Lisa Brennan

Establishment of antitumor memory in humans using in vitro-educated CD8+ T cells.

Antitumor CD8+ T cells educated in vitro can persist as memory T cells and induce antitumor responses in humans without prior conditioning or cytokine treatment. Memory that Keeps Going and Going Senior scientists consoling their trainees about failed experiments tout the value of persistence. Yet, persistence is not only important for the scientist tirelessly pipetting at two in the morning, it is key for immunological memory as well. Melanoma is a malignant tumor of melanocytes, the pigment cells found in the skin. Advanced-stage melanoma has a poor prognosis, with patients on average surviving less than a year. A new promising therapy for advanced-stage melanoma patients harnesses the immune system to attack the tumor cells. In adoptive T cell therapy, cytotoxic cells specific for the tumor are transferred into patients, where they then traffic to and destroy the tumor cells. However, one limitation of this therapy is keeping the tumor-specific T cells alive in the patient, which has been largely unsuccessful in the absence of extensive treatment of the patient. Butler et al. now report a means to expand these T cells that allows them to persist in the absence of extra patient manipulation. The authors use artificial antigen-presenting cells to turn antitumor T cells into memory T cells, which survive longer and respond more quickly than normal effector T cells. These artificial antigen-presenting cells express molecules that “costimulate” the T cells, resulting in T cells that both look and act like memory T cells in the culture dish. These educated tumor-specific cells were then introduced into patients with advanced-stage melanoma. These cells functioned as memory cells in the patients as well: persisting for long periods of time, homing to the tumor site, and demonstrating tumor-specific activation and function, all in the absence of further patient manipulation. Although this is an early clinical trial with a small number of patients, there is some indication that this treatment may have clinical benefit for patients with this devastating disease. Persistence, both of the T cells and the scientists running the study, has finally paid off. Although advanced-stage melanoma patients have a median survival of less than a year, adoptive T cell therapy can induce durable clinical responses in some patients. Successful adoptive T cell therapy to treat cancer requires engraftment of antitumor T lymphocytes that not only retain specificity and function in vivo but also display an intrinsic capacity to survive. To date, adoptively transferred antitumor CD8+ T lymphocytes (CTLs) have had limited life spans unless the host has been manipulated. To generate CTLs that have an intrinsic capacity to persist in vivo, we developed a human artificial antigen-presenting cell system that can educate antitumor CTLs to acquire both a central memory and an effector memory phenotype as well as the capacity to survive in culture for prolonged periods of time. We examined whether antitumor CTLs generated using this system could function and persist in patients. We showed that MART1-specific CTLs, educated and expanded using our artificial antigen-presenting cell system, could survive for prolonged periods in advanced-stage melanoma patients without previous conditioning or cytokine treatment. Moreover, these CTLs trafficked to the tumor, mediated biological and clinical responses, and established antitumor immunologic memory. Therefore, this approach may broaden the availability of adoptive cell therapy to patients both alone and in combination with other therapeutic modalities.

Severe ototoxicity following carboplatin-containing conditioning regimen for autologous marrow transplantation for neuroblastoma

Children with neuroblastoma receiving high-dose carboplatin as part of their conditioning regimen for autologous marrow transplantation have a high incidence of speech frequency hearing loss. We evaluated hearing loss in 11 children with advanced stage neuroblastoma who underwent autologous marrow transplantation, following a conditioning regimen containing high-dose carboplatin (2 g/m2, total dose). Audiometric evaluations were obtained at diagnosis, prior to and following transplant. Exposure to other known ototoxins also was assessed. All patients sustained worsening of hearing following high-dose carboplatin. Nine of the 11 children (82%) had evidence of speech frequency hearing loss post transplant for which hearing aids were recommended (grades 3–4). Three of the nine children had speech frequency loss prior to transplant which progressed following transplant. The entire group was heavily pre-treated with platinum-containing chemotherapy pre-BMT and had extensive exposure to other ototoxins, including aminoglycoside antibiotics, diuretics, and noise exposure – all of which could have exacerbated the effects of carboplatin. High-dose carboplatin is ototoxic, particularly in patients who have been primed with previous platinum therapy or other ototoxic agents. We conclude that further efforts are needed to monitor and minimize this complication. In cases where hearing loss is inevitable due to cumulative ototoxic exposures, families need to be adequately prepared for the tradeoffs of potentially curable therapy.

