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Background Interferon (IFN)-α is considered a key modulator of immunopathological processes through a signature-specific activation of mononuclear phagocytes (MPs). This study utilized global transcript analysis to characterize the effects of the entire type I IFN family in comparison to a broad panel of other cytokines on MP previously exposed to Lipopolysaccharide (LPS) stimulation in vitro. Results Immature peripheral blood CD14+ MPs were stimulated with LPS and 1 hour later with 42 separate soluble factors including cytokines, chemokines, interleukins, growth factors and IFNs. Gene expression profiling of MPs was analyzed 4 and 9 hours after cytokine stimulation. Four hours after stimulation, the transcriptional analysis of MPs revealed two main classes of cytokines: one associated with the alternative and the other with the classical pathway of MP activation without a clear polarization of type I IFNs effects. In contrast, after 9 hours of stimulation most type I IFN isoforms induced a characteristic and unique transcriptional pattern separate from other cytokines. These "signature" IFNs included; IFN-β, IFN-α2b/α2, IFN-αI, IFN-α2, IFN-αC, IFN-αJ1, IFN-αH2, and INF-α4B and induced the over-expression of 44 genes, all of which had known functional relationships with IFN such as myxovirus resistance (Mx)-1, Mx-2, and interferon-induced hepatitis C-associated microtubular aggregation protein. A second group of type I IFNs segregated separately and in closer association with the type II IFN-γ. The phylogenetic relationship of amino acid sequences among type I IFNs did not explain their sub-classification, although differences at positions 94 through 109 and 175 through 189 were present between the signature and other IFNs. Conclusion Seven IFN-α isoforms and IFN-β participate in the late phase polarization of MPs conditioned by LPS. This information broadens the previous view of the central role played by IFN-α in autoimmunity and tumor rejection by including and/or excluding an array of related factors likely to be heterogeneously expressed by distinct sub-populations of individuals in sickness or in response to biological therapy.

Objective To identify an immunodominant HLA-A33-restricted epitope within the CMV matrix phosphoprotein 65 (pp65) that could be used to elicit CMV-specific CTLs. Methods A computer algorithm was used to identify pp65 peptides that were expected to bind to HLA-A33. The peptides were screened for their ability to reactivate memory T lymphocytes from CMV-seropositive HLA-A33 donors by using quantitative real-time RT-PCR. The most promising peptides were then tested for their ability to expand a CD8 + population of HLA-A33 CTLs that produced interferon-γ (IFN-γ) and were cytotoxic to either peptide-loaded or CMV-infected target cells. Results Sixteen out of 250 peptides were selected using a computer algorithm and peptide stimulation by 8 out of the 16 induced a significant quantity of IFN-γ mRNA transcript from CMV-seropositive HLA-A33 peripheral blood mononuclear cells. All the eight peptides induced consistent T-cell reactivation. One specifically, the peptide pp65 91-100 (SVNVHNPTGR), proved to be more active. T cells in vitro sensitized for 2 or 3 weeks with pp65 91-100 were cytotoxic to both HLA-A33 peptide-loaded and CMV-infected target cells. Conclusions CMV pp65 91-100 is a new HLA-A33-restricted peptide that broadens the list of antigenic determinants that can be used for CMV adoptive immunotherapy.

This pilot study was carried out to gain a first insight into the effects of peptide vaccination in melanoma patients in the high-risk adjuvant disease setting. From the adjuvant peptide vaccination studies carried out in our institution since 1998, we identified all melanoma patients with a history of at least 3 completely resected metastases during the year preceding enrollment into the trial and describe the clinical and immunologic observations. Out of a total of 44 patients with resected cutaneous melanoma entered into adjuvant peptide vaccination trials, 9 patients were identified with more than 3 metastases in the year before vaccination. After initiation of vaccination, 2 patients remained relapse-free for 27 and 42+ months, 2 patients experienced single or several initial relapses and subsequent relapse-free intervals of 18 and 65+ months, whereas 5 patients progressed. In both patients with relapse after prolonged relapse-free intervals, the relapses were initially confined to the small intestine and could be resected. Induction or boosting of functional tyrosinase peptide-specific T cells was noted in 6 of 8 patients, including all 4 patients with prolonged relapse-free intervals. In conclusion, adjuvant peptide vaccination was associated with cessation of recurrences in 4 of 9 patients, of whom all 4 had an immunologic response to the vaccine.

Background Endogenous reference genes are commonly used to normalize expression levels of other genes with the assumption that the expression of the former is constant in different tissues and in different physiopathological conditions. Whether this assumption is correct it is, however, still matter of debate. In this study, we searched for stably expressed genes in 384 cDNA array hybridization experiments encompassing different tissues and cell lines. Results Several genes were identified whose expression was highly stable across all samples studied. The usefulness of 8 genes among them was tested by normalizing the relative gene expression against test genes whose expression pattern was known. The range of accuracy of individual endogenous reference genes was wide whereas consistent information could be obtained when information pooled from different endogenous reference genes was used. Conclusions This study suggests that even when the most stably expressed genes in array experiments are used as endogenous reference, significant variation in test gene expression estimates may occur and the best normalization is achieved when data from several endogenous reference genes are pooled together to minimize minimal but significant variation among samples. We are presently optimizing strategies for the preparation of endogenous reference gene mixtures that could yield information comparable to that of data pooled from individual endogenous reference gene normalizations.

Tumor immunology embraces an extensive array of biological phenomena that include interactions between neoplastic cells and the innate and adaptive immune response. Among immune cells, T cells have taken the center stage because they can be easily demonstrated to specifically recognize autologous cancer cells. However, their role is limited and other components of the immune response are likely necessary for the completion of cancer rejection. Metastatic melanoma and renal cell carcinoma (RCC) are malignancies strongly predisposed to regress in response to the systemic administration of high-dose interleukin (IL)-2. Several clinical Studies in extensive cohorts of patients have shown that this treatment can induce complete or partial clinical regressions of metastatic disease in 15 to 20% of patients who receive this treatment. Although IL-2 has direct pluri-potent effects on cells with immune and inflammatory function, it remains unexplained which cell subset is implicated in mediating tumor regression. In a quest to characterize the mechanism of action of IL-2 during the course of immunotherapy, we have investigated the early changes in transcriptional profiles of circulating mononuclear cells and microenvironment of melanoma metastases following high dose IL-2 administration (720,000 IU/kg) by serial sampling of blood cells and tumors in the form of fine needle aspirate (FNA). Furthermore, studies are currently ongoing to characterize the proteomic profiling of RCC patients undergoing the same treatment using protein arrays (manuscript in preparation). The predominant activation of genes related to inflammation and activation of mononuclear phagocytes lead us to further characterize this cell subset in the context of stimulation with a panel of soluble factors potentially present in the circulation and tumor microenvironment.

Among human cancers, melanoma is characterized by an almost unique predisposition to regress in response to immune therapy. Recent clinical studies suggest that the frequency of this favorable event is enhanced by combining T-cell-directed active specific immunization with the systemic administration of interleukin (IL)-2. While waiting for additional clinical experience to confirm this observation, we embraced the working hypothesis that this combination provides superior response rates than either treatment alone. In particular, we have focused our interest on the paradoxical observation that active specific immunization consistently induces circulating CD8+ T cells capable of recognizing in ex vivo assays tumor cells, but cannot induce tumor regression alone. In these settings, it appears that combining the systemic administration of IL-2 is almost an absolute requirement for the induction of clinical responses. Here, we will expand on previous speculations on the postulated mechanism(s) of action of systemic IL-2 administration and, based on original data recently derived through high-throughput transcriptional and post-translational analysis, we will suggest an explanation for the eclectic effects of IL-2 administration in the context of active specific immunization.

