Synaptotagmin-11 regulates the functions of caveolae and responds to mechanical stimuli in astrocytes.

Caveolae play crucial roles in intracellular membrane trafficking and mechanosensation. In this study, we report that synaptotagmin-11 (Syt11), a synaptotagmin isoform associated with Parkinson's disease and schizophrenia, regulates both caveolae-mediated endocytosis and the caveolar response to mechanical stimuli in astrocytes. Syt11-knockout (KO) accelerated caveolae-mediated endocytosis. Interestingly, the caveolar structures on the cell surface were markedly fewer in the absence of Syt11. Caveolar disassembly in response to hypoosmotic stimuli and astrocyte swelling were both impaired in Syt11-KO astrocytes. Live imaging revealed that Syt11 left caveolar structures before cavin1 during hypoosmotic stress and returned earlier than cavin1 after isoosmotic recovery. Chronic hypoosmotic stress led to proteasome-mediated Syt11 degradation. In addition, Syt11-KO increased the turnover of cavin1 and EH domain-containing protein 2 (EHD2), accompanied by compromised membrane integrity, suggesting a mechanoprotective role of Syt11. Direct interactions between Syt11 and cavin1 and EHD2, but not caveolin-1, are found. Altogether, we propose that Syt11 stabilizes caveolar structures on the cell surface of astrocytes and regulates caveolar functions under physiological and pathological conditions through cavin1 and EHD2.

Synaptotagmin-11 inhibits cytokine secretion and phagocytosis in microglia.

Cytokine secretion and phagocytosis are key functions of activated microglia. However, the molecular mechanisms underlying their regulation in microglia remain largely unknown. Here, we report that synaptotagmin-11 (Syt11), a non-Ca2+ -binding Syt implicated in Parkinson disease and schizophrenia, inhibits cytokine secretion and phagocytosis in microglia. We found Syt11 expression in microglia in brain slices and primary microglia. Interestingly, Syt11-knockdown (KD) increased cytokine secretion and NO release in primary microglia both in the absence and presence of lipopolysaccharide. NF-κB was activated in untreated KD microglia together with enhanced synthesis of IL-6, TNF-α, IL-1ß, and iNOS. When the release capacity was assessed by the ratio of extracellular to intracellular levels, only the IL-6 and TNF-α secretion capacity was increased in Syt11-KD cells, suggesting that Syt11 specifically regulates conventional secretion. Consistently, Syt11 localized to the trans-Golgi network and recycling endosomes. In addition, Syt11 was recruited to phagosomes and its deficiency enhanced microglial phagocytosis. All the KD phenotypes were rescued by expression of an shRNA-resistant Syt11, while overexpression of Syt11 suppressed cytokine secretion and phagocytosis. Importantly, Syt11 also inhibited microglial phagocytosis of α-synuclein fibrils, supporting its association with Parkinson disease. Taken together, we propose that Syt11 suppresses microglial activation under both physiological and pathological conditions through the inhibition of cytokine secretion and phagocytosis.

CD62L- memory T cells enhance T-cell regeneration after allogeneic stem cell transplantation by eliminating host resistance in mice.

A major challenge in allogeneic hematopoietic cell transplantation is how to transfer T-cell immunity without causing graft-versus-host disease (GVHD). Effector memory T cells (CD62L(-)) are a cell subset that can potentially address this challenge because they do not induce GVHD. Here, we investigated how CD62L(-) T cells contributed to phenotypic and functional T-cell reconstitution after transplantation. On transfer into allogeneic recipients, CD62L(-) T cells were activated and expressed multiple cytokines and cytotoxic molecules. CD62L(-) T cells were able to deplete host radioresistant T cells and facilitate hematopoietic engraftment, resulting in enhanced de novo T-cell regeneration. Enhanced functional immune reconstitution was demonstrated in CD62L(-) T-cell recipients using a tumor and an influenza virus challenge model. Even though CD62L(-) T cells are able to respond to alloantigens and deplete host radioresistant immune cells in GVHD recipients, alloreactive CD62L(-) T cells lost the reactivity over time and were eventually tolerant to alloantigens as a result of prolonged antigen exposure, suggesting a mechanism by which CD62L(-) T cells were able to eliminate host resistance without causing GVHD. These data further highlight the unique characteristics of CD62L(-) T cells and their potential applications in clinical hematopoietic cell transplantation.

