CD79A Monoclonal Antibody

What is CD79A and what role does it play in B-cell receptor signaling?

CD79A is a transmembrane glycoprotein in the immunoglobulin superfamily containing a single Ig-like domain in its extracellular region (spanning Leu33-Arg143) and one cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM). Alternative splicing can generate a short isoform with a 39 amino acid deletion in the extracellular domain. Within the extracellular domain, human CD79A shares 57% amino acid sequence identity with mouse and rat CD79A .

CD79A forms a heterodimer with CD79B (Ig beta) that associates with membrane-bound immunoglobulin to form the BCR complex. This heterodimer is essential for BCR-mediated signaling pathways that ultimately lead to B-cell activation, proliferation, and differentiation. CD79A and CD79B are required for both the development and activation of B lineage cells .

How can CD79A expression be detected in experimental settings?

CD79A expression can be detected through several methodological approaches:

Flow Cytometry: The most common method involves using mouse anti-human CD79A monoclonal antibodies (such as clone 706931) followed by detection with fluorochrome-conjugated secondary antibodies. For optimal results, co-staining with CD19 is recommended to accurately identify B-cell populations. When setting up flow cytometry experiments, quadrant markers should be established based on appropriate isotype control antibody staining .

Example protocol:

• Isolate peripheral blood mononuclear cells (PBMCs)

• Stain with Mouse Anti-Human CD79A Monoclonal Antibody (e.g., Catalog # MAB69201)

• Follow with Phycoerythrin-conjugated Anti-Mouse IgG Secondary Antibody

• Co-stain with Mouse Anti-Human CD19 APC-conjugated Monoclonal Antibody

• Analyze by flow cytometry using properly set quadrant markers

Alternative detection methods include immunohistochemistry for tissue sections, western blotting for protein expression analysis, and PCR techniques for mRNA quantification.

How do CD79A monoclonal antibodies compare with CD79B antibodies for targeting B-cell malignancies?

Research has demonstrated significant differences in the therapeutic potential of CD79A versus CD79B targeting:

Efficacy comparison: In xenograft models of B-cell malignancies, anti-CD79B antibody-drug conjugates (ADCs) consistently demonstrated superior efficacy compared to anti-CD79A ADCs. When testing MCC-DM1 conjugates of multiple antibodies (one anti-CD79B antibody (2F2) and three CD79A antibodies (7H7, 15E4, and 16C11)), only the anti-CD79B antibodies (SN8 and 2F2) caused tumor regression, while anti-CD79A antibodies merely slowed tumor growth .

Mechanistic differences: Interestingly, the superior efficacy of anti-CD79B antibodies does not correlate with binding affinity. The research data indicates that all three anti-CD79A antibodies bound cells with higher affinity than anti-CD79B (2F2) as measured by cell-based enzyme-linked immunosorbent assay. This suggests that factors beyond simple binding affinity determine therapeutic efficacy .

Internalization and trafficking: A key advantage of anti-CD79B antibodies is their efficient internalization and trafficking to lysosomal compartments. Anti-CD79B antibodies effectively downregulate the surface BCR and are specifically targeted to the lysosomal-like MIIC (MHC class II-containing compartment), which is essential for the release of active drug metabolites from antibody-drug conjugates .

These findings indicate that while both CD79A and CD79B can serve as targets, CD79B may offer superior characteristics for therapeutic development, particularly for ADC approaches.

What factors influence the sensitivity of lymphoma models to CD79A-targeted therapeutics?

Several important factors influence the sensitivity of lymphoma models to CD79A-targeted therapeutics:

Lymphoma subtype: Different lymphoma subtypes demonstrate variable sensitivity to CD79-targeted therapies. In xenograft studies, Burkitt lymphoma cells (BJAB) were most responsive to anti-CD79B ADCs, followed by follicular lymphoma cells (DoHH2), with mantle cell lymphoma cells (Granta-519) being least responsive .

Target expression levels: Surprisingly, efficacy does not necessarily correlate with CD79 expression levels. Flow cytometry analysis revealed that BJAB and Granta-519 cells had similar and much lower amounts of surface CD79B than DoHH2 cells, indicating that the difference in response did not correlate with target expression levels .

