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Purpose: Treatments are limited for metastatic melanoma and metastatic triple-negative breast cancer (mTNBC). This pilot phase I trial (NCT03060356) examined the safety and feasibility of intravenous RNA-electroporated chimeric antigen receptor (CAR) T cells targeting the cell-surface antigen cMET. Experimental Design: Metastatic melanoma or mTNBC subjects had at least 30% tumor expression of cMET, measurable disease and progression on prior therapy. Patients received up to six infusions (1 × 10e8 T cells/dose) of CAR T cells without lymphodepleting chemotherapy. Forty-eight percent of prescreened subjects met the cMET expression threshold. Seven (3 metastatic melanoma, 4 mTNBC) were treated. Results: Mean age was 50 years (35–64); median Eastern Cooperative Oncology Group 0 (0–1); median prior lines of chemotherapy/immunotherapy were 4/0 for TNBC and 1/3 for melanoma subjects. Six patients experienced grade 1 or 2 toxicity. Toxicities in at least 1 patient included anemia, fatigue, and malaise. One subject had grade 1 cytokine release syndrome. No grade 3 or higher toxicity, neurotoxicity, or treatment discontinuation occurred. Best response was stable disease in 4 and disease progression in 3 subjects. mRNA signals corresponding to CAR T cells were detected by RT-PCR in all patients’ blood including in 3 subjects on day +1 (no infusion administered on this day). Five subjects underwent postinfusion biopsy with no CAR T-cell signals seen in tumor. Three subjects had paired tumor tissue; IHC showed increases in CD8 and CD3 and decreases in pS6 and Ki67. Conclusions: Intravenous administration of RNA-electroporated cMET-directed CAR T cells is safe and feasible. Significance: Data evaluating CAR T therapy in patients with solid tumors are limited. This pilot clinical trial demonstrates that intravenous cMET-directed CAR T-cell therapy is safe and feasible in patients with metastatic melanoma and metastatic breast cancer, supporting the continued evaluation of cellular therapy for patients with these malignancies.

We conducted a phase I clinical trial of anti-BCMA chimeric antigen receptor T cells (CART-BCMA) with or without anti-CD19 CAR T cells (huCART19) in multiple myeloma (MM) patients responding to third- or later-line therapy (phase A, N = 10) or high-risk patients responding to first-line therapy (phase B, N = 20), followed by early lenalidomide or pomalidomide maintenance. We observed no high-grade cytokine release syndrome (CRS) and only one instance of low-grade neurologic toxicity. Among 15 subjects with measurable disease, 10 exhibited partial response (PR) or better; among 26 subjects responding to prior therapy, 9 improved their response category and 4 converted to minimal residual disease (MRD)–negative complete response/stringent complete response. Early maintenance therapy was safe, feasible, and coincided in some patients with CAR T-cell reexpansion and late-onset, durable clinical response. Outcomes with CART-BCMA + huCART19 were similar to CART-BCMA alone. Collectively, our results demonstrate favorable safety, pharmacokinetics, and antimyeloma activity of dual-target CAR T-cell therapy in early lines of MM treatment.Significance:CAR T cells in early lines of MM therapy could be safer and more effective than in the advanced setting, where prior studies have focused. We evaluated the safety, pharmacokinetics, and efficacy of CAR T cells in patients with low disease burden, responding to current therapy, combined with standard maintenance therapy.This article is highlighted in the In This Issue feature, p. 101

Chimeric antigen-receptor (CAR) T cells lead to high response rates in myeloma, but most patients experience recurrent disease. We combined several high-dimensional approaches to study tumor/immune cells in the tumor microenvironment (TME) of myeloma patients pre– and post–B-cell maturation antigen (BCMA)-specific CAR T therapy. Lower diversity of pretherapy T-cell receptor (TCR) repertoire, presence of hyperexpanded clones with exhaustion phenotype, and BAFF+PD-L1+ myeloid cells in the marrow correlated with shorter progression-free survival (PFS) following CAR T therapy. In contrast, longer PFS was associated with an increased proportion of CLEC9A+ dendritic cells (DC), CD27+TCF1+ T cells with diverse T-cell receptors, and emergence of T cells expressing marrow-residence genes. Residual tumor cells at initial response express stemlike genes, and tumor recurrence was associated with the emergence of new dominant clones. These data illustrate a dynamic interplay between endogenous T, CAR T, myeloid/DC, and tumor compartments that affects the durability of response following CAR T therapy in myeloma. Significance: There is an unmet need to identify determinants of durable responses following BCMA CAR T therapy of myeloma. High-dimensional analysis of the TME was performed to identify features of immune and tumor cells that correlate with survival and suggest several strategies to improve outcomes following CAR T therapy. See related commentary by Graham and Maus, p. 478. This article is highlighted in the In This Issue feature, p. 476

