A Massachusetts team has designed an in vivo shRNA screen to discover immunosuppressive tumor targets that can be blocked to improve the efficacy of T cell immunotherapies.1 The screening system shows that current checkpoint inhibitors are barely scratching the surface of potential targets to modulate and may enable new directions in immunotherapy enhancement.

Targets for modulating immune responses, such as CTLA-4 (CD152) and programmed cell death 1 (PDCD1; PD-1; CD279), are typically identified in vitro and tested in animal models later in the discovery process. The Massachusetts team thought it would be useful to identify targets directly in animals to account for the complex interactions of immune cells within tissues.

CTLA-4 and PD-1 are immunological checkpoint proteins. Signaling through these proteins can lead to T cell exhaustion and allow tumors to evade the immune response.

Yervoy ipilimumab, a human mAb against CTLA-4 from Bristol-Myers Squibb Co., is the first checkpoint inhibitor to reach the market. The antibody was approved to treat metastatic melanoma in 2011. Bristol-Myers and Ono Pharmaceutical Co. Ltd. have the most advanced PD-1 antibody, nivolumab, which is in Phase III testing for metastatic melanoma, non-small cell lung cancer (NSCLC) and renal cell carcinoma (RCC).

The Massachusetts group used two separate shRNA libraries-one focused on genes associated with dysfunctional T cells and the other on kinase and phosphatase genes. The hypothesis was that when the shRNAs were introduced into T cells, only a small subset of the nucleic acids would restore T cell proliferation within the tumor microenvironment.

The result would be an enriched population of those shRNA-expressing T cells within the tumors.

Mice were injected with melanoma antigen-specific T cells infected with multiple shRNAs. Seven days later, T cells were purified from tumors and secondary lymphoid tissue.

Deep sequencing of the shRNAs found in T cells purified from tumors and secondary lymphoid organs identified the over-represented shRNAs (see "In vivo shRNA discovery of immunotherapy targets").

The researchers identified 43 genes with shRNAs that were increased more than fourfold in tumors compared with spleens. The set included new targets as well as genes known to inhibit T cell receptor (TCR) signaling or function.

Next, the targets were validated in the same system using antigen-specific T cells infected with only a single shRNA for each target gene. The goal was to directly determine which shRNAs increased T cell accumulation in tumors.

In mice, the researchers identified shRNAs targeting seven genes that led to a tenfold increase in T cell accumulation in tumors compared with spleens.

These genes included Ppp2r2d (protein phosphatase 2 regulatory subunit Bd), Perk (eukaryotic translation initiation factor 2α kinase 3;
Eif2ak3), Arhgap5 (rho GTPase activating protein 5), Smad2 (Smad family member 2; Madh2), Akap8l (a-kinase anchor protein 8-like), ribokinase (Rbks) and Erg2 (potassium channel Kv11.2; Kcnh6).

The greatest difference in accumulation was observed for Ppp2r2d.

In the melanoma mouse model, tumor antigen-specific T cells expressing Ppp2r2d-targeting shRNA increased T cell proliferation, survival and function compared with T cells expressing a control shRNA. Compared with mice given the control shRNA T cells, mice given the Ppp2r2d shRNA T cells had decreased tumor volume and increased survival.

Results were published in Nature. The team included researchers from the Dana-Farber Cancer Institute, Massachusetts Institute of Technology, Broad Institute of MIT and Harvard, Novartis Institutes for BioMedical Research and Genomics Institute of the Novartis Research Foundation.

Surprise players

Corresponding author Kai Wucherpfennig said that the in vivo assay is a first attempt to find previously unknown genes that could be regulated to help T cells circumvent the immunosuppressive tumor microenvironment.

"For future modification to our system, we are thinking about including fluorescent reporters for expression of cytokines such as interferon-g (IFNG; IFN-g) and cytotoxic molecules such as granzyme B
(GrB; GZMB) and perforin 1 (PRF1) so that we might discover genes that control these critical T cell effector functions," added Wucherpfennig, a professor of neurology at Harvard Medical School and a professor in the Department of Cancer Immunology and AIDS at Dana-Farber.

Drew Pardoll, a professor of oncology and co-director of the cancer immunology and hematopoiesis program at The Johns Hopkins University School of Medicine, said that the screening strategy is a good way to identify truly unexpected targets relative to conventional screening procedures.

"What I really like about the screen is that most of the genes identified are not on the list of the usual suspects," said Pardoll. "Their method emphasizes that current checkpoint inhibitors being developed for immunotherapy are barely scratching the surface of what is available to modulate therapeutically. The team will ultimately have to demonstrate, using multiple targets identified in their screening approach, that knockdown or inhibition of the targets enhances T cell antitumor activity."

Going forward, Wucherpfennig said that the team wants to find additional targets and include human tumors in xenotransplant mouse models. "Subcutaneous tumors are imperfect models, but they provide information regarding interactions between tumor cells, immune cells and stromal cells that in vitro screening methods cannot," he said.

Sriram Sathy, director of target and biomarker discovery at cancer immunotherapy company Jounce Therapeutics Inc., said that human tumors "display a high degree of heterogeneity in the composition of T cell infiltrates. To expand on these results, these analyses need to be extended to additional tumor models. Ideally, models where tumor rejection antigens that have been modified by the immune system would be included to help solidify these findings."

Tibor Keler, CSO and SVP at antibody and immunomodulation company Celldex Therapeutics Inc., said that it would be important to link the findings of the Massachusetts team to clinical situations.

"For example, are these proteins regulated in tumor-infiltrating T cells in patient tumor samples?" asked Keler. "Are they differentially expressed in tumor-infiltrating T cells of patients with a good outcome relative to those with a poor outcome?"

"Ultimately, validation will require translation to the human setting, which could be partially addressed in a humanized mouse model of cancer," continued Keler. "This would provide a better understanding of the regulation of a target in a human disease setting."

Michael Briskin, VP of discovery research at Jounce, noted that "targeting the gene product of Ppp2r2d with antibodies or small molecules will be challenging because it is expressed intracellularly and is part of a complex regulatory network. Ppp2r2d might be more interesting from an adoptive T cell therapy perspective, where knockdown of Ppp2r2d could be included."

In their Nature manuscript, the team proposed that "the efficacy of such T-cell-based therapies could be enhanced by shRNA-mediated silencing of genes that inhibit T-cell function in the tumor microenvironment."

The patent and licensing status of the findings are not disclosed.

Baas, T. SciBX 7(6); doi:10.1038/scibx.2014.162 Published online Feb. 13, 2014


1.   Zhou, P. et al. Nature; published online Jan. 29, 2014; doi:10.1038/nature12988 Contact: Kai W. Wucherpfennig, Dana-Farber Cancer Institute, Boston, Mass. e-mail: kai_wucherpfennig@dfci.harvard.edu


      Bristol-Myers Squibb Co. (NYSE:BMY), New York, N.Y.

      Broad Institute of MIT and Harvard, Cambridge, Mass.

      Celldex Therapeutics Inc. (NASDAQ:CLDX), Needham, Mass.

      Dana-Farber Cancer Institute, Boston, Mass.

      Genomics Institute of the Novartis Research Foundation, San Diego, Calif.

      Harvard Medical School, Boston, Mass.

      The Johns Hopkins University School of Medicine, Baltimore, Md.

      Jounce Therapeutics Inc., Cambridge, Mass.

      Massachusetts Institute of Technology, Cambridge, Mass.

      Novartis Institutes for BioMedical Research, Cambridge, Mass.

      Ono Pharmaceutical Co. Ltd. (Tokyo:4528), Osaka, Japan