Antibody selection: The specific antibody clone used significantly impacts efficacy, as demonstrated by the superior performance of anti-CD79B clones compared to anti-CD79A clones .

In vitro versus in vivo correlation: The ability of cell lines to respond to anti-CD79B ADCs in xenograft models did not correspond to their in vitro sensitivity to either free drug or anti-CD79B ADCs. This highlights the importance of in vivo testing alongside in vitro studies .

Linker-drug technology: Different linker-drug combinations can affect efficacy, though the search results indicated that MC-MMAF and MCC-DM1 ADCs showed similar efficacies regardless of the cell line used, which was unexpected given their different chemistries .

These findings underscore the complex nature of response to CD79-targeted therapeutics and the need for comprehensive preclinical testing across different lymphoma models.

What is the expression pattern of CD79A across normal and malignant tissues?

CD79A exhibits a highly specific expression pattern that makes it valuable for both diagnostic applications and targeted therapies:

B-cell lineage: CD79A expression is largely restricted to the B-cell lineage, from pre-B cells to mature B cells, though expression may be downregulated in some plasma cells .

Malignant B-cells: CD79A is expressed in various B-cell malignancies, including:

• Burkitt lymphoma (e.g., BJAB cell line)

• Follicular lymphoma (e.g., DoHH2 cell line)

• Mantle cell lymphoma (e.g., Granta-519 cell line)

• Other non-Hodgkin lymphomas

Negative cell types: CD79A is not expressed in T cells (e.g., Jurkat cells) or other non-B lymphoid cells, making it a specific marker for B-cell identification .

This restricted expression pattern makes CD79A an excellent marker for B-cell identification and a promising target for B-cell directed therapies. The B-cell specificity of CD79A ensures that targeting therapies would primarily affect B cells while sparing other cell types.

What mechanisms explain the trafficking of anti-CD79 antibodies to lysosomal compartments?

The trafficking of anti-CD79 antibodies to lysosomal compartments follows a specific mechanistic pathway essential for the function of antibody-drug conjugates:

BCR cross-linking: Anti-CD79 antibodies bind to and cross-link CD79 molecules on the B-cell surface, initiating a signaling cascade .

Receptor-mediated endocytosis: This cross-linking triggers internalization of the BCR complex along with the bound antibody through receptor-mediated endocytosis .

Trafficking to specialized compartments: The internalized complex is specifically directed to the MIIC (MHC class II-containing compartment), which is a specialized lysosomal-like compartment in B cells where antigen processing occurs .

Evidence of this mechanism is supported by experimental data showing surface downregulation of BCR components after anti-CD79B antibody treatment: "Surface expression of CD79A and sIgM were substantially lower in the tumors treated with anti-CD79B antibodies or anti-CD79B ADC compared with tumors treated with control antibodies or ADCs" .

Importantly, this downregulation is specific to the BCR complex, as "the expression of CD22, another cell surface marker, remained the same regardless" of treatment .

This trafficking mechanism is particularly critical for ADC approaches using stable linkers, as these conjugates "must be internalized and degraded, limiting potential targets to surface antigens that are trafficked to lysosomes" .

How do different linker chemistries affect CD79A-targeted antibody-drug conjugate performance?

Linker chemistry plays a crucial role in the performance of CD79-targeted ADCs, with different approaches offering distinct advantages:

MCC-DM1 linker: "The maytansinoid DM1 linked to the antibody through the ε-amino group of lysine using the thioester linker MCC." When targeted to the lysosome, MCC-DM1 ADCs release lysine-Nε-DM1, which functions as an effective antimitotic agent within the cell but is relatively nontoxic once released from the cell .

MC-MMAF linker: "The dolastatin-10 derivative MMAF linked to antibody cysteines with a cleavage-resistant MC linker." Upon trafficking to the lysosome and degradation, this releases cysteine-MC-MMAF .

Comparative in vitro efficacy data from multiple cell lines:

Numbers in parentheses represent standard deviation of quadruplicate assays

Interestingly, despite their different mechanisms, "the MC-MMAF and MCC-DM1 ADCs showed similar efficacies regardless of the cell line used, which is unexpected because the linker and drug chemistries are different" .

These comparisons provide essential insights for researchers designing optimal CD79-targeted ADCs for therapeutic applications.

