Fatal Neurotoxicity Research Articles

IMMU-18. TARGETING GD2 IN THALAMIC DIFFUSE MIDLINE GLIOMA WITH TRANSIENT MRNA CAR T CELLS

Abstract Chimeric antigen receptor (CAR) T cells targeting the disialoganglioside GD2 have shown promise as a therapeutic for diffuse midline glioma (DMG). However, thalamic tumors have been excluded from clinical trials due to prior studies showing neurotoxicity and fatality when using virally transduced CAR T cells. We hypothesized repeated dosing of transient GD2-directed mRNA CAR T cells could be employed for safe and effective treatment of thalamic DMG. GD2-directed CAR T cells were created using mRNA and first tested against the murine thalamic DMG xenograft 7316-6349 via locoregional delivery with an indwelling catheter for repeated dosing. The previously reported fatal neurotoxicity observed in mice using lentiviral CAR T cells could be recapitulated with aggressive dosing. Four doses of 5x106 mRNA CAR T cells delivered intratumorally twice a week resulted in median overall survival of 9 days for GD2-treated mice compared to >30 days for CD19-treated controls (p<0.01). This toxicity could be avoided by decreasing the dose and timing of infusions to 2x106 mRNA CAR T cells delivered once weekly. Bioluminescent imaging showed regression of tumor in GD2-treated mice compared to CD19-treated controls (radiance fold change -3x106 versus +20x106 p/sec/cm2/sr, p<0.01). To assess survival before the onset of graft-versus-host disease, we utilized the aggressive hypermutant thalamic DMG model 7316-3058, treating with 2x106 CAR T cells intratumorally once weekly. Overall survival was significantly prolonged for 7316-3058 mice treated with GD2-directed mRNA CAR T cells compared to CD19-directed controls (median OS 48 days versus 33 days, p=0.03). Notably, non-tumor bearing mice treated with GD2-directed CAR T cells developed fatal neurotoxicity within 14 days, suggesting a very narrow therapeutic window in the brain. These data highlight the utility of titratable mRNA-based CAR T cell therapy for CNS tumors and establish GD2-directed mRNA CAR T cells as a safe and effective method for treating thalamic DMG.

IMMU-22. Safely targeting GD2 in thalamic diffuse midline glioma with mRNA CAR T cells

Chimeric antigen receptor (CAR) T cells targeting the disialoganglioside GD2 have shown promise as a therapeutic for diffuse midline glioma (DMG). However, prior studies raised significant concerns of neurotoxicity and fatality when using virally transduced CAR T cells against midline thalamic tumors. Building upon our prior work optimizing mRNA for use in CAR T cells (Hum Gen Ther, 2019), we hypothesized repeated dosing of transient GD2-directed mRNA CAR T cells could be employed for safe and effective treatment of thalamic DMG. GD2-directed CAR T cells were created using mRNA encoding the 14G2a single chain variable fragment paired with 41BB and CD3-zeta co-stimulatory domains and transfected into human T cells. CAR T cells were tested against the murine thalamic DMG xenograft 7316-6349 via locoregional delivery with an indwelling infusion catheter for repeated dosing. The previously reported fatal neurotoxicity observed in mice using lentiviral CAR T cells could be recapitulated with aggressive dosing. Four doses of 5 x 106 mRNA CAR T cells delivered intratumorally twice a week resulted in median overall survival of 9 days for GD2-treated mice compared to >30 days for CD19-treated controls (p<0.01). This toxicity could be avoided by decreasing the dose and timing of infusions to 2 x 106 mRNA CAR T cells delivered once weekly. Bioluminescent imaging showed regression of tumor in GD2-treated mice compared to CD19-treated controls (radiance fold change -3 x106 versus +20x106 p/sec/cm2/sr, p<0.01). Notably, non-tumor bearing mice treated with GD2-directed CAR T cells quickly developed fatal neurotoxicity within 14 days, suggesting on-target/off-tumor effect of the CAR T cells and a very narrow therapeutic window in the brain. These data highlight the utility of titratable mRNA-based CAR T cell therapy for CNS tumors and establish GD2-directed mRNA CAR T cells as a safe and effective method for treating thalamic DMG.

