Aisha Hasan

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

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.

Use of humanized severe combined immunodeficient mice for human vaccine development

The severe combined immunodeficient (SCID) mouse has no adaptive immunity, lacking mature T and B cells in the peripheral blood or the lymphoid organs. It has been used extensively in biomedical research as a valuable translational model for xeno-engraftment of human tissues and cells. This review focuses on the engraftment of human peripheral blood cells and tissues in SCID mice, as well as in the newly established and more permissive SCID mice deficient in the IL-2 receptor γ-chain. Human immune responses could be elicited and assessed in these humanized SCID mice upon vaccination or sensitization with allogeneic tissues. A translational model is proposed to attain preclinical data for testing human vaccines.

Affinity maturation of T-cell receptor-like antibodies for Wilms tumor 1 peptide greatly enhances therapeutic potential

WT1126 (RMFPNAPYL) is a human leukocyte antigen-A2 (HLA-A2)-restricted peptide derived from Wilms tumor protein 1 (WT1), which is widely expressed in a broad spectrum of leukemias, lymphomas and solid tumors. A novel T-cell-receptor (TCR)-like single-chain variable fragment (scFv) antibody specific for the T-cell epitope consisting of the WT1/HLA-A2 complex was isolated from a human scFv phage library. This scFv was affinity-matured by mutagenesis combined with yeast display and structurally analyzed using a homology model. This monovalent scFv showed a 100-fold affinity improvement (dissociation constant (KD)=3 nm) and exquisite specificity towards its targeted epitope or HLA-A2+/WT1+ tumor cells. Bivalent scFv-huIgG1-Fc fusion protein demonstrated an even higher avidity (KD=2 pm) binding to the T-cell epitope and to tumor targets and was capable of mediating antibody-dependent cell-mediated cytotoxicity or tumor lysis by chimeric antigen receptor-expressing human T- or NK-92-MI-transfected cells. This antibody demonstrated specific and potent cytotoxicity in vivo towards WT1-positive leukemia xenograft that was HLA-A2 restricted. In summary, T-cell epitopes can provide novel targets for antibody-based therapeutics. By combining phage and yeast displays and scFv-Fc fusion platforms, a strategy for developing high-affinity TCR-like antibodies could be rapidly explored for potential clinical development.

Human herpesvirus 6B immediate-early I protein contains functional HLA-A*02, HLA-A*03, and HLA-B*07 class I restricted CD8+ T-cell epitopes

Human herpesvirus 6B (HHV‐6B) is a ubiquitous pathogen with frequent reactivation observed in immunocompromised patients such as BM transplant (BMT) recipients. Adoptive immunotherapy is a promising therapeutic avenue for the treatment of opportunistic infections, including herpesviruses. While T‐cell immunotherapy can successfully control CMV and EBV reactivations in BMT recipients, such therapy is not available for HHV‐6 infections, in part due to a lack of identified protective CD8+ T‐cell epitopes. Our goal was to identify CD8+ T‐cell viral epitopes derived from the HHV‐6B immediate‐early protein I and presented by common human leukocyte Ag (HLA) class I alleles including HLA‐A*02, HLA‐A*03, and HLA‐B*07. These epitopes were functionally tested for their ability to induce CD8+ T‐cell expansion and kill HHV‐6‐infected autologous cells. Cross‐reactivity of specific HHV‐6B‐expanded T cells against HHV‐6A‐infected cells was also confirmed for a conserved epitope presented by HLA‐A*02 molecule. Our findings will help push forward the field of adoptive immunotherapy for the treatment and/or the prevention of HHV‐6 reactivation in BMT recipients.

Novel strategies for adoptive therapy following HLA disparate transplants

Transplants of SBA-E- allogeneic marrow or G-CSF mobilized CD34+ (ISOLEX) E- peripheral blood progenitor cells which are adequately depleted of T-cells, when administered without post-transplant immunosuppression now induce consistent engraftment with low incidences of acute and chronic GVHD both in HLA matched and HLA disparate recipients. Furthermore, the incidence of relapse post transplant is not increased in patients transplanted for AML, MDS or ALL. In our series, the incidence of severe infections in HLA-matched recipients of such T-cell depleted grafts also does not differ from that detected following similarly matched unmodified grafts. However, in recipients of HLA-haplotype disparate T-cell depleted grafts, the risk of lethal viral infections is increased and prolonged. In many cases, this risk is closely correlated with failures of immunodominant virus-specific donor T-cells transferred in the graft to recognize infected host cells because they are restricted by HLA alleles not shared by the host. To address this limitation, we have developed a panel of artificial antigen presenting cells, each expressing a single prevalent HLA-allele. Using this panel, we are able to selectively generate virus-specific cytotoxic T-cells of desired HLA restriction, to insure their effectiveness in HLA haplotype-disparate transplant recipients. We have also shown that partially HLA-matched, third party-derived EBV-specific T-cells, selected from our bank of previously generated and characterized GMP-grade cell lines on the basis of their HLA restriction, can induce durable remissions of rituximab-refractory EBV lymphomas. These approaches may thus provide new, immediately accessible resources for the generation and broad application of immune cell therapies to treat and prevent severe viral diseases post transplant.

