PDPN P56F7AT Antibody

What is PDPN and why is it a significant target for cancer research?

Podoplanin (PDPN), also known as T1α/Aggrus/gp36, is a type I transmembrane sialo-glycoprotein that plays essential roles in cancer progression and metastasis. PDPN-expressing cancers exhibit aggressive phenotypes, including increased stemness, invasiveness, and epithelial-to-mesenchymal transition (EMT), which contribute to malignant progression. Additionally, PDPN-positive cancer-associated fibroblasts (CAFs) mediate an immunosuppressive tumor microenvironment, reducing antitumor immunity. These characteristics make PDPN an attractive therapeutic target for cancer treatment, particularly in squamous cell carcinomas, malignant gliomas, and mesotheliomas, where its overexpression is associated with poor clinical outcomes .

How does antibody defucosylation enhance PDPN antibody efficacy?

Defucosylation of antibodies involves removing the core fucose from the N-glycan in the Fc region of the antibody. This structural modification significantly enhances the interaction between the antibody's Fc region and FcγRIIIa on natural killer (NK) cells, which mediates antibody-dependent cellular cytotoxicity (ADCC). In the case of anti-PDPN antibodies, defucosylation has been shown to dramatically increase ADCC activity against PDPN-positive cancer cells. For example, a defucosylated humanized anti-PDPN antibody (humLpMab-23-f) demonstrated significantly higher cytotoxicity (51.6%) against PDPN-expressing cells compared to control human IgG (13.6%), highlighting how this modification substantially improves the therapeutic potential of PDPN antibodies .

What are the major differences between various anti-PDPN antibody clones?

Different anti-PDPN antibody clones target distinct epitopes and demonstrate varying functionalities. For instance, the NZ-1 clone exhibits neutralizing activity for CLEC-2 binding, preventing platelet aggregation and hematogenous metastasis to the lung. LpMab-2, developed through the cancer-specific monoclonal antibody (CasMab) method, recognizes a glycopeptide structure of PDPN from Thr55 to Leu64. In contrast, LpMab-23 recognizes a peptide structure from Gly54 to Leu64. These different epitope recognitions result in varied efficacy in applications such as immunohistochemistry, flow cytometry, and therapeutic potential. Additionally, chimeric versions like NZ-8 and chLpMab-7 have been engineered with human IgG1 to enhance ADCC and CDC activities while maintaining the original binding specificity .

How does humanized anti-PDPN antibody compare with mouse-human chimeric versions in preclinical models?

Humanized anti-PDPN antibodies like humLpMab-23 demonstrate superior binding affinity compared to mouse-human chimeric versions. The KD value of humLpMab-23 with PC-10 cells (human lung squamous cell carcinoma) is approximately 5.4 × 10^-9 M, whereas the chimeric version (chLpMab-23) showed a KD of 1.4 × 10^-7 M with the same cells. This 26-fold improvement in binding affinity contributes to enhanced efficacy in both in vitro and in vivo settings. In xenograft models, the humanized and defucosylated version (humLpMab-23-f) demonstrated more potent antitumor effects than its chimeric counterpart. Notably, while chLpMab-23-f showed moderate ADCC activity against CHO/PDPN cells, it failed to exhibit significant ADCC activity against PC-10 cells or CDC activity against PDPN-expressing tumor cells, limitations that were overcome by the humanized version .

What mechanisms contribute to anti-PDPN antibody-mediated tumor suppression in vivo?

Anti-PDPN antibodies suppress tumor growth through multiple mechanisms. First, they induce antibody-dependent cellular cytotoxicity (ADCC), wherein NK cells recognize antibody-bound tumor cells and trigger their destruction. Second, they activate complement-dependent cytotoxicity (CDC), in which the complement system is activated to form membrane attack complexes on tumor cells. In xenograft models, treatment with humLpMab-23-f resulted in significant tumor volume reduction (approximately 73% compared to control) and tumor weight reduction (90%) against CHO/PDPN tumors. Additionally, anti-PDPN antibodies may interfere with PDPN's role in enhancing tumor invasiveness, EMT, and cancer stemness. Some antibodies also block the interaction between PDPN and CLEC-2 on platelets, potentially reducing platelet aggregation-associated venous thromboembolism and immune evasion .

How can researchers optimize experimental design when evaluating anti-PDPN antibodies in xenograft models?