Immune responses against tumor-associated antigens rely on efficient epitope presentation. The melanoma-associated antigen (Ag) gp100 contains HLA-A*0201 ligands that are characterized by low to medium binding affinity, among which gp100(209-217) is the most prominent (Kawakami et al., J Immunol 154:3961-3968, 1995). While this epitope is a natural T-cell target, it primes with low-efficiency T-cell responses during immunization. A modified gp100 epitope, gp100(209-217T210M), that contains a Thr to Met substitution at position 2 of the antigenic nonamer is characterized by high binding affinity for HLA-A*0201 and elicits strong and clinically effective T-cell responses. This higher affinity is believed to represent the sole reason for enhanced immunogenicity. Contrasting with this observation is the unpredictable relationship between affinity and immunogenicity observed in other antigen systems. In addition, we noted a striking difference between the capability of endogenously processed gp100(209-217) and gp100(209-217T210M) to induce T-cell responses in an in vitro model. Therefore, we questioned whether factors other than HLA-affinity might play a role in determining the immunogenicity of these epitopes. In the present study, we evaluated the in vitro proteasomal cleavages of 23meric precursor peptides encompassing the native sequence (gp100(201-223)) or the modified sequence (gp100(201-223T210M)). Here we show that the standard proteasome liberates the C-termini of both antigenic peptides but not the N-termini. Quantitative analysis of the digestion products revealed that more of the fragments displaying the final C-termini were produced from the wild-type precursor. However, a stronger TCR engagement was observed when fractions of digested gp100(201-223T210M) were used to activate an HLA-A*0201-expressing target T-cell clone. This difference was also found using separately produced, synthetic nonamers. In conclusion, the high binding affinity of gp100(209-217T210M) seems to compensate for possible differences in proteasomal cleavage at the biological level. Since the final antigenic nonamer is not directly produced by the proteasome, additional further factors may influence the antigenic peptide availability, such as post-proteasomal processing and intracellular peptide transport.

Understanding the whole process of dendritic cell (DC) activation might help in the development of more efficient immunotherapeutic strategies for tumor patients. Part of this process is cytokine secretion, which has important effects on innate and adaptive immune response. Here, we cultured circulating monocytes for five days with interleukin-4 and GM-CSF followed by two-day culture with or without CD40 ligand and LPS to create a mature DC (mDC) and an immature DC (iDC) phenotype, respectively, characterized by differential expression of co-stimulatory molecules (CD80, CD83). We then compared the cytokine expression profile of the mDC and iDC using two protein platform arrays. Twelve supernatants from mDC paired with 12 from iDC were compared. The mDC protein expression profile showed significant increases in 16 out of 34 factors tested, including TNFalpha, IL-10, IL-12, IFNgamma, MIP1alpha, MIP1beta, IL-8, MDC, RANTES, and IL-6, which play a crucial role in the regulation of the innate immune response as well as the recruitment and activation of adaptive immune effectors. Interestingly, some of the cytokines expressed during maturation were also found in the gene expression profile identified in tumor metastases following IL-2 therapy using cDNA arrays. This finding suggests a possible role for resident DC maturation as a mediator of systemic IL-2 effects. Most important, the array of cytokines secreted during DC maturation may be considered an important component during adoptive transfer. Further characterization of the kinetics and persistence of their secretion should be undertaken in the future.

It has long been a matter of debate whether tumors are spontaneously immunogenic in patients. With the availability of sensitive methods, naturally occurring T cells directed against tumor-associated antigens (TAAs) can be frequently detected in cancer patients. In this review, we summarize the current data on T cell responses to TAAs in various malignancies, including melanoma, colorectal cancer, leukemia, and breast cancer. T cell responses against various antigens, including melanoma differentiation antigens, carcinoembryonic antigen, epithelial cell adhesion molecule, her-2/neu, Wilms' tumor protein, proteinase 3, NY-ESO-1, and surviving, have been reported in a substantial number of patients. In contrast, other TAAs, including most antigens of the MAGE family, do not usually elicit spontaneous T cell responses. A distinction between direct ex vivo T cell responses and in vitro-generated T cell responses is provided because in vitro stimulation results in quantitative and functional changes of T cell responses. The possible role of TAA-specific T cells in immunosurveillance and tumor escape and the implications for immunological treatment strategies are discussed. Naturally occurring T cells against TAAs are a common phenomenon in tumor patients. Understanding the mechanisms and behavior of natural TAA-specific T cells could provide crucial information for rational development of more efficient T cell-directed immunotherapy.

Immunologic adjuvants are used to augment the immunogenicity of MHC class I-restricted peptide vaccines, but this effect has rarely been systematically evaluated in a clinical trial. We have investigated, in a phase I study, whether addition of the 2 adjuvants GM-CSF and KLH can enhance the T-cell response to MHC class I peptide vaccines. Forty-three high-risk melanoma patients who were clinically free of disease received 6 vaccinations with MHC class I-restricted tyrosinase peptides alone, with either GM-CSF or KLH or with a combination of both adjuvants. The primary end point was induction of tyrosinase-specific T cells, and serial T-cell monitoring was performed in unstimulated peripheral blood samples before and after the second, fourth and sixth vaccinations by ELISPOT assay. Tyrosinase-specific IFN-gamma-producing T cells were detected as early as 2 weeks after the second vaccination in 5 of 9 patients vaccinated with tyrosinase peptides in combination with GM-CSF and KLH but not in any patient vaccinated with tyrosinase peptides without adjuvants or in combination with either adjuvant alone. After 6 vaccinations, tyrosinase-specific T cells were found in patients immunized with peptides either without adjuvants (3 of 9 patients) or in combination with the single adjuvant GM-CSF (4 of 9 patients) but not with KLH (0 of 10 patients). Our results suggest that addition of either GM-CSF or KLH as a single adjuvant has little impact on the immunogenicity of tyrosinase peptides. The combined application of GM-CSF and KLH was associated with early induction of T-cell responses.

The identification and characterization of viral epitopes across the Human Leukocyte Antigen (HLA) polymorphism is critical for the development of actives-specific or adoptive immunotherapy of virally-mediated diseases. This work investigates whether cytokine mRNA transcripts could be used to identify epitope-specific HLA-restricted memory T lymphocytes reactivity directly in fresh peripheral blood mononuclear cells (PBMCs) from viral-seropositive individuals in response to ex vivo antigen recall. PBMCs from HLA-A*0201 healthy donors, seropositive for Cytomegalovirus (CMV) and Influenza (Flu), were exposed for different periods and at different cell concentrations to the HLA-A*0201-restricted viral FluM158–66 and CMVpp65495–503 peptides. Quantitative real time PCR (qRT-PCR) was employed to evaluate memory T lymphocyte immune reactivation by measuring the production of mRNA encoding four cytokines: Interferon-γ (IFN-γ), Interleukin-2 (IL-2), Interleukin-4 (IL-4), and Interleukin-10 (IL-10). We could characterize cytokine expression kinetics that illustrated how cytokine mRNA levels could be used as ex vivo indicators of T cell reactivity. Particularly, IFN-γ mRNA transcripts could be consistently detected within 3 to 12 hours of short-term stimulation in levels sufficient to screen for HLA-restricted viral immune responses in seropositive subjects. This strategy will enhance the efficiency of the identification of viral epitopes independently of the individual HLA phenotype and could be used to follow the intensity of immune responses during disease progression or in response to in vivo antigen-specific immunization.

