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Relapse after allogeneic hematopoietic stem cell transplantation (HCT) is the leading cause of death in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Infusion of unselected donor lymphocytes (DLIs) enhances the graft-versus-leukemia (GVL) effect. However, because the infused lymphocytes are not selected for leukemia specificity, the GVL effect is often accompanied by life-threatening graft-versus-host disease (GVHD), related to the concurrent transfer of alloreactive lymphocytes. Thus, to minimize GVHD and maximize GVL, we selectively activated and expanded stem cell donor–derived T cells reactive to multiple antigens expressed by AML/MDS cells (PRAME, WT1, Survivin, and NY-ESO-1). Products that demonstrated leukemia antigen specificity were generated from 29 HCT donors. In contrast to DLIs, leukemia-specific T cells (mLSTs) selectively recognized and killed leukemia antigen–pulsed cells, with no activity against recipient's normal cells in vitro. We administered escalating doses of mLSTs (0.5 to 10 × 107 cells per square meter) to 25 trial enrollees, 17 with high risk of relapse and 8 with relapsed disease. Infusions were well tolerated with no grade >2 acute or extensive chronic GVHD seen. We observed antileukemia effects in vivo that translated into not-yet-reached median leukemia-free and overall survival at 1.9 years of follow-up and objective responses in the active disease cohort (1 complete response and 1 partial response). In summary, mLSTs are safe and promising for the prevention and treatment of AML/MDS after HCT. This trial is registered at www.clinicaltrials.com as #NCT02494167.

Hematopoietic stem cell transplant (HSCT) is a curative option for patients with high-risk acute lymphoblastic leukemia (ALL), but relapse remains a major cause of treatment failure. To prevent disease relapse, we prepared and infused donor-derived multiple leukemia antigen–specific T cells (mLSTs) targeting PRAME, WT1, and survivin, which are leukemia-associated antigens frequently expressed in B- and T-ALL. Our goal was to maximize the graft-versus-leukemia effect while minimizing the risk of graft-versus-host disease (GVHD). We administered mLSTs (dose range, 0.5 × 107 to 2 × 107 cells per square meter) to 11 patients with ALL (8 pediatric, 3 adult), and observed no dose-limiting toxicity, acute GVHD or cytokine release syndrome. Six of 8 evaluable patients remained in long-term complete remission (median: 46.5 months; range, 9-51). In these individuals we detected an increased frequency of tumor-reactive T cells shortly after infusion, with activity against both targeted and nontargeted, known tumor-associated antigens, indicative of in vivo antigen spreading. By contrast, this in vivo amplification was absent in the 2 patients who experienced relapse. In summary, infusion of donor-derived mLSTs after allogeneic HSCT is feasible and safe and may contribute to disease control, as evidenced by in vivo tumor-directed T-cell expansion. Thus, this approach represents a promising strategy for preventing relapse in patients with ALL.

Autologous T cells engineered to express a CD19-specific chimeric antigen receptor (CAR) mediate high rates of complete response (CR) in patients with B-cell malignancies. However, autologous cell therapy products are time- and resource-intensive to manufacture and vary in potency and toxicity. Unlike polymorphic HLA-restricted T cells, monomorphic CD1d-restricted Vα24-invariant natural killer T cells (NKTs) are not alloreactive, and therefore therapeutic NKTs can be generated from allogeneic donors without the risk of graft-versus-host disease (GvHD). Moreover, pre-clinical models suggest that CAR-NKTs have inherent advantages over CAR-T products including an ability to trans-activate NK cells, cross-prime tumor-specific CD8 T cells, and recognize CD1d-positive B-cell lymphoma cells via their endogenous NKT T cell receptor. We report interim results from five patients treated on a phase 1 dose-escalation trial of allogeneic NKTs engineered to co-express a CD19-specific CAR, IL-15, and shRNA targeting beta-2 microglobulin and CD74 for downregulation of HLA class I and class II molecules, respectively (ANCHOR, NCT00840853). Four patients with relapsed/refractory B-cell non-Hodgkin's lymphoma (NHL, cohort A) were enrolled on dose level (DL) 1 (NHL-1, -2, -3) and DL 2 (NHL-4), and 1 patient with relapsed acute B-lymphoblastic leukemia (ALL, cohort B) was enrolled on DL 1 (ALL-1). Primary and secondary objectives of the trial are to assess safety and anti-tumor responses; immune response evaluation is an additional objective. NKTs were isolated from the leukapheresis product of 1 HLA-unmatched healthy individual, transduced with the CAR, expanded ex vivo for 14 days to a total of 2.7×10 9 CAR-positive cells (99.8% NKT purity, 0.04% T cells), and cryopreserved. Patients received 10 7 (DL 1) or 3×10 7 (DL 2) CAR-NKT cells per square meter of body surface area following lymphodepleting conditioning with cyclophosphamide/fludarabine. Adverse events were evaluated per NCI criteria. When accessible, patients underwent core biopsies of an involved site at 2-5 weeks post-infusion. Response to therapy was assessed at 4 weeks per Lugano Criteria (for NHL) or NCCN guidelines (for ALL). The most common adverse effects observed were nausea and grade 3-4 hematologic toxicities related to the lymphodepletion chemotherapy. There were no early adverse events attributable to the cellular product except grade 1 cytokine release syndrome in 1 patient. Of the 4 NHL patients (all with diffuse large B cell lymphoma), 3 had a partial response (NHL-1, -2, and -4, Figure 1A) that evolved into a CR in 1 case (NHL-2). The ALL patient achieved a CR with incomplete hematologic recovery and showed no evidence of leukemia by morphology, flow cytometry, or next-generation sequencing at 4 weeks. We detected in vivo expansion of donor-derived NKT and CAR-NKT cells in the peripheral blood of NHL-4 and ALL-1 that peaked at 1 week post-infusion in both cases as determined by flow cytometry and qPCR. While CAR-NKTs were not detected in the peripheral blood of the first 3 NHL patients beyond 3 hours post-infusion, they were found in tumor tissues collected from the 2 biopsied NHL patients (Figure 1B). In patient NHL-2, we also observed a 2000-fold expansion of recipient NKTs with a skewed TCRβ repertoire; this population peaked at 6 weeks post-treatment and remained elevated through 12 weeks (Figure 1C). An ELISpot assay performed with peripheral blood from NHL-1 and NHL-2 before treatment and at 4 and 6 weeks post-therapy to detect changes in the frequency of T cells reactive to a set of tumor-associated antigens (TAAs) found no evidence for epitope spreading toward the tested TAAs. In conclusion, our data indicate that allogeneic CAR-NKT cells are well-tolerated and can mediate objective responses in relapsed/refractory NHL and ALL patients even at the low doses tested. Our initial results from this first-in-human clinical evaluation of allogeneic CAR-NKTs suggest that NKTs represent a promising platform for "off-the-shelf" cancer immunotherapy. Figure 1 Figure 1. Disclosures Ramos: Tessa Therapeutics: Patents & Royalties, Research Funding; Athenex: Research Funding; Genentech: Consultancy; Novartis: Consultancy. Courtney: Athenex: Patents & Royalties, Research Funding. Metelitsa: Athenex: Patents & Royalties, Research Funding. OffLabel Disclosure: Cyclophosphamide and fludarabine are being used as lymphodepleting agents before immune effector infusion.

Background: Hematopoietic stem cell transplant (HSCT) is a curative option for patients with high-risk Acute Lymphoblastic Leukemia (HR-ALL), but relapse remains a major cause of treatment failure. Strategies to enhance the graft-versus-leukemia (GVL) effect have been employed to prevent relapse, including modulating immune suppression post-HSCT to hasten immune reconstitution or with the use of donor lymphocyte infusions (DLIs). However, DLIs carry a significant risk of graft-versus-host disease (GVHD) due to the concurrent transfer of alloreactive T cells. To enhance the GVL effect while minimizing GVHD, we developed a protocol for the generation of ex vivo expanded, donor-derived T-cell lines targeting PRAME, WT1 and Survivin - tumor associated antigens that are frequently expressed in both B- and T-cell ALL. These multi-antigen-targeted T cells (multiTAAs) were adoptively transferred to pediatric and adult patients with HR-ALL who had undergone an allogeneic HSCT. Methods: Donor-derived multiTAA-specific T cells were generated by co-culturing PBMCs with autologous DCs loaded with pepmixes (15 mer peptides overlapping by 11 amino acids) spanning all 3 target antigens in the presence of a Th1-polarizing/pro-proliferative cytokine cocktail. Following 2-4 rounds of stimulation these multiTAA-specific T cells were infused to patients with ALL who had undergone an HSCT but remained at a high risk for disease relapse. Results: We have generated 15 clinical grade multiTAA-specific T cell lines comprising CD3+ T cells (mean 95.1±1.9%) with a mixture of CD4+ (mean 22.8±6.3%) and CD8+ (mean 52.5±5.3%) cells, which expressed central [CD45RO+/CD62L+: 13.5±2.8%] and effector memory markers [CD45RO+/CD62L-: 56.4±3.8%]. The expanded lines recognized the targeted antigens PRAME (range 0-370 SFC/2x10 5), WT1 (0-363 SFC/2x10 5), and Survivin (0-65 SFC/2x10 5) in an IFNg ELIspot. None of the lines reacted against non-malignant patient-derived cells (3.7±0.8% specific lysis; E: T 20:1) - a study release criterion indicating lack of alloreactivity. We have infused 11 HR-ALL patients (8 pediatric and 3 adult) with donor-derived multiTAA-specific T cells to prevent disease relapse (Table 1). Patients were administered with up to 4 infusions of cells at 3 escalating dose levels, ranging from 0.5 - 2x10 7 cells/m 2. Infusions were well tolerated with no dose-limiting toxicity, GVHD, cytokine release syndrome or other adverse events. Three patients were not evaluable per study criteria as they received >0.5mg/kg of steroids (2 patients received stress doses for septic shock and 1 for elevated liver enzymes presumed to be GVHD that was later ruled out) within 4 weeks of infusion and were replaced. Six of the 8 remaining patients infused remain in CR on long-term follow up at a median of 46.5 months post-infusion (range 9-51 months). In patients who remained in long term CR we detected an expansion of tumor-reactive T cells in their peripheral blood post-infusion against both targeted (WT1, Survivin, PRAME) and non-targeted antigens (SSX2, MAGE-A4, -A1, -A2B, -C1, MART1, AFP and NYESO1) reflecting epitope and antigen spreading, which correlated temporally (within 4 weeks) with multiTAA infusions. By contrast in the two patients who relapsed we saw no evidence of in vivo T cell amplification within the first 4 weeks after infusion. Conclusion: The preparation and infusion of donor-derived multiTAA-specific T cells to patients with B- and T-ALL post allogeneic HSCT is feasible, safe and as evidenced by in vivo tumor-directed T cell expansion and antigen spreading in patients, may contribute to disease control. This strategy may present a promising addition to current immunotherapeutic approaches for prophylaxis for leukemic relapse in HSCT recipients. Figure 1 Figure 1. Disclosures Vasileiou: Allovir: Consultancy. Tzannou: Gileas: Honoraria; Allovir: Current equity holder in publicly-traded company. Kuvalekar: Allovir: Consultancy. Watanabe: Allovir: Consultancy. Grilley: QB Regulatory Consulting: Other: Ownership, project management support, Research Funding; Marker: Consultancy, Other: Regulatory and project management support; Allovir: Current equity holder in publicly-traded company, Other: Leadership. Hill: Incyte: Membership on an entity's Board of Directors or advisory committees. Omer: Allovir: Research Funding. Gottschalk: Tessa Therapeutics: Consultancy; Immatics: Membership on an entity's Board of Directors or advisory committees; Other: Other: patents and patent applications in the field of cancer cell and gene therapy ; Tidal: Consultancy; Novartis: Consultancy; Catamaran Bio: Consultancy. Heslop: Gilead: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Kiadis: Membership on an entity's Board of Directors or advisory committees; Kuur Therapeutics: Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees; Allovir: Current equity holder in publicly-traded company; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Marker Therapeutics: Current equity holder in publicly-traded company; Fresh Wind Biotherapies: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Rooney: Allogene: Patents & Royalties; Bellicum: Patents & Royalties; Bluebird: Current equity holder in publicly-traded company; Allovir: Current equity holder in publicly-traded company; Alimera: Consultancy; Memgen: Consultancy; TScan Therapeutics: Consultancy; Takeda: Patents & Royalties; Marker: Current equity holder in publicly-traded company; Tessa: Consultancy, Other: Leadership, Research Funding. Vera: Allovir: Consultancy, Current equity holder in publicly-traded company, Other: Leadership, travel , accomodations, expenses, Patents & Royalties; Marker: Current Employment, Other: Travel, Accomodations, Expenses, Patents & Royalties, Research Funding. Leen: Allovir: Consultancy, Current equity holder in publicly-traded company; Marker: Current equity holder in publicly-traded company.

