ALBERT

Description: Combination therapy with purine analogs, alkylators, and monoclonal antibodies has transformed the treatment paradigm in patients with CLL by dramatically enhancing both the quality and frequency of responses that can be achieved in these patients. However, combinations utilizing fludarabine as the purine analog have augmented myelosuppression and immunosuppression requiring careful attention to dosing and schedule in order to minimize these complications. Even with these precautions many patients are unable to complete the entire treatment program at full dose and for the planned number of cycles. Comparative experience with pentostatin indicates that it is less myelosuppressive than either fludarabine or cladribine. We previously reported our experience with pentostatin and cyclophosphamide. Subsequently, we have added rituximab to this active combination (PCR regimen) and treated a second cohort of 46 patients with previously treated CLL (32 patients) and other low grade lymphoid neoplasms (14 patients). The PCR regimen consists of pentostatin 4mg/m2, cyclophosphamide 600mg/m2, and rituximab 375mg/m2 all given on a single day with anti-emetics, hydration, and careful monitoring of renal function. The treatment was administered every 3 weeks for a total of 6 treatments. Rituximab was not given during the first cycle to reduce the frequency and severity of infusion reactions. Filgrastim, sulfamethoxazole/trimethoprim, and acyclovir were administered prophylactically. The median age of the patients treated was 62 (range 44–80) and the median number of prior regimens was 2 (range 1–7). The overall frequency of response was 75% with 25% achieving a complete response, 3% a nodular response, and 47% a partial response. We have compared these results to the recently reported MD Anderson FCR regimen. In terms of pre-treatment characteristics the patient groups in both studies appear comparable with the exception of a higher proportion of high-risk patients treated with PCR (78%) compared to FCR (50%) (P=0.003). The response frequencies are virtually identical in both studies with responses seen 75% of PCR treated patients and 73% of FCR treated patients and CR achieved in 25% in both studies. In terms of toxicity, however, PCR compares favorably to FCR in the following categories: Grade 3/4 neutropenia PCR 53% vs FCR 81% (P=0.0007), thrombocytopenia PCR 16% vs FCR 34% (P=0.04), anemia PCR 9% vs FCR 24% (P=0.06), and grade 3/4 infections (including fever of unknown origin) PCR 28% vs FCR 47% (P=0.05). PCR also appeared to be better tolerated than FCR as indicated by the fraction of patients completing all planned cycles of chemotherapy at full dose 72% vs 38% (P=0.0004). An important caveat in these comparisons is that myeloid growth factor was routinely administered to patients on the PCR study but was not routinely administered to patients treated with FCR. In conclusion, PCR appears to be equivalent in activity to FCR but may be better tolerated and less toxic. These results indicate that a prospective randomized comparison is warranted.

Description: INTRODUCTION T Cell Receptor mimic (TCRm) antibodies to low-density peptide epitopes from undruggable intracellular proteins presented in the context of major histocompatibility (MHC) molecules are therapeutically effective in mouse models of human cancers. CD47 blockade by use of a high affinity SIRPα variant protein (CV1) has been shown to improve the effects of monoclonal antibodies to high-density antigens in tumor models by enhancement of antibody dependent cellular phagocytosis (ADCP). We asked if combination therapy with a TCRm antibody to Preferentially Expressed Antigen in Melanoma (PRAME) could enhance activity of both drugs in vitro and in vivo. Additionally, we explored the role of macrophage-secreted cytokines in the enhanced in vivo activity. METHODS We performed in vitro ADCP assays with human acute myeloid and acute lymphoid leukemia cell lines containing antigens of interest using the two agents alone and in combination. We performed therapy experiments in NSG mice using the same leukemia cell lines transformed with a luciferase vector and measured tumor burden through bioluminescent imaging. Survival was measured. We examined cell-surface expression of epitopes of interest and HLA on cell lines in vitro after incubation with IFNγ and TNFα using flow cytometry and performed in vitro ADCP assays with the leukemia cell lines after pretreatment with IFNγ. RESULTS CV1 and TCRm antibody showed additive effects in vitro with a statistically significant increase in phagocytosis in both antigen positive cell lines with combination therapy versus single agent therapy. CV1 and TCRm antibody showed greater than additive therapeutic effects in vivo with a 3-log reduction in leukemia burden relative to control untreated mice and a 5-10 fold reduction relative to single agent groups. After therapy was stopped, mice treated with the combination had statistically significant increases in survival (p

