IGSF11 Antibody

What is IGSF11 and why is it important in research?

IGSF11 is a type I transmembrane cell adhesion molecule that functions through homophilic interaction and stimulates cell growth. Recent research has identified IGSF11 as a novel immune checkpoint with significant implications in cancer biology. It has been described as a putative ligand of V-domain Ig suppressor of T cell activation (VISTA), making it particularly relevant for immunotherapy research . IGSF11 is abundantly expressed in testis and ovary, with lower expression in brain, kidney and skeletal muscle in healthy individuals, while being frequently upregulated in various cancer types .

What are the molecular characteristics of IGSF11 that influence antibody selection?

IGSF11 has a calculated molecular weight of approximately 46 kDa, though it is typically observed at 40-50 kDa in experimental conditions . This discrepancy is likely due to post-translational modifications, particularly N-glycosylation . IGSF11 is a single-pass type I membrane protein localized to the cell membrane. When selecting antibodies, researchers should consider the protein's cellular localization and potential epitope accessibility. The immunogen used for antibody production (e.g., IGSF11 fusion protein or synthesized peptides derived from human IGSF11) will determine epitope specificity and cross-reactivity .

Which experimental applications have IGSF11 antibodies been validated for?

Commercial IGSF11 antibodies have been validated for multiple applications with varying recommended dilutions:

Note that optimal dilutions should be determined empirically for each experimental system to obtain the best results .

What tissue preparation and antigen retrieval methods are recommended for IGSF11 immunohistochemistry?

For optimal IGSF11 detection by IHC, TE buffer pH 9.0 is the primary recommended antigen retrieval method. Alternatively, citrate buffer pH 6.0 may be used, though potentially with different results . When working with paraffin-embedded tissue sections, complete deparaffinization is essential before antigen retrieval. For frozen sections, fixation with acetone or 4% paraformaldehyde followed by permeabilization with 0.1-0.3% Triton X-100 may improve antibody access to the IGSF11 epitope. Always include positive control tissues known to express IGSF11 (testis, ovary, or brain) to validate staining procedures .

How should researchers optimize Western blot conditions for IGSF11 detection?

When detecting IGSF11 by Western blot, researchers should prepare samples under reducing conditions with standard SDS-PAGE protocols. Since the observed molecular weight (40-50 kDa) may differ from the calculated weight (46 kDa) due to post-translational modifications, appropriate molecular weight markers should be included . For optimal detection:

• Use 20-50 μg of total protein per lane

• Transfer to PVDF membrane (preferred over nitrocellulose for this protein)

• Block with 5% non-fat milk in TBST

• Incubate with primary antibody at 1:500-1:1000 dilution overnight at 4°C

• Wash thoroughly and use appropriate HRP-conjugated secondary antibody

• Visualize using enhanced chemiluminescence

Note that sample-dependent variations may require protocol adjustments, and researchers should consult the specific protocol provided by the antibody manufacturer .

What controls should be included when working with IGSF11 antibodies?

For rigorous experimental design, the following controls should be included:

• Positive tissue controls: Human testis or ovary tissues (highest expression), brain tissue (moderate expression)

• Negative tissue controls: Tissues with minimal IGSF11 expression

• Antibody controls: Include isotype controls to identify non-specific binding

• Knockdown/knockout controls: IGSF11 siRNA-treated cells or CRISPR-Cas9 knockout cell lines to validate antibody specificity

• Peptide competition: Pre-incubation of antibody with immunizing peptide to confirm specificity

• Multiple antibody validation: When possible, confirm findings with at least two different IGSF11 antibodies recognizing distinct epitopes

Including these controls helps distinguish specific from non-specific signals and validates experimental findings .

How can IGSF11 antibodies be utilized to investigate immune checkpoint regulation?

IGSF11 has been identified as a novel immune checkpoint target with significant therapeutic potential . Researchers can use IGSF11 antibodies to:

• Characterize expression patterns in the tumor microenvironment using IHC, IF, or flow cytometry

• Block IGSF11-VISTA interactions with function-blocking antibodies that inhibit the immunosuppressive pathway

• Monitor changes in immune activation following antibody-mediated IGSF11 blockade by measuring T cell proliferation, cytokine production, and cytotoxic activity

• Develop antibody-drug conjugates targeting IGSF11-expressing tumors

• Identify and validate novel IGSF11 binding partners using co-immunoprecipitation followed by mass spectrometry

Research has shown that blocking IGSF11 with monoclonal antibodies resulted in improved immune lysis of tumor cells in vitro, suggesting therapeutic applications .

