Development and clinical implementation of targeted T-cell therapy for AML has so far been impeded by the phenotypic similarities between malignant and normal myelopoiesis, the paucity of tumor-specific antigens, the complex bone marrow microenvironment, and AML disease heterogeneity. Now, Dao et al redirected T cells against Wilms tumor 1 (WT1) with a novel antibody T-cell receptor (AbTCR) construct and exploited CD33 as input to activate a chimeric costimulatory signaling receptor (CSR) to enhance specificity, safety and efficacy (see figure). From their phage-display library, the authors identified new antibodies (ESK2) recognizing the WT1 RMF (RMFPNAPYL) peptide/HLA-A∗02:01 complex with enhanced specificity compared with their previously identified ESK1 antibodies. The fragment antigen binding regions of 2 lead candidates were linked to the constant chains of the γδ T-cell receptor (TCR) and expressed in a viral vector along with a CSR recognizing CD33 through a single-chain variable fragment (scFv) linked to CD28. When human T cells transduced with AbTCR + CSR encountered AML cells expressing WT1 (RMF)/HLA-A∗02:01 and CD33, the T cells recognized both target antigens and received T-cell activation signals from the γδ TCR/CD3 complex (signal 1, by AbTCR) and CD28 costimulation (signal 2, by CSR) (see figure). Investigation of engineered T-cell function showed that AbTCR+ T cells specifically recognized and killed WT1 (RMF)/HLA-A∗02:01+ AML targets and that both killing and interferon-γ production were increased on delivery of CD28 costimulation through the CSR (see figure). The CSR acted in cis, requiring both the WT1 (RMF)/HLA-A∗02:01 complex and CD33 expressed on the same target cell. More important, no toxicity against normal peripheral blood mononuclear cells, granulocytes, or cord blood CD34+ cells was detected in vitro. In vivo in mouse xenograft models, strongest antileukemic activity was observed when mice were treated with T cells expressing both AbTCR + CSR. Thus, the proposed AbTCR + CSR combination resulted in increased AML specificity and enhanced antileukemic activity while sparing normal hematopoiesis and reducing on-target off-tumor activity of the engineered T cells (see figure).
Being highly overexpressed in hematologic and solid tumors, with restricted low expression in some adult normal tissues (reproductive organs, kidney podocytes, mesothelial lining, and CD34+ cells), and exerting critical biological functions in tumors, WT1 is an intensively investigated highly prioritized oncofetal tumor-associated antigen. WT1 has several immunogenic epitopes presented in the context of human leukocyte antigen (HLA) class I. The WT1 (RMF)/HLA-A∗02:01 epitope targeted in the study by Dao et al is historically the most investigated WT1 epitope in vaccine or TCR-based adoptive cell transfer studies. Safety and therapeutic benefit have been reported in 12 patients with AML infused prophylactically with WT1 TCR-transgenic T cells after allogeneic hematopoietic stem cell transplant.2 However, the RMF epitope requires the immunoproteasome for natural processing,3 introducing some uncertainties about cell surface presentation in malignant cells. Some recent analyses are pointing toward a potential immune escape mechanism after WT1 TCR–T-cell therapy, where reduced levels of specific immunoproteasome subunits were reported in 2 resistant patients.4 Immunoproteasome-independent WT1 epitopes and corresponding class I restricted αβ TCRs have also been identified, opening new interesting therapeutic prospects for targeting WT1.4-6 Eventually, an AbTCR against a WT1 epitope processed by standard proteasomes could be developed.
The concept of splitting the T-cell activation and costimulation signals to enhance target specificity and engineered T-cell function has been explored in the field for over a decade,7 but has recently gained momentum in various combinations with conventional chimeric antigen receptors (CARs) or native or transgenic αβ TCRs.8,9 The possibility of simultaneously targeting both intracellular and cell surface antigens in a cooperative manner, with αβ TCRs or with AbTCRs, significantly broadens the number of potentially targetable antigens and allows the exploitation of their different signaling sensitivity thresholds. A novel dual-antigen targeting strategy for AML was recently presented, targeting 2 cell surface receptors with scFvs, combining a CAR with a chimeric costimulatory receptor.10 Target antigens were chosen on the basis of differential antigen density between normal and malignant hematopoiesis identified on a set of samples from patients with AML and healthy donors.10 This T-cell engineering strategy allowed tuning of engineered T-cell sensitivity by targeting adhesion G protein-coupled receptor E2 (ADGRE2) and C-type lectin domain family 12 member A (CLEC12A), and improved antileukemic activity with reduced toxicity to normal hematopoiesis, compared with single-antigen targeting. The study by Dao et al combined WT1 with CD33 targeting, WT1 by itself already having an excellent safety and specificity profile. When combined with a CSR exploiting the highly expressed pan-myeloid antigen CD33 for the delivery of costimulation, enhanced leukemia specificity and antileukemic activity were demonstrated. Although CD33 antigen densities were characterized recently,10 we have no information available regarding the densities of WT1 (RMF)/HLA-A∗02:01 on AML and normal hematopoietic stem and progenitors. It would be interesting to evaluate whether the combination of AbTCR + CSR shifts the activation threshold of the AbTCR toward recognition of lower-density target cells.
In conclusion, Dao et al present a compelling preclinical proof of concept for a novel approach of dual-antigen targeting for AML with engineered T cells conferring enhanced specificity, safety, and antileukemic activity. Clinical translation is eagerly awaited.
Conflict-of-interest disclosure: C.A. receives licensing fees and royalties from Immatics (through previous institution, Baylor College of Medicine); participated in advisory boards for Kite/Gilead, Janssen, and Celgene/Bristol Myers Squibb; received sponsored travel from Gilead; and has several patents and pending patent applications in the field of engineered T-cell therapies.