Glycyrrhiza uralensis Fisch. and its active components mitigate Semen Strychni-induced neurotoxicity through regulating high mobility group box 1 (HMGB1) translocation

Semen Strychni has long been used for the treatment of rheumatoid arthritis, facioplegia and myasthenia gravis due to its anti-inflammation and anti-nociceptive properties in China. However, the fatal neurotoxicity of Semen Strychni has limited its wider clinical application. To investigate the acute toxicity induced by Semen Strychni and the detoxification of liquorice, we evaluated inflammation, oxidative stress and the translocation of high mobility group box 1 (HMGB1) in rats. As a result, there were obvious oxidative stress and inflammation in hippocampus after the Semen Strychni extracts (STR) treatment in rats. Liquorice extracts (LE) and its three active monomers – glycyrrhizic acid (GA), liquiritigenin (LIQ), isoliquiritigenin (ISL) showed the potential for mitigating STR-induced neurotoxicity. HMGB1 levels in cytoplasm and serum and the levels of two downstream receptors RAGE and TLR4 were significantly increased after STR treatment. Through using LE and the monomers, the nucleocytoplasmic transport and release of HMGB1 were inhibited. In addition, the binding between HMGB1 and TLR4 was weakened in detoxification groups comparing with the STR group. Taken together, these findings indicated that liquorice and its active components alleviated acute neurotoxicity induced by Semen Strychni partly via HMGB1-related pathway.

Varespladib (LY315920) rescued mice from fatal neurotoxicity caused by venoms of five major Asiatic kraits (Bungarus spp.) in an experimental envenoming and rescue model

The venoms of Asiatic kraits (Bungarus spp.) contain various neurotoxic phospholipases A2 (beta-bungarotoxins) which can irreversibly damage motor nerve terminals, resulting in rapidly fatal suffocation by respiratory muscle paralysis or oral airway obstruction. Hence, there is a need of adjunct therapy at the pre-hospital stage to prevent or delay the onset of neurotoxicity, so that antivenom can be given within golden hour before the envenoming becomes antivenom-resistant. This study investigated the efficacy of varespladib, a small molecule PLA2 (phospholipase A2) inhibitor, given as a bolus subcutaneously upon the onset of krait venom-induced paralysis in a mouse experimental envenoming and rescue model, where the severity of neurotoxicity was scored and the survival rate was monitored over 24 h. Varespladib at 10 mg/kg effectively alleviated the neurotoxicity of Bungarus sindanus, Bungarus multicinctus and Bungarus fasciatus venoms, and rescued all mice from venom-induced lethality (100% survival). Varespladib at this dose, however, only partially reduced the neurotoxicity of Bungarus caeruleus and Bungarus candidus venoms, while all challenged mice were dead by 23 h (B. caeruleus) and 12 h (B. candidus). An increased dose of varespladib at 20 mg/kg markedly abated the venom neurotoxicity past 8 h of envenoming, and protected the mice from venom lethality (B. caeruleus: 75% survival; B. candidus: 100% survival). The finding is consistent with previous studies which demonstrated varespladib's inhibitory effect against some snake venoms. The findings suggest varespladib could be repurposed as an emergency drug for prevention or rescue (if given early enough) from the acute, neurotoxic envenoming syndromes caused by various major krait species in Asia.

Preclinical assessment of the efficacy and specificity of GD2-B7H3 SynNotch CAR-T in metastatic neuroblastoma

The ability to utilize preclinical models to predict the clinical toxicity of chimeric antigen receptor (CAR) T cells in solid tumors is tenuous, thereby necessitating the development and evaluation of gated systems. Here we found that murine GD2 CAR-T cells, specific for the tumor-associated antigen GD2, induce fatal neurotoxicity in a costimulatory domain-dependent manner. Meanwhile, human B7H3 CAR-T cells exhibit efficacy in preclinical models of neuroblastoma. Seeking a better CAR, we generated a SynNotch gated CAR-T, GD2-B7H3, recognizing GD2 as the gate and B7H3 as the target. GD2-B7H3 CAR-T cells control the growth of neuroblastoma in vitro and in metastatic xenograft mouse models, with high specificity and efficacy. These improvements come partly from the better metabolic fitness of GD2-B7H3 CAR-T cells, as evidenced by their naïve T-like post-cytotoxicity oxidative metabolism and lower exhaustion profile.