Description: Peptides derived from BCR-ABL have been used to trigger immune responses in CML pts. The native peptides may have low immunogenicity because they bind with low affinity to the major histocompatibility complex (MHC) molecules. Synthetic peptides containing mutations in the sequences of these peptides can bind with higher affinity than parental peptides and may generate a cross-reactive T cell response to the native sequence (known as a heteroclitic response). We used a mixture of heteroclitic and native peptides derived from both b3a2 and b2a2 sequences in a pilot study to vaccinate pts with CML in complete cytogenetic remission on imatinib (IM) therapy with stable bcr-abl transcript levels. Montanide ISA51 and GM-CSF were used as immunological adjuvants. Pts were required to have received IM for ≥12 mo with no change in dose for at least 6 mo and were not allowed any dose increases after registration. Pts were vaccinated with the peptides corresponding to their BCR-ABL breakpoint as follows: every 2 wks x4, then once 3 wks later, followed by 10 monthly vaccinations for a total of 15 vaccinations in 12 mo. After registration, pts had 3 additional measures of bcr-abl transcripts to better define baseline values. 8 pts have been vaccinated. At enrollment, pts had a median age of 45 yrs (range 29–63) and had been receiving IM for a median of 63 mo (range 35–68 mo) on a median dose of 600mg (range 300–800mg). Pts have been followed for a median of 15 wks (range 2–20) and have received a median of 7 vaccinations (range 1 to 8). To determine the effect of vaccination on immune leukocytes, we measured peripheral blood T-cell subsets, B, NK, and dendritic cells by FACS at baseline and at 3-month intervals thereafter. The percentage of myeloid DC (DC1) and plasmacytoid DC (DC2) cells were identified by the mutually exclusive expression of CD11c and CD123; Treg cells by the surface co-expression of CD3, CD4 and CD25 together with cytoplasmic expression of CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) and FoxP3 (forkhead/winged helix transcription factor). Vaccinations have been well tolerated. The only adverse event attributable to the vaccine is local site reactions grade 1 or 2 in 5 pts. Among the 5 pts who have had an evaluation at 3 mo from the 1st vaccination, 1 has achieved a major molecular response (bcr-abl/abl

Description: Abstract 3054 Poster Board II-1030 Cancer therapies targeting tumor endothelium have shown promising results when combined with chemotherapeutics. Vascular endothelial (VE) cadherin is an endothelial cell specific molecule that is expressed constitutively throughout the vasculature and participates in the formation of adherens junctions between adjacent endothelial cells. The monoclonal antibody E4G10 specifically binds to a cryptic epitope exposed only on the monomeric unengaged form of VE-Cadherin found in neovasculature. We chemically conjugated E4G10 with the cytotoxic, short-range αa-particle emitter, 225Ac, and show that the agent dampens tumor growth as a single agent and this effect is enhanced when given in combination with chemotherapy in a xenograft mouse model. The therapeutic efficacy of the combination therapy is sequence dependent and most pronounced when 225Ac-E4G10 is administered prior to the chemotherapy. Immunohistochemical and immunofluorescence studies for endothelial cells (Meca32), vascular basement membrane (Collagen IV), and pericytes (alpha-smooth muscle actin (alpha-SMA), nerve/glial antigen 2 (NG2) ) showed that the vasculature of 225Ac-E4G10 treated tumors was depleted and the remaining vessels appeared normalized as evidenced by morphological changes, increased pericyte density and coverage. Fluorescence studies with Hoechst 33342 dye i.v. injected showed more homogenous distribution of the dye within 225Ac-E4G10 treated tumors, suggesting improved perfusion, and biodistribution studies with i.v. injected 111In-Diethylene triamine pentaacetic acid (DTPA), a freely diffusible small molecule, showed significantly (p=0.03) increased passive tumor accumulation of 111In-DTPA in 225Ac-E4G10-treated tumors when compared to controls. Quantitative autoradiographic studies on tumor sections also showed both stronger and more homogenous signal distribution in 225Ac-E4G10-treated tumors. These results show that 225Ac-E4G10 treatment leads to ablation and remodeling of the tumor vasculature allowing improved delivery of subsequent small molecules, such as chemotherapeutics, and thereby improves therapeutic outcome. Disclosures McDevitt: Actinium Pharma Inc.: Patents & Royalties. Scheinberg:Actinium Pharma Inc.: Patents & Royalties.