Purpose Improvement of cure rates for patients treated with allogeneic hematopoietic stem-cell transplantation (HSCT) will require efforts to decrease treatment-related mortality from severe viral infections. Adoptively transferred virus-specific T cells (VSTs) generated from eligible, third-party donors could provide broad antiviral protection to recipients of HSCT as an immediately available off-the-shelf product. Patient and Methods We generated a bank of VSTs that recognized five common viral pathogens: Epstein-Barr virus (EBV), adenovirus (AdV), cytomegalovirus (CMV), BK virus (BKV), and human herpesvirus 6 (HHV-6). The VSTs were administered to 38 patients with 45 infections in a phase II clinical trial. Results A single infusion produced a cumulative complete or partial response rate of 92% (95% CI, 78.1% to 98.3%) overall and the following rates by virus: 100% for BKV (n = 16), 94% for CMV (n = 17), 71% for AdV (n = 7), 100% for EBV (n = 2), and 67% for HHV-6 (n = 3). Clinical benefit was achieved in 31 patients treated for one infection and in seven patients treated for multiple coincident infections. Thirteen of 14 patients treated for BKV-associated hemorrhagic cystitis experienced complete resolution of gross hematuria by week 6. Infusions were safe, and only two occurrences of de novo graft-versus host disease (grade 1) were observed. VST tracking by epitope profiling revealed persistence of functional VSTs of third-party origin for up to 12 weeks. Conclusion The use of banked VSTs is a feasible, safe, and effective approach to treat severe and drug-refractory infections after HSCT, including infections from two viruses (BKV and HHV-6) that had never been targeted previously with an off-the-shelf product. Furthermore, the multispecificity of the VSTs ensures extensive antiviral coverage, which facilitates the treatment of patients with multiple infections.

BACKGROUND. Treatment of B cell malignancies with adoptive transfer of T cells with a CD19-specific chimeric antigen receptor (CAR) shows remarkable clinical efficacy. However, long-term persistence of T cells targeting CD19, a pan–B cell marker, also depletes normal B cells and causes severe hypogammaglobulinemia. Here, we developed a strategy to target B cell malignancies more selectively by taking advantage of B cell light Ig chain restriction. We generated a CAR that is specific for the κ light chain (κ.CAR) and therefore recognizes κ-restricted cells and spares the normal B cells expressing the nontargeted λ light chain, thus potentially minimizing humoral immunity impairment. METHODS. We conducted a phase 1 clinical trial and treated 16 patients with relapsed or refractory κ+ non-Hodgkin lymphoma/chronic lymphocytic leukemia (NHL/CLL) or multiple myeloma (MM) with autologous T cells genetically modified to express κ.CAR (κ.CARTs). Other treatments were discontinued in 11 of the 16 patients at least 4 weeks prior to T cell infusion. Six patients without lymphopenia received 12.5 mg/kg cyclophosphamide 4 days before κ.CART infusion (0.2 × 108 to 2 × 108 κ.CARTs/m2). No other lymphodepletion was used. RESULTS. κ.CART expansion peaked 1–2 weeks after infusion, and cells remained detectable for more than 6 weeks. Of 9 patients with relapsed NHL or CLL, 2 entered complete remission after 2 and 3 infusions of κ.CARTs, and 1 had a partial response. Of 7 patients with MM, 4 had stable disease lasting 2–17 months. No toxicities attributable to κ.CARTs were observed. CONCLUSION. κ.CART infusion is feasible and safe and can lead to complete clinical responses. TRIAL REGISTRATION. ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00881920","term_id":"NCT00881920"}}NCT00881920. FUNDING. National Cancer Institute (NCI) grants 3P50CA126752 and 5P30CA125123 and Leukemia and Lymphoma Society (LLS) Specialized Centers of Research (SCOR) grant 7018.

Background. On 11 March, 2020 the World Health Organization declared COVID-19, caused by SARS-CoV-2, a global pandemic with almost 17,000,000 confirmed cases worldwide by the end of July, of which 4,500,000 were in the US. Approximately 20% of patients develop severe disease that can evolve into acute respiratory distress syndrome leading to respiratory or multiorgan failure, with an overall mortality of up to 4%. Older age, comorbidities such as hypertension and diabetes, and immune compromise have been identified as major risk factors associated with poor prognosis. For example, in immunocompromised HSCT patients mortality rates as high as 20% have been reported (www.cibmtr.org/COVID19). Furthermore, there is accumulating evidence regarding the protective role of T cells, with reduced counts and dysregulation seen more prominently in individuals with severe rather than mild COVID-19. Our group has previously demonstrated the feasibility, safety and clinical efficacy of administering allogeneic ex vivo expanded multivirus-specific T cells (multi-VSTs) as a banked, off-the-shelf product for the treatment of EBV, CMV, BKV, HHV6 and AdV infections/disease in immunocompromised individuals. Given the lack of preventative or therapeutic agents and the emerging evidence of the pivotal protective role of SARS-CoV-2-specific CD4+ and CD8+ T cells, we sought to explore the feasibility of developing a banked, SARS-CoV-2-specific VST product to treat those at highest risk of severe COVID-19 disease (i.e. HSCT recipients, elderly individuals, patients with comorbidities). Methods. To first identify immunogenic and protective SARS-CoV-2 antigens we screened PBMCs from convalescent individuals with mild COVID-19 (not requiring hospitalization) for T cell activity against overlapping peptide libraries (pepmixes) spanning 18 structural and non-structural SARS-CoV-2 proteins of which 8 [structural proteins: Spike (S), Membrane (M) and Nucleoprotein (N); non-structural proteins (Nsps): 3, 4, 6, 12; and the accessory protein (AP) 7a] were identified as immunodominant and advanced for VST manufacturing. We subsequently utilized our optimized VST manufacturing process and culture in a G-Rex device in medium supplemented with activating cytokines to generate SARS-CoV-2-specific T cells with activity against this combination of immunodominant targets. Results. We achieved a mean 29±7 fold expansion (mean±SEM; n=5) of cells that were comprised almost exclusively of CD3+ T cells (97.1±0.7%; mean±SEM), with a mixture of cytotoxic (CD8+; 10.2±1.2%) and helper (CD4+; 85.5±1.8%) T cells. These cells had a phenotype consistent with effector function and memory potential, as evidenced by upregulation of the activation markers CD25, CD69, and CD28 and expression of central (CD45RO+/CD62L+) and effector memory markers (CD45RO+/CD62L−), with minimal PD1 or Tim3 expression. To confirm the anti-viral activity of our expanded cells we performed an IFNγ ELIspot using each of the individual stimulating antigens as an immunogen and all lines proved to be reactive against the target antigens [S: 2,118±479 SFC/2x105; M: 1,084±182; N: 1,124±335; Nsp3: 71±48.6; Nsp4: 68±30; Nsp6: 23±6.7; AP7a: 65±43; and Nsp12: 29±9]. As demonstrated by intracellular cytokine staining (ICS), the immune response was mediated by both CD4+ and CD8+ T cell subsets, and the majority of IFNg-producing cells also produced TNFa. Reactive cells exhibited a primarily Th1-polarized profile as measured by Granzyme B production, luminex array and single-cell protein analysis. In addition, the expanded cells were able to kill viral pepmix-loaded autologous PHA blasts with minimal/no activity against non-antigen-expressing autologous and allogeneic targets (Figure 1). Conclusion. SARS-CoV-2 VSTs generated from convalescent individuals are Th1-polarized, polyfunctional and selectively able to kill viral antigen-expressing targets with no auto- or alloreactivity, indicative of both their selectivity and safety for clinical use. We are rapidly advancing this product to the clinic for administration in a randomized clinical trial (VSTs+SOC vs SOC) to prevent the development of severe disease in high risk hospitalized patients such as those post-transplant or therapy for hematologic malignancy. Figure 1: SARS-CoV-2 Specific T cells Demonstrate Selective Cytolytic Activity against viral antigen-expressing targets. Figure 1 Disclosures Vasileiou: AlloVir: Consultancy. Kuvalekar:AlloVir: Consultancy. Workineh:AlloVir: Current Employment. Watanabe:AlloVir: Consultancy. Heslop:Novartis: Consultancy; Gilead Biosciences: Consultancy; PACT Pharma: Consultancy; Kiadis: Consultancy; Tessa Therapeutics: Consultancy, Research Funding; AlloVir: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Marker Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Hill:Incyte: Membership on an entity's Board of Directors or advisory committees. Leen:AlloVir: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Marker Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.

