Augmentation of CAR T-cell Trafficking and Antitumor Efficacy by Blocking Protein Kinase A Localization

Our plain-English summary of Augmentation of CAR T-cell Trafficking and Antitumor Efficacy by Blocking Protein Kinase A Localization, by K. Newick, S. O'Brien, J. Sun, et al (Cancer Immunology Research, 2016).

The study

• Authors: K. Newick, S. O'Brien, J. Sun, V. Kapoor, S. Maceyko, A. Lo, E. Puré, E. Moon, S.M. Albelda
• Journal: Cancer Immunology Research
• Published: 2016;4(6):541-51
• Cited by: 168 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Antitumor treatments based on the infusion of T cells expressing chimeric antigen receptors (CAR T cells) are still relatively ineffective for solid tumors, due to the presence of immunosuppressive mediators [such as prostaglandin E2 (PGE2) and adenosine] and poor T-cell trafficking. PGE2 and adenosine activate protein kinase A (PKA), which then inhibits T-cell receptor (TCR) activation. This inhibition process requires PKA to localize to the immune synapse via binding to the membrane protein ezrin. We generated CAR T cells that expressed a small peptide called the "regulatory subunit I anchoring disruptor" (RIAD) that inhibits the association of PKA with ezrin, thus blunting the negative effects of PKA on TCR activation. After exposure to PGE2 or adenosine in vitro, CAR-RIAD T cells showed increased TCR signaling, released more cytokines, and showed enhanced killing of tumor cells compared with CAR T cells. When injected into tumor-bearing mice, the antitumor efficacy of murine and human CAR-RIAD T cells was enhanced compared with that of CAR T cells, due to resistance to tumor-induced hypofunction and increased T-cell infiltration of established tumors. Subsequent in vitro assays showed that both mouse and human CAR-RIAD cells migrated more efficiently than CAR cells did in response to the chemokine CXCL10 and also had better adhesion to various matrices. Thus, the intracellular addition of the RIAD peptide to adoptively transferred CAR T cells augments their efficacy by increasing their effector function and by improving trafficking into tumor sites. This treatment strategy, therefore, shows potential clinical application for treating solid tumors. Cancer Immunol Res; 4(6); 541-51. ©2016 AACR.

©2016 American Association for Cancer Research.

In plain English

This was a laboratory study in cultured cells and mice testing whether adding a small peptide (RISR-RIAD), which blocks protein kinase A from docking at the immune synapse, improves chimeric antigen receptor (CAR) T cells. The authors put the RIAD construct into T cells also carrying a CAR against human mesothelin (mesoCAR) or mouse fibroblast activation protein (FAPCAR), using human T cells and mouse T cells. In culture, mesoCAR-RIAD T cells generally killed mesothelin-expressing mesothelioma cells better, made more IFN-gamma, and their killing was "virtually unaffected" by adenosine or PGE2, while mesoCAR killing fell dose-dependently. Immunoblots showed reduced baseline phosphorylation of Csk S364 and Lck Y505. In NSG mice bearing human EMmeso tumours, mesoCAR slowed growth by about 40% (P less than or equal to 0.01) versus untreated, and mesoCAR-RIAD gave tumours 70% smaller than untreated (P less than or equal to 0.0001), and smaller than mesoCAR (P less than or equal to 0.05). RIAD cells infiltrated tumours more, migrated better toward CXCL10 and adhered better to matrices. The authors concluded RIAD could augment CAR efficacy in solid tumours.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory experiments (in vitro cell assays) plus mouse tumour experiments using genetically modified CAR T cells
• Who was studied: Primary human and mouse T cells and mesothelioma/pancreatic tumour cell lines; female mice aged 10-12 weeks: NOD/scid/IL2rγ-/- (NSG) mice bearing human EMmeso tumours and wild-type C57BL/6 mice bearing AE17meso or PDA4662 tumours, minimum 5 mice per group
• Exposure: Co-expression of the RISR-RIAD peptide with a mesothelin-directed CAR or a FAP-directed CAR in T cells; in vitro exposure of these cells to PGE2 and adenosine
• Main results:
  • In culture, both human and murine mesoCAR-RIAD T cells generally killed mesothelin-expressing tumour cells better and made more IFN-gamma than mesoCAR T cells; non-mesothelin tumour cells were little killed
  • Killing by mesoCAR T cells fell dose-dependently with adenosine or PGE2, whereas killing by mesoCAR-RIAD T cells was essentially unchanged
  • After mesothelin-bead stimulation of human T cells, 8.2% of mesoCAR-RIAD cells made IFN-gamma versus 2.4% of mesoCAR T cells; no such difference was seen with murine T cells
  • Baseline phosphorylation of Csk S364 and Lck Y505 was reduced in mesoCAR-RIAD T cells compared with mesoCAR T cells (human and mouse)
  • In NSG mice with human EMmeso tumours: mesoCAR slowed tumour progression by about 40% (P ≤ 0.01) versus untreated; mesoCAR-RIAD produced tumours 70% smaller than untreated (P ≤ 0.0001) and smaller than mesoCAR (P ≤ 0.05); neither cured established tumours
  • In C57BL/6 mice with murine AE17meso tumours, mesoCAR-RIAD T cells reduced tumour growth compared with mesoCAR T cells (P ≤ 0.05)
  • In the PDA4662 pancreatic tumour model, only FAPCAR-RIAD T cells showed significant antitumor activity at the dose used, fourteen days after transfer
  • mesoCAR-RIAD T cells were ineffective against AE17ova tumours, which do not express mesothelin
  • Tumours from mesoCAR-RIAD-treated mice contained a higher percentage of human CD3+, CD8+ and CD4+ cells (P < 0.05), with roughly a 10:1 CD8 to CD4 ratio; greater murine CD8+ infiltration was also seen at 3 days (P ≤ 0.05)
  • Freshly harvested mesoCAR TILs were hypofunctional for killing and IFN-gamma, while freshly harvested mesoCAR-RIAD TILs retained nearly full cytolytic capability
  • Human and mouse mesoCAR-RIAD T cells migrated more toward CXCL10, expressed more CXCR3 (P ≤ 0.05), adhered better to fibronectin, ICAM-1 and VCAM-1, and showed increased CD49a and CD29
• The authors' conclusion: The authors concluded that adding the RIAD peptide to CAR T cells increased their effector function and trafficking into tumours, and proposed it as a strategy to augment CAR efficacy in solid tumours
• Limitations the authors state:
  • PGE2 concentrations were relatively high in the models used, and other tumours may differ, which could change the degree of RIAD-induced benefit
  • Possible immunogenicity of the RISR-RIAD peptide for clinical translation, although in silico analysis showed no immunogenic neoepitopes for common HLA types
  • Intrinsic limitations of the animal models; no animal model fully predicts patient responses, and the NSG model lacks elements of a human tumour microenvironment
  • The relative importance of CD8+ versus CD4+ T cells was not directly studied
  • The cell lines used were not authenticated

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: PGE2, Cytokine.

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