Anti-Mouse CD19 [Clone 1D3] — Purified in vivo PLATINUM™ Functional Grade

Common In Vivo Applications of Clone 1D3 in Mice

Clone 1D3 is a rat monoclonal antibody that specifically targets mouse CD19, a key marker expressed on B cells. Its epitope is cross-competitive with clone 6D5, confirming its specificity for CD19. In vivo, it is widely used due to its ability to bind and modulate CD19-expressing B cells in mice.

Main In Vivo Applications

B Cell Depletion
The most prominent in vivo application of 1D3 is targeted B cell depletion. Administering 1D3 antibody to mice results in effective removal of B cells from the circulation and lymphoid tissues, a process critical for studies of immune system function, autoimmunity, and B cell malignancies. Notably, some protocols combine 1D3 with an anti-mouse B220 antibody (clone RA3) for enhanced B cell depletion.

CD19 Neutralization
1D3 is used to block CD19 signaling in vivo, which can modulate B cell responses, including proliferation, survival, and antibody production.

Chimeric Antigen Receptor (CAR) T Cell Studies
The variable regions of 1D3 have been cloned and incorporated into mouse CAR-T constructs. For instance, 1D3-28Z (a CAR construct using 1D3’s antigen-binding domain) has been tested in mouse lymphoma models, where adoptive transfer of 1D3 CAR-T cells led to effective elimination of both lymphoma cells and normal B cells, but with a lack of overt systemic toxicity—only B cell depletion was observed. These models are valuable for preclinical evaluation of CAR-T therapies targeting B cell malignancies.

Development of Switchable CAR Systems
The 1D3 Fab has also been used to develop switchable CAR-T platforms, where the presence of a CD19-targeting switch (derived from 1D3) controls CAR-T activity in vivo, offering a way to temporally regulate B cell depletion and CAR-T expansion.

B Cell Functional Studies
1D3 is employed in studies investigating B cell biology, such as the role of CD19 in immune responses and the effects of CD19 modulation on B cell subsets. Its use often involves analysis by flow cytometry of blood, spleen, lymph nodes, and bone marrow following antibody administration.

Typical Experimental Procedures

• Dosing: Common doses include intraperitoneal injection (e.g., 2 mg per mouse in CAR-T studies) or lower doses for depletion/neutralization.
• Schedule: Dosing may be single or repeated (e.g., every other day for multiple cycles in switchable CAR-T models).
• Outcome Measurement: Efficacy is typically monitored by flow cytometry (B cell counts in blood and tissues), tumor regression (in cancer models), or functional immune assays.

Summary Table: Key In Vivo Uses of Clone 1D3

Conclusion

Clone 1D3 is a versatile tool in mouse immunology, primarily used for in vivo B cell depletion and neutralization, as well as in the construction and evaluation of murine CAR-T and switchable CAR-T therapies targeting CD19. Its applications are foundational for studies of B cell biology, autoimmune disease, and B cell malignancies in preclinical mouse models.

Commonly used antibodies or proteins employed together with 1D3 (anti-mouse CD19 monoclonal antibody) in the literature are anti-mouse B220 (clone RA3-6B2), anti-mouse CD22 (clone Cy34.1), and anti-rat λ (lambda) light chain (clone MAR 18.5).

Supporting details:

• Anti-mouse B220 (RA3-6B2): A pan-B cell marker often used alongside 1D3 to define B-cell subpopulations and for more reliable B cell identification in mouse samples.
• Anti-mouse CD22 (Cy34.1): Another B cell marker, CD22 is useful in combination with 1D3 to further characterize B cell subsets and their activation states.
• Anti-rat λ light chain (MAR 18.5): Used in some flow cytometry protocols to distinguish specific B cell receptor isotypes or for gating strategies in conjunction with anti-CD19.

Additional context:

• 1D3 (anti-CD19) is most frequently used to label B cells in mouse models, commonly in flow cytometry, immunohistochemistry, and depletion studies.
• In some B cell depletion protocols, 1D3 is used in combination with anti-B220 to maximize B cell removal from a given sample.
• 1D3 is frequently used with panels of antibodies for multiparametric flow cytometry, which may also include markers for CD21, CD23, CD45R/B220, IgM, and IgD, depending on the specific experimental goals (e.g., to define B cell developmental stages or subtypes).

These combinations are widely adopted to ensure accurate and comprehensive phenotyping, depletion, or tracking of B cells in a variety of research applications, particularly in immunology and hematology.

Clone 1D3 is a monoclonal antibody most widely cited for its specificity to mouse CD19, a transmembrane glycoprotein critical for B cell development, signaling, and function. Key findings from scientific literature on clone 1D3 are as follows:

• B Cell Detection and Characterization: Clone 1D3 is extensively used in immunology for flow cytometry, immunoprecipitation, and in vivo B cell depletion studies, helping to track B cell populations throughout mammalian development except terminally differentiated plasma cells. Its specificity for CD19 makes it a gold standard for identifying and distinguishing B cells from other cell types.

