Depletion of high-content CD14+ cells from apheresis products is critical for successful transduction and expansion of CAR T cells during large-scale cGMP manufacturing.

With the US Food and Drug Administration (FDA) approval of four CD19- and one BCMA-targeted chimeric antigen receptor (CAR) therapy for B cell malignancies, CAR T cell therapy has finally reached the status of a medicinal product. The successful manufacturing of autologous CAR T cell products is a key requirement for this promising treatment modality. By analyzing the composition of 214 apheresis products from 210 subjects across eight disease indications, we found that high CD14+ cell content poses a challenge for manufacturing CAR T cells, especially in patients with non-Hodgkin's lymphoma and multiple myeloma caused by the non-specific phagocytosis of the magnetic beads used to activate CD3+ T cells. We demonstrated that monocyte depletion via rapid plastic surface adhesion significantly reduces the CD14+ monocyte content in the apheresis products and simultaneously boosts the CD3+ content. We established a 40% CD14+ threshold for the stratification of apheresis products across nine clinical trials and demonstrated the effectiveness of this procedure by comparing manufacturing runs in two phase 1 clinical trials. Our study suggests that CD14+ content should be monitored in apheresis products, and that the manufacturing of CAR T cells should incorporate a step that lessens the CD14+ cell content in apheresis products containing more than 40% to maximize the production success.

Preclinical Efficacy and Safety of a Human Embryonic Stem Cell-Derived Midbrain Dopamine Progenitor Product, MSK-DA01.

Parkinson's disease is characterized by the loss of dopaminergic neurons in the substantia nigra leading to disabling deficits. Dopamine neuron grafts may provide a significant therapeutic advance over current therapies. We have generated midbrain dopamine neurons from human embryonic stem cells and manufactured large-scale cryopreserved dopamine progenitors for clinical use. After optimizing cell survival and phenotypes in short-term studies, the cell product, MSK-DA01, was subjected to an extensive set of biodistribution, toxicity, and tumorigenicity assessments in mice under GLP conditions. A large-scale efficacy study was also performed in rats with the same lot of cells intended for potential human use and demonstrated survival of the grafted cells and behavioral amelioration in 6-hydroxydopamine lesioned rats. There were no adverse effects attributable to the grafted cells, no obvious distribution outside the brain, and no cell overgrowth or tumor formation, thus paving the way for a future clinical trial.

CD19 CAR T cells following autologous transplantation in poor-risk relapsed and refractory B-cell non-Hodgkin lymphoma.

High-dose chemotherapy and autologous stem cell transplantation (HDT-ASCT) is the standard of care for relapsed or primary refractory (rel/ref) chemorefractory diffuse large B-cell lymphoma. Only 50% of patients are cured with this approach. We investigated safety and efficacy of CD19-specific chimeric antigen receptor (CAR) T cells administered following HDT-ASCT. Eligibility for this study includes poor-risk rel/ref aggressive B-cell non-Hodgkin lymphoma chemosensitive to salvage therapy with: (1) positron emission tomography-positive disease or (2) bone marrow involvement. Patients underwent standard HDT-ASCT followed by 19-28z CAR T cells on days +2 and +3. Of 15 subjects treated on study, dose-limiting toxicity was observed at both dose levels (5 × 106 and 1 × 107 19-28z CAR T per kilogram). Ten of 15 subjects experienced CAR T-cell-induced neurotoxicity and/or cytokine release syndrome (CRS), which were associated with greater CAR T-cell persistence (P = .05) but not peak CAR T-cell expansion. Serum interferon-γ elevation (P < .001) and possibly interleukin-10 (P = .07) were associated with toxicity. The 2-year progression-free survival (PFS) is 30% (95% confidence interval, 20% to 70%).  Subjects given decreased naive-like (CD45RA+CCR7+) CD4+ and CD8+ CAR T cells experienced superior PFS (P = .02 and .04, respectively). There was no association between CAR T-cell peak expansion, persistence, or cytokine changes and PFS. 19-28z CAR T cells following HDT-ASCT were associated with a high incidence of reversible neurotoxicity and CRS. Following HDT-ASCT, effector CD4+ and CD8+ immunophenotypes may improve disease control. This trial was registered at www.clinicaltrials.gov as #NCT01840566.

Safety and tolerability of conditioning chemotherapy followed by CD19-targeted CAR T cells for relapsed/refractory CLL.