Chimeric Antigen Receptor T Cell-Related Neurotoxicity: Mechanisms, Clinical Presentation, and Approach to Treatment.

Chimeric antigen receptor T cell (CAR-T) adoptive cell therapy is an effective treatment for patients with refractory B cell malignancies. As its use has grown, there has been an increase in the incidence of a serious, potentially fatal neurotoxicity known as immune effector cell-associated neurotoxicity syndrome (ICANS). This review discusses the clinical manifestations of this neurotoxicity syndrome, current grading systems, management strategies, and proposed biologic mechanisms leading to neurotoxicity. Current research suggests that patients with a higher disease burden and higher CAR-T cell doses are positively associated with the development of ICANS, as are elevated serum levels of proinflammatory cytokines and the presence of cytokine release syndrome (CRS). While patterns observed on neuroimaging and electroencephalogram (EEG) are non-specific for the diagnosis of ICANS, each modality may provide helpful clinical information such as the detection of cerebral edema, the most serious of associated symptoms. Anti-epileptic medications and corticosteroids may ameliorate the symptoms of ICANS. The mechanism for ICANS is currently unknown; however, systemic inflammation and cytokine production triggering a cascade of endothelial activation and BBB disruption likely contribute. With limited treatment options available, further clinical research into the precise mechanism and treatment is urgently needed as the use of CAR-T and other adoptive cell therapies continues to grow.

Abstract 2317: Dose control of CAR-like T cell activity through post-translational covalent loading of ligands to a universal immune receptor

Abstract The use of chimeric antigen receptor (CAR) T cells for the treatment of CD19+ hematologic malignancies has shown great success in the clinic, leading to FDA approval for various forms of leukemia. However, their broad use is limited since a CAR targets a single tumor associated antigen (TAA), which is not effective against tumors with heterogeneous TAA expression or emerging antigen loss variants. Further, stably engineered CAR T cells can continually proliferate and activate in response to antigen, potentially causing fatal cytokine release syndrome (CRS) or neurotoxicity without a means of quantifiable control. These toxicities and limitations have prompted the development of an alternative CAR format called universal immune receptors (UIRs). UIRs allow multi-antigen targeting with a single receptor to prevent and address antigen escape due to heterogeneity and loss, targeting ligand (TL) dose dependent control of CAR activity, control of CRS, and normal B cell reconstitution following CD19 targeting. However, all current formats rely on non-covalent engagement of the TL which can lead to issues with sub-optimal affinity and specificity. We have developed a next generation UIR system that allows for post-translational covalent attachment of TLs to the receptor. This is accomplished through the use of the SpyCatcher-SpyTag protein system. When combined at physiologic pH and temperature, these two proteins form a covalent bond. The SpyCatcher immune receptor (Spy-IR) contains the SpyCatcher protein as its extracellular domain attached to standard second-generation CAR intracellular signaling domains. Addition of TAA-specific TLs labeled with SpyTag allows for on-demand CAR generation through covalent bond formation between the TL and the receptor at the T cell surface. Here, primary human T cells expressing the Spy-IR are quantitatively loaded with targeting ligands, allowing for dose-titratable control of redirected T cell effector function and tumor cell lysis. Even as a single cell product, Spy-IR T cells can recognize an array of tumor antigens, (e.g. Her2, EGFR, EpCAM, and CD20) either simultaneously or sequentially to address the evolving tumor antigen landscape. Further, dose-dependent arming of Spy-IR T cells with TL allows for controlled immune recognition of cells expressing either high or low level antigen to distinguish between tumor and normal cells, thus providing an additional means of safety. Finally, preclinical efficacy of the Spy-IR system was achieved in NSG mouse models of aggressive solid tumor and intraperitoneal disease. In conclusion, the SpyCatcher immune receptor is the first UIR designed for post-translational covalent attachment of targeting ligands that permits timed “on demand” redirection of T cells against a diverse array of antigens, to address limitations of safety and antigen diversity by conventional CAR T cell therapy. Citation Format: Nicholas G. Minutolo, Prannda Sharma, Andrew Tsourkas, Daniel J. Powell. Dose control of CAR-like T cell activity through post-translational covalent loading of ligands to a universal immune receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2317.

