Engineering CAR-T cells to activate small-molecule drugs in situ

2021 ◽  
Author(s):  
Thomas J. Gardner ◽  
J. Peter Lee ◽  
Christopher M. Bourne ◽  
Dinali Wijewarnasuriya ◽  
Nihar Kinarivala ◽  
...  
Keyword(s):  
T Cells ◽  
Small Molecule ◽  
Car T Cells ◽  
Small Molecule Drugs ◽  
Car T

scholarly journals Nanovesicles derived from bispecific CAR-T cells targeting the spike protein of SARS-CoV-2 for treating COVID-19

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Tianchuan Zhu ◽  
Yuchen Xiao ◽  
Xiaojun Meng ◽  
Lantian Tang ◽  
Bin Li ◽  
...  
Keyword(s):  
T Cells ◽  
Small Molecule ◽  
Neutralizing Antibodies ◽  
Antiviral Drugs ◽  
Spike Protein ◽  
Viral Resistance ◽  
Single Chain ◽  
Car T Cells ◽  
Small Molecule Drugs ◽  
Car T

Abstract Background Considering the threat of the COVID-19 pandemic, caused by SARS-CoV-2, there is an urgent need to develop effective treatments. At present, neutralizing antibodies and small-molecule drugs such as remdesivir, the most promising compound to treat this infection, have attracted considerable attention. However, some potential problems need to be concerned including viral resistance to antibody-mediated neutralization caused by selective pressure from a single antibody treatment, the unexpected antibody-dependent enhancement (ADE) effect, and the toxic effect of small-molecule drugs. Results Here, we constructed a type of programmed nanovesicle (NV) derived from bispecific CAR-T cells that express two single-chain fragment variables (scFv), named CR3022 and B38, to target SARS-CoV-2. Nanovesicles that express both CR3022 and B38 (CR3022/B38 NVs) have a stronger ability to neutralize Spike-pseudovirus infectivity than nanovesicles that express either CR3022 or B38 alone. Notably, the co-expression of CR3022 and B38, which target different epitopes of spike protein, could reduce the incidence of viral resistance. Moreover, the lack of Fc fragments on the surface of CR3022/B38 NVs could prevent ADE effects. Furthermore, the specific binding ability to SARS-CoV-2 spike protein and the drug loading capacity of CR3022/B38 NVs can facilitate targeted delivery of remdesiver to 293 T cells overexpressing spike protein. These results suggest that CR3022/B38 NVs have the potential ability to target antiviral drugs to the main site of viral infection, thereby enhancing the antiviral ability by inhibiting intracellular viral replication and reducing adverse drug reactions. Conclusions In summary, we demonstrate that nanovesicles derived from CAR-T cells targeting the spike protein of SARS-COV-2 have the ability to neutralize Spike-pseudotyped virus and target antiviral drugs. This novel therapeutic approach may help to solve the dilemma faced by neutralizing antibodies and small-molecule drugs in the treatment of COVID-19. Graphical Abstract

Identification of Small Molecule Modulators to Enhance the Therapeutic Properties of Chimeric Antigen Receptor T Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4712-4712
Author(s):  
Jonathan Rosen ◽  
Betsy Rezner ◽  
David Robbins ◽  
Ian Hardy ◽  
Eigen Peralta ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Small Molecules ◽  
Small Molecule ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T ◽  
Equity Ownership