What experimental approaches can be used to study IGSF11 in tumor immunology?

Several sophisticated experimental approaches can be employed:

• Genetic manipulation: CRISPR-Cas9 knockout of IGSF11 in tumor cell lines followed by functional assays. Studies have shown that knockout of Igsf11 in tumor cells increases T cell activity and tumor lysis .

• Antibody-based blocking: Utilize fully human monoclonal antibodies against IGSF11 that exhibit varying capacities to inhibit IGSF11-VISTA interactions. Researchers have developed antibodies with different binding profiles:

  Antibody Family	Characteristics	Application
  IOMX-0168 family	IGSF11-specific, human/mouse cross-reactive	Blocks IGSF11-VISTA interaction
  IOMX-0242 family	IGSF11-specific, human/mouse cross-reactive	Variable blocking activity

• Co-culture systems: Establish co-cultures of IGSF11-expressing tumor cells with T cells to study immune synapse formation and cytotoxic responses in the presence or absence of blocking antibodies .

• In vivo models: Utilize syngeneic mouse models with IGSF11-expressing tumors to evaluate the efficacy of anti-IGSF11 antibodies alone or in combination with other immunotherapies .

How does IGSF11 expression differ between normal tissues and cancer, and how can this be studied?

IGSF11 expression is largely restricted to immune-privileged sites (testis, ovary, brain) in healthy individuals but is frequently upregulated in various cancers, including intestinal-type gastric cancers, colorectal cancers, and hepatocellular carcinomas . This expression pattern suggests IGSF11 may function as a cancer-testis antigen, making it an attractive target for cancer immunotherapy .

To study these differential expression patterns:

• Tissue microarrays: Utilize IGSF11 antibodies for IHC analysis of multiple tissue types simultaneously to compare expression patterns

• Quantitative analysis: Employ digital pathology tools to quantify staining intensity across different tissues and cancer grades

• Co-localization studies: Combine IGSF11 staining with markers of immune cell infiltration to assess correlation with immunosuppressive microenvironments

• Transcriptional profiling: Correlate protein expression with mRNA expression data from cancer genomics databases

Northern blot analysis has demonstrated abundant IGSF11 expression in testis and ovary, with elevated expression in multiple cancer types compared to corresponding non-cancerous tissues .

How are IGSF11-targeting peptides being developed for cancer immunotherapy?

Researchers have identified specific IGSF11 epitope peptides that can elicit IGSF11-specific cytotoxic T lymphocytes (CTLs) with potential application in cancer immunotherapy:

• Wild-type epitope: IGSF11-9-207 (ALSSGLYQC) was identified as capable of inducing CTLs

• Anchor-modified peptides: To enhance immunogenicity, researchers developed modified peptides:

  • IGSF11-9V (ALSSGLYQV): Cysteine at position nine replaced by valine

  • IGSF11-9L (ALSSGLYQL): Cysteine at position nine replaced by leucine

These modified peptides demonstrated improved HLA-A*0201-binding scores compared to the wild-type IGSF11-9-207 peptide based on epitope prediction algorithms .

The anchor-modified IGSF11-9V peptide successfully established oligo-clonal CTLs that killed both peptide-pulsed T2 cells and SNU475 cells naturally expressing IGSF11, demonstrating its potential utility in cancer immunotherapy .

What methodological approaches are used to assess IGSF11 antibody effects on tumor immunity?

Researchers employ several sophisticated approaches to evaluate anti-IGSF11 antibody effects:

• T cell activation assays: Measure IFN-γ production, proliferation, and cytolytic activity of T cells co-cultured with tumor cells in the presence of anti-IGSF11 antibodies

• Tumor cell viability assays: Assess the impact of IGSF11 blockade on immune-mediated tumor cell killing using methods such as:

  • Luciferase-based cytotoxicity assays

  • Flow cytometry-based killing assays

  • Real-time cell analysis systems

• Immune synapse visualization: Use confocal microscopy to visualize changes in immune synapse formation between T cells and tumor cells following IGSF11 blockade

• Biochemical interaction studies: Employ techniques such as surface plasmon resonance or bio-layer interferometry to characterize antibody binding kinetics to IGSF11 and the impact on IGSF11-VISTA interactions

Experimental data has demonstrated that IGSF11 pathway blockade can enhance T cell activity against tumor cells, making this a promising approach for cancer immunotherapy .

