performed experiments

performed experiments. number of potential mAb-binding tumor antigens to include intracellular peptides bound to a surface MHC molecule. TCR-like antibodies are attractive because they can bind targets Anle138b with affinities that are logs higher than the TCR [12, 13]. The PR1-targeting humanized 8F4, h8F4, will enter an early phase clinical trial for myeloid leukemia patients next year [14]. Herein, we report the incorporation of the h8F4 scFv into a 2nd generation retroviral TCR-like CAR construct and demonstrate efficient transduction of the CAR vector into human healthy donor (HD) peripheral blood mononuclear cells (PBMCs). We show consistent, high expression of the h8F4-CAR on the surface of both CD4+ and CD8+ T-cells. We also show specific binding of the h8F4-CAR to the HLA-A2/PR1 antigen and demonstrate preferential cytotoxicity of h8F4-CAR-T cells against human AML cell lines and primary AML blasts [10, 11]. Mouse 8F4 was humanized – human IgG1 8F4 (h8F4) – and maintains specificity for PR1/HLA-A2 and activity against AML [14]. The h8F4 mAb has high affinity (Kd = 6.5 nM) for the PR1/HLA-A2 conformational epitope [14]. Thus, it is an ideal TCR-like mAb for constructing an 8F4-CAR to transduce T-cells and redirect them to mediate GVL activity against myeloid malignancies. Potential advantages of CAR-modified T cells over monoclonal antibodies Anle138b include greater cytotoxic potency, active trafficking, passage through the blood-brain barrier, fewer required doses, the potential for long-lived memory and protection against relapse, and increased sensitivity to low antigen density [24, 25]. Disadvantages include the greater potential for on-target, off-tissue toxicity and less control over the dose and schedule as CARs have the potential for immense proliferation and that this novel adoptive T cell approach merits further investigation. Considerable research is focused on identifying and targeting extracellular proteins on the surface of AML blasts using CAR-T cells, and strong preclinical evidence exists for CAR-T cells that target the antigens CD123 and CD33 [28C30]. However, these antigens are coexpressed on the surface of myeloid progenitor cells and additional safeguards will need to be implemented to prevent on-target, off-tissue toxicity, long-term myelosuppression, and consequent infections [30]. Additionally, a predictable challenge to the success of targeting specific leukemia antigens is clonal evolution and intratumoral heterogeneity, from which immune escape variants can emerge to comprise relapsed disease [31, 32]. Anle138b One solution is to target multiple leukemia antigens simultaneously; however, the limited number of effective antigens currently hinders this approach. We identified 8F4 as the first TCR-like mAb against a leukemia antigen [10]. The successful development and testing of the h8F4-CAR shown here supports the promise of developing a novel T-cell therapy directed against an endogenous self-antigen that is differentially expressed on the surface of leukemia stem cells. We also demonstrated that T cells derived from UCB could be efficiently transduced with the h8F4-CAR and were capable of killing leukemia cells in a PR1/HLA-A2-dependent manner. UCB lymphocytes are mostly naive T-cells and may be an ideal source for generating h8F4-CAR T cells [15, 16]. Since the first UCB transplant (CBT) was performed in 1988 by Gluckman et al. [33], more than 40,000 patients have received CBT for malignant and non-malignant diseases [34C40]. Importantly, UCB has improved the likelihood of finding a SCT donor for minority populations, who are under-represented in donor registries [41]. Of note, the HLA-A*02:01 allele is common among US African Americans (34C40% of individuals) and US Hispanic individuals (19C23%) in addition to being present at high rates in Caucasians (47% of individuals)[42C44]. To improve the outcome of CBT for patients with AML, the most common disease treated with CBT, graft engineering of donor T cells as demonstrated herein using UCB-derived h8F4-CAR-T cells could increase graft-versus-leukemia (GVL) without increasing graft-versus-host disease (GVHD). In support of this strategy, allogeneic CAR-T cells have already been used in at least 4 different clinical trials without any reports of acute GVHD [1, 2, 45, 46], and we anticipate UCB derived CAR-T cells will carry less risk of GVHD in this setting [47]. In summary, we have successfully developed a CAR-T cell based on the construct of Vamp3 a TCR-like monoclonal antibody that targets an intracellular leukemia-associated antigen. We showed that this approach is feasible using peripheral blood as well as UCB T cells. This method could improve upon the.