Anti-Mouse NKG2A/C/E – Purified in vivo PLATINUM™ Functional Grade

In Vivo Applications of Clone 20D5 in Mice

Clone 20D5 is a monoclonal antibody that recognizes mouse NKG2A, NKG2C, and NKG2E, which are isoforms of the CD94/NKG2 heterodimer expressed primarily on natural killer (NK) cells, NKT cells, and activated CD8+ T cells. These receptors play critical roles in immune surveillance, particularly in detecting and responding to cells with altered MHC class I expression—a common feature of viral infections and cancer. In in vivo mouse studies, clone 20D5 has two principal applications.

Immune Checkpoint Inhibition in Cancer Immunotherapy

• Blockade of NKG2A Signaling: Clone 20D5 is used to block NKG2A-mediated inhibitory signals in vivo, thereby enhancing the anti-tumor activity of both T cells and NK cells in murine cancer models. This approach exploits NKG2A as a novel immune checkpoint, analogous to PD-1/PD-L1 or CTLA-4 inhibitors, with the goal of "unleashing" immune effector cells to attack tumors more effectively.
• Promotion of Anti-Tumor Immunity: Preclinical studies have demonstrated that administering anti-NKG2A (clone 20D5) in vivo promotes robust anti-tumor immunity, making it a candidate for immune checkpoint inhibitor therapy in cancer. This application is especially relevant in the context of exploring new immunotherapeutic strategies for cancers where classical checkpoint inhibitors may be less effective.

Research Tool for Studying Immune Cell Function

• Investigation of NKG2/CD94 Biology: Clone 20D5 is widely used as a research tool to study the role of NKG2/CD94 receptors in immune cell activation, inhibition, and immune surveillance. By modulating these pathways in vivo, researchers can dissect the contributions of NKG2A (inhibitory) versus NKG2C/E (stimulatory) signaling in immune responses to infection, autoimmunity, and cancer.
• Strain-Specific Studies: The antibody can be used in most common mouse strains except DBA/2J, which is CD94-deficient and does not express the CD94/NKG2 heterodimer.

Summary Table

Conclusion

In vivo, clone 20D5 is primarily used to block NKG2A-mediated immune inhibition in mouse models of cancer, thereby enhancing anti-tumor immunity, and as a research tool to study the biology of NKG2/CD94 receptors in immune responses. These applications have significant implications for both basic immunology and the development of next-generation cancer immunotherapies.

Based on research applications, several antibodies and proteins are commonly used in conjunction with 20D5 (anti-mouse NKG2A/C/E) in the literature. These include anti-PD-1, anti-PD-L1, anti-asialo-GM1, and anti-CD8α antibodies, which are utilized to study immune modulation and tumor immunity.

The combination of 20D5 with anti-PD-L1 antibodies has been particularly explored in cancer immunotherapy research. Studies have examined treatment regimens where mice received IgG antibody controls, anti-PD-L1 antibody alone, anti-NKG2A antibody alone, or a combination of anti-NKG2A and anti-PD-L1 antibodies. This combination approach reflects growing interest in targeting multiple immune checkpoint pathways simultaneously to enhance anti-tumor responses.

Additionally, research has demonstrated that intratumoral co-injection of pre-activated NK cells with NKG2A neutralizing antibodies achieves synergistic anti-tumor activity against solid mouse tumor models. This suggests that 20D5 is often used alongside cellular immunotherapy approaches, specifically with natural killer (NK) cells, to boost therapeutic efficacy in experimental tumor models.

The clone 20D5 anti-mouse NKG2A/C/E monoclonal antibody has been utilized in scientific research to investigate NK cell biology and immune checkpoint inhibition, with several notable findings emerging from its use in the literature.

Anti-Tumor Immunity Enhancement

Blocking NKG2A signaling using clone 20D5 has demonstrated significant promise in promoting anti-tumor immunity in murine tumor models. This effect operates through a dual mechanism, enhancing the activity of both T cells and NK cells to mount more effective anti-cancer responses. The therapeutic potential of this approach has positioned NKG2A targeting as a novel immune checkpoint inhibitory strategy being explored for treating human cancers.

