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Supplemental Table 1. Targets identified in protein microarray screening with post-vaccination sera from AML patient 12.

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Purpose: The graft-versus-leukemia (GVL) reaction is an important example of immune-mediated tumor destruction. A coordinated humoral and cellular response accomplishes leukemia cell killing, but the specific targets remain largely uncharacterized. To learn more about the antigens that elicit antibodies during GVL reactions, we analyzed patients with advanced myelodysplasia (MDS) and acute myelogenous leukemia (AML) who received an autologous, granulocyte-macrophage colony-stimulating factor (GM-CSF)–secreting tumor cell vaccine early after allogeneic hematopoietic stem cell transplantation (HSCT).Experimental Design: A combination of tumor-derived cDNA expression library screening, protein microarrays, and antigen-specific ELISAs were used to characterize sera obtained longitudinally from 15 patients with AML/MDS who were vaccinated early after allogeneic HSCT.Results: A broad, therapy-induced antibody response was uncovered, which primarily targeted intracellular proteins that function in growth, transcription/translation, metabolism, and homeostasis. Unexpectedly, antibodies were also elicited against eight secreted angiogenic cytokines that play critical roles in leukemogenesis. Antibodies to the angiogenic cytokines were evident early after therapy, and in some patients manifested a diversification in reactivity over time. Patients that developed antibodies to multiple angiogenic cytokines showed prolonged remission and survival.Conclusions: These results reveal a potent humoral response during GVL reactions induced with vaccination early after allogeneic HSCT and raise the possibility that antibodies, in conjunction with natural killer cells and T lymphocytes, may contribute to immune-mediated control of myeloid leukemias. Clin Cancer Res; 21(5); 1010–8. ©2014 AACR.

Purpose: Invariant NKT cells (iNKT) are innate-like CD1d-restricted T cells with immunoregulatory activity in diseases including cancer. iNKT from advanced cancer patients can have reversible defects including IFNγ production, and iNKT IFNγ production may stratify for survival. Previous clinical trials using iNKT cell activating ligand α-galactosylceramide have shown clinical responses. Therefore, a phase I clinical trial was performed of autologous in vitro expanded iNKT cells in stage IIIB–IV melanoma.Experimental Design: Residual iNKT cells [in vitro to obtain up to approximately 109 cells.Results: Expanded iNKT cells produced IFNγ, but limited or undetectable IL4 or IL10. Three iNKT infusions each were completed on 9 patients, and produced only grade 1–2 toxicities. The 4th patient onward received systemic GM-CSF with their second and third infusions. Increased numbers of iNKT cells were seen in PBMCs after some infusions, particularly when GM-CSF was also given. IFNγ responses to α-galactosylceramide were increased in PBMCs from some patients after infusions, and delayed-type hypersensitivity responses to Candida increased in 5 of 8 evaluated patients. Three patients have died, three were progression-free at 53, 60, and 65 months, three received further treatment and were alive at 61, 81, and 85 months. There was no clear correlation between outcome and immune parameters.Conclusions: Autologous in vitro expanded iNKT cells are a feasible and safe therapy, producing Th1-like responses with antitumor potential. Clin Cancer Res; 23(14); 3510–9. ©2017 AACR.

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Purpose: Invariant NKT cells (iNKT) are innate-like CD1d-restricted T cells with immunoregulatory activity in diseases including cancer. iNKT from advanced cancer patients can have reversible defects including IFNγ production, and iNKT IFNγ production may stratify for survival. Previous clinical trials using iNKT cell activating ligand α-galactosylceramide have shown clinical responses. Therefore, a phase I clinical trial was performed of autologous in vitro expanded iNKT cells in stage IIIB–IV melanoma. Experimental Design: Residual iNKT cells [ Results: Expanded iNKT cells produced IFNγ, but limited or undetectable IL4 or IL10. Three iNKT infusions each were completed on 9 patients, and produced only grade 1–2 toxicities. The 4th patient onward received systemic GM-CSF with their second and third infusions. Increased numbers of iNKT cells were seen in PBMCs after some infusions, particularly when GM-CSF was also given. IFNγ responses to α-galactosylceramide were increased in PBMCs from some patients after infusions, and delayed-type hypersensitivity responses to Candida increased in 5 of 8 evaluated patients. Three patients have died, three were progression-free at 53, 60, and 65 months, three received further treatment and were alive at 61, 81, and 85 months. There was no clear correlation between outcome and immune parameters. Conclusions: Autologous in vitro expanded iNKT cells are a feasible and safe therapy, producing Th1-like responses with antitumor potential. Clin Cancer Res; 23(14); 3510–9. ©2017 AACR.

Purpose: Alveolar soft-part sarcoma (ASPS) and clear cell sarcoma (CCS) are rare mesenchymal malignancies driven by chromosomal translocations that activate members of the microphthalmia transcription factor (MITF) family. However, in contrast to malignant melanoma, little is known about their immunogenicity. To learn more about the host response to ASPS and CCS, we conducted a phase I clinical trial of vaccination with irradiated, autologous sarcoma cells engineered by adenoviral-mediated gene transfer to secrete granulocyte–macrophage colony-stimulating factor (GM-CSF). Experimental Design: Metastatic tumors from ASPS and CCS patients were resected, processed to single-cell suspensions, transduced with a replication-defective adenoviral vector encoding GM-CSF, and irradiated. Immunizations were administered subcutaneously and intradermally weekly three times and then every other week. Results: Vaccines were successfully manufactured for 11 of the 12 enrolled patients. Eleven subjects received from three to 13 immunizations. Toxicities were restricted to grade 1–2 skin reactions at inoculation sites. Vaccination elicited local dendritic cell infiltrates and stimulated T cell–mediated delayed-type hypersensitivity reactions to irradiated, autologous tumor cells. Antibody responses to tissue-type plasminogen activator (tTPA) and angiopoietins-1/2 were detected. Tumor biopsies showed programmed death-1 (PD-1)–positive CD8+ T cells in association with PD ligand-1 (PD-L1)–expressing sarcoma cells. No tumor regressions were observed. Conclusions: Vaccination with irradiated, GM-CSF–secreting autologous sarcoma cell vaccines is feasible, safe, and biologically active. Concurrent targeting of angiogenic cytokines and antagonism of the PD-1–negative regulatory pathway might intensify immune-mediated tumor destruction. Clin Cancer Res; 21(14); 3178–86. ©2015 AACR.

Background: Vaccination with tumor cells engineered to secrete granulocyte-macrophage colony stimulating factor (GM-CSF) generates potent, specific, and long-lasting anti-tumor immunity in multiple tumor models. Here, we present analysis on the safety, feasibility, and biologic activity of an autologous GM-CSF-secreting breast cancer vaccine in the high-risk adjuvant setting. Methods: Following IRB approval and patient consent, 18 patients with stage II-III breast cancer underwent tumor procurement for vaccine development at the time of breast surgery. Patients were required to have at least 4 cm of primary tumor, or have received neoadjuvant chemotherapy with at least 2 cm of residual tumor. 11 patients had insufficient tumor following neoadjuvant chemotherapy. 7 patients had sufficient tumor harvested to produce and subsequently receive vaccine therapy. Breast cancer cells were transduced with a replication defective adenoviral vector encoding GM-CSF and irradiated. Vaccinations were started 4-12 weeks after completion of all chemotherapy, immunotherapy, and radiation therapy. Vaccines were delivered subcutaneously (sc) and intradermally (id) weekly for three weeks, then every other week for a total of 6 doses. Immune monitoring included skin biopsies of vaccine sites, measurement of leukocyte populations, and proteomic-based assessment of antibody responses. Results: Tumor cell yields ranged from 0.07-31.6 x 106 cells. Dose levels were based on cellular yield, ranging from 105- 4 x106 cells/dose for the 7 patients with sufficient cell numbers. Vaccinated patients were 32-65 years of age, and all received 6 vaccines total. Three patients developed relapse within one year of the start of vaccinations, one of whom died at 14 months. The remaining four patients remained disease-free 23-34 months from start of vaccine. Toxicities related to treatment were mild and included Grade I/II local injection-site reactions, as well as grade I/II fatigue, fever, upper respiratory symptoms, cough, and joint pain. One episode of grade 3 fatigue was observed. Increases in antibody responses (p < .05) were observed for 17 antigens in at least 4 out of 5 patients evaluated. Conclusion: For larger tumors, breast cancer cells can be harvested from a subset of patients in sufficient number for autologous vaccine production at the time of breast surgery. Autologous vaccination can induce immune responses with limited toxicity. The proteomic-based identification of antigen-specific immune responses following vaccination will be presented. Citation Format: Karen S Anderson, Beth Overmoyer, Christine Canning, Jennifer Savoie, Garrick Wallstrom, Eric P Winer, Glenn Dranoff. A Phase Ib Study of an adjuvant GM-CSF-Secreting Breast Cancer Vaccine [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-15-03.

