Retraction Note: A homing system targets therapeutic T cells to brain cancer.

This Article has been retracted; see accompanying Retraction.

A homing system targets therapeutic T cells to brain cancer.

Successful T cell immunotherapy for brain cancer requires that the T cells can access tumour tissues, but this has been difficult to achieve. Here we show that, in contrast to inflammatory brain diseases such as multiple sclerosis, where endothelial cells upregulate ICAM1 and VCAM1 to guide the extravasation of pro-inflammatory cells, cancer endothelium downregulates these molecules to evade immune recognition. By contrast, we found that cancer endothelium upregulates activated leukocyte cell adhesion molecule (ALCAM), which allowed us to overcome this immune-evasion mechanism by creating an ALCAM-restricted homing system (HS). We re-engineered the natural ligand of ALCAM, CD6, in a manner that triggers initial anchorage of T cells to ALCAM and conditionally mediates a secondary wave of adhesion by sensitizing T cells to low-level ICAM1 on the cancer endothelium, thereby creating the adhesion forces necessary to capture T cells from the bloodstream. Cytotoxic HS T cells robustly infiltrated brain cancers after intravenous injection and exhibited potent antitumour activity. We have therefore developed a molecule that targets the delivery of T cells to brain cancer.

Autologous HER2-specific CAR T cells after lymphodepletion for advanced sarcoma: a phase 1 trial.

In this prospective, interventional phase 1 study for individuals with advanced sarcoma, we infused autologous HER2-specific chimeric antigen receptor T cells (HER2 CAR T cells) after lymphodepletion with fludarabine (Flu) ± cyclophosphamide (Cy): 1 × 108 T cells per m2 after Flu (cohort A) or Flu/Cy (cohort B) and 1 × 108 CAR+ T cells per m2 after Flu/Cy (cohort C). The primary outcome was assessment of safety of one dose of HER2 CAR T cells after lymphodepletion. Determination of antitumor responses was the secondary outcome. Thirteen individuals were treated in 14 enrollments, and seven received multiple infusions. HER2 CAR T cells expanded after 19 of 21 infusions. Nine of 12 individuals in cohorts A and B developed grade 1-2 cytokine release syndrome. Two individuals in cohort C experienced dose-limiting toxicity with grade 3-4 cytokine release syndrome. Antitumor activity was observed with clinical benefit in 50% of individuals treated. The tumor samples analyzed showed spatial heterogeneity of immune cells and clustering by sarcoma type and by treatment response. Our results affirm HER2 as a CAR T cell target and demonstrate the safety of this therapeutic approach in sarcoma. ClinicalTrials.gov registration: NCT00902044 .

A subset of cytotoxic effector memory T cells enhances CAR T cell efficacy in a model of pancreatic ductal adenocarcinoma.

In humans, the natural killer (NK) cell marker CD161 identifies several subsets of T cells, including a polyclonal CD8 αß T cell receptor-expressing subset with characteristic specificity for tissue-localized viruses. This subset also displays enhanced cytotoxic and memory phenotypes. Here, we characterized this unique T cell subset and determined its potential suitability for use in chimeric antigen receptor (CAR) T cell therapy. In mice, gene expression profiling among the CD161-equivalent CD8+ T cell populations (CD8+NK1.1+) revealed substantial up-regulation of granzymes, perforin, killer lectin-like receptors, and innate signaling molecules in comparison to CD8+NK1.1- T cells. Adoptive transfer of CD8+NK1.1+ cells from previously exposed animals offered substantially enhanced protection and improved survival against melanoma tumors and influenza infection compared to CD8+NK1.1- cells. Freshly isolated human CD8+CD61+ T cells exhibited heightened allogeneic killing activity in comparison to CD8+CD61- T cells or total peripheral blood mononuclear cells (PBMCs). To determine whether this subset might improve the antitumor efficacy of CAR T cell therapy against solid tumors, we compared bulk PBMCs, CD8+CD161-, and CD8+CD161+ T cells transduced with a human epidermal growth factor receptor-2 (HER2)-specific CAR construct. In vitro, CD8+CD161+ CAR-transduced T cells killed HER2+ targets faster and with greater efficiency. Similarly, these cells mediated enhanced in vivo antitumor efficacy in xenograft models of HER2+ pancreatic ductal adenocarcinoma, exhibiting elevated expression of granzymes and reduced expression of exhaustion markers. These data suggest that this T cell subset presents an opportunity to improve CAR T cell therapy for the treatment of solid tumors.

