Daniel J. Landsburg

Intraventricular Brain Injection of Adeno-Associated Virus Type 1 (AAV1) in Neonatal Mice Results in Complementary Patterns of Neuronal Transduction to AAV2 and Total Long-Term Correction of Storage Lesions in the Brains of β-Glucuronidase-Deficient Mice

ABSTRACT Inherited metabolic disorders that affect the central nervous system typically result in pathology throughout the brain; thus, gene therapy strategies need to achieve widespread delivery. We previously found that although intraventricular injection of the neonatal mouse brain with adeno-associated virus serotype 2 (AAV2) results in dispersed gene delivery, many brain structures were poorly transduced. This limitation may be overcome by using different AAV serotypes because the capsid proteins use different cellular receptors for entry, which may allow enhanced global targeting of the brain. We tested this with AAV1 and AAV5 vectors. AAV5 showed very limited brain transduction after neonatal injection, even though it has different transduction patterns than AAV2 in adult brain injections. In contrast, AAV1 vectors, which have not been tested in the brain, showed robust widespread transduction. Complementary patterns of transduction between AAV1 and AAV2 were established and maintained in the adult brain after neonatal injection. In the majority of structures, AAV1 transduced many more cells than AAV2. Both vectors transduced mostly neurons, indicating that differential expression of receptors on the surfaces of neurons occurs in the developing brain. The number of cells positive for a vector-encoded secreted enzyme (β-glucuronidase) was notably greater and more widespread in AAV1-injected brains. A comprehensive analysis of AAV1-treated brains from β-glucuronidase-deficient mice (mucopolysaccharidosis type VII) showed complete reversal of pathology in all areas of the brain for at least 1 year, demonstrating that the combination of this serotype and experimental strategy is therapeutically effective for treating global neurometabolic disorders.

Impact of induction regimen and stem cell transplantation on outcomes in double-hit lymphoma: A multicenter retrospective analysis

Patients with double-hit lymphoma (DHL), which is characterized by rearrangements of MYC and either BCL2 or BCL6, face poor prognoses. We conducted a retrospective multicenter study of the impact of baseline clinical factors, induction therapy, and stem cell transplant (SCT) on the outcomes of 311 patients with previously untreated DHL. At median follow-up of 23 months, the median progression-free survival (PFS) and overall survival (OS) rates among all patients were 10.9 and 21.9 months, respectively. Forty percent of patients remain disease-free and 49% remain alive at 2 years. Intensive induction was associated with improved PFS, but not OS, and SCT was not associated with improved OS among patients achieving first complete remission (P = .14). By multivariate analysis, advanced stage, central nervous system involvement, leukocytosis, and LDH >3 times the upper limit of normal were associated with higher risk of death. Correcting for these, intensive induction was associated with improved OS. We developed a novel risk score for DHL, which divides patients into high-, intermediate-, and low-risk groups. In conclusion, a subset of DHL patients may be cured, and some patients may benefit from intensive induction. Further investigations into the roles of SCT and novel agents are needed.

Structure-Based Mutagenesis of Herpes Simplex Virus Glycoprotein D Defines Three Critical Regions at the gD-HveA/HVEM Binding Interface