Purpose: The graft-versus-leukemia (GVL) reaction is an important example of immune-mediated tumor destruction. A coordinated humoral and cellular response accomplishes leukemia cell killing, but the specific targets remain largely uncharacterized. To learn more about the antigens that elicit antibodies during GVL reactions, we analyzed patients with advanced myelodysplasia (MDS) and acute myelogenous leukemia (AML) who received an autologous, granulocyte-macrophage colony-stimulating factor (GM-CSF)–secreting tumor cell vaccine early after allogeneic hematopoietic stem cell transplantation (HSCT). Experimental Design: A combination of tumor-derived cDNA expression library screening, protein microarrays, and antigen-specific ELISAs were used to characterize sera obtained longitudinally from 15 patients with AML/MDS who were vaccinated early after allogeneic HSCT. Results: A broad, therapy-induced antibody response was uncovered, which primarily targeted intracellular proteins that function in growth, transcription/translation, metabolism, and homeostasis. Unexpectedly, antibodies were also elicited against eight secreted angiogenic cytokines that play critical roles in leukemogenesis. Antibodies to the angiogenic cytokines were evident early after therapy, and in some patients manifested a diversification in reactivity over time. Patients that developed antibodies to multiple angiogenic cytokines showed prolonged remission and survival. Conclusions: These results reveal a potent humoral response during GVL reactions induced with vaccination early after allogeneic HSCT and raise the possibility that antibodies, in conjunction with natural killer cells and T lymphocytes, may contribute to immune-mediated control of myeloid leukemias. Clin Cancer Res; 21(5); 1010–8. ©2014 AACR.

Background Patients with advanced hematologic malignancies remain at risk for relapse following reduced-intensity conditioning (RIC) allogeneic hematopoietic stem cell transplantation (allo-HSCT). We conducted a prospective clinical trial to test whether vaccination with whole leukemia cells early after transplantation facilitates the expansion of leukemia-reactive T cells and thereby enhances antitumor immunity. Methods We enrolled 22 patients with advanced chronic lymphocytic leukemia (CLL), 18 of whom received up to 6 vaccines initiated between days 30 and 45 after transplantation. Each vaccine consisted of irradiated autologous tumor cells admixed with GM-CSF-secreting bystander cells. Serial patient PBMC samples following transplantation were collected, and the impact of vaccination on T cell activity was evaluated. Results At a median follow-up of 2.9 (range, 1-4) years, the estimated 2-year progression-free and overall survival rates of vaccinated subjects were 82% (95% CI, 54%-94%) and 88% (95% CI, 59%-97%), respectively. Although vaccination only had a modest impact on recovering T cell numbers, CD8+ T cells from vaccinated patients consistently reacted against autologous tumor, but not alloantigen-bearing recipient cells with increased secretion of the effector cytokine IFN-γ, unlike T cells from nonvaccinated CLL patients undergoing allo-HSCT. Further analysis confirmed that 17% (range, 13%-33%) of CD8+ T cell clones isolated from 4 vaccinated patients by limiting dilution of bulk tumor-reactive T cells solely reacted against CLL-associated antigens. Conclusion Our studies suggest that autologous tumor cell vaccination is an effective strategy to advance long-term leukemia control following allo-HSCT. Trial registration Clinicaltrials.gov NCT00442130. Funding NCI (5R21CA115043-2), NHLBI (5R01HL103532-03), and Leukemia and Lymphoma Society Translational Research Program.

Purpose: The target antigens of graft-versus-leukemia that are tumor associated are incompletely characterized. Experimental Design: We examined responses developing against CML66, an immunogenic antigen preferentially expressed in myeloid progenitor cells identified from a patient with chronic myelogenous leukemia who attained long-lived remission following CD4+ donor lymphocyte infusion (DLI). Results: From this patient, CML66-reactive CD8+ T-cell clones were detected against an endogenously presented HLA-B*4403–restricted epitope (HDVDALLW). Neither CML66-specific antibody nor T-cell responses were detectable in peripheral blood before DLI. However, by 1 month after DLI, CD8+ T cells were present in peripheral blood and at 10-fold higher frequency in marrow. Subsequently, plasma antibody to CML66 developed in association with disease remission. Donor-derived CML66-reactive T cells were detected at low levels in vivo in marrow before DLI by ELISpot and by a nested PCR-based assay to detect clonotypic T-cell receptor sequences but not in blood of the patient pre-DLI nor of the graft donor. Conclusions: CD4+ DLI results in rapid expansion of preexisting marrow-resident leukemia-specific donor CD8+ T cells, followed by a cascade of antigen-specific immune responses detectable in blood. Our single-antigen analysis thus shows that durable posttransplant tumor immunity is directed in part against nonpolymorphic overexpressed leukemia antigens that elicit coordinated cellular and humoral immunity. Clin Cancer Res; 16(10); 2729–39. ©2010 AACR.

Patients with chronic lymphocytic leukemia (CLL) who relapse after allogeneic transplant may achieve durable remission following donor lymphocyte infusion (DLI), showing the potency of donor-derived immunity in eradicating tumors. We sought to elucidate the antigenic basis of the effective graft-versus-leukemia (GvL) responses associated with DLI for the treatment of CLL by analyzing the specificity of plasma antibody responses developing in two DLI-treated patients who achieved long-term remission without graft-versus-host disease. By probing high-density protein microarrays with patient plasma, we discovered 35 predominantly intracellular antigens that elicited high-titer antibody reactivity greater in post-DLI than in pre-DLI plasma. Three antigens—C6orf130, MDS032, and ZFYVE19—were identified by both patients. Along with additional candidate antigens DAPK3, SERBP1, and OGFOD1, these proteins showed higher transcript and protein expression in B cells and CLL cells compared with normal peripheral blood mononuclear cells. DAPK3 and the shared antigens do not represent minor histocompatibility antigens, as their sequences are identical in both donor and tumor. Although ZFYVE19, DAPK3, and OGFOD1 elicited minimal antibody reactivity in 12 normal subjects and 12 chemotherapy-treated CLL patients, 5 of 12 CLL patients with clinical GvL responses were serologically reactive to these antigens. Moreover, antibody reactivity against these antigens was temporally correlated with clinical disease regression. These B-cell antigens represent promising biomarkers of effective anti-CLL immunity. Cancer Res; 70(4); 1344–55

