IGF2R Antibody

What is IGF2R and why is it significant for antibody-based research?

IGF2R (Insulin-like Growth Factor 2 Receptor), also known as M6P/IGF2R, CIMPR, CD222, CI-M6PR, and M6P-R, is a multifunctional type I transmembrane protein with a molecular mass of approximately 274.4 kilodaltons . It has significant research importance due to its:

• Role in IGF2 signaling and cancer biology

• Expression in multiple cell types including monocytes, glioblastoma, and lung carcinoma cells

• Potential as a therapeutic target for osteosarcoma and other cancers

• Complex structure with 15 cation-independent mannose receptor (CIMR) domains and one FNII domain

The extracellular domain of IGF2R contains binding sites for both IGF-II and mannose-6-phosphate moieties, making it a complex target requiring careful antibody selection and validation .

What specific applications are optimized for IGF2R antibodies?

IGF2R antibodies have been validated for numerous experimental applications, with optimization parameters varying by application:

Most protocols emphasize the importance of proper controls and secondary antibody selection for optimal results .

How should researchers approach IGF2R antibody validation?

Comprehensive validation of IGF2R antibodies requires multiple complementary approaches:

• Specificity assessment: Compare staining patterns with multiple antibodies targeting different epitopes of IGF2R

• Positive controls: Use cell lines with confirmed IGF2R expression (A172, A549, 143B)

• Negative controls: Use IGF2R-negative cell lines (K7M2) or isotype control antibodies (e.g., palivizumab)

• Knockdown validation: Confirm reduced signal in IGF2R knockdown models

• Cross-reactivity testing: Evaluate binding to recombinant human, murine, and canine IGF2R proteins via ELISA

For flow cytometry, compare staining patterns with established antibodies such as Goat Anti-Human IGF-II R/IGF2R Antigen Affinity-purified Polyclonal Antibody (AF2447) versus control antibody (AB-108-C) .

What are the critical parameters for successful IGF2R immunostaining?

Successful immunostaining of IGF2R requires careful attention to several technical parameters:

• Fixation method: Immersion fixation shows optimal results for both cytoplasmic and cell surface staining

• Antibody concentration: Typically 3-5 µg/mL for immunocytochemistry applications

• Incubation time and temperature: 3 hours at room temperature is standard for most protocols

• Counterstaining: DAPI is commonly used to visualize nuclei

• Secondary antibody selection: NorthernLights™ 557-conjugated Anti-Goat/Mouse IgG (red; NL001/NL007) shows excellent results

Different cell types show distinct subcellular localization patterns:

• A172 glioblastoma cells: predominantly cytoplasmic staining

• A549 lung carcinoma cells: both cell surface and cytoplasmic staining

These differences may reflect cell-type specific trafficking of IGF2R between late endosomal/prelysosomal compartments and the plasma membrane .

How can researchers accurately quantify IGF2R expression levels?

Accurate quantification of IGF2R expression requires a multi-method approach:

RNA-level quantification:

• qPCR measurement of Igf2r mRNA expression

• Normalization to appropriate housekeeping genes

• Time-course analysis to detect transient changes (30 min, 1 hour, 2 days, 1 week, 2 weeks post-treatment)

Protein-level quantification:

• Western blot analysis comparing total vs. synaptoneurosomal extracts

• Flow cytometry with calibration beads for cell surface expression

• ELISA with serial dilutions (3-300 nM) of antibody against recombinant IGF2R

Subcellular localization assessment:

• Immunofluorescence microscopy with quantitative image analysis

• Fractionation studies comparing membrane vs. cytoplasmic compartments

Research has shown that IGF2R levels remain relatively stable over time in some experimental conditions, with validated changes in control proteins like Egr1 .

How can researchers develop cross-reactive antibodies for comparative IGF2R studies?

