CD74 Recombinant Monoclonal Antibody

What is CD74 and where is it normally expressed in human tissues?

CD74, also known as the MHC class II associated invariant chain, is a type II transmembrane glycoprotein that plays a critical role in the presentation of peptides by MHC class II antigens to CD4-positive lymphocytes . It is expressed primarily on MHC class II-positive cells including B cells, a subset of activated T cells, monocytes, and dendritic cells . CD74 is expressed broadly in normal B-cell compartments including primary and secondary lymphoid follicles and in the thymic medulla . Expression of CD74 is predominantly intracellular with moderate expression at the cell surface of B cells and monocytes .

What are the different isoforms of CD74 recognized by antibodies?

Multiple isoforms of CD74 exist, with molecular weights of 33, 35, and 41 kDa recognized by antibodies such as the Bu45 clone . These isoforms result from alternative splicing and post-translational modifications of the CD74 protein. Researchers should be aware of which isoforms their chosen antibody recognizes when designing experiments, as this may impact interpretation of results in different tissue or cell types.

What applications are CD74 antibodies suitable for in research settings?

CD74 antibodies have been validated for multiple research applications including:

• Flow cytometry for detection on cell lines and primary cells

• Immunohistochemistry on tissue microarrays and sections

• Western blotting for protein expression analysis

• Immunofluorescence for localization studies

• Cell binding assays for functional studies

The choice of application should be guided by the specific research question and the validated applications for the particular antibody clone being used.

How is CD74 expression determined in cell lines and patient samples?

CD74 expression can be quantitatively assessed using flow cytometry to determine copy numbers on the cell surface. In research studies, CD74 copy numbers in DLBCL cell lines have been found to vary widely, from high levels (51,000-77,000 copies per cell) to below detection limits . In tissue samples, immunohistochemistry is commonly used to assess expression patterns, with studies showing CD74 expression in 100% (100/100) of DLBCL, 100% (28/28) of FL, and 94% (73/78) of MCL human tissue microarray samples . For detailed expression analysis, researchers can use antibodies conjugated to fluorophores (such as DBCO-Alexa 647) to stain cells isolated from various tissues and compare mean fluorescence intensity ratios against appropriate isotype controls .

How does CD74 expression differ between normal B cells and malignant B cells?

While CD74 is expressed on normal B cells, its expression is significantly upregulated in various B-cell malignancies including non-Hodgkin lymphomas (NHL) and multiple myeloma (MM) . Research has shown near-ubiquitous expression of CD74 in diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL) tissue microarrays . High-level expression (in >70% of cells) was observed in 86% of DLBCL, 79% of FL, and 63% of MCL samples . This differential expression makes CD74 an attractive target for both diagnostic applications and targeted therapies.

What experimental controls should be included when using CD74 antibodies?

Robust experimental design with appropriate controls is crucial when working with CD74 antibodies:

• Isotype controls: Use matched isotype antibodies (e.g., Mouse IgG1 for clone Bu45) to control for non-specific binding

• Positive controls: Include known CD74-positive cell lines (e.g., Raji, OPM2, SU-DHL-6) or transfected cells (CHO-human-CD74)

• Negative controls: Use CD74-negative cell lines or tissues lacking MHC class II expression

• Secondary antibody-only controls: For indirect detection methods

• Blocking controls: Pre-incubation with recombinant CD74 protein to demonstrate specificity

For flow cytometry applications, researchers have used Mouse IgG1 isotype control followed by anti-Mouse IgG APC-conjugated Secondary Antibody to establish background staining levels .

How can researchers troubleshoot variable CD74 staining results across different cell lines?

Variable CD74 staining can result from several factors:

• Heterogeneous expression: CD74 expression varies considerably between cell lines and even within the same tumor type. For example, in DLBCL cell lines, CD74 copy numbers range from very high (77,000 for OCI-Ly3) to below detection limits

• Epitope accessibility: CD74 is primarily intracellular, with moderate surface expression . Ensuring appropriate permeabilization for intracellular staining is critical

• Antibody clone specificity: Different clones (e.g., 332516, Bu45) recognize different epitopes and may show variable binding patterns

• Fixation methods: Optimal antigen retrieval methods (e.g., EDTA/Tris at pH 9 for 10 minutes) should be established for each application

To address variability, researchers should:

• Determine optimal antibody concentration through titration experiments

• Validate antibody performance in multiple positive and negative control cell lines

• Consider using multiple antibody clones that recognize different epitopes

• Standardize fixation and permeabilization protocols across experiments

What is the significance of using recombinant monoclonal antibodies versus traditional hybridoma-derived antibodies for CD74 research?

