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

Clone RMT3-23 is a rat monoclonal antibody against mouse TIM-3 (CD366), and is widely used in mice for in vivo functional blockade of the TIM-3 immune checkpoint receptor, particularly to investigate TIM-3’s role in immune regulation, tumor immunity, and autoimmunity.

Key in vivo applications include:

• Immune checkpoint blockade for cancer immunotherapy research: RMT3-23 is administered to mice to block TIM-3, releasing inhibition on T cell responses, and is used to examine antitumor immunity, either alone or in combination with other checkpoint inhibitors. This can lead to suppression of tumor cell growth in murine cancer models.

• Study of autoimmunity and inflammatory diseases: RMT3-23 is applied to assess TIM-3's function as a negative regulator of Th1-mediated diseases (e.g., experimental autoimmune encephalomyelitis for multiple sclerosis models, graft-versus-host disease, type 1 diabetes, and inflammatory bowel disease) by promoting or suppressing specific immune responses.

• Alloimmunity and transplantation tolerance: In mouse models of transplant (e.g., allogeneic skin or cardiac allografts), RMT3-23 is used to modulate T cell responses, test for enhanced alloimmune activation, and to study immune tolerance mechanisms. RMT3-23 treatment can increase the frequency of effector cytokine-producing T cells and regulate Treg induction in transplant models.

• Regulation of phagocytosis and innate immunity: RMT3-23 has been used to inhibit TIM-3-mediated phagocytosis of apoptotic cells by Mac1+ peritoneal exudate cells, probing TIM-3’s role on myeloid cells and innate immune tolerance.

• Investigations of other TIM-3–expressing cell types: TIM-3 is also expressed on regulatory T cells, NK cells, mast cells, and myeloid cells, broadening the application of RMT3-23 to various immunological models in vivo.

RMT3-23 is frequently administered intraperitoneally (i.p.) in these studies at defined doses (e.g., 250–500 µg per injection), and its effects are monitored via flow cytometry, cytokine analysis, and functional immune assays.

In summary:
The most common in vivo applications of clone RMT3-23 in mice are for functional blockade of TIM-3 in studies of tumor immunity, autoimmunity (including EAE and diabetes), transplantation, and regulation of phagocytosis, helping to dissect TIM-3's role as an inhibitory immune checkpoint in diverse immunological contexts.

Commonly used antibodies and proteins in the literature alongside RMT3-23 (an anti-mouse TIM-3/CD366 antibody) include other immune checkpoint inhibitors such as anti-PD-1 and anti-PD-L1 antibodies, as well as antibodies targeting LAG-3 and alternative anti-TIM-3 clones like B8.2C12. These combinations are frequently used to study synergistic effects on immune modulation, especially in tumor immunity research.

Key points:

• Anti-PD-1 (e.g., clones like RMP1-14 for mouse), anti-PD-L1, and sometimes anti-LAG-3 antibodies are commonly paired with RMT3-23 in combination immunotherapy or mechanistic studies, based on their roles as distinct but complementary immune checkpoint inhibitors.
• Alternative anti-TIM-3 clones, such as B8.2C12, are often used in comparative studies to validate findings or assess differences in efficacy and specificity between different clones.
• Proteins or ligands relevant to the TIM-3 pathway, such as galectin-9, PtdSer (phosphatidylserine), and CEACAM1, are also studied in conjunction with RMT3-23 for understanding signaling mechanisms.

These combinations are especially important in preclinical tumor models, autoimmune disease studies, and immune exhaustion research, reflecting the central role of RMT3-23 within broader immunoregulatory investigations.

Clone RMT3-23 is a widely used rat IgG2a monoclonal antibody targeting mouse Tim-3 (CD366), an immunoregulatory receptor involved in T cell exhaustion and tumor immunity. Key findings from scientific citations using clone RMT3-23 are:

• Epitope specificity: RMT3-23 binds a unique, non-overlapping epitope on mouse Tim-3 distinct from other anti-Tim-3 clones (such as B8.2C12 and 5D12), facilitating reliable detection and mechanistic studies, even during combination antibody therapy or tracking Tim-3+ cells in treated animals.

