Abstract
Background: The outcome for patients with high risk acute myeloid leukemia (AML) remains poor. Thus new targeted therapies are needed and immunotherapies have the potential to fulfill this need. Adoptive transfer of tumor-specific T cells is one promising approach; however infused T cells do not redirect the large reservoir of resident T cells to tumors. To overcome this limitation we have recently developed a new approach to render T cells specific for tumor cells, which relies on genetically modifying T cells with a secretable, bispecific T cell engager (ENG-T cells). Secretion of bispecific protein should activate infused cells as well as bystander T cells against tumor. Consistent and prolonged synthesis of engagers by T cells should be superior to the intermittent, direct infusion of the recombinant bispecific antibody, not only because these recombinant proteins have short half-lives but also because they do not accumulate at tumor sites. The goal of this project was to generate T cells secreting IL3Rα (CD123) and CD3 bispecific T cell engagers (CD123-ENG T cells) and to evaluate their effector function in vitro and in vivo.
Methods: CD123-ENG T cells were generated by transducing T cells with a retroviral vector encoding a CD123-specific T cell engager consisting of an scFv recognizing CD123 linked to an scFv recognizing CD3. The retroviral vector was also fashioned to include an mOrange gene downstream of an IRES element. The effector function of CD123-ENG T cells was evaluated in vitro and in a xenograft model.
Results: Transduction of CD3/CD28-activated T cells resulted in mOrange expression in transduced T cells (median transduction efficiency 78%, range 49-92%) The presence of CD123-ENG molecules on the cell surface of both transduced and non-transduced T cells was demonstrated by FACS analysis using an F(ab) antibody that recognizes the CD123 scFv. Coculture of CD123+ AML cells (MV-4-11, MOLM-1, KG1a) and K562 cells genetically modified to express CD123 (K562-CD123) with engager T cells resulted in robust T-cell activation as judged by IFNγ and IL2 secretion. In contrast CD123-negative cells (K562) did not activate T cells. Likewise, control engager T cells (targeting an irrelevant antigen) were not activated when cultured with CD123+ cells. Antigen-dependent recognition was confirmed with cytotoxicity assays, in which engager T cells specifically killed CD123+ AML cells at an effector:target ratios ranging from 40:1-5:1 (p<0.05) Since CD123 is expressed at low levels on normal hematopoietic progenitor cells (HPCs), we evaluated the ability of CD123-ENG T cells to recognize normal HPCs in colony formation assays. Only at high CD123-ENG to HPC ratios did we observe a decline in colony formation, indicating that CD123+ AML cells can be targeted while preserving normal HPCs. In vivo anti-tumor activity was assessed using a modified KG1a AML cell line expressing firefly luciferase (KG1a.ffluc) to allow for serial bioluminescence imaging. NSG (NOD-SCID, IL2γR deficient) mice were sublethally irradiated 24 hours prior to leukemia infusion and were then treated with two intravenous doses of CD123-ENG T cells or control T cells on days 7 and 14. CD123-ENG T cells had potent anti-leukemia activity resulting in a significant survival advantage of treated animals (p=0.002; n=5 CD123-ENG, n=5 Control-ENG, n=10 control animals).
Conclusions: We have generated CD123-ENG T cells with the potential to direct bystander T cells to CD123+ AML in a tumor antigen-specific manner. These CD123-ENG T cells have potent anti-AML activity in vivo, presenting a promising addition to currently available AML therapies.
Disclosures
Bonifant: Celgene, Bluebird bio: Baylor College of Medicine has a Research Collaboration with Celgene and Bluebirdbio to develop gene-modified T-cell Therapies. MPV, KI, XT, and SG have patent applications in the field of T-cell and gene-modified T-cell Therapy for cancer Other. Torres:Celgene, Bluebird bio: Baylor College of Medicine has a Research Collaboration with Celgene and Bluebirdbio to develop gene-modified T-cell Therapies. MPV, KI, XT, and SG have patent applications in the field of T-cell and gene-modified T-cell Therapy for cancer Other. Velasquez:Celgene, Bluebird bio: Baylor College of Medicine has a Research Collaboration with Celgene and Bluebirdbio to develop gene-modified T-cell Therapies. MPV, KI, XT, and SG have patent applications in the field of T-cell and gene-modified T-cell Therapy for cancer Other. Iwahori:Celgene, Bluebird bio: Baylor College of Medicine has a Research Collaboration with Celgene and Bluebirdbio to develop gene-modified T-cell Therapies. MPV, KI, XT, and SG have patent applications in the field of T-cell and gene-modified T-cell Therapy for cancer Other. Arber:Celgene, Bluebird bio: Baylor College of Medicine has a Research Collaboration with Celgene and Bluebirdbio to develop gene-modified T-cell Therapies. MPV, KI, XT, and SG have patent applications in the field of T-cell and gene-modified T-cell Therapy for cancer Other. Song:Celgene, Bluebird bio: Baylor College of Medicine has a Research Collaboration with Celgene and Bluebirdbio to develop gene-modified T-cell Therapies. MPV, KI, XT, and SG have patent applications in the field of T-cell and gene-modified T-cell Therapy for cancer Other. Gottschalk:Celgene, Bluebird bio: Baylor College of Medicine has a Research Collaboration with Celgene and Bluebirdbio to develop gene-modified T-cell Therapies. MPV, KI, XT, and SG have patent applications in the field of T-cell and gene-modified T-cell Therapy for cancer Other.
An evaluation of the potential to use tumor-associated antigens as targets for antitumor T cell therapy using transgenic mice expressing a retroviral tumor antigen in normal lymphoid tissues.