BACKGROUND: Subgroups of patients with relapsed or refractory (R/R) chronic lymphocytic leukemia (CLL) exhibit suboptimal outcomes after standard therapies, including oral kinase inhibitors. We and others have previously reported on safety and efficacy of autologous CD19-targeted CAR T-cells for these patients; here we report safety and long-term follow-up of CAR T-cell therapy with or without conditioning chemotherapy for patients with R/R CLL and indolent B-cell non-Hodgkin lymphoma (B-NHL). METHODS: We conducted a phase 1 clinical trial investigating CD19-targeted CAR T-cells incorporating a CD28 costimulatory domain (19-28z). Seventeen of 20 patients received conditioning chemotherapy prior to CAR T-cell infusion. Five patients with CLL received ibrutinib at the time of autologous T-cell collection and/or CAR T-cell administration. RESULTS: This analysis included 16 patients with R/R CLL and 4 patients with R/R indolent B-NHL. Cytokine release syndrome (CRS) was observed in all 20 patients but grades 3 and 4 CRS and neurological events were uncommon (10% for each). Ex vivo expansion of T-cells and proportions of CD4+/CD8+ CAR T-cells with CD62L+CD127+ immunophenotype were significantly greater in patients on ibrutinib at leukapheresis. Three of 12 evaluable CLL patients receiving conditioning chemotherapy achieved CR (two had minimal residual disease-negative CR). All patients achieving CR remained progression-free at median follow-up of 53 months. CONCLUSION: Conditioning chemotherapy and 19-28z CAR T-cells were acceptably tolerated across investigated dose levels in heavily pretreated patients with R/R CLL and indolent B-NHL, and a subgroup of patients achieved durable CR. Ibrutinib therapy may modulate autologous T-cell phenotype. TRIAL REGISTRATION: ClinicalTrials.gov NCT00466531. FUNDING: Juno Therapeutics.

Autologous CD19-Targeted CAR T Cells in Patients with Residual CLL following Initial Purine Analog-Based Therapy.

Patients with residual chronic lymphocytic leukemia (CLL) following initial purine analog-based chemoimmunotherapy exhibit a shorter duration of response and may benefit from novel therapeutic strategies. We and others have previously described the safety and efficacy of autologous T cells modified to express anti-CD19 chimeric antigen receptors (CARs) in patients with relapsed or refractory B cell acute lymphoblastic leukemia and CLL. Here we report the use of CD19-targeted CAR T cells incorporating the intracellular signaling domain of CD28 (19-28z) as a consolidative therapy in 8 patients with residual CLL following first-line chemoimmunotherapy with pentostatin, cyclophosphamide, and rituximab. Outpatients received low-dose conditioning therapy with cyclophosphamide (600 mg/m2), followed by escalating doses of 3 × 106, 1 × 107, or 3 × 107 19-28z CAR T cells/kg. An objective response was observed in 3 of 8 patients (38%), with a clinically complete response lasting more than 28 months observed in two patients. Self-limited fevers were observed post-CAR T cell infusion in 4 patients, contemporaneous with elevations in interleukin-6 (IL-6), IL-10, IL-2, and TGF-α. None developed severe cytokine release syndrome or neurotoxicity. CAR T cells were detectable post-infusion in 4 patients, with a longest observed persistence of 48 days by qPCR. Further strategies to enhance CAR T cell efficacy in CLL are under investigation.

Long-Term Follow-up of CD19 CAR Therapy in Acute Lymphoblastic Leukemia.

BACKGROUND: CD19-specific chimeric antigen receptor (CAR) T cells induce high rates of initial response among patients with relapsed B-cell acute lymphoblastic leukemia (ALL) and long-term remissions in a subgroup of patients. METHODS: We conducted a phase 1 trial involving adults with relapsed B-cell ALL who received an infusion of autologous T cells expressing the 19-28z CAR at the Memorial Sloan Kettering Cancer Center (MSKCC). Safety and long-term outcomes were assessed, as were their associations with demographic, clinical, and disease characteristics. RESULTS: A total of 53 adults received 19-28z CAR T cells that were manufactured at MSKCC. After infusion, severe cytokine release syndrome occurred in 14 of 53 patients (26%; 95% confidence interval [CI], 15 to 40); 1 patient died. Complete remission was observed in 83% of the patients. At a median follow-up of 29 months (range, 1 to 65), the median event-free survival was 6.1 months (95% CI, 5.0 to 11.5), and the median overall survival was 12.9 months (95% CI, 8.7 to 23.4). Patients with a low disease burden (<5% bone marrow blasts) before treatment had markedly enhanced remission duration and survival, with a median event-free survival of 10.6 months (95% CI, 5.9 to not reached) and a median overall survival of 20.1 months (95% CI, 8.7 to not reached). Patients with a higher burden of disease (≥5% bone marrow blasts or extramedullary disease) had a greater incidence of the cytokine release syndrome and neurotoxic events and shorter long-term survival than did patients with a low disease burden. CONCLUSIONS: In the entire cohort, the median overall survival was 12.9 months. Among patients with a low disease burden, the median overall survival was 20.1 months and was accompanied by a markedly lower incidence of the cytokine release syndrome and neurotoxic events after 19-28z CAR T-cell infusion than was observed among patients with a higher disease burden. (Funded by the Commonwealth Foundation for Cancer Research and others; ClinicalTrials.gov number, NCT01044069 .).

Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia.

We report on 16 patients with relapsed or refractory B cell acute lymphoblastic leukemia (B-ALL) that we treated with autologous T cells expressing the 19-28z chimeric antigen receptor (CAR) specific to the CD19 antigen. The overall complete response rate was 88%, which allowed us to transition most of these patients to a standard-of-care allogeneic hematopoietic stem cell transplant (allo-SCT). This therapy was as effective in high-risk patients with Philadelphia chromosome-positive (Ph(+)) disease as in those with relapsed disease after previous allo-SCT. Through systematic analysis of clinical data and serum cytokine levels over the first 21 days after T cell infusion, we have defined diagnostic criteria for a severe cytokine release syndrome (sCRS), with the goal of better identifying the subset of patients who will likely require therapeutic intervention with corticosteroids or interleukin-6 receptor blockade to curb the sCRS. Additionally, we found that serum C-reactive protein, a readily available laboratory study, can serve as a reliable indicator for the severity of the CRS. Together, our data provide strong support for conducting a multicenter phase 2 study to further evaluate 19-28z CAR T cells in B-ALL and a road map for patient management at centers now contemplating the use of CAR T cell therapy.

AtNOS/AtNOA1 is a functional Arabidopsis thaliana cGTPase and not a nitric-oxide synthase.

AtNOS1 was previously identified as a potential nitric-oxide synthase (NOS) in Arabidopsis thaliana, despite lack of sequence similarity to animal NOSs. Although the dwarf and yellowish leaf phenotype of Atnos1 knock-out mutant plants can be rescued by treatment with exogenous NO, doubts have recently been raised as to whether AtNOS1 is a true NOS. Moreover, depending on the type of physiological responses studied, Atnos1 is not always deficient in NO induction and/or detection, as previously reported. Here, we present experimental evidence showing that AtNOS1 is unable to bind and oxidize arginine to NO. These results support the argument that AtNOS1 is not a NOS. We also show that the renamed NO-associated protein 1 (AtNOA1) is a member of the circularly permuted GTPase family (cGTPase). AtNOA1 specifically binds GTP and hydrolyzes it. Complementation experiments of Atnoa1 mutant plants with different constructs of AtNOA1 show that GTP hydrolysis is necessary but not sufficient for the physiological function of AtNOA1. Mutant AtNOA1 lacking the C-terminal domain, although retaining GTPase activity, failed to complement Atnoa1, suggesting that this domain plays a crucial role in planta. cGTPases appear to be RNA-binding proteins, and the closest homolog of AtNOA1, the Bacillus subtilis YqeH, has been shown to participate in ribosome assembly and stability. We propose a similar function for AtNOA1 and discuss it in the light of its potential role in NO accumulation and plant development.

Tobamovirus infection is independent of HSP101 mRNA induction and protein expression.

Heat shock protein 101 (HSP101) has been implicated in tobamovirus infections by virtue of its ability to enhance translation of mRNAs possessing the 5'Omega-leader of Tobacco mosaic virus (TMV). Enhanced translation is mediated by HSP101 binding to a CAA-repeat motif in TMV Omega leader. CAA repeat sequences are present in the 5' leaders of other tobamoviruses including Oilseed rape mosaic virus (ORMV), which infects Arabidopsis thaliana. HSP101 is one of eight HSP100 gene family members encoded by the A. thaliana genome, and of these, HSP101 and HSP98.7 are predicted to encode proteins localized to the cytoplasm where they could potentially interact with TMV RNA. Analysis of the expression of the HSP100s showed that only HSP101 mRNA transcripts were induced significantly by ORMV in A. thaliana. The induction of HSP101 mRNA was also correlated with an increase in its protein levels and was independent of defense-related signaling pathways involving salicylic acid, jasmonic acid, or ethylene. A. thaliana mutants lacking HSP101, HSP98.7, or both supported wild-type levels of ORMV replication and movement. Similar results were obtained for TMV infection in Nicotiana benthamiana plants silenced for HSP101, demonstrating that HSP101 is not necessary for efficient tobamovirus infection.

Transgenic cucumbers harboring the 54-kDa putative gene of Cucumber fruit mottle mosaic tobamovirus are highly resistant to viral infection and protect non-transgenic scions from soil infection.

