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

Clone MD5-1 is an agonistic antibody against mouse DR5 (CD262/TRAIL-R2) with several key in vivo applications in preclinical mouse models, primarily focused on cancer research and immunotherapy.

Cancer Therapy and Tumor Rejection

MD5-1 has been extensively tested as a cancer therapeutic agent in mouse models. The antibody induces TRAIL-mediated apoptosis in tumor cells when administered in vivo. Studies have demonstrated its efficacy against various tumor types, including mammary carcinoma (4T1) and renal carcinoma (R331) in syngeneic tumor models. When administered at doses of 200 μg on days 0, 4, and 8 following tumor inoculation, MD5-1 can promote complete tumor rejection in immunocompetent mice.

Induction of Tumor-Specific T Cell Immunity

Beyond direct cytotoxic effects, MD5-1 generates lasting antitumor immunity. Mice that reject tumors after MD5-1 treatment develop immunological memory, becoming resistant to rechallenge with the same tumor cells. This protective immunity is mediated by both CD8+ and CD4+ T cells and involves death ligands including FasL and TRAIL. The antibody effectively primes T cell responses, with the kinetics of this priming critical for establishing durable antitumor immunity.

Selective Depletion of Myeloid-Derived Suppressor Cells

A particularly important application is the selective depletion of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. A single dose of MD5-1 significantly reduces MDSC accumulation, decreasing granulocytic MDSCs by approximately 40% and monocytic MDSCs by approximately 76% in tumor tissues. This depletion also occurs systemically in the spleen, reducing gMDSCs by about 26% and mMDSCs by about 34%. Importantly, MD5-1 selectively targets MDSCs without affecting other myeloid populations such as macrophages and dendritic cells. The reduction in immunosuppressive MDSCs leads to increased CD3+ T cell infiltration in tumors, thereby enhancing antitumor immune responses.

Combination Therapies

MD5-1 has been investigated in combination with other therapeutic agents, including histone deacetylase inhibitors like panobinostat, in multiple myeloma models. These combination approaches aim to enhance the therapeutic efficacy by targeting multiple pathways simultaneously.

Commonly used antibodies or proteins that are co-administered or studied along with MD5-1 (an anti-mouse DR5 monoclonal antibody) include:

• DNA vaccines encoding tumor antigens, particularly HPV-16 E7 antigen fused to calreticulin (CRT/E7), are frequently combined with MD5-1 in cancer immunotherapy studies. This combination boosts antigen-specific CD8+ T cell responses and enhances antitumor effects.
• Isotype-matched control antibodies are routinely used alongside MD5-1 as negative controls in flow cytometry or in vivo experiments to validate the specificity of MD5-1.
• Other DR5 antibodies such as AMG655 and KMTR2 have been engineered in dual specificity constructs with MD5-1 for comparative or synergistic studies targeting DR5-mediated apoptosis pathways.
• Antibodies against apoptosis pathway proteins, such as caspase-8, are used in immunoprecipitation or western blotting to assess downstream signaling and apoptotic activity when cells are treated with MD5-1.
• E7-specific cytotoxic T lymphocytes (CTLs) (not antibodies, but relevant immune effectors) are often examined for their activity in conjunction with MD5-1 treatment, as MD5-1 can sensitize tumor cells to CTL-mediated lysis.

Additional frequently used reagents in experiments involving MD5-1 include:

• Antibodies against mouse DR5/CD262 for flow cytometric detection.
• TRAIL (TNF-related apoptosis-inducing ligand), the endogenous ligand for DR5, as a positive control or comparative agent in apoptosis studies.

These combinations are common in experimental oncology, particularly when studying synergistic or additive effects on tumor rejection, apoptosis, or immune responses.

The key findings from scientific literature citing clone MD5-1—an agonistic anti-mouse DR5 (Death Receptor 5, CD262) antibody—are as follows:

• Induction of DR5-mediated apoptosis and cytotoxicity: MD5-1 efficiently induces apoptosis in DR5-expressing murine tumor cells, but is typically effective when Fc crosslinking occurs, mimicking immune effector cell engagement. MD5-1 cytotoxicity is strictly caspase-dependent and closely parallels sensitivity to TRAIL-mediated apoptosis.