The development of specific immunotherapeutic approaches to cancer treatment is making progress. However, a crucial clinical break-through in cancer vaccination is still missing. Spontaneous clinical remissions of malignancies are a rare phenomenon. The mechanisms are not clear but there are remissions described in correlation with bacterial infections. The use of bacteria or bacterial components might be a promising path for non-specific immunotherapy of tumors and might be a helpful adjuvant for specific immunotherapy. Here, we briefly review spontaneous remissions of various malignancies in humans, the impact of bacterial infection on tumor regression and the utilization of bacterial components in clinical trials.

Wilms tumor gene product WT1 and proteinase 3 are overexpressed antigens in acute myeloid leukemia (AML), against which cytotoxic T lymphocytes can be elicited in vitro and in murine models. We performed this study to investigate whether WT1- and proteinase 3-specific CD8 T cells spontaneously occur in AML patients. T cells recognizing HLA-A2.1-binding epitopes from WT1 or proteinase 3 could be detected ex vivo in 5 of 15 HLA-A2–positive AML patients by interferon-γ (IFN-γ) ELISPOT assay and flow cytometry for intracellular IFN-γ and in 3 additional patients by flow cytometry only. T cells producing IFN-γ in response to proteinase 3 were further characterized in one patient by 4-color flow cytometry, identifying them as CD3+CD8+CD45RA+ CCR7−T cells, resembling cytotoxic effector T cells. In line with this phenotype, most of the WT1- and proteinase-reactive T cells were granzyme B+. These results provide for the first time evidence for spontaneous T-cell reactivity against defined antigens in AML patients. These data therefore support the immunogenicity of WT1 and proteinase 3 in acute leukemia patients and the potential usefulness of these antigens for leukemia vaccines.

We report here on 2 patients who received adjuvant vaccination with an HLA-A2- or HLA-A24-restricted tyrosinase peptide, respectively, and GM-CSF for frequently relapsing stage IV melanoma. Following resection of metastases and irradiation of brain metastases in 1 patient, both patients were without evidence of disease when receiving the first vaccination. While the patients had had 9 and 12, respectively, mostly s.c., relapses during the 3 years before vaccination, they experienced freedom from relapse for more than 2 years after vaccination. We found a T-cell response to the vaccine peptide in both patients in the peripheral blood by ex vivo IFN-gamma ELISPOT assay. The T-cell population could be further characterized by 4-color flow cytometry in 1 patient, showing that the majority of the peptide-specific CD3(+)CD8(+)IFN-gamma(+) T cells were granzyme B-positive and CCR-7-negative, characterizing them as effector T cells with the ability to mediate cytotoxicity and migrate to inflamed tissues. In this patient also, augmentation of the T-cell response to autologous tumor cells by vaccination could be detected. A single-site postvaccination relapse occurred in both patients, showing downregulation of tyrosinase expression in 1 patient, while normal expression levels for tyrosinase, MHC class I antigens and components of the antigen-processing machinery were found in the other patient. These results suggest that peptide vaccination resulted in a prolonged relapse-free interval in these high-risk patients.

In previous studies CD8+ T cells specific for melanocyte antigens have been frequently found in melanoma patients responding to interleukin-2 (IL-2)-based therapies. In our study we analyzed the suitability of using circulating T cells from melanoma patients with clinical response after IL-2-based therapy to identify novel T-cell epitopes from defined tumor antigens. Using unstimulated peripheral blood mononuclear cells and the interferon-gamma (IFN-gamma) ELISPOT assay, we studied CD8(+) T-cell responses against 5 peptides from the tumor antigen tyrosinase (Tyr) selected by epitope prediction using an HLA-A1-binding computer algorithm. T cells specifically secreting IFN-gamma in response to 3 of these 5 peptides, namely, Tyr (454-463), Tyr (146-156) and Tyr (243-251), could be detected in 4 of 4 HLA-A1-positive patients with clinical response. In contrast, no T-cell responses against these peptides were seen in 6 HLA-A1-positive melanoma patients with progressive disease and in 8 healthy subjects. We could generate specific cytotoxic T lymphocytes (CTL) against Tyr (454-463) using peptide-pulsed autologous dendritic cells as antigen-presenting cells. The induced CTLs efficiently killed melanoma cells that express HLA-A1 and tyrosinase. The peptides Tyr (146-156) and Tyr (243-251) had recently been identified as CTL epitopes by other groups. Further ex vivo characterization of the T cells reactive against the novel epitope Tyr (454-463) in 1 patient by multicolor flow cytometry showed specific CD3+/CD8+/IFN-gamma+ T cells with frequencies of up to 0.41% of the CD3+/CD8+ T-cell population. Most of this T-cell population also expressed granzyme B. Our data confirm that in patients with tumor regressions induced by immunotherapy or chemoimmunotherapy circulating T cells reactive with tyrosinase epitopes can frequently be detected. Peripheral blood T cells from such patients are a valuable source for screening peptides selected by epitope prediction This strategy facilitates the rapid identification of immunogenic T-cell epitopes that are probable targets of immune-mediated tumor rejection.

To defend the host from malignancies, the immune system can spontaneously raise CD8(+) T-cell responses against tumor antigens. Investigating the functional state of tumor-reactive cytolytic T cells in cancer patients is a key step for understanding the role of these cells in tumor immunosurveillance and for evaluating the potential of immunotherapeutic approaches of vaccination against cancer. In this study we identified a subset of circulating tumor-reactive CD8(+) T lymphocytes, which specifically secreted IFN-gamma after exposition to autologous tumor cell lines in stage IV metastatic melanoma patients. Additional phenotypic characterization using multicolor flow cytometry revealed that a significant fraction of these cells were CD45RA(+)CCR7(-), a phenotype that has been proposed recently to characterize cytolytic effectors potentially able to home into inflamed tissues. In the case of an HLA-A2-expressing patient, the antigen specificity of this population was identified by using HLA-A2/peptide multimers incorporating a tyrosinase-derived peptide. Consistently with their phenotypic characteristics, A2/tyrosinase peptide multimer(+) CD8(+) T cells, isolated by cell sorting, were directly lytic ex vivo and able to specifically recognize tyrosinase-expressing tumor cells. Overall, these results provide the first evidence that a proportion of melanoma patients have circulating tumor-reactive T cells, which are lytic effectors cells.

The functional status of circulating vaccine-induced, tumor-specific T cells has been questioned to explain their paradoxical inability to inhibit tumor growth. We enumerated with HLA-A*0201/peptide tetramers (tHLA) vaccine-elicited CD8+ T cell precursor frequency among PBMC in 13 patients with melanoma undergoing vaccination with the HLA-A*0201-associated gp100:209–217(210 M) epitope. T cell precursor frequency increased from undetectable to 12,400 ± 3,600 × 106 CD8+ T cells after vaccination and appeared heterogeneous according to previously described functional subtypes: CD45RA+CD27+ (14 ± 2.6% of tHLA-staining T cells), naive; CD45RA−CD27+ (14 ± 3.2%), memory; CD45RA+CD27− (43 ± 6%), effector; and CD45RA−CD27− (30 ± 4.1%), memory/effector. The majority of tHLA+CD8+ T cells displayed an effector, CD27− phenotype (73%). However, few expressed perforin (17%). Epitope-specific in vitro stimulation (IVS) followed by 10-day expansion in IL-2 reversed this phenotype by increasing the number of perforin+ (84 ± 3.6%; by paired t test, p < 0.001) and CD27+ (from 28 to 67%; by paired t test, p = 0.01) tHLA+ T cells. This conversion probably represented a change in the functional status of tHLA+ T cells rather than a preferential expansion of a CD27+ (naive and/or memory) PBMC, because it was reproduced after IVS of a T cell clone bearing a classic effector phenotype (CD45RA+CD27−). These findings suggest that circulating vaccine-elicited T cells are not as functionally active as inferred by characterization of IVS-induced CTL. In addition, CD45RA/CD27 expression may be more informative about the status of activation of circulating T cells than their status of differentiation.