Through an immune-mediated graft-versus-leukemia effect, allogeneic hematopoietic stem cell transplantation (HSCT) affords durable clinical benefits for many patients with hematologic malignancies. Nonetheless, subjects with high-risk acute myeloid leukemia or advanced myelodysplasia often relapse, underscoring the need to intensify tumor immunity within this cohort. In preclinical models, allogeneic HSCT followed by vaccination with irradiated tumor cells engineered to secrete GM-CSF generates a potent antitumor effect without exacerbating the toxicities of graft-versus-host disease (GVHD). To test whether this strategy might be similarly active in humans, we conducted a Phase I clinical trial in which high-risk acute myeloid leukemia or myelodysplasia patients were immunized with irradiated, autologous, GM-CSF-secreting tumor cells early after allogeneic, nonmyeloablative HSCT. Despite the administration of a calcineurin inhibitor as prophylaxis against GVHD, vaccination elicited local and systemic reactions that were qualitatively similar to those previously observed in nontransplanted, immunized solid-tumor patients. While the frequencies of acute and chronic GVHD were not increased, 9 of 10 subjects who completed vaccination achieved durable complete remissions, with a median follow-up of 26 months (range 12–43 months). Six long-term responders showed marked decreases in the levels of soluble NKG2D ligands, and 3 demonstrated normalization of cytotoxic lymphocyte NKG2D expression as a function of treatment. Together, these results establish the safety and immunogenicity of irradiated, autologous, GM-CSF-secreting leukemia cell vaccines early after allogeneic HSCT, and raise the possibility that this combinatorial immunotherapy might potentiate graft-versus-leukemia in patients.

CD4(+)CD25(+)FOXP3(+) regulatory T cells (Treg) successfully control graft-versus-host-disease (GVHD) in animal models. In humans, incomplete reconstitution of Treg after allogeneic hematopoietic stem cell transplantation (HSCT) has been associated with chronic GVHD (cGVHD). Recent studies have demonstrated that interleukin (IL)-2 infusions expand Treg in vivo. However, the effectiveness of this therapy depends on the number of cells capable of responding to IL-2. We examined the effect of low-dose IL-2 infusions on Treg populations after HSCT in patients who also received infusions of donor CD4(+) lymphocytes. Utilizing FOXP3 as a Treg marker, we found that patients who received CD4+DLI concomitantly with IL-2 had greater expansion of Treg compared to patients who received IL-2 (P = .03) or CD4(+)DLI alone (P = .001). FOXP3 expression correlated with absolute CD4(+)CD25(+) cell counts. Moreover, expanded CD4(+)CD25(+) T cells displayed normal suppressive function and treatment with CD4(+)DLI and IL-2 was not associated with GVHD. This study suggests that administration of low-dose IL-2 combined with adoptive CD4(+) cellular therapy may provide a mechanism to expand Treg in vivo.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) functions as a negative regulator of endogenous and vaccine-induced antitumor immunity. The administration of fully human anti-CTLA-4 blocking monoclonal antibodies to advanced-cancer patients increases immune-mediated tumor destruction in some subjects. Nonetheless, patients that respond also frequently manifest serious inflammatory pathologies, raising the possibility that the therapeutic and toxic effects of CTLA-4 blockade might be linked. Here we show that periodic infusions of anti-CTLA-4 antibodies after vaccination with irradiated, autologous tumor cells engineered to secrete GM-CSF (GVAX) generate clinically meaningful antitumor immunity without grade 3 or 4 toxicity in a majority of metastatic melanoma patients. The application of this sequential immunotherapy to advanced ovarian carcinoma patients also revealed that tumor destruction and severe inflammatory pathology could be dissociated, although further refinements are required to increase clinical responses and to minimize toxicity in this population. The extent of therapy-induced tumor necrosis was linearly related to the natural logarithm of the ratio of intratumoral CD8 + effector T cells to FoxP3 + regulatory T cells (Tregs) in posttreatment biopsies. Together, these findings help clarify the immunologic and clinical effects of CTLA-4 antibody blockade in previously vaccinated patients and raise the possibility that selective targeting of antitumor Tregs may constitute a complementary strategy for combination therapy.

Donor lymphocyte infusions (DLIs) induce effective graft-versus-tumor responses in patients with multiple myeloma who relapse after allogeneic hematopoietic stem-cell transplantation. The graft-versus-myeloma response is presumably mediated primarily by donor T cells, but recent studies have also demonstrated the presence of antibodies specific for a variety of myeloma-associated antigens in patients who achieve complete remission after DLI. One of the B-cell antigens identified in these studies was B-cell maturation antigen (BCMA), a transmembrane receptor of the tumor necrosis factor (TNF) superfamily that is selectively expressed by mature B cells. The present studies were undertaken to characterize the functional significance of antibodies to BCMA in vivo. Using transfected cells expressing BCMA, antibodies in patient serum were found to react with the cell-surface domain of BCMA. Post-DLI patient serum was able to induce complement-mediated lysis and antibody-dependent cellular cytotoxicity (ADCC) of transfected cells and primary myeloma cells expressing BCMA. BCMA antibodies were only found in post-DLI responders and not in other allogeneic transplant patients or healthy donors. These results demonstrate that BCMA is a target of donor B-cell immunity in patients with myeloma who respond to DLI. Antibody responses to cell-surface BCMA may contribute directly to tumor rejection in vivo.

A CD8 murine monoclonal antibody-coated high-density microparticle (HDM) has been developed, which allows for the rapid depletion of CD8+ T cells from apheresis products by gravity sedimentation. We conducted a study to determine the efficacy and safety of CD8 depletion of donor lymphocyte infusions (DLI) to treat relapse after stem cell transplantation using the Eligix CD8-HDM Cell Separation System. Patients were targeted to receive 3 x 10(7) CD4+ T cells/kg. Nine patients were enrolled, three with CML, three myeloma, two CLL, and one NHL. A median of 1 x 10(10) mononuclear cells were obtained by apheresis and processed. The median depletion of CD8+ cells was 99.3% (97.8->99.5%). CD8 depletion was highly specific, with a median recovery of CD4+ cells of 75%. A median of 2.9 x 10(7) CD4+ cells/kg was infused. No infusional toxicity was noted. All CML patients achieved a complete molecular remission. A CLL patient demonstrated a complete response. One patient developed GVHD (grade II acute GVHD and subsequently chronic GVHD). The CD8-HDM Cell Separation System appears to be highly selective and effective in depleting CD8+ T cells from DLI apheresis products, and CD8-depleted DLI is capable of mediating a graft-versus-leukemia effect while minimizing GVHD.

A total of 228 patients with multiple myeloma (MM), 166 patients receiving autologous transplantation (124 PBSC and 38 BM) and 66 patients receiving T-cell-depleted allogeneic transplantation were analyzed to compare overall survival (OS), progression-free survival (PFS) and risk of relapse. Patients receiving autologous transplantation had a significantly improved OS (P=0.006) and PFS (P=0.002) at 2 years with OS and PFS for autologous transplant 74% and 48%, respectively, compared with 51% and 28% for allogeneic transplantation. By 4 years after transplantation, outcome was similar with OS and PFS for autologous transplantation 41% and 23%, respectively, compared with 39% and 18% for allogeneic transplantation. The 4-year cumulative incidence of nonrelapse mortality was significantly higher in patients receiving allogeneic transplantation (24% vs 13%) (P=0.004). Relapse was the principle cause of treatment failure for both groups; however, there was a significantly reduced risk of relapse associated with allogeneic transplantation at 4 years: 46% for allograft vs 56% for autograft (P=0.02). Despite a lower risk of relapse after allogeneic transplantation, autologous transplantation is associated with improved OS and PFS compared with allogeneic transplantation in patients with MM. Strategies focused on reducing nonrelapse mortality in allogeneic transplantation may translate into an improved outcome for patients receiving allogeneic transplantation.

