Anti-Mouse CD366 (Tim-3) [Clone RMT3-23] — Purified in vivo PLATINUM™ Functional Grade

Clone RMT3-23 is a rat monoclonal antibody widely used in in vivo studies with mice to functionally block TIM-3 (CD366), a key immune checkpoint receptor. Its main applications target the modulation and investigation of immune cell regulation, tolerance, and anti-tumor immunity in murine models.

Key in vivo applications of RMT3-23 in mice include:

• Functional Blockade of TIM-3 in Immunoregulation and Tumor Models: RMT3-23 is administered (typically by intraperitoneal injection) to mice to inhibit TIM-3, allowing researchers to study its role in controlling T cell activity and tumor immunity, as well as its potential as a checkpoint blockade therapy in cancer immunotherapy studies.

• Study of T Cell Responses:

  • Regulation of Th1 and Tc1 Cells: TIM-3 is predominantly expressed on differentiated Th1 (CD4⁺) and Tc1 (CD8⁺) cells. RMT3-23 helps dissect TIM-3's role in restricting Th1-mediated inflammatory disease models such as experimental autoimmune encephalomyelitis (EAE), graft-versus-host disease (GVHD), and type I diabetes.
  • Induction and Regulation of Tregs: Used in transplantation and tolerance models (e.g., skin or cardiac allografts), RMT3-23 administration modifies the balance between effector T cells and regulatory T cells (Tregs), influencing immune regulation and tolerance induction.

• Inflammatory and Autoimmune Disease Models:

  • Experimental Autoimmune Encephalomyelitis (EAE) and Type I Diabetes: RMT3-23 is employed to study exacerbation or amelioration of Th1-driven autoimmunity by blocking TIM-3-mediated restraint.
  • Inflammatory Bowel Disease (IBD): TIM-3 blockade can help determine its suppressive effect on intestinal inflammation.

• Transplantation and Alloimmunity:

  • In allograft models, RMT3-23 enhances the alloimmune response, promotes effector T cell expansion, and increases the frequency of inflammatory T cells producing IFN-γ, IL-6, IL-17, and granzyme B, providing insight into graft rejection and tolerance mechanisms.
  • Facilitates analysis of conversion and expansion of Treg cells in the context of transplant tolerance and in combination with other immunomodulatory agents.

• Study of Macrophage and Myeloid Cell Function:

  • Functional blockade by RMT3-23 reveals TIM-3's role as a phagocytic receptor on macrophages and its involvement in the recognition and clearance of apoptotic cells.

• Cancer Models:

  • Used in conjunction with or compared to other checkpoint inhibitors to assess the effect of TIM-3 blockade on tumor growth, anti-tumor immune responses, and immunotherapeutic synergy.

Additional applications include flow cytometry and immunohistochemistry for detection/staining of TIM-3 on immune cell populations in mouse tissues during or after in vivo functional studies.

In summary, RMT3-23 is predominantly used to block TIM-3 signaling in mice, thereby enabling the study of immune checkpoint function in models of cancer, transplantation, autoimmunity, inflammation, and T cell regulation.

Other commonly used antibodies or proteins applied with RMT3-23 (anti-mouse TIM-3/CD366) in the literature include immune checkpoint inhibitors targeting PD-1 and PD-L1, as well as alternative anti-TIM-3 antibody clones and proteins or ligands interacting with the TIM-3 pathway.

Key combinations seen in studies are:

• Anti-PD-1 antibodies: Many preclinical tumor immunology studies investigate the synergy between TIM-3 and PD-1 blockade, since dual inhibition can overcome adaptive resistance and improve antitumor immunity.
• Anti-PD-L1 antibodies: These are also used in combination with RMT3-23 to evaluate broader checkpoint blockade effects.
• Alternative anti-TIM-3 clones: Other anti-mouse TIM-3 monoclonal antibodies, such as B8.2C12, are used alongside RMT3-23 for comparative or confirmatory purposes in blocking and binding studies.
• Ligands of TIM-3: Proteins such as galectin-9, CEACAM1, and phosphatidylserine (PtdSer), which are known to interact with TIM-3, are often used to probe the specificity and blocking efficiency of RMT3-23 in functional assays.
• Oligonucleotide aptamers and small domains (sdAbs): Engineered molecules targeting TIM-3 (e.g., aptamers, sdAbs like R23 and R53) are sometimes used as alternatives or comparators to RMT3-23 in studies on T cell function and tumor immunity.

RMT3-23 is also frequently used together with markers for T cell subsets (such as anti-CD8 or anti-CD4) and activation markers in multicolor flow cytometry, as well as in functional experiments investigating macrophage activation, phagocytosis, and Th1-mediated immunity.

