Key Points• BI 836858, an Fc-engineered anti-CD33 antibody, mediates autologous and allogeneic NK cell-mediated ADCC.• Decitabine increases ligands for activating NK receptors potentiating BI 836858 activity, providing a rationale for combination therapy.Acute myeloid leukemia (AML) is the most common type of acute leukemia, affecting older individuals at a median age of 67 years. Resistance to intensive induction chemotherapy is the major cause of death in elderly AML; hence, novel treatment strategies are warranted. CD33-directed antibody-drug conjugates (gemtuzumab ozogamicin) have been shown to improve overall survival, validating CD33 as a target for antibody-based therapy of AML.Here, we report the in vitro efficacy of BI 836858, a fully human, Fc-engineered, anti-CD33 antibody using AML cell lines and primary AML blasts as targets. BI 836858-opsonized AML cells significantly induced both autologous and allogeneic natural killer (NK)-cell degranulation and NK-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). In vitro treatment of AML blasts with decitabine (DAC) or 5-azacytidine, 2 hypomethylating agents that show efficacy in older patients, did not compromise BI 836858-induced NK-cellmediated ADCC. Evaluation of BI 836858-mediated ADCC in serial marrow AML aspirates in patients who received a 10-day course of DAC (pre-DAC, days 4, 11, and 28 post-DAC) revealed significantly higher ADCC in samples at day 28 post-DAC when compared with pre-DAC treatment. Analysis of ligands to activating receptors (NKG2D) showed significantly increased NKG2D ligand [NKG2DL] expression in day 28 post-DAC samples compared with pre-DAC samples; when NKG2DL receptor was blocked using antibodies, BI 836858-mediated ADCC was significantly decreased, suggesting that DAC enhances AML blast susceptibility to BI 836858 by upregulating NKG2DL. These data provide a rationale for combination therapy of Fc-engineered antibodies such as BI 836858 with azanucleosides in elderly patients with AML.

The identification of novel tumor antigens is of extreme importance for effective immunotherapy against cancer. A major obstacle in this field is the limited life span of tumorspecific cytotoxic T lymphocytes (CTLs) in vitro. Therefore we searched for a method to isolate the tumor specificity of these CTLs, i.e., their T-cell receptors (TCRs) and transfer it to an immortalized T-cell line. For this purpose, a TCRnegative Jurkat T-cell line was equipped with a nuclear factor of activated T cells ( Key words: T-cell receptor; tumor antigen; NFAT; CAMEL; cDNA expression cloning; CD8; luciferase; retrovirus; JurkatSince the characterization of the first human tumor-specific antigen in 1991, MAGE-1, 1 a considerable number of other targets for tumor-specific cytotoxic T cells (CTLs) have been identified. Most of these antigens have been cloned from melanoma cells 2 and less frequently from other types of cancer. 3,4 For the development of effective immunotherapy against cancer, the identification of a larger variety of tumor-associated antigens is of great importance, to broaden the range of different tumors that can be treated as well as to prevent the outgrowth of antigen-loss tumor cells after vaccination.Several strategies have led to the identification of tumor-specific T-cell epitopes. Most frequently, cDNA expression cloning has been employed, in which the gene encoding the antigen is identified by transfecting pools of tumor-derived cDNA into recipient cells, followed by screening with the tumor-specific CTL. [5][6][7][8] In another approach, the peptide epitope for the tumor-specific CTL is identified, either by peptide elution and mass spectrometry 9 or by mimicry from designated peptide libraries, 10 although with the latter method no tumor antigens have been identified yet. Several tumor antigens have been found by the method of "reverse immunology." These antigens were initially cloned on the basis of their differential expression in tumors versus healthy tissues and antigenic peptides were subsequently identified by performing in vitro T-cell inductions. 11,12 For efficient elucidation of novel physiologically relevant tumor-specific antigens, large quantities of tumor-specific CTLs are required to allow for multiple screening rounds. In practice, the limited amount of CTLs available often constitutes the major obstacle for the cloning of new tumor antigens. We have sought to circumvent this issue by cloning and transferring the T-cell receptor (TCR) of a tumor-specific T cell to an immortalized T-cell line. In this way, an unlimited source of tumor-specific T cells can be obtained, which facilitates the identification of novel tumor antigens. The feasibility of TCR cloning and transfer has already been demonstrated by others. [13][14][15][16][17] We studied the properties of a TCR, isolated from a characterized CAMEL (CTL-recognized antigen on melanoma)-specific CTL clone, 8 which was transferred into a TCR-negative human leukemia Jurkat T-cell line. To detect TCRmediated activation, the cells were tr...

