Purpose This study examines cell-surface ROR1 expression in human tumors and normal tissues. ROR1 is considered a promising target for cancer therapy due to putative tumor-specific expression and multiple groups are developing antibodies and/or chimeric antigen receptor-modified T cells to target ROR1. On-target, off-tumor toxicity is a challenge for most non-mutated tumor antigens, however prior studies suggest that ROR1 is absent on most normal tissues. Experimental Design Our studies show that published antibodies lack sensitivity to detect endogenous levels of cell-surface ROR1 by immunohistochemistry (IHC) in FFPE tissues. We developed a ROR1-specific monoclonal antibody (mAb) targeting the carboxy-terminus of ROR1, and evaluated its specificity and sensitivity in IHC. Results The 6D4 mAb is a sensitive and specific reagent to detect cell-surface ROR1 by IHC. The data shows that ROR1 is homogenously expressed on a subset of ovarian cancer, triple negative breast cancer and lung adenocarcinomas. Contrary to previous findings, we found ROR1 is expressed on several normal tissues including parathyroid, pancreatic islets and regions of the esophagus, stomach and duodenum. The 6D4 mAb recognizes rhesus ROR1, and ROR1 expression was similar in human and macaque tissues suggesting that the macaque is a suitable model to evaluate safety of ROR1 targeted therapies. Conclusions ROR1 is a promising immunotherapeutic target in many epithelial tumors, however high cell-surface ROR1 expression in multiple normal tissues raises concerns for on-target off-tumor toxicities. Clinical translation of ROR1 targeted therapies warrants careful monitoring of toxicities to normal organs, and may require strategies to ensure patient safety.

Genetic engineering of T cells for adoptive transfer by introducing a tumor-targeting chimeric antigen receptor (CAR) is a new approach to cancer immunotherapy. A challenge for the field is to define cell surface molecules that are both preferentially expressed on tumor cells and can be safely targeted with T cells. The orphan tyrosine kinase receptor ROR1 is a candidate target for T-cell therapy with CAR-modified T cells (CAR-T cells) since it is expressed on the surface of many lymphatic and epithelial malignancies and has a putative role in tumor cell survival. The cell surface isoform of ROR1 is expressed in embryogenesis but absent in adult tissues except for B-cell precursors, and low levels of transcripts in adipocytes, pancreas, and lung. ROR1 is highly conserved between humans and macaques and has a similar pattern of tissue expression. To determine if low-level ROR1-expression on normal cells would result in toxicity or adversely affect CAR-T cell survival and/or function, we adoptively transferred autologous ROR1 CAR-T cells into nonhuman primates. ROR1 CAR-T cells did not cause overt toxicity to normal organs and accumulated in bone marrow and lymph node sites where ROR1-positive B cells were present. The findings support the clinical evaluation of ROR1 CAR-T cells for ROR1+ malignancies and demonstrate the utility of nonhuman primates for evaluating the safety of immunotherapy with engineered T cells specific for tumor-associated molecules that are homologous between humans and nonhuman primates.

The ability to engineer T cells to recognize tumor cells through genetic modification with a synthetic chimeric antigen receptor has ushered in a new era in cancer immunotherapy. The most advanced clinical applications are in in targeting CD19 on B cell malignancies. The clinical trials of CD19 CAR therapy have thus far not attempted to select defined subsets prior to transduction or imposed uniformity of the CD4 and CD8 cell composition of the cell products. This review will discuss the rationale for and challenges to utilizing adoptive therapy with genetically modified T cells of defined subset and phenotypic composition.

Purpose: The outgrowth of antigen-negative variants is a significant challenge for adoptive therapy with T cells that target a single specificity. Chimeric antigen receptors (CAR) are typically designed with one or two scFvs that impart antigen specificity fused to activation and costimulation domains of T-cell signaling molecules. We designed and evaluated the function of CARs with up to three specificities for overcoming tumor escape using Designed Ankyrin Repeat Proteins (DAR-Pins) rather than scFvs for tumor recognition.Experimental Design: A monospecific CAR was designed with a DARPin binder (E01) specific for EGFR and compared with a CAR designed using an anti-EGFR scFv. CAR constructs in which DARPins specific for EGFR, EpCAM, and HER2 were linked together in a single CAR were then designed and optimized to achieve multispecific tumor recognition. The efficacy of CAR-T cells bearing a multispecific DARPin CAR for treating tumors with heterogeneous antigen expression was evaluated in vivo.Results: The monospecific anti-EGFR E01 DARPin conferred potent tumor regression against EGFR þ targets that was comparable with an anti-EGFR scFv CAR. Linking three separate DARPins in tandem was feasible and in an optimized format generated a single tumor recognition domain that targeted a mixture of heterogeneous tumor cells, each expressing a single antigen, and displayed synergistic activity when tumor cells expressed more than one target antigen.Conclusions: DARPins can serve as high-affinity recognition motifs for CAR design, and their robust architecture enables linking of multiple binders against different antigens to achieve functional synergy and reduce antigen escape.

