Anti-Human CD3 [Clone UCHT-1] — Purified in vivo GOLD™ Functional Grade

Clone UCHT-1 is most commonly used in vivo in mice to target human CD3 for the activation, depletion, tracking, or therapeutic manipulation of human T cells—but only in models where mice contain or express human immune components, such as humanized or xenograft mouse models. Its specificity for human CD3 makes it unsuitable for use on the endogenous mouse immune system.

Key in vivo applications of UCHT-1 in mice include:

• Activation of human T cells: UCHT-1 crosslinks the CD3 complex on human T cells, triggering T cell activation, proliferation, and cytokine release for mechanistic and functional studies in vivo.
• Depletion of human T cells: In some settings, UCHT-1 can induce T cell depletion through antibody-dependent cell-mediated cytotoxicity (ADCC) or similar mechanisms.
• Testing immunotherapies: UCHT-1 is widely used to evaluate monoclonal antibodies (including bispecifics), cell therapies, and other immunotherapeutics targeting human T cells in humanized or xenograft mouse models, such as for tumor immunotherapy.
• Manipulating tumor growth: Studies show UCHT-1 can be used in combination with bispecific antibodies (e.g., blinatumomab) to redirect T cells toward tumors or, conversely, to modulate immune responses in cancer models featuring implanted human tumors.
• Phototargeting and conjugate studies: UCHT-1 has been chemically conjugated for in vivo delivery and targeted photolysis to study localized activation within tumors in xenograft models.
• Safety and toxicity testing: Because UCHT-1 is a prototypical anti-CD3, it is also used for safety and cytokine release studies, mirroring clinical trial risk assessments.

Critical context and limitations:

• Mouse CD3 is not recognized: UCHT-1 is strictly specific for human CD3 epsilon and will not bind mouse CD3, so it is used in mice only when human T cells or CD3E expression is present—commonly in humanized (CD3E knock-in) or xenograft (PBMC-engrafted or tissue-engrafted) models.
• Preparation: The antibody must be administered in endotoxin-free, in vivo-grade formulations to avoid nonspecific immune reactions and to ensure accurate modeling of human T cell responses.

In summary, common in vivo applications of UCHT-1 in mice are limited to humanized or xenograft models and center on the targeted activation or manipulation of human T cells for immuno-oncology, basic T cell biology, or preclinical drug testing.

The most commonly used antibodies or proteins studied alongside UCHT-1 in the literature are other immune cell markers and related monoclonal antibodies, particularly in immunophenotyping and functional assays.

Commonly co-used antibodies or proteins include:

• CD19: This antibody is frequently paired with UCHT-1 to distinguish T cells (CD3+, recognized by UCHT-1) from B cells (CD19+) in mixed lymphocyte populations.
• OKT3: Another monoclonal antibody targeting CD3ε, often compared or used alongside UCHT-1 in studies of T cell receptor complex function and antibody binding.
• CD4 and CD8: Antibodies against these molecules are typically used in flow cytometry to further subset T cells into helper (CD4+) and cytotoxic (CD8+) populations.
• CD45: A pan-leukocyte marker, frequently combined with CD3 and others to discriminate various immune cell types in assays.

In functional or activation studies, UCHT-1 is often used with:

• CD69 and CD25: These are activation markers assessed in combination with CD3 antibodies to measure T cell activation status.
• IFNγ and TNFα: Cytokine production is often analyzed in parallel to CD3 engagement for functional studies of T cell responses.

When examining γδ T cells or tumor-killing assays, UCHT-1 Fab fragments are tested with:

• Tumor cell lines such as Daudi and Raji
• Ligand proteins such as phosphoantigens and butyrophilin 3A1, which interact with the γδ TCR.

In summary, the most typical antibodies or proteins used with UCHT-1 across published literature are CD19, OKT3, CD4, CD8, CD45, CD69, CD25, IFNγ, and TNFα—all vital for immune cell phenotyping and T cell function assessment. UCHT-1’s direct comparison and cross-validation with OKT3 is particularly notable in T cell biology studies.

Key findings from scientific literature citing clone UCHT-1 center on its role as a monoclonal antibody targeting human CD3ε, its utility in T cell research, and structural as well as functional consequences of its binding:

• Target and Epitope: UCHT-1 is a monoclonal antibody specific for the human CD3ε chain, a component of the T cell receptor (TCR)/CD3 complex present on most mature T cells. It binds a large, conformational epitope on the CD3ε ectodomain, predominantly involving the B-C, C′-D, and F-G loops as well as C′, D, and F strands, engaging a broad acidic surface opposite the CD3 heterodimer interface.