Breast cancer exploits neural signaling pathways for bone-to-meninges metastasis.

The molecular mechanisms that regulate breast cancer cell (BCC) metastasis and proliferation within the leptomeninges (LM) are poorly understood, which limits the development of effective therapies. In this work, we show that BCCs in mice can invade the LM by abluminal migration along blood vessels that connect vertebral or calvarial bone marrow and meninges, bypassing the blood-brain barrier. This process is dependent on BCC engagement with vascular basement membrane laminin through expression of the neuronal pathfinding molecule integrin α6. Once in the LM, BCCs colocalize with perivascular meningeal macrophages and induce their expression of the prosurvival neurotrophin glial-derived neurotrophic factor (GDNF). Intrathecal GDNF blockade, macrophage-specific GDNF ablation, or deletion of the GDNF receptor neural cell adhesion molecule (NCAM) from BCCs inhibits breast cancer growth within the LM. These data suggest integrin α6 and the GDNF signaling axis as new therapeutic targets against breast cancer LM metastasis.

Monoclonal antibody blockade of IL-2 receptor α during lymphopenia selectively depletes regulatory T cells in mice and humans.

Lymphodepletion augments adoptive cell transfer during antitumor immunotherapy, producing dramatic clinical responses in patients with malignant melanoma. We report that the lymphopenia induced by the chemotherapeutic agent temozolomide (TMZ) enhances vaccine-driven immune responses and significantly reduces malignant growth in an established model of murine tumorigenesis. Unexpectedly, despite the improved antitumor efficacy engendered by TMZ-induced lymphopenia, there was a treatment related increase in the frequency of immunosuppressive regulatory T cells (T(Regs); P = .0006). Monoclonal antibody (mAb)-mediated inhibition of the high-affinity IL-2 receptor α (IL-2Rα/CD25) during immunotherapy in normal mice depleted T(Regs) (73% reduction; P = .0154) but also abolished vaccine-induced immune responses. However, during lymphodepletion, IL-2Rα blockade decreased T(Regs) (93% reduction; P = .0001) without impairing effector T-cell responses, to augment therapeutic antitumor efficacy (66% reduction in tumor growth; P = .0024). Of clinical relevance, we also demonstrate that anti-IL-2Rα mAb administration during recovery from lymphodepletive TMZ in patients with glioblastoma reduced T(Reg) frequency (48% reduction; P = .0061) while permitting vaccine-stimulated antitumor effector cell expansion. To our knowledge, this is the first report of systemic antibody-mediated T(Reg) depletion during lymphopenia and the consequent synergistic enhancement of vaccine-driven cellular responses, as well as the first demonstration that anti-IL-2Rα mAbs function differentially in nonlymphopenic versus lymphopenic contexts.

CD8+ T cells maintain killing of MHC-I-negative tumor cells through the NKG2D-NKG2DL axis.

The accepted paradigm for both cellular and anti-tumor immunity relies upon tumor cell killing by CD8+ T cells recognizing cognate antigens presented in the context of target cell major histocompatibility complex (MHC) class I (MHC-I) molecules. Likewise, a classically described mechanism of tumor immune escape is tumor MHC-I downregulation. Here, we report that CD8+ T cells maintain the capacity to kill tumor cells that are entirely devoid of MHC-I expression. This capacity proves to be dependent instead on interactions between T cell natural killer group 2D (NKG2D) and tumor NKG2D ligands (NKG2DLs), the latter of which are highly expressed on MHC-loss variants. Necessarily, tumor cell killing in these instances is antigen independent, although prior T cell antigen-specific activation is required and can be furnished by myeloid cells or even neighboring MHC-replete tumor cells. In this manner, adaptive priming can beget innate killing. These mechanisms are active in vivo in mice as well as in vitro in human tumor systems and are obviated by NKG2D knockout or blockade. These studies challenge the long-advanced notion that downregulation of MHC-I is a viable means of tumor immune escape and instead identify the NKG2D-NKG2DL axis as a therapeutic target for enhancing T cell-dependent anti-tumor immunity against MHC-loss variants.