ABSTRACT Herpes simplex virus (HSV) entry into cells requires the binding of glycoprotein D (gD) to one of several cell surface receptors. The crystal structure of gD bound to one of these receptors, HveA/HVEM, reveals that the core of gD comprises an immunoglobulin fold flanked by a long C-terminal extension and an N-terminal hairpin loop. HveA is a member of the tumor necrosis factor receptor family and contains four cysteine-rich domains (CRDs) characteristic of this family. Fourteen amino acids within the gD N-terminal loop comprise the entire binding site for HveA. To determine the contribution of each gD contact residue to virus entry, we constructed gD molecules mutated in these amino acids. We determined the abilities of the gD mutants to bind receptors, facilitate virus entry, and mediate cell-cell fusion. Seven of the gD mutants exhibited wild-type levels of receptor binding and gD function. Results from the other seven gD mutants revealed three critical regions at the gD-HveA interface. (i) Several gD residues that participate in an intermolecular β-sheet with HveA were found to be crucial for HveA binding and entry into HveA-expressing cells. (ii) Two gD residues that contact HveA-Y23 contributed to HveA binding but were not required for mediating entry into cells. HveA-Y23 fits into a crevice on the surface of gD and was previously shown to be essential for gD binding. (iii) CRD2 was previously shown to contribute to gD binding, and this study shows that one gD residue that contacts CRD2 contributes to HveA binding. None of the gD mutations prevented interaction with nectin-1, another gD receptor. However, when cotransfected with the other glycoproteins required for fusion, two gD mutants gained the ability to mediate fusion of cells expressing nectin-2, a gD receptor that interacts with several laboratory-derived gD mutants but not with wild-type gD. Thus, results from this panel of gD mutants as well as those of previous studies (A. Carfi, S. H. Willis, J. C. Whitbeck, C. Krummenacher, G. H. Cohen, R. J. Eisenberg, and D. C. Wiley, Mol. Cell 8:169-179, 2001, and S. A. Connolly, D. J. Landsburg, A. Carfi, D. C. Wiley, R. J. Eisenberg, and G. H. Cohen, J. Virol. 76:10894-10904, 2002) provide a detailed picture of the gD-HveA interface and the contacts required for functional interaction. The results demonstrate that of the 35 gD and HveA contact residues that comprise the gD-HveA interface, only a handful are critical for complex formation.

Structure-Based Analysis of the Herpes Simplex Virus Glycoprotein D Binding Site Present on Herpesvirus Entry Mediator HveA (HVEM)

ABSTRACT Binding of herpes simplex virus (HSV) envelope glycoprotein D (gD) to a cell surface receptor is an essential step of virus entry. We recently determined the crystal structure of gD bound to one receptor, HveA. HveA is a member of the tumor necrosis factor receptor family and contains four characteristic cysteine-rich domains (CRDs). The first two CRDs of HveA are necessary and sufficient for gD binding. The structure of the gD-HveA complex reveals that 17 amino acids in HveA CRD1 and 4 amino acids in HveA CRD2 directly contact gD. To determine the contribution of these 21 HveA residues to virus entry, we constructed forms of HveA mutated in each of these contact residues. We determined the ability of the mutant proteins to bind gD, facilitate virus entry, and form HveA oligomers. Our results point to a binding hot spot centered around HveA-Y23, a residue that protrudes into a crevice on the surface of gD. Both the hydroxyl group and phenyl group of HveA-Y23 contribute to HSV entry. Our results also suggest that an intermolecular β-sheet formed between gD and HveA residues 35 to 37 contributes to binding and that a C37-C19 disulfide bond in CRD1 is a critical component of HveA structure necessary for gD binding. The results argue that CRD2 is required for gD binding mainly to provide structural support for a gD binding site in CRD1. Only one mutant, HveA-R75A, exhibited enhanced gD binding. While some mutations influenced complex formation, the majority did not affect HSV entry, suggesting that most contact residues contribute to HveA receptor function collectively rather than individually. This structure-based dissection of the gD-HveA binding site highlights the contribution of key residues within HveA to gD binding and HSV entry and defines a target region for the design of small-molecule inhibitors.