Treatment of cytomegalovirus retinitis with cytomegalovirus-specific T-lymphocyte infusion.

Cytomegalovirus (CMV) retinitis is a potentially blinding infection that affects immunocompromised patients who are unable to generate a T-cell response against the organism. Infusion of CMV-specific leukocytes has been shown to be effective in patients with systemic CMV infection, especially those resistant to standard therapies. The authors report a case of a patient with CMV viremia with progressive retinitis in whom infusion of third-party donor-derived CMV pp65-specific T cells alone prompted resolution of CMV retinitis. This case suggests a potential role for CMV-specific leukocyte infusion in the treatment of CMV retinitis, especially in cases resistant or refractory to antiviral therapies.

Abstract 5515: WT1 specific T cells can efficiently eliminate tumorigenic ovarian carcinoma cells and prevent or inhibit the tumor growth in NOD/SCID model of ovarian carcinoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The Wilms tumor protein, WT-1 is expressed in over 60% of serous adenocarcinomas of the ovary. Its expression has been hypothesized to be critical for the growth or survival of tumorigenic stem cells. In this study, we have assessed the capacity of in vitro generated T-cells specific for a series of immunogenic WT1 peptide epitopes presented by different HLA class I alleles to prevent the outgrowth of two human ovarian adenocarcinoma cell lines expressing either low (SKOV3-A2) or high (OVCAR3) levels of WT1 by FACS in NOD/SCID mice. For this study, epitope-specific HLA restricted WT1-CTLs were generated from PBMC of 4 normal donors by in vitro sensitization with autologous EBV BLCL loaded with a pool of 141 15-mers overlapping by 11aa and spanning the entire sequence of the WT1 protein. WT1-CTL restricted by HLA alleles expressed on the OVCAR3 and SKOV-3 lines were pre-incubated in vitro for 8 hours at different E:T ratios (0:1, 5:1, 10:1, 50:1, 100:1) with 0.05×10⁁6 ovarian carcinoma cells transduced to express a luciferase reporter gene. The cell mixtures were injected i.p. into NOD/SCID mice. Tumor growth was monitored weekly by intensity of their bioluminescent signal. In all animals injected with the tumor cells alone the bioluminescent signal could be detected in the abdomen by day 10-15 and increased steadily through 60 days of observation (by which time, all mice died). The tumor engraftment was either markedly inhibited (SKOV3-A2WT1low) or completely abrogated (in OVCAR-3WT1high) by pre-incubation at 100:1 E:T ratio and was correlated with higher survival of the animals (80%) over a period of 120 days. In the animals injected with tumor cells pre-incubated with the WT1-CTLs at a 50:1 and 10:1 E:T ratios, tumor growth was suppressed as reflected by weaker bioluminescent signal in the abdomen which increased much later in the course of the study. Pre-incubation of the tumor cells with WT1-CTL at a 5:1 E:T ratio did not significantly affect tumor engraftment and growth or survival of the mice. WT1-CTL specific for the 398-406LKTHTTRTHT epitope presented by the A0201 allele induced significantly greater suppression of tumor growth than T-cells specific for the (−125)-(−117)RQRPHPGAL peptide that can be presented by either HLA-A0301 or B0702. T cells specific for different WT1 epitopes administered i.v. at a dose of 2.5×10⁁6 cells/animal inhibited the growth of the pre-established OVCAR3 tumor xenografts inoculated i.p. as compared to the growth of the same tumors in control animals. Mice treated with WT-1 specific T-cells also had a significantly extended survival. Our results provide evidence that tumorigenic ovarian carcinoma cells expressing WT1+ are susceptible to eradication in vivo by high doses of WT1-CTL. However, the effectiveness of these T cells may differ depending on the level of WT1 expressed by the tumor cells and peptide epitope of WT1 targeted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5515. doi:10.1158/1538-7445.AM2011-5515