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The emergence of immune escape is a significant roadblock to developing effective chimeric antigen receptor (CAR) T cell therapies against hematological malignancies, including acute myeloid leukemia (AML). Here, we demonstrate feasibility of targeting two antigens simultaneously by combining a GRP78-specific peptide antigen recognition domain with a CD123-specific scFv to generate a peptide-scFv bispecific antigen recognition domain (78.123). To achieve this, we test linkers with varying length and flexibility and perform immunophenotypic and functional characterization. We demonstrate that bispecific CAR T cells successfully recognize and kill tumor cells that express GRP78, CD123, or both antigens and have improved antitumor activity compared to their monospecific counterparts when both antigens are expressed. Protein structure prediction suggests that linker length and compactness influence the functionality of the generated bispecific CARs. Thus, we present a bispecific CAR design strategy to prevent immune escape in AML that can be extended to other peptide-scFv combinations., Competing Interests: Declaration of interests J.T.Z., P.J.C., D.M.L., G.K., S.G., and M.P.V. have patent applications in the field of immunotherapy. S.G. is a member of the Data and Safety Monitoring Board of Immatics, is on the Scientific Advisory Board of Be Biopharma, and has received honoraria from TESSA Therapeutics, Tidal, Catamaran Bio, and Sanofi within the last 2 years., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lack of targetable antigens is a key limitation for developing successful T cell-based immunotherapies. Members of the unfolded protein response (UPR) represent ideal immunotherapy targets because the UPR regulates the ability of cancer cells to resist cell death, sustain proliferation, and metastasize. Glucose-regulated protein 78 (GRP78) is a key UPR regulator that is overexpressed and translocated to the cell surface of a wide variety of cancers in response to elevated endoplasmic reticulum (ER) stress. We show that GRP78 is highly expressed on the cell surface of multiple solid and brain tumors, making cell surface GRP78 a promising chimeric antigen receptor (CAR) T cell target. We demonstrate that GRP78-CAR T cells can recognize and kill GRP78+ brain and solid tumors in vitro and in vivo. Additionally, our findings demonstrate that GRP78 is upregulated on CAR T cells upon T cell activation; however, this expression is tumor-cell-line specific and results in heterogeneous GRP78-CAR T cell therapeutic response., Competing Interests: Declaration of interests N.H. and M.P.V. are named inventors on a patent application related to GRP78-CAR. N.H., G.K., and M.P.V. have other patent applications in the field of immunotherapy unrelated to this work., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The bone marrow microenvironment (BME) drives drug resistance in acute lymphoblastic leukemia (ALL) through leukemic cell interactions with bone marrow (BM) niches, but the underlying mechanisms remain unclear. Here, we show that the interaction between ALL and mesenchymal stem cells (MSCs) through integrin β1 induces an epithelial-mesenchymal transition (EMT)-like program in MSC-adherent ALL cells, resulting in drug resistance and enhanced survival. Moreover, single-cell RNA sequencing analysis of ALL-MSC co-culture identifies a hybrid cluster of MSC-adherent ALL cells expressing both B-ALL and MSC signature genes, orchestrated by a WNT/β-catenin-mediated EMT-like program. Blockade of interaction between β-catenin and CREB binding protein impairs the survival and drug resistance of MSC-adherent ALL cells in vitro and results in a reduction in leukemic burden in vivo. Targeting of this WNT/β-catenin-mediated EMT-like program is a potential therapeutic approach to overcome cell extrinsically acquired drug resistance in ALL., Competing Interests: Declaration of interests I.I. received honoraria from Mission Bio. C.G.M. received research funding from AbbVie and Pfizer, honoraria from Amgen and Illumina, and royalty payments from Cyrus. C.G.M. is on an advisory board for Illumina. N.H. and M.P.V. have patent applications in the field of immunotherapy., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)