Immunotherapy with Donor T Cells Sensitized with Overlapping Pentadecapeptides for Treatment of Persistent Cytomegalovirus Infection or Viremia

Abstract

We conducted a phase I trial of allogeneic T cells sensitized in vitro against a pool of pentadecapeptides (15-mer peptides) spanning the sequence of CMVpp65 for adoptive therapy of 17 allogeneic hematopoietic cell transplant recipients with cytomegalovirus (CMV) viremia or clinical infection persisting despite prolonged treatment with antiviral drugs. All but 3 of the patients had received T cell-depleted transplants without graft-versus-host disease (GVHD) prophylaxis with immunosuppressive drugs after transplantation. The CMVpp65-specific T cells (CMVpp65CTLs) generated were oligoclonal and specific for only 1 to 3 epitopes, presented by a limited set of HLA class I or II alleles. T cell infusions were well tolerated without toxicity or GVHD. Of 17 patients treated with transplant donor (n = 16) or third-party (n = 1) CMVpp65CTLs, 15 cleared viremia, including 3 of 5 with overt disease. In responding patients, the CMVpp65CTLs infused consistently proliferated and could be detected by T cell receptor Vβ usage in CMVpp65/HLA tetramer + populations for period of 120 days to up to 2 years after infusion. Thus, CMVpp65CTLs generated in response to synthetic 15-mer peptides of CMVpp65 are safe and can clear persistent CMV infections in the post-transplantation period.

Keywords: Adoptive immunotherapy; Epitope-specific and HLA-restricted T cell responses; Overlapping pentadecapeptides; Persistent cytomegalovirus (CMV) infection.

Figure 1. Induction of Clinical Response after infusion of Donor-Derived HLA-A*0201 Restricted NLV Epitope-Specific CMV CTLs in a Viremic Patient

A–C. CMV CTL Characterization prior to infusion for UPN#2 is shown. A. Aliquots of 10⁵ T-cells labeled with anti-CD3 FITC and anti-CD8 PE as well as APC conjugated HLA-A*0201-NLV or HLA-B*0702-TPR (control) tetramers were analyzed via FACS. B. Functional characterization and epitope mapping was performed for the CTLs prior to infusion by quantitating the proportion of CD8+ T-cells generating IFN-γ upon overnight stimulation with aliquots of autologous PBMC, each loaded with one of 24 individual subpools containing specific CMVpp65 pentadecapeptides. An overlapping grid of the peptide subpools permitted epitope identification. As shown, IFN-γ+ CD8+ T-cells were seen in response to targets loaded with pool 3 and 23 corresponding to the NLV peptide. C. The in-vitro cytotoxicity and the HLA allele restriction of the T-cell cytotoxic activity are shown. A panel of peptide loaded EBV BLCLs sharing a single HLA allele with the T-cell donor were used in a 4 hour ⁵¹chromium release assay to define the HLA restriction. The T-cells shown were exclusively cytotoxic against HLA-A*0201 sharing targets loaded with CMVpp65 peptide pool or the NLV peptide. D–F. UPN#2. Analysis of CMVpp65 specific T-cells post CMV CTL infusion. D. Tetramer analysis is shown for T-cells directly obtained from patient’s peripheral blood at day 28 and 210 post infusion. Distinct populations of HLA-A*0201-NLV tetramer binding T-cells are demonstrated in comparison to a control HLA-B*0702-TPR tetramer. E. Cytokine release assay is shown at day 45 post CTL infusion demonstrating CD8+ IFN-γ+ T-cells in response to overnight stimulation with autologous NLV peptide loaded PBMC. F. The cytotoxic activity of T-cells recovered from patient’s blood 45 days after CTL infusion was tested in-vitro in a chromium release assay against an HLA-A*0201 [+] human fibroblast cell line (MRC5) either uninfected, or CMV AD169 infected or loaded with the NLV peptide. PHA blasts from the T-cell donor as well as HLA mismatched BLCLs either alone or loaded with the NLV peptide were used as controls. G. Clinical response and in-vivo kinetics of CMV CTLs after infusion is shown for UPN#2. The arrow indicates the time of infusion of the CMV CTLs. The clinical response was followed by CMV antigenemia assay (■) performed twice a week. The number of CMV specific T-cells detected after infusion is plotted as the absolute number of HLA A*0201-NLV tetramer [+] T-cells/10⁶ PBMC of blood (◆) and IFNγ+ CD3+ T-cells/10⁶ PBMC of blood (Δ)detected at day 0, 1, 7 and weekly thereafter.