CAR T-cells that target acute B-lineage leukemia irrespective of CD19 expression.

Chimeric antigen receptor (CAR) T-cells targeting CD19 demonstrate remarkable efficacy in treating B-lineage acute lymphoblastic leukemia (BL-ALL), yet up to 39% of treated patients relapse with CD19(-) disease. We report that CD19(-) escape is associated with downregulation, but preservation, of targetable expression of CD20 and CD22. Accordingly, we reasoned that broadening the spectrum of CD19CAR T-cells to include both CD20 and CD22 would enable them to target CD19(-) escape BL-ALL while preserving their upfront efficacy. We created a CD19/20/22-targeting CAR T-cell by coexpressing individual CAR molecules on a single T-cell using one tricistronic transgene. CD19/20/22CAR T-cells killed CD19(-) blasts from patients who relapsed after CD19CAR T-cell therapy and CRISPR/Cas9 CD19 knockout primary BL-ALL both in vitro and in an animal model, while CD19CAR T-cells were ineffective. At the subcellular level, CD19/20/22CAR T-cells formed dense immune synapses with target cells that mediated effective cytolytic complex formation, were efficient serial killers in single-cell tracking studies, and were as efficacious as CD19CAR T-cells against primary CD19(+) disease. In conclusion, independent of CD19 expression, CD19/20/22CAR T-cells could be used as salvage or front-line CAR therapy for patients with recalcitrant disease.

Tumor response and endogenous immune reactivity after administration of HER2 CAR T cells in a child with metastatic rhabdomyosarcoma.

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.

Locoregional delivery of CAR T cells to the cerebrospinal fluid for treatment of metastatic medulloblastoma and ependymoma.

Recurrent medulloblastoma and ependymoma are universally lethal, with no approved targeted therapies and few candidates presently under clinical evaluation. Nearly all recurrent medulloblastomas and posterior fossa group A (PFA) ependymomas are located adjacent to and bathed by the cerebrospinal fluid, presenting an opportunity for locoregional therapy, bypassing the blood-brain barrier. We identify three cell-surface targets, EPHA2, HER2 and interleukin 13 receptor α2, expressed on medulloblastomas and ependymomas, but not expressed in the normal developing brain. We validate intrathecal delivery of EPHA2, HER2 and interleukin 13 receptor α2 chimeric antigen receptor T cells as an effective treatment for primary, metastatic and recurrent group 3 medulloblastoma and PFA ependymoma xenografts in mouse models. Finally, we demonstrate that administration of these chimeric antigen receptor T cells into the cerebrospinal fluid, alone or in combination with azacytidine, is a highly effective therapy for multiple metastatic mouse models of group 3 medulloblastoma and PFA ependymoma, thereby providing a rationale for clinical trials of these approaches in humans.

Trivalent CAR T cells overcome interpatient antigenic variability in glioblastoma.

Background: Glioblastoma (GBM) is the most common primary malignant brain cancer, and is currently incurable. Chimeric antigen receptor (CAR) T cells have shown promise in GBM treatment. While we have shown that combinatorial targeting of 2 glioma antigens offsets antigen escape and enhances T-cell effector functions, the interpatient variability in surface antigen expression between patients hinders the clinical impact of targeting 2 antigen pairs. This study addresses targeting 3 antigens using a single CAR T-cell product for broader application. Methods: We analyzed the surface expression of 3 targetable glioma antigens (human epidermal growth factor receptor 2 [HER2], interleukin-13 receptor subunit alpha-2 [IL13Rα2], and ephrin-A2 [EphA2]) in 15 primary GBM samples. Accordingly, we created a trivalent T-cell product armed with 3 CAR molecules specific for these validated targets encoded by a single universal (U) tricistronic transgene (UCAR T cells). Results: Our data showed that co-targeting HER2, IL13Rα2, and EphA2 could overcome interpatient variability by a tendency to capture nearly 100% of tumor cells in most tumors tested in this cohort. UCAR T cells made from GBM patients' blood uniformly expressed all 3 CAR molecules with distinct antigen specificity. UCAR T cells mediated robust immune synapses with tumor targets forming more polarized microtubule organizing centers and exhibited improved cytotoxicity and cytokine release over best monospecific and bispecific CAR T cells per patient tumor profile. Lastly, low doses of UCAR T cells controlled established autologous GBM patient derived xenografts (PDXs) and improved survival of treated animals. Conclusion: UCAR T cells can overcome antigenic heterogeneity in GBM and lead to improved treatment outcomes.