Description: Background: Lintuzumab, a humanized anti-CD33 monoclonal antibody, targets myeloid leukemia cells but has only modest activity in AML. To increase the antibody’s potency yet avoid nonspecific cytotoxicity of β-emitting isotopes, 225Ac (t½=10 d), a radiometal that yields 4 α-particles, was conjugated to lintuzumab. A phase I trial showed that 225Ac-lintuzumab is safe at doses ≤ 3 µCi/kg and has anti-leukemic activity across all dose levels studied (Jurcic et al. ASH, 2011). We are conducting a multicenter, phase I dose-escalation trial to determine the maximum tolerated dose (MTD), toxicity, and biological activity of fractionated-dose 225Ac-lintuzumab in combination with LDAC. Patients and Methods: Patients ≥ 60 yrs who had untreated AML with poor prognostic factors, e.g., an antecedent hematologic disorder, unfavorable cytogenetic or molecular abnormalities, and significant comorbidities, were eligible. Patients received LDAC 20 mg twice daily for 10 d every 4-6 wks for up to 12 cycles. During Cycle 1, beginning 4-7 days after completion of LDAC, two doses of 225Ac-lintuzumab were given approximately one week apart. To prevent radiation-induced nephrotoxicity, patients were given furosemide while receiving 225Ac-lintuzumab and spironolactone for one year afterward. Results: Nine patients (median age, 76 yrs; range, 73-81 yrs) were treated. Seven patients (78%) had a history of myelodysplastic syndromes (MDS), for which five (56%) received prior therapy with hypomethylating agents (n=4) or allogeneic hematopoietic cell transplantation (n=1). One patient (11%) had chronic myeloid leukemia in a molecularly undetectable state at the time of AML diagnosis. Six patients (67%) had intermediate-risk cytogenetics, and three (33%) had unfavorable cytogenetics. The median CD33 expression was 76% (range, 45-100%). Patients received 225Ac-lintuzumab at doses of 0.5 (n=3) or 1 (n=6) μCi/kg/fraction. Total administered activity ranged from 68-199 μCi. The median number of cycles administered was 2 (range, 1-4). Dose-limiting toxicity was seen in one patient receiving 1 µCi/kg/fraction who had grade 4 thrombocytopenia with bone marrow aplasia persisting 〉 6 wks after receiving 225Ac-lintuzumab. Hematologic toxicities included grade 4 neutropenia (n=1) and thrombocytopenia (n=3). Grade 3/4 non-hematologic toxicities included febrile neutropenia (n=6), pneumonia (n=2), bacteremia (n=1), cellulitis (n=1), transient increase in creatinine (n=1), hypokalemia (n=1), and generalized weakness (n=1). Bone marrow blast reductions were seen in 5 of 7 patients (71%) evaluated after Cycle 1. Mean blast reduction was 61% (range, 34-100%). Three of the 7 patients (43%) had marrow blast reductions of ≥ 50%; however, no remissions were observed. Median progression-free survival (PFS) was 2.5 mos (range, 1.7-15.7+ mos). Median overall survival (OS) from study entry was 5.4 mos (range, 2.2-24 mos). For the 7 patients with prior MDS, median OS was 9.1 mos (range 2.3-24 mos). Conclusions: Fractionated-dose 225Ac-linutuzmab in combination with LDAC is feasible, safe, and has anti-leukemic activity. Dose escalation continues to define the MTD, with planned doses up to 2 µCi/kg/fraction. Additional patients will be treated at the MTD in the phase II portion of this trial to determine response rate, PFS, and OS. Disclosures Ravandi: Actinium Pharmaceuticals, Inc.: Research Funding. Pagel:Actinium Pharmaceuticals, Inc.: Equity Ownership, Research Funding. Park:Actinium Pharmaceuticals, Inc.: Research Funding. Wahl:Actinium Pharmaceuticals, Inc.: Research Funding. Earle:Actinium Pharmaceuticals, Inc.: Employment, Equity Ownership. Cicic:Actinium Pharmaceuticals, Inc.: Employment, Equity Ownership. Scheinberg:Actinium Pharmaceuticals, Inc.: Equity Ownership, Research Funding.