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/m2 ×3 per week), starting Results: No grade 3/4 toxicities were associated with ULD IL-2. CD4+CD25+FoxP3+ Tregs increased from a mean of 4.8% (range, 0%–11.0%) pre IL-2 to 11.1% (range, 1.2%–31.1%) following therapy, with the greatest change occurring in the recipients of matched related donor (MRD) transplants. No IL-2 patients developed grade 2–4 acute GVHD (aGVHD), compared with 4 of 33 (12%) of the comparator group who did not receive IL-2. IL-2 recipients retained T cells reactive to viral and leukemia antigens, and in the MRD recipients, only 2 of 13 (15%) of the IL-2 patients developed viral infections versus 63% of the comparator group (P = 0.022). Conclusions: Hence, ULD IL-2 is well tolerated, expands a Treg population in vivo, and may be associated with a lower incidence of viral infections and GVHD. Clin Cancer Res; 20(8); 2215–25. ©2014 AACR.

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.

Myelodysplastic syndromes (MDS) are a heterogeneous group of disorders characterized by bone marrow failure and a propensity to progress to acute myeloid leukemia (AML). A core component of the underlying pathogenesis in MDS is deregulation of inflammatory cytokines, such as tumor growth factor-β (TGFβ), which impact the function of immune cells and hence their capacity to mount anti-infective or anti-tumor responses. However, little is known about antigen-specific T cell function in patients with MDS. We hypothesized that virus-specific T cell (VST) function might be preserved in patients with MDS, and that the functional capacity of T cells reactive against tumor-associated antigens aberrantly overexpressed by clonal MDS cells such as Cyclin A1 (CCNA1) and Proteinase (PR3) might also be preserved and exploited for immunotherapeutic purposes. Following informed consent, we collected peripheral blood samples from 10 patients (pts) with MDS and 17 healthy donors. Most pts (9 out of 10) were transfusion dependent and 3 subsequently underwent an allogeneic HSCT. Table 1 summarizes the other clinical characteristics, karyotypic and mutational profile at the time of blood collection. Compared with T cells isolated from healthy donors, MDS patient-derived T cells had a similar CD4 to CD8 ratio (1.5-2.5:1 for healthy donors and 3:1 for MDS pts), but displayed a more exhausted profile at baseline (CD3+TIM3+: 1% in healthy donors and 5% in MDS pts) and produced higher levels of inflammatory cytokines [IFNγ (18±3pg/ml vs 36±16pg/ml, healthy donor vs MDS; p=0.12), and IL-8 (56±32 vs 704±446 pg/ml, p=0.01)]. Next, to assess the capacity of MDS pts to mount ex vivo functional virus-directed responses, we stimulated patient-derived PBMCs (n=5) with overlapping peptide libraries (pepmixes) spanning immunogenic AdV, CMV, EBV, BK and HHV-6 antigens. Similar to healthy donor-derived T cell lines (n=5, 3 specific for 4 viruses and 2 for 5 viruses), all 5 MDS patient-derived lines demonstrated specificity for one or more of the target viruses (1 for 5 viruses, 1 for 4, 2 for 3 and 1 for 1 virus) as observed by IFNγ ELISpot assay with comparable magnitude (range Adv: 43-730 vs 384-941 in healthy donors, CMV: 0-1599 vs 0-3002, EBV: 0-1486 vs 0-541, BK: 0-839 vs 38-275 and HHV6: 0-794 vs 5-407 SFU/2x105 cells, respectively). We next examined the feasibility of expanding autologous MDS-antigen directed T cell products (n=10) to determine whether an adoptive immunotherapeutic approach might be applicable for MDS treatment. Thus, we exposed patient-derived PBMCs to autologous dendritic cells (DC) loaded with pepmixes spanning 6 MDS-associated antigens (CCNA1, survivin, WT1, PRAME, PR3 and NYESO1). After 3 rounds of stimulation, the products obtained were predominantly CD3+ T cells (mean 88±1.3%) that were polyclonal (CD4: 46±5% and CD8: 41±4%) containing predominantly memory T cells (TEM: 36±6% TCM: 37±5% and Tnaïve =13±3%). Six lines (60%) showed specific recognition to at least one of the target antigens: 4 lines specific for PRAME, 1 for CCNA1, 1 for WT1 and 1 for NYESO1 (range 0-40, 0-184, 0-1386 and 0-179 SFU/2x105 cells, respectively by IFNγ ELIspot). T cell lines were capable of specifically secreting multiple effector cytokines in response to targets (TNFα: 12% and IFNγ: 16% in response to PRAME in a representative patient-derived T cell line). Furthermore, this line was capable of killing PRAME+ targets in a 4hr 51Cr release assay [60% specific lysis, E:T 20:1]. In conclusion, functional virus-directed T cell immunity in patients with MDS is preserved, potentially explaining the lower rates of viral reactivation seen in these patients compared with other infections. Moreover, T cells specific for MDS-expressed tumor antigens can also be successfully expanded ex vivo from patients. Taken together this raises the possibility of applying an adoptive immunotherapeutic approach for the treatment of MDS. Disclosures Ramos: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Research Funding. Leen:Allovir: Consultancy, Other: Cofounder, Ownership Interest; Marker Therapeutics: Consultancy, Other: Cofounder, Ownership Interest.

Background The use of hematopoietic progenitor cell (HPC) transplantation has rapidly expanded in recent years. Currently, several sources of HPCs are available for transplantation including peripheral blood HPCs (PBPCs), cord blood cells, and marrow cells. Of these, PBPC collection has become the major source of HPCs. An important variable in PBPC collection is the response to PBPC mobilization, which varies significantly and sometime causes mobilization failure. Study Design and Methods A retrospective study of 69 healthy donors who underwent PBPC donation by leukapheresis was performed. All of these donors received 10 μg/kg/day or more granulocyte–colony-stimulating factor (G-CSF) for 5 days before PBPC harvest. Donor factors were evaluated and correlated with mobilization responses, as indicated by the precollection CD34 count (pre-CD34). Results Donors with a pre-CD34 of more than 100 × 106/L had higher body mass index (BMI) compared with donors whose pre-CD34 was 38 × 106 to 99 × 106/L or less than 38 × 106/L (32.0 ± 1.04 kg/m2 vs. 28.7 ± 0.93 kg/m2 vs. 25.9 ± 1.27 kg/m2, respectively; p

Immunotherapy is emerging as a potent therapy for a range of hematologic malignancies including lymphomas. Indeed adoptive transfer of T cells genetically engineered to express the CD19 chimeric antigen receptor (CAR) has now received FDA approval for the treatment of patients with refractory diffuse large B cell lymphomas (DLBCL). We have developed a non-engineered T cell-based therapy to treat patients with all types of lymphomas: Hodgkin's (HL) and non-Hodgkin's lymphoma (NHL). The approach uses single T cell lines that simultaneously target a range of tumor-associated antigens (TAAs) that are frequently expressed by these tumors, including PRAME, SSX2, MAGEA4, NY-ESO-1 and Survivin. We can consistently prepare these lines by culturing PBMCs in the presence of a Th1-polarizing/pro-proliferative cytokine cocktail, and adding autologous DCs as APCs that are loaded with pepmixes (15mer peptides overlapping by 11 amino-acids) spanning all 5 target antigens. The use of whole antigen should remove the HLA restriction imposed by the use of transgenic TCRs specific for single peptides, while targeting multiple antigens simultaneously would reduce the risk of tumor immune evasion. We have generated 42 clinical-grade multiTAA-specific T cell lines, comprising CD3+ T cells (mean 98±1.1%) with a mixture of CD4+ (mean 48±4.3%) and CD8+ (mean 37±4%) T cells, which expressed central and effector memory markers (CD45RO+/CD62L+/CCR7+ -- mean 14±3%; CD45RO+/CD62L+/CCR7- -- 10±2.2%; CD45RO+/CD62L-/CCR7- -- 28.3±3.6%) (n=42). The expanded lines recognized the targeted antigens PRAME, SSX2, MAGEA4, NY-ESO-1 and Survivin (range 0-463, 0-496, 0-330, 0-379 and 0-304 spot forming units (SFU)/2x105 input cells, respectively in IFNg ELIspot, n=34). None of the lines reacted against non-malignant autologous recipient cells (3±3.8% specific lysis; E:T 20:1). We have treated 33 patients: 13 with HL, 17 with aggressive NHL (diffuse large B-cell, mantle cell, or T cell lymphomas) and 3 with indolent NHLs (FL and marginal zone lymphoma). Patients received 0.5-2x107 multiTAA-T cells/m2. Of 18 patients who were infused as adjuvant therapy all but 2 remain in remission (range 3-42 months post-infusion). Fifteen patients have received multiTAA-specific T cells to treat active disease, all of whom had failed a median of 4 lines of prior therapy. Of these, 5 had transient disease stabilization followed by disease progression, 4 have ongoing stable disease, 3-18 months post-multiTAA-specific T cells while the remaining 6 (3 with HL and 3 with DLBCL) have all had complete and durable responses ( 4 to 41 months), as assessed by PET imaging. These clinical responses correlated with the detection of tumor-reactive T cells in patient peripheral blood post-infusion directed against both targeted antigens as well as non-targeted TAAs including MAGEA2B and MAGE C1, indicating induction of antigen/epitope spreading. Notably, no patient, including the complete responders, had infusion-related systemic- or neuro-toxicity. Thus, infusion of autologous multiTAA-targeted T cells directed to PRAME, SSX2, MAGEA4, NY-ESO-1 and Survivin has been safe and provided durable clinical benefit to patients with lymphomas. Disclosures Brenner: Marker: Equity Ownership. Heslop:Marker: Equity Ownership; Cell Medica: Research Funding; Tessa Therapeutics: Research Funding; Viracyte: Equity Ownership; Gilead Biosciences: Membership on an entity's Board of Directors or advisory committees; Cytosen: Membership on an entity's Board of Directors or advisory committees. Rooney:Marker: Equity Ownership. Vera:Marker: Equity Ownership. Leen:Marker: Equity Ownership.

