Refractory metastatic rhabdomyosarcoma is largely incurable. Here we analyze the response of a child with refractory bone marrow metastatic rhabdomyosarcoma to autologous HER2 CAR T cells. Three ...cycles of HER2 CAR T cells given after lymphodepleting chemotherapy induces remission which is consolidated with four more CAR T-cell infusions without lymphodepletion. Longitudinal immune-monitoring reveals remodeling of the T-cell receptor repertoire with immunodominant clones and serum autoantibodies reactive to oncogenic signaling pathway proteins. The disease relapses in the bone marrow at six months off-therapy. A second remission is achieved after one cycle of lymphodepletion and HER2 CAR T cells. Response consolidation with additional CAR T-cell infusions includes pembrolizumab to improve their efficacy. The patient described here is a participant in an ongoing phase I trial (NCT00902044; active, not recruiting), and is 20 months off T-cell infusions with no detectable disease at the time of this report.
Antigen-independent tonic signaling by chimeric antigen receptors (CARs) can increase differentiation and exhaustion of T cells, limiting their potency. Incorporating 4-1BB costimulation in CARs may ...enable T cells to resist this functional exhaustion; however, the potential ramifications of tonic 4-1BB signaling in CAR T cells remain unclear. Here, we found that tonic CAR-derived 4-1BB signaling can produce toxicity in T cells via continuous TRAF2-dependent activation of the nuclear factor κB (NF-κB) pathway and augmented FAS-dependent cell death. This mechanism was amplified in a non-self-inactivating gammaretroviral vector through positive feedback on the long terminal repeat (LTR) promoter, further enhancing CAR expression and tonic signaling. Attenuating CAR expression by substitution with a self-inactivating lentiviral vector minimized tonic signaling and improved T cell expansion and anti-tumor function. These studies illuminate the interaction between tonic CAR signaling and the chosen expression platform and identify inhibitory properties of the 4-1BB costimulatory domain that have direct implications for rational CAR design.
Display omitted
•Tonic 4-1BB signaling in CARs causes T cell apoptosis, impeding antitumor activity•Tonic 4-1BB signaling enhances CAR expression from gammaretroviral LTR promoters•Reducing CAR expression in retro- or SIN lentiviral vectors attenuates this toxicity
Gomes-Silva et al. model tonic signaling from chimeric antigen receptors (CARs) harboring the 4-1BB endodomain and describe a mechanism through which this signaling produces toxicity in T cells. CAR-driven tonic 4-1BB signaling activates the LTR promoter in gammaretroviral vectors, thus further amplifying the toxicity and undermining CAR T cell anti-tumor activity.
Marked reactive stroma formation is associated with poor outcome in clinically localized prostate cancer. We have previously identified genes with diverse functions that are upregulated in reactive ...stroma. This study tests the hypothesis that expression of these genes in stromal cells enhances prostate cancer growth in vivo.
The expression of reactive stroma genes in prostate stromal cell lines was evaluated by reverse transcriptase (RT)-PCR and qRT-PCR. Genes were knocked down using stable expression of short-hairpin RNAs (shRNA) and the impact on tumorigenesis assessed using the differential reactive stroma (DRS) system, in which prostate stromal cell lines are mixed with LNCaP prostate cancer cells and growth as subcutaneous xenografts assessed.
Nine of 10 reactive stroma genes tested were expressed in one or more prostate stromal cell lines. Gene knockdown of c-Kit, Wnt10B, Bmi1, Gli2, or COMP all resulted in decreased tumorigenesis in the DRS model. In all tumors analyzed, angiogenesis was decreased and there were variable effects on proliferation and apoptosis in the LNCaP cells. Wnt10B has been associated with stem/progenitor cell phenotype in other tissue types. Using a RT-PCR array, we detected downregulation of multiple genes involved in stem/progenitor cell biology such as OCT4 and LIF as well as cytokines such as VEGFA, BDNF, and CSF2 in cells with Wnt10B knockdown.
These findings show that genes upregulated in prostate cancer-reactive stroma promote progression when expressed in prostate stromal cells. Moreover, these data indicate that the DRS model recapitulates key aspects of cancer cell/reactive stroma interactions in prostate cancer.
Prostate cancer is the most common visceral malignancy and the second leading cause of cancer deaths in US men. There is broad evidence that fibroblast growth factor (FGF) receptors are important in ...prostate cancer initiation and progression, but the contribution of particular FGFs in this disease is not fully understood. The FGF family members FGF19, FGF21, and FGF23 comprise a distinct subfamily that circulate in serum and act in an endocrine manner. These endocrine FGFs require α-Klotho (KL) and/or β-Klotho (KLB), two related single-pass transmembrane proteins restricted in their tissue distribution, to act as coreceptors along with classic FGF receptors (FGFR) to mediate potent biologic activity. Here we show that FGF19 is expressed in primary and metastatic prostate cancer tissues, where it functions as an autocrine growth factor. Exogenous FGF19 promoted the growth, invasion, adhesion, and colony formation of prostate cancer cells at low ligand concentrations. FGF19 silencing in prostate cancer cells expressing autocrine FGF19 decreased invasion and proliferation in vitro and tumor growth in vivo. Consistent with these observations, KL and/or KLB were expressed in prostate cancer cells in vitro and in vivo, raising the possibility that additional endocrine FGFs may also exert biologic effects in prostate cancer. Our findings support the concept that therapies targeting FGFR signaling may have efficacy in prostate cancer and highlight FGF19 as a relevant endocrine FGF in this setting.
