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A cancer and subject technology, applied in chemical instruments and methods, biochemical equipment and methods, pharmaceutical formulations, etc., can solve problems such as the increase in the prevalence of hematological cancers
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[0432] Example 1. Preparation of 2E4 antibody and design of 5T4CAR construct
[0433] Preparation of 2E4 Antibody
[0434] A recombinant protein purified from CHO cells using column chromatography (nickel beads) comprising the extracellular domain of 5T4 with a C-terminal enterokinase cleavage sequence, the myc epitope and six histidines was used as the immunogen Label. Immuno-Precise (Canada) fast primary protocol (immunization of 3 mice followed by 4 fast boosts) was used to prepare mAbs in mice.
[0435] Donor splenocytes were fused with SP2 / 0 myeloma IgG hybridomas. mAbs were screened against the recombinant protein and counter-screened by ELISA against the extracellular domain of different leucine-rich proteins bearing the same C-terminal tag. Ten supernatants were selected for further study.
[0436] All supernatants recognized recombinant human 5T4 by Western blot, although an additional band was detected by some supernatants in cell lysates. Using the intensity of r...
Embodiment 2
[0448] Example 2. In vitro activity of CAR-5T4T cells- Figure 2 to Figure 4
[0449] Tumor cell lines:
[0450] The tumor cell lines studied were NCI-H226 (Mesothelioma, ATCC; Middlesex, UK), SKOV-3 (Ovarian Adenocarcinoma, ATCC; Middlesex, UK), BT20 (Breast Cancer, ATCC; Middlesex, UK), OVCAR- 3 (ovarian adenocarcinoma, ATCC; Middlesex, UK), LS174T (colorectal adenocarcinoma, ATCC; Middlesex, UK), DLD-1 (colorectal adenocarcinoma, ATCC; Middlesex, UK) and NCI-H929 (myeloma, ATCC ; Middlesex, UK).
[0451] The medium used to cultivate each cell line is as follows: NCI-H226 medium: RPMI 1640 (SIGMA, DORSET, UK), 1% glutamine (SIGMA, DORSET, UK), 1% HEPES (Gibco; Paisley, UK ), 1% sodium pyruvate (SIGMA, DORSET, UK) and 10% FBS (SeraLabs; West Sussex, UK). SKOV-3 medium: McCoys 5a medium (Life Technologies; Carlsbad CA, US; Carlsbad CA, US), 1% glutamine (SIGMA, DORSET, UK), 10% FBS (SeraLabs; West Sussex, UK). BT20 medium: Eagles Minimum Essential Medium (SIGMA, DORSET, U...
Embodiment 3
[0464] Example 3. CAR-5T4T cell specificity- Figure 5
[0465] The specificity of the 5T4CAR construct was determined by blocking experiments in which 5T4-specific monoclonal antibodies or soluble 5T4 protein were incubated with tumor cell lines and CAR-5T4T cells. Both blockers were titrated and the effect on IFNγ secretion was assessed.
[0466] tumor cell line
[0467]The tumor cell lines studied were NCI-H226 (Mesothelioma, ATCC; Middlesex, UK) and LS174T (Colorectal adenocarcinoma, ATCC; Middlesex, UK). The medium used for culturing NCI-H226 cells was: RPMI 1640 (SIGMA, DORSET, UK), 1% glutamine (SIGMA, DORSET, UK), 1% HEPES (Gibco; Paisley, UK), 1% pyruvate Sodium (SIGMA, DORSET, UK) and 10% FBS (SeraLabs; West Sussex, UK). The medium used to culture LS174T cells was: Eagles minimal essential medium (SIGMA, DORSET, UK), 10% FBS (SeraLabs; West Sussex, UK), 1% glutamine (SIGMA, DORSET, UK). NCI-H929: RPMI1640 (SIGMA, DORSET, UK), 10% FBS (SeraLabs; West Sussex, UK) ...
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