SIRPA Recombinant Monoclonal Antibody

What is SIRPA and what are its primary biological functions?

SIRPA (Signal Regulatory Protein Alpha) is an immunoinhibitory receptor primarily expressed by myeloid lineage immune cells, including neutrophils, monocytes, and macrophages. Its main function is to regulate immune responses and phagocytosis by interacting with CD47 and other ligands. SIRPA prevents unnecessary phagocytosis of healthy cells while facilitating clearance of pathogens and damaged cells by immune cells. This receptor plays a crucial role in immune regulation with implications for both normal immune function and potential therapeutic strategies for cancer and inflammatory diseases .

Functionally, SIRPA contains immunoreceptor tyrosine-based inhibition motifs (ITIMs) in its cytoplasmic domain that, upon ligand binding, recruit and activate protein tyrosine phosphatases. This activation leads to inhibitory signaling cascades that modulate cellular responses, particularly in contexts of inflammation and immune surveillance .

How are SIRPA recombinant monoclonal antibodies produced for research applications?

SIRPA recombinant monoclonal antibodies are typically produced through a sophisticated multi-step process:

• Initial extraction of SIRPA antibody genes from B cells isolated from immunoreactive animals (commonly rabbits)

• Amplification of these genes followed by cloning into suitable phage vectors

• Introduction of these vectors into mammalian cell expression systems (such as HEK293F or CHO cells)

• Production of functional antibodies in serum-free conditions

• Purification through affinity chromatography from the culture supernatant

• Rigorous quality control including SDS-PAGE, SEC-HPLC analysis, and functional validation

This recombinant technology provides significant advantages over traditional hybridoma-derived antibodies, including better consistency, reduced batch variation, and the ability to engineer specific modifications to enhance performance for particular applications .

What are the primary research applications for SIRPA recombinant monoclonal antibodies?

SIRPA recombinant monoclonal antibodies serve multiple purposes in research settings:

These antibodies are particularly valuable for investigating SIRPA expression in inflammatory conditions, as SIRPA levels have been found to be significantly elevated in tissues from patients with rheumatoid arthritis (RA) and inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn's disease (CD) .

How do researchers determine the optimal conditions for using SIRPA antibodies in immunohistochemistry?

Optimizing SIRPA antibody use in immunohistochemistry requires methodical approach:

• Begin with a titration experiment using recommended dilution ranges (typically 1:50-1:200) on known positive control tissues

• Evaluate different antigen retrieval methods, as SIRPA detection may require heat-induced epitope retrieval in citrate or EDTA buffers

• Systematically test incubation times and temperatures (typically 1-2 hours at room temperature or overnight at 4°C)

• Include appropriate negative controls (isotype-matched antibodies) to assess specificity

• For inflamed tissues, consider dual staining with markers for neutrophils or monocytes to confirm colocalization with SIRPA

This systematic approach is essential as studies have demonstrated that SIRPA+ mononuclear cells show increased frequency in RA synovium and CD-derived inflamed colon biopsies, making proper staining optimization critical for accurate assessment .

How can SIRPA antibodies with different binding properties be used to investigate myeloid cell function in inflammatory diseases?

The functional consequences of SIRPA antibody binding depend significantly on whether the antibody acts as an agonist or antagonist:

Agonistic SIRPA Antibodies:
These antibodies mimic natural ligand binding and induce SIRPA receptor phosphorylation, activating inhibitory signaling cascades. Research has demonstrated that agonistic anti-SIRPA antibodies exhibit potent anti-inflammatory effects by:

• Reducing neutrophil and monocyte chemotaxis and tissue infiltration

• Inhibiting integrin-dependent migration of myeloid cells to inflamed tissues

• Suppressing pro-inflammatory cytokine production (e.g., TNFα, G-CSF)

• Redistributing neutrophils and monocytes away from inflamed tissues to secondary lymphoid organs

These agonistic antibodies have shown therapeutic potential in preclinical models of arthritis and colitis, where they ameliorated autoimmune joint inflammation and inflammatory colitis by reducing neutrophil and monocyte tissue infiltration .

