Clinical Implications and Dynamics of Clonal Hematopoiesis in Anti-CD19 CAR T-cell Treated Patients.

Recent evidence revealed important interactions between clonal hematopoiesis (CH) and cellular therapies established for the treatment of hematologic malignancies. The impact of CH on safety, efficacy, and outcome of chimeric antigen receptor (CAR) T-cell therapy is currently under investigation. We analyzed 110 patients with relapsed/refractory B-cell non-Hodgkin lymphoma (n = 105) or acute lymphoblastic leukemia (ALL) (n = 5), treated with Axicabtagene-Ciloleucel (39%), Tisagenlecleucel (51%), or Brexucabtagene autoleucel (10%). Using error-corrected targeted sequencing, a high CH prevalence of 56.4% (variant allele frequency [VAF] ≥1%) at the time of CAR T-cell infusion was detected. The most frequently mutated gene was PPM1D followed by DNMT3A, TET2, ASXL1, and TP53. Variant allele frequencies were significantly lower in B and T cells compared with monocytes and granulocytes. CH did not increase the risk of CAR T-related toxicities. The incidences of cytokine release syndrome and immune effector-cell-associated neurotoxicity syndrome were similar between CHpos and CHneg patients, regardless of clone size, age, or CAR T product. Prolonged cytopenias were not associated with CH. Best overall response rates (ORRs) were numerically but not significantly higher in CHpos patients (ORR 76.7% versus 62.2%; P = 0.13). Furthermore, CH status did not predict progression-free survival or overall survival. Lastly, sequential analysis showed a modest VAF increase of 1.3% and acquisition of novel mutations within 100 days postinfusion. CH was frequent in large B-cell lymphoma/ALL patients receiving CAR T-cells but did not affect toxicity nor treatment response or outcome.

Donor Lymphocyte Infusion and Molecular Monitoring for Relapsed Myelofibrosis After Hematopoietic Cell Transplantation.

Hematopoietic cell transplantation (HCT) is a curative approach for myelofibrosis patients, but relapse is a major cause of treatment failure. We investigated the effect of donor lymphocyte infusion (DLI) in 37 patients with molecular (n = 17) or hematological relapse (n = 20) after HCT. Patients received median of 2 (range, 1-5) cumulative DLI (total of 91 infusions). Median starting dose was 1 × 106 cells/kg, escalated by half-log ≥6 weeks if no response nor graft-versus-host disease (GvHD) occurred. Median time to first DLI was 40 weeks for molecular relapse versus 145 weeks for hematological relapse. Overall molecular complete response (mCR) at any time was 73% (n = 27) and was significantly higher for initial molecular relapse (88%) versus hematological relapse (60%; P = 0.05). The 6-year overall survival was 77% versus 32% (P = 0.03). Acute GvHD 2-4 occurred in 22% and half of the patients achieved mCR without any GvHD. All patients who relapsed from mCR achieved after first DLI could be salvaged with subsequent DLI, showing long-term survival. No second HCT was needed for molecular relapse versus 6 for hematological relapse. This comprehensive and largest study to date suggests molecular monitoring together with DLI as standard of care and a crucial approach to achieve excellent outcomes in relapsed myelofibrosis.

Comparison of single copy gene­based duplex quantitative PCR and digital droplet PCR for monitoring of expansion of CD19­directed CAR T cells in treated patients.

Chimeric antigen receptor (CAR) T cell therapy with axicabtagene ciloleucel, tisagenlecleucel and brexucabtagen ciloleucel has been adopted as the standard of care for patients with refractory and/or relapsed CD19­positive lymphoid malignancies. Monitoring of kinetics of CAR T cells after administration is crucial for patient follow­up and important to guide clinical decisions for patients subjected to CAR T cell therapy. Information of transgene copies within a CAR T cell product prior to administration, i.e. vector copy numbers, is of high importance to warrant patient safety. However, experimental assays for quantitative CAR T cell monitoring in the open domain are currently lacking. Several institutions have established in­house assays to monitor CAR T cell frequencies. In the present study, the quantitative (q)PCR assay established at the Heidelberg University Hospital (Heidelberg, Germany), i.e. single copy gene­based duplex qPCR, was compared with the digital droplet PCR assay established at the University Medical Center Hamburg­Eppendorf (Hamburg, Germany). Both methods that were independently developed enable accurate and comparable CAR T cell frequency assessment and are useful in the clinical setting.

