Hypoxia Inducible Factor-1α binds and activates γ-secretase for Aß production under hypoxia and cerebral hypoperfusion.

Hypoxic-ischemic injury has been linked with increased risk for developing Alzheimer's disease (AD). The underlying mechanism of this association is poorly understood. Here, we report distinct roles for hypoxia-inducible factor-1α (Hif-1α) in the regulation of BACE1 and γ-secretase activity, two proteases involved in the production of amyloid-beta (Aß). We have demonstrated that Hif-1α upregulates both BACE1 and γ-secretase activity for Aß production in brain hypoxia-induced either by cerebral hypoperfusion or breathing 10% O2. Hif-1α binds to γ-secretase, which elevates the amount of active γ-secretase complex without affecting the level of individual subunits in hypoxic-ischemic mouse brains. Additionally, the expression of full length Hif-1α increases BACE1 and γ-secretase activity in primary neuronal culture, whereas a transcriptionally incompetent Hif-1α variant only activates γ-secretase. These findings indicate that Hif-1α transcriptionally upregulates BACE1 and nontranscriptionally activates γ-secretase for Aß production in hypoxic-ischemic conditions. Consequently, Hif-1α-mediated Aß production may be an adaptive response to hypoxic-ischemic injury, subsequently leading to increased risk for AD. Preventing the interaction of Hif-1α with γ-secretase may therefore be a promising therapeutic strategy for AD treatment.

Generation of T-cell-redirecting bispecific antibodies with differentiated profiles of cytokine release and biodistribution by CD3 affinity tuning.

T-cell-redirecting bispecific antibodies have emerged as a new class of therapeutic agents designed to simultaneously bind to T cells via CD3 and to tumor cells via tumor-cell-specific antigens (TSA), inducing T-cell-mediated killing of tumor cells. The promising preclinical and clinical efficacy of TSAxCD3 antibodies is often accompanied by toxicities such as cytokine release syndrome due to T-cell activation. How the efficacy and toxicity profile of the TSAxCD3 bispecific antibodies depends on the binding affinity to CD3 remains unclear. Here, we evaluate bispecific antibodies that were engineered to have a range of CD3 affinities, while retaining the same binding affinity for the selected tumor antigen. These agents were tested for their ability to kill tumor cells in vitro, and their biodistribution, serum half-life, and anti-tumor activity in vivo. Remarkably, by altering the binding affinity for CD3 alone, we can generate bispecific antibodies that maintain potent killing of TSA + tumor cells but display differential patterns of cytokine release, pharmacokinetics, and biodistribution. Therefore, tuning CD3 affinity is a promising method to improve the therapeutic index of T-cell-engaging bispecific antibodies.

Targeting Solid Tumors Using CD3 Bispecific Antibodies.

Immunotherapies to treat cancer have made tremendous progress over the past decade. In particular, T cell-directed therapies have gained considerable attention with CD3 bispecific antibodies and CAR T cells showing potent responses against hematologic tumors. At present, the ability to adapt these therapeutics to treat solid tumors is less established. Herein, we discuss recent advances in T cell-engaging CD3 bispecific antibodies targeting solid tumors, potential mechanisms of resistance, and future prospects. A better understanding of the mechanisms of immune evasion in solid tumors will enable the development of strategies to overcome this resistance and inform choices of therapeutic combinations.

A PSMA-Targeting CD3 Bispecific Antibody Induces Antitumor Responses that Are Enhanced by 4-1BB Costimulation.

Patients with hematologic cancers have improved outcomes after treatment with bispecific antibodies that bind to CD3 on T cells and that redirect T cells toward cancer cells. However, clinical benefit against solid tumors remains to be shown. We made a bispecific antibody that targets both the common prostate tumor-specific antigen PSMA and CD3 (PMSAxCD3) and provide evidence for tumor inhibition in several preclinical solid tumor models. Mice expressing the human extracellular regions of CD3 and PSMA were generated to examine antitumor efficacy in the presence of an intact immune system and PSMA expression in normal tissues. PSMAxCD3 accumulated in PSMA-expressing tissues and tumors as detected by immuno-PET imaging. Although PSMAxCD3 induced T-cell activation and showed antitumor efficacy in mice with low tumor burden, PSMAxCD3 lost efficacy against larger solid tumors, mirroring the difficulty of treating solid tumors in the clinic. Costimulatory receptors can enhance T-cell responses. We show here that costimulation can enhance the antitumor efficacy of PSMAxCD3. In particular, 4-1BB stimulation in combination with PSMAxCD3 enhanced T-cell activation and proliferation, boosted efficacy against larger tumors, and induced T-cell memory, leading to durable antitumor responses. The combination of CD3 bispecific antibodies and anti-4-1BB costimulation represents a therapeutic approach for the treatment of solid tumors.

A class of costimulatory CD28-bispecific antibodies that enhance the antitumor activity of CD3-bispecific antibodies.