Purpose: To study the biology and identify markers of severe cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS) in children after chimeric antigen receptor T-cell (CAR T) treatment. Experimental Design: We used comprehensive proteomic profiling to measure over 1,400 serum proteins at multiple serial timepoints in a cohort of patients with B-cell acute lymphoblastic leukemia treated with the CD19-targeted CAR T CTL019 on two clinical trials. Results: We identified fms-like tyrosine kinase 3 (FLT3) and mast cell immunoglobulin-like receptor 1 (MILR1) as preinfusion predictive biomarkers of severe CRS. We demonstrated that CRS is an IFNγ-driven process with a protein signature overlapping with hemophagocytic lymphohistiocytosis (HLH). We identified IL18 as a potentially targetable cytokine associated with the development of ICANS. Conclusions: We identified preinfusion biomarkers that can be used to predict severe CRS with a sensitivity, specificity, and accuracy superior to the current gold standard of disease burden. We demonstrated the fundamental role of the IFNγ pathway in driving CRS, suggesting CRS and carHLH are overlapping rather than distinct phenomena, an observation with important treatment implications. We identified IL18 as a possible targetable cytokine in ICANS, providing rationale for IL18 blocking therapies to be translated into clinical trials in ICANS.

Study procedures. Participant study procedures including cell collection/manufacturing timeline as well as infusion schedule from eligibility confirmation onward.

Purpose:Treatments are limited for metastatic melanoma and metastatic triple-negative breast cancer (mTNBC). This pilot phase I trial (NCT03060356) examined the safety and feasibility of intravenous RNA-electroporated chimeric antigen receptor (CAR) T cells targeting the cell-surface antigen cMET.Experimental Design:Metastatic melanoma or mTNBC subjects had at least 30% tumor expression of cMET, measurable disease and progression on prior therapy. Patients received up to six infusions (1 × 10e8 T cells/dose) of CAR T cells without lymphodepleting chemotherapy. Forty-eight percent of prescreened subjects met the cMET expression threshold. Seven (3 metastatic melanoma, 4 mTNBC) were treated.Results:Mean age was 50 years (35–64); median Eastern Cooperative Oncology Group 0 (0–1); median prior lines of chemotherapy/immunotherapy were 4/0 for TNBC and 1/3 for melanoma subjects. Six patients experienced grade 1 or 2 toxicity. Toxicities in at least 1 patient included anemia, fatigue, and malaise. One subject had grade 1 cytokine release syndrome. No grade 3 or higher toxicity, neurotoxicity, or treatment discontinuation occurred. Best response was stable disease in 4 and disease progression in 3 subjects. mRNA signals corresponding to CAR T cells were detected by RT-PCR in all patients’ blood including in 3 subjects on day +1 (no infusion administered on this day). Five subjects underwent postinfusion biopsy with no CAR T-cell signals seen in tumor. Three subjects had paired tumor tissue; IHC showed increases in CD8 and CD3 and decreases in pS6 and Ki67.Conclusions:Intravenous administration of RNA-electroporated cMET-directed CAR T cells is safe and feasible.Significance:Data evaluating CAR T therapy in patients with solid tumors are limited. This pilot clinical trial demonstrates that intravenous cMET-directed CAR T-cell therapy is safe and feasible in patients with metastatic melanoma and metastatic breast cancer, supporting the continued evaluation of cellular therapy for patients with these malignancies.

mRNA signals corresponding to CAR T cells in peripheral blood

Supplementary Figure 1.: Representative immunohistochemical staining for Subject 74 with metastatic melanoma; lymph node biopsy. Panel A: pS6 staining prior to infusion; Panel B: pS6 staining at post-infusion biopsy; Panel C: Ki-67 staining prior to infusion; Panel D: Ki-67 staining at post-infusion biopsy.