Outcome of alloanergized haploidentical bone marrow transplantation after ex vivo costimulatory blockade: results of 2 phase 1 studies

We report the outcomes of 24 patients with high-risk hematologic malignancies or bone marrow failure (BMF) who received haploidentical bone marrow transplantation (BMT) after ex vivo induction of alloantigen-specific anergy in donor T cells by allostimulation in the presence of costimulatory blockade. Ninety-five percent of evaluable patients engrafted and achieved full donor chimerism. Despite receiving a median T-cell dose of 29 x10(6)/kg, only 5 of 21 evaluable patients developed grade C (n = 4) or D (n = 1) acute graft-versus-host disease (GVHD), with only one attributable death. Twelve patients died from treatment-related mortality (TRM). Patients reconstituted T-cell subsets and immunoglobulin levels rapidly with evidence of in vivo expansion of pathogen-specific T cells in the early posttransplantation period. Five patients reactivated cytomegalovirus (CMV), only one of whom required extended antiviral treatment. No deaths were attributable to CMV or other viral infections. Only 1 of 12 evaluable patients developed chronic GVHD. Eight patients survive disease-free with normal performance scores (median follow-up, 7 years). Thus, despite significant early TRM, ex vivo alloanergization can support administration of large numbers of haploidentical donor T cells, resulting in rapid immune reconstitution with very few viral infections. Surviving patients have excellent performance status and a low rate of chronic GVHD.

Favorable outcome of allogeneic hematopoietic stem cell transplantation for relapsed or refractory acute promyelocytic leukemia in childhood

Summary:The optimal therapy for children with relapsed or refractory acute promyelocytic leukemia (APL) is unclear. We therefore reviewed our institutional outcomes for children undergoing allogeneic hematopoietic stem cell transplantation (HSCT) for advanced APL. Between 1986 and 2003, 12 allogeneic HSCTs (five related donor, seven unrelated donor) were performed for 11 patients (median age, 13 years) with relapsed (n=8) or refractory (n=3) APL. All patients engrafted, after a median of 18.5 days. Grade B–D acute graft-versus-host disease (GVHD) developed after five transplants (42%; 90% CI, 18–68%), and the cumulative incidence of chronic GVHD was 45% (90% CI, 19–71%). The cumulative incidence of overt relapse post-HSCT was 10% (90% CI, 0–28%). The overall 5-year survival was 73% (90% confidence interval (CI), 51–95%), with a median post-HSCT follow-up of 64 months. The Lansky/Karnofsky performance scores are 100% in six of eight survivors. In view of the low risk of subsequent relapse and favorable survival suggested by other reports and our own experience, we continue to recommend allogeneic HSCT for children with advanced APL for whom a suitably HLA-matched donor is identified.

Alloanergization of Human T Cells Results in Expansion of Alloantigen-Specific CD8+CD28− Suppressor Cells

Allostimulation with concurrent costimulatory blockade induces alloantigen‐specific hyporesponsiveness in responder T cells (“alloanergization”). Alloanergized responder cells also acquire alloantigen‐specific suppressive activity, suggesting this strategy induces active immune tolerance. While this acquired suppressive activity is mediated primarily by CD4+FOXP3+ cells, other cells, most notably CD8+ suppressor cells, have also been shown to ameliorate human alloresponses. To determine whether alloanergization expands CD8+ cells with allosuppressive phenotype and function, we used mixed lymphocyte cultures in which costimulatory blockade was provided by belatacept, an FDA‐approved, second‐generation CTLA‐4‐immunoglobulin fusion protein that blocks CD28‐mediated costimulation, as an in vitro model of HLA‐mismatched transplantation. This strategy resulted in an eightfold expansion of CD8+CD28− T cells which potently and specifically suppressed alloresponses of both CD4+ and CD8+ T cells without reducing the frequency of a range of functional pathogen‐specific T cells. This CD8‐mediated allosuppression primarily required cell–cell contact. In addition, we observed expansion of CD8+CD28− T cells in vivo in patients undergoing alloanergized HLA‐mismatched bone marrow transplantation. Use of costimulatory blockade‐mediated alloanergization to expand allospecific CD8+CD28− suppressor cells merits exploration as an approach to inducing or supporting immune tolerance to alloantigens after allogeneic transplantation.