Background: Leukemic relapse remains the major cause of treatment failure in hematopoietic stem cell transplant (HSCT) recipients. While the infusion of donor lymphocytes to prevent and treat relapse has been clinically implemented this strategy does not provide durable remissions and carries the risk of life-threatening graft-versus-host disease (GVHD). More recently the adoptive transfer of T cells that have been engineered to express CD19-targeted chimeric antigen receptors (CARs), has shown potent anti-leukemic activity in HSCT recipients with recurrent disease. However, disease relapse with the emergence of CD19 negative tumors is an emerging clinical issue post-administration of these mono-targeted T cells. To overcome these limitations, we developed a protocol for the generation of donor-derived T cell lines that simultaneously targeted a range of tumor associated antigens (multiTAAs) that are frequently expressed by B- and T-cell ALL including PRAME, WT1 and Survivin for adoptive transfer to high risk recipients transplanted for ALL. Methods/Results: We were consistently able to generate donor-derived multiTAA-specific T cells by culturing PBMCs in the presence of a Th1-polarizing/pro-proliferative cytokine cocktail, using autologous DCs as APCs and loading them with pepmixes (15 mer peptides overlapping by 11 amino acids) spanning all 3 target antigens. The use of whole antigen increases the range of patient HLA polymorphisms that can be exploited beyond those matched to single peptides, while targeting multiple antigens simultaneously reduces the risk of tumor immune evasion. To date, we have generated 14 clinical grade multiTAA-specific T cell lines comprising CD3+ T cells (mean 94±9%) with a mixture of CD4+ (mean 21±28%) and CD8+ (mean 52±24 %) cells, which expressed central [CD45RO+/CD62L+: 14±9%] and effector memory markers [CD45RO+/CD62L-: 80±11%] associated with long term in vivo persistence. The expanded lines recognized the targeted antigens WT1, PRAME and Survivin by IFNg ELIspot with activity against >1 targeted antigens in all cases. None of the lines reacted against non-malignant patient-derived cells (4±3% specific lysis; E: T 20:1) - a study release criterion. Thus far we have treated 8 high risk ALL patients with donor derived TAA T cells post-transplant to prevent disease relapse (Table 1). Infusions were well tolerated with no dose-limiting toxicity, GVHD, CRS or other adverse events. Two patients were not evaluable per study criteria as they received >0.5mg/kg of steroids within 4 weeks of infusion and were replaced. Five of the 6 remaining patients infused remain in CR a median of 11.2 months post-infusion (range 9-22 months). We detected the expansion of tumor-reactive T cells in patient peripheral blood post-infusion against both targeted (WT1, Survivin, PRAME) and non-targeted antigens (SSX2, MAGE-A4, -A1, -A2B, -C1, MART1, AFP and NYESO1) reflecting epitope and antigen spreading. The single patient who relapsed showed no evidence of tumor-directed T cell expansion despite receiving 3 additional infusions at 4 week intervals. Conclusion: In summary, infusion of donor multi-TAA-specific T cells to patients with ALL post allogeneic HSCT is feasible, safe and as evidenced by expansion and antigen spreading in patients, may contribute to disease control. This strategy may present a promising addition to current immunotherapeutic approaches for prophylaxis for leukemic relapse in HSCT recipients. Table 1. Table 1. Disclosures Vera: Marker: Equity Ownership. Heslop:Marker: Equity Ownership; Cytosen: Membership on an entity's Board of Directors or advisory committees; Cell Medica: Research Funding; Gilead Biosciences: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Research Funding; Viracyte: Equity Ownership. Leen:Marker: Equity Ownership.

Despite an array of approved agents for the treatment of multiple myeloma (MM), most patients eventually relapse after conventional treatments. The adoptive transfer of tumor-targeted T cells has demonstrated efficacy in the treatment of patients with chemo-refractory hematological malignancies including MM. While the majority of T cell-based immunotherapeutic studies in the clinic explore genetically modified T cells that target a single tumor-expressed antigen, we have developed a strategy to generate non-engineered T cell lines that simultaneously target multiple MM-expressed antigens, thereby reducing the risk of tumor immune evasion. We manufacture multiTAA-specific T cells targeting the tumor-associated antigens PRAME, SSX2, MAGEA4, NY-ESO-1 and Survivin by culturing patient-derived PBMCs with autologous DCs loaded with pepmixes (15mer peptides overlapping by 11 aminos acids) spanning all 5 target antigens in the presence of a Th1-polarizing/pro-proliferative cytokine cocktail. In our current clinical trial (NCT02291848), we have successfully generated multi-antigen-targeted lines from 18/ of 19 patients thus far, with one in production. The T cell lines comprise of CD3+ T cells (mean 95.6±2.2%) with a mixture of CD4+ (28.9±7.2%) and CD8+ (56.6±7.2%) T cells, which express central and effector memory markers (CD45RO+/CD62L+/CCR7+ -- 1.21±0.2%; CD45RO+/CD62L+/CCR7- -- 15.16±2.5%; CD45RO+/CD62L-/CCR7- -- 56.9±6.3%). All the expanded lines were specific for two to five target antigens with the majority of lines (13 of 18) specific for ≥3, (PRAME: Mean 45, range: 0 to 205 spot forming units (SFU)/2x105 input cells ; SSX2 mean: 57, 0 to 583, NYESO1: mean: 51, 0 to 125 , MAGE-A4 Mean: 67, 0 to 377 and Survivin mean: 53, 0 to 51), and did not react against non-malignant autologous recipient cells (2±3% specific lysis; E:T 20:1). We assessed the clonal diversity of the clinical product using TCR vβ deep sequencing analysis. We found both polyclonality and that the majority (mean 79%; range: 59 to 95%) represented rare T cell clones that were unique to the ex vivo expanded cell line and below levels of detection in the patients peripheral blood prior to infusion, thereby enabling in vivo tracking studies.. To date we have infused 18 patients with at least 2 infusions, 2 weeks apart of doses ranging from 0.5 to 2x107/m2. These patients had received a median of 4 lines of prior therapy including high dose chemotherapy with autologous stem cell rescue. Ten patients were refractory to their latest therapy and had active MM, while 8 were in remission at the time of infusion. At the 6 week evaluation period, of the 10 patients receiving multiTAA-specific T cells to treat active disease, 1 had a complete remission (CR) by the international myeloma working group (IMWG) response criteria, 1 had a partial remission (PR) and 8 others had stable disease (SD). Seven of these 10 patients were infused more than 1 year ago. Although 2 of the 7 subsequently had disease progression, the remaining 5 continue to respond, with sustained CR (1), PR (2) or SD (2). Of the 8 patients in CR at the time of T cell infusion, all remained in CR at the week 6 disease assessment and of the 6 evaluable patients who are >1 year post T cells, only one patient has relapsed, at 7 months after T cell infusion. These clinical responses correlated with the emergence and persistence (>6 months) of "line-exclusive" tumor-reactive T cells in patient peripheral blood, as assessed by longitudinal tracking of infused T cell clones using TCR deep sequencing. These infused product-derived T cells were detected in both peripheral blood (mean 0.43% ±SD of 0.3 of the total repertoire) and the marrow (mean 0.61%±0.24% of total repertoire). The expansion of product-derived T cell clones was higher among patients with active MM than in patients treated in remission (active: 0.60±0.39%, remission: 0.2±0.08%, p=0.048). Notably, no patient, including the complete responder, had infusion-related systemic- or neuro-toxicity. Thus, autologous multiTAA-targeted T cells directed to PRAME, SSX2, MAGEA4, NY-ESO-1 and Survivin can be safely administered to patients with MM, in whom they can subsequently be detected long-term in peripheral blood and marrow, and where they produce sustained tumor responses including CR. It will be of interest to discover whether larger or more frequent doses of these T cells can produce further benefit with maintained safety. Disclosures Brenner: Marker: Equity Ownership. Heslop:Marker: Equity Ownership; Viracyte: Equity Ownership; Cell Medica: Research Funding; Gilead Biosciences: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Research Funding; Cytosen: Membership on an entity's Board of Directors or advisory committees. Vera:Marker: Equity Ownership. Leen:Marker: Equity Ownership.

Background: Amyotrophic lateral sclerosis (ALS), an inexorably progressive motoneuron disease, is accompanied by significantly increased markers of inflammation. These inflammatory constituents could protect, harm, do neither, or do both. Objective: Allogeneic hematopoietic stem cell transplantation (HSCT) was performed in patients with sporadic ALS to suppress neuroinflammation and improve clinical outcomes after CNS engraftment. Methods: Six patients with definite ALS received total body irradiation followed by peripheral blood HSCT infusion from human leukocyte antigen identically matched sibling donors. Disease progression and survival were assessed monthly and compared with matched historic database patients. Autopsy samples from brain and spinal cord were examined immunohistochemically and by quantitative reverse-transcriptase polymerase chain reaction. Donor-derived DNA in brain and spinal cord tissue was evaluated for the extent of chimerism. Results: No clinical benefits were evident. Four patients were 100% engrafted; postmortem tissue examination in two of the 100% engrafted patients demonstrated 16% to 38% donor-derived DNA at sites with motoneuron pathology, which may correspond to the observed increased CD68 or CD1a-positive cells. Neither donor DNA nor increased cell numbers were found in several unaffected brain regions. A third minimally engrafted patient had neither donor DNA nor increased infiltrating cells in the CNS. Conclusions: This study demonstrates that peripheral cells derived from donor hematopoietic stem cells can enter the human CNS primarily at sites of motoneuron pathology and engraft as immunomodulatory cells. Although unmodified hematopoietic stem cells did not benefit these sporadic amyotrophic lateral sclerosis patients, such cells may provide a cellular vehicle for future CNS gene therapy.

Background: Recent studies using qualitative analysis of flow cytometry data have demonstrated various im- munophenotypic abnormalities associated with myelodysplastic syndromes (MDS). However, there are limited reports as- sessing the ability of quantitative immunophenotypic analysis to discriminate MDS from other cytopenic conditions. Design: Using flow cytometry, we studied 37 bone marrow specimens from 23 patients with MDS and 14 cytopenic pa- tients with non-clonal hematologic disorders. Samples were analyzed quantitatively for percentages of T-cells, B-cells, NK cells, granulocytes, monocytes, blasts, erythroid precursors, and plasma cells; CD4:CD8 ratio; % granulocyte subsets; % CD56+ monocytes; and % erythroid precursor subsets. Results: Quantitative analysis of immunophenotypic data in MDS patients compared to controls showed decreases in total granulocytes (p=0.037) and more mature subsets of CD11b + CD16 bright granulocytes (p=0.0046) and CD10 + granulocytes (p=0.022). MDS patients also showed a trending increase in subset percentage of CD56+monocytes (p=0.056). Using re- ceiver operating characteristic (ROC) analysis, cut-off values for these parameters favoring a diagnosis of MDS were identified as follows: total granulocytes 10%. Subsequently, a scoring system was proposed whereby a score of one was assigned for the presence of each quantitative abnormality. Using this system on the original study population, the pres- ence of at least two abnormalities (score 2) revealed optimal sensitivity (69.6%) and specificity (71.4%) for a diagnosis of MDS. Conclusion: These findings suggest that quantitative analysis of immunophenotypic data can be complementary to quali- tative interpretation. These data may be useful for distinguishing MDS from non-clonal cytopenic disorders and warrant prospective study in additional MDS patients.