Patient population

Response

Supplementary Figure 2: Representative immunohistochemical staining for Subject 27 with metastatic triple-negative breast cancer; lymph node biopsy. Panel A shows CD8 staining prior to infusion and Panel B shows CD8 staining at the time of post-infusion biopsy.

IHC analysis of paired tumor tissue

IHC analysis of CD8 in preinfusion and postinfusion tumor tissue. Three subjects had preinfusion and postinfusion tumor tissue available for analysis; postinfusion tumor tissue demonstrated an increase in cytoxic CD8+ cells in all 3 subjects.

Adverse events, clinically relevant (# subjects includes those with the given toxicity at least possibly related to study treatment)

Supplementary Figure from Modulation of BCL-2 in Both T Cells and Tumor Cells to Enhance Chimeric Antigen Receptor T-cell Immunotherapy against Cancer

Supplementary Data from Changes in Bone Marrow Tumor and Immune Cells Correlate with Durability of Remissions Following BCMA CAR T Therapy in Myeloma

Chimeric antigen receptor T-cell (CART) immunotherapy led to unprecedented responses in patients with refractory/relapsed B-cell non-Hodgkin lymphoma (NHL); nevertheless, two thirds of patients experience treatment failure. Resistance to apoptosis is a key feature of cancer cells, and it is associated with treatment failure. In 87 patients with NHL treated with anti-CD19 CART, we found that chromosomal alteration of B-cell lymphoma 2 (BCL-2), a critical antiapoptotic regulator, in lymphoma cells was associated with reduced survival. Therefore, we combined CART19 with the FDA-approved BCL-2 inhibitor venetoclax and demonstrated in vivo synergy in venetoclax-sensitive NHL. However, higher venetoclax doses needed for venetoclax-resistant lymphomas resulted in CART toxicity. To overcome this limitation, we developed venetoclax-resistant CART by overexpressing mutated BCL-2(F104L), which is not recognized by venetoclax. Notably, BCL-2(F104L)-CART19 synergized with venetoclax in multiple lymphoma xenograft models. Furthermore, we uncovered that BCL-2 overexpression in T cells intrinsically enhanced CART antitumor activity in preclinical models and in patients by prolonging CART persistence.Significance:This study highlights the role of BCL-2 in resistance to CART immunotherapy for cancer and introduces a novel concept for combination therapies—the engineering of CART cells to make them resistant to proapoptotic small molecules, thereby enhancing the therapeutic index of these combination therapies.This article is highlighted in the In This Issue feature, p. 2221

Supplementary Figure from Changes in Bone Marrow Tumor and Immune Cells Correlate with Durability of Remissions Following BCMA CAR T Therapy in Myeloma

Chimeric antigen-receptor (CAR) T cells lead to high response rates in myeloma, but most patients experience recurrent disease. We combined several high-dimensional approaches to study tumor/immune cells in the tumor microenvironment (TME) of myeloma patients pre– and post–B-cell maturation antigen (BCMA)-specific CAR T therapy. Lower diversity of pretherapy T-cell receptor (TCR) repertoire, presence of hyperexpanded clones with exhaustion phenotype, and BAFF+PD-L1+ myeloid cells in the marrow correlated with shorter progression-free survival (PFS) following CAR T therapy. In contrast, longer PFS was associated with an increased proportion of CLEC9A+ dendritic cells (DC), CD27+TCF1+ T cells with diverse T-cell receptors, and emergence of T cells expressing marrow-residence genes. Residual tumor cells at initial response express stemlike genes, and tumor recurrence was associated with the emergence of new dominant clones. These data illustrate a dynamic interplay between endogenous T, CAR T, myeloid/DC, and tumor compartments that affects the durability of response following CAR T therapy in myeloma.Significance:There is an unmet need to identify determinants of durable responses following BCMA CAR T therapy of myeloma. High-dimensional analysis of the TME was performed to identify features of immune and tumor cells that correlate with survival and suggest several strategies to improve outcomes following CAR T therapy.See related commentary by Graham and Maus, p. 478.This article is highlighted in the In This Issue feature, p. 476