What experimental approaches are recommended for optimizing CD79A antibody use in flow cytometry?

Optimizing CD79A monoclonal antibodies for flow cytometry requires systematic attention to several experimental variables:

Antibody selection and titration:

• Test multiple clones (such as clone 706931) to identify optimal signal-to-noise ratio

• Conduct careful titration experiments to determine the optimal antibody concentration

• As noted in the search results: "Optimal dilutions should be determined by each laboratory for each application"

Co-staining optimization:

• CD19 provides an excellent co-staining marker with CD79A for B-cell identification

• Detailed protocol from search results: "PBMC with CD19 costain were stained with Mouse Anti-Human CD19 APC‐conjugated Monoclonal Antibody (Catalog # FAB4867A) and either Mouse Anti-Human CD79A Monoclonal Antibody or Mouse IgG..."

Controls implementation:

• Always include proper isotype controls (Mouse IgG is recommended)

• Set quadrant markers "based on control antibody staining"

• Include known positive (B cells) and negative (T cells) populations

Sample preparation considerations:

• For human samples, peripheral blood monocytes can be treated with recombinant human IL-2 to maintain cell viability

• Fresh samples typically yield better results than frozen ones

• Proper fixation protocols may be necessary depending on experimental design

Detection system selection:

• Secondary detection can use "Phycoerythrin-conjugated Anti-Mouse IgG Secondary Antibody"

• Direct conjugates may be preferable for multicolor panels

• Choose fluorochromes based on expected expression level and other panel markers

By systematically addressing these factors, researchers can achieve highly sensitive and specific detection of CD79A in flow cytometry experiments, enabling more precise characterization of B-cell populations in both research and clinical applications.

What are the key considerations for validating CD79A antibody specificity in research applications?

Rigorous validation of CD79A antibody specificity is essential for generating reliable research data. A comprehensive validation approach should include:

Positive and negative controls:

• Positive controls: B-cell lines known to express CD79A

• Negative controls: Non-B-cell lines (e.g., T-cell lines like Jurkat, as mentioned in the search results)

• The research demonstrates this approach by showing that anti-CD79B ADCs effectively killed CD79-positive cell lines (BJAB-luc, SU-DHL-4, DoHH2, Granta-519, and Ramos) but not Jurkat cells (a T-cell line) or Raji cells (which do not express CD79)

Isotype control verification:

• Use appropriate isotype control antibodies (such as Mouse IgG)

• The protocol from the search results specifies: "Quadrant markers were set based on control antibody staining (MAB002)"

Co-expression analysis:

• Validate with co-staining using established B-cell markers like CD19

• The search results describe detection using "Mouse Anti-Human CD79A Monoclonal Antibody followed by Phycoerythrin-conjugated Anti-Mouse IgG Secondary Antibody and Mouse Anti-Human CD19 APC-conjugated Monoclonal Antibody"

Cross-reactivity assessment:

• Test on multiple cell types to confirm B-cell specificity

• Examine potential cross-reactivity with other immunoglobulin superfamily members

Functional validation:

• Confirm that antibody binding affects expected downstream events (e.g., internalization)

• The research shows that anti-CD79B antibodies caused "surface expression of CD79A and sIgM were substantially lower in the tumors treated with anti-CD79B antibodies"

Epitope characterization:

• Identify the specific region recognized by the antibody (the search results mention an antibody binding to the extracellular domain spanning Leu33-Arg143)

• Consider how epitope location may affect antibody function and accessibility

Species cross-reactivity:

• Human CD79A shares 57% amino acid sequence identity with mouse and rat CD79A

• Species-specific validation is necessary when working across different model systems

These validation approaches ensure that experimental results accurately reflect CD79A biology rather than non-specific binding or artifacts.

How do xenograft models inform the development of CD79A-targeted therapeutics for lymphoma treatment?