Abstract Adoptive cellular therapies using engineered chimeric antigen receptor T cells (CAR-T cells) are rapidly emerging as a highly effective treatment option for a variety of life-threatening hematological malignancies. Small molecule-mediated modulation of T cell differentiation during the in vitro CAR-T manufacturing process has great potential as a method to optimize the therapeutic potential of cellular immunotherapies. In animal models, T cells with a central or stem memory (TCM/SCM) phenotype display enhanced in vivoefficacy and persistence relative to other T cell subpopulations. We sought to identify small molecules that promote skewing towards a TCM/SCM phenotype during the CAR-T manufacturing process, with associated enhanced viability, expansion and metabolic profiles of the engineered cells. To this end, we developed a high-throughput functional screening platform with primary human T cells using a combination of high-content immunophenotyping and gene expression-based readouts to analyze cells following a high-throughput T cell culture platform that represents a scaled-down model of clinical CAR-T cell production. Multicolor flow cytometry was used to measure expansion, cell viability and the expression levels of cell surface proteins that define TCM cells (e.g., CCR7, CD62L and CD27) and markers of T cell exhaustion (e.g., PD1, LAG3, and TIM3). In parallel, a portion of each sample was evaluated using high content RNA-Seq based gene expression analysis of ~100 genes representing key biological pathways of interest. A variety of known positive and negative control compounds were incorporated into the high-throughput screens to validate the functional assays and to assess the robustness of the 384-well-based screening. The ability to simultaneously correlate small molecule-induced changes in protein and gene expression levels with impacts on cell proliferation and viability of various T cell subsets, enabled us to identify multiple classes of small molecules that favorably enhance the therapeutic properties of CAR-T cells. Consistent with results previously presented by Perkins et al. (ASH, 2015), we identified multiple PI3K inhibitors that could modify expansion of T cells while retaining a TCM/SCM phenotype. In addition, we identified small molecules, and small molecule combinations, that have not been described previously in the literature that could improve CAR-T biology. Several of the top hits from the screens have been evaluated across multiple in vitro (e.g., expansion, viability, CAR expression, serial restimulation/killing, metabolic profiling, and evaluation of exhaustion markers) and in vivo (e.g., mouse tumor models for persistence and killing) assays. Results from the initial screening hits have enabled us to further refine the optimal target profile of a pharmacologically-enhanced CAR-T cell. In addition, we are extending this screening approach to identify small molecules that enhance the trafficking and persistence of CAR-T cells for treating solid tumors. In conclusion, the approach described here identifies unique small molecule modulators that can modify CAR-T cells during in vitro expansion, such that improved profiles can be tracked and selected from screening through in vitro and in vivo functional assays. Disclosures Rosen: Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics, Inc: Employment, Equity Ownership. Robbins:Fate Therapeutics: Employment, Equity Ownership. Hardy:Fate Therapeutics: Employment, Equity Ownership. Peralta:Fate Therapeutics: Employment, Equity Ownership. Maine:Fate Therapeutics: Employment, Equity Ownership. Sabouri:Fate Therapeutics: Employment, Equity Ownership. Reynal:Fate Therapeutics: Employment. Truong:Fate Therapeutics: Employment, Equity Ownership. Moreno:Fate Therapeutics, Inc.: Employment, Equity Ownership. Foster:Fate Therapeutics: Employment, Equity Ownership. Borchelt:Fate Therapeutics: Employment, Equity Ownership. Meza:Fate Therapeutics: Employment, Equity Ownership. Thompson:Juno Therapeutics: Employment, Equity Ownership. Fontenot:Juno Therapeutics: Employment, Equity Ownership. Larson:Juno Therapeutics: Employment, Equity Ownership. Mujacic:Juno Therapeutics: Employment, Equity Ownership. Shoemaker:Fate Therapeutics: Employment, Equity Ownership.

scholarly journals A conformation-specific ON-switch for controlling CAR T cells with an orally available drug

2020 ◽  
Vol 117 (26) ◽  
pp. 14926-14935 ◽  
Author(s):  
Charlotte U. Zajc ◽  
Markus Dobersberger ◽  
Irene Schaffner ◽  
Georg Mlynek ◽  
Dominic Pühringer ◽  
...  
Keyword(s):  
T Cells ◽  
Small Molecule ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Fold Increase ◽  
Retinol Binding Protein ◽  
Dependent Manner ◽  
Car T Cells ◽  
Car T