Pathogenic Mutations Differentially Regulate Cell-to-Cell Transmission of α-Synuclein.

Recent studies suggest that the cell-to-cell spread of pathological α-synuclein (α-syn) plays important roles in the development of Parkinson's disease (PD). PD patients who carry α-syn gene mutations often have an earlier onset and more severe clinical symptoms and pathology than sporadic PD cases who carry the wild-type (WT) α-syn gene. However, the molecular mechanism by which α-syn gene mutations promote PD remains unclear. Here, we hypothesized that pathogenic mutations facilitate the intercellular transfer and cytotoxicity of α-syn, favoring an early disease onset and faster progression. We investigated the effects of eight known pathogenic mutations in human α-syn (A18T, A29S, A30P, E46K, H50Q, G51D, A53E, and A53T) on its pathological transmission in terms of secretion, aggregation, intracellular level, cytotoxicity, seeding, and induction of neuroinflammation in SH-SY5Y neuroblastoma cells, cultured rat neurons, and microglia, and the rat substantia nigra pars compacta. We found that 2 of the 8 mutations (H50Q and A53T) significantly increased α-syn secretion while 6 mutations (A18T, A29S, A30P, G51D, A53E, and E46K) tended to enhance it. In vitroα-syn aggregation experiments showed that H50Q promoted while G51D delayed aggregation most strongly. Interestingly, 3 mutations (E46K, H50Q, and G51D) greatly increased the intracellular α-syn level when cultured cells were treated with preformed α-syn fibrils (PFFs) compared with the WT, while the other 5 had no effect. We also demonstrated that H50Q, G51D, and A53T PFFs, but not E46K PFFs, efficiently seeded in vivo and acutely induced neuroinflammation in rat substantia nigra pars compacta. Our data indicate that pathogenic mutations augment the prion-like spread of α-syn at different steps and blockade of this pathogenic propagation may serve as a promising therapeutic intervention for PD.

4-1BB Agonism Averts TIL Exhaustion and Licenses PD-1 Blockade in Glioblastoma and Other Intracranial Cancers.

PURPOSE: The success of checkpoint blockade against glioblastoma (GBM) has been disappointing. Anti-PD-1 strategies may be hampered by severe T-cell exhaustion. We sought to develop a strategy that might license new efficacy for checkpoint blockade in GBM. EXPERIMENTAL DESIGN: We characterized 4-1BB expression in tumor-infiltrating lymphocytes (TIL) from human GBM. We implanted murine tumor models including glioma (CT2A), melanoma (B16), breast (E0771), and lung carcinomas intracranially and subcutaneously, characterized 4-1BB expression, and tested checkpoint blockade strategies in vivo. RESULTS: Our data reveal that 4-1BB is frequently present on nonexhausted CD8+ TILs in human and murine GBM. In murine gliomas, 4-1BB agonism and PD-1 blockade demonstrate a synergistic survival benefit in a CD8+ T-cell-dependent manner. The combination decreases TIL exhaustion and improves TIL functionality. This strategy proves most successful against intracranial CT2A gliomas. Efficacy in all instances correlates with the levels of 4-1BB expression on CD8+ TILs, rather than with histology or with intracranial versus subcutaneous tumor location. Proffering 4-1BB expression to T cells licenses combination 4-1BB agonism and PD-1 blockade in models where TIL 4-1BB levels had previously been low and the treatment ineffective. CONCLUSIONS: Although poor T-cell activation and severe T-cell exhaustion appear to be limiting factors for checkpoint blockade in GBM, 4-1BB agonism obviates these limitations and produces long-term survival when combined with anti-PD-1 therapy. Furthermore, this combination therapy is limited by TIL 4-1BB expression, but not by the intracranial compartment, and therefore may be particularly well-suited to GBM.