• Structural Insights:

  • Binding Interface: The structure of UCHT-1 (particularly as an scFv fragment) in complex with CD3ε/δ reveals a highly stable 1:1:1 tripartite structure.
  • Contact Residues and Interactions: The UCHT-1–CD3ε interaction is extensive—~1,789 Ų of buried surface (greater than the average for antibody-protein contacts)—involving salt bridges, hydrogen bonds, and van der Waals contacts.
  • Epitope Breadth: Compared to the widely used anti-CD3 antibody OKT3, UCHT-1 engages a broader section of CD3ε, with partial overlap, but distinct extension into the groove between structural strands.

• Functional Effects:

  • T Cell Activation: UCHT-1 binding has been shown to activate human T cells, though its mechanism differs from OKT3. It can induce upregulation of activation markers (CD69, CD25) and cytokines (such as IFNγ, TNFα) on γδ T cells.
  • Enhancement of Cytotoxicity: The addition of UCHT-1 to human γδ T cells enhances their tumor cell killing capacity in vitro for various cancer cell lines, likely by stabilizing the TCR/CD3 complex in an active conformation.
  • Comparison with OKT3: While both OKT3 and UCHT-1 recognize CD3ε and can trigger T cell proliferation, UCHT-1's mitogenic effects and signaling pathways may differ, producing distinct IL-2-mediated responses.

• Preclinical and Diagnostic Use:

  • Immuno-Oncology: UCHT-1, including forms conjugated to other molecules, is used to investigate T-cell-mediated tumor rejection.
  • Human Immune Engraftment: UCHT-1 is commonly used in studies of adoptive transfer or engraftment of human immune cells into mouse models for immune system tracking and manipulation.

• Technical Notes:

  • UCHT-1 is used in functional, flow cytometry, and immunohistochemistry applications for identifying and isolating human T cells via CD3ε expression.

In summary, clone UCHT-1 is a central reagent in immunology for probing human T cell biology, with structural studies revealing a complex and distinctive binding footprint on CD3ε that underlies its utility in both basic and translational research.

Dosing regimens of clone UCHT-1 (anti-human CD3 monoclonal antibody) vary widely across different mouse models based on several key experimental parameters, including the model system used (especially the type of humanized or immunodeficient mouse), the antibody’s formulation or conjugation, the tumor or disease context, and the mechanistic goals of the study.

Key factors affecting dosing regimens include:

• Mouse model type: Regimens differ across immunodeficient (e.g., NSG, NOG) and humanized mouse models due to variations in immune cell reconstitution and tolerance to human antibodies.
• Tumor or disease context: Doses may be adjusted for hematologic vs. solid tumors, xenograft vs. syngeneic models, or non-tumor immune modulation studies.
• Conjugation and formulation: The pharmacokinetics and bioactivity of UCHT-1 can shift if the antibody is unmodified, fluorescently tagged, or otherwise conjugated, impacting optimal dosing.

Examples of dosing regimens:

• Adoptive Cell Transfer Studies: In studies evaluating engineered T cells (UCHT1-TAC), NSG mice with leukemia or solid tumors were treated with varying doses of T cells expressing UCHT1-based constructs:
  • For leukemia (NALM6): Doses ranged from 1.0 × 10⁶ to 1.2 × 10⁷ engineered T cells per mouse, with higher doses leading to complete attenuation of tumor growth.
  • For solid tumors (e.g., OE19 gastric tumors): Effective doses included 6.0 × 10⁶ engineered T cells per mouse.
  • Lower doses (1.0 × 10⁶ engineered T cells) revealed efficacy differences between wild-type and mutant UCHT1 constructs, indicating the importance of both dose and variant.
• General Mouse Studies: Suppliers of functional-grade anti-human CD3 (UCHT-1) antibodies emphasize that precise dosing must be empirically determined for each mouse model and experimental aim. There is no single standardized dosing protocol; published studies often optimize dose and schedule for therapeutic effect, toxicity, and immune activation profiles.

Summary Table: Effects on UCHT-1 Dosing Regimens

Important Caveat: The lack of a universal dosing guideline means that published and vendor protocols should be consulted for each specific experimental setup, often starting with pilot titration studies.

In summary: UCHT-1 dosing regimens are highly model and application dependent, ranging from single, low-dose injections to high and repeated doses, with specific regimens reported in the literature determined by experimental context and mouse immune reactivity. Validation and adjustment are recommended for each new study.