7005^ Background: Blinatumomab, an investigational bispecific T-cell engaging (BiTE) antibody that directs cytotoxic T-cells to CD19-expressing target cells, has shown antileukemia activity in an e...

Meeting abstracts BiTE® antibodies are novel recombinant single chain Ig domain constructs that leverage the endogenous cytotoxic potential of polyclonal T cells to target malignant cells by utilizing the specific binding properties of variable domains from two different antibodies. Antibody-based

The detection of antigen-induced IFNgamma secretion at the single cell level can be used to identify and enumerate antigen-reactive T cells from peripheral blood. This study was performed to analyze the suitability of T cell enumeration by flow cytometry in comparison with the ELISPOT assay. Peripheral blood mononuclear cell (PBMC) samples from six HLA-A2+ healthy subjects were analysed for the frequency of influenza-reactive CD8+ T cells by flow cytometry detecting either intracellular IFNgamma (IC-FC) or secreted IFNgamma (S-FC). All samples were also analysed by IFNgamma ELISPOT assay. The frequency of influenza peptide-reactive T cells determined by IC-FC was 0.01 to 0.34% of CD8+ T cells and by ELISPOT assay 0.02 to 0.23% of CD8+ T cells (n=6 subjects) with a high inter-assay reproducibility and a close correlation between the assays (r=0.77, P

To determine whether circulating tumor-reactive T cells are present in melanoma patients, unstimulated T cells from peripheral blood were tested for recognition of HLA-A2- or HLA-A1-matched melanoma cell lines using the ELISPOT assay. Eleven out of 19 patients with metastatic melanoma had a T-cell response with up to 0.81%, 0.78%, 0.53%, 0.12%, 0.10%, 0.09%, 0.07%, 0.06%, 0.06%, 0.04%, and 0.04% of peripheral blood mononuclear cells (PBMC) secreting IFNγ upon exposure to various HLA-A2- or HLA-A1-matched melanoma cell lines. These T-cell responses were mediated by CD8+ T cells and could specifically be blocked by an anti-HLA-A2 antibody in HLA-A2-positive patients. Separation experiments performed in one melanoma patient showed tumor-reactive T cells in both the CD8+ effector T cell (CD45RA+/IFNγ+) as well as the CD8+ memory T-cell compartment (CD45RO+/IFNγ+). In 3 out of 5 patients, in whom autologous cell lines were available, similar frequencies of T cells in response to HLA-A1- or HLA-A2-matched allogeneic and autologous tumor cells were observed, while 2 patients had a T-cell response restricted to either the autologous or the allogeneic cell lines. These results give evidence for the presence of tumor-reactive CD8+ T cells in more than half of melanoma patients tested. Although some of these patients have clinical evidence for an immunological-mediated tumor control, several patients have growing tumors suggesting presence of escape mechanisms. Int. J. Cancer 87:659–664, 2000. © 2000 Wiley-Liss, Inc.

Introduction Blinatumomab, a bispecific T-cell engager (BiTE®) that redirects cytotoxic T cells to CD19+ B-lineage cells, has shown anticancer activity and acceptable toxicity in a phase 1 study in patients with relapsed/refractory non-Hodgkin lymphoma, including diffuse large B-cell lymphoma (DLBCL). Using a stepwise dose escalation treatment, the overall response rate (ORR) in patients with DLBCL was 55% (Goebeler et al. Hematol Oncol 2013;31[suppl 1]:197). This open-label phase 2 study has been initiated to investigate the efficacy and tolerability of blinatumomab in patients with relapsed/refractory DLBCL, comparing stepwise dose escalation with constant target dosing. Methods In this ongoing study, eligible patients must be ≥18 years of age, must have relapsed/refractory DLBCL and Eastern Cooperative Oncology Group performance status ≤2. Blinatumomab is administered by continuous intravenous infusion over 8 weeks. In part 1 of the study, two cohorts were evaluated using a double-step or flat dose escalation regimen, respectively, in order to achieve the target dose of 112 μg/d. Part 2 will investigate the selected treatment schedule from part 1. Data from part 1 (cohorts I and II) are presented herein. Patients in cohort I received stepwise blinatumomab dosing of 9, 28, and 112 μg/d during weeks 1, 2, and thereafter, respectively; patients in cohort II received blinatumomab at 112 μg/d throughout. After a 4-week treatment-free period, patients achieving an objective response were permitted to receive a 4-week consolidation cycle. All patients received prophylactic dexamethasone. The primary endpoint is ORR by Cheson (2007) revised response criteria for malignant lymphomas. Results To date, 11 patients have been enrolled and treated in part 1 of the study: nine in cohort I and two in cohort II. The median age was 73 years (range, 55–85); 64% of patients were women. Six (55%) patients had received ≥3 lines of previous systemic antitumor therapy; study treatment was given as fourth-line (median) systemic treatment. Three patients had received autologous hematopoietic stem cell transplantation. At the time of this analysis, seven patients were evaluable for response (cohort I, n=6; cohort II, n=1). The ORR based on independent radiological assessment was 57% (cohort I: complete response, n=1; partial response, n=2; cohort II: partial response, n=1). Three patients had progressive disease (all in cohort I). Four patients were not evaluable for ORR per protocol definition: early treatment discontinuation after Conclusions In this ongoing phase 2 study, blinatumomab was tolerable and showed antitumor activity in adult, heavily pretreated patients with relapsed/refractory DLBCL. Part 1 of the study established a recommended blinatumomab dose for this patient population. The study continues to enroll patients in part 2 (cohort III). Disclosures: Libicher: Amgen Inc.: Consultancy. Degenhard:Amgen Research (Munich) GmbH: Employment; Amgen Inc.: Equity Ownership. Stieglmaier:Amgen Research (Munich) GmbH: Employment; Amgen Inc.: Equity Ownership. Zhang:Amgen Inc.: Employment; Amgen Inc.: Equity Ownership. Nagorsen:Amgen Inc.: Employment; Amgen Inc.: Equity Ownership. Bargou:University of Würzburg: Consultancy; University of Würzburg: Honoraria; Amgen Inc.: patent, patent Other.

