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1.
Sci Transl Med ; 16(750): eadk7640, 2024 Jun 05.
Article in English | MEDLINE | ID: mdl-38838132

ABSTRACT

Approximately 50% of patients with hematologic malignancies relapse after chimeric antigen receptor (CAR) T cell treatment; mechanisms of failure include loss of CAR T persistence and tumor resistance to apoptosis. We hypothesized that both of these challenges could potentially be overcome by overexpressing one or more of the Bcl-2 family proteins in CAR T cells to reduce their susceptibility to apoptosis, both alone and in the presence of BH3 mimetics, which can be used to activate apoptotic machinery in malignant cells. We comprehensively investigated overexpression of different Bcl-2 family proteins in CAR T cells with different signaling domains as well as in different tumor types. We found that Bcl-xL and Bcl-2 overexpression in CAR T cells bearing a 4-1BB costimulatory domain resulted in increased expansion and antitumor activity, reduced exhaustion, and decreased apoptotic priming. In addition, CAR T cells expressing either Bcl-xL or a venetoclax-resistant Bcl-2 variant led to enhanced antitumor efficacy and survival in murine xenograft models of lymphoma and leukemia in the presence or absence of the BH3 mimetic venetoclax, a clinically approved BH3 mimetic. In this setting, Bcl-xL overexpression had stronger effects than overexpression of Bcl-2 or the Bcl-2(G101V) variant. These findings suggest that CAR T cells could be optimally engineered by overexpressing Bcl-xL to enhance their persistence while opening a therapeutic window for combination with BH3 mimetics to prime tumors for apoptosis.


Subject(s)
Apoptosis , Bridged Bicyclo Compounds, Heterocyclic , Proto-Oncogene Proteins c-bcl-2 , Receptors, Chimeric Antigen , Sulfonamides , Humans , Animals , Proto-Oncogene Proteins c-bcl-2/metabolism , Receptors, Chimeric Antigen/metabolism , Sulfonamides/pharmacology , Apoptosis/drug effects , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Xenograft Model Antitumor Assays , Mice , T-Lymphocytes/metabolism , T-Lymphocytes/immunology , Cell Line, Tumor , Immunotherapy, Adoptive/methods , bcl-X Protein/metabolism , Peptide Fragments , Proto-Oncogene Proteins
2.
bioRxiv ; 2024 Feb 11.
Article in English | MEDLINE | ID: mdl-38370665

ABSTRACT

Chimeric antigen receptor (CAR) T cells are an effective treatment for some blood cancers. However, the lack of tumor-specific surface antigens limits their wider use. We identified a set of surface antigens that are limited in their expression to cancer and the central nervous system (CNS). We developed CAR T cells against one of these antigens, LINGO1, which is widely expressed in Ewing sarcoma (ES). To prevent CNS targeting, we engineered LINGO1 CAR T cells lacking integrin α4 (A4ko), an adhesion molecule essential for migration across the blood-brain barrier. A4ko LINGO1 CAR T cells were efficiently excluded from the CNS but retained efficacy against ES. We show that altering adhesion behavior expands the set of surface antigens targetable by CAR T cells.

3.
Sci Transl Med ; 15(705): eadd7900, 2023 07 19.
Article in English | MEDLINE | ID: mdl-37467316

ABSTRACT

T cells expressing chimeric antigen receptors (CARs) have shown remarkable therapeutic activity against different types of cancer. However, the wider use of CAR T cells has been hindered by the potential for life-threatening toxicities due to on-target off-tumor killing of cells expressing low amounts of the target antigen. CD229, a signaling lymphocyte-activation molecule (SLAM) family member, has previously been identified as a target for CAR T cell-mediated treatment of multiple myeloma (MM) due to its high expression on the surfaces of MM cells. CD229 CAR T cells have shown effective clearance of MM cells in vitro and in vivo. However, healthy lymphocytes also express CD229, albeit at lower amounts than MM cells, causing their unintended targeting by CD229 CAR T cells. To increase the selectivity of CD229 CAR T cells for MM cells, we used a single amino acid substitution approach of the CAR binding domain to reduce CAR affinity. To identify CARs with increased selectivity, we screened variant binding domains using solid-phase binding assays and biolayer interferometry and determined the cytotoxic activity of variant CAR T cells against MM cells and healthy lymphocytes. We identified a CD229 CAR binding domain with micromolar affinity that, when combined with overexpression of c-Jun, confers antitumor activity comparable to parental CD229 CAR T cells but lacks the parental cells' cytotoxic activity toward healthy lymphocytes in vitro and in vivo. The results represent a promising strategy to improve the efficacy and safety of CAR T cell therapy that requires clinical validation.