Axicabtagene ciloleucel in vivo expansion and treatment outcome in aggressive B-cell lymphoma in a real-world setting.

Data on the association between chimeric antigen receptor (CAR)-T-cell kinetics and patient outcome in the nontrial setting are missing, mainly due to the lack of broadly available CAR-T-cell diagnostic quantification tools. We performed prospective quantification of axicabtagene ciloleucel (axi-cel) in 21 patients treated for aggressive B-cell lymphoma at our clinic. Median peak CAR-T-cell count was 16.14 CAR-T cells/µL. Patients with 16.14/µL or higher peak CAR-T cells (strong expanders) had more day-30 objective responses (91% vs 40%, P = .02). In univariate analysis, peak CAR-T cell ≥ 16.14 (P < .001), normal platelet counts at start of lymphodepletion (P < .001), no prior stem cell transplant (P = .04), and peak CAR-T cells as continuous variable (P = .03) were associated with better progression-free survival (PFS). After adjusting for platelet counts and prior stem cell transplantation, peak CAR-T cells below median was still associated with shorter PFS (relative risk, 0.15, 95% confidence interval, 0.04-0.59, P = .007). Low platelet counts also maintained significant impact on PFS. Our data demonstrate association of axi-cel levels and outcome in a nontrial setting and for the first time use a cutoff to segregate weak and strong expanders with respective outcomes.

CAR-HEMATOTOX: a model for CAR T-cell-related hematologic toxicity in relapsed/refractory large B-cell lymphoma.

Hematotoxicity represents a frequent chimeric antigen receptor (CAR) T-cell-related adverse event and remains poorly understood. In this multicenter analysis, we studied patterns of hematopoietic reconstitution and evaluated potential predictive markers in 258 patients receiving axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel) for relapsed/refractory large B-cell lymphoma. We observed profound (absolute neutrophil count [ANC] <100 cells per µL) neutropenia in 72% of patients and prolonged (21 days or longer) neutropenia in 64% of patients. The median duration of severe neutropenia (ANC < 500 cells per µL) was 9 days. We aimed to identify predictive biomarkers of hematotoxicity using the duration of severe neutropenia until day +60 as the primary end point. In the training cohort (n = 58), we observed a significant correlation with baseline thrombocytopenia (r = -0.43; P = .001) and hyperferritinemia (r = 0.54; P < .0001) on univariate and multivariate analysis. Incidence and severity of cytokine-release syndrome, immune effector cell-associated neurotoxicity syndrome, and peak cytokine levels were not associated with the primary end point. We created the CAR-HEMATOTOX model, which included markers associated with hematopoietic reserve (eg, platelet count, hemoglobin, and ANC) and baseline inflammation (eg, C-reactive protein and ferritin). This model was validated in independent cohorts, one from Europe (n = 91) and one from the United States (n = 109) and discriminated patients with severe neutropenia ≥14 days to <14 days (pooled validation: area under the curve, 0.89; sensitivity, 89%; specificity, 68%). A high CAR-HEMATOTOX score resulted in a longer duration of neutropenia (12 vs 5.5 days; P < .001) and a higher incidence of severe thrombocytopenia (87% vs 34%; P < .001) and anemia (96% vs 40%; P < .001). The score implicates bone marrow reserve and inflammation prior to CAR T-cell therapy as key features associated with delayed cytopenia and will be useful for risk-adapted management of hematotoxicity.

Immunogenic Chemotherapy Enhances Recruitment of CAR-T Cells to Lung Tumors and Improves Antitumor Efficacy when Combined with Checkpoint Blockade.