Supplemental tables: (1) Subject characteristics. (2) Cytogenetic profiles and high-risk features. (3) Prior treatment exposures and refractoriness. (4) CAR T cell product characteristics. (5) Products that did not meet target dose. (6) Adverse events of grade 3-4. (7) Cytokine release syndrome and ICANS. (8). Maintenance therapy. Supplemental Figures: (1) Study schematic and subject disposition, (2) Correlates of manufacturing success, (3) Hematopoietic recovery, (4) Post-infusion T cell phenotypes, (5) Correlates of in vivo expansion and manufacturing success, (6) Late post-infusion CAR T cell re-expansion, (7) Soluble BCMA, (8) Late-onset clinical responses, (9) MM cell BCMA expression, (10) Pre- and post-treatment Sox2-specific T cell responses in CART-BCMA monotherapy patients, (11) Pre- and post-treatment Sox2-specific T cell responses in CART-BCMA + huCART19 combination therapy patients, (12) Sustained post-treatment SOX2-specific T-cell responses.

We conducted a phase I clinical trial of anti-BCMA chimeric antigen receptor T cells (CART-BCMA) with or without anti-CD19 CAR T cells (huCART19) in multiple myeloma (MM) patients responding to third- or later-line therapy (phase A, N = 10) or high-risk patients responding to first-line therapy (phase B, N = 20), followed by early lenalidomide or pomalidomide maintenance. We observed no high-grade cytokine release syndrome (CRS) and only one instance of low-grade neurologic toxicity. Among 15 subjects with measurable disease, 10 exhibited partial response (PR) or better; among 26 subjects responding to prior therapy, 9 improved their response category and 4 converted to minimal residual disease (MRD)–negative complete response/stringent complete response. Early maintenance therapy was safe, feasible, and coincided in some patients with CAR T-cell reexpansion and late-onset, durable clinical response. Outcomes with CART-BCMA + huCART19 were similar to CART-BCMA alone. Collectively, our results demonstrate favorable safety, pharmacokinetics, and antimyeloma activity of dual-target CAR T-cell therapy in early lines of MM treatment.Significance:CAR T cells in early lines of MM therapy could be safer and more effective than in the advanced setting, where prior studies have focused. We evaluated the safety, pharmacokinetics, and efficacy of CAR T cells in patients with low disease burden, responding to current therapy, combined with standard maintenance therapy.This article is highlighted in the In This Issue feature, p. 101

Supplementary Table from Changes in Bone Marrow Tumor and Immune Cells Correlate with Durability of Remissions Following BCMA CAR T Therapy in Myeloma

Supplementary Table 1. DNA fragments synthesized and inserted into MSCV-CD19-IRES-DsRedFP via EcoRI/BglII or BglII/XhoI digestion, and later moved into pMX-IRES-Blast via EcoRI/XhoI cloning. Supplementary Table 2. Sets of primers used for PCR amplification and Sanger sequencing of CD19 gene and the upstream -1.2Kb enhancer and promoter regions. Supplementary Table 3. Sets of primers for the analysis of CpG-Methylation status after bisulfite modification of genomic DNA. Supplementary Table 4. Sets of primers used for radioactive semiquantitative PCR analysis of CD19 mRNA isoforms. Supplementary Table 5. Sequence of the mini-gene expanding exon2 and 220nt of its flanking introns that was synthesized (Genewiz) and inserted into pBSKii+ via XhoI/NotI cloning. Supplementary Table 6. Antibodies used for pull down assays for the identification of splicing factors that bind to the CD19-int1-exon2-int2 mini-gene. Supplementary Table 7. Primary and secondary antibodies used for immunoblotting and immunoprecipitations after protein separation by SDS-PAGE and transference to PVDF membrane (N/A, not applicable). Supplementary Table 8. Pairs of oligos for semiquantitative PCR amplification of CD19 cDNA followed by visualization in agarose gel. Same primers were used for sequencing after gel purification of specific bands. Supplementary Table 9. Pairs of oligos used for Real Time-qPCR amplification of cDNA.

