Preparation and Characterization of the Nanobody
The metabotropic glutamate receptor 2 (mGlu2) homodimer is a key regulator of glutamatergic activity in the brain and has been clinically validated as a target for schizophrenia and related disorders. However, previous drugs targeting mGlu2 failed due to insufficient efficacy or side effects. Thus, the research team focused on developing a novel therapeutic antibody capable of crossing the BBB, specifically targeting mGlu2, and possessing a long half-life and high therapeutic potency.
The team had previously developed the nanobody DN13, which specifically binds to and positively modulates the mGlu2 receptor. To enhance its efficacy and brain-targeting potential, the researchers innovatively conjugated DN13 with another nanobody, DN1, which binds to mGlu2 without activating it, using a specialized linker to construct the bivalent bispecific nanobody DN13-DN1.
As illustrated, this bivalent design conferred significant advantages, validated in subsequent experiments. Saturation binding and functional activity assays showed that in the presence of the agonist LY379268, DN13-DN1 exhibited a 10-fold increase in apparent affinity for mGlu2 (Kd = 0.50 nM) compared to the monomeric DN13 (Kd = 4.10 nM). Its positive allosteric modulator (PAM) potency (EC50 = 1.76 nM) was nearly two orders of magnitude higher than that of DN13 (EC50 = 101 nM). Moreover, DN13-DN1 specifically bound to mGlu2 homodimers without cross-reacting with other mGlu receptors, establishing a foundation of high specificity and efficacy for further studies.
Figure 1: DN13–DN1 nanobody enhances receptor activation
Validation of Brain Penetration
To confirm whether DN13-DN1 could enter the brain following peripheral administration, the team conducted studies using IP injection combined with tritium labeling and immunohistochemistry. After labeling DN13-DN1 as [³H]DN13-DN1 and administering it via IP injection, quantitative analysis revealed that 0.08% of the injected dose reached the brain within 4 hours, corresponding to an estimated brain concentration of 9.2 nM (≈20×Kd). Even after 7 days, the brain concentration remained at 1.5 nM (≈3×Kd), both above the effective concentration.
Immunohistochemistry further confirmed that 24 hours post-injection, DN13-DN1 was specifically distributed in mGlu2-high regions such as the ventral tegmental area (VTA) and cerebral cortex, while the control group (saline injection) showed no positive signal. Autoradiography experiments also demonstrated similar regional distribution, unequivocally proving that DN13-DN1 crosses the BBB and accumulates in specific brain regions.
Figure 2: DN13–DN1 crosses the blood-brain barrier and exhibits long-lasting efficacy
Behavioral Improvement in Animal Models
To further validate the therapeutic efficacy of DN13-DN1, the research team utilized two classic mouse models of NMDA receptor hypofunction: the neonatal PCP-induced model (simulating schizophrenia) and the GluN1-KD genetic model (simulating GRIN1 disorder).
Neonatal PCP-Induced Model:
NMDA receptor hypofunction is a core pathological mechanism in schizophrenia. The team subcutaneously administered the NMDA receptor antagonist PCP to neonatal mice (on P7, P9, and P11) to induce schizophrenia-like cognitive deficits. In adulthood, the novel object recognition (NOR) test was used to evaluate recognition memory, with both 24-hour and 7-day retention intervals to assess short- and long-term effects. Results showed that a single IP injection of 10 mg/kg DN13-DN1 significantly restored recognition memory in PCP-treated mice after 24 hours, with efficacy comparable to the mGlu2/3 agonist LY379268 (1 mg/kg IP). In contrast, the control nanobody DN1 showed no improvement, and the mGlu2 antagonist LY341495 completely blocked the effect of DN13-DN1, confirming its dependency on mGlu2 activation. Importantly, in the 7-day retention test, LY379268 lost its therapeutic effect, while DN13-DN1 continued to improve recognition memory, highlighting its long-lasting efficacy.