Molecular ON-switches in which a chemical compound induces protein–protein interactions can allow cellular function to be controlled with small molecules. ON-switches based on clinically applicable compounds and human proteins would greatly facilitate their therapeutic use. Here, we developed an ON-switch system in which the human retinol binding protein 4 (hRBP4) of the lipocalin family interacts with engineered hRBP4 binders in a small molecule-dependent manner. Two different protein scaffolds were engineered to bind to hRBP4 when loaded with the orally available small molecule A1120. The crystal structure of an assembled ON-switch shows that the engineered binder specifically recognizes the conformational changes induced by A1120 in two loop regions of hRBP4. We demonstrate that this conformation-specific ON-switch is highly dependent on the presence of A1120, as demonstrated by an ∼500-fold increase in affinity upon addition of the small molecule drug. Furthermore, the ON-switch successfully regulated the activity of primary human CAR T cells in vitro. We anticipate that lipocalin-based ON-switches have the potential to be broadly applied for the safe pharmacological control of cellular therapeutics.

scholarly journals A CAR RNA FISH assay reveals functional and spatial heterogeneity of chimeric antigen receptor T cells in tissue

2020 ◽  
Author(s):  
Karsten Eichholz ◽  
Alvason Zhenhua Li ◽  
Kurt Diem ◽  
Semih U. Tareen ◽  
Michael C. Jensen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T Cell ◽  
Rna Fish ◽  
Car T

AbstractChimeric antigen receptor (CAR) T cells are engineered cells used in cancer therapy and are studied to treat infectious diseases. Trafficking and persistence of CAR T cells is an important requirement for efficacy to target cancer and HIV sanctuary sites. Here, we describe a CAR RNA FISH histocytometry platform combined with a dnnRRS image analysis algorithm to quantitate spatial distribution and in vivo functional ability of a CAR T cell population at a single cell resolution. In situ, CAR T cell exhibited a heterogenous effector gene expression and this was related to the distance from tumor cells, allowing a quantitative assessment of the potential in vivo effectiveness. The platform offers the potential to study immune functions engineered cells in situ with their target cells in tissues with high statistical power and thus, as an important tool for preclinical and potentially clinical assessment of CAR T cell effectiveness.One Sentence SummaryWe developed a CAR RNA FISH assay to study chimeric antigen receptor T cell trafficking and function in human and mouse tissue.Impact statementWe developed an imaging platform and analysis pipeline to study large populations of engineered cells on a single cell level in situ.

Abstract 2193: Small molecule inducible MyD88/CD40 (iMC) in CAR-T cells can repolarize M2 macrophage to an anti-tumor M1 phenotype

2020 ◽  
Author(s):  
Xiaohong Wang ◽  
Konrad Gabrusiewicz ◽  
David M. Spencer ◽  
Aaron E. Foster ◽  
J. Henri Bayle
Keyword(s):  
T Cells ◽  
Small Molecule ◽  
M2 Macrophage ◽  
Car T Cells ◽  
Car T ◽  
M1 Phenotype

scholarly journals A CAR RNA FISH assay to study functional and spatial heterogeneity of chimeric antigen receptor T cells in tissue

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Karsten Eichholz ◽  
Alvason Zhenhua Li ◽  
Kurt Diem ◽  
Michael Claus Jensen ◽  
Jia Zhu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T Cell ◽  
Rna Fish ◽  
Car T

AbstractChimeric antigen receptor (CAR) T cells are engineered cells used in cancer therapy and are studied to treat infectious diseases. Trafficking and persistence of CAR T cells is an important requirement for efficacy to target cancer. Here, we describe a CAR RNA FISH histo-cytometry platform combined with a random reaction seed image analysis algorithm to quantitate spatial distribution and in vivo functional activity of a CAR T cell population at a single cell resolution for preclinical models. In situ, CAR T cell exhibited a heterogenous effector gene expression and this was related to the distance from tumor cells, allowing a quantitative assessment of the potential in vivo effectiveness. The platform offers the potential to study immune functions of genetically engineered cells in situ with their target cells in tissues with high statistical power and thus, can serve as an important tool for preclinical assessment of CAR T cell effectiveness.