Rituximab has significant activity as a single agent in the treatment of follicular non-Hodgkin's lymphoma (NHL). Interleukin 2 (IL-2) is a lymphokine that increases effector cell number. In an effort to augment antibody-dependent cell-mediated cytotoxicity (ADCC) associated with rituximab therapy, low-dose IL-2 was added to a standard rituximab regimen and patients were evaluated for safety and efficacy. Twenty patients with relapsed or refractory follicular NHL were treated with IL-2 (1.2 MIU/m(2)/d for 56 d subcutaneously) as outpatients. Rituximab (375 mg/m(2)) was given on d 15, 22, 29 and 36. The regimen was well tolerated and only three patients required dose adjustments in IL-2. Infusional toxicity associated with rituximab was not exacerbated by IL-2. Peripheral blood immunophenotyping demonstrated significant increases in circulating CD8+ and CD56+ lymphocytes in all evaluable patients (P = 0.0002). Increases in total eosinophil number were observed in all patients. Eleven patients responded to therapy, for an overall response rate of 55%. Four additional patients had stable disease. For these 15 patients, the median time to progression exceeded 13 months. We conclude concomitant cytokine therapy to enhance ADCC with monoclonal antibody therapy was well tolerated and did not exacerbate antibody-related infusional toxicity. Further studies of this rational combination are warranted and ongoing.

Previous trials of allogeneic bone marrow transplantation (BMT) in patients with multiple myeloma (MM) have demonstrated high response rates but also high transplantation-related mortality (TRM) and high relapse rates. Exploitation of this strategy remains of interest because donor lymphocyte infusions (DLIs) can induce a potent graft-versus-myeloma (GVM) effect. CD6 T-cell--depleted allogeneic BMT was combined with prophylactic CD4(+) DLI administered 6 to 9 months after BMT in an effort to reduce TRM and to induce a GVM response after BMT. Twenty-four patients with matched sibling donors and chemotherapy-sensitive disease underwent BMT. CD6 T-cell depletion of donor bone marrow was the sole method of graft-versus-host disease (GVHD) prophylaxis. GVHD after BMT was minimal, 1 (4%) grade III and 4 (17%) grade II GVHD. Fourteen patients received DLI, 3 in complete response and 11 with persistent disease after BMT. Significant GVM responses were noted after DLI in 10 patients with persistent disease, resulting in 6 complete responses and 4 partial responses. After DLI, 50% of patients developed acute (or = II) or extensive chronic GVHD. Two-year estimated overall survival and current progression-free survival (PFS) for all 24 patients is 55% and 42%, respectively. The 14 patients receiving DLI had an improved 2-year current PFS (65%) when compared with a historical cohort of MM patients who underwent CD6-depleted BMT survived 6 months with no GVHD and did not receive DLI (41%) (P =.13). Although this study suggests that prophylactic DLI induces significant GVM responses after allogeneic BMT, only 58% of patients were able to receive DLI despite T-cell--depleted BMT. Therefore, less toxic transplantation strategies are needed to allow a higher proportion of patients to receive DLI and the benefit from the GVM effect after transplantation. (Blood. 2001;98:934-939)

This report describes a tumor-associated antigen, termed CML66, initially cloned from a chronic myelogenous leukemia (CML) cDNA expression library. CML66 encodes a 583-aa protein with a molecular mass of 66 kDa and no significant homology to other known genes. CML66 gene is localized to human chromosome 8q23, but the function of this gene is unknown. CML66 is expressed in leukemias and a variety of solid tumor cell lines. When examined by Northern blot, expression in normal tissues was restricted to testis and heart, and no expression was found in hematopoietic tissues. When examined by quantitative reverse transcription–PCR, expression in CML cells was 1.5-fold higher than in normal peripheral blood mononuclear cells. The presence of CML66-specific antibody in patient serum was confirmed by Western blot and the development of high titer IgG antibody specific for CML66 correlated with immune induced remission of CML in a patient who received infusion of normal donor lymphocytes for treatment of relapse. CML66 antibody also was found in sera from 18–38% of patients with lung cancer, melanoma, and prostate cancer. These findings suggest that CML66 may be immunogenic in a wide variety of malignancies and may be a target for antigen-specific immunotherapy.

Background. In patients with chronic myelocytic leukemia (CML), the breakpoint cluster region and fusion between the BCR and the c-ABL genes (BCR-ABL) oncogen product is a potential tumor-specific antigen. Previous studies have shown that T cells specific for the junctional region peptides of the BCR-ABL oncoprotein can be detected in healthy individuals as well as in patients with CML in chronic phase. We assessed whether BCR-ABL- specific T cells could be found in a patient achieving a complete cytogenetic remission after CD4+ donor lymphocyte infusion. Methods. Using dendritic cells pulsed with BCR-ABL breakpoint peptides as antigen-presenting cells, we stimulated patient peripheral blood lymphocytes to isolate peptide-specific T cell clones present at the time of the cytogenetic response. T cell clones were isolated and the cellular specificity of these cells was examined. Results. A CD3+ CD4+ T cell clone (1F7) that recognizes overlapping p210 junctional peptides presented by HLA-DR molecules was identified and expanded in vitro. Clone 1F7 failed to recognize autologous tumor cells as well as dendritic cells derived from patient CML cells. Clone 1F7 did not inhibit the growth and differentiation of CML precursor cells in a standard colony formation assay. Finally, using a clone-specific probe, 1F7 cells could not be detected in patient peripheral blood at the time of the donor lymphocyte infusion response. Conclusions. These results suggest that clone 1F7 was selected in vitro using highly potent peptide pulsed dendritic cells but was not representative of the anti-leukemia immune response in vivo. Based on these findings, CD4+ T cells with BCR-ABL specificity do not appear to be mediators of the anti-leukemia response in vivo after donor lymphocyte infusion.

Donor lymphocyte infusions (DLI) can induce remissions in patients who have relapsed after allogeneic bone marrow transplantation (BMT). However, DLI frequently also result in significant acute and/or chronic graft-versus-host disease (GVHD). Several clinical and experimental lines of evidence have suggested that CD8+ T cells play a critical role in the pathogenesis of GVHD. To develop methods to reduce the incidence of GVHD associated with DLI, we administered defined numbers of CD4+ donor T cells after ex vivo depletion of CD8+ lymphocytes to 40 patients with relapsed hematologic malignancies after allogeneic BMT. Cohorts of patients received 0.3, 1.0, or 1.5 × 108CD4+ cells/kg. Overall, 12 of 38 patients (32%) evaluable for toxicity developed acute or chronic GVHD. However, 6 of 27 patients (22%) receiving 0.3 × 108 CD4 cells/kg developed GVHD compared with 6 of 11 patients (55%) who received ≥1.0 × 108 CD4 cells/kg (P = .07). Treatment-related mortality was low (3%), with 1 death related to infection in the setting of immunosuppression for GVHD. Disease responses after CD4+ DLI were documented in 15 of 19 patients (79%) with early-phase chronic myelogenous leukemia (CML) relapse, 5 of 6 patients (83%) with relapsed multiple myeloma, and 1 patient with myelodysplasia. For patients with early-phase CML relapse, the Kaplan-Meier probability of achieving complete cytogenetic remission was 87% and the probability of complete molecular response was 78% at 1 year after DLI. The median time to complete cytogenetic response and molecular response in patients with CML was 13 weeks (range, 9 to 30 weeks) and 34 weeks (range, 10 to 56 weeks), respectively. The median time to response in patients with multiple myeloma was 26 weeks (range, 15 to 62 weeks). All patients in this trial who developed GVHD demonstrated tumor regression, but the presence of GVHD was not required for patients to achieve a response, because 48% of responding patients never developed evidence of GVHD. Two patients with CML who did not respond at dose level 1 subsequently achieved complete cytogenetic remission after a second infusion of CD8-depleted cells at dose level 2. In patients with evidence of mixed hematopoietic chimerism who achieved a complete remission after DLI, cytogenetic analysis of marrow cells also demonstrated conversion to complete donor hematopoiesis in all evaluable patients. These studies suggest that relatively low numbers of CD8-depleted donor lymphocytes are effective in inducing complete remissions in patients with stable-phase CML and multiple myeloma who have relapsed after allogeneic BMT. Because of the relatively low risk of toxicity associated with the infusion of defined numbers of CD4+donor cells, further studies can be undertaken in the setting of persistent minimal residual disease to prevent relapse after allogeneic BMT.