• CD19 Functional Studies: CD19, targeted by 1D3, forms a complex with CD21, CD81, and MHC class II at the B cell surface and associates with the B cell receptor (BCR). Engaging this complex via 1D3 induces downstream effects such as tyrosine phosphorylation, calcium flux, and B cell proliferation, which are fundamental to immune signaling.

• B Cell Depletion: Many studies use 1D3 in vivo to deplete B cells in murine models, enabling research into B cell roles in autoimmune diseases, lymphoma, and antibody production. This depletion is often leveraged to establish causal links between B cells and disease states or immune responses.

• Functional Assays and Epitope Mapping: Clone 1D3 is validated for high performance in flow cytometric analyses and is utilized to provide discrete epitope binning for expanded B cell clones, revealing insights into B cell diversity and epitope convergence during immune responses.

• Antibody Engineering and Modification: Recent efforts include the de novo sequencing and recombinant expression of 1D3, allowing custom modifications, affinity maturation, and production of functionally identical surrogates for advanced applications, such as elucidating the alternative conformations of specific proteins and probing new aspects of cellular signaling. This expands the utility of 1D3 beyond B cell detection into biochemical and biophysical investigations.

• Reagent Reliability: Numerous manufacturers confirm that clone 1D3 provides consistent results across various conjugates (PerCP, APC, PE) and applications (ELISA, immunoprecipitation, depletion, functional assays), with recommendations for titration to optimize performance in specific experimental contexts.

In summary, clone 1D3 is a foundational tool for mouse B cell research, enabling mechanistic studies, functional manipulations, and detailed immunophenotyping, with ongoing improvements for recombinant engineering and expanded assay compatibility.

Dosing regimens of the anti-mouse CD19 monoclonal antibody clone 1D3 vary significantly across mouse models, depending on the strain, immune context, experimental goals, and the desired degree and duration of B cell depletion.

Key variations in dosing regimens include:

• Dose Amount:
  For B cell depletion, reported doses of clone 1D3 typically range from single low doses (e.g., 50–100 µg per mouse) for short-term labeling, to higher repeated doses (e.g., 300 µg per mouse, every few days) for sustained depletion or functional studies.
  Some protocols recommend 300 µg of 1D3 per mouse, often in combination with 300 µg of anti-mouse B220 (RA3.3A1/6.1) for efficient B cell depletion in standard C57BL/6 mice.

• Dosing Schedule & Frequency:
  Schedules range from a single administration for brief depletion to multiple doses every other day or weekly for ongoing depletion in chronic or tumor settings. In complex regimens (such as switch-based CAR-T therapies), the 1D3-based switch is dosed every other day for up to 8 doses per cycle, with rest periods between cycles to allow for immune recovery or monitoring.

• Mouse Model Dependency:
  The immune background of the mouse strain (e.g., C57BL/6 vs. BALB/c vs. genetically engineered or tumor-bearing models) affects both responsiveness to 1D3 and required dosing. For example, syngeneic tumor models and genetically engineered strains often require adjustment in both dose and frequency to achieve the targeted degree and duration of B cell depletion.
  Some strains may be more resistant or sensitive to antibody-mediated depletion, necessitating titration before experimental use.

• Route of Administration:
  Most commonly used routes are intraperitoneal (i.p.) or intravenous (i.v.) injection, with the route affecting the kinetics and perhaps the efficiency of cell depletion.

Example Regimens by Model/System:
| Mouse System | Typical Dose & Schedule | Notes ||----------------------------------------|-------------------------------------------------------------------|----------------------------------------|| Standard B cell depletion (C57BL/6) | 300 µg 1D3 ± 300 µg anti-B220, i.p., 1–3 times per week | For sustained depletion || Tumor-bearing (CAR-T switch model) | 1 mg/kg (~20–30 µg), i.v., every other day ×8 per cycle, 2–3 cycles | For iterative B cell control || Syngeneic or GE strains | Dose/frequency adjusted experimentally | Varies by immune sensitivity || Flow cytometry labeling | Single low dose (e.g., 10–50 µg) | Not intended for depletion |

Essentially, the optimum 1D3 dosing regimen must be titrated for each mouse model and experimental endpoint. Researchers are advised to monitor the degree of B cell depletion by flow cytometry or similar methods and adjust accordingly.

References to combination approaches:
Maximal B cell depletion often leverages combination antibodies (e.g., 1D3 with anti-B220) or additional immune modulating therapies.

Conclusion:
There is no universal 1D3 dosing protocol; regimens must be tailored to mouse strain, immune status, and experimental design, with ranges from low, single doses for labeling to high, repeated doses (e.g., 200–300 µg per mouse) for efficient B cell depletion.