Development of cross-reactive antibodies targeting IGF2R from multiple species requires strategic approaches:

• Target conserved epitopes: Target the IGFII binding region (domains 11-13) which shows 82% sequence identity across human, mouse, and canine species

• Use alternating selection pressure:

  • Round 1: Human IGF2R fragment selection

  • Round 2: Murine IGF2R fragment selection

  • Round 3: Canine IGF2R fragment selection

  • Round 4: Human IGF2R fragment selection

• Library generation and screening:

  • For naïve libraries: Amplify antibody repertoire from pooled peripheral blood monocyte cDNA

  • For synthetic libraries: Use structure-guided mutagenesis with trastuzumab 4D5-8 clone as template

  • Focus on VH3 family and kappa light chain genes as these are most abundant

• Validation across species:

  • Perform competitive ELISA to confirm binding to human, murine, and canine IGF2R with similar affinities

  • Conduct flow cytometry on species-specific cell lines

  • Test across patient-derived cell lines from human and canine sources

This approach successfully generated cross-reactive antibodies (IF1 and IF3) with nanomolar affinity to IGF2R from multiple species .

What strategies exist for evolving high-affinity IGF2R antibodies?

Several sophisticated approaches have been developed to create high-affinity IGF2R antibodies:

• In vitro directed evolution combined with structural-directed mutagenesis:

  • Initial phage library panning with selective pressure

  • Structural-guided mutagenesis of binding loops

  • Surface plasmon resonance (SPR) affinity selection

  • Iterative combination of individually identified loop mutants

• Specific mutation strategies:

  • Target AB, CD, and FG loops for optimization

  • Introduce mutations that reduce k_off values by approximately twofold

  • Combine mutations for additive improvements (up to 100-fold in affinity)

• Converting Fab fragments to full IgG:

  • Conversion of Fab fragments to full-length human IgG formats

  • Expression in human origin Expi293F or Chinese hamster ovary GS knockout cells

  • Purification by protein A affinity chromatography

• Conjugation optimization:

  • Use of bifunctional linkers like CHXA" at optimal molar ratios (2.5:1 preferred over 10:1)

  • Radiolabeling with diagnostic (111In) or therapeutic (225Ac) isotopes

  • Validation of immunoreactivity post-conjugation

These strategies have yielded domain 11 variants with improved binding kinetics, thermodynamic properties, and IGF2 antagonist functionality in vivo .

How can IGF2R antibodies be employed for therapeutic applications?

IGF2R antibodies have significant therapeutic potential through multiple mechanisms:

• Radioimmunoimaging applications:

  • Radiolabeling with 111In for biodistribution studies

  • Monitoring with gamma counters to calculate percent injected dose per gram (%ID/g)

  • Tumor visualization in xenograft models

• Radioimmunotherapy approaches:

  • Conjugation with alpha-emitters like 225Ac

  • Dose-dependent cytotoxicity in IGF2R-positive cells (e.g., 143B cells)

  • Specificity confirmed by lack of effect on IGF2R-negative cells (K7M2)

• IGF2 antagonist development:

  • Creation of high-affinity soluble IGF2 traps (IGF2-TRAP)

  • Ability to deplete both mature and higher molecular weight IGF2 isoforms from serum

  • Specific targeting of pathological IGF2 isoforms

• Synergistic therapeutic strategies:

  • Combination with conventional oncology drugs

  • Screening for synergistic lethality with 307-compound oncology drug libraries

  • Targeting both primary tumors and metastases

Recent in vivo studies demonstrate that IGF2-TRAP can deplete IGF2 from both binary complexes with IGFBP3 and from ternary complexes with IGFBP3 and acid labile subunit .

What methodological approaches are used for studying IGF2R function in neurological contexts?

Research on IGF2R's role in neurological function employs several methodological approaches:

• Memory consolidation assessment:

  • Inhibitory avoidance (IA) training paradigms

  • Comparison of trained versus untrained controls

  • Time-course analysis (30 min, 1 hr, 2 days, 1 week, 2 weeks post-training)

• Protein expression analysis:

  • Western blot comparison of total versus synaptoneurosomal extracts

  • Validation using immediate early gene (IEG) Egr1 induction

  • Perinuclear versus cytoplasmic distribution patterns

• Antibody-based interventions:

  • Targeted knockdown of IGF2R in specific brain regions

  • Neutralizing antibodies to block receptor function

  • Temporal control of antibody administration to distinguish between roles in acquisition, consolidation, and retrieval

Research has demonstrated that CIM6P/IGF2R is required for memory consolidation but not learning or memory retrieval, highlighting the importance of timing in experimental design .