Xenograft models provide critical insights into the development of CD79A-targeted therapeutics through several key research applications:

Model diversity for therapeutic evaluation:

• The research utilized xenograft models representative of different types of NHL: "The Burkitt lymphoma cell line BJAB contains the t(2;8)(p12;q24) translocation resulting in the overexpression of myc; the mantle cell lymphoma cell line Granta-519 contains the t(11;14)(q13;q32) translocation resulting in the overexpression of cyclin D1 (BCL1); and the follicular lymphoma cell line DoHH2 contains the t(14;18)(q32;q21) translocation resulting in the overexpression of Bcl-2"

Differential response assessment:

• Xenograft studies revealed varying sensitivities across lymphoma subtypes: "The BJAB tumors proved the most responsive to the anti-CD79B ADCs, followed by DoHH2, with Granta-519 tumors being the least responsive"

• This differential response informs potential patient selection strategies for clinical development

In vivo expression analysis:

Antibody comparison studies:

• Xenograft models facilitated direct comparison between different antibodies: "Each of the anti-CD79 ADCs was effective in slowing tumor growth, but only the 2 anti-CD79B antibodies (SN8 and 2F2) caused tumor regression"

Dosing and efficacy relationship:

• The remarkable finding that "a single dose of either anti-CD79B-MC-MMAF or anti-CD79B-MCC-DM1 ADCs resulted in tumor regression or complete remission in all of the tumor models" provides important dosing insights

In vivo/in vitro correlation analysis:

• Xenograft models revealed that "the ability of the cell lines to respond to the anti-CD79B ADCs in the xenograft models did not correspond to the in vitro sensitivity of a given cell line to free drug or anti-CD79B ADCs"

• This emphasizes the importance of in vivo testing alongside in vitro studies

These xenograft studies provide a crucial bridge between in vitro research and clinical development, enabling more informed design of human clinical trials for CD79-targeted therapies in lymphoma.

What methodological approaches should be used to investigate resistance mechanisms to CD79A-targeted therapies?

Investigation of resistance mechanisms to CD79A-targeted therapies requires systematic methodological approaches spanning multiple research disciplines:

Sequential sampling analysis:

• Collect samples from xenograft models before treatment, during response, and upon progression

• Analyze changes in CD79A/B expression, localization, and associated signaling pathways

• Such analysis could help determine whether the target downregulation observed in the research ("surface expression of CD79A and sIgM were substantially lower in the tumors treated with anti-CD79B antibodies") contributes to resistance development

Development of resistant cell lines:

• Generate resistant variants through continuous or intermittent exposure to CD79A-targeted agents

• Characterize molecular changes associated with acquired resistance

• Test cross-resistance patterns to different CD79-targeting modalities

Genomic and transcriptomic profiling:

• Compare sensitive and resistant models using next-generation sequencing

• Identify mutations, expression changes, or splice variants in CD79A/B and related pathway components

• Investigate alternative splicing of CD79A, as the research notes "alternate splicing generates a short isoform with a 39 aa deletion in the ECD"

Trafficking and internalization studies:

• Utilize fluorescently-labeled antibodies to track internalization kinetics

• Compare endocytosis and lysosomal targeting between sensitive and resistant cells

• Build on the finding that "anti-CD79B ADCs cross-link the BCR, causing the internalization of the BCR and delivery of the BCR–ADC complex to the lysosome-like MIIC compartment"

Combination therapy evaluation:

• Test rational combinations targeting complementary pathways

• Evaluate sequencing strategies to prevent or overcome resistance

• Investigate combinations with standard lymphoma therapies

Computational modeling:

• Develop predictive models of resistance based on molecular profiles

• Simulate the impact of different dosing schedules on resistance development

• Incorporate pharmacokinetic and pharmacodynamic data

Patient-derived xenograft (PDX) models:

• Establish PDX models from treatment-naïve and post-progression patient samples

• Characterize response patterns and molecular features

• Identify biomarkers predictive of primary or acquired resistance

These methodological approaches provide a comprehensive framework for understanding resistance mechanisms, ultimately informing strategies to optimize CD79A-targeted therapies and improve patient outcomes.

How can CD79A antibodies be used to investigate B-cell receptor signaling pathways?