Abstract 1637 Blinatumomab (MT103) is a single-chain bispecific antibody construct with specificity for CD19 and CD3 belonging to the class of bispecific T cell engager (BiTE®). A phase I trial with indolent and mantle cell lymphoma patients established a maximal tolerable dose (MTD) at 60 μg/m2/d. The trial was subsequently amended to evaluate blinatumomab in patients with diffuse large B cell lymphoma (DLBCL). Patients were treated by 4–8-week continuous i.v. administration with the following dosing regimen: first week at 5 μg/m2/d, second week at 15 μg/m2/d and for the remaining treatment period at 60 μg/m2/d. Two cohorts each with 6 DLBCL patients were enrolled. The two cohorts solely differed by the dose and schedule of corticosteroid medication administered at the beginning of blinatumomab infusion for mitigation of adverse events. In the first cohort 100 mg prednisolone was applied 1 hour prior to start; and in the second cohort patients received dexamethasone on days 1, 2, and 3. Three sequential patients received dexamethasone also 6–12 hours prior to start of infusion. Out of the twelve patients, 5 were male and 7 female. The median age was 57 years (range from 26 to 78 years). Patients had received a median of 4 prior regimens (range from 2–6). All patients had been exposed to rituximab. Eight of the 12 patients had undergone autologous stem cell transplantation (ASCT). International prognostic index (IPI) at screening ranged from 1 to 3 with a median of 2. The most common clinical adverse events (AEs) regardless of causality (>30%) were pyrexia (81.8%), fatigue (54.5%), constipation (36.4%), headache (36.4%), tremor (36.4%) and weight increase (36.4%). The most frequent laboratory AEs regardless of causality (>30%) were hyperglycemia (63.6%), lymphopenia (54.5%), C-reactive protein increase (45.5%), gamma-glutamyltransferase increase (45.5%) and thrombocytopenia (36.4%). Most AEs occurred early and were reversible. Four of 12 patients discontinued infusion due to fully reversible CNS events, 2 of which qualified as dose limiting toxicities (DLTs). Although just one DLT (reversible CNS event grade 3) occurred in the prednisolone cohort, a further cohort applying prophylactic dexamethasone was opened to optimize management of CNS events. A further refinement of the dexamethasone schedule, starting longer time prior to start of blinatumomab, was introduced after one early patient in the cohort receiving dexamethasone had experienced a reversible CNS event leading to discontinuation. All three patients treated in this manner completed the first blinatumomab cycle without discontinuations. Only one showed a grade 1 tremor, and no other CNS AEs were reported in these three patients. Two of 12 patients were not exposed to 60 μg/m2/d due to early discontinuations and 1 patient is too early in treatment for response evaluation. Five out of the remaining 9 evaluable patients (56%) showed objective clinical responses (4 CR/CRu; 1 PR). Three out of the 5 patients with CR/CRu or PR had prior ASCT. Two patients achieved objective responses (1 CR, 1 PR) despite of discontinuation at 60 μg/m2/d. The median response duration is +182 days (longest current duration +428 days), with 4 out of 5 responses still ongoing. Further evaluation of the last cohort will refine the recommended phase II dose, and the intensity and timing of dexamethasone comedication. The observation of lasting CRs after blinatumomab monotherapy in DLBCL patients is promising and warrants further exploration in a phase II study. Disclosures: Krause: Micromet: Research Funding. Mackensen:Micromet Inc.: Research Funding. Topp:Micromet: Consultancy, Honoraria. Scheele:Micromet Inc.: Employment, Equity Ownership, Patents & Royalties. Nagorsen:Micromet Inc.: Employment, Equity Ownership, Patents & Royalties. Zugmaier:Micromet: Employment. Degenhard:Micromet Inc: Employment. Schmidt:Micromet AG: Employment. Kufer:Micromet Inc: Employment, Equity Ownership. Libicher:Micromet Inc.: Consultancy, Honoraria. Bargou:Micromet: Consultancy, Honoraria.

Abstract 2880 Blinatumomab (MT103) is a single-chain bispecific antibody construct with specificity for CD19 and CD3 belonging to the class of bispecific T cell engager (BiTE®). We have previously reported that blinatumomab delivered as single agent to patients with relapsed NHL and B-precursor acute lymphoblastic leukemia by continuous intravenous (CIV) infusion over 4–8 weeks depleted peripheral B cells, expanded T effector cells, and resulted in clinical responses. In this phase I study 52 patients (41 males, 11 females) have been treated, 21 with FL, 21 with MCL and 10 with other subtypes of lymphoma (MZL, SLL, LPL, CLL). Patients have received a median of 3 prior regimens (range 1 to 12). Ninety percent of the patients had prior exposure to rituximab and 45% to fludarabine. Patients were treated at a dose range from 0.5 to 90 μg/m2/d. The most common adverse events (AEs) occurred early, were transient, reversible and did not require discontinuation of treatment. The most common clinical AEs regardless of causality were pyrexia (75%), headache (45%) and fatigue (37%). The most common laboratory abnormality AEs regardless of causality were lymphopenia (75%), leukopenia (57%), thrombocytopenia (39%), C-reactive protein increase (53%) and fibrin D dimer increase (37%). The medically most important AEs that resulted in permanent discontinuation were CNS events. Signs and symptoms observed included kinetic tremor, speech impairment, disorientation, apraxia and seizure. All CNS events were fully reversible without sequelae and no pathological findings by MRI imaging were reported. Out of the 52 patients treated, 9 had to discontinue treatment permanently in the first cycle due to these CNS events. At a dose of 90 μg/m2/d two DLTs were observed which were CNS events during the DLT period of the first 2 weeks of treatment. Therefore 60μg/m2/d is the currently recommended dose. A low B to T cell ratio ( Overall 18 patients with FL or MCL were treated with constant or step dosing regimens at or reaching 60 μg/m2/d dose level. Eight out of the 9 patients with constant dosing showed an objective response by Cheson criteria. All responders had a high B:T cell ratio. One patient with a low B:T cell ratio was discontinued due to a CNS AE. Six out of 9 patients with low B:T cell ratio enrolled for step dosing showed an objective response. One patient (single step dosing) discontinued treatment because of a CNS AE and 2 patients discontinued because of tumor progression. As of June 15th 2010, response duration ranged from 1 to 30+ months. Median for response duration was 26 months with 5 out of 14 responses ongoing. Table 1: Response evaluation in patients enrolled in 60 μg/m2/d cohorts. Dose Level Cohort Patients Complete Response Partial Response Overall Response FL MCL Total FL MCL Total FL MCL Total FL MCL Total 60 μg/m2/d Constant 6 3 9 2 1 3 4 1 5 6 2 8 5 or 15 then 60 μg/m2/d Single step 3 3 6 2 0 2 1 1 2 3 1 4 5/15/60 μg/m2/d Double Step 2 1 3 0 0 0 2 0 2 2 0 2 Total 11 7 18 4 1 5 7 2 9 11 3 14 These data confirm high single-agent activity of blinatumomab with long lasting remissions. Initial data with double step dosing demonstrate that this approach maintains clinical activity without discontinuations due to CNS AEs. Evaluation of safety and clinical efficacy of this uniform double step schedule including other subtypes of NHL is ongoing. Disclosure: Scheele: Micromet Inc.: Employment. Zugmaier:Micromet Inc.: Employment. Nagorsen:Micromet Inc.: Employment. Klinger:Micromet Inc.: Employment. Schmidt:Micromet Inc.: Employment. Klappers:Micromet. Inc: Employment. Kufer:Micromet Inc.: Employment. Bargou:Micromet Inc.: Consultancy.