Multivariable Modeling of Disease and Treatment Characteristics of Adults with B-ALL in MRD-Negative CR after CD19 CAR-T Cells Identifies Factors Impacting Disease-Free Survival

Multivariable Modeling of Disease and Treatment Characteristics of Adults with B-ALL in MRD-Negative CR after CD19 CAR-T Cells Identifies Factors Impacting Disease-Free Survival

Concurrent 5-fluorouracil and carboplatin for the treatment of canine carcinomas.

5-Fluorouracil (5-FU) is used in combination chemotherapy protocols for human head and neck cancer and other epithelial neoplasms. However, a paucity of literature describing use of this drug in veterinary oncology exists, likely due to previous reports of fatal neurotoxicity in both dogs and cats, mainly due to ingestion of human creams. The primary aim of this retrospective study was to report the safety of concurrent 5-FU and carboplatin in canine carcinomas. Secondarily, we aimed to look at the efficacy of the combination using overall response rate in treated dogs. Medical records were searched from 2007 to 2017 for dogs treated with both agents; 24 dogs met inclusion criteria. Carboplatin dosages ranged from 180 to 250 mg/m2 (median 200 mg/m2 ); 5-FU dosage was 150 mg/m2 . Fourteen dogs had myelosuppression, ranging from Grade I to asymptomatic Grade IV; thrombocytopenia was more common than neutropenia. Gastrointestinal upset was uncommon, with only seven dogs having Grade I or II nausea, vomiting or diarrhoea. No cases were hospitalized for any of the above toxicities. One dog had an episode of ataxia, which could not be differentiated between otitis and 5-FU neurotoxicity. This protocol is well tolerated. Response rate in the gross disease setting was 43% (three complete responses, three partial responses). Prospective analysis of this combination protocol, and potentially 5-FU with other platinum agents, is warranted in the treatment of canine carcinomas.

Human interleukin-10 delivered intrathecally by self-complementary adeno-associated virus 8 induces xenogeneic transgene immunity without clinical neurotoxicity in swine.

Intrathecal interleukin (IL)-10 delivered by plasmid or viral gene vectors has been proposed for clinical testing because it is effective for chronic pain in rodents, is a potential therapeutic for various human diseases, and was found to be nontoxic in dogs, when the human IL-10 ortholog was tested. However, recent studies in swine testing porcine IL-10 demonstrated fatal neurotoxicity. The present study aimed to deliver vector-encoded human IL-10 in swine, measure expression of the transgene in cerebrospinal fluid and monitor animals for signs of neurotoxicity. Human IL-10 levels peaked 2weeks after vector administration followed by a rapid decline that occurred concomitant with the emergence of anti-human IL-10 antibodies in the cerebrospinal fluid and serum. Animals remained neurologically healthy throughout the study period. The findings of the present study suggest that swine are not idiosyncratically sensitive to intrathecal IL-10 because, recapitulating previous reports in dogs, they suffered no clinical neurotoxicity from the human ortholog. These results strongly infer that toxicity of intrathecal IL-10 in large animal models was previously overlooked because of a species mismatch between transgene and host. The present study further suggests that swine were protected from interleukin-10 by a humoral immune response against the xenogeneic cytokine. Future safety studies of IL-10 or related therapeutics may require syngeneic large animal models.

Prophylactic Neuroprotection of Total Glucosides of Paeoniae Radix Alba against Semen Strychni-Induced Neurotoxicity in Rats: Suppressing Oxidative Stress and Reducing the Absorption of Toxic Components.

Strychnos alkaloids (SAs) are the main toxic constituents in Semen Strychni, a traditional Chinese medicine, which is known for its fatal neurotoxicity. Hence, the present study was carried out to evaluate the neurotoxicity induced by SAs and the pre-protective effects of the total glucosides of Paeoniae Radix Alba (TGP). An SA brain damage model was firstly established. The neurotoxicity induced by SAs and the pre-protective effects of TGP were confirmed by physical and behavioral testing, biochemical assay, and histological examination. Then, a liquid chromatography-tandem mass spectrometry method was developed and validated to investigate the time-course change and distribution of strychnine and brucine (two main SAs) in the brain after oral SA administration with or without TGP pretreatment. Biochemical analysis results indicated that TGP could ameliorate the oxidative stress status caused by SAs. Time-course change and distribution studies demonstrated that strychnine and brucine were rapidly absorbed into the brain, peaked early at 0.5 h, and were mainly located in the hippocampus and cerebellum. TGP showed a pre-protective effect against neurotoxicity by reducing the absorption of toxic alkaloids into the brain. These findings could provide beneficial information in facilitating future studies of Semen Strychni neurotoxicity and developing herbal medicines to alleviate neurotoxicity in the clinic.