Human herpesvirus 6 (HHV-6) viremia, as detected by polymerase chain amplification, occurs in approximately half of allogeneic hematopoietic stem cell transplant recipients. The significance of such viremia is incompletely understood, but HHV-6 encephalitis and bone marrow suppression are increasingly being recognized in patients with high viral DNA. We report two patients in whom donor-to-recipient transmission occurred through hematopoietic transplant by means of chromosomally integrated (CI) HHV-6. Iatrogenic transmission manifested at engraftment as asymptomatic elevation of HHV-6 viral DNA of 3600 and 15 400 DNA copies/ml in plasma and 6.1 x 10(6) and 9.7 x 10(5) DNA copies/ml in the whole blood. Both donors had elevated plasma HHV-6 PCR at 5.6 x 10(4) and 1.3 x 10(5) DNA copies/ml and strikingly elevated whole blood HHV-6 levels at 4.1 x 10(6) and 4.7 x 10(6) DNA copies/ml, respectively. CI of the virus was traced to the mother of one patient and his donor. CI of HHV-6 may confound the interpretation of HHV-6 viremia after stem cell transplantation; consideration of the possibility of CI HHV-6 will avoid unnecessary antiviral therapy.

Immunocompromised individuals are at high risk for life-threatening diseases, especially those caused by cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus. Conventional therapeutics are primarily active only against CMV, and resistance is frequent. Adoptive transfer of polyclonal cytotoxic T lymphocytes (CTLs) specific for CMV or EBV seems promising, but it is unclear whether this strategy can be extended to adenovirus, which comprises many serotypes. In addition, the preparation of a specific CTL line for each virus in every eligible individual would be impractical. Here we describe genetic modification of antigen-presenting cell lines to facilitate the production of CD4(+) and CD8(+) T lymphocytes specific for CMV, EBV and several serotypes of adenovirus from a single cell culture. When administered to immunocompromised individuals, the single T lymphocyte line expands into multiple discrete virus-specific populations that supply clinically measurable antiviral activity. Monoculture-derived multispecific CTL infusion could provide a safe and efficient means to restore virus-specific immunity in the immunocompromised host.

We studied the incidence and recurrence of Cytomegalovirus (CMV) infection and reactivation in 38 recipients of Alemtuzumab reduced intensity conditioning-stem cell transplantation, and used CMV-HLA tetramer studies to discover if these events correlated with recovery of circulating CMV-specific CD8+ T cells (cytotoxic T lymphocyte (CTLs)). The cumulative incidence of CMV infection was 60% at 1 year (95% CI, 45-78%) with a median reactivation time of 24 days (range 5-95 days). All patients with CMV reactivation received Ganciclovir or Foscarnet, and only one developed CMV disease. More strikingly, only 8/21 patients had relapse of CMV antigenemia. Tetramer analysis in 13 patients showed that 11 reconstituted CMV CTLs (7/11 by day 30 and 10/11 by day 90). The development of CMV infection was accompanied by a >5-fold rise of CMV CTLs. Recurrence of CMV infection occurred only in the patients who failed to generate a CTL response to the virus. Hence, recipients of SCT using Alemtuzumab-RIC are initially profoundly immunosuppressed and have a high incidence of early CMV reactivation. However, in the majority of patients, infection is transient, and antiviral T cell reconstitution is rapid. Monitoring with CMV-specific CTLs may help identify the subset of patients at risk from recurrent infection or disease.

The majority of children and some adults with acute lymphocytic leukaemia (ALL) can be cured with current intensive chemotherapy regimens. For those patients who relapse or who do not achieve remission, allogeneic haemopoietic stem cell transplantation (HSCT) offers the best chance for long-term disease control. Different sources of haemopoietic stem cells including marrow, peripheral blood, and cord blood are now available and the introduction of subablative regimens has increased the number of patients who are transplant candidates. Relapse remains the major cause of transplant failure and immunotherapy strategies post-transplant to augment the graft versus leukaemia effect are being explored.

It remains difficult to treat the multiplicity of distinct viral infections that afflict immunocompromised patients. Adoptive transfer of virus-specific T cells (VSTs) can be safe and effective, but such cells have been complex to prepare and limited in antiviral range. We now demonstrate the feasibility and clinical utility of rapidly generated single-culture VSTs that recognize 12 immunogenic antigens from five viruses (Epstein-Barr virus, adenovirus, cytomegalovirus, BK virus, and human herpesvirus 6) that frequently cause disease in immunocompromised patients. When administered to 11 recipients of allogeneic transplants, 8 of whom had up to four active infections with the targeted viruses, these VSTs proved safe in all subjects and produced an overall 94% virological and clinical response rate that was sustained long-term.

Viral infections remain a significant cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Adoptive immunotherapy with donor-derived virus-specific T cells (VSTs) has proven safe and effective for the prophylaxis and treatment of EBV, CMV, AdV, BKV and HHV-6 infections post-HSCT. However, broader application is restricted by the time taken to prepare patient-specific products and the lack of virus-specific T cell precursors in cord blood and seronegative donors. Thus, to assess whether 3rd party multivirus-directed VSTs could produce clinical benefit when administered as an "off the shelf" product to allogeneic HSCT recipients with refractory BKV, EBV, CMV, AdV and/or HHV-6 infections we initiated a Phase II clinical trial and now report interim results for 22 patients infused to date. We prepared a bank of 58 VST lines from individuals with common HLA polymorphisms by exposing donor PBMCs (3x107) to overlapping peptide libraries spanning AdV (Hexon, Penton), EBV (LMP2, EBNA1, BZLF1), CMV (pp65, IE1), BKV (Large T, VP1) and HHV-6 (U11, U14, U90) antigens followed by a 9-11 day expansion phase in a G-Rex device in the presence of IL4 and IL7. This produced a mean of 4.2±1x108 VSTs that were polyclonal, comprising both CD4+ (61±2.4%) and CD8+ (34±2.1%) cells that expressed central (CD45RO+/CD62L+/CCR7+ - 43±3.9%) and effector memory markers (CD45RO+/CD62L- - 10±1%). 56/58 lines had activity against AdV, 50/58 against EBV, 35/58 against BKV, 34/58 against HHV-6, and 33/58 against CMV. To date, 62 HSCT recipients have been screened for study participation and a potential line, based on target virus specificity through a shared allele and overall HLA match, was identified for 58. Of these, 22 patients with infections that were unresponsive to at least 2 lines of antiviral treatment have been infused (fixed dose level - 2x107 VSTs/m2) with VST lines matched at 1 to 6 HLA antigens. Seventeen received just 1 infusion, while 5 patients required 2 or more infusions for sustained benefit. Eight patients received VSTs for CMV infections, including 3 cases of CMV colitis, 8 for BKV (6 for cystitis, 2 for nephritis), 1 for HHV-6, 1 for EBV-PTLD, 1 for AdV, 1 for BKV and EBV, 1 for CMV and AdV and 1 for CMV and BKV infections. There were no immediate adverse effects related to infusion. Based on viral load measurements by quantitative PCR a single VST infusion successfully controlled active infections in 19/21 evaluable patients: CMV (4 CR, 5 PR, 1NR); EBV (2 CR); AdV (1 CR, 1 NR); BKV (1 CR, 7 PR); and HHV-6 (1 PR). Of note, all 6 patients with BK hemorrhagic cystitis and 2/3 patients with CMV colitis had marked improvement/resolution of symptoms following VST treatment. In 8 subjects who responded to VST therapy we saw an increase in virus-specific T cells post-infusion. These expanded cells were confirmed to be of 3rd party VST origin in 3 patients and persisted for up to 6 weeks post-infusion. Finally, despite the HLA disparity of VSTs and recipients, de novo GvHD occurred in only one subject, who developed Grade I skin GVHD 1 week post-infusion, which resolved with the administration of topical steroids. One additional patient had a flare of chronic skin GVHD coincident with tapering of immunosuppression and 1 patient developed a transient fever 5 hours post-infusion, which spontaneously resolved. These results demonstrate the feasibility and safety of 3rd party multivirus-directed VSTs, generated by direct stimulation of PBMCs with synthetic peptides and administered as an "off the shelf" product. The infused cells were capable of in vivo expansion in allogeneic HSCT recipients and proved clinically effective against refractory EBV, CMV and AdV infections and also controlled HHV-6 reactivation and BKV-associated hemorrhagic cystitis. Disclosures Off Label Use: Adoptively transferred T cells administered under an IND. Brenner:Bluebird Bio: Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; Celgene: Other: Collaborative Research Agreement; Cell Medica: Other: Licensing Agreement. Rooney:Celgene: Other: Collaborative research agreement; Cell Medica: Other: Licensing Agreement. Heslop:Celgene: Other: Collaborative research agreement; Cell Medica: Other: Licensing Agreement.