Abstract 174 Blinatumomab, a bispecific, T cell-engaging (BiTE®) antibody, can effectively redirect T cells for highly selective lysis of CD19+ target cells. The B-cell differentiation antigen CD19 is a marker for B-ALL cells. In B-lineage acute lymphoblastic leukemia (ALL), persistence or relapse of minimal residual disease (MRD) is an independent poor prognostic factor, and new treatments are urgently needed. MRD relapse during or after maintenance treatment in adult standard risk patients generally heralds a hematological relapse in 90% of patients. A phase 2 study was conducted to determine the efficacy of blinatumomab in ALL patients with MRD persistence or relapse (MRD level Between May 2008 and November 2009, 21 patients (16 Ph-negative; 2 patients with MLL-AF4; 5 patients with Ph+ ALL) were enrolled. The cut-off date for data analysis was May 15, 2010. Patients received between 1 and 7 cycles of blinatumomab (total of 66 cycles). Transient pyrexia (100%) and chills (43%) were the most common clinical AEs. There were no blinatumomab related deaths. Sixteen patients became MRD-negative. One patient was not evaluable due to a grade 3 adverse event (AE) leading to treatment discontinuation. Of the responding patients, 13 had never before achieved a negative MRD status on chemotherapy. Regardless of their MRD level prior to study treatment, all 16 (13/15 patients with Ph− and 3/5 patients with Ph+ ALL) became MRD-negative after the first cycle of blinatumomab. Nine patients were enrolled with a MRD load >10-2 prior to study treatment and all reached complete MRD response. Thirteen out of 16 patients with persisting MRD prior to study treatment and 3 out of 4 patients with MRD relapse showed complete MRD response. Overall relapse-free survival (RFS) currently is 78% at a median follow up of 405 days. RFS is 100% for the 8 patients who received subsequent allogeneic stem cell transplantation (median follow up 434 days). Blinatumomab is a highly active treatment for patients with MRD-positive B-lineage ALL after intensive chemotherapy and has an acceptable safety profile. T cells engaged by blinatumomab seem capable of eradicating chemotherapy-resistant tumor cells in bone marrow that otherwise might cause clinical relapse. A long RFS suggests that blinatumomab may improve outcome in patients with B-precursor ALL. A multicenter international study of blinatumomab in patients with MRD-positive B-lineage ALL has been initiated. Disclosures: Zugmaier: Micromet Inc.: Employment. Degenhard:Micromet Inc.: Employment. Schmidt:Micromet Inc.: Employment. Scheele:Micromet Inc.: Employment. Kufer:Micromet Inc.: Employment. Klinger:Micromet Inc.: Employment. Nagorsen:Micromet Inc.: Employment. Bargou:Micromet Inc.: Consultancy.

Abstract 840 Introduction: In patients with B-precursor ALL, presence of MRD after induction therapy or at any time point later predicts a hematological relapse, despite continued intensive chemotherapy or/and an allogeneic hematological stem cell transplantation (HSCT). Blinatumomab (MT103) targets the CD19 antigen, and is a member of a novel class of bispecific BiTE® antibodies that redirect T cells for lysis of target cells. A phase II study was conducted in collaboration with the German Multicenter Study Group on Adult Lymphoblastic Leukemia (GMALL) in patients with MRD-positive B precursor ALL. Methods: B–precursor ALL patients in complete hematological remission with either persistent or reappeared MRD at any time after consolidation I of front-line therapy were included. One treatment cycle of blinatumomab is a 4-week continuous i.v. infusion, which can be followed by allogeneic HSCT or in case of response by repeated consolidation cycles of blinatumomab with 2-week treatment-free intervals. The dose level at enrollment is 15 μg/m2/day. In patients, who do not respond within four cycles of treatment, the dose can be increased to 30 μg/m2/day. Molecular response is assessed by quantitative PCR of either individual rearrangements of immunoglobulin/TCR-genes or specific genetic aberrations such as bcr/abl or MLL-AF4. Results: Nineteen patients have been treated to date and 16 patients are already evaluable for response. Thirteen of 16 evaluable patients went into molecular complete remission (CR) already after one cycle of blinatumomab. Three patients had a stable MRD level. Of note, 10 of the responding 13 patients had never achieved a molecular CR before blinatumomab treatment despite multiple treatment cycles including tyrosine kinase inhibitors in case of Ph-positive ALL. Two patients in molecular CR had an extra-medullary relapse one in testis and one in cerebro-spinal fluid, both representing immunological niches with limited accessibility for T cells. One patient with stable MRD level had a medullary relapse. All other patients are still relapse-free. None of the patients with molecular CR has shown a medullary relapse to date. The maximum follow-up of molecular CR has been 12 months. Most common adverse events (AEs) included lymphopenia, pyrexia, leucopenia and hypoimmunoglobulinemia. Only one patient had to be discontinued because of a fully reversible epileptical seizure. All other AEs resolved during treatment. Overall, treatment with blinatumomab was well tolerated. Response data of all 21 patients will be presented at ASH. Conclusions: Treatment with blinatumomab converted MRD-positive B–precursor ALL into molecular CR in 13 of 16 evaluable patients with refractory disease as indicated by persistent MRD after intensive chemotherapy. This amounts to a response rate of 81% providing thus the rationale for introducing blinatumomab as a novel agent in the treatment of B–precursor ALL. Disclosures: Zugmaier: Micromet: Employment, Equity Ownership. Goekbuget:Micromet: Consultancy, Research Funding. Kufer:Micromet: Employment, Equity Ownership, Patents & Royalties. Klinger:Micromet: Employment, Equity Ownership. Degenhard:Micromet: Employment, Equity Ownership. Baeuerle:Micromet: Employment, Equity Ownership. Schmidt:Micromet: Employment, Equity Ownership. Nagorsen:Micromet: Employment, Equity Ownership. Riethmueller:Micromet: Consultancy, Equity Ownership. Bargou:Micromet: Consultancy, Equity Ownership, Patents & Royalties.

Abstract 4793 Blinatumomab is a CD19/CD3-bispecific antibody construct of the bispecific T cell engager (BiTE®) class showing as single agent a high rate and duration of responses in patients with relapsed non-Hodgkin lymphoma (NHL) and B-precursor acute lymphocytic leukemia (ALL). Blinatumomab has a favorable safety profile with exception of a subset of patients developing neurological adverse events (AEs) during the first days of treatment, such as confusion, speech impairment or cerebellar symptoms. Thus far, all relevant neurological AEs (11 out of 48 patients) were transient, fully reversible and resolved without sequelae within 3 to 72 hours after stop of infusion. In no case, pathological findings were seen upon cranial magnetic resonance imaging. Despite treatment discontinuation, 4 patients with neurological AEs have achieved an objective lymphoma remission. Analysis of cerebrospinal fluid (CSF) taken within hours after stop of infusion showed detectable levels of blinatumomab in the majority of affected patients, while in one patient without neurological symptoms no blinatumomab was detectable in CSF during infusion. Moreover, increased levels of albumin and T lymphocytes in CSF support a disturbance of the blood brain barrier (BBB) as a possible underlying event. Analyses of patient serum samples for angiopoetin-2 and S100b are ongoing to investigate whether levels of the endothelial stress and BBB integrity marker, respectively, correlate with neurological AEs. In a retrospective analysis of 39 NHL patients, a baseline B cell to T cell (B:T) ratio in peripheral blood at or below 1:10 was identified as the only predictive factor for the subsequent occurrence of neurological AEs. The predictive value was then prospectively confirmed in 8 additional patients. Of note, ALL patients –despite very low B:T ratios– rarely showed neurological AEs, which may relate to previous intrathecal chemotherapy depleting target cells in the brain. Potential mechanisms for the neuroprotective effect of peripheral B cells are being investigated. In conclusion, we identified a simple measure to prospectively identify patients at risk of developing neurological AEs after onset of blinatumomab treatment. Mitigating measures are currently tested in these high-risk patients in order to avoid discontinuation of treatment. Disclosures: Zugmaier: Micromet: Employment, Equity Ownership. Nagorsen:Micromet: Employment, Equity Ownership. Klinger:Micromet: Employment, Equity Ownership. Bargou:Micromet: Consultancy, Patents & Royalties. Baeuerle:Micromet: Employment, Equity Ownership. Kufer:Micromet: Employment, Equity Ownership, Patents & Royalties.