Ex Vivo Costimulatory Blockade to Generate Regulatory T Cells From Patients Awaiting Kidney Transplantation.

Short‐term outcomes of kidney transplantation have improved dramatically, but chronic rejection and regimen‐related toxicity continue to compromise overall patient outcomes. Development of regulatory T cells (Tregs) as a means to decrease alloresponsiveness and limit the need for pharmacologic immunosuppression is an active area of preclinical and clinical investigation. Nevertheless, the immunomodulatory effects of end‐stage renal disease on the efficacy of various strategies to generate and expand recipient Tregs for kidney transplantation are incompletely characterized. In this study, we show that Tregs can be successfully generated from either freshly isolated or previously cryopreserved uremic recipient (responder) and healthy donor (stimulator) peripheral blood mononuclear cells using the strategy of ex vivo costimulatory blockade with belatacept during mixed lymphocyte culture. Moreover, these Tregs maintain a CD3+CD4+CD25+CD127lo surface phenotype, high levels of intracellular FOXP3 and significant demethylation of the FOXP3 Treg‐specific demethylation region on allorestimulation with donor stimulator cells. These data support evaluation of this simple, brief Treg production strategy in clinical trials of mismatched kidney transplantation.

Identification of single nucleotide polymorphisms in hematopoietic cell transplant patients affecting early recognition of, and response to, endotoxin

Hematopoietic cell transplant (HCT) is a life-saving therapy for many malignant and non-malignant bone marrow diseases. Associated morbidities are often due to transplant-related toxicities and infections, exacerbated by regimen-induced immune suppression and systemic incursion of bacterial products. Patients undergoing myeloablative conditioning for HCT become endotoxemic and display blood/plasma changes consistent with lipopolysaccharide (LPS)-induced systemic innate immune activation. Herein, we addressed whether patients scheduled for HCT display differences in recognition/response to LPS ex vivo traceable to specific single nucleotide polymorphisms (SNPs). Two SNPs of LPS binding protein (LBP) were associated with changes in plasma LBP levels, with one LBP SNP also associating with differences in efficiency of extraction and transfer of endotoxin to myeloid differentiation factor-2 (MD-2), a step needed for activation of TLR4. None of the examined SNPs of CD14, bactericidal/permeability-increasing protein (BPI), TLR4 or MD-2 were associated with corresponding protein plasma levels or endotoxin delivery to MD-2, but CD14 and BPI SNPs significantly associated with differences in LPS-induced TNF-α release ex vivo and infection frequency, respectively. These findings suggest that specific LBP, CD14 and BPI SNPs might be contributory assessments in studies where clinical outcome may be affected by host response to endotoxin and bacterial infection.

Effect of weight on outcomes of children undergoing hematopoietic cell transplantation.

Chemotherapy dosing in hematopoietic cell therapy (HCT) conditioning regimens is based on patient weight. We hypothesized that potential underdosing or overdosing of patients with significant deviation of weight from normal might alter HCT outcomes, such as early mortality, overall or organ-specific toxicity, and/or relapse. We therefore conducted a retrospective analysis of 400 children between the ages of 2 and 18 years who underwent HCT for malignant or nonmalignant disease at Boston Childrens Hospital over a 10-year period. Using the Centers for Disease Control and Prevention standard weight classification schema, we found no evidence to suggest a difference in survival or in time to engraftment or in relapse in patients with malignant disease. In the subgroups of patients either receiving autologous HCT or with underlying malignancy, combined overweight and obese patients had a higher rate of any organ, but not organ-specific, Grade 3–5 toxicity compared with the normal weight group. The study was not powered to detect a difference between underweight and normal weight patients. These data suggest that multiple outcome measures over the first year after HCT are unaffected by weight.

