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532. Donor T Cells Specific for Multiple Leukemia Antigens for Adoptive Immunotherapy of Myeloid Leukemias after Stem Cell Transplantation
CANCER – IMMUNOTHERAPY: GENETIC MODIFICATION OF T CELLS Cancer – Immunotherapy: Genetic Modification of T Cells 531. TCR Gene Editing Results in Effective Immunotherapy of Leukemia without the Development of GvHD
Elena Provasi,1 Pietro Genovese,2 Angelo Lombardo,2 Zulma Magnani,1 Pei-Qi Liu,3 Andreas Reik,3 Victoria Chu,3 David E. Paschon,3 Lei Zhang,3 Jurgen Kuball,4 Attilio Bondanza,1 Giulia Casorati,1 Fabio Ciceri,1 Cladio Bordignon,1 Philip D. Greenberg,5 Michael C. Holmes,3 Philip D. Gregory,3 Luigi Naldini,2 Chiara Bonini.1 1 San Raffaele Scientific Institute, Milan, Italy; 2Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy; 3 Sangamo Biosciences, Richmond; 4University Medical Center Utrecht, Netherlands; 5Fred Hutchinson Cancer Research Center, Seattle. T cell receptor (TCR) gene-transfer into T cells is a valuable tool for adoptive immunotherapy of cancer patients for whom natural tumor-specific lymphocytes cannot be isolated. Nevertheless, TCRtransferred T lymphocytes differ from their natural counterparts in carrying two different TCRs and this can result in lower efficacy and potential toxicity due to reduced expression of tumor-specific TCR and inappropriate pairing of TCR chains. To address these issues, we developed a novel strategy based on zinc finger nucleases (ZFNs) that allows editing of T cell specificity at the DNA level, combining the disruption of the endogenous TCR chain genes with the transfer of a tumor-specific TCR. Two sets of ZFNs targeting the constant regions of the α and the β TCR chain genes, respectively, were designed. Transient ZFN delivery into activated T lymphocytes disrupted the TCR genes and abrogated cell surface expression of CD3. The introduction of exogenous TCR chains rescued CD3 surface expression. As a model TCR for gene transfer, we selected an HLA-A2 restricted TCR specific for Wilms’ tumor antigen 1 (WT1). To achieve complete editing of T cell specificity, we established a protocol that sequentially disrupted the endogenous TCR α and β chains with high efficiency (34%±11 and 16%±10), followed by lentiviral transfer of the WT1-specific TCR α and β chains (53%±25 and 21%±15). Sequential sorting for CD3 negative and CD3 positive cells resulted in a population of TCR-edited lymphocytes encoding only the tumor-specific TCR that, in the absence of competition from the endogenous receptor, was expressed at high physiological levels. Accordingly, TCR-edited lymphocytes were superior to conventional TCR-transferred cells in promoting specific recognition of WT1-expressing targets, including primary leukemias, and most importantly, were devoid of residual endogenous TCR reactivity including alloreactivity. Finally, for a comprehensive assessment of safety and anti-tumor efficacy, we treated immunodeficient mice, infused with primary human leukemic blasts, with matched TCRtransferred, TCR-edited, and unmanipulated cells and monitored mice for Graft-versus-host disease and the appearance of leukemia. Mice treated with TCR-edited cells showed higher event free survival than TCR-transferred (p<0.05) or unmanipulated lymphocytes (p<0.01). These data demonstrate that the genetic re-programming of T cell specificity in primary lymphocytes results in a functionally superior target specific activity and thus has the potential to greatly improve the safety and efficacy of cancer immunotherapy. (Provasi and Genovese: equal contribution).
Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
532. Donor T Cells Specific for Multiple Leukemia Antigens for Adoptive Immunotherapy of Myeloid Leukemias after Stem Cell Transplantation John A. Barrett,1 Gerrit Weber,1 Ulrike Gerdemann,2 Sid Kerkar,1 Pawel Muranski,1 Nancy F. Hensel,1 Ann Leen,2 Jos J. Melenhorst,1 Cath M. Bollard.2 1 Stem Cell Transplant, Hematology Branch, NHLBI, NIH, Bethesda, MD; 2Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX.