Adoptive therapy using chimeric antigen receptor-modified T cells (CAR-T cells) is effective in hematologic but not epithelial malignancies, which cause the greatest mortality. In breast and lung cancer patients, CAR-T cells targeting the tumor-associated antigen receptor tyrosine kinase-like orphan receptor 1 (ROR1) infiltrate tumors poorly and become dysfunctional. To test strategies for enhancing efficacy, we adapted the KrasLSL-G12D/+;p53f/f autochthonous model of lung adenocarcinoma to express the CAR target ROR1. Murine ROR1 CAR-T cells transferred after lymphodepletion with cyclophosphamide (Cy) transiently control tumor growth but infiltrate tumors poorly and lose function, similar to what is seen in patients. Adding oxaliplatin (Ox) to the lymphodepletion regimen activates tumor macrophages to express T-cell-recruiting chemokines, resulting in improved CAR-T cell infiltration, remodeling of the tumor microenvironment, and increased tumor sensitivity to anti-PD-L1. Combination therapy with Ox/Cy and anti-PD-L1 synergistically improves CAR-T cell-mediated tumor control and survival, providing a strategy to improve CAR-T cell efficacy in the clinic.

Accurate In-Vivo Quantification of CD19 CAR-T Cells after Treatment with Axicabtagene Ciloleucel (Axi-Cel) and Tisagenlecleucel (Tisa-Cel) Using Digital PCR.

Immunotherapy with CD19-specific chimeric antigen receptor (CAR-) T cells has shown excellent efficacy in relapsed/refractory B-cell cancers. The in vivo expansion and persistence of CAR-T cells after infusion are important response- and toxicity-determining variables, but diagnostic tools are largely missing. We showed previously for axi-cel that digital PCR (dPCR) is excellently suited to monitoring CAR-T cells in vivo. Here, we aimed to develop an analogous dPCR assay for tisa-cel. To do so, we cloned and sequenced the CAR construct from the lentiviral tisa-cel vector and designed primers and Black hole quencher (BHQ) probes complimentary to sequences present in the FMC63 scFv part of axi-cel (assay A), tisa-cel (T), and both constructs (U = "universal"). In conjunction with excellent specificity, all assays have a detection limit of one single CAR copy, corresponding to a sensitivity of approximately 1 in 5000 cells (0.02%) for 100 ng genomic DNA (for one vector copy per transduced cell). The new universal assay was first validated using patient samples previously quantified with the axi-cel-specific dPCR and thereafter applied to quantify and monitor adoptively transferred axi-cel and tisa-cel T cells in post-infusion samples (peripheral blood, bone marrow, liquor, and ascites). Actual CAR-T counts per µl were calculated, taking into account vector copy and peripheral blood mononuclear cell (PBMC) numbers, and showed very good correlation with flow cytometry results. We conclude that our novel dPCR assay is optimally suited to monitoring tisa-cel and axi-cel CAR-T cells in real-time in various body fluids.

Development of CAR-T cell therapies for multiple myeloma.

Currently available data on chimeric antigen receptor (CAR)-T cell therapy has demonstrated efficacy and manageable toxicity in heavily pretreated multiple myeloma (MM) patients. The CAR-T field in MM is rapidly evolving with >50 currently ongoing clinical trials across all phases, different CAR-T design, or targets. Most of the CAR-T trials are performed in China and the United States, while European centers organize or participate in only a small fraction of current clinical investigations. Autologous CAR-T cell therapy against B cell maturation antigen shows the best evidence of efficacy so far but main issues remain to be addressed: duration of response, longer follow-up, prolonged cytopenia, patients who may benefit the most such as those with extramedullary disease, outcome prediction, and the integration of CAR-T cell therapy within the MM treatment paradigm. Other promising targets are, i.a.,: CD38, SLAMF7/CS1, or GPRC5D. Although no product has been approved to date, cost and production time for autologous products are expected to be the main obstacles for broad use, for which reason allogeneic CAR-T cells are currently explored. However, the inherent risk of graft-versus-host disease requires additional modification which still need to be validated. This review aims to present the current status of CAR-T cell therapy in MM with an overview on current targets, designs, and stages of CAR-T cell development. Main challenges to CAR-T cell therapy will be highlighted as well as strategies to structurally improve the CAR-T cell product, and thereby its efficacy and safety. The need for comparability of the most promising therapies will be emphasized to balance risks and benefits in an evidence-based but personalized approach to further improve outcome of patients with MM.