The success of chimeric antigen receptor (CAR)–mediated immunotherapy in acute lymphoblastic leukemia (ALL) highlights the potential of T-cell therapies with directed cytotoxicity against specific tumor antigens. The efficacy of CAR T-cell therapy depends on the engraftment and persistence of T cells following adoptive transfer. Most protocols for T-cell engineering routinely expand T cells ex vivo for 9 to 14 days. Because the potential for engraftment and persistence is related to the state of T-cell differentiation, we hypothesized that reducing the duration of ex vivo culture would limit differentiation and enhance the efficacy of CAR T-cell therapy. We demonstrated that T cells with a CAR-targeting CD19 (CART19) exhibited less differentiation and enhanced effector function in vitro when harvested from cultures at earlier (day 3 or 5) compared with later (day 9) timepoints. We then compared the therapeutic potential of early versus late harvested CART19 in a murine xenograft model of ALL and showed that the antileukemic activity inversely correlated with ex vivo culture time: day 3 harvested cells showed robust tumor control despite using a 6-fold lower dose of CART19, whereas day 9 cells failed to control leukemia at limited cell doses. We also demonstrated the feasibility of an abbreviated culture in a large-scale current good manufacturing practice–compliant process. Limiting the interval between T-cell isolation and CAR treatment is critical for patients with rapidly progressing disease. Generating CAR T cells in less time also improves potency, which is central to the effectiveness of these therapies. Cancer Immunol Res; 6(9); 1100–9. ©2018 AACR.

Supplementary Figure 1. Enhanced skipping of exons 2 and 5-6 in post-CART-19 leukemias. Supplementary Figure 2. Analysis of splicing factors involved in alternative splicing of CD19-exon2.

Supplementary Figure 1. : Representative immunohistochemical staining for Subject 74 with metastatic melanoma; lymph node biopsy. Panel A: pS6 staining prior to infusion; Panel B: pS6 staining at post-infusion biopsy; Panel C: Ki-67 staining prior to infusion; Panel D: Ki-67 staining at post-infusion biopsy.

Supplementary Figure 2: Representative immunohistochemical staining for Subject 27 with metastatic triple-negative breast cancer; lymph node biopsy. Panel A shows CD8 staining prior to infusion and Panel B shows CD8 staining at the time of post-infusion biopsy.

Purpose: Treatments are limited for metastatic melanoma (MM) and triple-negative breast cancer (mTNBC). This pilot phase I trial (NCT03060356) examined the safety and feasibility of intravenous RNA- electroporated chimeric antigen receptor (CAR) T cells targeting the cell-surface antigen cMET. Patients and Methods: MM or mTNBC subjects had at least 30% tumor expression of cMET, measurable disease and progression on prior therapy. Patients received up to 6 infusions (1x10e8 T cells / dose) of CAR T cells without lymphodepleting chemotherapy. Forty-eight percent of prescreened subjects met the cMET expression threshold. Seven (3 MM, 4 mTNBC) were treated. Results: Mean age was 50 years (35-64); median ECOG 0 (0-1); median prior lines of chemotherapy/immunotherapy were 4/0 for TNBC and 1/3 for melanoma subjects. Six patients experienced grade (G) 1 or 2 toxicity. Toxicities in at least 1 patient included anemia, fatigue and malaise. One subject had grade 1 cytokine release syndrome. No G 3 or higher toxicity, neurotoxicity, or treatment discontinuation occurred. Best response was stable disease in 4 and disease progression in 3 subjects. Messenger RNA signals corresponding to CAR T cells were detected by RT-PCR in all patients’ blood including in 3 subjects on Day +1 (no infusion administered on this day). Five subjects underwent post-infusion biopsy with no CAR T cell signals seen in tumor. Three subjects had paired tumor tissue; immunohistochemistry showed increases in CD8 and CD3 and decreases in pS6 and Ki67. Conclusions: Intravenous administration of RNA-electroporated cMET-directed CAR T cells is safe and feasible.

Supplemental Information and Legends

S3. Serial quantification of serum IL6 from a patient with prolonged subjectove myalgia post IT injection of mRNA c-Met CAR T cells.