Several lines of evidence indicate that a subset of murine intestinal intraepithelial lymphocytes (iIEL), particularly those which express the CD8 alpha alpha homodimer, mature extrathymically. This study confirms that a small fraction of adult human iIEL also express the CD8 alpha alpha homodimer and demonstrates that most of these cells in the small intestine are T cells using the alpha beta T-cell receptor (TCR). Whether these cells or other subsets of adult human iIEL mature extrathymically in the intestine was assessed by measuring the expression of terminal deoxynucleotidyltransferase (TdT), an enzyme expressed exclusively by immature lymphocytes. Very low levels of TdT message could be detected by polymerase chain reaction (PCR) amplification in some iIEL samples. The level of TdT expression was assayed by competitive PCR amplification and compared with thymocytes and peripheral blood lymphocytes. These measurements indicated that the number of immature T cells expressing TdT in the intestinal epithelium was less than one cell per 10(7) lymphocytes. This demonstrates that there are few if any TdT expressing immature T cells in the adult human intestinal mucosa and indicates, therefore, that T-cell development in the intestinal mucosa does not contribute significantly to the T-cell repertoire of the adult human intestine.

BcR-aBL + K562 cells hold clinical promise as a component of cancer vaccines, either as bystander cells genetically modified to express immunostimulatory molecules, or as a source of leukemia antigens. To develop a method for detecting T-cell reactivity against K562 cell-derived antigens in patients, we exploited the dendritic cell (D c)-mediated cross-presentation of proteins generated from apoptotic cells. We used UVB irradiation to consistently induce apoptosis of K562 cells, which were then fed to autologous D cs. These D cs were used to both stimulate and detect antigen-specific cD8 + T-cell reactivity. a s proof-of-concept, we used cross-presented apoptotic influenza matrix protein-expressing K562 cells to elicit reactivity from matrix protein-reactive T cells. Likewise, we used this assay to detect increased anticML antigen T-cell reactivity in c ML patients that attained long-lasting clinical remissions following immunotherapy (donor lymphocyte infusion), as well as in 2 of 3 c ML patients vaccinated with lethally irradiated K562 cells that were modified to secrete high levels of granulocyte macrophage colony-stimulating factor (GM- csF). This methodology can be readily adapted to examine the effects of other whole tumor cell-based vaccines, a scenario in which the precise tumor antigens that stimulate immune responses are unknown.

The most important problem in the therapy of patients with acute myeloid leukemia (AML) is relapse after intensive therapy. We sought to determine if interleukin-2 (low-dose with intermittent boluses) administration could be feasibly administered after standard therapy to potentiate anti-tumor immunity in a fashion analogous to the post-allogeneic stem cell transplant "graft-vs-leukemic" effect. Adults with de novo AML received daunorubicin and cytosine arabinoside induction therapy. Patients achieving complete remission received high dose ara-C (HIDAC) for three courses followed by low dose rIL-2 (Amgen), administered by continuous infusion (450,000 U/m(2)/day) for 10 weeks with intermittent boluses (500,000/U/m(2) over 2 hr) given in weekly intervals starting on Week 4. Of the 32 enrolled patients, 27 achieved CR; 8/11 who received rIL-2 completed therapy. 6/11 are long term survivors (median follow-up, 139 months). rIL-2 was well tolerated and associated with a 5-fold increase in circulating NK-lymphocytes and a 3-fold increase in circulating T-cells. Mononuclear cells from patients receiving rIL-2 exhibited enhanced cytolytic activity in vitro against cryopreserved autologous leukemia cells. This study supports further investigation of immunotherapy in the post-intensive chemotherapy setting in the management of patients with AML.

IL-2 plays a critical role in the maintenance of CD4+CD25+ FOXP3+ regulatory T cells (Tregs) in vivo. We examined the effects of IL-2 signaling in human Tregs. In vitro, IL-2 selectively up-regulated the expression of FOXP3 in purified CD4+CD25+ T cells but not in CD4+CD25- cells. This regulation involved the binding of STAT3 and STAT5 proteins to a highly conserved STAT-binding site located in the first intron of the FOXP3 gene. We also examined the effects of low-dose IL-2 treatment in 12 patients with metastatic cancer and 9 patients with chronic myelogenous leukemia after allogeneic hematopoietic stem cell transplantation. Overall, IL-2 treatment resulted in a 1.9 median fold increase in the frequency of CD4+CD25+ cells in peripheral blood as well as a 9.7 median fold increase in FOXP3 expression in CD3+ T cells. CD56+CD3- natural killer (NK) cells also expanded during IL-2 therapy but did not express FOXP3. In vitro treatment of NK cells with 5-aza-2′-deoxycytidine restored the IL-2 signaling pathway leading to FOXP3 expression, suggesting that this gene was constitutively repressed by DNA methylation in these cells. Our findings support the clinical evaluation of low-dose IL-2 to selectively modulate CD4+CD25+ Tregs and increase expression of FOXP3 in vivo.

The application of DLI is limited by the potential development of GVHD. Results of single-arm trials suggest that CD8+ depletion of DLI may reduce the incidence of GVHD while still inducing pathologic and cytogenetic remissions. To test the impact of CD8 depletion on GVHD, we initiated a randomized trial comparing outcome among patients receiving unselected donor lymphocytes or CD8+-depleted cells. DLI was administered to patients with disease in remission to prevent relapse 6 months after T-cell-depleted allogeneic BMT. CD8 depletion was performed with monoclonal antibody and rabbit complement. Donor lymphocytes obtained from the original donor were infused fresh without cryopreservation. Infusions were adjusted so that all patients received 1.0 x 107 CD4+ cells/kg. Patients randomized to CD8 depletion received a median of 0.7 x 105 versus 32.0 x 105 CD8+ cells/kg in the unmanipulated cohort. Six (67%) of 9 patients receiving unselected DLI developed acute GVHD compared with 0 (0%) of 9 recipients of CD8-depleted DLI (P = .009). In the unselected group, 2 patients died while the disease was in remission, and 3 patients had relapses. In the CD8-depleted cohort, there were no toxic deaths and only 1 relapse. Measures of immunologic reconstitution by T-cell receptor excision circle analysis and T-cell receptor spectratyping demonstrated similar patterns of T-cell recovery in both the CD8-depleted and the unselected cohorts. Both groups converted from mixed to full donor hematopoietic chimerism after DLI. Our results indicate that CD8 depletion reduces the incidence of GVHD associated with DLI without adversely affecting conversion to donor hematopoiesis or immunologic recovery. Biol Blood Marrow Transplant 2002;8(11):625-32.

Donor lymphocyte infusions (DLIs) provide effectivetherapy for patients with relapsed chronic myeloid leukemia after allogeneic bone marrow transplantation. Previous studies have suggested that depletion of CD8+ T cells from the infused donor lymphocytes can reduce the incidence of graft-versus-host disease associated with DLI without reducing antileukemia activity. In this situation however, the immune effector cells responsible for tumor rejection have not been identified. The goal of this study was to characterize these effector populations.We studied three representative patients with relapsed chronic myeloid leukemia who achieved complete molecular remission after receiving CD8+ T-cell-depleted DLI from HLA-identical sibling donors. Effector T cells were characterized in patient samples after in vitro stimulation and functional assessment. T-cell clones relevant to the immune response were then isolated and further characterized.Analysis of peripheral blood samples collected after DLI indicated the presence of a high frequency of circulating host-reactive cytolytic CD8+ T cells secreting IFN-gamma. These HLA class I-restricted CTLs were specific for recipient minor histocompatibility antigens (mHAs) because they did not recognize target cells of donor origin. One CTL clone was further expanded in vitro and shown to recognize a broadly expressed mHA presented by HLA-B5701. Using a molecular approach, we demonstrated that this clone was expanded in peripheral blood and marrow after DLI. It was not detected before DLI.These results indicate that CD4+ DLI elicits a potent allogeneic response mediated by mHA-specific CD8+ T cells.