Targeting PD-L1 Initiates Effective Antitumor Immunity in a Murine Model of Cushing Disease.

PURPOSE: Although pituitary adenoma is classified as benign, Cushing disease is associated with significant morbidity due to the numerous sequelae of elevated cortisol levels. Successful therapy for Cushing disease remains elusive due to high rates of treatment-refractory recurrence. The frequent emergence of lymphocytic hypophysitis following checkpoint blockade for other cancers, as well as the expression of PD-L1 on pituitary adenomas, suggest a role for immunotherapy. EXPERIMENTAL DESIGN: This study confirms PD-L1 expression on functioning pituitary adenomas and is the first to evaluate the efficacy of checkpoint blockade (anti-PD-L1) therapy in a preclinical model of Cushing disease. RESULTS: Herein, treatment with anti-PD-L1 was successful in reducing adrenocorticotropic hormone plasma levels, decreasing tumor growth, and increasing survival in our model. Furthermore, tumor-infiltrating T cells demonstrated a pattern of checkpoint expression similar to other checkpoint blockade-susceptible tumors. CONCLUSIONS: This suggests that immunotherapy, particularly blockade of the PD1/PD-L1 axis, may be a novel therapeutic option for refractory Cushing disease. Clinical investigation is encouraged.

Imbalance between excitatory and inhibitory amino acids at spinal level is associated with maintenance of persistent pain-related behaviors.

Although the postsynaptic events responsible for development of pathological pain have been intensively studied, the relative contribution of presynaptic neurotransmitters to the whole process remains less elucidated. In the present investigation, we sought to measure temporal changes in spinal release of both excitatory amino acids (EAAs, glutamate and aspartate) and inhibitory amino acids (IAAs, glycine, ?-aminobutyric acid and taurine) in response to peripheral inflammatory pain state. The results showed that following peripheral chemical insult induced by subcutaneous bee venom (BV) injection, there was an initial, parallel increase in spinal release of both EAAs and IAAs, however, the balance between them was gradually disrupted when pain persisted longer, with EAAs remaining at higher level but IAAs at a level below the baseline. Moreover, the EAAs-IAAs imbalance at the spinal level was dependent upon the ongoing activity from the peripheral injury site. Intrathecal blockade of ionotropic (NMDA and non-NMDA) and metabotropic (mGluRI, II, III) glutamate receptors, respectively, resulted in a differential inhibition of BV-induced different types of pain (persistent nociception vs. hyperalgesia, or thermal vs. mechanical hyperalgesia), implicating that spinal antagonism of any specific glutamate receptor subtype fails to block all types of pain-related behaviors. This result provides a new line of evidence emphasizing an importance of restoration of EAAs-IAAs balance at the spinal level to prevent persistence or chronicity of pain.

Phospholipase C and protein kinase A mediate bradykinin sensitization of TRPA1: a molecular mechanism of inflammatory pain.

Bradykinin is an inflammatory mediator that plays a pivotal role in pain and hyperalgesia in inflamed tissues by exciting and/or sensitizing nociceptors. TRPA1 is an important component of the transduction machinery through which environmental irritants and endogenous proalgesic agents depolarize nociceptors to elicit inflammatory pain. Here, using electrophysiological, immunocytochemical and behavioural analyses, we showed a functional interaction of these two inflammation-related molecules in both heterologous expressing systems and primary sensory neurons. We found that bradykinin increased the TRPA1 currents evoked by allyl isothiocyanate (AITC) or cinnamaldehyde in HEK293 cells expressing TRPA1 and bradykinin receptor 2 (B2R). This potentiation was inhibited by phospholipase C (PLC) inhibitor or protein kinase A (PKA) inhibitor, and mimicked by PLC or PKA activator. The functional interaction between B2R and TRPA1, as well as the modulation mechanism, was also observed in rat dorsal root ganglia neurons. In an occlusion experiment, the PLC activator could enhance AITC-induced TRPA1 current further even in saturated PKA-mediated potentiation, indicating the additive potentiating effects of the PLC and PKA pathways. These data for the first time indicate that a cAMP-PKA signalling is involved in the downstream from B2R in dorsal root ganglia neurons in addition to PLC. Finally, subcutaneous pre-injection of a sub-inflammatory dose of bradykinin into rat hind paw enhanced AITC-induced pain behaviours, which was consistent with the observations in vitro. Collectively, these results represent a novel mechanism through which bradykinin released in response to tissue inflammation might trigger the sensation of pain by TRPA1 activation.