Digital PCR Assays for Precise Quantification of CD19-CAR-T Cells after Treatment with Axicabtagene Ciloleucel.

Treatment with axicabtagene ciloleucel (Axi-cel) CD19-CAR-T (chimeric antigen receptor T) cells has been approved for refractory/relapsed diffuse large B cell lymphoma (DLBCL) and primary mediastinal large B cell lymphoma (PMBCL). Because treatment success as well as side effects might depend on CAR-T cell expansion in vivo, we aimed at developing digital PCR (dPCR) assays for detection and quantification of CAR-T cells. To this end, we cloned and sequenced the complete cDNA of the CAR construct. We designed different combinations of primers and dual-labeled hydrolysis probes located in various CAR regions. Three combinations were successfully tested on CAR-positive and -negative cells in duplex reactions with a reference gene (REF) to concomitantly assess cell numbers. All assays demonstrated excellent specificity and reproducibility with neglectable inter-assay variations. For all three assays, almost perfect correlation between the two dPCRs (Axi-cel versus REF) was observed, and the limit of detection was one single CAR-transduced cell corresponding to a sensitivity of 0.01% for 100 ng genomic DNA. After cross-validation, we used one assay to monitor Axi-cel CAR-T numbers in patients. CAR-T expansion and contraction followed the expected kinetics with median peak value of 11.2 Axi-cel CAR-T cells/µL at 11.3 days (median). Clinically, we observed only two partial responses (PRs) in the five patients with CAR-T cell peak numbers below median, whereas four of the five patients with comparatively good expansion showed clinical responses (two complete responses [CRs] and two PRs) on day 30. In conclusion, we established a novel dPCR assay for the sensitive detection of transgenic CAR-T cells, which should be very useful in the context of Axi-cel treatment.

Chimeric Antigen Receptor T Cell-Mediated Neurotoxicity in Nonhuman Primates.

Chimeric antigen receptor (CAR) T-cell immunotherapy has revolutionized the treatment of refractory leukemias and lymphomas, but is associated with significant toxicities, namely cytokine release syndrome (CRS) and neurotoxicity. A major barrier to developing therapeutics to prevent CAR T cell-mediated neurotoxicity is the lack of clinically relevant models. Accordingly, we developed a rhesus macaque (RM) model of neurotoxicity via adoptive transfer of autologous CD20-specific CAR T cells. Following cyclophosphamide lymphodepletion, CD20 CAR T cells expand to 272 to 4,450 cells/µL after 7 to 8 days and elicit CRS and neurotoxicity. Toxicities are associated with elevated serum IL6, IL8, IL1RA, MIG, and I-TAC levels, and disproportionately high cerebrospinal fluid (CSF) IL6, IL2, GM-CSF, and VEGF levels. During neurotoxicity, both CD20 CAR and non-CAR T cells accumulate in the CSF and in the brain parenchyma. This RM model demonstrates that CAR T cell-mediated neurotoxicity is associated with proinflammatory CSF cytokines and a pan-T cell encephalitis.Significance: We provide the first immunologically relevant, nonhuman primate model of B cell-directed CAR T-cell therapy-mediated CRS and neurotoxicity. We demonstrate CAR and non-CAR T-cell infiltration in the CSF and in the brain during neurotoxicity resulting in pan-encephalitis, accompanied by increased levels of proinflammatory cytokines in the CSF. Cancer Discov; 8(6); 750-63. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 663.

Photolytic insertion of albumin on activated carbon modified with ozone.