T cell activation is initiated upon binding of the T cell receptor (TCR)/CD3 complex to peptide-major histocompatibility complexes ("signal 1"); activation is enhanced by engagement of a second "costimulatory" receptor, such as the CD28 receptor on T cells binding to its cognate ligand(s) on the target cell ("signal 2"). CD3-based bispecific antibodies act by replacing conventional signal 1, linking T cells to tumor cells by binding a tumor-specific antigen (TSA) with one arm of the bispecific and bridging to TCR/CD3 with the other. Although some of these so-called TSAxCD3 bispecifics have demonstrated promising antitumor efficacy in patients with cancer, their activity remains to be optimized. Here, we introduce a class of bispecific antibodies that mimic signal 2 by bridging TSA to the costimulatory CD28 receptor on T cells. We term these TSAxCD28 bispecifics and describe two such bispecific antibodies: one specific for ovarian and the other for prostate cancer antigens. Unlike CD28 superagonists, which broadly activate T cells and resulted in profound toxicity in early clinical trials, these TSAxCD28 bispecifics show limited activity and no toxicity when used alone in genetically humanized immunocompetent mouse models or in primates. However, when combined with TSAxCD3 bispecifics, they enhance the artificial synapse between a T cell and its target cell, potentiate T cell activation, and markedly improve antitumor activity of CD3 bispecifics in a variety of xenogeneic and syngeneic tumor models. Combining this class of CD28-costimulatory bispecific antibodies with the emerging class of TSAxCD3 bispecifics may provide well-tolerated, off-the-shelf antibody therapies with robust antitumor efficacy.

A Mucin 16 bispecific T cell-engaging antibody for the treatment of ovarian cancer.

Advanced ovarian cancer is frequently treated with combination chemotherapy, but high recurrence rates show the need for therapies that can produce durable responses and extend overall survival. Bispecific antibodies that interact with tumor antigens on cancer cells and activating receptors on immune cells offer an innovative immunotherapy approach. Here, we describe a human bispecific antibody (REGN4018) that binds both Mucin 16 (MUC16), a glycoprotein that is highly expressed on ovarian cancer cells, and CD3, thus bridging MUC16-expressing cells with CD3+ T cells. REGN4018 induced T cell activation and killing of MUC16-expressing tumor cells in vitro. Binding and cytotoxicity of REGN4018 in vitro were minimally affected by high concentrations of CA-125, the shed form of MUC16, which is present in patients. In preclinical studies with human ovarian cancer cells and human T cells in immunodeficient mice, REGN4018 potently inhibited growth of intraperitoneal ovarian tumors. Moreover, in a genetically engineered immunocompetent mouse expressing human CD3 and human MUC16 [humanized target (HuT) mice], REGN4018 inhibited growth of murine tumors expressing human MUC16, and combination with an anti-PD-1 antibody enhanced this efficacy. Immuno-PET imaging demonstrated localization of REGN4018 in MUC16-expressing tumors and in T cell-rich organs such as the spleen and lymph nodes. Toxicology studies in cynomolgus monkeys showed minimal and transient increases in serum cytokines and C-reactive protein after REGN4018 administration, with no overt toxicity. Collectively, these data demonstrate potent antitumor activity and good tolerability of REGN4018, supporting clinical evaluation of REGN4018 in patients with MUC16-expressing advanced ovarian cancer.

Nontranscriptional role of Hif-1α in activation of γ-secretase and notch signaling in breast cancer.

γ-Secretase is composed of four proteins that are obligatory for protease activity: presenilin, nicastrin, Aph1, and Pen-2. Despite the progress toward understanding the function of these individual subunits, there is no information available pertaining to the modulation of γ-secretase in response to environmental changes in cells. Here, we show that hypoxia upregulates γ-secretase activity through a direct interaction with Hif-1α, revealing an unconventional function for Hif-1α as an enzyme subunit, which is distinct from its canonical role as a transcription factor. Moreover, hypoxia-induced cell invasion and metastasis are alleviated by either γ-secretase inhibitors or a dominant-negative Notch coactivator, indicating that γ-secretase/Notch signaling plays an essential role in controlling these cellular processes. The present study reveals a mechanism in which γ-secretase can achieve temporal control through conditional interactions with regulatory proteins, such as Hif-1α, under select physiological and pathological conditions.

Complex regulation of γ-secretase: from obligatory to modulatory subunits.

γ-Secretase is a four subunit, 19-pass transmembrane enzyme that cleaves amyloid precursor protein (APP), catalyzing the formation of amyloid beta (Aß) peptides that form amyloid plaques, which contribute to Alzheimer's disease (AD) pathogenesis. γ-Secretase also cleaves Notch, among many other type I transmembrane substrates. Despite its seemingly promiscuous enzymatic capacity, γ-secretase activity is tightly regulated. This regulation is a function of many cellular entities, including but not limited to the essential γ-secretase subunits, nonessential (modulatory) subunits, and γ-secretase substrates. Regulation is also accomplished by an array of cellular events, such as presenilin (active subunit of γ-secretase) endoproteolysis and hypoxia. In this review we discuss how γ-secretase is regulated with the hope that an advanced understanding of these mechanisms will aid in the development of effective therapeutics for γ-secretase-associated diseases like AD and Notch-addicted cancer.