Biology drives the discovery of bispecific antibodies as innovative therapeutics.

A bispecific antibody (bsAb) is able to bind two different targets or two distinct epitopes on the same target. Broadly speaking, bsAbs can include any single molecule entity containing dual specificities with at least one being antigen-binding antibody domain. Besides additive effect or synergistic effect, the most fascinating applications of bsAbs are to enable novel and often therapeutically important concepts otherwise impossible by using monoclonal antibodies alone or their combination. This so-called obligate bsAbs could open up completely new avenue for developing novel therapeutics. With evolving understanding of structural architecture of various natural or engineered antigen-binding immunoglobulin domains and the connection of different domains of an immunoglobulin molecule, and with greatly improved understanding of molecular mechanisms of many biological processes, the landscape of therapeutic bsAbs has significantly changed in recent years. As of September 2019, over 110 bsAbs are under active clinical development, and near 180 in preclinical development. In this review article, we introduce a system that classifies bsAb formats into 30 categories based on their antigen-binding domains and the presence or absence of Fc domain. We further review the biology applications of approximately 290 bsAbs currently in preclinical and clinical development, with the attempt to illustrate the principle of selecting a bispecific format to meet biology needs and selecting a bispecific molecule as a clinical development candidate by 6 critical criteria. Given the novel mechanisms of many bsAbs, the potential unknown safety risk and risk/benefit should be evaluated carefully during preclinical and clinical development stages. Nevertheless we are optimistic that next decade will witness clinical success of bsAbs or multispecific antibodies employing some novel mechanisms of action and deliver the promise as next wave of antibody-based therapeutics.

Cognitive impairments associated with alterations in synaptic proteins induced by the genetic loss of adenosine A2A receptors in mice.

The study of psychiatric disorders usually focuses on emotional symptoms assessment. However, cognitive deficiencies frequently constitute the core symptoms, are often poorly controlled and handicap individual's quality of life. Adenosine receptors, through the control of both dopamine and glutamate systems, have been implicated in the pathophysiology of several psychiatric disorders such as schizophrenia and attention deficit/hyperactivity disorder. Indeed, clinical data indicate that poorly responsive schizophrenia patients treated with adenosine adjuvants show improved treatment outcomes. The A2A adenosine receptor subtype (A2AR) is highly expressed in brain areas controlling cognition and motivational responses including the striatum, hippocampus and cerebral cortex. Accordingly, we study the role of A2AR in the regulation of cognitive processes based on a complete cognitive behavioural analysis coupled with the assessment of neurogenesis and sub-synaptic protein expression in adult and middle-aged A2AR constitutional knockout mice and wild-type littermates. Our results show overall cognitive impairments in A2AR knockout mice associated with a decrease in new-born hippocampal neuron proliferation and concomitant changes in synaptic protein expression, in both the prefrontal cortex and the hippocampus. These results suggest a deficient adenosine signalling in cognitive processes, thus providing new opportunities for the therapeutic management of cognitive deficits associated with psychiatric disorders.

Maternal alcohol binge drinking induces persistent neuroinflammation associated with myelin damage and behavioural dysfunctions in offspring mice.

Alcohol binge drinking is on the increase in the young adult population, and consumption during pregnancy can be deleterious for foetal development. Maternal alcohol consumption leads to a wide range of long-lasting morphological and behavioural deficiencies known as foetal alcohol spectrum disorders (FASD), associated with neurodevelopmental disabilities. We sought to test the effects of alcohol on neuroimmune system activation and its potential relation to alcohol-induced neurodevelopmental and persistent neurobehavioural effects in offspring after maternal alcohol binge drinking during the prenatal period or in combination with lactation. Pregnant C57BL/6 female mice underwent a procedure for alcohol binge drinking either during gestation or both the gestation and lactation periods. Adult male offspring were assessed for cognitive functions and motor coordination. Early alcohol exposure induced motor coordination impairments in the rotarod test. Object recognition memory was not affected by maternal alcohol binge drinking, but Y-maze performance was impaired in pre- and early postnatal alcohol-exposed mice. Behavioural effects were associated with an upregulation of pro-inflammatory signalling (Toll-like receptor 4, nuclear factor-kappa B p65, NOD-like receptor protein 3, caspase-1, and interleukin-1ß), gliosis, neuronal cell death and a reduction in several structural myelin proteins (myelin-associated glycoprotein, myelin basic protein, myelin proteolipid protein and myelin regulatory factor) in both the prefrontal cortex and hippocampus of adult mice exposed to alcohol. Altogether, our results reveal that maternal binge-like alcohol consumption induces neuroinflammation and myelin damage in the brains of offspring and that such effects may underlie the persistent cognitive and behavioural impairments observed in FASD.