Figure 3: DN13–DN1 exerts significant therapeutic effects in a neonatal PCP-induced schizophrenia mouse model
GluN1-KD Genetic Model:
This model mimics GRIN1 disorder by reducing GluN1 subunit expression, leading to partial loss of NMDA receptor function. The team used the Y-maze test to assess working memory and prepulse inhibition (PPI) to evaluate sensorimotor gating. Y-maze results showed that a single IP injection of 20 mg/kg DN13-DN1 significantly improved working memory in GluN1-KD mice within 3 hours, comparable to LY379268. After 7 days, the DN13-DN1 group maintained improvement, while the LY379268 group showed no significant effect.
PPI tests indicated that DN13-DN1 improved PPI deficits at 81 dB prepulse intensity 3 hours post-administration. After 7 days, the effect extended to both 69 dB and 81 dB prepulse intensities without affecting acoustic startle response (ASR), demonstrating effective restoration of sensorimotor gating.
Figure 4: DN13–DN1 improves working memory and sensorimotor gating in GluN1-KD mice
Subchronic Treatment Safety and Comparative Advantages
To simulate long-term clinical use, the team designed a subchronic dosing regimen: an initial IP injection of 10 mg/kg DN13-DN1, followed by 1 mg/kg injections once per week for a total of four doses. Results showed that subchronic treatment stably maintained improvements in recognition memory, working memory, and sensorimotor gating in both models, with no gender differences and no effect on body weight.
Safety assessments confirmed that DN13-DN1 did not alter mGlu2 receptor expression in the brain, nor did it affect motor coordination, balance, or catalepsy. The endotoxin (LPS) content was below the neurotoxicity threshold, and further purification did not alter therapeutic efficacy, ruling out endotoxin interference and confirming its safety.
Figure 5: Subchronic administration of DN13–DN1 ameliorates behavioral deficits in two schizophrenia mouse models
To highlight the advantages of nanobodies, the team compared DN13-DN1 with the small-molecule drug LY379268 and the IgG-class antibody DN13-Fc. Results demonstrated that a single dose of DN13-DN1 maintained efficacy for 7 days, whereas LY379268 was only acutely effective. Additionally, DN13-Fc (80 kDa) showed no behavioral improvement after IP injection, while DN13-DN1’s smaller size enabled superior BBB penetration, underscoring the unique advantages of nanobodies in treating brain disorders.
Figure 6: DN13–Fc shows no acute behavioral effects in two schizophrenia mouse models
The core advantages of DN13-DN1 stem from its unique molecular design and mechanism of action. Structurally, its small size (15 kDa) enables BBB penetration and accumulation in specific brain regions—a feat unattainable by traditional IgG antibodies. Its specificity for mGlu2 homodimers without cross-reactivity avoids off-target effects. As a positive allosteric modulator (PAM), it enhances mGlu2 signaling only in the presence of endogenous glutamate, preserving spatiotemporal specificity of physiological signals and avoiding receptor desensitization due to overactivation. Furthermore, the bivalent design significantly enhances its affinity and potency for mGlu2, allowing prolonged retention in the brain for up to 7 days after a single dose. This enables long-lasting treatment and greatly improves patient compliance.
This research represents a major breakthrough in nanobody technology for brain disorders. It offers a revolutionary therapeutic strategy for various central nervous system diseases caused by NMDA receptor hypofunction, particularly cognitive impairments and negative symptoms in schizophrenia, as well as genetic GRIN1 disorders, bringing new hope to millions of patients worldwide.
Wuhan Nano Body Life Science and Technology Co. Ltd. (NBLST) is a nanobody industry platform established under the initiative of the Wuhan Industrial Innovation and Development Research Institute. Its headquarters is located in the main building of the Wuhan Industrial Innovation and Development Research Institute in the East Lake High-tech Development Zone, Wuhan. It boasts a 1400 m² independent laboratory in the Precision Medicine Industrial Base of Wuhan Biolake. Additionally, NBLST has established alpaca experimental and transfer bases in Zuoling, Wuhan, and Tuanfeng, Huanggang, both compliant with laboratory animal standards. These bases currently house over 600 alpacas, providing "zero-immunization-background" guaranteed alpaca immunization services for research institutions and antibody drug development companies.
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