Tumor Associated Antigen Reactive Donor T Cells As Elementary Components of Streptamer Isolated Multi-Antigen Specific T Cells

Patients with hematological malignancies can be successfully treated with allogeneic stem cell transplantation (alloSCT). The therapeutic potential of alloSCT in the HLA-matched setting is primarily mediated by donor T cells directed against minor histocompatibility antigens (MiHA) presented in self-HLA. In addition, tumor associated antigens (TAA) represent a diversity of proteins overexpressed by various malignancies (self-peptide recognized in self-HLA) that may be recognized by donor T cells. Early application of donor T cells after alloSCT often results in graft versus host disease (GVHD) besides graft versus leukemia (GVL) responses. After T cell depleted transplantation, patients are vulnerable to opportunistic viral infections and disease relapses. Adoptive transfer of a multi-antigen specific T cell product containing T cells directed against viruses, TAA and MiHA may restore anti-viral immunity and support the GVL effect early after alloSCT. Detectable frequencies of high avidity memory virus specific T cells can be easily enriched from blood of seropositive healthy donors. However, due to the extremely low precursor frequencies of TAA and MiHA specific T cells, the isolation of these cells is more complicated. Moreover, high avidity, self-HLA restricted TAA specific T cells may have been deleted during thymic selection.

In this study (T-control, EU FP7), we aim to treat patients with a prophylactic infusion of a multi-antigen specific T cell product consisting of T cells directed against MHC class I restricted peptides of CMV, EBV and adenovirus, HLA-A2 restricted peptides from the TAA WT1, RHAMM, NY-eso and the peptide PR1 derived from proteinase 3, and the MiHA HA-1h. The reversible streptamer technology allows purification of minimally manipulated T cells from donor peripheral blood mononuclear cells (PBMC) and is based on strep-tag labeled peptide/MHC complexes coupled to Strep-Tactin coated magnetic nanobeads, followed by purification using the CliniMACS under Good Manufacturing Practice (GMP) conditions.

In large scale validation runs (n=3) we purified up to 12 viral, TAA and MiHA specificities out of ≥500*10^6 donor PBMC in one procedure. This resulted in an immediately clinical applicable multi-antigen specific T cell product with a purity of ≥98% within the T cell compartment. As expected the the different viral specificities comprised the main portion of the product (98%). To confirm that the remaining part of the product contains T cells directed against TAA and MiHA, we performed subsequent individual streptamer enrichments. After each enrichment the isolated cells were non-specifically expanded with allogeneic feeders, IL-2 and PHA.

Starting with 100*10^6 PBMC (n=4), we could detect in every donor ≥ 3 different TAA and/or MiHA specific T cell populations after 2 or 3 enrichments. Moreover, when starting with 500*10^6 PBMC (n=3) all TAA and MiHA specific T cells could be enriched to detectable frequencies (1-78%) after 2 or 3 enrichments. This indicates that TAA and MiHA specific T cells are commonly present in healthy donors, although at very low frequencies.

The TAA and MiHA specific T cell populations were clonally isolated and expanded, evaluated for CD8 and tetramer positivity and screened for antigen specific reactivity measured by cytokine release (IFN-g and GM-CSF) after 24 hours of stimulation with TAP deficient T2 cells loaded with 10^-5M of the relevant peptide.

From the tetramer positive T cell clones 8/132 NY-eso clones, 8/17 RHAMM clones, 31/47 WT1 clones and 13/16 PR1 clones showed peptide recognition. Among these were TAA clones of intermediate avidity recognizing up to 10^-7M peptide, despite the anticipated self-tolerance. As expected, the HA-1h clones recognizing a foreign MiHA in self HLA displayed high functional avidity reflected by recognition of peptide-loaded T2 cells in the pM range. This illustrates that intermediate avidity TAA-specific and high avidity HA-1h specific T cell clones can be enriched from healthy donors and that these cells may have therapeutic potential.

In conclusion, we are able to generate from donor PBMC a highly purified multi-antigen specific T cell product for clinical application. Besides the main component of MHC class I restricted viral specific T cells, we have demonstrated that this product also contains highly proliferative high avidity MiHA specific T cells and TAA specific T cells of intermediate avidity.