Although donor lymphocyte infusions can prevent or treat relapse in myeloid malignancies after SCT, they are complicated by GVHD and show limited antileukemic efficacy. To improve the efficacy of adoptive T cell therapy in this context, we used peptide libraries of four antigens often preferentially expressed by leukemia cells to generate multipeptide specific CD4+ and CD8+ CTL targeting AML. To broaden the applicability and enhance the potency of DLI for patients with myeloid malignancies, we obtained 15mer peptide libraries overlapping by 11 aa for WT1, Proteinase 3 (Pr3), MAGE-A3, PRAME and human neutrophil elastase (HNE). Healthy donor PBMC were stimulated with DC, pulsed with peptide libraries of one LAA or a mix (LAA mix), and expanded in IL7, IL12, IL15 and/or IL2. Cultures were re-stimulated weekly with peptide-pulsed DC and peptide-recognition was tested by IFNγ-ELIspot after 2-3 stimulations. Positive cultures were further characterized by intracellular cytokine staining, CD107a release, and cytolytic activity against peptide-pulsed autologous PHA blasts in a flow cytometric killing assay. Of 10 healthy donors, 9 responded to the LAA mix by IFNγ-ELIspot (mean: 471 SFU/200.000 cells, range: 296-968). Expanded CTL specifically recognized a mean of 3/5 antigens, with PRAME being most frequently recognized (9/ 10 donors). Single antigen stimulations did not induce higher specific responses than the LAA mix (28 vs. 27 responses). No competitive suppression by any LAA was observed. Correlating with ELIspot results, LAA CTL were cytotoxic to PHAB pulsed with the LAA mix (E:T=40:1; mean: 33%, range: 12-80%,; negative control: PHAB: mean: 4.2%, range: 0-13%, n=7), as well as with individual antigens (WT1: mean:10.6%, range: 0-26%; HNE: mean: 35.4%, range: 0-85%, Pr3: mean: 7.3%, range: 0-14%, MAGE-A3: mean: 14%, range: 0-28%; PRAME: mean: 14%, range: 0-37.5%). Epitope mapping was performed using peptide pools where each peptide was represented twice. Peptide specificity of CTL was confirmed by testing against the identified 15mer peptide. In addition to known peptides, we identified 16 new MHC class I and II-restricted peptides (WT1: 8, HNE: 6 and Pr3: 2). WT1 peptides showed class I and class II restriction in antibody blocking experiments. HNE peptides were mainly class I restricted, with a dominant response against RRLACLFLACVLPAL in 5 of 7 donors. Cytotoxicity against the peptide library or the LAA mix was always greater than against targets pulsed with the immunodominant 15mer peptide, demonstrating the benefit of a broad immune response. Cytolytic activity was a predominantly CD4+ response, indicating a role for CD4+ T cells in tumor lysis. Finally we showed that multipeptide-induced CTL express CD107a on recognition of myeloid leukemia blast cells. These results suggest that the generation of HLAunrestricted multi-LAA–specific CD4 and CD8 T cells is a practical approach for adoptive immunotherapy of myeloid leukemia after SCT.
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Elena Provasi,1 Pietro Genovese,2 Angelo Lombardo,2 Zulma Magnani,1 Pei-Qi Liu,3 Andreas Reik,3 Victoria Chu,3 David E. Paschon,3 Lei Zhang,3 Jurgen Kuball,4 Attilio Bondanza,1 Giulia Casorati,1 Fabio Ciceri,1 Cladio Bordignon,1 Philip D. Greenberg,5 Michael C. Holmes,3 Philip D. Gregory,3 Luigi Naldini,2 Chiara Bonini.1 1 San Raffaele Scientific Institute, Milan, Italy; 2Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy; 3 Sangamo Biosciences, Richmond; 4University Medical Center Utrecht, Netherlands; 5Fred Hutchinson Cancer Research Center, Seattle. T cell receptor (TCR) gene-transfer into T cells is a valuable tool for adoptive immunotherapy of cancer patients for whom natural tumor-specific lymphocytes cannot be isolated. Nevertheless, TCRtransferred T lymphocytes differ from their natural counterparts in carrying two different TCRs and this can result in lower efficacy and potential toxicity due to reduced expression of tumor-specific TCR and inappropriate pairing of TCR chains. To address these issues, we developed a novel strategy based on zinc finger nucleases (ZFNs) that allows editing of T cell specificity at the DNA level, combining the disruption of the endogenous TCR chain genes with the transfer of a tumor-specific TCR. Two sets of ZFNs targeting the constant regions of the α and the β TCR chain genes, respectively, were designed. Transient ZFN delivery into activated T lymphocytes disrupted the TCR genes and abrogated cell surface expression of CD3. The introduction of exogenous TCR chains rescued CD3 surface expression. As a model TCR for gene transfer, we selected an HLA-A2 restricted TCR specific for Wilms’ tumor antigen 1 (WT1). To achieve complete editing of T cell specificity, we established a protocol that sequentially disrupted the endogenous TCR α and β chains with high efficiency (34%±11 and 16%±10), followed by lentiviral transfer of the WT1-specific TCR α and β chains (53%±25 and 21%±15). Sequential sorting for CD3 negative and CD3 positive cells resulted in a population of TCR-edited lymphocytes encoding only the tumor-specific TCR that, in the absence of competition from the endogenous receptor, was expressed at high physiological levels. Accordingly, TCR-edited lymphocytes were superior to conventional TCR-transferred cells in promoting specific recognition of WT1-expressing targets, including primary leukemias, and most importantly, were devoid of residual endogenous TCR reactivity including alloreactivity. Finally, for a comprehensive assessment of safety and anti-tumor efficacy, we treated immunodeficient mice, infused with primary human leukemic blasts, with matched TCRtransferred, TCR-edited, and unmanipulated cells and monitored mice for Graft-versus-host disease and the appearance of leukemia. Mice treated with TCR-edited cells showed higher event free survival than TCR-transferred (p<0.05) or unmanipulated lymphocytes (p<0.01). These data demonstrate that the genetic re-programming of T cell specificity in primary lymphocytes results in a functionally superior target specific activity and thus has the potential to greatly improve the safety and efficacy of cancer immunotherapy. (Provasi and Genovese: equal contribution).
Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
532. Donor T Cells Specific for Multiple Leukemia Antigens for Adoptive Immunotherapy of Myeloid Leukemias after Stem Cell Transplantation John A. Barrett,1 Gerrit Weber,1 Ulrike Gerdemann,2 Sid Kerkar,1 Pawel Muranski,1 Nancy F. Hensel,1 Ann Leen,2 Jos J. Melenhorst,1 Cath M. Bollard.2 1 Stem Cell Transplant, Hematology Branch, NHLBI, NIH, Bethesda, MD; 2Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX.
Although donor lymphocyte infusions can prevent or treat relapse in myeloid malignancies after SCT, they are complicated by GVHD and show limited antileukemic efficacy. To improve the efficacy of adoptive T cell therapy in this context, we used peptide libraries of four antigens often preferentially expressed by leukemia cells to generate multipeptide specific CD4+ and CD8+ CTL targeting AML. To broaden the applicability and enhance the potency of DLI for patients with myeloid malignancies, we obtained 15mer peptide libraries overlapping by 11 aa for WT1, Proteinase 3 (Pr3), MAGE-A3, PRAME and human neutrophil elastase (HNE). Healthy donor PBMC were stimulated with DC, pulsed with peptide libraries of one LAA or a mix (LAA mix), and expanded in IL7, IL12, IL15 and/or IL2. Cultures were re-stimulated weekly with peptide-pulsed DC and peptide-recognition was tested by IFNγ-ELIspot after 2-3 stimulations. Positive cultures were further characterized by intracellular cytokine staining, CD107a release, and cytolytic activity against peptide-pulsed autologous PHA blasts in a flow cytometric killing assay. Of 10 healthy donors, 9 responded to the LAA mix by IFNγ-ELIspot (mean: 471 SFU/200.000 cells, range: 296-968). Expanded CTL specifically recognized a mean of 3/5 antigens, with PRAME being most frequently recognized (9/ 10 donors). Single antigen stimulations did not induce higher specific responses than the LAA mix (28 vs. 27 responses). No competitive suppression by any LAA was observed. Correlating with ELIspot results, LAA CTL were cytotoxic to PHAB pulsed with the LAA mix (E:T=40:1; mean: 33%, range: 12-80%,; negative control: PHAB: mean: 4.2%, range: 0-13%, n=7), as well as with individual antigens (WT1: mean:10.6%, range: 0-26%; HNE: mean: 35.4%, range: 0-85%, Pr3: mean: 7.3%, range: 0-14%, MAGE-A3: mean: 14%, range: 0-28%; PRAME: mean: 14%, range: 0-37.5%). Epitope mapping was performed using peptide pools where each peptide was represented twice. Peptide specificity of CTL was confirmed by testing against the identified 15mer peptide. In addition to known peptides, we identified 16 new MHC class I and II-restricted peptides (WT1: 8, HNE: 6 and Pr3: 2). WT1 peptides showed class I and class II restriction in antibody blocking experiments. HNE peptides were mainly class I restricted, with a dominant response against RRLACLFLACVLPAL in 5 of 7 donors. Cytotoxicity against the peptide library or the LAA mix was always greater than against targets pulsed with the immunodominant 15mer peptide, demonstrating the benefit of a broad immune response. Cytolytic activity was a predominantly CD4+ response, indicating a role for CD4+ T cells in tumor lysis. Finally we showed that multipeptide-induced CTL express CD107a on recognition of myeloid leukemia blast cells. These results suggest that the generation of HLAunrestricted multi-LAA–specific CD4 and CD8 T cells is a practical approach for adoptive immunotherapy of myeloid leukemia after SCT.
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