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Introduction:Myeloid azurophil granules provide a rich source of intracellular leukemia antigens. Cathepsin G (CG) is a serine protease that has higher expression in AML blasts in comparison to normal myeloid progenitors. Based on the unique biology of HLA-A*0201 (HLA-A2), in which presentation of leader sequence (LS)-derived peptides is favored, we focused on the LS-CG-derived peptide CG1 (FLLPTGAEA). LS peptides are naturally processed and loaded on HLA-A2, providing an abundant source of surface peptide/HLA (pHLA) targets. We eluted CG1 from the surface of primary HLA-A2 +AML blasts and AML cell lines, and demonstrated CG1-targeting immunity in leukemia patients following allogeneic stem cell transplant, hence highlighting the potential for CG1 to be a promising immunotherapeutic target in AML.

Background

No relevant conflicts of interest to declare.

Daver: Incyte: Consultancy; BMS: Research Funding; ImmunoGen: Consultancy; ARIAD: Research Funding; Sunesis: Consultancy; Alexion: Consultancy; Novartis: Research Funding; Daiichi-Sankyo: Research Funding; Sunesis: Research Funding; Incyte: Research Funding; Karyopharm: Research Funding; Otsuka: Consultancy; Novartis: Consultancy; Kiromic: Research Funding; Karyopharm: Consultancy; Pfizer: Consultancy; Pfizer: Research Funding.

Introduction

The azurophil granule proteases neutrophil elastase (NE) and proteinase 3 (P3) have been established as effective immunotherapeutic targets in hematological malignancies. Expression of PR1, an HLA-A2 restricted nonameric peptide derived from NE and P3, has been demonstrated in acute myeloid leukemia (AML). We have successfully targeted PR1 in AML patients using PR1-peptide vaccine, anti-PR1/HLA-A2 antibody and PR1-specific cytotoxic T-lymphocytes (PR1-CTL). We have also identified the uptake and cross-presentation of NE and P3 by breast cancer, lung cancer and melanoma, which underlines their potential as immunotherapeutic targets in non-myeloid malignancies.

Sergeeva: Astellas Pharma: Patents & Royalties. Molldrem:Astellas Pharma: Patents & Royalties.

Introduction

No relevant conflicts of interest to declare.

Introduction

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

PR1 is an HLA-A2-retricted, nonameric peptide that is derived from the azurophil granule proteases neutrophil elastase (NE) and proteinase 3 (P3). PR1 has been targeted successfully in acute (AML) and chronic (CML) myeloid leukemia using anti-PR1/HLA-A2 antibody (8F4), PR1-peptide vaccine and PR1-specific cytotoxic T lymphocytes (PR1-CTL). We have previously reported that NE and P3 are cross-presented by normal B cells and dendritic cells (DC), leading to PR1 expression by HLA-A2. Since multiple myeloma (MM) is a B cell malignancy, we investigated whether MM cells can cross-present PR1 as a possible target for immunotherapy.

Immunotherapy targeting individual antigens in acute myeloid leukemia (AML) has shown promise. However, in view of leukemia heterogeneity and the loss of tumor antigen expression by AML, it is unlikely that any single antigen will be consistently expressed by all leukemia cells. This highlights the need to identify additional antigens in AML that can be targeted.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Abstract 2502