Mediating roles of the vanilloid receptor TRPV1 in activation of rat primary afferent nociceptive neurons by formaldehyde.

The formalin test is a commonly used animal model of acute and tonic pain. However, the molecular targets of formaldehyde (FA, the main ingredient of the formalin solution) on primary nociceptor cells remain controversial. In this report, the effects of FA on electrophysiologically-identified primary nociceptor cells were evaluated in vitro and the roles of the vanilloid receptor TRPV1 in FA-produced activation of primary nociceptors were also examined at both cellular and behavioral levels. Of 92 acutely dissociated dorsal root ganglion (DRG) cells recorded by current patch-clamp technique, 34% were discharged by FA application with the mean onset latencies of the first action potential (AP) being (367.34+/-32.96) s. All the FA-sensitive cells were identified as nociceptor cells by their distinguishable features of AP including longer duration, existence of a hump (a shoulder or inflection) on the repolarizing phase, and longer after-hyperpolarization of APs. Co-application of capsazepine (CPZ), a competitive antagonist of TRPV1 receptors, could block FA-evoked firing with partial inhibition on the membrane depolarization of all cells tested. Of another 160 cells examined by confocal calcium imaging, 32% were shown to respond to FA with an intracellular Ca(2+) rise. Of 51 FA-sensitive cells, 67% were suppressed by CPZ, suggesting partial involvement of TRPV1 in mediation of the FA-evoked intracellular Ca(2+) rise. Under voltage-clamp mode, 41% of DRG cells were evoked to give rise to inward current with the remaining 59% being unchanged. In separate experiments on the other 56 FA-sensitive cells, concentration-dependent increase in the FA-evoked current amplitude was demonstrated. In comparison with controls, the FA-evoked inward current could be significantly suppressed by CPZ that was further enhanced by HC-030031, a TRPA1 selective antagonist. Finally, local effects of CPZ were confirmed in the formalin test and it was shown that the formalin-induced paw flinches were strongly suppressed by CPZ in phase 1 but with phase 2 being significantly suppressed only during 25-55 min. It is therefore concluded that FA can directly activate a subpopulation of primary nociceptor cells and the FA-induced AP discharges are likely to contribute mainly to phase 1, but not phase 2 of the formalin-induced nociception. The activation of primary nociceptor cells by FA is likely to be mediated, at least in part, through TRPV1 and/or TRPA1 receptors.

Erratum: Histological characteristics following a long-term nitrate-rich diet in miniature pigs with parotid atrophy.

[This corrects the article on p. 6225 in vol. 8, PMID: 26261499.].

Plasmonic gold nanostar-mediated photothermal immunotherapy for brain tumor ablation and immunologic memory.

Brain tumors present unique therapeutic challenges and they include glioblastoma (GBM) and metastases from cancers of other organs. Current treatment options are limited and include surgical resection, radiation therapy, laser interstitial thermal therapy and chemotherapy. Although much research has been done on the development of immune-based treatment platforms, only limited success has been demonstrated. Herein, we demonstrate a novel treatment of GBM through the use of plasmonic gold nanostars (GNS) as photothermal inducers for synergistic immuno photothermal nanotherapy (SYMPHONY), which combines treatments using gold nanostar and laser-induced photothermal therapy with checkpoint blockade immunotherapy. In the treatment of a murine flank tumor model with the CT-2A glioma cell line, SYMPHONY demonstrated the capability of producing long-term survivors that rejects rechallenge with cancer cells, heralding the successful emergence of immunologic memory. This study is the first to investigate the use of this novel therapy for the treatment of GBM in a murine model.