Severe and fatal viral infections remain common after HSCT. Adoptive transfer of cytotoxic T lymphocytes (CTLs) specific for EBV, CMV and Adv antigens can treat infections that are impervious to conventional therapies, but extension to additional viruses has been limited by competition between virus-derived antigens and laborious manufacturing procedures. We are now evaluating whether infusion of rapidly-generated donor-derived pentavalent T cell lines (pCTL), stimulated just once with peptide libraries spanning immunogenic EBV (LMP2, EBNA1, BZLF1), Adv (Hexon, Penton), CMV (pp65, IE1), BK (Large T, VP1) and HHV6 (U90, U11, U14) antigens, and expanded in the presence of IL4+7 in a G-Rex device, is safe and effective in HSCT recipients with active infections. With NHLBI-PACT support, 35 clinical-grade pCTL lines have been generated. From 30x106 PBMCs we prepared a mean of 374x106 cells (range 99-713x106) over 9-11 days. The lines were polyclonal, comprising both CD4+ (57±5%) and CD8+ (35±5%) cells, with specificity for CMV (IE1: 337±141; pp65: 1059±479 SFC/2x105), EBV (LMP2: 175±87; EBNA1: 116±44; BZLF1: 129±88), Adv (Hexon: 446±153; Penton: 317±108), BK (Large T: 130±67; VP1: 231±104) and HHV6 (U90: 66±50; U11: 36±18; U14: 82±21) and no alloreactivity against recipient PHA blasts (mean Cr51 release 1% 20:1 E:T). To date we have infused 10 patients on study; 4 on DL1 (5x106/m2), 4 on DL2 (1x107/m2) and 2 on DL3 (2x107/m2), with no adverse events. Three patients were infused prophylactically (38-43 days post-HSCT), while 7 received the cells as treatment for one or more active infections at 59-139 days post-HSCT. Based on viral load measurements by day 42, the pCTLs were successful in controlling active CMV (1 complete (CR) and 1 partial response (PR)), EBV (2 CRs, including a case of frank PTLD); Adv (1 CR); BK (1 CR, 3 PR) and HHV6 (1 CR) infections. Additionally, 1 patient received cells off study as treatment for widespread rituximab-resistant EBV-PTLD. Post-infusion there was an immediate decline in her EBV viral load with PTLD resolution, coincident with an increase in circulating EBV-specific T cells. However, the profound anti-tumor activity mediated by the rapidly-expanding EBV-directed T cells also produced a transient systemic inflammatory response syndrome in this patient, which was controlled with steroids and anti-TNFR antibody, with no long term adverse effects. In summary, infusion of pCTLs has thus far proven safe and is associated with the appearance of virus-reactive T cells in peripheral blood and subsequent virus clearance.

Background It is common for diffuse large B-cell lymphoma (DLBCL) patients following autologous hematopoietic transplant (auto-HCT) to undergo surveillance imaging to monitor for lymphoma progression/relapse. Although a standard at most centers, this practice is not evidence-based, has not been shown to improve survival, and may pose a risk to patients due to radiation exposure. We studied DLBCL patients who underwent auto-HCT at three transplant centers to define how many surveillance imaging studies were obtained, how often relapses were detected by surveillance imaging versus clinically (based on signs, symptoms or laboratory findings), and whether survival was improved if relapse was detected by imaging versus clinically. Methods DLBCL patients who underwent auto-HCT during 2000-2013 were identified using clinical databases. Pre-transplant patient and disease characteristics were collected. Progression-free survival (PFS) and overall survival (OS) were determined. Patients were classified as having "radiographic" or "clinical" relapses. PFS and OS were compared for these two groups. Results A total of 209 patients with DLBCL who underwent auto-HCT between 2000 and 2013 were identified. There was insufficient follow-up information on 15 patients and hence only 194 patients were evaluable. Of these, 22 patients relapsed or progressed prior to or at their initial post-transplant disease assessment (day 100 evaluation). The remaining 172 patients were then analyzed to determine which patients relapsed and how relapses were detected. 48 patients relapsed at a median time of 287 days post-transplant. Of these patients, 14 (29%) had relapse detected clinically and 34 (71%) had relapse detected by surveillance imaging. Comparing the radiographic and clinically detected groups, median PFS post auto-HCT was 218 vs 402 days respectively (p= 0.49, log rank test, Figure 1) and median survival post auto-HCT was 643 vs 615 days respectively (p= 0.44, log rank test, Figure 1). For patients who never relapsed after auto-HCT, a median of 4 surveillance imaging studies were performed (with median follow up of 3.0 years post-transplant). Conclusions In DLBCL patients post auto-HCT, the majority of relapses were detected by surveillance imaging. However, the survival of patients with relapse detected by surveillance imaging was not superior to those whose relapse was detected clinically. Given this, combined with the radiation exposure and cost associated with surveillance imaging, the practice of surveillance imaging post auto-HCT appears to have limited utility in DLBCL. Disclosures Hari: Janssen: Consultancy; Novartis: Consultancy; Spectrum: Consultancy; Sanofi: Consultancy; Takeda: Consultancy; Celgene: Consultancy; BMS: Consultancy. Hamadani:Takeda: Research Funding; Cellerant: Consultancy; Celgene: Consultancy; MedImmune: Consultancy. Fenske:Celgene: Honoraria; Pharmacyclics: Honoraria; Seattle Genetics: Honoraria; Millennium/Takeda: Research Funding.

Abstract 4489 Dominant cardiac involvement by primary systemic amyloidosis (AL) precludes effective AL treatment and is associated with short survival (median survival for stage-III AL= 3–4 months). Three patients who presented with severe cardiac dysfunction as their major AL manifestation underwent orthotopic heart transplantation (OHT) followed by autologous hematopoietic stem cell transplantation (ASCT). The clinicopathological characteristics of these patients form the basis of this report. Age at AL presentation was 63, 62 and 44 years in 2 females and 1 male. The time from initial symptoms of AL to diagnosis was 12, 24 and 12 months. Diagnosis of cardiac AL was established via endomyocardial biopsy, Congo red staining and immunohistochemistry. Standard work-up for plasma cell dyscrasia included; serum and urine electrophoresis, immunofixation, measurement of serum-free light-chain ratio and bone marrow biopsy. All patients had stage III AL based on cardiac troponin I and NT-pro-BNP. All had end stage heart failure with New York Heart Association class 4 symptoms and developed cardiogenic shock requiring intra-aortic balloon pump support as a bridge to OHT. All were considered unsuitable for ASCT. Initial treatment was with lenalidomide, dexamethasone in two patients while the third received melphalan and dexamethasone. Table -1 shows the timeline of diagnosis and dual transplants. Table-1: . Orthotropic cardiac transplantation (OHT) followed by ASCT Age/Sex Time to AL diagnosis (months) Organ involvement (months) Time * to OHT (months) Time to ASCT s/p OHT (months) Survival (months) 63/F 12 Heart, Kidney, PN 7 13 30+ 62/F 24 Heart, Kidney, PN 5 16 26+ 44/M 12 Heart, Kidney, tongue 4 13 14+ * Time to OHT from onset of heart failure symptoms, PN= peripheral nerve, F = female, M= male, s/p= status post ASCT was performed 13, 16 and 13 months after OHT. All 3 patients were on tacrolimus and prednisone at the time of stem cell mobilization and hematopoietic transplant; two patients were also receiving mycophenolate mofetil and valganciclovir. We collected 6.1 and 6.2 × 106/kg CD-34+ cells in 2 days with filgrastim (5 ug/kg, twice, daily) and plerixafor (16 mg/kg based on day- 4 CD-34+ counts) in two subjects. The third initially failed to mobilize but 4.3×106/kg CD-34+ cells were subsequently obtained when she (patient #2) was remobilized with filgrastim and plerixafor 4 weeks after stopping mycophenolate mofetil. The creatinine clearance at the time of high-dose chemotherapy was 36, 30 and 43 ml/minute. All 3 patients received renal adjusted dose of melphalan at 140mg/m2. Mycophenolate mofetil and Valganciclovir were withheld during neutropenia until engraftment. Time to engraftment for neutrophils (ANC of >500/uL x3 days) was 13, 11 and 14 days. Platelets engraftment (untransfused >20 × 109 for 3 consecutive days) occurred in 14, 13 and 16 days. No patients received post-transplant filgrastim. Patients were hospitalized for 17, 18 and 18 days. Renal function remained stable during ASCT and non-hematological toxicity was limited to grade I-II apart from one grade III oral mucositis and colitis. One patient achieved hematologic complete remission (patient#2) while 2 patients had partial response following ASCT. Post OHT and ASCT all patients are alive and well at follow-up of 14, 26 and 30 months from OHT. OHT should be considered in select patients with dominant cardiac involvement and advanced heart failure to allow subsequent ASCT and improve survival. Disclosures: No relevant conflicts of interest to declare.

We herein document a complete response of extensive chronic graft-versus-host disease (cGvHD) to a Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib. A 41 years old female with primary refractory MCL underwent mismatched unrelated donor (MMUD) allogeneic hematopoietic stem cell transplantation in December 2011 (conditioning with CY/TBI and alemtuzumab, graft=6.6x 106/kg CD-34+ cells, tacrolimus alone for GVHD prevention). Following engraftment on day 11, she developed grade III acute GvHD involving the skin and gut on day 17 of transplantation that persisted beyond 100 days post-transplant. Her cGvHD was treated with steroids, but remained active and extensive. Despite persistent cGvHD and 100% donor chimerism she relapsed in July 2012. Treatment with radiation and bendamustine with rituximab failed. By December 2013, the patient had extensive cGvHD manifesting as scleromatous skin thickening, oral ulcers and sclerosis of the buccal mucosa, ocular dryness and diarrhea, and was started on ibrutinib1 560 mg once daily for relapsed MCL. After 8 weeks of therapy, cGvHD had begun to improve. Oral steroids were reduced and ultimately stopped after 26 weeks of ibrutinib; after 30 weeks treatment all cGvHD manifestations resolved completely. A complete remission for MCL was documented at 8 weeks of ibrutinib initiation. Currently she continues to be on 560 mg daily ibrutinib without cGvHD exacerbation or MCL relapse for 22 weeks and 52 weeks, respectively. Chronic graft versus host disease (cGvHD) is mediated donor T cells. The role of B cells in the pathogenesis of cGvHD is increasingly recognized. Two murine studies have explored the role of ibrutinib in cGVHD-like syndromes, one in which there is T cell driven sclerodermatous cGvHD and a second in which there is Ab driven multiorgan system cGvHD that includes bronchiolitis obliterans (BO). Administration of ibrutinib decreased the incidence and severity of sclerodermatous, and improved pre-existing lesions and also improved pulmonary fibrosis and reduced BO. Animals lacking BTK and ITK did not develop cGVHD, indicating that these molecules are critical to cGVHD development. Our report provides the evidence that BTK inhibition led to complete resolution of cGvHD and supports exploration of its role in future clinical trials. Disclosures No relevant conflicts of interest to declare.