S1. Clinical trial schema. S2. Representative immunohistochemistry (IHC). S3. Histology and IHC of tumor tissue pre and post intratumoral injection of RNA CAR T c- Met from one patient. S4. Histology and IHC of tumor tissue pre and post intratumoral injection with lidocaine (1 mL) and RNA CAR T c-Met (1 mL) from another patient. S5. Characterization of CD68+ tumor infiltrated immune cells using multiplex IHC analyses as described (1) in pre and post IT injection of RNA CAR T c-Met tumor tissue sections from two patients.

Chimeric antigen receptors (CAR) are synthetic molecules that provide new specificities to T cells. Although successful in treatment of hematologic malignancies, CAR T cells are ineffective for solid tumors to date. We found that the cell-surface molecule c-Met was expressed in ∼50% of breast tumors, prompting the construction of a CAR T cell specific for c-Met, which halted tumor growth in immune-incompetent mice with tumor xenografts. We then evaluated the safety and feasibility of treating metastatic breast cancer with intratumoral administration of mRNA-transfected c-Met-CAR T cells in a phase 0 clinical trial (NCT01837602). Introducing the CAR construct via mRNA ensured safety by limiting the nontumor cell effects (on-target/off-tumor) of targeting c-Met. Patients with metastatic breast cancer with accessible cutaneous or lymph node metastases received a single intratumoral injection of 3 × 107 or 3 × 108 cells. CAR T mRNA was detectable in peripheral blood and in the injected tumor tissues after intratumoral injection in 2 and 4 patients, respectively. mRNA c-Met-CAR T cell injections were well tolerated, as none of the patients had study drug–related adverse effects greater than grade 1. Tumors treated with intratumoral injected mRNA c-Met-CAR T cells were excised and analyzed by immunohistochemistry, revealing extensive tumor necrosis at the injection site, cellular debris, loss of c-Met immunoreactivity, all surrounded by macrophages at the leading edges and within necrotic zones. We conclude that intratumoral injections of mRNA c-Met-CAR T cells are well tolerated and evoke an inflammatory response within tumors. Cancer Immunol Res; 5(12); 1152–61. ©2017 AACR.

Supplementary methods, statistical methods, results, tables, and figures. Supplementary Table 1. CRS grading system. Supplementary Table 2. Samples collected and time points on the trials. Supplementary Table 3. HLH diagnostic criteria. Supplementary Table 4. Timing of severe CRS relative to CRS onset and time from infusion. Supplementary Table 5. Patient demographics and baseline characteristics. Supplementary Table 6. Co-morbid infections. Supplementary Table 7. Median (range) for baseline cytokine values comparing normal subjects and total study population, children on the trials, and adults on the trials. Supplementary Table 8. Median (range) baseline cytokine values comparing baseline disease burden. Supplementary Table 9. Median (range) one-month peak cytokine values in children and adults comparing CRS grades (0-3 vs 4-5). Supplementary Table 10. Time points of target cytokine assessment listed as day since CTL019 infusion, with the acceptable windows provided by the study protocols. Supplementary Table 11. Median (range) day 1-3 peak cytokine values for those with severe (grade 4-5) and without severe (grade 0-3) CRS for total study population, and split into adults and children. Supplementary Table 12. Summary of all predictive models generated by discovery cohort and validated. Supplementary Table 13. Clinical details on the validation cohort. Supplementary Table 14. Therapies used for CRS. Supplementary Table 15. Tocilizumab use by grade and age, as well as, clinical effects of tocilizumab on CRS. Supplementary Table 16. Lee CRS grading scale. Supplementary Table 17. Davila CRS grading scale. Supplementary Table 18. Reclassification of CRS severity by dividing grade 3 into 3a and 3b. Supplementary Table 19. Summary of top predictive models with alternate determination of CRS severity. Supplementary Figure 1. CRP and ferritin are poor early predictors of severe CRS. Supplementary Figure 2. Early increases in IFNγ and sgp130 after CTL019 infusion are associated with development of severe CRS. Supplementary Figure 3. Severity of CRS correlates moderately with CAR T cell expansion but early expansion of CAR T cells does not predict CRS severity. Supplementary Figure 4. Cytokine profiles predict CRS. Supplementary Figure 5. Alternate CRS prediction models.