CD79A antibodies serve as valuable tools for investigating B-cell receptor signaling pathways through various methodological approaches:

BCR complex isolation and characterization:

• Immunoprecipitation using CD79A antibodies to isolate the intact BCR complex

• Mass spectrometry analysis to identify associated proteins and post-translational modifications

• Investigation of heterodimer formation with CD79B, as "heterodimers of CD79A and CD79B/Ig beta associate with a membrane bound immunoglobulin on the B cell surface to form the B cell antigen receptor complex (BCR)"

Signaling pathway activation and inhibition:

• Use anti-CD79A antibodies to stimulate or block BCR signaling

• Monitor downstream effects on tyrosine phosphorylation cascades initiated through the ITAM motif

• Compare with anti-IgM stimulation to identify CD79A-specific signaling events

Trafficking and internalization studies:

• Track CD79A dynamics during BCR activation using fluorescently-labeled antibodies

• Monitor trafficking to the "lysosomal-like major histocompatibility complex class II–positive compartment MIIC"

• Analyze how CD79A trafficking influences antigen presentation and downstream signaling

Genetic modification coupled with antibody detection:

• Use CRISPR/Cas9 to modify CD79A sequence or expression

• Apply CD79A antibodies to detect changes in localization, complex formation, or signaling

• Investigate the functional consequences of "alternate splicing generates a short isoform with a 39 aa deletion in the ECD"

Live-cell imaging techniques:

• Utilize anti-CD79A antibody fragments for real-time visualization of BCR dynamics

• Combine with calcium flux indicators to correlate BCR movement with signaling events

• Examine how CD79A clustering influences signal propagation

Analysis of conformational changes:

• Develop conformation-specific antibodies that recognize active versus inactive BCR states

• Use these antibodies to monitor BCR activation status in different cellular compartments

• Correlate with downstream signaling events

Model system comparisons:

• Apply CD79A antibodies across different model systems (human, mouse, cell lines)

• Investigate species-specific differences, considering that "human CD79A shares 57% aa sequence identity with mouse and rat CD79A"

• Correlate findings with clinical observations in B-cell disorders

These methodological approaches leverage CD79A antibodies as powerful tools for dissecting the complex signaling networks involved in B-cell activation, differentiation, and dysfunction in disease states.

What are the technical challenges in developing bispecific antibodies targeting CD79A and other B-cell markers?

Developing bispecific antibodies targeting CD79A and other B-cell markers presents several technical challenges that require sophisticated approaches:

Target biology considerations:

• Ensuring compatible internalization kinetics between CD79A and the second target

• The research indicates CD79B may be superior to CD79A for ADC approaches due to better internalization properties: "anti-CD79B antibodies downregulated surface B-cell receptor and were trafficked to the lysosomal-like MIIC compartment"

• Accounting for potential differential expression of targets across B-cell malignancy subtypes

Epitope selection challenges:

• Identifying optimal epitopes on CD79A that don't interfere with BCR complex formation

• Considering the extracellular domain (Leu33-Arg143) and potential splice variants with "39 aa deletion in the ECD"

• Ensuring epitopes remain accessible in the context of the intact BCR

Bispecific format optimization:

• Selecting appropriate bispecific formats (e.g., diabodies, tandem scFv, CrossMAb)

• Optimizing domain orientation to maintain binding to both targets

• Addressing stability challenges associated with complex antibody formats

Affinity balancing:

• Calibrating binding affinities for CD79A and the second target to achieve desired selectivity

• The research notes that efficacy "did not correlate with the affinity of the antibodies," suggesting complex relationships between binding and functional outcomes

Manufacturing complexities:

• Developing consistent production processes for complex bispecific molecules

• Addressing chain association issues to minimize mispaired species

• Implementing purification strategies to isolate correctly assembled bispecifics

Functional characterization:

• Developing assays to verify simultaneous binding to both targets

• Assessing whether dual targeting enhances internalization compared to monospecific antibodies

• Investigating how bispecific binding affects BCR signaling and B-cell activation

Payload attachment strategies:

• For bispecific ADCs, determining optimal conjugation sites that preserve dual binding

• Selecting appropriate linker-drug combinations based on findings that "the MC-MMAF and MCC-DM1 ADCs showed similar efficacies regardless of the cell line used"

In vivo validation complexity:

• Addressing potential differences between xenograft models and human disease

• The research revealed that "the ability of the cell lines to respond to the anti-CD79B ADCs in the xenograft models did not correspond to the in vitro sensitivity"

• Developing predictive preclinical models for bispecific antibody efficacy

Addressing these technical challenges requires multidisciplinary expertise spanning protein engineering, cell biology, and immunology to develop effective bispecific antibodies targeting CD79A and other B-cell markers for therapeutic applications.