Subject(s)
Antineoplastic Agents , Multiple Myeloma , Receptors, Chimeric Antigen , Humans , Multiple Myeloma/pathology , Amino Acids/metabolism , T-Lymphocytes , Receptors, Chimeric Antigen/metabolism , Immunotherapy, Adoptive/methods , Antineoplastic Agents/metabolism , Xenograft Model Antitumor Assays , Cell Line, Tumor
4.
Cell Death Dis ; 14(4): 267, 2023 04 13.
Article in English | MEDLINE | ID: mdl-37055388

ABSTRACT

Chimeric antigen receptor (CAR) T cell therapy is an FDA-approved treatment for several hematologic malignancies, yet not all patients respond to this treatment. While some resistance mechanisms have been identified, cell death pathways in target cancer cells remain underexplored. Impairing mitochondrial apoptosis via knockout of Bak and Bax, forced Bcl-2 and Bcl-XL expression, or caspase inhibition protected several tumor models from CAR T killing. However, impairing mitochondrial apoptosis in two liquid tumor cell lines did not protect target cells from CAR T killing. We found that whether a cell was Type I or Type II in response to death ligands explained the divergence of these results, so that mitochondrial apoptosis was dispensable for CART killing of cells that were Type I but not Type II. This suggests that the apoptotic signaling induced by CAR T cells bears important similarities to that induced by drugs. Combinations of drug and CAR T therapies will therefore require tailoring to the specific cell death pathways activated by CAR T cells in different types of cancer cells.


Subject(s)
Neoplasms , Receptors, Chimeric Antigen , Humans , Apoptosis , Caspases/metabolism , Cell Line, Tumor , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2 Homologous Antagonist-Killer Protein/metabolism , Neoplasms/therapy
6.
Nat Commun ; 11(1): 798, 2020 02 07.
Article in English | MEDLINE | ID: mdl-32034142

ABSTRACT

Multiple myeloma (MM) is a plasma cell malignancy and most patients eventually succumb to the disease. Chimeric antigen receptor (CAR) T cells targeting B-Cell Maturation Antigen (BCMA) on MM cells have shown high-response rates, but limited durability. CD229/LY9 is a cell surface receptor present on B and T lymphocytes that is universally and strongly expressed on MM plasma cells. Here, we develop CD229 CAR T cells that are highly active in vitro and in vivo against MM plasma cells, memory B cells, and MM-propagating cells. We do not observe fratricide during CD229 CAR T cell production, as CD229 is downregulated in T cells during activation. In addition, while CD229 CAR T cells target normal CD229high T cells, they spare functional CD229neg/low T cells. These findings indicate that CD229 CAR T cells may be an effective treatment for patients with MM.


Subject(s)
Immunotherapy, Adoptive/methods , Multiple Myeloma/therapy , Receptors, Antigen, T-Cell/immunology , Signaling Lymphocytic Activation Molecule Family/metabolism , Animals , Antibodies/immunology , B-Lymphocytes/metabolism , Humans , K562 Cells/immunology , Male , Mice, Inbred NOD , Multiple Myeloma/pathology , Receptors, Antigen, T-Cell/metabolism , Signaling Lymphocytic Activation Molecule Family/genetics , Signaling Lymphocytic Activation Molecule Family/immunology , T-Lymphocytes/immunology , T-Lymphocytes/transplantation , Xenograft Model Antitumor Assays
7.
Biochemistry ; 54(27): 4163-6, 2015 Jul 14.
Article in English | MEDLINE | ID: mdl-26115234

ABSTRACT

CCL19 and CCL21 are chemokines involved in the trafficking of immune cells, particularly within the lymphatic system, through activation of CCR7. Concurrent expression of PSGL-1 and CCR7 in naive T-cells enhances recruitment of these cells to secondary lymphoid organs by CCL19 and CCL21. Here the solution structure of CCL19 is reported. It contains a canonical chemokine domain. Chemical shift mapping shows the N-termini of PSGL-1 and CCR7 have overlapping binding sites for CCL19 and binding is competitive. Implications for the mechanism of PSGL-1's enhancement of resting T-cell recruitment are discussed.


Subject(s)
Chemokine CCL19/chemistry , Chemokine CCL19/metabolism , Membrane Glycoproteins/metabolism , Receptors, CCR7/metabolism , Binding Sites , Humans , Models, Molecular , Protein Conformation
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