How should researchers interpret conflicting IGSF11 expression data across different tumor types?

When faced with inconsistent IGSF11 expression data:

• Consider technical variables: Different antibodies may recognize distinct epitopes affected by post-translational modifications or protein conformation

• Evaluate tumor heterogeneity: IGSF11 expression may vary within tumors and across patients with the same cancer type

• Assess tumor microenvironment: IGSF11 expression might be influenced by inflammatory signals or hypoxic conditions

• Compare detection methods: Transcriptomic data may not always correlate with protein expression due to post-transcriptional regulation

• Stratify by molecular subtypes: Different molecular subtypes within a cancer type may show varying IGSF11 expression patterns

Current evidence indicates IGSF11 expression is elevated in intestinal-type gastric cancers, colorectal cancers, and hepatocellular carcinomas, but comprehensive analysis across all cancer types has not been completed .

What are the emerging combination strategies involving IGSF11 antibodies?

Based on current understanding of IGSF11 as a novel immune checkpoint, several promising combination approaches are being explored:

• Dual checkpoint blockade: Combining anti-IGSF11 with established checkpoint inhibitors (anti-PD-1/PD-L1, anti-CTLA-4) to overcome resistance mechanisms

• Targeting the IGSF11-VISTA axis: Simultaneous blockade of both IGSF11 and VISTA to more completely disrupt this immunosuppressive pathway

• Antibody-drug conjugates: Leveraging IGSF11's tumor-specific expression to deliver cytotoxic payloads

• Bispecific antibodies: Developing constructs that simultaneously target IGSF11 and engage T cells

• Combination with conventional therapies: Exploring synergies between IGSF11 blockade and chemotherapy, radiation, or targeted therapies

Early research suggests that targeting IGSF11 may be effective in cancer models that fail to respond to PD-L1 inhibition, highlighting the potential for addressing treatment-resistant populations .

What technical challenges must be overcome in developing therapeutic IGSF11 antibodies?

Several technical hurdles require consideration:

• Epitope selection: Identifying epitopes that optimally block IGSF11-VISTA interactions while minimizing off-target effects

• Antibody format optimization: Determining whether full IgG, F(ab')2, Fab, or alternative formats provide optimal tumor penetration and efficacy

• Cross-reactivity concerns: Developing antibodies with appropriate species cross-reactivity for preclinical testing while ensuring human specificity

• Immunogenicity assessment: Evaluating potential anti-drug antibody responses, particularly for novel epitope-targeting antibodies

• Manufacturability: Addressing stability, aggregation, or other chemistry, manufacturing, and controls (CMC) challenges

Current research has already yielded fully human monoclonal antibodies against IGSF11 that exhibit improved immune lysis of tumor cells, demonstrating progress in addressing some of these challenges .

How can functional IGSF11 antibody screening be optimized for research applications?

For researchers developing or selecting IGSF11 antibodies for specific applications, consider these methodological approaches:

• Epitope binning: Use competitive binding assays to group antibodies based on the epitopes they recognize, especially identifying those that interfere with VISTA binding

• Species cross-reactivity profiling: Systematically test reactivity against human, mouse, and other relevant species' IGSF11 to enable translational research:

  Antibody Example	Human IGSF11 Reactivity	Mouse IGSF11 Reactivity
  14003-1-AP	Positive	Positive
  DF15961	Positive	Positive
  IOMX-0168 family	Positive	Positive

• Functional screening cascades: Implement hierarchical screening approaches:

  • Primary screen: Binding to recombinant IGSF11

  • Secondary screen: Cellular binding and internalization

  • Tertiary screen: Functional activity (blocking VISTA interaction, T cell activation)

• Quantitative binding analysis: Use surface plasmon resonance or bio-layer interferometry to determine binding kinetics (kon, koff) and affinity (KD) to select optimal antibodies

By implementing these methodological approaches, researchers can select the most appropriate IGSF11 antibodies for their specific research applications and accelerate the development of potential therapeutic agents.