High-Affinity GD2-Specific CAR T Cells Induce Fatal Encephalitis in a Preclinical Neuroblastoma Model.

The GD2 ganglioside, which is abundant on the surface of neuroblastoma cells, is targeted by an FDA-approved therapeutic monoclonal antibody and is an attractive tumor-associated antigen for cellular immunotherapy. Chimeric antigen receptor (CAR)-modified T cells can have potent antitumor activity in B-cell malignancies, and trials to harness this cytolytic activity toward GD2 in neuroblastoma are under way. In an effort to enhance the antitumor activity of CAR T cells that target GD2, we generated variant CAR constructs predicted to improve the stability and the affinity of the GD2-binding, 14G2a-based, single-chain variable fragment (scFv) of the CAR and compared their properties in vivo We included the E101K mutation of GD2 scFv (GD2-E101K) that has enhanced antitumor activity against a GD2+ human neuroblastoma xenograft in vivo However, this enhanced antitumor efficacy in vivo was concomitantly associated with lethal central nervous system (CNS) toxicity comprised of extensive CAR T-cell infiltration and proliferation within the brain and neuronal destruction. The encephalitis was localized to the cerebellum and basal regions of the brain that display low amounts of GD2. Our results highlight the challenges associated with target antigens that exhibit shared expression on critical normal tissues. Despite the success of GD2-specific antibody therapies in the treatment of neuroblastoma, the fatal neurotoxicity of GD2-specific CAR T-cell therapy observed in our studies suggests that GD2 may be a difficult target antigen for CAR T-cell therapy without additional strategies that can control CAR T-cell function within the CNS. Cancer Immunol Res; 6(1); 36-46. ©2017 AACR.

Endothelial Activation and Blood-Brain Barrier Disruption in Neurotoxicity after Adoptive Immunotherapy with CD19 CAR-T Cells.

Lymphodepletion chemotherapy followed by infusion of CD19-targeted chimeric antigen receptor-modified T (CAR-T) cells can be complicated by neurologic adverse events (AE) in patients with refractory B-cell malignancies. In 133 adults treated with CD19 CAR-T cells, we found that acute lymphoblastic leukemia, high CD19+ cells in bone marrow, high CAR-T cell dose, cytokine release syndrome, and preexisting neurologic comorbidities were associated with increased risk of neurologic AEs. Patients with severe neurotoxicity demonstrated evidence of endothelial activation, including disseminated intravascular coagulation, capillary leak, and increased blood-brain barrier (BBB) permeability. The permeable BBB failed to protect the cerebrospinal fluid from high concentrations of systemic cytokines, including IFNγ, which induced brain vascular pericyte stress and their secretion of endothelium-activating cytokines. Endothelial activation and multifocal vascular disruption were found in the brain of a patient with fatal neurotoxicity. Biomarkers of endothelial activation were higher before treatment in patients who subsequently developed grade ≥4 neurotoxicity.Significance: We provide a detailed clinical, radiologic, and pathologic characterization of neurotoxicity after CD19 CAR-T cells, and identify risk factors for neurotoxicity. We show endothelial dysfunction and increased BBB permeability in neurotoxicity and find that patients with evidence of endothelial activation before lymphodepletion may be at increased risk of neurotoxicity. Cancer Discov; 7(12); 1404-19. ©2017 AACR.See related commentary by Mackall and Miklos, p. 1371This article is highlighted in the In This Issue feature, p. 1355.

Enhancing the Oncolytic Activity of CD133-Targeted Measles Virus: Receptor Extension or Chimerism with Vesicular Stomatitis Virus Are Most Effective.