Cucumber fruit mottle mosaic tobamovirus (CFMMV) causes severe mosaic symptoms and yellow mottling on leaves and fruits and, occasionally, severe wilting of cucumber (Cucumis sativus L.) plants. No genetic source of resistance against this virus has been identified in cucumber. The gene coding for the putative 54-kDa replicase gene of CFMMV was cloned into an Agrobacterium tumefaciens binary vector, and transformation was performed on cotyledon explants of a parthenocarpic cucumber cultivar. R1 seedlings were screened for resistance to CFMMV by symptom expression, back inoculation on an alternative host and ELISA. From a total of 14 replicase-containing R1 lines, eight resistant lines were identified. Line 144--homozygous for the putative 54-kDa replicase gene--was immune to CFMMV infection by mechanical and graft inoculation, and to root infection following planting in CFMMV-infested soil. A substantial delay of symptom appearance was observed following infection by three additional cucurbit-infecting tobamoviruses. When used as a rootstock, line I44 protected susceptible cucumber scions from soil infection by CFMMV. This paper is the first report on protection of a susceptible cultivar against a soil-borne viral pathogen, by grafting onto a transgenic rootstock.

Functional analysis of the Cucumber mosaic virus 2b protein: pathogenicity and nuclear localization.

The 2b protein encoded by Cucumber mosaic virus (CMV) has been shown to be a silencing suppressor and pathogenicity determinant in solanaceous hosts, but a movement determinant in cucumber. In addition, synergistic interactions between CMV and Zucchini yellow mosaic virus (ZYMV) have been described in several cucurbit species. Here, it was shown that deletion of the 2b gene from CMV prevented extensive systemic movement of the virus in zucchini squash, which could not be complemented by co-infection with ZYMV. Thus, ZYMV expressing a silencing suppressor with a different target could not complement the CMV 2b-specific movement function. Expression of the 2b protein from an attenuated ZYMV vector resulted in a synergistic response, largely restoring infection symptoms of wild-type ZYMV in several cucurbit species. Deletion or alteration of either of two nuclear localization signals (NLSs) did not affect nuclear localization in two assays, but did affect pathogenicity in several cucurbit species, whilst deletion of both NLSs led to loss of nuclear localization. The 2b protein interacted with an Arabidopsis thaliana karyopherin alpha protein (AtKAPalpha) in the yeast two-hybrid system, as did each of the two single NLS-deletion mutants. However, 2b protein containing a deletion of both NLSs was unable to interact with AtKAPalpha. These data suggest that the 2b protein localizes to the nucleus by using the karyopherin alpha-mediated system, but demonstrate that nuclear localization was insufficient for enhancement of the 2b-mediated pathogenic response in cucurbit hosts. Thus, the sequences corresponding to the two NLSs must have another role leading to pathogenicity enhancement.

Roles for host factors in plant viral pathogenicity.

The simple, obligate nature of viruses requires them to usurp or divert cellular resources, including host factors, away from their normal functions. The characterization of host proteins, membranes, and nucleic acids that are implicated in viral infection cycles, together with other recent discoveries, is providing fundamental clues about the molecular bases of viral susceptibility. As viruses invade susceptible plants, they create conditions that favor systemic infections by suppressing multiple layers of innate host defenses. When viruses meddle in these defense mechanisms, which are interlinked with basic cellular functions, phenotypic changes can result that contribute to disease symptoms.

Characterization of Synergy Between Cucumber mosaic virus and Potyviruses in Cucurbit Hosts.

ABSTRACT Mixed infections of cucurbits by Cucumber mosaic virus (CMV) and potyviruses exhibit a synergistic interaction. Zucchini squash and melon plants coinfected by the potyvirus Zucchini yellow mosaic virus (ZYMV) and either Fny-CMV (subgroup IA) or LS-CMV (subgroup II) displayed strong synergistic pathological responses, eventually progressing to vascular wilt and plant death. Accumulation of Fny- or LS-CMV RNAs in a mixed infection with ZYMV in zucchini squash was slightly higher than infection with CMV strains alone. There was an increase in CMV (+) strand RNA levels, but no increase in CMV (-) RNA3 levels during mixed infection with ZYMV. Moreover, only the level of capsid protein from LS-CMV increased in mixed infection. ZYMV accumulated to similar levels in singly and mixed infected zucchini squash and melon plants. Coinfection of squash with the potyvirus Watermelon mosaic virus (WMV) and CMV strains increased both the Fny-CMV RNA levels and the LS-CMV RNA levels. However, CMV (-) strand RNA3 levels were increased little or not at all for CMV on coinfection with WMV. Infection of CMV strains (LS and Fny) containing satellite RNAs (WL47-sat RNA and B5*-sat RNA) reduced the accumulation of the helper virus RNA, except when B5*-sat RNA was mixed with LS- CMV. However, mixed infection containing ZYMV and the CMV strains with satellites reversed the suppression effect of satellite RNAs on helper virus accumulation and increased satellite RNA accumulation. The synergistic interaction between CMV and potyviruses in cucurbits exhibited different features from that documented in tobacco, indicating there are differences in the mechanisms of potyvirus synergistic phenomena.