Antagonistic SIRPA Antibodies:
In contrast, antagonistic antibodies block the interaction between SIRPA and its ligands (particularly CD47), which can:

• Enhance phagocytic activity of macrophages against target cells

• Synergize with tumor-specific monoclonal antibodies to increase phagocytosis of cancer cells

• Augment anti-tumor immune responses in vivo

This approach has shown particular promise in oncology research, where antagonizing SIRPA can overcome the "don't eat me" signal provided by CD47 overexpression on cancer cells .

What methodological approaches are used to engineer high-affinity SIRPA variants with enhanced therapeutic potential?

Engineering high-affinity SIRPA variants involves sophisticated protein engineering techniques:

• In vitro evolution via yeast surface display: This approach involves:

  • Creation of mutant libraries of the N-terminal V-set Ig domain of SIRPA

  • Conjugation to Aga2p for yeast surface-display

  • Multiple rounds of selection using CD47 IgSF domain as a selection reagent

  • Progressive enrichment for higher affinity variants

• Targeted mutagenesis of key residues:

  • Focusing on residues that directly contact CD47

  • Modifying residues within the hydrophobic core

  • Creating combinatorial libraries with mutations at multiple positions

Through these approaches, researchers have generated SIRPA variants with remarkable 50,000-fold increases in affinity for human CD47 compared to wild-type SIRPA. These high-affinity variants function as potent CD47 antagonists and demonstrate superior performance compared to CD47-Fc fusion proteins, likely due to their much higher binding affinity (9 nM binding Kd for engineered variants versus 2 μM for natural CD47-SIRPA interactions) .

What are the key technical considerations when designing experiments to assess the functional effects of SIRPA antibodies on neutrophil and monocyte migration?

When designing experiments to evaluate SIRPA antibody effects on myeloid cell migration, researchers should consider:

• In vitro migration assays:

  • Transwell migration assays with purified neutrophils or monocytes

  • Include appropriate chemoattractants (e.g., CXCL1, CXCL8)

  • Test multiple antibody concentrations to establish dose-response relationships

  • Include controls for integrin dependency (e.g., LFA-1 and MAC-1 blocking)

• In vivo migration models:

  • Peritoneal chemotaxis models using CXCL1 injection

  • Air pouch models for localized inflammation

  • Careful timing of antibody administration (prophylactic vs. therapeutic)

  • Flow cytometric analysis of both target tissues and secondary lymphoid organs to assess redistribution effects

• Critical controls:

  • Isotype-matched control antibodies

  • Blocking antibodies against integrins to distinguish between adhesion-dependent and independent effects

  • Evaluation of systemic effects using blood counts and analysis of secondary lymphoid organs

• Assessment of downstream signaling:

  • Immunoprecipitation and phospho-Western blotting to confirm SIRPA receptor phosphorylation

  • Analysis of SHP1/SHP2 recruitment to phosphorylated ITIM domains

  • Evaluation of downstream effects on integrin activation

Research has demonstrated that agonistic anti-SIRPA antibodies can reduce CXCL1-mediated neutrophil and monocyte chemotaxis to the peritoneal cavity by up to 80%, but this effect is dependent on integrin-mediated adhesion mechanisms. Blockade of LFA-1 and MAC-1-dependent endothelial cell adhesion abrogated the difference between isotype control and anti-SIRPA antibody treatments .

How can researchers troubleshoot inconsistent results when using SIRPA antibodies in flow cytometry?

Flow cytometry with SIRPA antibodies may present several technical challenges. A methodical troubleshooting approach includes:

• Sample preparation issues:

  • Ensure fresh samples or proper storage conditions to maintain SIRPA epitope integrity

  • Optimize fixation protocols (paraformaldehyde concentration and time)

  • Test multiple permeabilization reagents if intracellular staining is required

• Antibody-specific considerations:

  • Titrate antibody concentration (typical range 1:50-1:200)

  • Test different incubation temperatures and times

  • Consider using directly conjugated antibodies to eliminate secondary antibody variability

  • Evaluate potential for internalization of SIRPA upon antibody binding

• Panel design challenges:

  • Test for spectral overlap with other fluorophores in your panel

  • Consider SIRPA expression levels when selecting fluorophore brightness

  • Include proper FMO (Fluorescence Minus One) controls

• Data analysis approach:

