Infusion of CD19-Directed and Multivirus Specific Cytotoxic T Lymphocytes After Allogeneic Hematopoietic Stem Cell Transplantation for B Cell Malignancies,
Authors: Cruz Conrad Russell Y. , Micklethwaite Kenneth P. , Savoldo Barbara , Ku Stephanie , Krance Robert A. , Diouf Oumar , Kamble Rammurti , Barrett A. John , Shpall Elizabeth J. , Heslop Helen E. , Rooney Cliona M. , Brenner Malcolm K. , Bollard Catherine M. , Dotti Gianpietro 
Published: October 2019

Abstract
Abstract 4083
Allogeneic hematopoietic stem cell transplant (HSCT) may increase long term disease free survival in patients with high risk B-cell malignancies. The delayed immune reconstitution associated with the procedure is, however, associated with significant morbidity and mortality from infections with cytomegalovirus (CMV), Epstein Barr virus (EBV), and adenoviruses (AdV). Disease relapse is another common cause of treatment failure. We reasoned that a single T cell platform mediating both antiviral and antileukemic activity could benefit these patients. We prepared cytotoxic T lymphocytes with specificities through their native receptors that were directed toward EBV, CMV and adenovirus; we then engineered these cells to express a chimeric antigen receptor (CAR) directed against CD19 (an antigen commonly present on B cell malignancies) and containing the co-stimulatory endodomain derived from CD28. We used donor derived antigen presenting cells (monocytes for the initial stimulation, EBV- lymphoblastoid cell lines, EBV-LCLs, for subsequent stimulations) that expressed adenovirus antigens and transgenic CMVpp65 following transduction with the adenoviral vector Ad5f35CMVpp65. After the third stimulation, multivirus-specific T cells were transduced with a retroviral vector encoding the CAR-CD19.28zeta transgene. Ten CTL cultures have been generated for clinical use, and contain a median of 71% (range 3–98%) CD8+ and 23% (range 0.1–92%) CD4+ cells, with a median of 14% (range 1–83%) CD45RA-/CD62L+ T cells. Transduction efficiency ranged from 20–55% (median 41%). These CTLs showed specific activity against B-cell leukemia/lymphoma targets in 51Cr release and/or IFNγ Elispot assays (mean specific lysis 48% at E:T ratio of 20:1, range 4–87%) and against CMV, EBV, and adenovirus peptide expressing target cells (mean specific lysis 28% at E:T ratio of 20:1, range 4–100%) but not recipient PHA blasts (<10%). Three patients (23/F, 58/M, and 56/M) with relapsed Ph+ALL (n=1) or B-CLL (n=2) have been treated with CAR-CD19.28z-CTLs. Patients received between 1.5 to 3 × 107CTLs/m2 without infusion related toxicity. In patient 1, who has the longest follow up, a three-fold increase in CMV–specific T cells was seen by 4 weeks post CTL infusion as detected by IFNγ Elispot assay. Responses to CMV peptides increased from 225 IFNγ-secreting cells (SFC/2.5 × 105 PBMC) pre-T cells to 782 SFC/2.5×105PBMC 6 weeks post CTLs and responses to CD19+ targets increased from 3 SFC/2.5×105 PBMC 1 week post T cells to 35 SFC/2.5×105 PBMC 6 weeks post CTLs. Detection of Transgenic T cells: CAR-CD19-transduced T cells have been detected by quantitative real-time PCR amplification in the peripheral blood of Patients 1, 2, and 3 up to 4, 1, and 0 weeks post infusion, respectively. Although already absent from peripheral blood, the transgenic cells were found at sites of disease. In Patient 1, T cells were detected in the bone marrow (44.8 copies/1000 ng of DNA) 9 weeks post T cell transfer. Patient 2 developed fever, diarrhea and hypotension 4 weeks post T cell therapy. Findings were consistent with ileitis at a known previous site of disease. Biopsy of the gut showed an abnormal absence of normal and malignant B cells, but significant levels of CAR-CD19.28z T cells by quantitative PCR (55.44 copies/1000 ng of DNA). Anti-Viral Activity of Transgenic Cells: No patient developed viral infection post CTL. Anti-Tumor Activity of Transgenic Cells: Patient 1 with Ph+ ALL had 4% blasts detectable in the peripheral blood at the time of the first infusion of CTLs which cleared within two weeks. She received a second infusion two months later. She became bcr abl negative but subsequently relapsed and died of progressive disease seven months post CTL. Patient 2 with CLL had resolution of lymphadenopathy within two weeks but following the disappearance of CTLs from peripheral blood showed evidence of disease progression two months post CTL. Patient 3 is too early in the course of therapy to evaluate for disease response. In summary, our initial findings show that immunotherapy with CTL targeting both CD19 through a chimeric receptor and multiple viral antigens through their native receptor can be well tolerated after allogeneic stem cell transplantation and may have both anti-viral and anti-tumor activity.

Disclosures:
No relevant conflicts of interest to declare.