Rituximab has significant activity as a single agent in the treatment of follicular non-Hodgkin's lymphoma (NHL). Interleukin 2 (IL-2) is a lymphokine that increases effector cell number. In an effort to augment antibody-dependent cell-mediated cytotoxicity (ADCC) associated with rituximab therapy, low-dose IL-2 was added to a standard rituximab regimen and patients were evaluated for safety and efficacy. Twenty patients with relapsed or refractory follicular NHL were treated with IL-2 (1.2 MIU/m(2)/d for 56 d subcutaneously) as outpatients. Rituximab (375 mg/m(2)) was given on d 15, 22, 29 and 36. The regimen was well tolerated and only three patients required dose adjustments in IL-2. Infusional toxicity associated with rituximab was not exacerbated by IL-2. Peripheral blood immunophenotyping demonstrated significant increases in circulating CD8+ and CD56+ lymphocytes in all evaluable patients (P = 0.0002). Increases in total eosinophil number were observed in all patients. Eleven patients responded to therapy, for an overall response rate of 55%. Four additional patients had stable disease. For these 15 patients, the median time to progression exceeded 13 months. We conclude concomitant cytokine therapy to enhance ADCC with monoclonal antibody therapy was well tolerated and did not exacerbate antibody-related infusional toxicity. Further studies of this rational combination are warranted and ongoing.

Reconstitution of T-cell immunity after bone marrow transplantation (BMT) is often delayed, resulting in a prolonged period of immunodeficiency. Donor lymphocyte infusion (DLI) has been used to enhance graft-versus-leukemia activity after BMT, but the effects of DLI on immune reconstitution have not been established. We studied 9 patients with multiple myeloma who received myeloablative therapy and T-cell–depleted allogeneic BMT followed 6 months later by infusion of lymphocytes from the same donor. DLI consisted of 3 × 107 CD4+ donor T cells per kilogram obtained after in vitro depletion of CD8+ cells. Cell surface phenotype of peripheral lymphocytes, T-cell receptor (TCR) Vβ repertoire, TCR rearrangement excision circles (TRECs), and hematopoietic chimerism were studied in the first 6 months after BMT and for 1 year after DLI. These studies were also performed in 7 patients who received similar myeloablative therapy and BMT but without DLI. Phenotypic reconstitution of T and natural killer cells was similar in both groups, but patients who received CD4+ DLI developed increased numbers of CD20+ B cells. TCR Vβ repertoire complexity was decreased at 3 and 6 months after BMT but improved more rapidly in patients who received DLI (P = .01). CD4+ DLI was also associated with increased numbers of TRECs in CD3+ T cells (P

Following myeloablative therapy, it is unknown to what extent age-dependent thymic involution limits the generation of new T cells with a diverse repertoire. Normal T-cell receptor gene rearrangement in T-cell progenitors results in the generation of T-cell receptor rearrangement excision circles (TRECs). In this study, a quantitative assay for TRECs was used to measure T-cell neogenesis in adult patients with leukemia who received myeloablative therapy followed by transplantation of allogeneic hematopoietic stem cells. Although phenotypically mature T cells had recovered by 1 to 2 months after bone marrow transplantation (BMT), TREC levels remained low for 3 months after BMT. T-cell neogenesis became evident by 6 months, and normal levels of adult thymic function were restored at 6 to 12 months after BMT. Subsequent leukemia relapse in some patients was associated with reduced TREC levels, but infusion of mature donor CD4(+) T cells resulted in rapid restoration of thymic function. These studies demonstrate that T-cell neogenesis contributes to immune reconstitution in adult patients and suggest that thymic function can be manipulated in vivo. (Blood. 2001;98:1116-1121)

The effectiveness of donor-lymphocyte infusion (DLI) for treatment of relapsed chronic myelogenous leukemia (CML) after allogeneic bone marrow transplantation is a clear demonstration of the graft-versus-leukemia (GVL) effect. T cells are critical mediators of GVL, but the antigenic targets of this response are unknown. To determine whether patients who respond to DLI also develop B-cell immunity to CML-associated antigens, we analyzed sera from three patients with relapsed CML who achieved a complete molecular remission after infusion of donor T cells. Sera from these individuals recognized 13 distinct gene products represented in a CML-derived cDNA library. Two proteins, Jkappa-recombination signal-binding protein (RBP-Jkappa) and related adhesion focal tyrosine kinase (RAFTK), were recognized by sera from three of 19 DLI responders. None of these antigens were recognized by sera from healthy donors or patients with chronic graft-versus-host disease. Four gene products were recognized by sera from CML patients treated with hydroxyurea and nine were detected by sera from CML patients who responded to IFN-alpha. Antibody titers specific for RAFTK, but not for RBP-Jkappa, were found to be temporally associated with the response to DLI. These results demonstrate that patients who respond to DLI generate potent antibody responses to CML-associated antigens, suggesting the development of coordinated T- and B-cell immunity. The characterization of B cell-defined antigens may help identify clinically relevant targets of the GVL response in vivo.

Donor lymphocyte infusions (DLIs) have been demonstrated to induce clinical responses in patients with relapsed multiple myeloma after allogeneic bone marrow transplantation, but the immunologic mechanisms involved have not been well characterized. In patients with chronic myelocytic leukemia (CML), remissions following DLI are invariably associated with conversion to complete donor hematopoiesis, suggesting that the target antigens of this response are expressed on both normal and CML-derived hematopoietic stem cells. In the present study, we examined hematopoietic chimerism and the complexity of the T-cell receptor (TCR) repertoire in 4 patients with relapsed multiple myeloma who received infusions of donor CD4+ lymphocytes. Three of 4 patients had a clinical response that began 1 to 2 months after DLI. All 3 responding patients developed lymphocytosis at the initiation of response that was due to a 2- to 4.5-fold increase in the number of CD3+ T cells. In 1 patient, this was due primarily to increases in CD3+ and CD8+ cells; in 2 patients, to increased numbers of CD3+ and CD8+ and CD3+ and CD4+ T cells. In all responding patients, conversion to complete donor hematopoiesis occurred in the first 2 months after DLI. The single nonresponding patient remained it 100% recipient hematopoiesis. The TCR repertoire complexity was examined by polymerase chain reaction amplification of complementary-determining region 3 (CDR3) derived from 24 Vbeta gene subfamilies. In 2 patients, the initiation of myeloma response and conversion to complete donor hematopoiesis was associated with normalization of TCR complexity. Complete donor chimerism and normal TCR complexity remained stable in all patients and did not change with subsequent relapse or development of graft-versus-host disease (GVHD). Thus, conversion to full donor chimerism was temporally associated with the antimyeloma effect of DLI but not with the development of GVHD. Nevertheless, the maintenance of stable donor hematopoiesis did not prevent disease relapse and was not associated with prolonged remission. The selective relapse of myeloma cells without concomitant return of mixed hematopoietic chimerism suggests that myeloma tumor cells in some patients develop resistance to immune destruction.Biol Blood Marrow Transplant 2000;6(4):375-86.