What are common pitfalls in IGF2R antibody experiments and how can they be mitigated?

Several challenges are frequently encountered in IGF2R antibody research:

For optimal results, researchers should:

• Validate antibodies on known positive and negative cell lines

• Include appropriate isotype controls (e.g., AB-108-C, palivizumab)

• Use human, murine, and canine-specific positive controls when testing cross-reactivity

• Consider epitope accessibility in different experimental conditions

How should researchers interpret contradictory results from different IGF2R antibody clones?

When facing contradictory results with different IGF2R antibody clones:

• Compare epitope specificity:

  • Different antibodies target distinct epitopes (e.g., extracellular epitope between amino acids 192-697 vs. domains 11-13)

  • Some clones recognize only specific conformational states of IGF2R

• Consider subcellular localization patterns:

  • Different antibodies may preferentially detect different pools of IGF2R (membrane vs. cytoplasmic)

  • The majority of CD222/IGF2R is found in late endosomal/prelysosomal compartments (90-95%), with only 5-10% in plasma membrane

• Evaluate species-specific differences:

  • Some antibodies have limited cross-reactivity across species despite targeting conserved regions

  • Human and non-human primate IGF2R may show differences in glycosylation patterns affecting antibody binding

• Assess truncated forms:

  • Consider that truncated (220 kDa) forms of IGF2R are present in human and bovine serum

  • Some antibodies may differentially detect truncated versus full-length forms

• Perform orthogonal validation:

  • Combine results from multiple techniques (WB, ICC, FACS, IHC)

  • Use genetic approaches (siRNA, CRISPR) to confirm specificity

Understanding the specific characteristics of each antibody clone is essential for proper interpretation of experimental results.

What emerging technologies are advancing IGF2R antibody development and applications?

Several cutting-edge technologies are transforming IGF2R antibody research:

• Advanced library generation methods:

  • Cost-effective custom codon approaches using (N1)HT that encode only 12 amino acids

  • Bias for tyrosine and serine residues in CDRs

  • Generation of libraries with >5 × 10^9 variants

• Combinatorial optimization strategies:

  • Iterative combination of individually identified loop mutants

  • Novel structural mechanisms of ligand-receptor binding site interactions

  • Improved thermodynamic properties through rational design

• Multimodal imaging applications:

  • Dual-labeled antibodies for both PET and optical imaging

  • Species-specific validation in human and canine patient-derived xenografts

  • "One-health" approach targeting both human and companion animal diseases

• Novel therapeutic formats:

  • High-specificity M6P/IGF2R domain 11 binding sites for IGF2

  • IgG1 and IgG2 Fc fusion domain constructs

  • Bispecific antibodies targeting multiple signaling pathways

These technologies offer promising avenues for developing next-generation IGF2R targeting strategies with applications in cancer therapy, neurodegenerative diseases, and companion animal medicine .

What are the most promising research avenues for therapeutic IGF2R antibodies?

Current research highlights several promising directions for therapeutic IGF2R antibodies:

• Osteosarcoma targeted therapies:

  • Dual targeting of human and canine osteosarcoma

  • Potential for radioimmunoimaging and radioimmunotherapy applications

  • Particular promise for patients with distant metastases who have poor prognosis (survival rate ~67%)

• IGF2 trap development:

  • Fc fusion proteins that function as IGF2 antagonists

  • Ability to target higher molecular weight pro-IGF2 isoforms associated with human cancer

  • Potential for treating non-islet cell tumor-associated hypoglycemia (NICTH)

• Combination therapy approaches:

  • Synergistic effects with conventional oncology drugs

  • IGF2-TRAP combined with targeted therapies

  • Overcoming IGF1R-mediated resistance to EGFR inhibitors in breast cancer

• Neurological applications:

  • Memory consolidation and cognitive enhancement

  • Potential role in neurodegenerative diseases

  • Targeting specific temporal windows of IGF2R function