Therapy resistance and tumor recurrence are often linked to a small refractory and highly tumorigenic subpopulation of neoplastic cells, known as cancer stem cells (CSCs). A putative marker of CSCs is CD133 (prominin-1). We have previously described a CD133-targeted oncolytic measles virus (MV-CD133) as a promising approach to specifically eliminate CD133-positive tumor cells. Selectivity was introduced at the level of cell entry by an engineered MV hemagglutinin (H). The H protein was blinded for its native receptors and displayed a CD133-specific single-chain antibody fragment (scFv) as targeting domain. Interestingly, MV-CD133 was more active in killing CD133-positive tumors than the unmodified MV-NSe despite being highly selective for its target cells. To further enhance the antitumoral activity of MV-CD133, we here pursued arming technologies, receptor extension, and chimeras between MV-CD133 and vesicular stomatitis virus (VSV). All newly generated viruses including VSV-CD133 were highly selective in eliminating CD133-positive cells. MV-CD46/CD133 killed in addition CD133-negative cells being positive for the MV receptors. In an orthotopic glioma model, MV-CD46/CD133 and MVSCD-CD133, which encodes the super cytosine deaminase, were most effective. Notably, VSV-CD133 caused fatal neurotoxicity in this tumor model. Use of CD133 as receptor could be excluded as being causative. In a subcutaneous tumor model of hepatocellular cancer, VSV-CD133 revealed the most potent oncolytic activity and also significantly prolonged survival of the mice when injected intravenously. Compared to MV-CD133, VSV-CD133 infected a more than 104-fold larger area of the tumor within the same time period. Our data not only suggest new concepts and approaches toward enhancing the oncolytic activity of CD133-targeted oncolytic viruses but also raise awareness about careful toxicity testing of novel virus types.

Open label, non-randomized, multi-cohort pilot study of genetically engineered NY-ESO-1 specific NY-ESO-1c259t in HLA-A2+ patients with synovial sarcoma (NCT01343043).

3000 Background: NY-ESO-1 is expressed in ~70% of synovial sarcomas (SS). NY-ESO-1c259T cells recognizing an NY-ESO-1 derived peptide complexed with HLA-A*02 are being studied in SS. Methods: Eligible patients (pt) are HLA-A*02:01, 02:05 or 02:06, with unresectable, metastatic or recurrent SS expressing NY-ESO-1. Primary endpoint of ORR (CR+PR) is evaluated in high (≥ 50% tumor cells express 2+/3+) and low (≥ 1+ in ≥ 1% cells, not exceeding 2+/3+ in ≥ 50% cells) NY-ESO-1 expressers with different lymphodepleting regimens. Secondary endpoints are safety, DOR, PFS, OS, and gene-marked cell persistence. Lymphocytes are obtained by leukapheresis, isolated, activated, transduced to express NY-ESO-1c259T, and expanded. Target dose is 1–6 × 109cells. Disease is assessed at wk 4, 8 and 12 post-T-cell infusion, and then every 3 months. Results: 34 pt have been enrolled with 24 treated. 50% are male; median age is 30 yr (range 15 – 73). 12/15 pt in cohort 1 were treated. ORR was 50% (1 CR; 5 PR). Time to response was 6 wk (range 4-9) and median DOR 31 wk (range 13-72). Cohort 3 was closed due to only 1 PR out of 5 pt. Evaluation is ongoing in cohorts 2 (6 enrolled; 5 treated) and 4 (8 enrolled; 2 treated) as of 1/9/17. The most common AE are leukopenia (96%), nausea and pyrexia (88%), neutropenia (88%), lymphopenia (83%), anemia (79%), and thrombocytopenia (79%). 11 events of CRS were reported (3 G3; 1 G4), with no events of seizure, cerebral edema or fatal neurotoxicity; all resolved with supportive therapy. One fatal SAE (bone marrow failure) occurred in cohort 2; investigations have not identified a mechanism by which NY-ESO-1c259T may have caused this event. Conclusions: NY-ESO-1c259T has promising efficacy and acceptable safety. CRS is not associated with severe neurotoxicity and appears manageable with appropriate supportive care. Cohort 3 data indicate that Flu may be important for efficacy. Efficacy and safety data will be further evaluated and presented. Clinical trial information: NCT01343043. [Table: see text]

ZW38, a Novel Azymetric Bispecific CD19-Directed CD3 T Cell Engager Antibody Drug Conjugate with Controlled T Cell Activation and Improved B Cell Cytotoxicity