Recent reports of clinical responses following donor lymphocyte infusions (DLI) in patients with relapsed multiple myeloma (MM) after allogeneic BMT have demonstrated the ability of allogeneic cells to mediate a graft-versus-myeloma (GVM) effect, but the mechanisms involved have not been determined. To identify changes in the T cell compartment associated with DLI, we performed a molecular analysis of the T cell receptor (TCR) repertoire in four patients with relapsed MM who received infusions of CD4+ lymphocytes from HLA-identical sibling donors. Three of the four patients demonstrated a clinical anti-myeloma response following DLI but also developed graft-versus-host disease (GVHD). The TCR repertoire was examined after PCR amplification of 24 Vbeta gene subfamilies. This method determines the relative utilization of each Vbeta gene subfamily and also allows the identification of clonal and oligoclonal T cell populations through analysis of CDR3 regions for each TCR Vbeta gene subfamily. Serial blood samples were obtained over at least a 1 year period before and after DLI and results compared to 10 normal donors. Serial analysis of CDR3 size profiles demonstrated the appearance of clonal T cell populations after DLI in each of the three responding patients. The appearance of some clones was noted within the first 3 months after DLI and coincided with decreasing levels of monoclonal paraprotein indicating an ongoing GVM response. Other T cell clones appeared at later time points and coincided with the development of GVHD. These findings demonstrate that T cell clones with different patterns of onset can be identified in the peripheral blood of MM patients following DLI. Further functional characterization of these distinct clonal expansions will be required to determine whether these T cell clones are mediators of either anti-myeloma or anti-host activity.

PURPOSE: Donor lymphocyte infusion (DLI) can restore complete remission in patients with chronic myelogenous leukemia (CML) who have relapsed after T-cell–depleted (TCD) allogeneic bone marrow transplantation (BMT). The existence of salvage treatment for patients with DLI after TCD allogeneic BMT prompted an evaluation of overall outcome after CD6+-TCD allogeneic BMT for patients treated during the time when DLI has been available. PATIENTS AND METHODS: We performed a retrospective analysis of outcomes of 46 patients who underwent TCD allogeneic BMT for stable-phase CML and compared these outcomes with those of 40 patients who underwent non-TCD allogeneic BMT. All subjects were patients at one of two neighboring institutions during a period when DLI was available. All patients received marrow from HLA-identical sibling donors, underwent similar myeloablative regimens, and had similar pretreatment characteristics. RESULTS: After BMT, the TCD group had a lower incidence of grade 2 to 4 acute (15% v 37%, P = .026) and chronic graft-versus-host disease (GVHD) (18% v 42%, P = .024) than did the non-TCD group. The 1-year treatment-related mortality rates for the TCD group and the non-TCD group were 13% and 29%, respectively (P = .07). The estimated 3-year probability of relapse (cytogenetic or hematologic) was higher for patients in the TCD group than for patients in the non-TCD group (62% v 24%, P = .0003). Twenty-three patients (20 in the TCD group and three in the non-TCD group) received and were assessable for response to DLI. After DLI, 17 of 20 patients in the TCD group and two of three patients in the non-TCD group achieved complete remission. Donor lymphocyte infusion induced GVHD in nine of 23 patients. Thirty (65%) of 46 patients in the TCD group and 27 (69%) of 39 assessable patients in the non-TCD group remained alive without evidence of disease. The estimated 3-year overall survival rates were similar for the TCD group and the non-TCD group (72% v 68%, respectively; P = .38). At last follow-up, there was no difference in the overall prevalence of GVHD or the proportion of patients requiring immunosuppressive agents between groups. CONCLUSION: These results suggest that the combination of T-cell depletion and post-BMT DLI is a viable treatment option for patients undergoing allogeneic BMT for CML and should be prospectively compared with traditional forms of GVHD prophylaxis.

Abstract 1892 Patients with advanced hematological malignancies remain at high risk for eventual disease progression following reduced intensity conditioning (RIC) allogeneic hematopoietic stem cell transplantation (allo-HSCT). We hypothesized that vaccination with whole leukemia cells during the critical period of immune reconstitution early after transplant may enhance antitumor immunity and facilitate expansion of leukemia-reactive T cell responses. We tested this hypothesis in a prospective clinical trial, in which patients with advanced chronic lymphocytic leukemia (CLL) received up to 6 vaccine doses initiated between day 30–45 following RIC allo-HSCT. Each vaccine consisted of 1×107 irradiated autologous tumor cells admixed with 1×107 irradiated K562 bystander cells secreting GM-CSF (GM-K562). All patients received tacrolimus and mini-methotrexate as graft-versus-disease (GvHD) prophylaxis. Tacrolimus was maintained at therapeutic levels during the vaccination period without taper. Twenty-two patients were enrolled, all with advanced disease (median number of prior therapies 3; range 2–11). Many of the leukemias expressed markers associated with aggressive disease (e.g. unmutated IgVH - 68%) and displayed high-risk cytogenetic abnormalities (sole del(11q) - 41%; sole del(17p) - 23%; del(11q and 17p) - 18%). Greater than 50% (n=13) of patients had persistent marrow involvement (≥10%) at time of allo-HSCT. Eighteen of 22 subjects were vaccinated after allo-HSCT and received a median of 6 (range 1–6) vaccines. The remaining 4 patients were precluded from vaccination due to development of acute GvHD before day 45. Vaccines were generally well tolerated, but mild, transient injection site erythema was common. Only one grade 4 event (neutropenia) with a possible attribution to treatment occurred. We observed a similar incidence of grade II-IV aGvHD at 1 year in the 18 vaccinated patients (39%; 95% CI: 17–61%) and 42 control CLL patients that underwent RIC allo-HSCT at our institution from 2004–2009 (31%; 95%CI: 18–46%). At a median follow-up of 2.9 (range 1–4) years, the estimated 2-year rates of progression-free survival and overall survival of vaccinated study participants were 80% (95% CI: 54–92%) and 84% (95% CI: 58–95%). With these promising clinical results, we next focused on gaining insight into the mechanism that generated the observed clinical graft-versus-leukemia (GvL) responses. To delineate the specific contribution of vaccination to the overall GvL effect, we performed T cell assays to detect CLL-specific reactivity in serial pre- and post-HSCT samples obtained from vaccinated patients (n=9) who received median of 6 vaccines (range 3–6). In comparison, we examined T cell responses in study subjects (n=4) that developed aGvHD at a median of 44.5 days (range 26–56) after HSCT; and control CLL patients (n=4; no vaccine, no GvHD in the early post-transplant period) that were not enrolled in the study. Although early post-transplant vaccination had no impact on recovering absolute T cell numbers, reactivity of CD8+ T cells from the vaccinated patients was consistently directed against autologous tumor cells but not alloantigen bearing-recipient cells (PHA T cell blasts and fibroblasts) in IFNγ ELISpot assays. A peak response against autologous tumor cells was reached at day 60 after allo-HSCT (average 221 SFC/5×105 cells vs. 29 and 33 average SFC/5×105cells for PHA blasts and fibroblasts, respectively). CD8+ T cell clones were isolated from 4 vaccinated study subjects by limiting dilution and 17% (range 13–33%) reacted solely against CLL-associated antigens. In contrast, broad CD8+ T cell reactivity indicating an alloantigen response was observed in GvHD patients, while no increase in T cell reactivity against tumor-associated or alloantigens was seen in control patients. Tumor-reactive CD8+ T cells isolated from vaccinated patients secreted a broad profile of effector cytokines (GM-CSF, TNFα and IP10). Moreover, the amount of cytokines secreted by these CLL-specific CD8+ T cells steadily increased following early post-transplant vaccination, but not after allo-HSCT alone or in relation to GvHD. Our studies reveal that vaccination with autologous whole CLL/GM-K562 cells between days 30–100 after allo-HSCT is associated with induction of immunity against recipient CLL cells, and suggest that this is an effective strategy for promoting GvL following RIC allo-HSCT. Disclosures: Brown: Genzyme, Celgene: Research Funding; Calistoga, Celgene, Genentech, Pharmacyclics, Novartis, Avila: Consultancy. Cutler:Pfizer, inc: Research Funding; Astellas, Inc: Consultancy, Research Funding.

