Andreas Loew

Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma.

A chimeric antigen receptor redirects T cells to treat glioblastoma. CAR T cells drive glioblastoma therapy Immunotherapy with chimeric antigen receptor (CAR) T cells can successfully treat B cell malignancies, but expansion into solid tumors has been limited by the lack of availability of tumor-specific antigens. Now, Johnson et al. target CAR T cells to a variant III mutation of the epidermal growth factor receptor (EGFRvIII), which is thought to be enriched in glioblastoma stem cells. They found that a low-affinity single-chain variable fragment was specific for EGFRvIII over wild-type EGFR and that CAR T cells transduced with this fragment were able to target antigen-expressing cells in vitro and in vivo in multiple mouse xenograft models of human glioblastoma. These cells are currently being moved into the clinic in a phase 1 clinical trial. Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv–based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII+ glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376).

Affinity-Tuned ErbB2 or EGFR Chimeric Antigen Receptor T Cells Exhibit an Increased Therapeutic Index against Tumors in Mice

Target-mediated toxicity is a major limitation in the development of chimeric antigen T-cell receptors (CAR) for adoptive cell therapy of solid tumors. In this study, we developed a strategy to adjust the affinities of the scFv component of CAR to discriminate tumors overexpressing the target from normal tissues that express it at physiologic levels. A CAR-expressing T-cell panel was generated with target antigen affinities varying over three orders of magnitude. High-affinity cells recognized target expressed at any level, including at levels in normal cells that were undetectable by flow cytometry. Affinity-tuned cells exhibited robust antitumor efficacy similar to high-affinity cells, but spared normal cells expressing physiologic target levels. The use of affinity-tuned scFvs offers a strategy to empower wider use of CAR T cells against validated targets widely overexpressed on solid tumors, including those considered undruggable by this approach.

Interaction with both ZNRF3 and LGR4 is required for the signalling activity of R‐spondin

R‐spondin proteins sensitize cells to Wnt signalling and act as potent stem cell growth factors. Various membrane proteins have been proposed as potential receptors of R‐spondin, including LGR4/5, membrane E3 ubiquitin ligases ZNRF3/RNF43 and several others proteins. Here, we show that R‐spondin interacts with ZNRF3/RNF43 and LGR4 through distinct motifs. Both LGR4 and ZNRF3 binding motifs are required for R‐spondin‐induced LGR4/ZNRF3 interaction, membrane clearance of ZNRF3 and activation of Wnt signalling. Importantly, Wnt‐inhibitory activity of ZNRF3, but not of a ZNRF3 mutant with reduced affinity to R‐spondin, can be strongly suppressed by R‐spondin, suggesting that R‐spondin primarily functions by binding and inhibiting ZNRF3. Together, our results support a dual receptor model of R‐spondin action, where LGR4/5 serve as the engagement receptor whereas ZNRF3/RNF43 function as the effector receptor.

Pre-clinical validation of a humanized anti-EGFR variant III chimeric antigen receptor and phase I trial of CART-EGFRvIII in glioblastoma

Chimeric antigen receptors are synthetic molecules designed to re-direct T cells to specific surface antigens; CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of novel surface targets with limited expression. The variant III mutation of the epidermal growth factor receptor (EGFR variant III) is the most common variant of the EGF receptor observed in human tumors, and results from an in-frame deletion of a portion of the extracellular domain. In glioblastoma, the EGFRvIII mutation is oncogenic, portends a poor prognosis, and is thought to be enriched in glioblastoma stem cells. However, because the neoepitope of EGFR variant III is based on a small peptide sequence, an antibody or single-chain variable fragment (scFv) directed to this epitope must be rigorously tested to confirm lack of cross-reactivity to the ubiquitously expressed normal EGFR. Having selected a candidate murine scFv directed to EGFRvIII and a vector backbone encoding a second generation CAR, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to kill antigen-bearing targets effectively, and proliferate and secrete cytokines specifically in response to antigen. We further evaluated the specificity of the lead candidate CAR by comparing it to a cetuximab-based CAR which does not discriminate between EGFR and EGFR variant III; the two CARs, along with negative controls, were tested in vitro against primary cells derived from a panel of normal tissues, and in vivo in immunodeficient mice grafted with normal human skin, which naturally expresses EGFR. CAR-T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFR variant III+ glioblastoma. We have designed a Phase I clinical study of CAR T cells transduced with humanized scFv directed to EGFR variant III in patients with glioblastoma.