Description: Adoptive transfer therapy of T cells expressing chimeric antigen receptors (CAR) against tumor-associated antigens has been shown to be clinically successful in a limited set of leukemia. However, novel antigen targets for both hematological and solid malignancies are required. Most CARs described thus far are targeted against external antigens on particular cell types. We have designed and engineered the first CAR T cell against a human intracellular protein, WT1. WT1 is overexpressed in many cancers, including acute and chronic leukemias and numerous solid tumors. Our TCRm CAR, derived from the ESK1 TCRm mAb, termed WT1 28z, is reactive with the RMFPNAPYL peptide of the WT1 protein that is processed and presented on the surface of cells in the context of HLA-A*02:01. WT1 28z expressing T cells have high expression of the CAR on their surface. They are cytotoxic in standard 51Cr assays against a range of cancer cell lines, including the megakaryoblastic cell line SET2, the acute myeloid leukemia (AML) cell line AML14, the multiple myeloma cell line KARPAS, and the ovarian cancer line, OVCAR3, as compared to CAR T cells against an irrelevant antigen. The WT1 28z CAR T cells are also cytotoxic against primary AML bone marrow blasts in this assay. When co-cultured with these primary cells or cancer cell lines, the WT1 28z CAR T cells have enhanced production of proinflammatory cytokines such as IFN-g, IL-2, and GM-CSF, as compared to irrelevant CAR T cells. Importantly, WT1 28z T cells are specific for the WT1-HLA-A*02:01 complex. The cells do not show cytotoxicity against cell lines or primary cells that are not both HLA-A*02:01- positive and WT1 positive. WT1 28z T cells are currently being tested alongside irrelevant antigen CAR T cells in AML and ovarian cancer murine models in vivo to assess efficacy, with the ultimate goal of translating this novel approach into the clinical setting for both hematological and solid cancers. The data provide the proof-of-concept that CAR T cells also may be directed at intracellular antigens. Disclosures Dao: Novartis: Patents & Royalties. Liu:Eureka: Employment, Inventor Other. Scheinberg:Novartis: Patents & Royalties. Brentjens:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other.

Description: Background: The use of short-ranged (50-80 µm), high-energy (~100 keV/µm) α particle-emitting isotopes for radioimmunotherapy may result in more specific tumor cell kill and less damage to normal tissues than β-emitters. 225Ac-lintuzumab consists of a radiometal that emits four α-particles linked to an anti-CD33 antibody. A phase I trial showed that 225Ac-lintuzumab is safe at doses ≤ 3 µCi/kg and has anti-tumor activity against relapsed/refractory AML across all dose levels studied (Jurcic et al. ASH, 2011). We are conducting a multicenter, phase I dose-escalation trial to determine the maximum tolerated dose (MTD), toxicity, and biological activity of fractionated-dose 225Ac-lintuzumab in combination with LDAC. Patients and Methods: Patients ≥ 60 years with untreated AML not suitable for standard induction chemotherapy (e.g., antecedent hematologic disorder, unfavorable cytogenetic or molecular abnormalities, and significant comorbidities) were eligible. Patients received LDAC 20 mg twice daily for 10 days every 4-6 weeks for up to 12 cycles. During Cycle 1, two fractions of 225Ac-lintuzumab were given one week apart, beginning 4-7 days following completion of LDAC. To prevent radiation-induced nephrotoxicity, patients were given furosemide while receiving 225Ac-lintuzumab and spironolactone for one year afterward. 225Ac doses were escalated using a 3+3 design. Four dose levels were studied with a total accrual of up to 24 patients. In planned analyses, dose escalation proceeded if 〈 33% of patients in a cohort experienced dose-limiting toxicity (DLT). Results: Fourteen patients (median age, 77 years; range, 68-87 years) completed therapy. An additional patient received only one of two planned fractions of 225Ac-lintuzumab due to technical issues and is excluded from analysis. Nine (64%) had prior myelodysplastic syndrome, for which seven received prior therapy with hypomethylating agents (n=6) or allogeneic hematopoietic cell transplantation (n=1). One patient (7%) had chronic myeloid leukemia in molecular remission prior to development of AML. Nine patients (64%) had intermediate-risk and five (36%) had unfavorable cytogenetics. Median CD33 expression was 81% (range, 45-100%). 