When designing xenograft experiments to evaluate anti-PDPN antibodies, researchers should consider several critical factors. First, appropriate cell line selection is essential, including both PDPN-overexpressing engineered lines (like CHO/PDPN) and endogenous PDPN-expressing cancer lines (such as PC-10 for lung squamous cell carcinoma or LN319 for glioblastoma). Second, the administration schedule is crucial – in published studies, antibodies were administered intraperitoneally on days 6, 14, and 21 following tumor cell inoculation. Third, for evaluating ADCC activity, human NK cells should be injected around the tumors concurrently with antibody administration. Fourth, comprehensive assessment should include tumor volume measurements (taken at regular intervals, e.g., days 6, 11, 14, 18, 21, and 25), tumor weight at study endpoint, and animal body weight monitoring to assess potential toxicity. Finally, researchers should consider including appropriate controls such as non-defucosylated versions and non-targeting human IgG .

What assays are essential for characterizing the functional properties of anti-PDPN antibodies?

Comprehensive characterization of anti-PDPN antibodies requires multiple complementary assays. Flow cytometry is fundamental for confirming binding to PDPN-positive cells and determining binding affinity (KD values). For example, humLpMab-23 demonstrated KD values of 4.7 × 10^-9 M, 4.9 × 10^-9 M, and 5.4 × 10^-9 M against CHO/PDPN, LN319, and PC-10 cells, respectively. ADCC assays using human NK cells as effectors are critical for evaluating cytotoxic potential against PDPN-expressing target cells. Similarly, CDC assays with human complement are essential for assessing complement-activated killing. Additionally, immunohistochemistry can characterize reactivity patterns in tumor tissues versus normal PDPN-expressing cells. For therapeutic potential, xenograft models with tumor volume and weight measurements provide crucial in vivo efficacy data. Researchers should also consider evaluating potential neutralizing activity for PDPN-CLEC-2 interaction if studying effects on metastasis or platelet aggregation .

How can researchers accurately quantify ADCC and CDC activities of anti-PDPN antibodies?

Accurate quantification of ADCC and CDC activities requires standardized experimental protocols. For ADCC, researchers should isolate NK cells from human peripheral blood and use a consistent effector-to-target cell ratio (e.g., 100:1 or 50:1). Cytotoxicity can be measured using lactate dehydrogenase (LDH) release assays or fluorescent dye-based viability assays after incubation periods of 4-6 hours. For CDC assays, human complement serum is typically used at 10% (v/v), and cytotoxicity is similarly measured after 2-4 hours of incubation. In both assays, percent cytotoxicity should be calculated using the formula: [(experimental release - spontaneous release)/(maximum release - spontaneous release)] × 100. Control antibodies of matching isotype but non-specific binding should be included to establish baseline activity. When testing against multiple cell lines, it's important to maintain consistent antibody concentrations (typically 1-10 μg/mL) to enable direct comparisons. Data reporting should include both raw cytotoxicity percentages and statistical analysis of differences between test and control antibodies .

What techniques are available for evaluating anti-PDPN antibody binding in heterogeneous tumor samples?

Heterogeneous tumor samples present challenges for evaluating anti-PDPN antibody binding that can be addressed through multiple complementary techniques. Immunohistochemistry remains the gold standard for visualizing PDPN expression in formalin-fixed paraffin-embedded (FFPE) tissue sections, allowing assessment of expression in both tumor cells and surrounding stroma, including cancer-associated fibroblasts (CAFs). Flow cytometry of dissociated tumor samples can quantify binding affinity across different cell populations when combined with cell-type-specific markers. Single-cell RNA sequencing paired with protein analysis can correlate PDPN expression with specific cell phenotypes. For in vivo imaging, fluorescently labeled or radiolabeled antibodies can trace biodistribution within heterogeneous tumors. Importantly, researchers should validate findings using multiple patient-derived samples, as PDPN expression varies significantly among tumor types and individual patients. Multiplexed immunofluorescence can be particularly valuable for simultaneously visualizing PDPN expression alongside markers for tumor cells, CAFs, immune cells, and vascular structures .

How might anti-PDPN antibodies synergize with immune checkpoint inhibitors?

