T?cells were then washed and stained for CD3 expression

T?cells were then washed and stained for CD3 expression. Flow Cytometry and Binding Experiments Flow cytometry experiments were performed on a Bio-Rad S3E or a BD Celesta equipped with a high-throughput sampler (HTS). of putative T?cell epitopes and investigate the binding kinetics and affinity requirements of the resultant D domain CARs. Finally, we extended the utility of D domains Rabbit Polyclonal to SGOL1 by generating functional, bi-specific CARs comprised of a CD123-specific D domain and a CD19-specific scFv. The properties of D domains suggest that this class of targeting domain may facilitate the development of multi-functional CARs where conventional, scFv-based designs may be suboptimal. and findings suggest that the D domain as a targeting element does not promote inhibitory signaling. The extracellular domain of human CD123 is 87% and 31% identical to that of cynomolgus and mouse CD123, respectively. Thus, species selectivity for CD123 was characterized through the use of HEK293T target cells transfected with CD123 orthologs. Human T?cells transduced with Dd-cg06-CAR are effective in killing HEK cells expressing human and cynomolgus monkey CD123, but not mouse CD123 or mock-transfected cells (Figures 3A and S7). Notably, 32716 scFv does not demonstrate cross-reactivity against cynomolgus CD123. Open in a separate window Figure?3 Dd-cg06-CAR T Cells Kill Target Cells and efficacy of the Dd-cg06 CAR was assessed using a?MOLM14 tumor model. Engrafted tumors were treated with Dd-cg06-CAR, Amprenavir 32716-CAR, and FLAG-CAR (a negative control construct, expressing only the FLAG epitope sequence as the extracellular domain). Mice receiving Dd-cg06-CAR or 32716 scFv CAR demonstrated regression between days 18 and 24 and remained tumor-free by day 31 (Figure?3C) with comparable kinetics of tumor clearance by both CARs and no signs of graft versus host disease (GvHD). Taken together, the and results indicate that the Dd-cg06 can direct target-specific, potent T?cell activation resulting in eradication of tumor xenografts. Deimmunization of Dd-cg06 through Amprenavir Rational Design The safety and efficacy of any protein therapeutic may be compromised by potential host-mediated immunogenic responses. Therefore, we employed a virtual matrix-based T?cell epitope prediction algorithm27 in the assessment of D domain sequences. While Dd-cg06 exhibited potent and activity, it nevertheless harbored three putative promiscuous T?cell epitopes, which we endeavored to remove via targeted mutation. Based on the structure of 3D and our D domain library design, we identified mutagenic sites that we hypothesized would be less likely to be involved in either target binding (i.e., opposite or adjacent to the F3 face of the domain) or the formation of the domains hydrophobic corethe primary driver of the folding and stability.15 The introduction of three mutations (R17Q, S24T, and S65E) into Dd-cg06 resulted in a new sequence, Dd-cg77, in which Amprenavir the number of promiscuous epitopes had been reduced to zero (Figure?S9). To assess the impact of these mutations on binding, we generated Fc fusions of Dd-cg06 and Dd-cg77 and measured the KD of their mono-valent interactions with CD123 to be 14.4 and 6.4?nM, respectively (Figure?S10; Table?S1). Nevertheless, the approximately 2-fold increase in mono-valent affinity of Dd-cg77 did not affect the CAR activity, as demonstrated by comparable cytokine release, degranulation (Figure?4A), and killing of CD123+ target cells (Figure?4B). Notably, the KD of the FLAG-less, Dd-cg77-Fc fusion (6.4?nM) is similar to that of the FLAG-tagged Dd-cg77 MBP fusion (7.2?nM), suggesting the presence of the tag has little impact on the binding of this D domain. and xenograft model. NSG mice received 6? 106 KG1a cells on day 0 and various doses of Dd-cg77 or FLAG CAR T?cells on day 29. Expression data (FLAG positive) for CAR T?cells and statistical analysis of bioluminescent signal flux data can be found in Figure?S8. D Domain CAR Activity Can Be Tuned through Affinity Mutations The ability of lower affinity CARs to discriminate between low (normal) and high (neoplastic) target densities has been advocated as a mechanism for reducing on-target, off-tumor toxicities.6, 28, 29 In an effort to reduce the relative high affinity of Dd-cg77, we targeted the 13 library positions (i.e., those most likely involved in binding), and in an effort to maximize proper folding, mutated them to the corresponding residue found in Dd-a3D. For 7 of the 13 mutants, surface plasmon resonance (SPR) assessment of CD123-HIS binding to MBP-captured Dd-MBP fusion proteins revealed KD values ranging from 3.2?nM (N66E) to 100?nM (W16T), with lower.