225Ac-lintuzumab was given at 0.5 (n=3), 1 (n=6), 1.5 (n=3), or 2 (n=2) μCi/kg/fraction. Up to 4 cycles of LDAC were administered. DLT was seen in one patient at 1 µCi/kg/fraction who had grade 4 thrombocytopenia with marrow aplasia for more than 6 weeks following therapy. Hematologic toxicities included grade 4 neutropenia (n=4) and thrombocytopenia (n=6). Grade 3/4 non-hematologic toxicities included febrile neutropenia (n=7), pneumonia (n=4), bacteremia (n=1), cellulitis (n=1), transient creatinine increase (n=1), hypokalemia (n=1), rectal hemorrhage (n=1), and generalized weakness (n=2). Eight of 11 patients (73%) evaluated after Cycle 1 had bone marrow blast reductions (mean reduction, 72%; range, 34-100%). Seven (64%) had blast reductions of at least 50%. Objective responses (1 CR, 1 CRp, 2 CRi) were seen in four of the 14 patients (29%) after one cycle of therapy (Table 1). Responses were seen only at doses ≥ 1 µCi/kg/fraction (4 of 11 patients, 36%). Median progression-free survival (PFS) was 2.7 months (range, 1.7-16.9 months). Median overall survival (OS) was 5.5 months (range, 2.2-24 months). Conclusions: Fractionated-dose 225Ac-linutuzmab can be safely combined with LDAC and produce remission in older patients with untreated AML. Dose escalation continues to define the MTD. Additional patients will be treated at the MTD in the phase II portion of this trial to determine response rate, PFS, and OS. Table 1. Objective Responses Response Dose Level (μCi/kg/fraction) Total (n=14) 0.5 (n=3) 1 (n=6) 1.5 (n=3) 2 (n=2) CR 0 0 1 (33%) 0 1 (7%) CRp 0 0 0 1 (50%) 1 (7%) CRi 0 1 (17%) 1 (33%) 0 2 (14%) Overall Response 0 1 (17%) 2 (67%) 1 (50%) 4 (29%) Abbreviations: CR, complete remission; CRp, CR with incomplete platelet recovery; CRi, CR with incomplete count recovery. Disclosures Jurcic: Ambit Biosciences: Research Funding; Astellas Pharma US, Invc.: Research Funding; Tetralogic Pharmaceuticals: Research Funding; Sunesis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Bayer Pharmaceuticals: Consultancy; Merck and Co.: Consultancy; Celgene Corp.: Research Funding; Actrinium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Off Label Use: Ac-225-lintuzumab is an investigational agent being developed for the treatment of acute myeloid leukemia.. Pagel:Actinium Pharmacetuicals, Inc.: Equity Ownership. Park:Actinium Pharmaceuticals, Inc.: Research Funding; Juno Therapeutics: Consultancy. Levy:Takeda: Consultancy. Perl:Actinium Pharmaceuticals, Inc.: Research Funding. Earle:Actinium Pharmaceuticals, Inc.: Employment. Cicic:Actinium Pharmaceuticals, Inc.: Employment, Equity Ownership. Scheinberg:Actinium Pharmaceuticals, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Description: Wilms tumor protein (WT1) is a transcription factor selectively over expressed in several types of leukemia and solid tumors, making it a promising potential target antigen for immunotherapy. Several open clinical trials use native or altered peptide sequences derived from the WT1 protein in order to overcome the weak immunogenicity of the self-antigen. Here we report a new strategy to circumvent tolerance by designing peptides that incorporate non-natural amino acids into the native sequence of WT1 peptides. Starting from the nonamer sequences WT1 187–195 and WT1 235–243, eight peptides containing natural amino acids and nine peptides in which different chemical modifications (fluorination, photo-reactive azido groups or benzophenone groups) were introduced at major histocompatibility complex (MHC) and T cell receptor binding positions, were synthesized. The new non-natural peptides could stabilize MHC class I molecules better than the native sequences and were also able to elicit strong specific T-cell responses. Photo-reactive peptides were additionally modified with biotin handles to allow streptavidin-biotin pull down and western blot analysis of kinetics of binding and catabolism. Upon UV irradiation, these peptides covalently bound to MHC molecules on the live cells; clearance of the peptide-MHC covalent complex occurred over 24 hours, consistent with the T2 thermo-stabilization data for the same peptide. Further catabolic studies may elucidate the important or novel cellular proteins involved in antigenic peptide processing and cross presentation and should aid in vaccine development. We are investigating whether covalent interaction with the MHC may lead to alterations in immune responses as well. T cells stimulated with one of the synthetic peptides (WT1J-W4WF) cross-reacted with the native WT1J sequence and were able to kill WT1 positive HLA-A0201 matched acute lymphoblastic leukemia cell lines. In conclusion, this study shows that peptides with non-natural amino acids can be successfully incorporated into T cell epitopes to provide increased immunogenicity and novel biological information.