Although blinatumomab is an approved treatment for Philadelphia chromosome negative relapsed or refractory (r/r) precursor-B cell ALL and is under development for r/r B cell NHL, blinatumomab has several limitations impacting fuller therapeutic utility. For instance, blinatumomab therapy requires continuous infusion due to its rapid clearance owing to its size and lack of an antibody Fc and has been associated with potentially life-threatening CNS toxicities and cytokine release syndrome (Viardot A et al, Blood 2016; Goebeler ME et al, J Clin Oncol 2016; Topp et al, Lancet Oncol 2015). In addition, blinatumomab treatment is associated with higher incidence of relapse in patients with high disease burden, and its T cell redirected killing is limited by T cell immunosuppression (e.g. PD-1/PD-L1 up-regulation [Köhnke et al, J Hematol & Oncol 2015]; Treg suppression [Duell et al, ASH abstract 2014]). ZW38 is designed to address each of these limitations and represents a best in class CD19-directed CD3 T cell engager and a novel class of bispecific antibody drug conjugate (ADC).ZW38 contains an Azymetric IgG1-like Fc that carries mutations in the CH2 domain preventing FcgR dependent ADCC and ADCP and exhibits typical IgG1-like PK in rodent studies. Transient expression in mammalian CHO cells demonstrate ZW38 can be expressed at a titre of hundreds mg/L and can be purified using conventional IgG antibody methods and resins with typical IgG step purification yields and high heterodimer purity. Additionally, ZW38 has been conjugated to a microtubule inhibitor that lacks bystander killing.ZW38 antibody paratopes have been engineered to favor T cell-B cell functional engagement and selective target B cell cytotoxicity. In vitro studies demonstrated that ZW38 binds to human CD19+ B cells with >30-fold higher affinity than to human CD3+ T cells. Similar to blinatumomab, ZW38 can redirect the killing of target cancer B cells via T cell subtypes from human PBMC and its cytotoxicity is target B cell dependent. At concentrations that result in efficacious B cell depletion, ZW38 does not overly activate T cells. ZW38 is specifically engineered to induce more ’controlled’ T cell activation than blinatumomab while still mediating sufficient T cell redirected target B cell depletion. ZW38 mediates T cell activation, cytokine release, and proliferation at nanomolar potency. By design, the sufficient, lower cytokine levels necessary for B cell killing may reduce the risk of cytokine release syndrome and T cell anergy. ZW38 exhibited potent growth inhibition in a panel of different B cell ALL and NHL cancer lines including but not limited to: G2, Nalm-6, RS4-11, Daudi, SUDHL-4 and SUDHL-6. In comparison to blinatumomab, ZW38 exhibits superior target B cell depletion in in vitro co-cultures of Raji lymphoma cells and human PBMC (ZW38 depletes > 90% B cells; blinatumomab depletes 20-90% B cells depending on the PBMC donor). In addition, ZW38 was effective in killing target B cells in PBMC cultures in which PD-1 has been up-regulated, which were resistant to blinatumomab killing in this assay. The dual mechanisms of action of ZW38, redirected T cell cytotoxicity and ADC cytotoxicity, may prolong and/or boost response rates, lower the incidence of relapse, and reduce the likelihood of acquired resistance while its ’controlled’ T cell activation profile may reduce the risk of life-threatening and potentially fatal neurotoxicity and CRS. DisclosuresNg:Zymeworks: Employment, Patents & Royalties. Spreter:Zymeworks: Employment, Patents & Royalties. Davies:Zymeworks Biopharmaceuticals: Employment. Wickman:Zymeworks: Employment.

Pediatric Scorpion Envenomation in the United States

In the Southwestern United States, the venom of the scorpion Centruroides sculpturatus (common name bark scorpion) can cause serious and potentially fatal neurotoxicity, with young children most vulnerable to its effects. Historically, advances in the quality of supportive care have made significant improvements in morbidity and mortality. In recent years, the development of effective antivenom therapies has changed the landscape of caring for these patients. This article reviews the background, pathophysiology, diagnosis, and treatment options for C. sculpturatus envenomation. Recent advances in immunotherapy and subsequent implications for pediatric emergency care providers are discussed.