Protection against filarial infection by 45-49 kDa molecules of Brugia malayi via IFN-γ-mediated iNOS induction.

Nitric oxide (NO) mediated mechanisms have been implicated in killing of some life-stages of Brugia malayi/Wuchereria bancrofti and protect the host through type 1 responses and IFN-γ stimulated toxic mediators' release. However, the identity of NO stimulating molecules of the parasites is not known. Three predominantly NO-stimulating SDS-PAGE resolved fractions F8 (45.24-48.64 kDa), F11 (33.44-38.44 kDa) and F12 (28.44-33.44 kDa) from B. malayi were identified and their proteins were analyzed by 2-DE and MALDI-TOF/TOF. Tropomyosin, calponin and de novo peptides were identified by 2-DE and MALDI-TOF/TOF in F8 and immunization with F8 conferred most significant protection against L3-initiated infection in Mastomys coucha. Immunized animals showed upregulated F8-induced NO, IFN-γ, TNF-α, IL-1ß, IL-10, TGF-ß release, cellular proliferative responses and specific IgG and IgG1. Anti-IFN-γ, anti-TNF-α, and anti-IL-1ß significantly reduced F8-mediated NO generation and iNOS induction at protein levels. Anti-IFN-γ treated cells showed maximum reduction (>74%) in NO generation suggesting a predominant role of IFN-γ in iNOS induction. In conclusion, the findings suggest that F8 which contains tropomyosin, calponin and de novo peptides protects the host via IFN-γ mediated iNOS induction and may hold promise as vaccine candidate(s). This is also the first report of identification of tropomyosin and calponin in B. malayi.

Cross reactive molecules of human lymphatic filaria Brugia malayi inhibit Leishmania donovani infection in hamsters.

Coinfections are common in natural populations and the outcome of their interactions depends on the immune responses of the host elicited by the parasites. Earlier we showed that immunization with BmAFII (Sephadex G-200 eluted) fraction of human lymphatic filaria Brugia malayi inhibited progression of Leishmania donovani infection in golden hamsters. In the present study we identified cross reactive molecules of B. malayi, and investigated their effect on L. donovani infection and associated immune responses in the host. The sequence alignment and sharing of linear T- and B-cell epitopes in protein molecules of B. malayi and L. donovani counterparts were studied in silico. Hamsters were immunized with robustly cross reactive SDS-PAGE resolved fractions F6 (54.2-67.8kDa) and F9 (41.3-45.0kDa) of B. malayi and subsequently inoculated with amastigotes of L. donovani intracardially. F6 inhibited (∼72%) L. donovani infection and upregulated Th1 cytokine expression, lymphoproliferation, IgG2, IgG2/3 levels and NO production, and downregulated Th2 cytokine expression. Sequences in HSP60 and EF-2 of F6 and L. donovani counterparts were conserved and B- and T-cell epitopes in the proteins shared antigenic regions. In conclusion, leishmania-cross reactive molecules of filarial parasite considerably inhibited leishmanial infection via Th1-mediated immune responses and NO production. Common B- and T-cell epitope regions in HSP60 and EF-2 of the parasites might have contributed to the inhibitory effect on the L. donovani infection. Thus, leishmania-cross reactive filarial parasite molecules may help in designing prophylactic(s) against L. donovani.

Anti-inflammatory BmAFI of Brugia malayi modulates IgE, histamine and histamine receptor responses in Mastomys coucha.

We recently reported that BmAFI, an anti-inflammatory fraction of Brugia malayi adult worm supports parasite development in the hostile peritoneal cavity (p.c.) of Mastomys coucha through a modified Th2 type of response that includes IL-13 and IgE response and anti-inflammatory IL-10 cytokine milieu. In the present study we investigated IgE related responses such as histamine release and modulation of histamine receptors 1 and 2 (HR1 and HR2) by presensitization with BmAFI of M. coucha infected with B. malayi. Sensitization with BmAFI alone enhanced IgE, histamine and HR2, but decreased HR1. Exposure of these animals to infection produced an IgE response that was inversely related to the parasite burden, and decreased histamine conc., and HR1 and HR2 expression. However, there was an early small increase in HR1 expression for a short period after exposure to infection. As expected, BmAFI sensitization supported parasite survival and development in the hostile p.c. of the host. These findings further establish that BmAFI decreases inflammatory/Th1 response and modulates Th2 responses to favour survival and development of the parasite in the hostile p.c. of the host and that IgE and histamine play an important role in this.