• Receptor antagonism and mechanism: RMT3-23 acts as a TIM-3 receptor antagonist, effectively blocking Tim-3 function without depleting Tim-3+ cells in vivo. Unlike some antibodies that induce target cell depletion via Fc effector functions, RMT3-23 primarily down-modulates Tim-3 surface expression, especially when crosslinking occurs, but does not significantly reduce CD4+ or CD8+ T cell numbers, nor Treg frequencies in the tumor microenvironment.

• Allelic specificity: RMT3-23 binds both BALB/c and C57BL/6 alleles of mouse Tim-3, in contrast to clone B8.2C12, which binds only BALB/c Tim-3. This makes RMT3-23 particularly versatile for murine immunology studies.

• Preclinical efficacy: In mouse models of cancer (e.g., WT3 sarcoma), RMT3-23 demonstrates monotherapeutic antitumor efficacy and can enhance antitumor immunity when used alone or in combination with anti-PD-1 therapy.

• Functional studies: RMT3-23 is used to:

  • Block Tim-3:phosphatidylserine (PS) and Tim-3:CEACAM1 interactions, complicating T cell–APC crosstalk and effecting antitumor immunity.
  • Inhibit Tim-3 mediated T cell trogocytosis, which is a process that limits antitumor responses.
  • Study the dynamics and regulation of Tim-3+ immune cells during experimental therapies and infection processes.

• Usage and detection: RMT3-23 is compatible with a variety of detection platforms (flow cytometry, in vivo blockade, functional assays), facilitating both mechanistic and therapeutic studies in mouse models.

• Summary Table

These findings position RMT3-23 as a gold-standard tool for dissecting Tim-3 mediated immunoregulation and for developing combinatorial immunotherapy strategies in preclinical mouse models.

Dosing regimens of clone RMT3-23 (anti-TIM-3) in mouse models vary significantly based on the experimental context, disease model, and therapeutic goals. The protocols differ primarily in dose amounts, frequency, and timing of administration.

Maternal Tolerance and Pregnancy Models

In pregnancy and maternal tolerance studies, RMT3-23 follows a specific declining dose schedule. Pregnant mice receive four intraperitoneal injections at 500 μg, 500 μg, 250 μg, and 250 μg on gestational days 6.5, 8.5, 10.5, and 12.5 respectively. This protocol uses higher initial doses that taper over time, reflecting the critical windows of immune regulation during early pregnancy. This dosing schedule has been well-documented and represents the most robust published protocol for RMT3-23.

Cancer Immunotherapy Models

Tumor models employ different dosing strategies compared to pregnancy studies. In checkpoint inhibitor combination therapy experiments using CT26 and MC38 tumor xenografts, mice receive 250 μg of anti-TIM-3 (RMT3-23) on days 3, 6, and 9 following tumor inoculation. This regimen uses consistent doses throughout treatment and is administered in combination with other checkpoint inhibitors like anti-PD-1 (200 μg) as part of combination immunotherapy protocols. The dosing is designed to approximate the 3-10 mg/kg range used in human clinical studies when scaled appropriately for mice.

Key Dosing Considerations

The choice of dosing regimen depends on several factors:

• Disease model: Pregnancy models use higher initial doses with tapering, while tumor models maintain consistent doses
• Treatment duration: Pregnancy protocols span approximately one week across critical gestational periods, whereas cancer models follow tumor growth dynamics
• Combination therapy: When used with other checkpoint inhibitors, TIM-3 blockade doses are coordinated with companion antibodies
• Route of administration: All documented protocols use intraperitoneal injection as the standard delivery method

The variation in these protocols reflects the distinct immunological mechanisms at play—immune tolerance in pregnancy versus anti-tumor immunity—requiring tailored approaches to effectively modulate TIM-3 signaling in each context.