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Myelin-reactive T-cells (Opexa Therapeutics)

When myelin-reactive T-cells (MRTC) are attenuated by irradiation, autologous T-cell vaccination (TCV) provides an antigen-specific therapeutic means of inducing immunity against disease-associated T-cells.

All vaccines were ≥90% T-cells expressed as total CD3 with the majority of the remainder being T-cell fragments.

Peripheral blood mononuclear cells (PBMC) were isolated from whole blood by density gradient centrifugation and T-cells selected for vaccine production were identified with an Epitope Identification Assay (EIA) based on the split-well technique. For all patients, three 96-well plates were seeded with 2x105 PBMCs/well into 60 wells per plate in AIM V medium, and myelin basic protein (MBP), proteolipid protein (PLP) or myelin oligodendrocyte glycoprotein (MOG) peptides as pairs were added to separate plates at a final peptide concentration of 20 μg/mL. Plates were incubated at 37 °C for 7 to 10 days, adding 20 IU/mL rhIL-2 on day two, and then wells were split to generate “test plates” in the presence of 1x105 antigen presenting cells (APC; PBMC irradiated at 3500 rads) per well. The cells remaining in the three original stimulation plates were continued using RPMI containing 10% human AB serum and 100 IU/mL rhIL-2. Peptides were added to one well of the two replicate test plate wells for 48–72 h, with reactivity of the cells assessed by a [methyl-3H]Thymidine incorporation for the last 6–18 h. An original stimulation plate well was initially defined based on the work of others as being positive when both the quotient of the counts per minute (CPM) of a myelin peptide containing well divided by the CPM of control wells gave a stimulation index (SI) ≥3.0 and the total CPM of a myelin peptide-containing well was ≥1000. Cells from the EIA with SI≥1.6 were expanded in a Good Manufacturing Practices (GMP) facility by direct expansion in the presence of IL-2 and alternating stimulations with peptide antigens and phytohemagglutinin (PHA) repeated each 7 to 14 days and aided by the addition of 100,000 APCs/well. Cells were harvested and frozen until needed for final vaccine expansion, at which time they were thawed and activated with PHA prior to the preparation of Tcelna (Tovaxin). The cells were harvested, pooled and washed with sterile physiological saline with the low- midhigh-dose cell concentrations adjusted to 106 ,206 or 406 cells/mL.

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Primary Cell
Homo sapiens
Myelin-reactive T-cells