Fatal Neurotoxicity in a Patient With Down Syndrome Treated With Chemotherapy, Irradiation, Stem Cell Transplant, and Clofarabine

Clofarabine is an effective therapy of pediatric patients with relapsed acute lymphoblastic leukemia (ALL). We present a child with Down syndrome who had received previous chemotherapy, cranial radiation, and a stem cell transplant with total body irradiation for her acute lymphoblastic leukemia. She subsequently relapsed and was treated with clofarabine. After her third course, she had a stroke that was felt to be secondary to dehydration and radiation vasculitis. After her subsequent course of clofarabine, she developed fatal neurotoxicity.

Safety Studies on Intrahepatic or Intratumoral Injection of Oncolytic Vesicular Stomatitis Virus Expressing Interferon-β in Rodents and Nonhuman Primates

Toxicology studies were performed in rats and rhesus macaques to establish a safe starting dose for intratumoral injection of an oncolytic vesicular stomatitis virus expressing human interferon-beta (VSV-hIFNbeta) in patients with hepatocellular carcinoma (HCC). No adverse events were observed after administration of 7.59 x 10(9) TCID(50) (50% tissue culture infective dose) of VSV-hIFNbeta into the left lateral hepatic lobe of Harlan Sprague Dawley rats. Plasma alanine aminotransferase and alkaline phosphatase levels increased and platelet counts decreased in the virus-treated animals on days 1 and 2 but returned to pretreatment levels by day 4. VSV-hIFNbeta was also injected into normal livers or an intrahepatic McA-RH7777 HCC xenograft established in Buffalo rats. Buffalo rats were more sensitive to neurotoxic effects of VSV; the no observable adverse event level (NOAEL) of VSV-hIFNbeta in Buffalo rats was 10(7) TCID(50). Higher doses were associated with fatal neurotoxicity and infectious virus was recovered from tumor and brain. Compared with VSV-hIFNbeta, toxicity of VSV-rIFNbeta (recombinant VSV expressing rat IFN-beta) was greatly diminished in Buffalo rats (NOAEL, >10(10) TCID(50)). Two groups of two adult male rhesus macaques received 10(9) or 10(10) TCID(50) of VSV-hIFNbeta injected directly into the left hepatic lobe under computed tomographic guidance. No neurological signs were observed at any time point. No abnormalities (hematology, clinical chemistry, body weights, behavior) were seen and all macaques developed neutralizing anti-VSV antibodies. Plasma interleukin-6, tumor necrosis factor-alpha, and hIFN-beta remained below detection levels by ELISA. On the basis of these studies, we will be proposing a cautious approach to dose escalation in a phase I clinical trial among patients with HCC.

Neurotoxicity upon infusion of dimethylsulfoxide‐cryopreserved peripheral blood stem cells in patients with and without pre‐existing cerebral disease

Toxicity related to the infusion of dimethylsulfoxide (DMSO)-cryopreserved peripheral blood stem cells (DMSO-PBSC) mainly comprises cardiovascular events. Fatal neurotoxicity has been reported in a few cases. DMSO represents the putative causative agent of such rare toxicities and elaborate strategies to replace DMSO would benefit from the identification of predisposing factors for DMSO-related toxicities. Here, we report on DMSO-related neurotoxicity in a series of patients (n = 51) receiving DMSO-PBSC. The analyzed patient-series included eight patients with pre-existing cerebral disease, partially with a history of epileptic seizures. Neurotoxicity was observed in only one patient who suffered from a generalized tonic seizure upon infusion of DMSO-PBSC and for which the clinical course is reported herein. No neurotoxicity was observed in the group of patients with pre-existing neurological disease. Furthermore, no neurotoxicity was observed in patients who received particularly large volumes of DMSO. In all patients, mild non-neurological side effects occurred but besides the reported seizure no other severe adverse events were observed upon PBSC-infusion. To our knowledge, this is the first report addressing the identification of predisposing factors for DMSO-related neurotoxicty. We conclude that infusion of DMSO-PBSC can be performed safely in patients with pre-existing cerebral disease despite the rare occurrence of severe neurotoxicity. Retrospective multicenter studies are warranted to identify patients who would benefit from elaborate methods of DMSO-replacement.