Chimeric Antigen Receptor T Cells in Refractory B-Cell Lymphomas

Background Patients with diffuse large B‐cell lymphoma or follicular lymphoma that is refractory to or that relapses after immunochemotherapy and transplantation have a poor prognosis. High response rates have been reported with the use of T cells modified by chimeric antigen receptor (CAR) that target CD19 in B‐cell cancers, although data regarding B‐cell lymphomas are limited. Methods We used autologous T cells that express a CD19‐directed CAR (CTL019) to treat patients with diffuse large B‐cell lymphoma or follicular lymphoma that had relapsed or was refractory to previous treatments. Patients were monitored for response to treatment, toxic effects, the expansion and persistence of CTL019 cells in vivo, and immune recovery. Results A total of 28 adult patients with lymphoma received CTL019 cells, and 18 of 28 had a response (64%; 95% confidence interval [CI], 44 to 81). Complete remission occurred in 6 of 14 patients with diffuse large B‐cell lymphoma (43%; 95% CI, 18 to 71) and 10 of 14 patients with follicular lymphoma (71%; 95% CI, 42 to 92). CTL019 cells proliferated in vivo and were detectable in the blood and bone marrow of patients who had a response and patients who did not have a response. Sustained remissions were achieved, and at a median follow‐up of 28.6 months, 86% of patients with diffuse large B‐cell lymphoma who had a response (95% CI, 33 to 98) and 89% of patients with follicular lymphoma who had a response (95% CI, 43 to 98) had maintained the response. Severe cytokine‐release syndrome occurred in 5 patients (18%). Serious encephalopathy occurred in 3 patients (11%); 2 cases were self‐limiting and 1 case was fatal. All patients in complete remission by 6 months remained in remission at 7.7 to 37.9 months (median, 29.3 months) after induction, with a sustained reappearance of B cells in 8 of 16 patients and with improvement in levels of IgG in 4 of 10 patients and of IgM in 6 of 10 patients at 6 months or later and in levels of IgA in 3 of 10 patients at 18 months or later. Conclusions CTL019 cells can be effective in the treatment of relapsed or refractory diffuse large B‐cell lymphoma and follicular lymphoma. High rates of durable remission were observed, with recovery of B cells and immunoglobulins in some patients. Transient encephalopathy developed in approximately one in three patients and severe cytokine‐release syndrome developed in one in five patients. (Funded by Novartis and others; ClinicalTrials.gov number, NCT02030834.)

Potential Nectin-1 Binding Site on Herpes Simplex Virus Glycoprotein D

ABSTRACT Four glycoproteins (gD, gB, gH, and gL) are essential for herpes simplex virus (HSV) entry into cells. An early step of fusion requires gD to bind one of several receptors, such as nectin-1 or herpesvirus entry mediator (HVEM). We hypothesize that a conformational change in gD occurs upon receptor binding that triggers the other glycoproteins to mediate fusion. Comparison of the crystal structures of gD alone and gD bound to HVEM reveals that upon HVEM binding, the gD N terminus transitions from a flexible stretch of residues to a hairpin loop. To address the contribution of this transition to the ability of gD to trigger fusion, we attempted to “lock” the gD N terminus into a looped conformation by engineering a disulfide bond at its N and C termini. The resulting mutant (gD-A3C/Y38C) failed to trigger fusion in the absence of receptor, suggesting that formation of the loop is not the sole fusion trigger. Unexpectedly, although gD-A3C/Y38C bound HVEM, it failed to bind nectin-1. This was due to the key role played by Y38 in interacting with nectin-1. Since tyrosines are often “hot spot” residues at the center of protein-protein interfaces, we mutated residues that surround Y38 on the same face of gD and tested their binding and functional properties. Our results suggest that this region of gD is important for nectin-1 interaction and is distinct from but partially overlaps the site of HVEM binding. Unique gD mutants with altered receptor usage generated in this study may help dissect the roles played by various HSV receptors during infection.

Progressive multifocal leukoencephalopathy associated with brentuximab vedotin therapy: a report of 5 cases from the Southern Network on Adverse Reactions (SONAR) project.

Brentuximab vedotin (BV) is an anti‐CD30 monoclonal antibody‐drug conjugate that was approved in 2011 for the treatment of patients with anaplastic large cell and Hodgkin lymphomas. The product label indicates that 3 patients who were treated with BV developed progressive multifocal leukoencephalopathy (PML), a frequently fatal JC virus‐induced central nervous system infection. Prior immunosuppressive therapy and compromised immune systems were postulated risk factors. In the current study, the authors reported 5 patients who developed BV‐associated PML, including 2 immunocompetent patients.