Abstract 4973

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Immunotherapy targeting aberrantly expressed leukemia associated antigens (LAA) has shown promising results in the management of myeloid leukemia. However, because of the heterogeneity and clonal evolution that is a feature of myeloid leukemia, targeting single peptide epitopes has had limited success, highlighting the need for novel antigen discovery. PR1 is a well-characterized LAA, is derived from the azurophil granule proteases neutrophil elastase (NE) and proteinase-3 (P3), and was effectively targeted in myeloid leukemia. We have previously shown that NE and P3 are aberrantly localized outside azurophil granules in myeloid leukemia and that their aberrant expression facilitates PR1 antigen presentation. Similar to NE and P3, cathepsin G (CG) is a serine protease located within azurophil granules in neutrophils. Because azurophil granules are disrupted in myeloid leukemia and since CG was shown to be immunogenic through its association with autoimmune disease, we hypothesized that CG may be aberrantly expressed in myeloid leukemia, thereby facilitating its presentation on HLA class I molecules and rendering it a novel target for myeloid leukemia immunotherapy.SYFPEITHI and IEDB binding algorithms were used to identify CG peptides with highest binding affinities. Flow cytometry based binding assays were performed using the T2 cell line followed by HLA-A0201 staining to confirm peptide binding and determine affinities of CG-derived peptides for HLA-A0201. Peptide/HLA-A0201 complexes were released from HLA-A0201 transfected U937 cells and purified using immunoaffinity chromatography with the anti-HLA-A0201 antibody BB7.2. The peptides were dissociated from HLA-A0201 by weak acid elution and analyzed using reverse-phase HPLC-tandem mass spectrometry. Tandem mass spectra were analyzed using the Mascot sequencing algorithm. Western blots (WB) and immunoflouresence confocal microscopy were used to determine CG expression by primary leukemia. Peptide-pulsed T2 cells were used to expand CG1- and PR1-cytotoxic T lymphocytes (CTLs) for use in calcein AM cytotoxicity assays. Peptide-pulsed T2 cells and primary leukemia from patients were used as target cells. Tetramer staining and cytokine flow cytometry (CFC) assays were used to show frequency and function of CG1-CTLs, respectively. CG1- and PP65- pulsed T2 cells were used as stimulators in CFC assays. Patient samples were collected after informed consent.Five CG derived nonameric peptides were identified using SYFPEITHI and IEDB binding algorithms. CG1 peptide (FLLPTGAEA) was identified as having the highest binding affinity to HLA-A0201 in comparison with other CG-derived peptides and with PR1 peptide (CG1 IC50=1 uM vs. PR1=9 uM). The dissociation t1/2 for CG1 and PR1 peptides was > 8 hours. CG1 peptide was eluted from the surface of U937-A2 cell line, and using reverse-phase HPLC-tandem mass spectrometry, we confirmed CG1 antigen presentation by leukemia. WB analysis for CG showed high CG expression in 9 of 11 AML patient samples and along with immunofluorescent cell imaging, showed localization of CG outside granules in compartments that could render CG susceptible to proteasomal degradation and antigen presentation. Killing of primary AML blasts by CG1-CTL was shown in 6 of 8 AML patient samples and directly correlated with CG expression and HLA-A0201 status. Blocking HLA-A0201 with BB7.2 antibody abrogated CG1-CTL mediated cytotoxicity, indicating HLA-A0201 dependent killing by CG1-CTLs. CG1-specific CTLs were detected in AML patient samples at frequencies ranging between 0.6% to 1.4% of total CD8+ cells. Furthermore, following stem cell transplant (SCT), 4–10 fold higher frequencies of functional CG1-CTLs were detected in patient samples when compared with pre-SCT samples from the same patient, as measured by IFN-gamma and/or TNF-alpha CFC.We demonstrate that CG is a novel immunotherapeutic target in myeloid leukemia. We show that CG is a LAA that is aberrantly expressed in leukemia, is presented on HLA-A0201 molecule and can be effectively targeted by CG1-CTLs. Since immunity to CG has been previously reported in autoimmune diseases, our findings show that the immunogenic potential of CG can be redirected therapeutically to target leukemia. Cumulatively, our data suggest that further development of CG-targeting therapies in myeloid leukemia is warranted.No relevant conflicts of interest to declare.

Abstract 766

Abstract 2986

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No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Abstract 4301

