464491A61K39/4611A61K2239/38C12N2510/00A61K38/00C07K2319/20A61K35/17C12N15/85
Inventor E·M·因德贝格G·高德纳克S·瓦赤莉G·科瓦尔海姆
Owner UNIV OSLO HF
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