Maternal Separation Impairs Cocaine-Induced Behavioural Sensitization in Adolescent Mice.

Adverse early-life conditions induce persistent disturbances that give rise to negative emotional states. Therefore, early life stress confers increased vulnerability to substance use disorders, mainly during adolescence as the brain is still developing. In this study, we investigated the consequences of maternal separation, a model of maternal neglect, on the psychotropic effects of cocaine and the neuroplasticity of the dopaminergic system. Our results show that mice exposed to maternal separation displayed attenuated behavioural sensitization, while no changes were found in the rewarding effects of cocaine in the conditioned place preference paradigm and in the reinforcing effects of cocaine in the self-administration paradigm. The evaluation of neuroplasticity in the striatal dopaminergic pathways revealed that mice exposed to maternal separation exhibited decreased protein expression levels of D2 receptors and increased levels of the transcriptional factor Nurr1. Furthermore, animals exposed to maternal separation and treated with cocaine exhibited increased DA turnover and protein expression levels of DAT and D2R, while decreased Nurr1 and Pitx3 protein expression levels were observed when compared with saline-treated mice. Taken together, our data demonstrate that maternal separation caused an impairment of cocaine-induced behavioural sensitization possibly due to a dysfunction of the dopaminergic system, a dysfunction that has been proposed as a factor of vulnerability for developing substance use disorders.

Genetic blockade of adenosine A2A receptors induces cognitive impairments and anatomical changes related to psychotic symptoms in mice.

Schizophrenia is a chronic severe mental disorder with a presumed neurodevelopmental origin, and no effective treatment. Schizophrenia is a multifactorial disease with genetic, environmental and neurochemical etiology. The main theories on the pathophysiology of this disorder include alterations in dopaminergic and glutamatergic neurotransmission in limbic and cortical areas of the brain. Early hypotheses also suggested that nucleoside adenosine is a putative affected neurotransmitter system, and clinical evidence suggests that adenosine adjuvants improve treatment outcomes, especially in poorly responsive patients. Hence, it is important to elucidate the role of the neuromodulator adenosine in the pathophysiology of schizophrenia. A2A adenosine receptor (A2AR) subtypes are expressed in brain areas controlling motivational responses and cognition, including striatum, and in lower levels in hippocampus and cerebral cortex. The aim of this study was to characterize A2AR knockout (KO) mice with complete and specific inactivation of A2AR, as an animal model for schizophrenia. We performed behavioral, anatomical and neurochemical studies to assess psychotic-like symptoms in adult male and female KO and wild-type (WT) littermates. Our results show impairments in inhibitory responses and sensory gating in A2AR KO animals. Hyperlocomotion induced by d-amphetamine and MK-801 was reduced in KO animals when compared to WT littermates. Moreover, A2AR KO animals show motor disturbances, social and cognitive alterations. Finally, behavioral impairments were associated with enlargement of brain lateral ventricles and decreased BDNF levels in the hippocampus. These data highlight the role of adenosine in the pathophysiology of schizophrenia and provide new possibilities for the therapeutic management of schizophrenia.

Maternal separation induces neuroinflammation and long-lasting emotional alterations in mice.

