Ultra low-dose IL-2 for GVHD prophylaxis after allogeneic hematopoietic stem cell transplantation mediates expansion of regulatory T cells without diminishing antiviral and antileukemic activity.

PURPOSE: GVHD after allogeneic hematopoietic stem cell transplantation (alloSCT) has been associated with low numbers of circulating CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs). Because Tregs express high levels of the interleukin (IL)-2 receptor, they may selectively expand in vivo in response to doses of IL-2 insufficient to stimulate T effector T-cell populations, thereby preventing GVHD. EXPERIMENTAL DESIGN: We prospectively evaluated the effects of ultra low-dose (ULD) IL-2 injections on Treg recovery in pediatric patients after alloSCT and compared this recovery with Treg reconstitution post alloSCT in patients without IL-2. Sixteen recipients of related (n = 12) or unrelated (n = 4) donor grafts received ULD IL-2 post hematopoietic stem cell transplantation (HSCT; 100,000-200,000 IU/m(2) ×3 per week), starting

Sustained complete responses in patients with lymphoma receiving autologous cytotoxic T lymphocytes targeting Epstein-Barr virus latent membrane proteins.

PURPOSE: Tumor cells from approximately 40% of patients with Hodgkin or non-Hodgkin lymphoma express the type II latency Epstein-Barr virus (EBV) antigens latent membrane protein 1 (LMP1) and LMP2, which represent attractive targets for immunotherapy. Because T cells specific for these antigens are present with low frequency and may be rendered anergic by the tumors that express them, we expanded LMP-cytotoxic T lymphocytes (CTLs) from patients with lymphoma using autologous dendritic cells and EBV-transformed B-lymphoblastoid cell lines transduced with an adenoviral vector expressing either LMP2 alone (n = 17) or both LMP2 and ΔLMP1 (n = 33). PATIENTS AND METHODS: These genetically modified antigen-presenting cells expanded CTLs that were enriched for specificity against type II latency LMP antigens. When infused into 50 patients with EBV-associated lymphoma, the expanded CTLs did not produce infusional toxicities. RESULTS: Twenty-eight of 29 high-risk or multiple-relapse patients receiving LMP-CTLs as adjuvant therapy remained in remission at a median of 3.1 years after CTL infusion. None subsequently died as a result of lymphoma, but nine succumbed to complications associated with extensive prior chemoradiotherapy, including myocardial infarction and secondary malignancies. Of 21 patients with relapsed or resistant disease at the time of CTL infusion, 13 had clinical responses, including 11 complete responses. T cells specific for LMP as well as nonviral tumor-associated antigens (epitope spreading) could be detected in the peripheral blood within 2 months after CTL infusion, but this evidence for epitope spreading was seen only in patients achieving clinical responses. CONCLUSION: Autologous T cells directed to the LMP2 or LMP1 and LMP2 antigens can induce durable complete responses without significant toxicity. Their earlier use in the disease course may reduce delayed treatment-related mortality.

Antigen presenting cell-mediated expansion of human umbilical cord blood yields log-scale expansion of natural killer cells with anti-myeloma activity.

Natural killer (NK) cells are important mediators of anti-tumor immunity and are active against several hematologic malignancies, including multiple myeloma (MM). Umbilical cord blood (CB) is a promising source of allogeneic NK cells but large scale ex vivo expansion is required for generation of clinically relevant CB-derived NK (CB-NK) cell doses. Here we describe a novel strategy for expanding NK cells from cryopreserved CB units using artificial antigen presenting feeder cells (aAPC) in a gas permeable culture system. After 14 days, mean fold expansion of CB-NK cells was 1848-fold from fresh and 2389-fold from cryopreserved CB with >95% purity for NK cells (CD56(+)/CD3(-)) and less than 1% CD3(+) cells. Though surface expression of some cytotoxicity receptors was decreased, aAPC-expanded CB-NK cells exhibited a phenotype similar to CB-NK cells expanded with IL-2 alone with respect to various inhibitory receptors, NKG2C and CD94 and maintained strong expression of transcription factors Eomesodermin and T-bet. Furthermore, CB-NK cells formed functional immune synapses with and demonstrated cytotoxicity against various MM targets. Finally, aAPC-expanded CB-NK cells showed significant in vivo activity against MM in a xenogenic mouse model. Our findings introduce a clinically applicable strategy for the generation of highly functional CB-NK cells which can be used to eradicate MM.

Infusion of donor-derived CD19-redirected virus-specific T cells for B-cell malignancies relapsed after allogeneic stem cell transplant: a phase 1 study.

Autologous T cells expressing a CD19-specific chimeric antigen receptor (CD19.CAR) are active against B-cell malignancies, but it is unknown whether allogeneic CD19.CAR T cells are safe or effective. After allogeneic hematopoietic stem cell transplantation (HSCT), infused donor-derived virus-specific T cells (VSTs) expand in vivo, persist long term, and display antiviral activity without inducing graft-vs-host disease; therefore, we determined whether donor VSTs, engineered to express CD19.CAR, retained the characteristics of nonmanipulated allogeneic VSTs while gaining antitumor activity. We treated 8 patients with allogeneic (donor-derived) CD19.CAR-VSTs 3 months to 13 years after HSCT. There were no infusion-related toxicities. VSTs persisted for a median of 8 weeks in blood and up to 9 weeks at disease sites. Objective antitumor activity was evident in 2 of 6 patients with relapsed disease during the period of CD19.CAR-VST persistence, whereas 2 patients who received cells while in remission remain disease free. In 2 of 3 patients with viral reactivation, donor CD19.CAR-VSTs expanded concomitantly with VSTs. Hence CD19.CAR-VSTs display antitumor activity and, because their number may be increased in the presence of viral stimuli, earlier treatment post-HSCT (when lymphodepletion is greater and the incidence of viral infection is higher) or planned vaccination with viral antigens may enhance disease control.