Teratomas are a unique class of tumors composed of ecto- meso- and endodermal tissues, all foreign to the site of origin. In humans, the most common teratoma is the ovarian teratoma. Not much is known about the molecular and genetic etiologies of these tumors. Female carriers of the Tgkd transgene are highly susceptible to developing teratomas. Ovaries of Tgkd/+ hemizygous female mice exhibit defects in luteinization, with numerous corpora lutea, some of which contain central trapped, fully-grown oocytes. Genetically, Tgkd teratomas originate from mature oocytes that have completed meiosis I, suggesting that Tgkd teratomas originate from these trapped oocytes. The insertion of Tgkd 3′ of the Inpp4b gene is associated with decreased expression of Inpp4b and changes in intracellular PI3 Kinase/AKT signaling in follicular granulosa cells. Because Inpp4b is not expressed in fully-grown wild-type or Tgkd oocytes, these findings suggest that hyperactivation of the PI3K/AKT pathway caused by the decrease in INPP4B in granulosa cells promotes an ovarian environment defective in folliculogenesis and conducive to teratoma formation.

BACKGROUND: ROR1 is a type 1 transmembrane tyrosine kinase receptor that plays a critical role in embryonic and fetal development. ROR1 has been described as a possible oncogene and is expressed in numerous malignancies including TNBC and non-small cell lung cancer (NSCLC). We are conducting a first-in-human trial targeting ROR1 with CAR-T cells in patients with advanced TNBC and NSCLC. The cellular construct employed targets the Ig/Fz portion of the extracellular domain of ROR1 and contains 4-1BB/CD3ζ intracellular signaling domain. The manufacturing process utilizes autologous peripheral blood lymphocytes, separated into CD4 and CD8 subsets, which are independently cultured with anti-CD3/anti-CD28 beads and IL-2, then transduced with a lentiviral vector encoding the ROR1 CAR. The CAR-T cell product is formulated in a 1:1 ratio of CD4+ and CD8+ CAR-T cells. METHODS: This ongoing phase I trial (NCT02706392) is evaluating the safety of administering ROR1 CAR-T cells in escalating doses (3.3x105, 1x106, 3.3x106 and 1x107 cells/kg) following lymphodepletion with cyclophosphamide-containing regimens using a continual reassessment method (CRM) for dose escalation. TNBC patients with adequate organ function and performance status, measurable disease, and tumors expressing ROR1 (>20% by IHC) are eligible for enrollment. Persistence of CAR-T cells in blood, cytokine levels, measures of immunogenicity and multi-parametric flow cytometry are being evaluated at multiple time points. Imaging assessments by RECIST 1.1 are performed day 28 - 90, then at 6 and 12 months, and every 6 months as clinically indicated to estimate efficacy. RESULTS: To date, 4 TNBC patients (age range 38-67) have been enrolled, treated and are evaluable for response. Patients had received prior therapies for metastatic disease (range 3-11). 3 of 4 had visceral metastases. No dose-limiting toxicities, severe neurotoxicity or severe cytokine release syndrome (sCRS) were observed at dose levels 1 and 2. Two patients experienced grade 1 CRS. 2 of 4 patients had evidence of CAR-T cell expansion between days 14 and 20, with peak CD8+ CAR-T up to 232.1 cells/uL. Analysis of surface phenotype revealed upregulation of inhibitory receptors on CAR-T cells at the peak of expansion, confirmed by RNA seq. Post-treatment tumor biopsy in patient with partial response revealed an influx of CD3+ T cells and macrophages suggesting ROR1 CAR-T cell trafficking. Two patients received 2 CAR-T cell infusions. Two patients had confirmed stable disease at 15 weeks and 19 weeks, respectively. One patient has stable disease after 1st CAR-T cell infusion, then confirmed partial response after 2nd CAR-T cell infusion which has persisted for 14 weeks. Results will be updated. CONCLUSIONS: ROR1+ CAR-T cells can be safely transferred, expand in vivo in patients with TNBC. Current efforts are directed at understanding and overcoming the mechanisms that limit homing, persistence, and/or function at tumor sites. The trial is continuing with dose-escalation. Funding provided by 8RO1 CA114536-11 and Juno Therapeutics. Citation Format: Specht JM, Lee SM, Turtle C, Berger C, Balakrishnan A, Srivastava S, Viollet V, Veatch J, Gooley T, Mullane E, Chaney CN, Rader C, Pierce RH, Gottardo R, Maloney DG, Riddell SR. A phase I study of adoptive immunotherapy for ROR1+ advanced triple negative breast cancer (TNBC) with defined subsets of autologous T cells expressing a ROR1-specific chimeric antigen receptor (ROR1-CAR) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-09-13.

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