Abstract 2723 Poster Board II-699 Indolent and mantle cell lymphoma (MCL) are predominantly treated with chemotherapy or a combination of chemotherapy with monoclonal antibodies. Despite high initial response rates, eventually almost all patients however relapse, leaving the disease incurable. Moreover, with increasing numbers of regimens administered, the responsiveness of patients is reduced. Blinatumomab is a single-chain bispecific antibody construct with specificity for CD19 and CD3, belonging to the class of bispecific T cell engager (BiTE®). Here, we report on patients in an ongoing phase 1 trial treated at a dose of 60 μg/m2/d for 4–8-week by continuous i.v. infusion with single-agent blinatumomab. In total, 12 patients with indolent mainly follicular lymphoma or MCL were treated at 60 μg/m2/d during the first treatment cycle. 11/12 patients showed an objective response (7 PR and 4 CR). As of July 2009, median response duration was 12 months with 6 out of 11 responses still ongoing. The single non-responding patient experienced a reversible, neurological adverse event leading to early discontinuation of treatment. Of the 11 responders, one patient developed a port infection and 4 patients showed neurological symptoms, which were all fully reversible. In order to mitigate neurological adverse events during first dosing, which can occur in a defined subset of patients, patients were treated for 1–2 weeks with a lower initial dose (5 and/or 15 μg/m2/d) followed by a maintenance dose of 60 μg/m2/d. A lower starting dose appeared to ameliorate initial adverse events to an extent that treatment could be continued without interruption. Taken together, our data confirm a high single-agent activity of 60 μg/m2/d blinatumomab infused for 4–8 week with long lasting remissions and a favorable risk/benefit profile. The confirmed dose will be considered for further clinical development of blinatumomab in follicular lymphoma and MCL. New data on patients treated with a dose of 90 μg/m2/d will be presented. Disclosures: Nagorsen: Micromet: Employment, Equity Ownership. Zugmaier:Micromet: Employment, Equity Ownership. Schmidt:Micromet: Employment, Equity Ownership. Klappers:Micromet: Employment, Equity Ownership. Baeuerle:Micromet: Employment, Equity Ownership. Kufer:Micromet: Employment, Equity Ownership, Patents & Royalties. Bargou:Micromet: Consultancy, Patents & Royalties.

Abstract 4798 Blinatumomab is a single-chain bispecific antibody construct with specificity for CD19 and CD3 belonging to the class of bispecific T cell engager (BiTE®). It has shown high response rates as single agent after treatment of patients with indolent and mantle cell lymphoma and B-precursor acute lymphocytic leukemia. We here report from an ongoing phase 1 trial in non-Hodgkin lymphoma patients on transient laboratory findings seen upon start of continuous i.v. infusion of blinatumomab. The vast majority of adverse events (AEs) during 4-8 weeks of infusion occurred within the first two days of treatment. Lymphopenia (up to CTCAE grade 4) was the most frequent early laboratory AE, and can be explained by rapid B cell depletion and initial redistribution of T cells. Except for B cell decline, all laboratory parameters were transient and normalized within days during continued treatment. Dose-dependent increases were seen for AST and ALT (maximally grade 1/2), CRP, and D-dimer during the first days of treatment. Dose-dependent declines during the first days of treatment were observed for platelets and hemoglobin, which normalized after a few days. Of note, no drug-related thromboembolic events were observed. Serum levels of cytokines IL-6, IL-2, IFNγ, and IL-10 showed small dose-dependent increases, which reversed to baseline within hours. No association between cytokine levels and thrombocytopenia was found. Moreover, there was no evident correlation between any laboratory abnormality and clinical adverse event observed during the first days of treatment. The laboratory AEs induced by blinatumomab appear to reflect consequences of the onset of redirected target cell lysis, T cell activation and bystander effects. In conclusion, blinatumomab causes characteristic laboratory findings during the first days of treatment, which are transient, not associated with clinically relevant AEs and do not require treatment interruption. Compared to AEs caused by chemotherapy regimens, the laboratory findings observed after start of blinatumomab treatment are considered rather mild. Disclosures: Nagorsen: Micromet: Employment, Equity Ownership. Zugmaier:Micromet: Employment, Equity Ownership. Kufer:Micromet: Employment, Equity Ownership, Patents & Royalties. Klinger:Micromet: Employment, Equity Ownership. Schmidt:Micromet: Employment, Equity Ownership. Klappers:Micromat: Employment, Equity Ownership. Wolf:Micromet: Employment, Equity Ownership. Brandl:Micromet: Employment, Equity Ownership. Baeuerle:Micromet: Employment, Equity Ownership. Bargou:Micromet: Consultancy, Patents & Royalties.

Blinatumomab is designed to link T cells with CD19-expressing target cells resulting in a non-restricted cytotoxic T-cell response and T-cell activation. Several preclinical studies showed that very low concentrations of blinatumomab mediate redirected lysis of human B lymphoma and leukemia cells. A phase I study has demonstrated significant clinical activity of the BiTE antibody in relapsed B-cell non-Hodgkin’s lymphoma (NHL). Based on these results, a phase II dose-escalating study was designed in collaboration with the German Multicenter Study Group on Adult Acute Lymphoblastic Leukemia to investigate efficacy, safety, and tolerability of blinatumomab in ALL patients who achieved a complete hematological remission, but remained MRD-positive. MRD is an independent prognostic factor that reflects primary drug resistance and is associated with a high relapse risk after start of consolidation. MRD was measured with standardized methods either by quantitative detection of individual rearrangements of immunoglobulin or T-cell receptor (TCR) rearrangements, or by bcr-abl fusion transcripts. The study population includes patients with acute B-precursor ALL at a minimum age of 18 years who show a bcr/abl signal above detection limit and/or at least one marker by rearrangement with a sensitivity of ≥10−4. Primary endpoint is the conversion rate to MRD negative status as defined by a bcr/abl signal below detection limit and/or by individual rearrangements of immunoglobulin or T-cell receptor (TCR) genes below 10−4. Secondary endpoints are time to hematological relapse, time to MRD progression, and time to molecular relapse. One treatment cycle of blinatumomab is a 4-week continuous intravenous infusion, which can be followed by allogeneic stem cell transplantation after the first cycle, or by repeated cycles after a 2-week treatment-free interval. MRD status is controlled after each treatment cycle. The starting dose level is 15 microgram/m2/24 hr, which may be escalated to 30 microgram/m2/24 hr and higher dose levels based on clinical activity and safety data. Four patients aged 31, 57, 62, and 65 years have so far been enrolled at the initial dose level of 15 microgram/m2/24 hr. Three out of the 4 patients had MRD by rearrangements at levels of 10−4, 10−3 and 10−1, and one patient had bcr/abl fusion transcripts at a level of 10−4. Three out of the 3 patients with rearrangements turned MRD negative after the first treatment cycle, independently from the level of MRD positivity at baseline. The other patient had stable bcr/abl level without signs of hematological relapse after the initial treatment cycle. Except for fever on the first 3 days of treatment, no clinically significant toxicities were recorded. Conclusion: These preliminary results indicate that treatment with blinatumomab is able to convert MRD positive ALL into an MRD negative status, and that this is well tolerated. Further exploration is warranted, as this may be a promising alternative treatment option especially for ALL refractory to chemotherapy.