Improving T-cell therapy for relapsed EBV-negative Hodgkin lymphoma by targeting upregulated MAGE-A4.

PURPOSE: Patients with Hodgkin lymphoma (HL) relapsing after hematopoietic stem cell transplant have limited options for long-term cure. We have shown that infused cytotoxic T cells (CTL) targeting Epstein Barr virus (EBV)-derived proteins induced complete remissions in EBV(+) HL patients. A limitation of this approach is that up to 70% of relapsed HL tumors are EBV-negative. For these patients, an alternative is to target the cancer/testis antigen MAGE-A4 present in EBV antigen-negative HL tumors. Furthermore, epigenetic modification by clinically available demethylating agents can enhance MAGE-A4 expression in previously MAGE-negative tumors. EXPERIMENTAL DESIGN: We explored the feasibility of combining adoptive T cell therapy with epigenetic modification of tumor antigen expression. We further characterized MAGE-A4-specific T-cell phenotype and function, and examined the effects of the epigenetic modifying drug decitabine on these T cells. RESULTS: Cytotoxic T cells were generated specifically recognizing MAGE-A4 expressed by autologous HL targets and tumor cell lines. Decitabine-previously shown to increase tumor antigen expression in HL-did not compromise MAGE-A4-specific T-cell phenotype and function. In patients treated with decitabine, expanded MAGE-A4-specific T cells had a broader antitumor T cell repertoire, consistent with increased antigen stimulation in vivo. CONCLUSIONS: Adoptive transfer of MAGE-A4-specific T cells, combined with epigenetic modifying drugs to increase expression of the protein, may improve treatment of relapsed HL.

Expansion of T cells targeting multiple antigens of cytomegalovirus, Epstein-Barr virus and adenovirus to provide broad antiviral specificity after stem cell transplantation.

BACKGROUND AIMS: Hematopoietic stem cell transplant (HSCT) is the treatment of choice for a proportion of patients with hematologic malignancies as well as for non-malignant diseases. However, viral infections, particularly Epstein-Barr virus (EBV), cytomegalovirus (CMV) and adenovirus (Ad), remain problematic after transplant despite the use of antiviral drugs. We have shown that cytotoxic T lymphocytes (CTL) generated against CMV-pp65, EBV and Ad antigens in a single culture are capable of controlling infections with all three viruses after HSCT. Although pp65-specific CTL have proved efficacious for the control of CMV infection, several reports highlight the importance of targeting additional CMV antigens. METHODS: To expand multivirus-specific T cells with activity against both CMV-pp65 and CMV-IE-1, peripheral blood mononuclear cells (PBMC) were transduced with the adenoviral vector (Ad5f35-IE-1-I-pp65). After 9-12 days the CTL were restimulated with autologous EBV-transformed B cells transduced with the same Ad vector. RESULTS: After 18 days in culture nine CTL lines expanded from less than 1.5 × 10(7) PBMC to a mean of 6.1 × 10(7) T cells that recognized CMV antigens pp65 [median 273 spot-forming cells (SFC), range 47-995] and IE-1 (median 154 SFC, range 11-505), the Ad antigens hexon (median 153 SFC, range 26-465) and penton (median 37 SFC, range 1-353), as well as EBV lymphoblastoid cell lines (median 55 SFC, range 9-301). Importantly, the T cells recognized at least two antigens per virus and lysed virus peptide-pulsed targets. CONCLUSIONS: CTL that target at least two antigens each of CMV, EBV and Ad should have clinical benefit with broad coverage of all three viruses and enhanced control of CMV infections compared with current protocols.

Antigen-specific cytotoxic T lymphocytes can target chemoresistant side-population tumor cells in Hodgkin lymphoma.

Side-population (SP) analysis has been used to identify progenitor cells from normal and malignant tissues as well as revealing tumor cells with increased resistance to radiation and chemotherapy. Despite enhanced chemoresistance, tumor SP cells may still express tumor-associated antigens (TAAs), which may render them susceptible to elimination by the immune system. In this study, we show that both Hodgkin lymphoma (HL) cell lines and primary HL tumor samples contain a distinct SP phenotype. Importantly, while these cells showed increased resistance to gemcitabine, a commonly used drug for the treatment of refractory HL, HL SP cells also expressed higher levels of the TAAs MAGEA4, SSX2, survivin, and NY-ESO-1, which allowed them to be specifically recognized and killed by TAA-specific cytotoxic T lymphocytes. This study suggests that chemoresistant HL SP cells can be targeted by the immune system, providing a rationale for combined chemotherapy and immunotherapy for the treatment of HL.