• Antimetastatic and antitumor effects: In murine models, MD5-1 significantly inhibits spontaneous metastasis and prolongs survival, even when administered after large tumor burdens have already formed. The antimetastatic effect is independent of endogenous TRAIL or perforin pathways, instead relying on DR5 death signaling.

• Induction of anti-tumor immunity: Treatment with MD5-1 eradicates susceptible tumors and induces a CD8+ T cell–mediated immune response. Remarkably, this immunization effect enables mice to subsequently reject not only the original tumor but also resistant variants, emphasizing its role in immunological memory.

• Selective depletion of immunosuppressive cells: MD5-1 selectively induces apoptosis in polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), sparing normal neutrophils (PMNs) and other myeloid cells. This reduces tumor-induced immune suppression and enhances CD8+ T cell-mediated antitumor activity in vivo. In tumor-bearing mice, administration of MD5-1 decreases MDSC numbers and delays tumor progression.

• Mechanistic insights and limitations:

  • The apoptotic effect of MD5-1 is lost in cells with blocked caspase 8 signaling or in the absence of Fc crosslinking.
  • While the antitumor effects are robust, they depend on the susceptibility of target cells to DR5-mediated apoptosis—cells overexpressing FLIP (an inhibitor of caspase-8) or otherwise resistant to DR5 signaling are not affected by MD5-1.

• Safety and targeting:

  • The antitumor and potential hepatotoxic effects of MD5-1 are context-dependent and require Fc receptor engagement.
  • MD5-1 specifically targets DR5-expressing cells, making it useful in dissecting the function of DR5 and studying DR5-targeted therapies in mouse models.

These conclusions are grounded in published mechanistic studies and preclinical mouse tumor models, which collectively establish MD5-1 as a valuable tool for exploring DR5 signaling, tumor immunotherapy, and selective depletion of immunosuppressive cells in vivo.

Dosing regimens of clone MD5-1, an anti-mouse DR5 monoclonal antibody, vary across mouse models primarily in dose amount, frequency, administration route, mouse strain, and combination with other agents.

Key dosing regimens in published studies include:

• Wild-type BALB/c, SCID, and immune-deficient models (tumor growth inhibition):

  • Dose: 200 μg per mouse, intraperitoneally (i.p.)
  • Schedule: Days 0, 4, and 8 after tumor cell inoculation.
  • Variants: Used alone or in combination with immune cell depletion or rechallenge experiments.

• C57BL/6 mice (TC-1 tumor model):

  • Dose: 250 μg per mouse, i.p. (as 100 μl of a 2.5 mg/ml solution)
  • Schedule: Single dose on day 8 after tumor inoculation.

• 615 mice (MFC tumor model):

  • Dose: 50 μg per mouse, i.p.
  • Schedule: Once every 3 days, for a total of three doses.

• C57BL/6 (dermal myofibroblast targeting):

  • Dose: 100 μg per mouse, i.p.
  • Schedule: Dosing frequency not always specified, but typically resembles the above repeat dosing strategies.

• Transgenic and syngeneic tumor models (combination therapy, e.g., panobinostat+MD5-1):

  • Dose: 50 μg per mouse, i.p.
  • Schedule: Specific timing varies depending on the study design and combination partner.

Factors influencing regimen differences:

• Mouse strain and genetic background: Some regimens use wild-type, immune-deficient (SCID, perforin knockout), or transgenic mice to test for on-target toxicity or immune dependency.
• Tumor model: The choice of tumor cell line (e.g., 4T1, TC-1, MFC, Vk*MYC) and tumor location affect the dose and schedule.
• Combination therapy: Lower doses are sometimes used in combination regimens due to additive toxicities (e.g., with histone deacetylase inhibitors like panobinostat or vorinostat).

General principles:

• The most common dose range for MD5-1 is 50–250 μg per mouse, usually administered i.p.
• Monotherapy regimens tend to use higher single or repeat doses; combination therapy regimens often reduce dose frequency or amount due to enhanced or synergistic toxicity.
• Treatment schedules typically start at or shortly after tumor inoculation, with injections spaced several days apart (often 3–4 days).

Summary Table: MD5-1 Dosing Regimens Across Mouse Models

Regimen selection should be tailored to the mouse strain, tumor type, and experimental goals, with close attention to toxicity, especially in combination therapy.