Anti-PDPN antibodies have significant potential to synergize with immune checkpoint inhibitors based on emerging understanding of PDPN's immunomodulatory functions. Research has revealed that PDPN acts as a co-inhibitory receptor on both CD4+ and CD8+ T lymphocytes. Studies in T lymphocyte-specific Pdpn-deleted mice showed significantly delayed growth of inoculated tumors, with PDPN-deficient CD8+ tumor-infiltrating lymphocytes exhibiting increased tumor necrosis factor production and decreased exhausted T lymphocyte populations. This suggests PDPN contributes to tumor immune suppression through mechanisms distinct from but potentially complementary to PD-1 and CTLA-4 pathways. Additionally, PDPN-positive cancer-associated fibroblasts (CAFs) promote an immunosuppressive tumor microenvironment, partly through increased TGF-β expression. Therefore, combining anti-PDPN antibodies with established checkpoint inhibitors could potentially address multiple immunosuppressive mechanisms simultaneously, potentially overcoming resistance to single-agent immunotherapy. Future research should explore optimal sequencing and dosing of such combinations across different tumor types .

How can researchers address variability in PDPN expression levels across tumor models?

Variability in PDPN expression across tumor models presents a significant challenge for researchers. To address this, implement a multi-faceted approach beginning with comprehensive baseline characterization. Quantify PDPN expression using both RNA-seq and protein-level analyses (Western blot, flow cytometry) across all cell lines or patient-derived samples. Consider developing standardized expression categories (high, medium, low, negative) based on quantitative thresholds. For xenograft studies, verify expression stability in vivo through immunohistochemistry of harvested tumors at different time points, as expression may evolve. When evaluating antibody efficacy, normalize results against PDPN expression levels to identify potential correlations between expression and response. If working with heterogeneous samples, consider cell sorting to create defined PDPN-high and PDPN-low populations for parallel testing. Additionally, researchers might consider developing CRISPR-engineered isogenic cell lines with controlled PDPN expression levels to eliminate confounding variables when comparing antibody effects across different genetic backgrounds .

What strategies can optimize the production of defucosylated antibodies for research applications?

Optimizing production of defucosylated antibodies requires attention to several critical factors. The preferred approach utilizes fucosyltransferase 8 (Fut8)-deficient Chinese hamster ovary (CHO) cell lines as expression hosts, as these produce completely defucosylated recombinant antibodies. Researchers should implement stable transfection of these specialized cell lines with optimized expression vectors containing the humanized variable regions and human IgG1 constant regions. For laboratory-scale production, consider using suspension culture in chemically defined, serum-free media to improve reproducibility and reduce purification challenges. Implement a two-step purification protocol, typically combining Protein A affinity chromatography followed by size exclusion chromatography. Quality control should include verification of defucosylation levels using mass spectrometry or lectin blotting, binding affinity assessment via surface plasmon resonance or bio-layer interferometry, and functional testing of ADCC activity compared to fucosylated counterparts. For comparative studies, produce matched fucosylated versions using standard CHO cells to isolate the specific effects of defucosylation on antibody performance .

How can researchers validate the specificity of anti-PDPN antibodies in complex experimental systems?

Validating anti-PDPN antibody specificity in complex systems requires a multi-layered approach. First, conduct basic validation using PDPN-knockout cell lines generated through CRISPR-Cas9 alongside PDPN-overexpressing lines as positive controls. This comparison establishes baseline specificity parameters. For tissue-based studies, include appropriate blocking experiments with recombinant PDPN protein or competing antibodies targeting known epitopes. When performing immunohistochemistry or immunofluorescence, validate staining patterns against multiple anti-PDPN antibody clones recognizing different epitopes to confirm consistent localization patterns. For complex systems like patient-derived xenografts or clinical samples, combine antibody staining with in situ hybridization for PDPN mRNA to confirm correlation between protein detection and gene expression. Additionally, mass spectrometry-based approaches can provide unbiased validation of antibody pull-down specificity. When testing in animal models, species cross-reactivity should be carefully assessed, as epitope conservation across species may vary. Finally, functional validation through expected biological effects (e.g., ADCC activity against PDPN-positive but not negative cells) provides critical confirmation of specificity in the experimental context .

What are the prospects for developing anti-PDPN antibody drug conjugates or radioimmunotherapies?