Table S1 and Figures S1 -S8

Purpose: Human neural stem cells (NSC) are inherently tumor tropic, making them attractive drug delivery vehicles. Toward this goal, we retrovirally transduced an immortalized, clonal NSC line to stably express cytosine deaminase (HB1.F3.CD.C21; CD-NSCs), which converts the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU).Experimental Design: Recurrent high-grade glioma patients underwent intracranial administration of CD-NSCs during tumor resection or biopsy. Four days later, patients began taking oral 5-FC every 6 hours for 7 days. Study treatment was given only once. A standard 3 + 3 dose escalation schema was used to increase doses of CD-NSCs from 1 × 107 to 5 × 107 and 5-FC from 75 to 150 mg/kg/day. Intracerebral microdialysis was performed to measure brain levels of 5-FC and 5-FU. Serial blood samples were obtained to assess systemic drug concentrations as well as to perform immunologic correlative studies.Results: Fifteen patients underwent study treatment. We saw no dose-limiting toxicity (DLT) due to the CD-NSCs. There was 1 DLT (grade 3 transaminitis) possibly related to 5-FC. We did not see development of anti-CD-NSC antibodies and did not detect CD-NSCs or replication-competent retrovirus in the systemic circulation. Intracerebral microdialysis revealed that CD-NSCs produced 5-FU locally in the brain in a 5-FC dose-dependent manner. Autopsy data indicate that CD-NSCs migrated to distant tumor sites and were nontumorigenic.Conclusions: Collectively, our results from this first-in-human study demonstrate initial safety and proof of concept regarding the ability of NSCs to target brain tumors and locally produce chemotherapy. Clin Cancer Res; 23(12); 2951–60. ©2016 AACR.

Supplementary Figure from Comprehensive Serum Proteome Profiling of Cytokine Release Syndrome and Immune Effector Cell–Associated Neurotoxicity Syndrome Patients with B-Cell ALL Receiving CAR T19

sTable 1 shows radiation characteristics of Group A, B, and C. sTable 2 shows characteristics of CART-BCMA cells during manufacturing and at peak expansion in vivo in Group A, B, and C. sTable 3 shows details of radiation received after CART-BCMA therapy. sFigure 1 shows soluble BCMA serum levels before cyclophosphamide lymphodepletion on Day -3. sFigure 2 shows that prior radiation exposure is not associated with the level of CART-BCMA cells in vivo at peak expansion. sFigure 3 shows radiation was not associated with the level of persistent CART-BCMA cells in peripheral blood at Day 28. sFigure 4 shows level of CART-BCMA cells at peak expansion and Day 28 correlated with clinical response, and within responders, receipt of prior radiation was not associated with differences in CART=BCMA levels.

Purpose: Responses to therapy with chimeric antigen receptor T cells recognizing CD19 (CART19, CTL019) may vary by histology. Mantle cell lymphoma (MCL) represents a B-cell malignancy that remains incurable despite novel therapies such as the BTK inhibitor ibrutinib, and where data from CTL019 therapy are scant. Using MCL as a model, we sought to build upon the outcomes from CTL019 and from ibrutinib therapy by combining these in a rational manner.Experimental Design: MCL cell lines and primary MCL samples were combined with autologous or normal donor-derived anti-CD19 CAR T cells along with ibrutinib. The effect of the combination was studied in vitro and in mouse xenograft models.Results: MCL cells strongly activated multiple CTL019 effector functions, and MCL killing by CTL019 was further enhanced in the presence of ibrutinib. In a xenograft MCL model, we showed superior disease control in the CTL019- as compared with ibrutinib-treated mice (median survival not reached vs. 95 days, P < 0.005) but most mice receiving CTL019 monotherapy eventually relapsed. Therefore, we added ibrutinib to CTL019 and showed that 80% to 100% of mice in the CTL019 + ibrutinib arm and 0% to 20% of mice in the CTL019 arm, respectively, remained in long-term remission (P < 0.05).Conclusions: Combining CTL019 with ibrutinib represents a rational way to incorporate two of the most recent therapies in MCL. Our findings pave the way to a two-pronged therapeutic strategy in patients with MCL and other types of B-cell lymphoma. Clin Cancer Res; 22(11); 2684–96. ©2016 AACR.