Author Correction: Sequestration of T cells in bone marrow in the setting of glioblastoma and other intracranial tumors.

In the version of this article originally published, the figure callout in this sentence was incorrect: "Furthermore, in S1P1-KI mice themselves, whereas PD-1 blockade was ineffectual as monotherapy, the effects of 4-1BB agonism and checkpoint blockade proved additive, with the combination prolonging median survival and producing a 50% long-term survival rate (Fig. 6f)." The callout should have been to Supplementary Fig. 6b. The error has been corrected in the PDF and HTML versions of the article.

Differential activation of p38 and extracellular signal-regulated kinase in spinal cord in a model of bee venom-induced inflammation and hyperalgesia.

BACKGROUND: Honeybee's sting on human skin can induce ongoing pain, hyperalgesia and inflammation. Injection of bee venom (BV) into the intraplantar surface of the rat hindpaw induces an early onset of spontaneous pain followed by a lasting thermal and mechanical hypersensitivity in the affected paw. The underlying mechanisms of BV-induced thermal and mechanical hypersensitivity are, however, poorly understood. In the present study, we investigated the role of mitogen-activated protein kinase (MAPK) in the generation of BV-induced pain hypersensitivity. RESULTS: We found that BV injection resulted in a quick activation of p38, predominantly in the L4/L5 spinal dorsal horn ipsilateral to the inflammation from 1 hr to 7 d post-injection. Phosphorylated p38 (p-p38) was expressed in both neurons and microglia, but not in astrocytes. Intrathecal administration of the p38 inhibitor, SB203580, prevented BV-induced thermal hypersensitivity from 1 hr to 3 d, but had no effect on mechanical hypersensitivity. Activated ERK1/2 was observed exclusively in neurons in the L4/L5 dorsal horn from 2 min to 1 d, peaking at 2 min after BV injection. Intrathecal administration of the MEK inhibitor, U0126, prevented both mechanical and thermal hypersensitivity from 1 hr to 2 d. p-ERK1/2 and p-p38 were expressed in neurons in distinct regions of the L4/L5 dorsal horn; p-ERK1/2 was mainly in lamina I, while p-p38 was mainly in lamina II of the dorsal horn. CONCLUSION: The results indicate that differential activation of p38 and ERK1/2 in the dorsal horn may contribute to the generation and development of BV-induced pain hypersensitivity by different mechanisms.

Flow Cytometric Identification of Tumor-Infiltrating Lymphocytes from Glioblastoma.

We describe an isolation method of tumor-infiltrating lymphocytes (TILs) from glioblastoma tumors for the purpose of analysis by flow cytometry. This protocol is unique from many others in that the use of a selective lymphocyte isolation procedure, such as a Ficoll or Percoll gradient, is not used. We find that staining of TILs and analysis by flow cytometry is not affected by the presence of heterogeneous populations, while other selective isolation procedures can significantly decrease lymphocyte yield from already rare populations.

T-Cell Exhaustion Signatures Vary with Tumor Type and Are Severe in Glioblastoma.

Purpose: T-cell dysfunction is a hallmark of glioblastoma (GBM). Although anergy and tolerance have been well characterized, T-cell exhaustion remains relatively unexplored. Exhaustion, characterized in part by the upregulation of multiple immune checkpoints, is a known contributor to failures amid immune checkpoint blockade, a strategy that has lacked success thus far in GBM. This study is among the first to examine, and credential as bona fide, exhaustion among T cells infiltrating human and murine GBM.Experimental Design: Tumor-infiltrating and peripheral blood lymphocytes (TILs and PBLs) were isolated from patients with GBM. Levels of exhaustion-associated inhibitory receptors and poststimulation levels of the cytokines IFNγ, TNFα, and IL2 were assessed by flow cytometry. T-cell receptor Vß chain expansion was also assessed in TILs and PBLs. Similar analysis was extended to TILs isolated from intracranial and subcutaneous immunocompetent murine models of glioma, breast, lung, and melanoma cancers.Results: Our data reveal that GBM elicits a particularly severe T-cell exhaustion signature among infiltrating T cells characterized by: (1) prominent upregulation of multiple immune checkpoints; (2) stereotyped T-cell transcriptional programs matching classical virus-induced exhaustion; and (3) notable T-cell hyporesponsiveness in tumor-specific T cells. Exhaustion signatures differ predictably with tumor identity, but remain stable across manipulated tumor locations.Conclusions: Distinct cancers possess similarly distinct mechanisms for exhausting T cells. The poor TIL function and severe exhaustion observed in GBM highlight the need to better understand this tumor-imposed mode of T-cell dysfunction in order to formulate effective immunotherapeutic strategies targeting GBM. Clin Cancer Res; 24(17); 4175-86. ©2018 AACRSee related commentary by Jackson and Lim, p. 4059.