Purpose: Marked reactive stroma formation, designated as grade 3 reactive stroma, is associated with poor outcome in clinically localized
prostate cancer. To understand the biological processes and ...signaling mechanisms underlying the formation of such reactive
stroma, we carried out microarray gene expression analysis of laser-captured reactive stroma and matched normal stroma.
Experimental Design: Seventeen cases of reactive stroma grade 3 cancer were used to laser-capture tumor and normal stroma. Expression analysis
was carried out using Agilent 44K arrays. Up-regulation of selected genes was confirmed by quantitative reverse transcription-PCR.
Expression data was analyzed to identify significantly up- and down-regulated genes, and gene ontology analysis was used to
define pathways altered in reactive stroma.
Results: A total of 544 unique genes were significantly higher in the reactive stroma and 606 unique genes were lower. Gene ontology
analysis revealed significant alterations in a number of novel processes in prostate cancer reactive stroma, including neurogenesis,
axonogenesis, and the DNA damage/repair pathways, as well as evidence of increases in stem cells in prostate cancer reactive
stroma.
Conclusions: Formation of reactive stroma in prostate cancer is a dynamic process characterized by significant alterations in growth factor
and signal transduction pathways and formation of new structures, including nerves and axons.
Activation of the inducible caspase 9 (iC9) safety gene by a dimerizing drug (chemical inducer of dimerization (CID) AP1903) effectively resolves the symptoms and signs of graft-versus-host disease ...(GvHD) in haploidentical stem cell transplant (HSCT) recipients. However, after CID treatment, 1% of iC9-T cells remain and can regrow over time; although these resurgent T cells do not cause recurrent GvHD, it remains unclear whether repeat CID treatments are a safe and feasible way to further deplete residual gene-modified T cells should any other adverse effects associated with them occur. Here, we report a patient who received an infusion of haploidentical iC9-T cells after HSCT and subsequently received three treatments with AP1903. There was a mild (grade 2) and transient pancytopenia following each AP1903 administration but no non-hematological toxicity. Ninety five percent of circulating iC9-T cells (CD3+CD19+) were eliminated after the first AP1903 treatment. Three months later, the residual cells had expanded more than eightfold and had a lower level of iC9 expression. Each repeated AP1903 administration eliminated a diminishing percentage of the residual repopulating cells, but elimination could be enhanced by T-cell activation. These data support the safety and efficiency of repeated CID treatments for persistent or recurring toxicity from T-cell therapies.
Prostate cancer-associated stroma (CAS) plays an active role in malignant transformation, tumor progression, and metastasis. Molecular analyses of CAS have demonstrated significant changes in gene ...expression; however, conflicting evidence exists on whether genomic alterations in benign cells comprising the tumor microenvironment (TME) underlie gene expression changes and oncogenic phenotypes. This study evaluates the nuclear and mitochondrial DNA integrity of prostate carcinoma cells, CAS, matched benign epithelium and benign epithelium-associated stroma by whole-genome copy-number analyses, targeted sequencing of TP53, and FISH. Array comparative genomic hybridization (aCGH) of CAS revealed a copy-neutral diploid genome with only rare and small somatic copy-number aberrations (SCNA). In contrast, several expected recurrent SCNAs were evident in the adjacent prostate carcinoma cells, including gains at 3q, 7p, and 8q, and losses at 8p and 10q. No somatic TP53 mutations were observed in CAS. Mitochondrial DNA (mtDNA) extracted from carcinoma cells and stroma identified 23 somatic mtDNA mutations in neoplastic epithelial cells, but only one mutation in stroma. Finally, genomic analyses identified no SCNAs, LOH, or copy-neutral LOH in cultured cancer-associated fibroblasts, which are known to promote prostate cancer progression in vivo
The gene expression changes observed in prostate cancer-adjacent stroma and the attendant contribution of the stroma to the development and progression of prostate cancer are not due to frequent or recurrent genomic alterations in the TME.