Combination Therapy Applications

Research has shown that combining NKG2A blockade with other therapeutic modalities yields improved outcomes. Studies have demonstrated that the combination of NKG2A and PD-1 blockade improves radiotherapy responses in tumor models. The mechanism involves radiotherapy's modulation of NKG2A ligand expression, which can otherwise inhibit radiation-induced T cell responses in tumors that fail to respond to combined radiotherapy and PD-1 treatment.

HCV Persistence and NK Cell Exhaustion

Clone 20D5 has been instrumental in identifying NKG2A as an NK cell exhaustion checkpoint in hepatitis C virus (HCV) persistence. This finding has expanded our understanding of how chronic viral infections evade immune surveillance through the upregulation of inhibitory receptors on effector cells.

Intratumoral NK Cell Therapy

Recent research has explored the intratumoral co-injection of NK cells with NKG2A-neutralizing antibodies using clone 20D5. These studies have contributed to understanding how local delivery of immune cells combined with checkpoint blockade can enhance therapeutic efficacy in solid tumors.

Engineered Variants

An important development emerging from the literature involves an engineered recombinant mouse variant of the original rat IgG2a clone 20D5 antibody, which incorporates mouse IgG constant domains. This modified version has been specifically designed to block NKG2A signals more effectively in vivo in murine tumor models, representing an advancement in the antibody's therapeutic application.

Overview of Clone 20D5 (anti-mouse NKG2A/C/E) and Its Use

Clone 20D5 is a monoclonal antibody that targets mouse NKG2A, NKG2C, and NKG2E—receptors primarily expressed on natural killer (NK) cells, NK T cells, and a subset of activated CD8⁺ T cells. These receptors, when complexed with CD94, play key roles in immune surveillance and modulation, particularly in contexts of cancer, infection, and autoimmunity.

Published Dosing Regimens in Mouse Models

Dosing of clone 20D5 is context-dependent and varies based on the disease model, therapeutic objective (e.g., checkpoint blockade vs. immune cell depletion), and the specific immune parameter being investigated. However, several published protocols provide insight into typical usage:

• Oncologic Models: In a B16F10 melanoma model, mice received 200 µg of anti-NKG2A (20D5) antibody via intraperitoneal (i.p.) injection on days 7, 10, 13, and 16 following tumor implantation. This regimen was used to assess the effect of NKG2A blockade on tumor growth, often in combination with other therapies (e.g., radiotherapy).
• Liver Injury Model: In a study investigating immune-mediated liver injury, a single 300 µg i.p. dose of 20D5 was administered two days before Concanavalin A (Con A) or α-galactosylceramide (α-GalCer) injection, to modulate NK cell activity prior to injury induction.
• Autoimmune Models: In experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, F(ab′)2 fragments of 20D5 (not full antibody) were used, with dosing and frequency tailored to assess impact on disease progression. Full dosing details for the intact antibody in this context are not specified in the cited literature.

Comparison with Other Immune Checkpoint Antibodies

While there is no comprehensive dosing guide specifically for 20D5, its use is broadly in line with other checkpoint blockade antibodies in mice, which typically involve doses of 100–500 µg per mouse, administered i.p., with frequencies ranging from single doses to repeated administrations every 3–4 days or 2–3 times per week. The 200 µg dose used in the B16F10 model falls within this standard range.

Key Variables Affecting Dosing

• Model Objective: Tumor models may use repeated dosing for sustained blockade, while acute injury models may use a single dose for transient effect.
• Combination Therapies: When combined with other agents (e.g., radiotherapy, PD-1 blockade), dosing may be adjusted to balance efficacy and toxicity.
• Antibody Format: Intact antibody vs. F(ab′)2 fragments may require different dosing due to differences in half-life and Fc-mediated effects.
• Route of Administration: Intraperitoneal injection is most common, but other routes (e.g., intratumoral) may be explored in specialized settings.

Summary Table: Reported Dosing Regimens for Clone 20D5

Conclusions

• Dosing of clone 20D5 in mouse models is not standardized and should be tailored to the experimental context, including the disease model, desired duration of NKG2A/C/E blockade, and potential combination therapies.
• Published regimens range from a single 300 µg dose to repeated 200 µg doses, with intraperitoneal injection as the standard route.
• Consultation of primary literature for the specific model of interest is essential, as subtle differences in strain, tumor type, or immune challenge can influence optimal dosing.

For novel applications, pilot dose-ranging studies are recommended to establish efficacy and tolerability in the specific experimental system.