Sequestration of T cells in bone marrow in the setting of glioblastoma and other intracranial tumors.

T cell dysfunction contributes to tumor immune escape in patients with cancer and is particularly severe amidst glioblastoma (GBM). Among other defects, T cell lymphopenia is characteristic, yet often attributed to treatment. We reveal that even treatment-naïve subjects and mice with GBM can harbor AIDS-level CD4 counts, as well as contracted, T cell-deficient lymphoid organs. Missing naïve T cells are instead found sequestered in large numbers in the bone marrow. This phenomenon characterizes not only GBM but a variety of other cancers, although only when tumors are introduced into the intracranial compartment. T cell sequestration is accompanied by tumor-imposed loss of S1P1 from the T cell surface and is reversible upon precluding S1P1 internalization. In murine models of GBM, hindering S1P1 internalization and reversing sequestration licenses T cell-activating therapies that were previously ineffective. Sequestration of T cells in bone marrow is therefore a tumor-adaptive mode of T cell dysfunction, whose reversal may constitute a promising immunotherapeutic adjunct.

A Rationally Designed Fully Human EGFRvIII:CD3-Targeted Bispecific Antibody Redirects Human T Cells to Treat Patient-derived Intracerebral Malignant Glioma.

Purpose: Conventional therapy for malignant glioma fails to specifically target tumor cells. In contrast, substantial evidence indicates that if appropriately redirected, T cells can precisely eradicate tumors. Here we report the rational development of a fully human bispecific antibody (hEGFRvIII-CD3 bi-scFv) that redirects human T cells to lyse malignant glioma expressing a tumor-specific mutation of the EGFR (EGFRvIII).Experimental Design: We generated a panel of bispecific single-chain variable fragments and optimized design through successive rounds of screening and refinement. We tested the ability of our lead construct to redirect naïve T cells and induce target cell-specific lysis. To test for efficacy, we evaluated tumor growth and survival in xenogeneic and syngeneic models of glioma. Tumor penetrance following intravenous drug administration was assessed in highly invasive, orthotopic glioma models.Results: A highly expressed bispecific antibody with specificity to CD3 and EGFRvIII was generated (hEGFRvIII-CD3 bi-scFv). Antibody-induced T-cell activation, secretion of proinflammatory cytokines, and proliferation was robust and occurred exclusively in the presence of target antigen. hEGFRvIII-CD3 bi-scFv was potent and target-specific, mediating significant lysis of multiple malignant glioma cell lines and patient-derived malignant glioma samples that heterogeneously express EGFRvIII. In both subcutaneous and orthotopic models, well-engrafted, patient-derived malignant glioma was effectively treated despite heterogeneity of EGFRvIII expression; intravenous hEGFRvIII-CD3 bi-scFv administration caused significant regression of tumor burden (P < 0.0001) and significantly extended survival (P < 0.0001). Similar efficacy was obtained in highly infiltrative, syngeneic glioma models, and intravenously administered hEGFRvIII-CD3 bi-scFv localized to these orthotopic tumors.Conclusions: We have developed a clinically translatable bispecific antibody that redirects human T cells to safely and effectively treat malignant glioma. On the basis of these results, we have developed a clinical study of hEGFRvIII-CD3 bi-scFv for patients with EGFRvIII-positive malignant glioma. Clin Cancer Res; 24(15); 3611-31. ©2018 AACR.