Anti-Mouse DR5 (CD262) (Clone MD5-1) – Purified in vivo GOLD™ Functional Grade

In Vivo Applications of Clone MD5-1 in Mice

Clone MD5-1 is a monoclonal antibody specifically targeting mouse Death Receptor 5 (DR5, also known as CD262 or TRAIL-R2), a member of the tumor necrosis factor receptor superfamily involved in apoptotic signaling pathways. Its primary in vivo applications in mice relate to cancer immunotherapy and basic research in tumor immunology.

Selective Depletion of Myeloid-Derived Suppressor Cells (MDSCs)

• MD5-1 has been used to selectively deplete MDSCs—immune cells that suppress anti-tumor immune responses—in tumor-bearing mice. When administered in vivo, MD5-1 significantly reduces the accumulation of both granulocytic (gMDSCs) and monocytic (mMDSCs) subsets in tumors and spleens, without affecting other myeloid populations like macrophages or dendritic cells.
• This selective depletion is associated with a boost in T-cell antitumor responses, as evidenced by increased numbers of CD3⁺ T cells within the tumor microenvironment after treatment.
• The antibody’s action is specific to DR5-expressing cells, making it a tool to study the role of MDSCs and DR5 signaling in tumor immune evasion and therapy resistance.

Induction of Tumor Cell Apoptosis

• MD5-1 acts as a DR5 agonist and, when cross-linked (e.g., by Fc receptors on immune cells), induces TRAIL-mediated apoptosis in DR5-expressing tumor cells both in vitro and in vivo.
• Tumor growth inhibition has been demonstrated in various syngeneic mouse tumor models. For example, combination therapy with MD5-1 and a DNA vaccine showed significant reduction in tumor volume compared to controls.
• The antibody’s cytotoxic activity is dependent on cross-linking and is abolished by pan-caspase inhibitors, confirming the apoptotic mechanism.
• MD5-1 has been tested in models of mammary carcinoma (4T1) and renal carcinoma (R331), among others, showing efficacy against TRAIL-sensitive tumors.

Investigation of Immune Memory and Rejection

• MD5-1 treatment can induce tumor-specific T cell immunity. Mice that reject tumors after MD5-1 therapy develop immune memory, as shown by their ability to reject subsequent tumor challenges.
• Depletion and antibody blocking studies (e.g., anti-CD8, anti-CD4, anti-FasL, anti-TRAIL) have been used alongside MD5-1 to dissect the contributions of different immune cell subsets and death pathways in tumor rejection.

Combination Therapies

• MD5-1 has been combined with other therapies such as DNA vaccines or histone deacetylase inhibitors (e.g., panobinostat) to evaluate synergistic effects and potential toxicities in preclinical models.
• These studies aim to enhance antitumor efficacy and understand the safety profile of combinatorial approaches targeting the DR5 pathway.

Summary Table: Key In Vivo Applications

Conclusion

Clone MD5-1 is widely used in mice to study DR5-mediated apoptosis, to deplete MDSCs and enhance antitumor immunity, and to evaluate combination cancer therapies. Its specificity and functional activity make it a valuable tool in preclinical cancer immunology research.

Commonly Used Antibodies and Proteins with MD5-1 in the Literature

MD5-1 is a hamster monoclonal antibody that specifically targets the murine death receptor 5 (DR5 or CD262), a key component of the TNF-related apoptosis-inducing ligand (TRAIL) pathway. In the literature, MD5-1 is frequently used in combination with other immunomodulatory biologics to enhance antitumor effects, particularly in preclinical cancer models.

Key Combinations with MD5-1

• DNA Vaccines (CRT/E7(detox)): MD5-1 is often combined with a DNA vaccine encoding calreticulin (CRT) linked to the human papillomavirus type 16 (HPV-16) E7 antigen. In these studies, mice bearing E7-expressing TC-1 tumors were treated sequentially with MD5-1 followed by the CRT/E7(detox) DNA vaccine, resulting in enhanced E7-specific immune responses and potent antitumor effects. Calreticulin acts as an adjuvant to boost antigen presentation, while E7 is the target antigen in HPV-associated cancers.
• Anti-PD-L1 (Avelumab): In studies investigating immune evasion mechanisms in triple-negative breast cancer (TNBC) and ovarian cancer, MD5-1 was used alongside the anti-PD-L1 antibody avelumab. These combination therapies were explored to overcome resistance mechanisms and improve immune-mediated tumor control.
• Engineering Chimeric Antibodies: MD5-1 has been used as a variable region template to generate chimeric antibodies with human Fc regions (e.g., WT hIgG1, N297A, S267E) for investigating FcγRIIB binding and its impact on in vivo potency. These engineered antibodies retain the specificity of MD5-1 but are tailored for enhanced immune engagement or reduced off-target effects.
• Other DR5 Agonist Antibodies: In studies comparing signal transduction pathways of different TRAIL receptors, MD5-1 has been used alongside other DR5-targeting antibodies such as Lexa, AMG655, and KMTR2. These comparisons help elucidate the mechanisms of DR5 activation and apoptotic signaling.

Additional Context

• Flow Cytometry and Functional Assays: MD5-1 is routinely used in flow cytometry to detect DR5 expression and in functional assays to measure apoptosis induction, often in combination with other apoptosis-related markers (e.g., cleaved caspase-8).
• Induction of Cytotoxicity: The in vitro and in vivo cytotoxic effects of MD5-1 are often assessed in concert with other agents that target the TRAIL pathway or immune checkpoints.