Recombinant monoclonal antibodies offer several advantages over traditional hybridoma-derived antibodies for CD74 research:

• Increased consistency: Recombinant antibodies show lower batch-to-batch variability due to defined genetic sequence and controlled expression systems

• Higher specificity: Engineered to target specific epitopes with reduced cross-reactivity

• Reproducibility: The defined genetic sequence ensures consistent performance across experiments

• Reduced background: Lower non-specific binding in complex samples

• Customization potential: Can be engineered with specific conjugates, fragments, or humanized versions for various applications

These advantages are particularly relevant for CD74 research where precise quantification and comparison across different cell types and tissues is essential.

What is the optimal sample preparation protocol for CD74 detection in primary tissues?

For optimal detection of CD74 in primary tissues, the following protocol has been validated in research settings:

• Tissue Fixation and Processing:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Process and embed in paraffin following standard protocols

  • Section tissues at 4-μm thickness

• Antigen Retrieval:

  • Deparaffinize in xylene and hydrate in graduated alcohols

  • Perform antigen retrieval by pressure cooker in EDTA (1 mM)/Tris (5 mM) at pH 9 for 10 minutes

• Staining Procedure:

  • Block endogenous peroxidase activity with 3% hydrogen peroxide

  • Apply primary anti-CD74 antibody at optimized dilution

  • Incubate at room temperature for 30-60 minutes or at 4°C overnight

  • Detect using appropriate secondary detection system

  • Counterstain, dehydrate, and mount

• Controls:

  • Include known CD74-positive tissues (lymph node, tonsil) as positive controls

  • Use isotype-matched irrelevant antibodies as negative controls

This protocol has been successfully used to characterize CD74 expression in large cohorts of normal and neoplastic human hematolymphoid specimens .

How should researchers optimize antibody concentration for flow cytometry applications?

For flow cytometry applications targeting CD74, optimization is critical due to variable expression levels across different cell types. The following methodological approach is recommended:

• Initial Titration:

  • Perform a broad-range antibody titration (e.g., 0.1-10 μg/mL)

  • Use a known CD74-positive cell line (e.g., Raji, SU-DHL-6)

  • Analyze signal-to-noise ratio at each concentration

• Refined Optimization:

  • Select the concentration range showing the best separation

  • Test on multiple cell types with varying CD74 expression levels

  • Compare results using different secondary detection reagents if using indirect staining

• Protocol Standardization:

  • Standardize cell numbers (typically 1×10^6 cells per test)

  • Use consistent staining buffer (PBS with 0.5-2% BSA and 0.1% sodium azide)

  • Maintain consistent incubation times and temperatures

• Validation:

  • Calculate the staining index (SI = [MFI positive - MFI negative]/2 × SD of negative)

  • Compare results with quantitative standards if available

  • Establish clear positive/negative cutoffs based on isotype controls

Research has shown that optimal dilutions vary by application and should be determined by each laboratory .

What methodology is used to quantify CD74 copy number on cell surfaces?

Accurate quantification of CD74 copy number on cell surfaces is essential for comparative studies across different cell types and disease states. The following methodology has been employed in research settings:

• Quantitative Flow Cytometry:

  • Use calibrated beads with known antibody binding capacity (ABC)

  • Create a standard curve correlating mean fluorescence intensity (MFI) to ABC

  • Use a 1:1 binding ratio of antibody to CD74 molecule

  • Calculate copy number based on the standard curve and cell MFI values

• Standard Protocol:

  • Label cells with saturating concentrations of anti-CD74

  • Include parallel samples with calibration beads

  • Process samples under identical conditions

  • Convert raw MFI to absolute copy number using the standard curve

Using this approach, researchers have determined that CD74 copy numbers vary significantly across cell lines, from high levels (77,000 copies per cell in ABC-like OCI-Ly3) to below detection limits in some DLBCL cell lines .

How does CD74 antibody binding affect receptor internalization and cell signaling?