Description: A tumor-specific, bcr-abl-derived fusion peptide vaccine can be safely administered to patients with chronic myelogenous leukemia (CML) and can elicit a bcr-abl peptide-specific T-cell immune response. In the present phase 2 trial, 14 patients with CML in chronic phase were vaccinated with 6 fusion peptides mixed with Quillaja saponaria (QS-21). No significant toxic effects were observed. In 14 of 14 patients, delayed-type hypersensitivity (DTH) and/or CD4 proliferative responses developed after beginning vaccinations, and 11 of 14 patients showed interferon-gamma (IFN-gamma) release by CD4 enzyme-linked immunospot (ELISPOT) at one or more time points. These responses were CD4+CD45RO+. A peptide-specific CD8+ interferon-gamma ELISPOT was found in 4 patients. Four patients in hematologic remission had a decrease in Philadelphia chromosome (Ph) percentage (3 concurrently receiving interferon-alpha and 1 on imatinib mesylate), and 3 patients in molecular relapse after allogenic transplantation became transiently polymerase chain reaction (PCR) negative after vaccination; 2 of these patients received concurrent donor lymphocyte infusion (DLI). All 5 patients on IFN-alpha ultimately reached a complete cytogenetic remission. In conclusion, a tumor-specific bcr-abl breakpoint peptide-derived vaccine can be safely administered and can reliably elicit measurable peptide-specific CD4 immune responses, including in patients after bone marrow transplantation, on interferon, or on imatinib mesylate. A relationship between the clinical responses and vaccination cannot be determined from this trial. (Blood. 2004;103:1037-1042)

Description: Unlike β particle–emitting isotopes, α emitters can selectively kill individual cancer cells with a single atomic decay. HuM195, a humanized anti-CD33 monoclonal antibody, specifically targets myeloid leukemia cells and has activity against minimal disease. When labeled with the β-emitters 131I and 90Y, HuM195 can eliminate large leukemic burdens in patients, but it produces prolonged myelosuppression requiring hematopoietic stem cell transplantation at high doses. To enhance the potency of native HuM195 yet avoid the nonspecific cytotoxicity of β-emitting constructs, the α-emitting isotope 213Bi was conjugated to HuM195. Eighteen patients with relapsed and refractory acute myelogenous leukemia or chronic myelomonocytic leukemia were treated with 10.36 to 37.0 MBq/kg 213Bi-HuM195. No significant extramedullary toxicity was seen. All 17 evaluable patients developed myelosuppression, with a median time to recovery of 22 days. Nearly all the 213Bi-HuM195 rapidly localized to and was retained in areas of leukemic involvement, including the bone marrow, liver, and spleen. Absorbed dose ratios between these sites and the whole body were 1000-fold greater than those seen with β-emitting constructs in this antigen system and patient population. Fourteen (93%) of 15 evaluable patients had reductions in circulating blasts, and 14 (78%) of 18 patients had reductions in the percentage of bone marrow blasts. This study demonstrates the safety, feasibility, and antileukemic effects of 213Bi-HuM195, and it is the first proof-of-concept for systemic targeted α particle immunotherapy in humans.