Figure 2

HLA-B*0701 Restricted T cells Induce Disease Clearance

Figure 3. Sequential Evaluation of T-cell Responses and CMV Antigenemia post Adoptive Therapy in a Patient with CMV Chorioretinitis

A. The clinical response and in-vivo kinetics of CMV CTLs after infusion is shown for UPN#5 inheriting HLA-A*0201 and HLA-B*0702. The arrow indicates the infusion of CMV CTLs. The clinical response was followed by CMV antigenemia assay (■) performed twice a week. The CMV CTLs detected after infusion are plotted as the absolute number of for HLA-B* 0702-TPR (◆) and HLA-A*0201-NLV tetramers (Δ) epitope specific tetramer[+] Tcells/10⁶ PBMC of blood and of IFNγ+ CD3+ (■) T-cells/10⁶ PBMC detected at day 0, 1, 7 and weekly thereafter. B. Tetramer analysis of the CMV CTLs infused to patient UPN#5 co-inheriting HLA-A*0201 and HLA-B*0702 is shown. The CTLs predominantly bound to tetramers for HLA-B*0702-TPR, and also for HLA-B*0702-RPHER, but did not bind to tetramers for HLA-A*2402-QYD (control) or HLA-A*0201-NLV. Tetramer analysis of patient’s T-cells performed at day 90 (C.), and at 2 years (D.) post infusion of CMV CTLs is shown, which demonstrates T-cells binding to tetramers for HLA-B* 0702-TPR, HLA-B*0702-RPHER, and emergence of T-cells binding to tetramers for HLA-A* 0201-NLV.

Fig. 4. Infused CMV CTLs Demonstrate an Oligoclonal TCR Vb Phenotype Represented in Specific Vb Families which are also Detected after Infusion

The TCR Vb phenotype of the infused tetramer positive CMV CTLs (top bar graph) was compared to the TCR Vb of the tetramer + CMV CTLs recovered from the patient’s blood after CTL infusion (bottom mirror image bar graph) at early and late time points.

Fig. 5. Clinical Response after Infusion of Epitope Specific CMV CTLs from HLA Partially Matched Third Party Donors

A. The HLA typing of the patient, the HSCT donor as well as the third party CMV CTL donor are shown. The third party donor was matched at HLA-A*0201 and HLA-DQB1*0202 and HLA-DQB1*0602 with the patient and donor. B. The clinical response and in-vivo kinetics of CMV CTLs at various time points after infusion of third party donor derived CMV CTLs is shown. The black arrows indicate the infusion of CTLs. The CMV copies/μl blood (■) was followed as marker for clinical response, and the CMV CTLs were detected in patient’s blood using HLA A*0201-NLV tetramers specific for the epitope to which the infused third party T-cells were responsive. The absolute number of A*0201-NLV tetramer+ T-cells/μl (◆) and of IFNγ+CD8+ T-cells/μl (Δ) detected post infusion at day 0, 1, 7 and weekly thereafter are plotted. C. Tetramer analysis of T-cells is shown from the patient prior to, days +14, +28, +90 and +150 after infusion of third party CMV CTLs. HLA-A*0201-NLV tetramer+ T-cells were not detected prior to CTL infusion, but were detectable thereafter with maximal response of 5.6% at day 150 post CMV CTL infusion. D. Intracellular IFN-γ production of CD8+ cells is shown from the patient prior to and on day +90 (1.5%) after infusion of third party CMV CTLs.