CD74 antibody binding has significant effects on receptor dynamics and downstream signaling:

• Receptor Internalization:

  • CD74 undergoes rapid internalization following antibody binding

  • The internalization rate is significantly faster than many other surface receptors

  • This property makes CD74 an excellent target for antibody-drug conjugates (ADCs)

• Signaling Effects:

  • Anti-CD74 antibodies can induce direct antiproliferative effects in NHL cell lines

  • CD74 engagement can influence NF-κB activation and interleukin-8 production

  • Antibody binding may interfere with macrophage migration inhibitory factor (MIF) binding to CD74

• Therapeutic Implications:

  • The rapid internalization of CD74-antibody complexes allows efficient delivery of cytotoxic payloads in ADCs like STRO-001

  • Anti-CD74 antibodies have shown efficacy in preclinical models of lymphoma

Understanding these mechanisms is crucial for developing effective CD74-targeted therapeutics and interpreting experimental results.

Researchers have utilized multiple experimental models to evaluate CD74-targeted therapies:

• Cell Line Models:

  • DLBCL cell lines: SU-DHL-6, U2932, WSU-DLCL-2, OCI-Ly3

  • MCL cell lines: Mino, Jeko-1

  • MM cell lines: ARP-1, MM.1S

  • These represent different lymphoma subtypes with varying CD74 expression levels

• Xenograft Models:

  • Subcutaneous xenografts: Mice implanted with human lymphoma cell lines

  • STRO-001 showed linear dose-response relationships in DLBCL models (SU-DHL-6, U2932)

  • Complete tumor regression was observed at doses ≥10 mg/kg

  • In MCL models (Mino, Jeko-1), STRO-001 at 3 mg/kg significantly prolonged survival or induced tumor regression

• Non-Human Primate Models:

  • Cynomolgus monkeys have been used to evaluate toxicity profiles

  • STRO-001 induced dose-responsive, reversible B-cell and monocyte depletion at doses up to 10 mg/kg

  • No evidence of off-target toxicity was observed

The choice of model should be guided by the specific research question, with consideration of CD74 expression levels and the mechanism of action of the therapeutic being evaluated.

What are the emerging applications for CD74 antibodies beyond cancer research?

While CD74 antibodies have been primarily studied in the context of B-cell malignancies, emerging research suggests potential applications in other areas:

• Autoimmune Diseases:

  • Anti-CD74 antibodies have been investigated for systemic lupus erythematosus treatment

  • CD74's role in antigen presentation makes it relevant in various autoimmune conditions

• Infectious Disease Research:

  • CD74 has been identified as a binding target for Helicobacter pylori urease B subunit

  • This interaction induces NF-κB activation and interleukin-8 production

  • Anti-CD74 antibodies could help elucidate host-pathogen interactions

• Immunomodulation:

  • CD74 plays roles in B-cell maturation and T-cell responses

  • Antibodies targeting specific epitopes might selectively modulate immune functions

• Diagnostic Applications:

  • CD74 expression patterns could serve as biomarkers in various inflammatory conditions

  • Imaging applications using radiolabeled anti-CD74 antibodies are being explored

These emerging applications represent promising areas for future research with CD74 antibodies.

How might combination therapies with CD74 antibodies be optimized for research purposes?

Optimizing combination therapies with CD74 antibodies requires systematic experimental approaches:

• Rational Combination Selection:

  • Combine with agents targeting complementary pathways (e.g., anti-CD20 antibodies)

  • Test combinations with different mechanisms of action (e.g., ADCs plus immune checkpoint inhibitors)

  • Consider tumor microenvironment modifiers to enhance antibody penetration

• Experimental Design Strategies:

  • Use factorial designs to systematically evaluate multiple combinations

  • Implement response surface methodology to optimize dosing ratios

  • Conduct time-course experiments to determine optimal sequencing

• Relevant Preclinical Models:

  • Use patient-derived xenografts to better reflect tumor heterogeneity

  • Implement syngeneic models with humanized CD74 to assess immune components

  • Consider 3D organoid cultures to evaluate penetration and efficacy

• Translational Considerations:

  • Develop pharmacodynamic markers to monitor target engagement

  • Establish predictive biomarkers for response to combination therapy

  • Design mechanistic studies to understand synergistic interactions