Abstract 1903 Donor lymphocyte infusion (DLI) can provide curative treatment for relapsed hematologic malignancies following allogeneic hematopoietic stem cell transplant (HSCT). However, the precise mechanism by which DLI eliminates leukemia cells in vivo has not been established. We hypothesized that marrow-infiltrating immune populations play a critical role in DLI responses since marrow is the primary site of disease and a reservoir of high-avidity antigen-specific memory T cells that can recognize tumor antigens, therefore potentially mediating graft-versus-leukemia (GvL) responses. We performed immunohistochemical staining of immune cells in serial marrow biopsies collected before and after DLI from 29 patients with relapsed CML. Twenty-two patients achieved cytogenetic remission within twelve months (‘responders’) while 7 patients demonstrated persistent disease (‘non-responders’). While no significant changes in the numbers of circulating T cells were seen between patient groups following DLI, the median number of marrow-infiltrating CD8+ T cells increased 2-fold in responders but remained unchanged in non-responders (P=0.02), demonstrating that clinical response to DLI is associated with T cell responses at the site of disease that may not be apparent in the peripheral blood. To investigate whether immune cell infiltration of the marrow could predict DLI response, we compared pre-treatment samples from both patient groups. Responders exhibited significantly higher proportions of CD8+ T cells in pre-DLI marrow compared to non-responders (5% vs 2.5%; P=0.01). Because disease burden is a known risk factor for ineffectual DLI response, we evaluated the interaction between disease burden and pre-existing CD8+ T cell infiltrate through the clinical course of 8 patients with high (≥70%) pre-treatment marrow cellularity. Three of 8 had ≥5% CD8+ T cell marrow infiltrates, and all 3 subsequently achieved cytogenetic remission. In contrast, 5 of 8 patients had To identify candidate mechanisms underlying T cell responses to DLI, we performed mRNA expression profiling of CD3+ T cells isolated from matched pre- and post-treatment marrows of 4 responders and 2 non-responders (U133+ Affymetrix cDNA microarrays). We first compared global gene expression patterns between pre-treatment marrow-infiltrating T cells of both groups. Notably, 28% of significantly upregulated genes in responders play critical roles in T cell exhaustion. This finding was strengthened by unbiased gene set enrichment analysis (GSEA) using 880 sets from the Molecular Signatures Database spiked with 17 additional specifically curated T cell exhaustion sets. Four of 15 positively enriched sets were T cell exhaustion-specific. We next compared differential gene expression in marrow-infiltrating T cells before and after therapy in responders compared to non-responders and found 21% of significantly down-regulated genes to be components within T cell exhaustion pathways along with repression of multiple, distinct T cell exhaustion gene sets. The robust reversibility of T cell exhaustion signatures after DLI therapy underscores this gene module as a likely therapeutic target of DLI. In conclusion, CD8+ T cell marrow infiltration may serve as a novel predictive marker of response to DLI, including patients with high disease burden. In addition, these data implicate T cell exhaustion in distinguishing responders from non-responders and, provocatively, propose reversal of T cell exhaustion as a potential marker of DLI responsiveness in patients with relapsed CML after HSCT. These studies illustrate the importance of local immune responses at the site of disease and suggest a potential tool to predict DLI response in other hematologic malignancies. The clinical debut of newer agents that reverse T cell exhaustion suggests their use in lieu of DLI to promote GvL responses after allogeneic HSCT. Disclosures: No relevant conflicts of interest to declare.

Abstract #4124 Background: Vaccination with tumor cells engineered to secrete granulocyte-macrophage colony stimulating factor (GM-CSF) generates potent, specific, and long-lasting anti-tumor immunity in multiple tumor models. At DF/HCC, over 100 patients with melanoma, ovarian cancer, and lung cancer have been vaccinated. These studies have led to the identification of targets of immune-mediated tumor destruction. Here, we present analysis on the safety, feasibility, and biologic activity of an autologous GM-CSF-secreting breast cancer vaccine. Methods:27 patients with metastatic breast cancer have undergone tumor procurement for vaccine from malignant pleural effusions (23), ascites (1), tumor nodules (2), lymph node (1), and 12 patients have received vaccine. Cells were transduced with a replication defective adenoviral vector encoding GM-CSF, irradiated, and GM-CSF secretion was measured by ELISA. Patients were required to have received at least one prior chemotherapy for metastatic disease and have an ECOG performance status 0-1. Vaccine was delivered s.c. and i.d. weekly for three weeks, then every other week. Immune monitoring included skin biopsies of vaccine sites, measurement of leukocyte populations, and proteomic-based assessment of antibody responses. Results: Sufficient viable tumor cell yields for vaccination were obtained from all patients, with cellular yields ranging from 1.1-679 x 106 cells and viabilities ranging from 71-100%. Dose levels were based on cellular yield, ranging from 105 -107 cells/dose. The average yield of GM-CSF was 838 ng/106 cells/24 hrs (range: 24.4-5696), higher than the average yields for lung cancer and melanoma. Vaccinated patients were 34-69 years, and received 3-23 vaccines. 9 patients had progressive disease after 3-10 vaccines (1-4 months). One patient had stable disease after 23 vaccines (11 months), resumed vaccination at 16 months for progression and remains on study. One other patient remains on study, and one patient did not have measurable disease. Toxicities related to treatment include grade I/II local injection-site reactions, grade I/II inflammation at tumor sites as well as grade I/II fatigue, fever, nausea, and edema. Skin biopsies of vaccination sites revealed mild-moderate infiltration of lymphocytes, granulocytes, and macrophages. Conclusion: Breast cancer cells can be harvested in sufficient number for autologous vaccine production from solid tumor as well as from malignant effusions. Autologous vaccination can induce coordinated immune responses with limited toxicity. The proteomic-based identification of antigen-specific immune responses following vaccination will be presented. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4124.

Eradication of residual disease remains a critical problem in patients with chronic myeloid leukemia (CML). Donor lymphocyte infusion (DLI) for the treatment of relapsed CML after transplantation is a robust example of the graft-versus-leukemia (GvL) effect, producing complete durable remission in 75% of patients. Since CML originates as a clonal stem cell disorder, we hypothesize that the curative effect of DLI results from immunologic targeting of the self-renewing CML progenitor cell. We have focused on B cell responses as a critical element in effective tumor immunity after observing significant B cell lymphocytosis in 7 responders of DLI at 6 and 9 months following DLI compared to pre-treatment levels (p=0.03; p=0.04, respectively), which was not seen in 5 DLI nonresponders. Moreover, some DLI responders showed marrow plasmacytosis at the time of cytogenetic response. To identify targets of this visible B cell response, we used post-DLI plasma as a source of immunoglobulin to probe two complementary immunoproteomic platforms and identify antigens eliciting increased reactivity compared to pre-DLI plasma. In the first approach, plasma from 7 individual responders was screened for binding to proteins expressed from a CML cDNA phage library (SEREX). In the second approach, plasma from 3 of the 7 responders was screened for binding to proteins expressed from approximately 5000 open reading frames spotted on a protein microarray (ProtoArray, Invitrogen). Screening by SEREX yielded 24 and by ProtoArray 58 candidate antigens. Analysis of datasets generated from normal and malignant myeloid cells on HG-Focus and HG-U133A Affymetrix microarrays confirmed that most candidate targets were expressed in CML progenitor cells. Of the antigens represented on the microarrays, 83% (10 of 12) and 64% (18 of 28) of the SEREX- and ProtoArray-identified antigens, respectively, were expressed in CML CD34+ cells. EXOSC5 was expressed on CML CD34+ cells and was identified by both platforms. Four of 16 ProtoArray-identified antigens (RAB38, TBCE, DUSP12, RPS6KC1) were expressed at higher levels in CML compared to normal CD34+ and mature myeloid lineage cells (p