Introduction: Blinatumomab (MT103/MEDI-538), a BiTE antibody targeting the CD19 antigen, is a member of a novel class of molecules that redirect T cells for lysis of target cells. A Phase 1 dose escalation study is being conducted in patients with advanced NHL. Methods: Relapsed NHL patients requiring treatment were included. Most patients were pre-treated, with a median of 3 previous chemo/immunotherapy regimens. To date, 7 dose levels ranging from 0.0005 to 0.09 mg/m2/24 h have been tested. Blinatumomab was continuously infused as single agent over a period of 4–8 weeks. Objective responses were assessed by Cheson criteria and centrally reviewed. Results: To date 39 patients have been treated. Most common adverse events (AEs) included lymphopenia, pyrexia, and leukopenia. The majority of AEs (>95%) improved or resolved during treatment. Permanent treatment discontinuation due to AEs occurred in a total of 8 patients, of which 6 had fully reversible CNS events. One patient with a history of nearly fatal sepsis, pre-existing hypogammaglobulinemia and bone marrow affection by chemotherapy, experienced a fatal sepsis 5 weeks after treatment start. Dose-dependent activity was observed in mantle cell lymphoma, follicular lymphoma and CLL with responses observed in 11 out of 27 patients treated at doses of 0.015 mg/m2/24 h and higher. Five of those patients had complete and six had partial responses. At the dose level of 0.060 mg/m2/24 h, 7 out of 7 patients have shown objective responses. Beside one relapse after 14 months, no treatment failure has so far been observed for responders at dose levels of 0.030 mg/m2/24 h and 0.060 mg/m2/24 h. Five patients at these dose levels have ongoing responses for more than 6 months. Interestingly, partial remissions converted into complete remissions in two patients four weeks after end of infusion suggesting either reduction in lesion size due to efflux of a previously expanded T cell pool or prolonged T cell activity. Conclusions: Blinatumomab as single agent induced apparently durable responses in pre-treated B-NHL patients with the highest response rate at a dose level of 0.06 mg/m2/24 h. Recruitment is ongoing.

2570 Background: This phase II study was undertaken to evaluate the immunogenicity and clinical efficacy of vaccination with tyrosinase peptides as adjuvant treatment in melanoma patients after multiple relapses. Methods: Stage III/IV melanoma patients after complete resection of relapses were vaccinated with tyrosinase peptides together with GM-CSF and KLH. Vaccination was given bi-weekly times 4, followed by monthly times 8 in absence of relevant disease progression. Tyrosinase-specific T cells were quantitated and characterized ex vivo by intracellular cytokine flow cytometry. Results: 44 patients were accrued with a median of 5.8 (range 2–25) previous relapses at cutaneous /s.c. only (n = 11), ln (n = 30) and/or visceral sites (n = 7). 11 patients received less than 6 vaccinations due to rapid progression. Of 32 patients evaluable for immunological response 16 (50%) displayed pre-vaccine spontaneous tyrosinase-specific T cells (median 0.32%). In 7 of the 15 patients without pre-existing T cells tyrosinase-specific T cells were induced after 6 vaccinations (median 0.08% CD3+CD8+ T cells) and in 10 of 12 patients after 12 vaccinations (median 0.36%, p = 0.03 compared to 6 vaccinations). Similarly, in 9 of the 16 patients with pre-existing T cell responses the frequency of tyrosinase-specific T cells did increase after 6 cycles (median 0.43 %), but in none of 10 patients with pre-existing T cells an increase was observed after 12 vaccinations (median 0.20%). Both, the spontaneous and the vaccine-induced T cells were predominantly of effector and effector-memory phenotype. With respect to clinical efficacy vaccination was terminated after 3 to 9 cycles due to disease progression in 19 of 44 patients. Of the remaining 25 patients receiving all 12 vaccinations 7 (16% of all 44 enrolled) had a favourable clinical course with cessation of recurrences. Conclusions: Taken together, this study shows high immunogenicity of the vaccine in patients with limited tumor burden associated with clinical efficacy. In case of a spontaneous pre-existing T cell response to the vaccine peptides, however, this could not, or only transiently be augmented. No significant financial relationships to disclose.

Background Mononuclear phagocytes (MPs) stand at the crossroads between the induction of acute inflammation to recruit and activate immune effector cells and the downmodulation of the inflammatory process to contain collateral damage. This decision is extensively modulated by the cytokine microenvironment, which includes a broad array of cytokines whose direct effect on MPs remains largely unexplored. Therefore, we tested whether polarized responses of MPs to pathogens are related to the influence of selected cytokines or represent a mandatory molecular switch through which most cytokines operate.

The identification and characterization of viral epitopes across the Human Leukocyte Antigen (HLA) polymorphism is critical for the development of actives-specific or adoptive immunotherapy of virally-mediated diseases. This work investigates whether cytokine mRNA transcripts could be used to identify epitope-specific HLA-restricted memory T lymphocytes reactivity directly in fresh peripheral blood mononuclear cells (PBMCs) from viral-seropositive individuals in response to ex vivo antigen recall. PBMCs from HLA-A*0201 healthy donors, seropositive for Cytomegalovirus (CMV) and Influenza (Flu), were exposed for different periods and at different cell concentrations to the HLA-A*0201-restricted viral FluM158–66 and CMVpp65495–503 peptides. Quantitative real time PCR (qRT-PCR) was employed to evaluate memory T lymphocyte immune reactivation by measuring the production of mRNA encoding four cytokines: Interferon-γ (IFN-γ), Interleukin-2 (IL-2), Interleukin-4 (IL-4), and Interleukin-10 (IL-10). We could characterize cytokine expression kinetics that illustrated how cytokine mRNA levels could be used as ex vivo indicators of T cell reactivity. Particularly, IFN-γ mRNA transcripts could be consistently detected within 3 to 12 hours of short-term stimulation in levels sufficient to screen for HLA-restricted viral immune responses in seropositive subjects. This strategy will enhance the efficiency of the identification of viral epitopes independently of the individual HLA phenotype and could be used to follow the intensity of immune responses during disease progression or in response to in vivo antigen-specific immunization.

Over the last decade, tumor immunology in general and tumor immunotherapy in particular have made important progress. Thanks to the discovery of tumor-associated antigens (TAA), and the consequent development of active-specific immunization protocols, TAA-specific T-cell responses can now be induced reproducibly in cancer patients. However, clinical responses directly ascribable to TAA-specific T cells occur only occasionally. It is not clear why TAA-specific T cells do not eliminate tumor cells in vivo. This paradoxical coexistence of antigen-bearing tumor cells and antigen-specific T cells constitutes a critical point of current investigation. In recent years, protein-ligand interaction-based methods aimed at the identification and characterization of T cells using antibodies (cell surface phenotyping, intracellular and secreted cytokine detection), or HLA-peptide multimers, have significantly improved the analysis of TAA-specific T cells. These methods, however, seem to have reached the limit of their usefulness. Transcriptional analysis may add sensitivity and resolution and may provide global pictures of the multifactorial requirements for an efficient immune response against tumors. In this review, we describe the use of molecular genetic methods, such as real time qRT-PCR, cDNA microarrays, and TCR repertoire analysis. In addition, we describe techniques for high-fidelity messenger RNA amplification that allow high-throughput analysis of samples obtained from minimal sources, such as HLA/peptide tetramer sorted antigen-specific T cells, laser capture dissection, or fine needle aspirates. Recent work discussed in this review summarizes the complementarity of transcriptional analysis as an essential tool that, in addition to conventional methods, may deepen and broaden the characterization of tumor-specific T cells.