Summary Table

Key Points

• MD5-1 is most commonly combined with DNA vaccines (e.g., CRT/E7), immune checkpoint inhibitors (e.g., avelumab), and other DR5 agonists in the literature to enhance anticancer immunity and apoptosis.
• Engineering of MD5-1 into chimeric antibodies with different Fc regions allows investigation of Fc receptor-dependent effects on apoptosis and immune modulation.
• Functional studies often use MD5-1 alongside apoptosis markers and other immunotherapies to dissect mechanisms of tumor cell killing.

This literature landscape reflects a strong emphasis on combination immunotherapy strategies, where MD5-1 serves as a backbone for exploring synergistic effects with vaccines, checkpoint inhibitors, and engineered biologics.

Key findings from the clone MD5-1 antibody in scientific literature are highlighted below:

1. Induction of Apoptosis and Cytotoxicity: MD5-1, an agonistic anti-DR5 antibody, induces apoptosis in cancer cells by activating caspase-dependent death signaling via DR5. This effect is similar to TRAIL-mediated cytotoxicity, suggesting a mechanism involving apoptosis pathways.

2. Antimetastatic Effects: MD5-1 has demonstrated significant antimetastatic effects in mouse models, particularly in reducing lung and liver metastases. This effect is independent of endogenous TRAIL or perforin-mediated cytotoxicity, highlighting its potential as an immunotherapeutic agent.

3. Targeting Myeloid-Derived Suppressor Cells (MDSCs): MD5-1 selectively targets and induces apoptosis in PMN-MDSCs, but not in polymorphonuclear neutrophils (PMNs). This targeting results in a decrease in MDSC survival, which can enhance antitumor immunity by reducing immune suppression.

4. Immune Response Induction: Treatment with MD5-1 can induce CD8+ T cell-mediated immunity, capable of eradicating not only initial tumor cells but also TRAIL-resistant variants. This suggests a role in tumor-specific immune response induction.

5. Mechanism of Action: MD5-1 requires cross-linking (e.g., by FcR-expressing cells) to effectively induce apoptosis in target cells. Its activity is abrogated by pan-caspase inhibitors, indicating a caspase-dependent mechanism.

Overall, MD5-1 is a potent tool for inducing apoptosis in DR5-expressing cells, offering potential therapeutic benefits in cancer treatment by both directly killing cancer cells and modulating the immune environment.

Dosing regimens of the anti-mouse DR5 antibody clone MD5-1 vary considerably across different mouse models and experimental contexts, reflecting adaptations based on tumor type, strain characteristics, and therapeutic combinations.

Standard Monotherapy Dosing

The most commonly employed dosing regimen for MD5-1 monotherapy involves 200 μg administered intraperitoneally on days 0, 4, and 8 following tumor inoculation. This schedule has been used successfully in multiple tumor models including R331 renal carcinoma and 4T1 mammary carcinoma in both SCID and wild-type BALB/c mice. An alternative regimen uses 250 μg (from a 2.5 mg/ml solution) administered intraperitoneally on day 8 after tumor inoculation, as demonstrated in C57BL/6 mice bearing TC-1 tumors.

Some protocols employ 50 μg per mouse when MD5-1 is used in combination therapies. However, this lower dose showed limited efficacy when administered alone in myeloma models, with MD5-1-treated mice showing no significant survival benefit over vehicle-treated controls (median survival 24 versus 26.5 days).

Combination Therapy Adjustments

When MD5-1 is combined with other therapeutic agents, dosing strategies are often modified to balance efficacy with toxicity. In ErbB-2/neuT transgenic mice with established tumors, MD5-1 was administered in combination with anti-ErbB-2 antibodies, achieving a 63.3% complete response rate in 60 treated mice. The specific dosing for this regimen began when tumors reached an average size of 9 mm².

Notably, combination therapy with histone deacetylase inhibitors revealed strain-dependent toxicity concerns. When MD5-1 (50 μg per mouse) was combined with panobinostat in wild-type mice, severe dose-limiting toxicity occurred, with all mice receiving combination therapy reaching endpoints by day 10 despite tumor burden reduction. Even when panobinostat dosage was reduced to 7.5 mg/kg, the combination remained intolerable (median survival 15 versus 18 days for vehicle). However, in C57BL/6.DR5⁻/⁻ mice, the same combination showed no dose-limiting toxicity and provided the greatest survival advantage (median survival 54 versus 30.5 days).

Tumor-Specific Variations

Different tumor models require tailored approaches. For dermal myofibroblast targeting in B6 mice, 100 μg of MD5-1 was administered to investigate potential toxicity. In female 615 mice bearing MFC tumors, 50 μg administered intraperitoneally once every 3 days for three doses significantly inhibited tumor growth.

Mechanistic Considerations

The varying dosing regimens reflect MD5-1's mechanism of action, which requires cross-linking for optimal activity. MD5-1 exhibited cytotoxic activity against 4T1 cells when cross-linked by FcR on P815 cells, but this was abolished by anti-FcR antibody. This cross-linking dependency suggests that in vivo efficacy may be enhanced by Fc receptor engagement on immune cells, potentially explaining why certain dosing schedules and combinations prove more effective in immunocompetent versus immunodeficient mice.

The substantial variation in MD5-1 dosing—ranging from 50 μg to 250 μg per administration—underscores the importance of considering tumor type, mouse strain, immune status, and concurrent therapies when designing experimental protocols.