Background: It is common for classical Hodgkin lymphoma (cHL) patients following auto-HCT to undergo surveillance imaging to monitor for disease progression or relapse. Although post auto-HCT surveillance imaging is employed at most centers, the literature remains unclear on how this practice affects overall survival. Furthermore, surveillance imaging results in significant amounts of radiation exposure as well as invasive procedures to work up false positive findings. We studied cHL patients who underwent auto-HCT at three transplant centers to define how many surveillance imaging studies were obtained, how often relapses were detected by surveillance imaging versus clinically (based on signs, symptoms or laboratory findings), and whether survival was improved if relapse was detected by surveillance imaging versus clinically. Methods: Classical HL patients who underwent auto-HCT between 2000 and 2013 were identified using clinical databases. Pre-transplant patient and disease characteristics were collected. Progression-free survival (PFS) and overall survival (OS) were determined. Patients were classified as having “radiographic” or “clinical” relapses. PFS and OS were compared for these two groups. Results: A total of 148 patients with cHL who underwent auto-HCT were identified. Patient characteristics are presented in Table 1. Of these 148 patients, 20 relapsed or progressed prior to or at their initial post-transplant disease assessment (day 100 evaluation). The remaining 128 patients were then analyzed to determine which patients had relapse/progression, and how relapses were detected. 31 patients relapsed post day 100 evaluation. The date of relapse for 2 patients was unknown, and these patients were excluded. For the 29 remaining patients, the median time to relapse was 389 days post-transplant. Of these patients, 14 (48%) had relapse detected clinically and 15 (52%) had relapse detected by surveillance imaging. Comparing the radiographic and clinically detected groups, median PFS post auto-HCT was 426 vs 318 days respectively (p= 0.62, log rank test) and median survival post auto-HCT was 1270 vs 931 days respectively (p= 0.71, log rank test, Figure 1). For patients who never relapsed after auto-HCT, a median of 6 surveillance imaging studies were performed (with a median follow up of 5 years post-transplant). Conclusions: In cHL patients post auto-HCT, the majority of relapses were detected by surveillance imaging. However, the survival of patients with relapse detected by surveillance imaging was not superior to those whose relapse was detected clinically. Given this, combined with the radiation exposure and cost associated with surveillance imaging, the practice of surveillance imaging post auto-HCT appears to have limited utility in cHL. Disclosures Hari: Janssen: Consultancy; Novartis: Consultancy; Spectrum: Consultancy; Sanofi: Consultancy; Takeda: Consultancy; Celgene: Consultancy; BMS: Consultancy. Hamadani:Takeda: Research Funding; Cellerant: Consultancy; MedImmune: Consultancy; Celgene: Consultancy. Fenske:Seattle Genetics: Honoraria; Celgene: Honoraria; Millennium/Takeda: Research Funding; Pharmacyclics: Honoraria.

Immunotherapy is emerging as a potent therapy for a range of hematologic malignancies. To extend this approach to individuals with Hodgkin's (HL) and non-Hodgkin's lymphoma (NHL) we developed a protocol for the generation of single T cell lines that simultaneously targeted a range of tumor associated antigens (TAAs) that are frequently expressed by these tumors, including PRAME, SSX2, MAGEA4, NY-ESO-1 and Survivin. We were consistently able to generate multiTAA-specific T cells by culturing PBMCs in the presence of a Th1-polarizing/pro-proliferative cytokine cocktail, using autologous DCs as APCs and loading them with pepmixes (15mer peptides overlapping by 11 aminos acids) spanning all 5 target antigens. The use of whole antigen increases the range of patient HLA polymorphisms that can be exploited beyond those matched to single peptides, while targeting multiple antigens simultaneously reduces the risk of tumor immune evasion. To date 28 clinical-grade multiTAA-specific T cell lines have been generated comprising CD3+ T cells (mean 94±2%) with a mixture of CD4+ (mean 46±7%) and CD8+ (mean 46±6%) T cells, which expressed central and effector memory markers (CD45RO+/CD62L+/CCR7+ -- mean 22±5%; CD45RO+/CD62L+/CCR7- -- 12±4%; CD45RO+/CD62L-/CCR7- -- 35±6%) (n=28). The expanded lines recognized the targeted antigens PRAME, SSX2, MAGEA4, NY-ESO-1 and Survivin (range 1-354, 0-496, 0-330, 0-379 and 0-304 spot forming units (SFU)/2x105 input cells, respectively in IFNg ELIspot, n=28). None of the lines reacted against non-malignant autologous recipient cells (3±3.7% specific lysis; E:T 20:1). We have treated 18 patients to date: 9 with HL, 8 with aggressive NHL (diffuse large B-cell, mantle cell, or peripheral T cell lymphomas), and 1 with a composite lymphoma. Patients have received 0.5-2x107 multiTAA-T cells/m2 without adverse events. Of 11 patients who were infused as adjuvant therapy all but 1 remain in remission (range 1-24 months post-infusion). Seven patients have received multiTAA-specific T cells to treat active disease. Of these, 1 had transient disease stabilization followed by disease progression, 3 have stable disease 3-6 months post-multiTAA-specific T cells while the remaining 3 (all with HL) have all had complete responses, as assessed by PET imaging. These clinical responses correlated with the detection of tumor-reactive T cells in patient peripheral blood post-infusion directed against both targeted antigens as well as non-targeted TAAs including MAGEA2B and MAGE C1, indicating antigen/epitope spreading. Thus, infusion of autologous multiTAA-targeted T cells directed to PRAME, SSX2, MAGEA4, NY-ESO-1 and Survivin has been safe and provided clinical benefit to patients with lymphomas. Disclosures Off Label Use: Tumor-specific T cells administered under an investigator-initiated IND. Brenner:Celgene: Other: Collaborative Research Agreement; Bluebird Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cell Medica: Other: Licensing Agreement. Heslop:Celgene: Other: Collaborative research agreement; Cell Medica: Other: Licensing Agreement. Rooney:Celgene: Other: Collaborative research agreement; Cell Medica: Other: Licensing Agreement.

Background aims. Several studies have demonstrated that the immunogenicity of chronic lymphocytic leukemia (CLL) cells can be increased by manipulation of the CD40/CD40-ligand (CD40L) pathway. Although immunologic, and perhaps clinical, benefits have been obtained with an autologous CLL tumor vaccine obtained by transgenic expression of CD40L and interleukin (IL)-2, there is little information about the optimal gene transfer strategies. Methods. We compared two different CLL vaccines prepared by adenoviral gene transfer and plasmid electroporation, analyzing their phenotype and immunostimulatory activity. Results. We found that higher expression of transgenic CD40L was mediated by adenoviral gene transfer than by plasmid transduction, and that adenoviral transfer of CD40L was associated with up-regulation of the co-stimulatory molecules CD80 and CD86 and adhesion molecule CD54. In contrast, transgenic IL-2 secretion was greater following plasmid transduction. These phenotypic differences in the vaccines were associated with different functionality, both ex vivo and following administration to patients. Thus adenoviral vaccines induced greater activation of leukemia-reactive T cells ex vivo than plasmid vaccines. In treated patients, specific T-cell (T helper 1 (Th1) and T helper 2 (Th2)) and humoral anti-leukemia responses were detected following administration of the adenoviral vaccine (n = 15), while recipients of the plasmid vaccine (n = 9) manifested only a low-level Th2 response. Progression-free survival at 2 years was 46.7% in the adenoviral vaccine recipients, versus 11.1 % in those receiving plasmid vaccine. Conclusions. CLL vaccines expressing the same transgenes but produced by distinct methods of gene transfer may differ in the polarity of the immune response they induce in patients

Targeted T cell immunotherapies using engineered T lymphocytes expressing tumor-directed chimeric antigen receptors (CARs) are designed to benefit patients with cancer. Although incorporation of costimulatory endodomains within these CARs increases the proliferation of CAR-redirected T lymphocytes, it has proven difficult to draw definitive conclusions about the specific effects of costimulatory endodomains on the expansion, persistence, and antitumor effectiveness of CAR-redirected T cells in human subjects, owing to the lack of side-by-side comparisons with T cells bearing only a single signaling domain. We therefore designed a study that allowed us to directly measure the consequences of adding a costimulatory endodomain to CAR-redirected T cells. Patients with B cell lymphomas were simultaneously infused with 2 autologous T cell products expressing CARs with the same specificity for the CD19 antigen, present on most B cell malignancies. One CAR encoded both the costimulatory CD28 and the ζ-endodomains, while the other encoded only the ζ-endodomain. CAR+ T cells containing the CD28 endodomain showed strikingly enhanced expansion and persistence compared with CAR+ T cells lacking this endodomain. These results demonstrate the superiority of CARs with dual signal domains and confirm a method of comparing CAR-modified T cells within individual patients, thereby avoiding patient-to-patient variability and accelerating the development of optimal T cell immunotherapies.

Side-population (SP) analysis identifies precursor cells in normal and malignant tissues. Cells with this phenotype have increased resistance to many cytotoxic agents, and may represent a primary drug-resistant population in malignant diseases. To discover whether drug-resistant malignant SP cells are nonetheless sensitive to immune-mediated killing, we first established the presence of a malignant CD5(+)CD19(+) SP subset in the blood of 18/21 subjects with B-cell chronic lymphocytic leukemia (B-CLL). We examined the fate of these cells in six of these individuals who received autologous human CD40 ligand and interleukin-2 (hCD40L/IL-2) gene-modified tumor cells as part of a tumor vaccine study. Vaccinated patients showed an increase in B-CLL-reactive T cells followed by a corresponding decline in circulating CD5(+)CD19(+) SP cells. T-cell lines and clones generated from vaccinated patients specifically recognized B-CLL SP tumor cells. Elimination of SP cells is likely triggered by their increased expression of target antigens, such as receptor for hyaluronan-mediated motility (RHAMM), after stimulation of the malignant cells by hCD40L, as CD8(+) RHAMM-specific T cells could be detected in the peripheral blood of immunized patients and were associated with the decline in B-CLL SP cells. Hence, malignant B cells with a primary drug-resistant phenotype can be targeted by T- cell-mediated effector activity after immunization of human subjects.

Epstein-Barr virus (EBV)–associated tumors developing in immunocompetent individuals present a challenge to immunotherapy, since they lack expression of immunodominant viral antigens. However, the tumors consistently express viral proteins including LMP2, which are immunologically “weak” but may nonetheless be targets for immune T cells. We previously showed that a majority of cytotoxic T lymphocytes (CTLs) reactivated using EBV-transformed B-lymphoblastoid cells lines (LCLs) contained minor populations of LMP2-specific T cells and homed to tumor sites. However, they did not produce remissions in patients with bulky disease. We have now used gene transfer into antigen-presenting cells (APCs) to augment the expression and immunogenicity of LMP2. These modified APCs increased the frequency of LMP2-specific CTLs by up to 100-fold compared with unmodified LCL-APCs. The LMP2-specific population expanded and persisted in vivo without adverse effects. Nine of 10 patients treated in remission of high-risk disease remain in remission, and 5 of 6 patients with active relapsed disease had a tumor response, which was complete in 4 and sustained for more than 9 months. It is therefore possible to generate immune responses to weak tumor antigens by ex vivo genetic modification of APCs and the CTLs so produced can have substantial antitumor activity. This study is registered at http://www.cancer.gov/clinicaltrials (protocol IDs: BCM-H-9936, NCT00062868, NCT00070226).