Purpose:B-cell maturation antigen (BCMA)-targeted chimeric antigen receptor (CAR) T cells (CART-BCMA) are a promising treatment for relapsed/refractory multiple myeloma (r/rMM). We evaluated the safety and feasibility of bridging radiation (RT) in subjects treated on a phase I trial of CART-BCMA.Experimental Design:Twenty-five r/rMM subjects were treated in three cohorts with two doses of CART-BCMA cells ± cyclophosphamide. We retrospectively analyzed toxicity, response, and CART manufacturing data based on RT receipt.Results:Thirteen subjects received no RT 18–35). Group C had qualitatively lower rates of grade 4 (G4) hematologic toxicities (25%) versus A (61.5%) and B (62.5%). G3–4 neurotoxicity occurred in 7.7%, 25%, and 25% in Group A, B, and C, respectively. G3–4 cytokine release syndrome was observed in 38.5%, 25%, and 25% in Group A, B, and C, respectively. Partial response or better was observed in 54%, 38%, and 50% of Group A, B, and C, respectively. RT administered P = 0.002) and P = 0.069) before apheresis was associated with lower in vitro proliferation during manufacturing; however, in vivo CART-BCMA expansion appeared similar across groups.Conclusions:Bridging-RT appeared safe and feasible with CART-BCMA therapy in our r/rMM patients, though larger future studies are needed to draw definitive conclusions.

Supplementary Methods from Neural Stem Cell–Based Anticancer Gene Therapy: A First-in-Human Study in Recurrent High-Grade Glioma Patients

Purpose:To study the biology and identify markers of severe cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS) in children after chimeric antigen receptor T-cell (CAR T) treatment.Experimental Design:We used comprehensive proteomic profiling to measure over 1,400 serum proteins at multiple serial timepoints in a cohort of patients with B-cell acute lymphoblastic leukemia treated with the CD19-targeted CAR T CTL019 on two clinical trials.Results:We identified fms-like tyrosine kinase 3 (FLT3) and mast cell immunoglobulin-like receptor 1 (MILR1) as preinfusion predictive biomarkers of severe CRS. We demonstrated that CRS is an IFNγ-driven process with a protein signature overlapping with hemophagocytic lymphohistiocytosis (HLH). We identified IL18 as a potentially targetable cytokine associated with the development of ICANS.Conclusions:We identified preinfusion biomarkers that can be used to predict severe CRS with a sensitivity, specificity, and accuracy superior to the current gold standard of disease burden. We demonstrated the fundamental role of the IFNγ pathway in driving CRS, suggesting CRS and carHLH are overlapping rather than distinct phenomena, an observation with important treatment implications. We identified IL18 as a possible targetable cytokine in ICANS, providing rationale for IL18 blocking therapies to be translated into clinical trials in ICANS.

Supplementary Methods: Fluorescence in situ hybridization (FISH); MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) enzymatic conversion assay; Western blot analysis; Multiparametric flow cytometry; Table S1. List of the antibodies used for multiparametric flow cytometry; Degranulation assay; Proliferation assay; Cytotoxicity assays; Cytokine measurements; Animal experiments; Figure S1. Different sensitivity to ibrutinib in hematological cell lines; Figure S2. MCL-RL engrafted mice: tumor burden and histology; Figure S3. CTL019 design and production; Figure S4. CTL019 cell production from primary MCL samples; Figure S5. Effect of ibrutinib in T cell function in vitro and in vivo; Figure S5. Effect of ibrutinib in T cell function in vitro and in vivo. A. CTL019 proliferation assay in the presence of ibrutinib;Figure S6. Peripheral blood T cell subsets in mice treated with CART19 or CART19-ibrutinib; Figure S7. CXCR4 and inhibitory/costimulatory receptor expression in peripheral blood of mice treated with CART19 or CART19-ibrutinib; Figure S8. A. Ibrutinib did not enhance the anti-tumor activity of control untransduced T cells.