Daniel Sommermeyer

CD19 CAR–T cells of defined CD4+:CD8+ composition in adult B cell ALL patients

BACKGROUNDnT cells that have been modified to express a CD19-specific chimeric antigen receptor (CAR) have antitumor activity in B cell malignancies; however, identification of the factors that determine toxicity and efficacy of these T cells has been challenging in prior studies in which phenotypically heterogeneous CAR-T cell products were prepared from unselected T cells.nnnMETHODSnWe conducted a clinical trial to evaluate CD19 CAR-T cells that were manufactured from defined CD4+ and CD8+ T cell subsets and administered in a defined CD4+:CD8+ composition to adults with B cell acute lymphoblastic leukemia after lymphodepletion chemotherapy.nnnRESULTSnThe defined composition product was remarkably potent, as 27 of 29 patients (93%) achieved BM remission, as determined by flow cytometry. We established that high CAR-T cell doses and tumor burden increase the risks of severe cytokine release syndrome and neurotoxicity. Moreover, we identified serum biomarkers that allow testing of early intervention strategies in patients at the highest risk of toxicity. Risk-stratified CAR-T cell dosing based on BM disease burden decreased toxicity. CD8+ T cell-mediated anti-CAR transgene product immune responses developed after CAR-T cell infusion in some patients, limited CAR-T cell persistence, and increased relapse risk. Addition of fludarabine to the lymphodepletion regimen improved CAR-T cell persistence and disease-free survival.nnnCONCLUSIONnImmunotherapy with a CAR-T cell product of defined composition enabled identification of factors that correlated with CAR-T cell expansion, persistence, and toxicity and facilitated design of lymphodepletion and CAR-T cell dosing strategies that mitigated toxicity and improved disease-free survival.nnnTRIAL REGISTRATIONnClinicalTrials.gov NCT01865617.nnnFUNDINGnR01-CA136551; Life Science Development Fund; Juno Therapeutics; Bezos Family Foundation.

Receptor Affinity and Extracellular Domain Modifications Affect Tumor Recognition by ROR1-Specific Chimeric Antigen Receptor T Cells

Purpose: The adoptive transfer of T cells modified to express a chimeric antigen receptor (CAR) comprised of an extracellular single-chain antibody (scFV) fragment specific for a tumor cell surface molecule, and linked to an intracellular signaling module, has activity in advanced malignancies. The receptor tyrosine kinase–like orphan receptor 1 (ROR1) is a tumor-associated molecule expressed in prevalent B-lymphoid and epithelial cancers and is absent on normal mature B cells and vital tissues, making it a candidate for CAR T-cell therapy. Experimental Design: We constructed ROR1-CARs from scFVs with different affinities and containing extracellular IgG4-Fc spacer domains of different lengths, and evaluated the ability of T cells expressing each CAR to recognize ROR1+ hematopoietic and epithelial tumors in vitro, and to eliminate human mantle cell lymphoma (MCL) engrafted into immunodeficient mice. Results: ROR1-CARs containing a short “Hinge-only” extracellular spacer conferred superior lysis of ROR1+ tumor cells and induction of T-cell effector functions compared with CARs with long “Hinge-CH2-CH3” spacers. CARs derived from a higher affinity scFV conferred maximum T-cell effector function against primary CLL and ROR1+ epithelial cancer lines in vitro without inducing activation-induced T-cell death. T cells modified with an optimal ROR1-CAR were equivalently effective as CD19-CAR–modified T cells in mediating regression of JeKo-1 MCL in immunodeficient mice. Conclusions: Our results show that customizing spacer design and increasing affinity of ROR1-CARs enhances T-cell effector function and recognition of ROR1+ tumors. T cells modified with an optimized ROR1-CAR have significant antitumor efficacy in a preclinical model in vivo, suggesting they may be useful to treat ROR1+ tumors in clinical applications. Clin Cancer Res; 19(12); 3153–64. ©2013 AACR.

The Nonsignaling Extracellular Spacer Domain of Chimeric Antigen Receptors Is Decisive for In Vivo Antitumor Activity

Hudecek, Sommermeyer, and colleagues show that modifications of the length and composition of the extracellular spacer of a chimeric antigen receptor (CAR) that abrogate its binding to Fc receptors can prevent off-target activation of CAR T cells and enhance their antitumor efficacy. The use of synthetic chimeric antigen receptors (CAR) to redirect T cells to recognize tumor provides a powerful new approach to cancer immunotherapy; however, the attributes of CARs that ensure optimal in vivo tumor recognition remain to be defined. Here, we analyze the influence of length and composition of IgG-derived extracellular spacer domains on the function of CARs. Our studies demonstrate that CD19-CARs with a long spacer from IgG4 hinge-CH2-CH3 are functional in vitro but lack antitumor activity in vivo due to interaction between the Fc domain within the spacer and the Fc receptor–bearing myeloid cells, leading to activation-induced T-cell death. We demonstrate that in vivo persistence and antitumor effects of CAR-T cells with a long spacer can be restored by modifying distinct regions in the CH2 domain that are essential for Fc receptor binding. Our studies demonstrate that modifications that abrogate binding to Fc receptors are crucial for CARs in which a long spacer is obligatory for tumor recognition as shown here for a ROR1-specific CAR. These results demonstrate that the length and composition of the extracellular spacer domain that lacks intrinsic signaling function can be decisive in the design of CARs for optimal in vivo activity. Cancer Immunol Res; 3(2); 125–35. ©2014 AACR.