Front‐line, dose‐escalated immunochemotherapy is associated with a significant progression‐free survival advantage in patients with double‐hit lymphomas: a systematic review and meta‐analysis

‘Double‐hit lymphomas’ (DHL), defined by concurrent MYC and BCL2 (or, alternatively, BCL6) rearrangements, have a very poor outcome compared to standard‐risk, diffuse large B‐cell lymphomas (DLBCL). Consequently, dose‐intensive (DI) therapies and/or consolidation with high‐dose therapy and transplant have been explored in DHL, although benefit has been debated. This meta‐analysis compared survival outcomes in DHL patients receiving dose‐escalated regimens [DI: R‐Hyper‐CVAD (rituximab, cyclophosphamide, vincristine, doxorubicin, dexamethasone) or R‐CODOX‐M/IVAC (rituximab, cyclophosphamide, doxorubicin, vincristine, methotrexate/ifosfamide, etoposide, high dose cytarabine); or intermediate‐dose: R‐EPOCH (rituximab, etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone)] versus standard‐dose regimens (R‐CHOP; rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) in the first‐line setting. Data were synthesized to estimate hazard ratios of dose‐escalated treatments versus R‐CHOP using a Weibull proportional hazards model within a Bayesian meta‐analysis framework. Eleven studies examining 394 patients were included. Patients were treated with either front‐line R‐CHOP (n = 180), R‐EPOCH (n = 91), or R‐Hyper‐CVAD/rituximab, methotrexate, cytarabine (R‐M/C), R‐CODOX‐M/R‐IVAC (DI) (n = 123). Our meta‐analysis revealed that median progression‐free survival (n = 350) for the R‐CHOP, R‐EPOCH and DI groups was 12·1, 22·2, and 18·9 months, respectively. First‐line treatment with R‐EPOCH significantly reduced the risk of a progression compared with R‐CHOP (relative risk reduction of 34%; P = 0·032); however, overall survival (n = 374) was not significantly different across treatment approaches. A subset of patients might benefit from intensive induction with/without transplant. Further investigation into the role of transplant and novel therapy combinations is necessary.

‘Double‐Hit’ cytogenetic status may not be predicted by baseline clinicopathological characteristics and is highly associated with overall survival in B cell lymphoma patients

‘Double‐Hit’ (DH) B cell non‐Hodgkin lymphomas are characterized by the presence of a MYC rearrangement and additional rearrangement(s) most commonly involving BCL2 and/or BCL6. Patients with DH lymphomas are unlikely to achieve long‐term survival when treated with standard immunochemotherapy alone. DH gene rearrangements can be identified through metaphase karyotyping or more sensitive fluorescence in situ hybridization (FISH), although the latter is not routinely performed. We report 53 cases of B cell lymphoma that underwent diagnostic metaphase karyotying or FISH for MYC rearrangements. DH lymphoma was detected in 17 cases. No baseline factor, including age, serum lactate dehydrogenase, stage, International Prognostic Index or histology predicted for DH status. The median overall survival was significantly shorter for DH compared to non‐DH lymphoma patients (8·2 vs. 56·8 months, P < 0·001). DH status retained the most statistically significant association with overall survival on multivariate Cox regression analysis. DH status could not be inferred by baseline disease‐ or patient‐related characteristics and was most predictive of overall survival in this cohort of B cell lymphoma patients. These findings support the practice of routine performance of FISH for DH gene rearrangements on B cell lymphoma specimens in order to effectively identify DH patients who may benefit from risk‐adapted therapy.

Impact of Pretransplant Therapy and Depth of Disease Response before Autologous Transplantation for Multiple Myeloma

Patients with multiple myeloma (MM) who are eligible for autologous stem cell transplantation (ASCT) typically receive a finite period of initial therapy before ASCT. It is not clear if patients with suboptimal (less than a partial) response to initial therapy benefit from additional alternative therapy with intent to maximize pretransplant response. We identified 539 patients with MM who had an ASCT after having achieved less than a partial response (PR) to first-line induction chemotherapy between 1995 and 2010. These patients were then divided into 2 groups: those who received additional salvage chemotherapy before ASCT (n = 324) and those who had no additional salvage chemotherapy immediately before ASCT (n = 215). Additional pretransplant chemotherapy resulted in deepening responses in 68% (complete response in 8% and PR in 60%). On multivariate analysis there was no impact of pretransplant salvage chemotherapy on treatment-related mortality, risk for relapse, progression-free survival, or overall survival. In conclusion, for patients achieving less than a PR to initial induction therapy, including with novel agent combinations, additional pre-ASCT salvage chemotherapy improved the depth of response and pre-ASCT disease status but was not associated with survival benefit.