  • Use biaxial plots comparing SIRPA to lineage markers for myeloid cells

  • Apply consistent gating strategies across experiments

  • Consider using median fluorescence intensity rather than percent positive for quantitative comparisons

• Biological variables:

  • SIRPA expression can vary significantly with activation states of myeloid cells

  • Expression may change during inflammation or disease states

  • Consider analyzing multiple myeloid populations separately (neutrophils vs. monocytes vs. macrophages)

Flow cytometric analysis of SIRPA expression has been crucial in demonstrating that agonistic anti-SIRPA antibody treatment can reduce neutrophils and inflammatory monocytes in joint synovial fluids by more than 80% while increasing their numbers in the spleen, confirming the redistribution effect of these antibodies .

How can SIRPA antibodies be combined with other therapeutic approaches to enhance efficacy in cancer immunotherapy?

The combination of high-affinity SIRPA variants with tumor-specific antibodies represents a promising approach for cancer immunotherapy:

• Synergistic mechanisms:

  • High-affinity SIRPA monomers block the "don't eat me" signal from CD47 on cancer cells

  • Tumor-specific antibodies provide an "eat me" signal through Fc receptor engagement

  • This "one-two punch" directs immune responses against tumor cells while lowering the threshold for macrophage activation

• Experimental evidence:

  • In studies with Her2/neu+ breast cancer cell lines, combining high-affinity SIRPA monomers with trastuzumab resulted in maximal levels of phagocytosis that were considerably higher than the additive effect of either agent alone

  • Similar synergistic effects were observed with rituximab against B-cell lymphoma cells

  • In vivo studies demonstrated that this combination therapy led to cures in the majority of animals with persistent effects long after treatment discontinuation

• Advantages over alternative approaches:

  • High-affinity SIRPA monomers (14 kDa) are smaller than antibodies, potentially improving tumor penetration

  • They are amenable to further engineering to alter efficacy, toxicity, or pharmacokinetic parameters

  • As adjuvants to existing approved antibodies, they may offer a faster pathway to clinical translation

This approach is particularly promising because many cancers overexpress CD47, making high-affinity SIRPA variants potentially applicable as universal adjuvants to monoclonal antibody therapies across multiple cancer types .

What are the key considerations for translating SIRPA-targeted therapies from preclinical models to clinical applications?

Translating SIRPA-targeted therapies to the clinic requires addressing several critical considerations:

• Target expression and heterogeneity:

  • Evaluate SIRPA expression across patient populations and disease subtypes

  • Assess potential for inter-patient and intra-patient heterogeneity

  • Consider temporal dynamics of SIRPA expression during disease progression

• Pharmacological properties:

  • Optimize pharmacokinetics through antibody engineering (e.g., Fc modifications)

  • Evaluate tissue penetration, particularly for solid tumors or inflamed tissues

  • Assess potential for immunogenicity of engineered SIRPA variants

• Mechanism-based toxicities:

  • Carefully evaluate potential for off-target effects on normal SIRPA-expressing cells

  • Monitor for excessive suppression of innate immunity (for agonistic antibodies)

  • Assess potential for enhanced autoimmunity (for antagonistic antibodies)

• Biomarker development:

  • Identify predictive biomarkers for response to SIRPA-targeted therapies

  • Research suggests that enhanced SIRPA expression in inflamed tissues correlates with other neutrophil and inflammatory monocyte-associated genes (e.g., S100A8, S100A9, FCGR2A, VNN2, NCF2)

  • Elevated SIRPA expression is associated with non-responsiveness to anti-TNF (infliximab) or anti-α4β7 (vedolizumab) therapy in IBD patients

• Combination strategies:

  • For inflammatory diseases: Consider combinations with existing anti-inflammatory agents

  • For cancer: Evaluate synergy with various tumor-specific antibodies and other immunotherapies

  • Develop rational sequencing of therapies based on mechanistic understanding

These considerations are supported by clinical observations that SIRPA expression is significantly elevated in RA patients compared to healthy controls and osteoarthritis patients, and in inflamed colon tissues of UC and CD patients. The correlation between SIRPA upregulation and treatment refractoriness suggests potential utility as both a therapeutic target and a biomarker .