Chimeric antigen receptor-modified T cells derived from defined CD8 + and CD4 + subsets confer superior antitumor reactivity in vivo

Adoptive T-cell therapy with gene-modified T cells expressing a tumor-reactive T-cell receptor or chimeric antigen receptor (CAR) is a rapidly growing field of translational medicine and has shown success in the treatment of B-cell malignancies and solid tumors. In all reported trials, patients have received T-cell products comprising random compositions of CD4+ and CD8+ naive and memory T cells, meaning that each patient received a different therapeutic agent. This variation may have influenced the efficacy of T-cell therapy, and complicates comparison of outcomes between different patients and across trials. We analyzed CD19 CAR-expressing effector T cells derived from different subsets (CD4+/CD8+ naive, central memory, effector memory). T cells derived from each of the subsets were efficiently transduced and expanded, but showed clear differences in effector function and proliferation in vitro and in vivo. Combining the most potent CD4+ and CD8+ CAR-expressing subsets, resulted in synergistic antitumor effects in vivo. We show that CAR-T-cell products generated from defined T-cell subsets can provide uniform potency compared with products derived from unselected T cells that vary in phenotypic composition. These findings have important implications for the formulation of T-cell products for adoptive therapies.

Safety of Targeting ROR1 in Primates with Chimeric Antigen Receptor–Modified T Cells

Berger and colleagues adoptively transferred autologous ROR1 chimeric antigen receptor-modified T (CAR-T) cells into nonhuman primates to demonstrate the safety, persistence, and function of ROR1 CAR-T cells in vivo, and the utility of the model for preclinical testing of novel CARs. 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) because 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. Cancer Immunol Res; 3(2); 206–16. ©2014 AACR.

Adoptive therapy with chimeric antigen receptor-modified T cells of defined subset composition.

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 targeting CD19 on B-cell malignancies. The clinical trials of CD19 chimeric antigen receptor therapy have thus far not attempted to select defined subsets before 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 using adoptive therapy with genetically modified T cells of defined subset and phenotypic composition.

Analysis of ROR1 protein expression in human cancer and normal tissues

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 nonmutated 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 formalin-fixed, paraffin-embedded 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 show 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. Clin Cancer Res; 23(12); 3061–71. ©2016 AACR.

Fully human CD19-specific chimeric antigen receptors for T-cell therapy

Impressive results have been achieved by adoptively transferring T-cells expressing CD19-specific CARs with binding domains from murine mAbs to treat B-cell malignancies. T-cell mediated immune responses specific for peptides from the murine scFv antigen-binding domain of the CAR can develop in patients and result in premature elimination of CAR T-cells increasing the risk of tumor relapse. As fully human scFv might reduce immunogenicity, we generated CD19-specific human scFvs with similar binding characteristics as the murine FMC63-derived scFv using human Ab/DNA libraries. CARs were constructed in various formats from several scFvs and used to transduce primary human T-cells. The resulting CD19-CAR T-cells were specifically activated by CD19-positive tumor cell lines and primary chronic lymphocytic leukemia cells, and eliminated human lymphoma xenografts in immunodeficient mice. Certain fully human CAR constructs were superior to the FMC63-CAR, which is widely used in clinical trials. Imaging of cell surface distribution of the human CARs revealed no evidence of clustering without target cell engagement, and tonic signaling was not observed. To further reduce potential immunogenicity of the CARs, we also modified the fusion sites between different CAR components. The described fully human CARs for a validated clinical target may reduce immune rejection compared with murine-based CARs.

Inclusion of Strep-tag II in design of antigen receptors for T-cell immunotherapy

Adoptive immunotherapy with genetically engineered T cells has the potential to treat cancer and other diseases. The introduction of Strep-tag II sequences into specific sites in synthetic chimeric antigen receptors or natural T-cell receptors of diverse specificities provides engineered T cells with a marker for identification and rapid purification, a method for tailoring spacer length of chimeric receptors for optimal function, and a functional element for selective antibody-coated, microbead-driven, large-scale expansion. These receptor designs facilitate cGMP manufacturing of pure populations of engineered T cells for adoptive T-cell therapies and enable in vivo tracking and retrieval of transferred cells for downstream research applications.

Development of T-cell immunotherapy for hematopoietic stem cell transplantation recipients at risk of leukemia relapse

Leukemia relapse remains the major cause of allogeneic hematopoietic stem cell transplantation (HCT) failure, and the prognosis for patients with post-HCT relapse is poor. There is compelling evidence that potent selective antileukemic effects can be delivered by donor T cells specific for particular minor histocompatibility (H) antigens. Thus, T-cell receptors (TCRs) isolated from minor H antigen-specific T cells represent an untapped resource for developing targeted T-cell immunotherapy to manage post-HCT leukemic relapse. Recognizing that several elements may be crucial to the efficacy and safety of engineered T-cell immunotherapy, we developed a therapeutic transgene with 4 components: (1) a TCR specific for the hematopoietic-restricted, leukemia-associated minor H antigen, HA-1; (2) a CD8 coreceptor to promote function of the class I-restricted TCR in CD4+ T cells; (3) an inducible caspase 9 safety switch to enable elimination of the HA-1 TCR T cells in case of toxicity; and (4) a CD34-CD20 epitope to facilitate selection of the engineered cell product and tracking of transferred HA-1 TCR T cells. The T-cell product includes HA-1 TCR CD4+ T cells to augment the persistence and function of the HA-1 TCR CD8+ T cells and includes only memory T cells; naive T cells are excluded to limit the potential for alloreactivity mediated by native TCR coexpressed by HA-1 TCR T cells. We describe the development of this unique immunotherapy and demonstrate functional responses to primary leukemia by CD4+ and CD8+ T cells transduced with a lentiviral vector incorporating the HA-1 TCR transgene construct.