Abstract 2090

Abstract 2089

Adoptive T-cell therapy using donor lymphocyte infusions is a promising approach for treating hematological malignancies. But, efficacy is limited by the induction of graft-versus-host disease. Transfer of tumor-specific T-cell clones could enhance the graft-versus-tumor effect and eliminate graft-versus-host disease. However, isolating antigen-specific T-cell clones by the traditional limiting dilution approach is a time-consuming and laborious process. Here, we describe a novel strategy for rapidly cloning tumor-specific T cells. Lymphoma-specific T-cell lines were generated from two follicular lymphoma patients by repeated in vitro stimulation of lymphocytes isolated from tumor or blood with autologous soluble CD40 ligand-activated tumor cells. After four in vitro stimulations at 10-day intervals in the presence of IL-2 and IL-15, T-cell lines were found to be predominantly CD4+ T cells and produced significant amounts of TNF-a, GM-CSF, and IFN-? in response to autologous tumor cells. The tumor reactivity was MHC class II restricted suggesting that it was mediated by CD4+ T cells. Staining with a TCR Vb antibody panel, a set of monoclonal antibodies against 24 human TCR Vb families, revealed that certain Vb families were overrepresented in each CD4+ T-cell line. In patient 1, 51% of CD4+ T cells were Vb1 positive, and in patient 2, 27% of CD4+ T cells were Vb8 positive. To clone lymphoma-specific T cells, CD4+ T-cell lines were labeled with CFSE and stimulated with autologous tumor cells. After 9 days of in vitro expansion in the presence of IL-2 and IL-15, CD4+ T-cell lines were stained with an anti-human CD4-APC monoclonal antibody and an anti-human TCR Vb-PE monoclonal antibody for each CD4+ T-cell line. Proliferating Vb1 cells from patient 1 and Vb8 cells from patient 2 were identified by their reduction in CFSE staining, and CD4+TCRV b +CFSEdim cells were sorted by flow cytometer. Monoclonality of the sorted cells was confirmed by PCR using a panel of optimized primers specific for 24 TCR Vb families, by TCR Vb spectratype analysis, and finally, by sequencing the TCR Vb gene used by each T-cell clone. Sorted tumor-specific T-cell clones could be expanded to large numbers using a 14-day rapid expansion protocol with allofeeder PBMCs, and confirmed to retain specificity against autologous tumor cells in a cytokine induction assay. This approach was also successfully used to isolate melanoma-specific CD8+ T-cell clones from two patients. We conclude that this approach is highly reproducible, rapid, and efficient for generating antigen-specific T-cell clones for adoptive T-cell therapy against human malignancies in the autologous or allogeneic setting.

Adoptive T-cell therapy using donor lymphocyte infusions is a promising approach for treating hematological malignancies. But, efficacy is limited by the induction of graft-versus-host disease. Transfer of tumor-specific T-cell clones could enhance the graft-versus-tumor effect and eliminate graft-versus-host disease. However, isolating antigen-specific T-cell clones by the traditional limiting dilution approach is a time-consuming and laborious process. Here, we describe a novel strategy for rapidly cloning tumor-specific T cells. Lymphoma-specific T-cell lines were generated from two follicular lymphoma patients by repeated in vitro stimulation of lymphocytes isolated from tumor or blood with autologous soluble CD40 ligand-activated tumor cells. After four in vitro stimulations at 10-day intervals in the presence of IL-2 and IL-15, T-cell lines were found to be predominantly CD4+T cells and produced significant amounts of TNF-a, GM-CSF, and IFN-γ in response to autologous tumor cells. The tumor reactivity was MHC class II restricted suggesting that it was mediated by CD4+T cells. Staining with a TCR Vb antibody panel, a set of monoclonal antibodies against 24 human TCR Vb families, revealed that certain Vb families were overrepresented in each CD4+T-cell line. In patient 1, 51% of CD4+T cells were Vb1 positive, and in patient 2, 27% of CD4+T cells were Vb8 positive. To clone lymphoma-specific T cells, CD4+T-cell lines were labeled with CFSE and stimulated with autologous tumor cells. After 9 days of in vitro expansion in the presence of IL-2 and IL-15, CD4+T-cell lines were stained with an anti-human CD4-APC monoclonal antibody and an anti-human TCR Vb-PE monoclonal antibody for each CD4+T-cell line. Proliferating Vb1 cells from patient 1 and Vb8 cells from patient 2 were identified by their reduction in CFSE staining, and CD4+TCRV b +CFSEdimcells were sorted by flow cytometer. Monoclonality of the sorted cells was confirmed by PCR using a panel of optimized primers specific for 24 TCR Vb families, by TCR Vb spectratype analysis, and finally, by sequencing the TCR Vb gene used by each T-cell clone. Sorted tumor-specific T-cell clones could be expanded to large numbers using a 14-day rapid expansion protocol with allofeeder PBMCs, and confirmed to retain specificity against autologous tumor cells in a cytokine induction assay. This approach was also successfully used to isolate melanoma-specific CD8+T-cell clones from two patients. We conclude that this approach is highly reproducible, rapid, and efficient for generating antigen-specific T-cell clones for adoptive T-cell therapy against human malignancies in the autologous or allogeneic setting.