Early life experiences play a key role in brain function and behaviour. Adverse events during childhood are therefore a risk factor for psychiatric disease during adulthood, such as mood disorders. Maternal separation is a validated mouse model for maternal neglect, producing negative early life experiences that result in subsequent emotional alteration. Mood disorders have been found to be associated with neurochemical changes and neurotransmitter deficits such as reduced availability of monoamines in discrete brain areas. Emotional alterations like depression result in reduced serotonin availability and enhanced kynurenine metabolism through the action of indoleamine 2, 3-dioxygenase in response to neuroinflammatory factors. This mechanism involves regulation of the neurotransmitter system by neuroinflammatory agents, linking mood regulation to neuroinmunological reactions. In this context, the aim of this study was to investigate the effects of maternal separation with early weaning on emotional behaviour in mice. We investigated neuroinflammatory responses and the state of the tryptophan-kynurenine metabolic pathway in discrete brain areas following maternal separation. We show that adverse events during early life increase risk of long-lasting emotional alterations during adolescence and adulthood. These emotional alterations are particularly severe in females. Behavioural impairments were associated with microglia activation and disturbed tryptophan-kynurenine metabolism in brain areas related to emotional control. This finding supports the preeminent role of neuroinflammation in emotional disorders.

Reduced Contextual Discrimination following Alcohol Consumption or MDMA Administration in Mice.

The recreational drugs, alcohol and 3,4-Methylenedioxymethamphetamine (MDMA, "Ecstasy") have both been shown to cause immune activation in vivo, and they are linked to cognitive impairment and anxiety-like behaviors in rodents. The neuronal effects of these drugs in the hippocampal area, an area that has been a focus of studies aiming to explain the mechanisms underlying anxiety related-disorders, remains poorly understood. Therefore we investigated the specific inflammatory impact of alcohol and MDMA on this area of the brain and on a hippocampal-related behavioral task. We centered our study on two inflammatory factors linked to anxiety-related disorders, namely Interleukin-1ß (IL-1ß) and brain-derived neurotrophic factor (BDNF). We subjected drug-consuming mice to a battery of behavioral tests to evaluate general activity, anxiety-like and depressive-live behaviors. We then introduced them to a contextual fear discrimination task and immune-related effects were examined by immunohistochemical and biochemical studies. Our results suggest that there is a relationship between the induction of immune activated pathways by voluntary alcohol consumption and a high-dose MDMA. Furthermore, the ability of mice to perform a contextual fear discrimination task was impaired by drug consumption and we report long term inflammatory alterations in the hippocampus even several weeks after drug intake. This information will be helpful for discovering new selective drug targets, and to develop treatments and preventive approaches for patients with anxiety-related disorders.

Memory impairment and hippocampus specific protein oxidation induced by ethanol intake and 3, 4-methylenedioxymethamphetamine (MDMA) in mice.

Ethanol and 3, 4-Methylenedioxymethamphetamine (MDMA) are popular recreational drugs widely abused by adolescents that may induce neurotoxic processes associated with behavioural alterations. Adolescent CD1 mice were subjected to ethanol intake using the drinking in the dark (DID) procedure, acute MDMA or a combination. Considering that both drugs of abuse cause oxidative stress in the brain, protein oxidative damage in different brain areas was analysed 72 h after treatment using a proteomic approach. Damage to specific proteins in treated animals was significant in the hippocampus but not in the prefrontal cortex. The damaged hippocampus proteins were then identified by mass spectrometry, revealing their involvement in energy metabolism, structural function, axonal outgrowth and stability, and neurotransmitter release. Mice treated with MDMA displayed higher oxidative damage than ethanol-treated mice. To determine whether this oxidative damage was affecting hippocampus activity, declarative memory was evaluated at 72 h after treatment using the object recognition assay and the radial arm maze. Although acquisition in the radial arm maze was not impaired by ethanol intake, MDMA treatment impaired long-term memory in both tests. Therefore, oxidative damage to specific proteins observed under MDMA treatment affects important cellular function on the hippocampus that may contribute to declarative memory deficits.