254nm photolyses of bovine serum albumin [BSA] in aqueous solutions, were carried out in the presence of activated carbons modified by reaction with ozone. The photolyses were monitored by fluorescence spectroscopy and UV spectrophotometry, and the products were characterized by elemental analysis, FTIR, TGA, total organic carbon analyses [TOC], and XPS. The ozonation reaction was carried out at room temperature with O3 under dry and wet conditions. The carbon characterization showed that the reaction increased the amount of epoxide and carbonyl groups on the carbon matrix. The activated carbon modified with dry O3 exhibited higher concentration of oxidized groups in its surface, smaller surface area and lower thermal stability. Characterization of the photolysis of ozonized carbons pointed to a small release of carbon organic groups during the reaction with elimination of epoxide groups and increase of carbonyl groups without change of thermal stability. Photolysis of BSA in aqueous solution occurred with fluorescence quenching due to changes of the local microenvironment and/or macromolecular conformational changes. Absorbance increase of the UV spectrum indicated a hyperchromic effect due to albumin structure modifications during photolysis. TGA analysis of the photolysed activated carbons in the presence of BSA suggested that ozonized carbon samples underwent insertion of BSA upon photolysis, in particular the sample ozonized under dry conditions. The changes observed for the FTIR and elemental analysis agreed with this conclusion, which was further supported by 13C SS-NMR, fluorescence emission and XPS.

Preclinical Assessment of CD171-Directed CAR T-cell Adoptive Therapy for Childhood Neuroblastoma: CE7 Epitope Target Safety and Product Manufacturing Feasibility.

PURPOSE: The identification and vetting of cell surface tumor-restricted epitopes for chimeric antigen receptor (CAR)-redirected T-cell immunotherapy is the subject of intensive investigation. We have focused on CD171 (L1-CAM), an abundant cell surface molecule on neuroblastomas and, specifically, on the glycosylation-dependent tumor-specific epitope recognized by the CE7 monoclonal antibody. EXPERIMENTAL DESIGN: CD171 expression was assessed by IHC using CE7 mAb in tumor microarrays of primary, metastatic, and recurrent neuroblastoma, as well as human and rhesus macaque tissue arrays. The safety of targeting the CE7 epitope of CD171 with CE7-CAR T cells was evaluated in a preclinical rhesus macaque trial on the basis of CD171 homology and CE7 cross reactivity. The feasibility of generating bioactive CAR T cells from heavily pretreated pediatric patients with recurrent/refractory disease was assessed. RESULTS: CD171 is uniformly and abundantly expressed by neuroblastoma tumor specimens obtained at diagnoses and relapse independent of patient clinical risk group. CD171 expression in normal tissues is similar in humans and rhesus macaques. Infusion of up to 1 × 108/kg CE7-CAR+ CTLs in rhesus macaques revealed no signs of specific on-target off-tumor toxicity. Manufacturing of lentivirally transduced CD4+ and CD8+ CE7-CAR T-cell products under GMP was successful in 4 out of 5 consecutively enrolled neuroblastoma patients in a phase I study. All four CE7-CAR T-cell products demonstrated in vitro and in vivo antitumor activity. CONCLUSIONS: Our preclinical assessment of the CE7 epitope on CD171 supports its utility and safety as a CAR T-cell target for neuroblastoma immunotherapy. Clin Cancer Res; 23(2); 466-77. ©2016 AACR.

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.

Immunotherapy of non-Hodgkin's lymphoma with a defined ratio of CD8+ and CD4+ CD19-specific chimeric antigen receptor-modified T cells.

CD19-specific chimeric antigen receptor (CAR)-modified T cells have antitumor activity in B cell malignancies, but factors that affect toxicity and efficacy have been difficult to define because of differences in lymphodepletion and heterogeneity of CAR-T cells administered to individual patients. We conducted a clinical trial in which CD19 CAR-T cells were manufactured from defined T cell subsets and administered in a 1:1 CD4(+)/CD8(+) ratio of CAR-T cells to 32 adults with relapsed and/or refractory B cell non-Hodgkin's lymphoma after cyclophosphamide (Cy)-based lymphodepletion chemotherapy with or without fludarabine (Flu). Patients who received Cy/Flu lymphodepletion had increased CAR-T cell expansion and persistence, and higher response rates [50% complete remission (CR), 72% overall response rate (ORR)] than patients who received Cy-based lymphodepletion without Flu (8% CR, 50% ORR). The CR rate in patients treated with Cy/Flu at the maximally tolerated dose was 64% (82% ORR; n = 11). Cy/Flu minimized the effects of an immune response to the murine single-chain variable fragment component of the CAR, which limited CAR-T cell expansion and clinical efficacy in patients who received Cy-based lymphodepletion without Flu. Severe cytokine release syndrome (sCRS) and grade ≥3 neurotoxicity were observed in 13 and 28% of all patients, respectively. Serum biomarkers, one day after CAR-T cell infusion, correlated with subsequent sCRS and neurotoxicity. Immunotherapy with CD19 CAR-T cells in a defined CD4(+)/CD8(+) ratio allowed identification of correlative factors for CAR-T cell expansion, persistence, and toxicity, and facilitated optimization of lymphodepletion that improved disease response and overall and progression-free survival.