Evaluating the reversible control of an engineered CAR T cell ON-switch.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e14550-e14550 ◽  
Author(s):  
Amy Gilbert ◽  
Stephen Santoro ◽  
Tiffany Tse ◽  
Tara Candelario-Chopra ◽  
Tina Gomes ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Small Molecule ◽  
Cytokine Production ◽  
Polypeptide Chain ◽  
Car T Cells ◽  
Small Molecule Drug ◽  
Car T Cell ◽  
Car T

e14550 Background: CAR T cell therapy holds enormous promise for many cancer types but its application may be limited by serious toxicities. To lower this hurdle, our aim is to engineer tunable cell therapies. One of our approaches includes a “ON-switch” chimeric antigen receptor (Wu et al., Science 2015) that requires the administration of a small molecule acting as a dimerizing agent between one polypeptide chain containing the antigen recognition domain and half of an inducible heterodimerization system and another polypeptide chain containing the second half of the inducible heterodimerization motif, the CD3ζ chain and a costimulatory motif. Using an FDA approved small molecule drug, we evaluate the reversibility of ON-switch CAR T cells in preclinical models. Methods: First, we evaluated the proliferation, cytotoxicity and cytokine production of several ON-switch constructs in human primary T cells. Next, to address the reversibility of the ON-switch (ON→OFF→ON), we performed a series of co-culture experiments where the small molecule drug was added to tumor cells and ON-switch CAR T cells, then washed out, and then re-introduced back into the co-cultures. We compared CAR T cell mediated killing and cytokine production from the On-switch CAR T cells relative to a canonical CAR T control. Results: Our On-switch CAR T cells were shown to proliferate, secrete cytokines as well as mediate dose dependent cytotoxicity in the presence of the small molecule drug. Importantly, in the presence of antigen but in absence of the small molecule drug we did not measure any significant functional activity in our ON-switch CARs. Additonally, following the removal of the small molecule drug over a period several days we did not observe any significant CAR mediated cytotoxicity. Following the subsequent re-addition of the small molecule, we observed further CAR T cell mediated cytotoxicity against tumor cells. Conclusions: These results show that the small molecule inducible On-switch CARs maintain functional activity as well as reversibility allowing for the tunable control of a CAR T cell.

scholarly journals Direct control of CAR T cells through small molecule-regulated antibodies

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Spencer Park ◽  
Edward Pascua ◽  
Kevin C. Lindquist ◽  
Christopher Kimberlin ◽  
Xiaodi Deng ◽  
...  
Keyword(s):  
T Cells ◽  
Adverse Events ◽  
Small Molecule ◽  
Cell Activity ◽  
Antibody Activity ◽  
Car T Cells ◽  
Antibody Therapeutics ◽  
Drug Conjugates ◽  
Car T ◽  
Specific Cytotoxicity

AbstractAntibody-based therapeutics have experienced a rapid growth in recent years and are now utilized in various modalities spanning from conventional antibodies, antibody-drug conjugates, bispecific antibodies to chimeric antigen receptor (CAR) T cells. Many next generation antibody therapeutics achieve enhanced potency but often increase the risk of adverse events. Antibody scaffolds capable of exhibiting inducible affinities could reduce the risk of adverse events by enabling a transient suspension of antibody activity. To demonstrate this, we develop conditionally activated, single-module CARs, in which tumor antigen recognition is directly modulated by an FDA-approved small molecule drug. The resulting CAR T cells demonstrate specific cytotoxicity of tumor cells comparable to that of traditional CARs, but the cytotoxicity is reversibly attenuated by the addition of the small molecule. The exogenous control of conditional CAR T cell activity allows continual modulation of therapeutic activity to improve the safety profile of CAR T cells across all disease indications.

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