re: MRKR
rfj1862,
My hematologist sent me this. (It's way beyond my feeble understanding.)
"i have tried to explain to people how the expansion of the T cells is provoked, but i do not think anyone even understands it. how the dendritic cell presents and antigenic peptide, (the epitope as it is called), to the naive T cell as it goes by inside the peripheral lymph nodes.
the way mrkr does this is to basically make a synthetic lymph node with the APC antigen presenting cells being some special selection of dendritic cells used to do the tissue culture expansion in the normal CAR or TCR expansion.
so they include a collection of peptides, that these APCs can present to the T cell. there is a series of 9 amino acids in the epitope that is bound up on the heavy chain of the MHC major histocompatability complex (HLA phenotype is not clear here) and then when the T cells in that small aliquot of peripheral blood is exposed to the peptide which makes the epitope rest in the curved arch of the MHC and presents itself to the passing T cell in this special tissue culture/expansion medium. that causes the T cells which recognizes the cancer neoantigen, in the tumor and fights it but then goes into anergy, expand again when it recognizes that same antigen in this tissue culture. that's what T cells do, when they see antigen they like. they multiply like crazy.
anyway this first 10 day culture forces the small (infinitely small ) amount of T cells in the peripheral blood, (that is not bound to the tumor so it can be found in the peripheral blood sample), that see that same epitope presented by these aAPC artificial APC like everyone uses already to expand the T cells after transfection with the CAR. that is part of the first batch that mrkr processes.
so that grows up a huge population of effector cells, then next, they culture for 7 days in the IL-7 which promotes the development of the memory stem cell type of CD4 cells that will maintain the memory of that tumor neoantigen.
so basically it uses the T cells that are a natural response to the tumor, but give up after fighting for a long time from anergy, acquired tolerance, and the other forms of Teff cell shutdown as the TME tumor micro environment uses the Th2 type phenotype to allow the tumor to escape by pretending to be wound healing, that's the best way to think of immune escape.
anyway after the T cells are cultured in the IL-7 they have enuff of both the Teff cells and Tmsc (for long term memory of tumor neoantigen) so after they reinfuse the cultured cells, there is enuff to restart the entire natural immune response to the tumor, just with a lot more of the Teff cells to work on the tumor than occurred in the body up until then.
the mixture of peptides is determined from looking at the mutation in the DNA sequence, which changed the amino acid, and they make a string of DNA with the code for the mutated spot included, and since the mutation could be anywhere in the full length of the peptide, they make a different peptide for each position of the mutation, so it shifts by one amino acid for each of the multimers created. this is what dr Rosenberg does, and has been doing since 2013. they have a dozen papers over the last 6 years about it, before getting to this last one in january.
anyway, so each of these little bit different peptides is presented by the APC, so one out of all of them will provoke the T cell activation and replication, and the others which were just one amino acid over from it not creating an antigenic response, because the epitope does not lay in the groove of the MHC in the same way as the true neoantigen does.
the epitope is held in place by ionic and hydrogen bonds to the MHC, a linear epitope is what it is called. antigen MHC (HLA) complex is what they call it. we each have different allotype, most of us are HLA2-02 i think (forgot how the labeling works), but about 70% of anglo, western europeans share the same haplotype, so that would have to be shared between the patients in dr Rosenberg's work in order for them to use the epitope they derive from the known mutation and carry in the 'off the shelf' bottle so when the patient comes in with a tumor, they take a blod sample and by reacting in these ELISPOT assay cells, they can see which of the epitope/HLA batch it works on, even before they examine the genome for a mutation. these common mutations will be recognized in the assay spots of the immunohistochemical reactions .
for dr Cooper to do this he would ceate a plasmid with the TCR for that hotspot neoantigen and transfect the naive T cell with it so the naive T cell now is a young and newly armed attack machine and goes after the tumor with gusto, expanding rapidly upon exposure to the tumor neoantigens. and the mbIL-15 forces some of the T cells to become memory T cells so the tumor wil be recognized if it comes back."
Bladerunner