Infusion of Alloanergized Donor Lymphocytes after CD34-selected Haploidentical Myeloablative Hematopoietic Stem Cell Transplantation

Purpose: Allogeneic hematopoietic stem-cell transplantation (HSCT) is a curative treatment for many hematologic cancers. Use of haploidentical (mismatched) donors increases HSCT availability but is limited by severe graft-versus-host disease (GvHD) and delayed immune reconstitution. Alloanergization of donor T cells is a simple approach to rebuild immunity while limiting GvHD after haploidentical HSCT, but the optimal T-cell dose and impact on immune reconstitution remain unknown. Patients and Methods: We performed a multicenter phase I trial of alloanergized donor lymphocyte infusion (aDLI) after CD34-selected myeloablative haploidentical HSCT. The primary aim was feasibility and safety with secondary aims of assessing the less frequently addressed issue of impact on immune reconstitution. Results: Nineteen patients with high-risk acute leukemia or myelodysplasia were enrolled. Engraftment occurred in 18 of 19 patients (95%). Pre-aDLI, 12 patients (63%) had bacteremia, nine of 17 at-risk patients (53%) reactivated CMV, and one developed acute GvHD. Sixteen patients received aDLI at dose levels 1 (103 T cells/kg, n = 4), 2 (104, n = 8), and 3 (105, n = 4). After aDLI, five patients developed clinically significant acute GvHD, and four of 14 at-risk patients (29%) reactivated CMV. T-cell recovery was significantly greater, and functional virus- and tumor-associated antigen-specific T cells were detectable earlier in patients receiving dose level 2 or 3 versus dose level 1/no aDLI. Alloanergization of donor cells expanded the CD4+ T-regulatory cell frequency within aDLI, which increased further in vivo without impeding expansion of virus- and tumor-associated antigen-specific T cells. Conclusions: These data demonstrate safety and a potential role for aDLI in contributing to immune reconstitution and expanding tolerogenic regulatory T cells in vivo after CD34-selected myeloablative haploidentical HSCT. Clin Cancer Res; 24(17); 4098–109. ©2018 AACR.

Pilot experience with opebacan/rBPI 21 in myeloablative hematopoietic cell transplantation

Bacterial infection and inflammation contribute significantly to the morbidity and mortality of myeloablative allogeneic hematopoietic cell transplantation (HCT). Endotoxin, a component of the outer membrane of Gram-negative bacteria, is a potent inflammatory stimulus in humans. Bactericidal/permeability increasing protein (BPI), a constituent of human neutrophil granules, binds endotoxin thereby precluding endotoxin-induced inflammation and also has direct anti-infective properties against bacteria. As a consequence of myeloablative therapy used in preparation for hematopoietic cell infusion, patients experience gastrointestinal leak of bacteria and bacterial toxins into the systemic circulation and a period of inflammatory cytokine elevation associated with subsequent regimen-related toxicities. Patients frequently become endotoxemic and febrile as well as BPI-deficient due to sustained neutropenia. To examine whether enhancing endotoxin-neutralizing and anti-infective activity by exogenous administration of a recombinant N-terminal fragment of BPI (rBPI 21, generic name opebacan) might ameliorate regimen-related toxicities including infection, we recruited patients scheduled to undergo myeloablative HCT to participate in a proof-of-concept prospective phase I/II trial. After the HCT preparative regimen was completed, opebacan was initiated 18-36 hours prior to administration of allogeneic hematopoietic stem cells (defined as Day 0) and continued for 72 hours. The trial was to have included escalation of rBPI 21 dose and duration but was stopped prematurely due to lack of further drug availability. Therefore, to better understand the clinical course of opebacan-treated patients (n=6), we compared their outcomes with a comparable cohort meeting the same eligibility criteria and enrolled in a non-interventional myeloablative HCT observational study (n = 35). Opebacan-treated participants had earlier platelet engraftment (p=0.005), mirroring beneficial effects of rBPI 21 previously observed in irradiated mice, fewer documented infections (p=0.03) and appeared less likely to experience significant regimen-related toxicities (p=0.05). This small pilot experience supports the potential utility of rBPI 21 in ameliorating HCT-related morbidity and merits further exploration.