In summary, the most commonly paired reagents with RMT3-23 are:

• Anti-PD-1
• Anti-PD-L1
• Other anti-TIM-3 clones (e.g., B8.2C12)
• TIM-3 ligands (galectin-9, CEACAM1, PtdSer)
• T cell and myeloid cell lineage/activation markers

These combinations enable comprehensive analysis of TIM-3-mediated immune regulation and checkpoint blockade effects in preclinical models.

The clone RMT3-23 is a rat IgG2a monoclonal antibody widely used to target murine TIM-3 (CD366) in immunology and oncology research. Key findings from scientific literature citing RMT3-23 are:

• Epitope Specificity: RMT3-23 binds a unique, non-overlapping epitope on mouse TIM-3, distinct from other common anti-TIM-3 clones such as B8.2C12 and 5D12. This allows for reliable detection and tracking of TIM-3-expressing cells in experimental contexts, even when used alongside other anti-TIM-3 antibodies.

• Allele Reactivity: Unlike B8.2C12 (which binds only to BALB/c TIM-3), RMT3-23 efficiently binds both BALB/c and C57BL/6 mouse TIM-3 alleles, making it broadly applicable across common mouse strains.

• Functional Properties: RMT3-23 antagonizes TIM-3 function and has demonstrated monotherapeutic efficacy in mouse cancer models, suppressing tumor growth by blocking TIM-3-mediated immunoregulation.

• Down-Modulation, Not Depletion: Administration of RMT3-23 does not deplete TIM-3\^+ T cells in vivo; rather, it causes significant down-modulation of TIM-3 surface expression on these cells, a process that requires antibody cross-linking. This distinguishes its mechanism from cell-depleting antibodies.

• Tumor Microenvironment Effects: RMT3-23 does not reduce the frequency of regulatory T cells (Tregs) in tumors, but its use in combination with anti-PD-1 antibodies can more robustly reduce immune suppressive cell populations and enhance anti-tumor immunity compared to either blockade alone.

• Target Site: RMT3-23 binds at or near the phosphatidylserine (PS) binding site of TIM-3, directly interfering with TIM-3:PS-dependent immune suppression and trogocytosis (exchange of membrane fragments) by T cells within tumors.

• Research Utility: Due to its specificity and cross-strain reactivity, RMT3-23 is the most commonly used functional anti-murine TIM-3 antibody in checkpoint blockade and mechanistic T cell studies. It is often used to test the impact of TIM-3 in T cell regulation, exhaustion, and tumor immunology.

In summary, clone RMT3-23 is validated as a functional and widely applicable reagent for antagonizing TIM-3 activity in mice, with key roles in studies of immune regulation, anti-tumor immunity, and T cell checkpoint blockade.

Dosing regimens of clone RMT3-23 (anti-TIM-3) in mouse models show substantial variation depending on the disease context, targeted cell types, and experimental objectives, but are most commonly administered intraperitoneally over a schedule of multiple days.

For example:

• Maternal tolerance (pregnancy) models: Pregnant CBA/CaJ and C57BL/6J female mice received a regimen of 4 intraperitoneal injections: 500 μg on day 6.5, 500 μg on day 8.5, 250 μg on day 10.5, and 250 μg on day 12.5 of gestation. This specific 500/500/250/250 μg protocol is the most rigorously documented in maternal immune tolerance research.

• Tumor and immunotherapy models: In syngeneic tumor models (CT26 and MC38), C57BL/6 or BALB/c mice received intraperitoneal injections of 250 μg of RMT3-23 on days 3, 6, and 9 following tumor cell inoculation, often in combination with other checkpoint inhibitors (e.g., anti-PD-1 at 200 μg). The rationale for these doses is to approximate efficacious ranges used in related checkpoint blockade studies.

General details:

• Route of administration is consistently intraperitoneal.
• Dose and schedule are adapted to match the disease model, immune cell kinetics, and experimental endpoint.
• Other models or applications (e.g., adoptive transfer, infection, or general immune studies) may use variations of the above regimens, but most published protocols fall within a similar range of 250–500 μg, typically split over several days.

If considering use in a new model:

• Published dosing can serve as a starting point, but optimization by pilot titration studies is recommended, as pharmacodynamics may differ with disease state, tissue distribution, and therapeutic goal.

In sum: 500/500/250/250 μg schedule in pregnancy models; 250 μg every 3 days in tumor studies—both via intraperitoneal injection—are the most prevalent regimens. Adjustments are made based on model and outcome measures.