Vα24-invariant natural killer T (NKT) cells have shown potent anti-tumor properties in murine tumor models and have been linked to favorable outcomes in patients with cancer. However, low numbers of ...these cells in humans have hindered their clinical applications. Here we report interim results from all three patients enrolled on dose level 1 in a phase 1 dose-escalation trial of autologous NKT cells engineered to co-express a GD2-specific chimeric antigen receptor (CAR) with interleukin-15 in children with relapsed or resistant neuroblastoma (NCT03294954). Primary and secondary objectives were to assess safety and anti-tumor responses, respectively, with immune response evaluation as an additional objective. We ex vivo expanded highly pure NKT cells (mean ± s.d., 94.7 ± 3.8%) and treated patients with 3 × 10
CAR-NKT cells per square meter of body surface area after lymphodepleting conditioning with cyclophosphamide/fludarabine (Cy/Flu). Cy/Flu conditioning was the probable cause for grade 3-4 hematologic adverse events, as they occurred before CAR-NKT cell infusion, and no dose-limiting toxicities were observed. CAR-NKT cells expanded in vivo, localized to tumors and, in one patient, induced an objective response with regression of bone metastatic lesions. These initial results suggest that CAR-NKT cells can be expanded to clinical scale and safely applied to treat patients with cancer.
Targeting disialoganglioside (GD2) on neuroblastoma (NB) with T cells expressing a first-generation chimeric antigen receptor (CAR) was safe, but the cells had poor expansion and long-term ...persistence. We developed a third-generation GD2-CAR (GD2-CAR3) and hypothesized that GD2-CAR3 T cells (CARTs) would be safe and effective. This phase 1 study enrolled relapsed or refractory NB patients in three cohorts. Cohort 1 received CART alone, cohort 2 received CARTs plus cyclophosphamide and fludarabine (Cy/Flu), and cohort 3 was treated with CARTs, Cy/Flu, and a programmed death-1 (PD-1) inhibitor. Eleven patients were treated with CARTs. The infusions were safe, and no dose-limiting toxicities occurred. CARTs were detectable in cohort 1, but the lymphodepletion induced by Cy/Flu increased circulating levels of the homeostatic cytokine interleukin (IL)-15 (p = 0.003) and increased CART expansion by up to 3 logs (p = 0.03). PD-1 inhibition did not further enhance expansion or persistence. Antitumor responses at 6 weeks were modest. We observed a striking expansion of CD45/CD33/CD11b/CD163+ myeloid cells (change from baseline, p = 0.0126) in all patients, which may have contributed to the modest early antitumor responses; the effect of these cells merits further study. Thus, CARTs are safe, and Cy/Flu can further increase their expansion.
The outcome of children with relapsed or refractory neuroblastoma remains poor. Because GD2 is a validated immunotherapeutic target of NB, we tested T cells expressing a third-generation GD2-CAR (GD2-CAR3) in three cohorts of patients with relapsed or refractory NB. Eleven patients were treated, the infusions were safe, and no dose-limiting toxicities occurred. CAR T cell expansion improved, and the OS of patients was longer after salvage therapies in Cy/Flu cohorts. A striking expansion of CD45/CD33/CD11b/CD163+ myeloid cells was detected in all patients, which may have contributed to the modest early antitumor responses; the effect of these cells merits further study.
Targeting CD30 with monoclonal antibodies in Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) has had profound clinical success. However, adverse events, mainly mediated by the toxin ...component of the conjugated antibodies, cause treatment discontinuation in many patients. Targeting CD30 with T cells expressing a CD30-specific chimeric antigen receptor (CAR) may reduce the side effects and augment antitumor activity.
We conducted a phase I dose escalation study in which 9 patients with relapsed/refractory HL or ALCL were infused with autologous T cells that were gene-modified with a retroviral vector to express the CD30-specific CAR (CD30.CAR-Ts) encoding the CD28 costimulatory endodomain. Three dose levels, from 0.2 × 108 to 2 × 108 CD30.CAR-Ts/m2, were infused without a conditioning regimen. All other therapy for malignancy was discontinued at least 4 weeks before CD30.CAR-T infusion. Seven patients had previously experienced disease progression while being treated with brentuximab.
No toxicities attributable to CD30.CAR-Ts were observed. Of 7 patients with relapsed HL, 1 entered complete response (CR) lasting more than 2.5 years after the second infusion of CD30.CAR-Ts, 1 remained in continued CR for almost 2 years, and 3 had transient stable disease. Of 2 patients with ALCL, 1 had a CR that persisted 9 months after the fourth infusion of CD30.CAR-Ts. CD30.CAR-T expansion in peripheral blood peaked 1 week after infusion, and CD30.CAR-Ts remained detectable for over 6 weeks. Although CD30 may also be expressed by normal activated T cells, no patients developed impaired virus-specific immunity.
CD30.CAR-Ts are safe and can lead to clinical responses in patients with HL and ALCL, indicating that further assessment of this therapy is warranted.
ClinicalTrials.gov NCT01316146.
National Cancer Institute (3P50CA126752, R01CA131027 and P30CA125123), National Heart, Lung, and Blood Institute (R01HL114564), and Leukemia and Lymphoma Society (LLSTR 6227-08).
PDF