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

BACKGROUND: T 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. METHODS: We 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. RESULTS: The 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. CONCLUSION: Immunotherapy 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. TRIAL REGISTRATION: ClinicalTrials.gov NCT01865617. FUNDING: R01-CA136551; Life Science Development Fund; Juno Therapeutics; Bezos Family Foundation.

Comparison of naïve and central memory derived CD8+ effector cell engraftment fitness and function following adoptive transfer.

Human CD8+ effector T cells derived from CD45RO+CD62L+ precursors enriched for central memory (TCM) precursors retain the capacity to engraft and reconstitute functional memory upon adoptive transfer, whereas effectors derived from CD45RO+CD62L- precursors enriched for effector memory precursors do not. Here we sought to compare the engraftment fitness and function of CD8+ effector T cells derived from CD45RA+CD62L+ precursors enriched for naïve and stem cell memory precursors (TN/SCM) with that of TCM. We found that cytotoxic T cells (CTLs) derived from TCM transcribed higher levels of CD28, FOS, INFγ, Eomesodermin (Eomes), and lower levels of BCL2L11, maintained higher levels of phosphorylated AKT, and displayed enhanced sensitivity to the proliferative and anti-apoptotic effects of γ-chain cytokines compared to CTLs derived from TN/SCM. Higher frequencies of CTLs derived from TCM retained CD28 expression and upon activation secreted higher levels of IL-2. In NOD/Scid IL-2RγCnull mice, CD8+ TCM derived CTLs engrafted to higher frequencies in response to human IL-15 and mounted robust proliferative responses to an immunostimulatory vaccine. Similarly, CD8+ TCM derived CD19CAR+ CTLs exhibited superior antitumor potency following adoptive transfer compared to their CD8+ TN/SCM derived counterparts. These studies support the use of TCM enriched cell products for adoptive therapy of cancer.

Safety of targeting ROR1 in primates with chimeric antigen receptor-modified T cells.

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.

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.

Combining a CD20 chimeric antigen receptor and an inducible caspase 9 suicide switch to improve the efficacy and safety of T cell adoptive immunotherapy for lymphoma.

Modification of T cells with chimeric antigen receptors (CAR) has emerged as a promising treatment modality for human malignancies. Integration of co-stimulatory domains into CARs can augment the activation and function of genetically targeted T cells against tumors. However, the potential for insertional mutagenesis and toxicities due to the infused cells have made development of safe methods for removing transferred cells an important consideration. We have genetically modified human T cells with a lentiviral vector to express a CD20-CAR containing both CD28 and CD137 co-stimulatory domains, a "suicide gene" relying on inducible activation of caspase 9 (iC9), and a truncated CD19 selectable marker. Rapid expansion (2000 fold) of the transduced T cells was achieved in 28 days after stimulation with artificial antigen presenting cells. Transduced T cells exhibited effective CD20-specific cytotoxic activity in vitro and in a mouse xenograft tumor model. Activation of the iC9 suicide switch resulted in efficient removal of transduced T cells both in vitro and in vivo. Our work demonstrates the feasibility and promise of this approach for treating CD20(+) malignancies in a safe and more efficient manner. A phase I clinical trial using this approach in patients with relapsed indolent B-NHL is planned.