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Vaccination against tumor Ags may become a promising treatment modality especially in cancer types where other therapeutic approaches fail. However, diversity of tumors requires that a multitude of Ags become available. Differential expression in normal vs cancerous tissues, both at the mRNA and the protein level, may identify Ag candidates. We have previously compared transcripts from squamous cell lung cancer and normal lung tissue using differential display analysis, and found a transcript that was overexpressed in malignant cells and was identical with the calcium-activated chloride channel 2 (CLCA2) gene. We have now selected HLA-A2-restricted peptides from CLCA2, and have generated T cell lines against the CLCA2-derived KLLGNCLPTV, LLGNCLPTV, and SLQALKVTV peptides using in vitro priming. Specificity of T cells was ascertained in ELISPOT assays. The primed T cells also recognized allogeneic tumor cells in an Ag-specific and HLA-restricted fashion. Moreover, peptide LLGNCLPTV was also independently recognized by CD8+ T cells expanded from pancreatic carcinoma/T cell cocultures. CLCA2-specific CD8+ T cells were absent from the peripheral blood of healthy donors. These data indicate that an immune response can be induced against CLCA2, which thus may become an important Ag for anti-tumor vaccination approaches.

1363 Introduction: Acute myeloid leukemia (AML), the most frequent form of adult acute leukemia, is a heterogeneous disease with respect to presentation and clinical outcome. Although there has been progress made in treatment for some subgroups of AML patients, there is still a high medical need to develop new anti-leukemic therapies. CD33 is a myeloid lineage-specific antigen which is expressed on the cell surface of normal myeloid cells and AML blasts. CD33 has been investigated as a target for antibody-based therapies for over a decade, leading to transient approval of Mylotarg®, an antibody-calicheamicin drug conjugate. Mylotarg®has been shown to substantially improve outcome of patients with AML in combination with chemotherapy, however is associated with additional toxicity. Here we describe a novel, unconjugated Fc-engineered antibody to CD33, mAb 33.1, which mediates potent antibody dependent cellular cytotoxicity (ADCC) against AML derived cell lines and malignant AML blasts. Methods: MAb 33.1 is a fully human, Fc-engineered, IgG1-type of antibody which has increased affinity to human Fc-gamma receptor IIIa and binds CD33 with low nanomolar affinity as determined by FACS scatchard analysis. ADCC of mAb 33.1 was determined on a panel of myeloid cell lines and primary AML cells. ADCC assays were performed using peripheral blood mononuclear cells (PBMC) from healthy donors as effector cells and cytotoxicity was determined by LDH release or FACS quantification of 7-AAD-positive cells. Internalization kinetics was assessed by FACS analysis using fluorochrome labelled antibody. Epitope mapping of mAb 33.1 was performed by direct competition experiments and hydrogen/deuterium exchange mass spectrometry (HXMS). Results: Antibody mAb 33.1 is able to mediate potent ADCC on a panel of AML derived cell lines and primary AML blasts in vitro. Direct comparison of mAb 33.1 to the humanized CD33 mAb lintuzumab (HuM195) demonstrates superior cytolytic activity on AML cell lines and primary AML cells. ADCC activity of mAb 33.1 is also evident on AML cell lines and primary AML blasts that do not respond to lintuzumab treatment in vitro. In contrast to lintuzumab, mAb 33.1 shows significantly slower internalization kinetics on HL60 cells, leading to prolonged exposure of antigen/antibody complexes on the cell surface. In direct competition experiments mAb 33.1 is competing with lintuzumab for antigen binding. HXMS experiments revealed that mAb 33.1 recognizes a novel epitope on CD33 different to that of lintuzumab. Conclusion: MAb 33.1 represents a novel, Fc-engineered antibody specific for CD33 which shows decelerated internalization compared to lintuzumab, and which recognizes a novel epitope on CD33 different to that of lintuzumab. MAb 33.1 displays strongly enhanced ADCC against leukemia cell lines and primary AML cells, resulting in significantly higher cytolysis than lintuzumab. MAb 33.1 is considered as a promising candidate for treatment of patients with CD33-positive myeloid malignancies and phase I clinical trials are currently under preparation. Disclosures: Heider: Boehringer Ingelheim: Employment. Konopitzky:Boehringer Ingelheim: Employment. Ostermann:Boehringer Ingelheim: Employment. Lamche:Boehringer Ingelheim: Employment. Kuepcue:Boehringer Ingelheim: Employment. Koessl:Boehringer Ingelheim: Employment. Adam:Boehringer Ingelheim: Employment. Borges:Boehringer Ingelheim: Employment.