Immunosuppressive monoclonal antibodies directed to immune system cells may reduce rejection and graft versus host disease (GvHD) after allogeneic stem cell transplantation (SCT), but can increase the risks of viral infection. Here, we report human herpes virus-6 (HHV-6) encephalitis despite antiviral prophylaxis in 5 of 43 (11.6%) patients receiving alemtuzumab supported conditioning. Encephalitis occurred at 41–103 days (median 60 days) presenting with confusion in all patients, combined with amnesia (n=3) or seizures (n=2). MRI revealed non-specific white matter changes in two and a non-enhancing medial temporal lobe lesion in three patients. Cerebrospinal fluid (CSF) PCR amplification for HHV-6 was positive in all five patients, (600–2 25 000 (median 4700) copies/ml CSF), while analysis of peripheral blood revealed 100–22 500 (median 1200) viral copies/ml plasma. CSF protein was elevated in four patients, with minimal CSF pleocytosis. Intravenous foscarnet produced neurological improvement at 8–13 (median 11) days and negative plasma PCR at 30–66 (median 50) days. Four patients had complete neurological recovery, but one patient with persistent viral DNA in the CSF succumbed to progressive encephalopathy. Given this high incidence of HHV-6 and the possibility of successful outcome with prompt treatment, a high index of suspicion of this disorder is required in recipients of monoclonal antibody supported allografts.

Clinical Lymphoma & Myeloma January 2006 Introduction Cellular immune responses have significant therapeutic potential as evidenced by the graft-versus-lymphoma effect and the use of donor lymphocyte infusions after allogeneic stem cell transplantation (SCT) for Hodgkin’s disease and non-Hodgkin’s lymphoma (NHL), which has resulted in reduced relapse rates.1-7 The potential for immunotherapy in Epstein-Barr virus (EBV)–positive Hodgkin’s disease and NHL lies in the fact that EBV-encoded antigens expressed in the Hodgkin’s ReedSternberg and NHL cells are a source of unique target epitopes. To enhance the tumor-specific immune response, the aim of this study is to generate cytotoxic T lymphocytes (CTLs), which are specific for the EBV tumor-associated antigen latent membrane protein–2 (LMP-2) and adoptively transfer them to patients with EBV-positive Hodgkin’s disease or EBV NHL.

Invasive fungal infections are a complication of allogeneic BMT. We report the first case of a Neosartorya fischeri fungal infection in a patient following allogeneic BMT. Neosartorya fischeri is related to Aspergillus fumigatus, but it is a distinct fungal species. Despite granulocytic engraftment and aggressive anti-fungal therapy with amphotericin B, the patient died of overwhelming fungal infection on day 60 post-BMT. Neosartorya fischeri is a pathogen that grows slowly in culture which can delay or confuse identification. This case further supports the need for more effective prophylaxis and treatment of non-Candida fungal infections in the allogeneic BMT population.

CD40L generates immune responses in leukemia-bearing mice, an effect that is potentiated by IL-2. We studied the feasibility, safety, and immunologic efficacy of an IL-2– and CD40L-expressing recipient-derived tumor vaccine consisting of leukemic blasts admixed with skin fibroblasts transduced with adenoviral vectors encoding human IL-2 (hIL-2) and hCD40L. Ten patients (including 7 children) with high-risk acute myeloid (n = 4) or lymphoblastic (n = 6) leukemia in cytologic remission (after allogeneic stem cell transplantation [n = 9] or chemotherapy alone [n = 1]) received up to 6 subcutaneous injections of the IL-2/CD40L vaccine. None of the patients were receiving immunosuppressive drugs. No severe adverse reactions were noted. Immunization produced a 10- to 890-fold increase in the frequencies of major histocompatibility complex (MHC)–restricted T cells reactive against recipient-derived blasts. These leukemia-reactive T cells included both T-cytotoxic/T-helper 1 (Th1) and Th2 subclasses, as determined from their production of granzyme B, interferon-γ, and interleukin-5. Two patients produced systemic IgG antibodies that bound to their blasts. Eight patients remained disease free for 27 to 62 months after treatment (5-year overall survival, 90%). Thus, even in heavily treated patients, including recipients of allogeneic stem cell transplants, recipient-derived antileukemia vaccines can induce immune responses reactive against leukemic blasts. This approach may be worthy of further study, particularly in patients with a high risk of relapse.

Purpose: Human CD40 ligand activates the malignant B-cell chronic lymphocytic leukemia cells and enhances their capacity to present tumor antigens. Human interleukin-2 further potentiates the immunogenicity of human CD40 ligand in preclinical murine models. Experimental Design: We prepared autologous B-cell chronic lymphocytic leukemia cells that expressed both human CD40 ligand (>90% positive) and human interleukin-2 (median secretion, 1,822 pg/mL/106 cells; range, 174-3,604 pg). Nine patients were enrolled in a phase I trial, receiving three to eight s.c. vaccinations. Results: Vaccinations were administered without evidence of significant local or systemic toxicity. A B-cell chronic lymphocytic leukemia–specific T-cell response was detected in seven patients. The mean frequencies of IFN-γ, granzyme-B, and IL-5 spot-forming cells were 1/1,230, 1/1,450, and 1/4,500, respectively, representing a 43- to 164-fold increase over the frequency before vaccine administration. Three patients produced leukemia-specific immunoglobulins. Three patients had >50% reduction in the size of affected lymph nodes. Nonetheless, the antitumor immune responses were observed only transiently once immunization ceased. High levels of circulating CD4+/CD25+/LAG-3+/FoxP-3+ immunoregulatory T cells were present before, during and after treatment and in vitro removal of these cells increased the antileukemic T-cell reactivity. Conclusions: These results suggest that immune responses to B-cell chronic lymphocytic leukemia can be obtained with human CD40 ligand/human interleukin-2–expressing s.c. vaccines but that these responses are transient. High levels of circulating regulatory T cells are present, and it will be of interest to see if their removal in vivo augments and prolongs the antitumor immune response.

Allogeneic hemopoietic stem cell transplantation (HSCT) is the only curative option for many patients with hematological malignancies. Since many of these patients lack HLA-identical sibling donors and are older or have comorbidity, a fully ablative HSCT is not feasible and an alternative approach is required. We studied 22 consecutive patients who could not have myeloablative conditioning because of comorbidity or age - 21/22 being over the age of 50 years (median 58 years range 20-70 years). A conditioning regimen consisting of fludarabine, total body radiation 450 cGy and alemtuzumab (CD52 mAb) was used for 15 patients. A second group of seven patients received CD45 monoclonal antibodies in addition. Unrelated donor stem cells were HLA matched (15 patients - 68%) or one locus mismatched (seven patients - 32%). In all, 16 patients had high-risk disease, including 12 with active malignancy at the time of transplant. With a median follow-up of 715 (216-1470) days, nonrelapse mortality, actuarial event-free and overall survival is 27, 45 and 45%, respectively. Hence the outcome of reduced intensity HSCT with lymphodepleting antibodies in older patients with intermediate/high-risk hematological malignancies appears comparable to that obtained with fully ablative transplantation in younger patients, even when these older recipients lack HLA-identical sibling donors.

Epstein Barr virus (EBV)+ Hodgkin's disease (HD) expresses clearly identified tumor antigens derived from the virus and could, in principle, be a target for adoptive immunotherapy with viral antigen–specific T cells. However, like most tumor-associated antigens in immunocompetent hosts, these potential targets are only weakly immunogenic, consisting primarily of the latent membrane protein (LMP)1 and LMP2 antigens. Moreover, Hodgkin tumors possess a range of tumor evasion strategies. Therefore, the likely value of immunotherapy with EBV-specific cytotoxic effector cells has been questioned. We have now used a combination of gene marking, tetramer, and functional analyses to track the fate and assess the activity of EBV cytotoxic T lymphocyte (CTL) lines administered to 14 patients treated for relapsed EBV+ HD. Gene marking studies showed that infused effector cells could further expand by several logs in vivo, contribute to the memory pool (persisting up to 12 mo), and traffic to tumor sites. Tetramer and functional analyses showed that T cells reactive with the tumor-associated antigen LMP2 were present in the infused lines, expanded in peripheral blood after infusion, and also entered tumor. Viral load decreased, demonstrating the biologic activity of the infused CTLs. Clinically, EBV CTLs were well tolerated, could control type B symptoms (fever, night sweats, and weight loss), and had antitumor activity. After CTL infusion, five patients were in complete remission at up to 40 mo, two of whom had clearly measurable tumor at the time of treatment. One additional patient had a partial response, and five had stable disease. The performance and fate of these human tumor antigen–specific T cells in vivo suggests that they might be of value for the treatment of EBV+ Hodgkin lymphoma.

Multiple sclerosis is thought to involve aberrant immune responses to myelin autoantigens. Haematopoietic stem-cell transplantation (HSCT) is in clinical trials for progressive multiple sclerosis based on the rationale that it destroys aberrant immune system, while recapitulation of lymphocyte ontogeny might alter the immune system and slow down disease progression. This study was undertaken to analyse characteristics of the T-cell receptor (TCR) repertoire, serum cytokine profile and the T-cell responses to myelin basic protein (MBP) in the reconstituted immune system in progressive multiple sclerosis. The study revealed that, following autologous HSCT, the T-cell immunity recovered in two distinctive phases. The first phase was characterized by limited T-cell immunity as a result of selective expansion of pre-existing T cells commonly expressing the TCR beta chain variable region (TCR BV) 20 and increased serum cytokine production during the first several months. The second phase of T-cell reconstitution coincided with increased thymic T-cell output 9-12 months after HSCT. T cells reconstituted from stem-cell grafts had the distinctive properties of comprehensive T-cell immunity and a broad TCR repertoire. T cells recognizing MBP were initially depleted by immunoablation and rapidly expanded from the reconstituted T-cell repertoire in 12 months. The reconstituted MBP-reactive T cells exhibited a broader epitope recognition repertoire while maintaining the same skewed reactivity pattern compared with that seen at baseline. The findings have important implications in the understanding of the role of HSCT as a potential treatment for multiple sclerosis.