Abstract B139: Toxicity testing of EGFRvIII CAR-based immunotherapy of glioblastoma: From bench to bedside

The purpose of this study was to generate a panel of donor-derived primary cells, expand them ex-vivo in to sufficient numbers to utilize as targets for evaluating potential normal tissue toxicity of epidermal growth factor receptor mutation variant three (EGFRvIII)-specific chimeric antigen receptors (CAR) prior to use in clinical trials for patients with glioblastoma (GBM). Nine cell types were obtained, including different epithelial, endothelial, bone, smooth muscle, cardiac, neural, hematopoetic, stem cells, and keratinocytes. Cells were expanded with individual specialized media and protocols for between 8-12 passages. After expansion, primary cell identity was confirmed by morphology, ICC and IHC for characteristic markers. Levels of EGFR and EGFRvIII in each cell type were determined by qRT-PCR. To evaluate potential normal-cell toxicity, CAR T cells were co-cultured with each type of primary cell and function was evaluated in two ways: i) T cell activation was measured by staining and flow cytometry of CD3+ T cells stained intracellularly for CD107a, or GzmB, TNFalpha, IFNgamma, IL-2 cytokines. ii) Target cell lysis was evaluated by labeling primary cells with 51Cr prior to 4 hour co-culture with increasing numbers of CAR T cells, and measuring chromium-release. None of the primary cells showed expression of EGFRvIII, although several, in particular keratinocytes and renal epithelial, had high levels of EGFR. In functional assays, while the EGFR CAR T cells recognized and lysed EGFR expressing cell types, EGFRvIII CARs showed T cell activation and target lysis only of EGFRvIII expressing tumors. EGFRvIII CAR 2173 was selected for use in clinical trials at UPENN and UCSD, treating patients with GBM. To date, 6 patients have been infused with 2173 EGFRvIII CAR T cells, with no observed toxicity. All patients had detectable expansion of CAR T cells in vivo in blood, and one patient with subsequent tumor resection had detectable intra-tumoral CAR T cells. These CARs appear to be safe, persist in vivo and traffic into GBM tumor. An update on the clinical trial will be presented at the conference. Note:This abstract was not presented at the conference. Citation Format: Alexandria P. Cogdill, Alina Boesteanu, Chong Xu, Kathleen Haines, John Scholler, Joseph Fraietta, Yangbing Zhao, Xiaojun Liu, Jennifer Morrissette, Bruce Levine, Simon Lacey, Andreas Loew, Reshma Singh, Jennifer Brogdon, Donald M. O9Rourke, Marcela V. Maus, Carl H. June, Laura A. Johnson. Toxicity testing of EGFRvIII CAR-based immunotherapy of glioblastoma: From bench to bedside. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B139.

Abstract B05: A biologic screen to evaluate potential toxicity of chimeric antigen receptor modified T cells against primary normal human tissues

Adoptive cell therapy with chimeric antigen receptor (CAR) engineered T lymphocytes has recently shown great success for the treatment of hematologic malignancies. Similar strategies based on enhancing T cell function and redirecting these lymphocytes against solid tumors are being developed, however, early attempts have resulted in unexpected toxicity of normal cells expressing shared antigen or cross-reactive antigens. A primary challenge in translating new CARs to therapeutic platforms is the lack of robust models to predict potential adverse events pre-clinically. To address this challenge, we developed an in vitro system consisting of thirteen expanded primary human cell types. To detect potential off-tumor on-target effects on normal tissues, we co-cultured CARs directed against human epidermal growth factor receptor (EGFR) in our primary cell panel. Flow cytometric and chromium release assays were used to measure intracellular cytokine production by effectors and specific cell lysis of targets, respectively. T cells expressing a CAR against wild-type EGFR elicited strong effector cytokine production and effectively lysed both tumor and normal cells expressing EGFR. In contrast, T cells engineered to express a humanized CAR against mutant EGFR variant III (EGFRvIII), a tumor-specific antigen, reacted only with tumors and did not react to the panel of primary cell targets. This demonstrated a lack of potential cross-reactivity related to off-tumor on-target effects in the normal cell types represented here, suggesting improved safety for clinical use. The thirteen primary cell types we have generated form a panel of normal cells to test novel CAR T cells for potential toxicity and an adequate therapeutic index prior to first-in-human studies. Citation Format: Alexandria P. Cogdill, Alina Boesteanu, Kathleen Haines, Joseph Fraietta, John Scholler, Andreas Loew, Pramod Thekkat, Jennifer Brogdon, Marcela V. Maus, Carl June, Laura A. Johnson. A biologic screen to evaluate potential toxicity of chimeric antigen receptor modified T cells against primary normal human tissues. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B05.