Generation and Characterization Of a Third Party GMP Grade Bank Of CMV Specific T-Cells For Adoptive Immunotherapy Of CMV Infections In Recipients Of HSCT From Cord Blood Or Seronegative Donors

Adoptive transfer of virus-specific T-cells (CTLs) derived from allogeneic HLA partially matched third party donors can also be effective in a proportion of patients developing EBV lymphomas, or infections due to CMV or adenovirus following transplants from seronegative donors. Such third party donor derived CTLs offer an off the shelf reagent for treatment of viral infections developing after transplant. However, the immunodominant cytotoxic activity exhibited by the CTLs is directed against specific epitopes of the viral protein and restricted by 1-2 HLA alleles. Therefore, it is critical that the T-cells administered from third party donors can recognize viral epitopes presented on shared HLA alleles. We have established a bank of 119 CMV specific T-cell lines (CMV CTLs) generated using autologous APCs loaded with a pool of overlapping peptides spanning the sequence of the dominant immunogenic protein CMVpp65. Each of these 119 CMV CTL lines has been characterized as to the epitope inducing T-cell response as well as the HLA allele restricting the epitope specific T-cell response. Epitopes were identified using an overlapping grid of peptide pools and the HLA restriction by cytotoxic activity against peptide loaded EBVBLCLs matched at a single HLA allele with the T-cell donor.

The distribution of the common HLA alleles among the donors for these CTL lines was predominantly within the distribution of HLA allele frequencies represented in the caucasian and black populations, except for HLA A0201 and B0702, which were over represented ( 33% vs 25% and 21% vs 8.7% respectively). In 54% of the CTL lines, the immunodominant T-cell responses were restricted by HLA A0201 (25%), B0702 (21%) and B 3501-11(8%), and in the remaining 50%, the responses were restricted by other HLA class-I alleles, while only 16/119 lines (13%) were restricted by HLA class-II alleles.

All 25 donors inheriting HLA B0702 (25/25) demonstrated HLA B0702 restricted CMV CTL responses, while 30/39 (77%) donors inheriting HLA A0201 and 9/19 (47%) donors inheriting HLA B3501-11 demonstrated HLA A0201 and B3501-11 restricted CMV CTL responses. Among all 9 donors co-inheriting HLA A0201 and B 0702, the immunodominant T-cell response was restricted by B0702. Among 12 donors co-inheriting A0201 and B 4401-04, 11/12 (91.6%) demonstrated immunodominant CMV CTL responses restricted by A0201; 1 donor also co-inherited HLA B0702 whose response was restricted by B0702. Therefore, an immunodominance hierarchy for HLA class-I alleles presenting the dominant CMVpp65 epitope was evident through this analysis among these 119 donors and was as follows: B 0702, A0201, B3501-11, A2601, B44, B40, B4201, A0101, B 1801. Strikingly, only 1 of 119 donors demonstrated T-cell responses restricted by A1101; a commonly inherited HLA class –I allele.

In a series of 239 consecutive HLA matched related or unrelated transplants (MUD) and 137 HLA mismatched unrelated (MMUD) transplants, and 100 cord blood transplants conducted at our center, in 86%, 89% and 80% of the cases respectively, we could identify a CMV CTL line restricted by a shared HLA allele and matched at 2-3 alleles within this GMP grade CTL bank that would be immediately available for treatment of CMV infection. Appropriately restricted CMV CTLs would only be available in 60-70% of MMUD transplant and none of the cord blood transplants without this approach. This CMV CTL bank therefore represents a readily available clinical reagent for the treatment of resistant CMV infections developing in post transplant patients. The characterization of the CTLs has also enabled the further elucidation of immunodominant CMVpp65 epitopes and hierarchies. Since we have previously shown that CMV CTLs can be generated against subdominant epitopes presented by both common HLA alleles as well as less prevalent HLA alleles using a panel of artificial antigen presenting cells (AAPCs), expansion of this bank using T-cell sensitized against CMVpp65 presented on such AAPCs should broaden the applicability of this bank to all HSCT recipients.