The development of anti-PDPN antibody drug conjugates (ADCs) and radioimmunotherapies represents a promising frontier, building on established precedent in the field. Previous research has already demonstrated the potential of such approaches with the development of NZ-1-R700 conjugates for near-infrared photoimmunotherapy and NZ-16 labeled with actinium-225 (225Ac) for radioimmunotherapy, both showing efficacy against malignant pleural mesothelioma xenograft models. The high binding specificity of humanized anti-PDPN antibodies like humLpMab-23, combined with their demonstrated tumor-targeting capabilities in vivo, makes them excellent candidates for developing next-generation ADCs. Potential payloads could include conventional cytotoxic agents, DNA-damaging compounds, or novel immunomodulatory molecules. The relatively selective expression of PDPN in tumors versus most normal tissues (with exceptions like lymphatic endothelium) provides a favorable therapeutic window for these targeted approaches. Future research should focus on optimizing drug-to-antibody ratios, linker chemistry, payload selection, and combinatorial approaches with other treatment modalities to maximize efficacy while minimizing off-target effects .

What is the potential for combining anti-PDPN antibodies with CAR-T cell therapies?

The combination of anti-PDPN antibodies with CAR-T cell therapies represents an innovative convergence of immunotherapeutic approaches with substantial promise. Previous research has successfully applied PDPN-targeting chimeric antigen receptor (CAR)-T therapy in preclinical studies of human glioblastoma, demonstrating the feasibility of targeting this antigen with cellular immunotherapy. Anti-PDPN antibodies could potentially enhance CAR-T efficacy through multiple mechanisms. First, by depleting PDPN-positive cancer-associated fibroblasts (CAFs), antibodies could reduce immunosuppressive signals in the tumor microenvironment, creating more favorable conditions for CAR-T cell infiltration and function. Second, antibody treatment might modulate PDPN's role as a co-inhibitory receptor on T lymphocytes, potentially enhancing the persistence and activity of the administered CAR-T cells. Additionally, sequential treatment with antibodies and CAR-T cells targeting different PDPN epitopes could reduce antigen escape, a common limitation of single-epitope targeted therapies. Further research should explore optimal timing and sequencing of these approaches, potential epitope competition, and synergistic effects on both direct tumor killing and microenvironment modulation .

How should researchers interpret differential responses to anti-PDPN antibodies across diverse tumor models?

Interpreting differential responses to anti-PDPN antibodies across tumor models requires systematic analysis of multiple variables. Begin by quantifying absolute PDPN expression levels across models using standardized methods (flow cytometry, Western blotting, immunohistochemistry with digital quantification) to determine if response correlates with expression intensity. Beyond simple expression levels, analyze PDPN glycosylation patterns, as glycoform differences can significantly impact epitope accessibility and antibody binding. Assess the tumor microenvironment composition, particularly the prevalence and activation state of NK cells and complement factors, which are essential for ADCC and CDC activities respectively. Consider genetic factors like FcγR polymorphisms in the model systems that might affect ADCC potential. Evaluate the infiltration capability of effector cells and antibodies into different tumor types, as physical barriers may limit efficacy despite target expression. Additionally, analyze expression of potential resistance factors, such as complement inhibitory proteins or anti-apoptotic molecules. Finally, develop multivariate models incorporating these parameters to predict response patterns, which can guide rational selection of tumor types and combination strategies for further development .

What factors should researchers consider when translating preclinical findings with anti-PDPN antibodies to clinical applications?

Translating preclinical findings with anti-PDPN antibodies to clinical applications requires consideration of multiple critical factors. First, species differences in PDPN expression patterns and immune system interactions must be addressed; while cynomolgus monkey studies showed no toxicity with mouse-human chimeric LpMab-23 at 20 mg/kg, human tissue cross-reactivity studies remain essential. Second, heterogeneity of PDPN expression in human tumors is considerably greater than in cell line-derived xenograft models, necessitating thorough expression profiling across patient samples. Third, effector cell function variability among patients (particularly NK cell abundance and activity) will impact ADCC potential, suggesting the need for potential biomarkers of immune effector status. Fourth, the tumor microenvironment differs significantly between mouse models and human cancers, particularly regarding immune cell composition and functionality, which may alter antibody penetration and activity. Fifth, pharmacokinetic and pharmacodynamic parameters must be carefully modeled, as these will determine dosing strategies. Finally, rational combination strategies should be considered based on identified resistance mechanisms in preclinical models, with particular attention to potential synergies with immune checkpoint inhibitors given PDPN's newly discovered role as a co-inhibitory receptor on T lymphocytes .

How can researchers distinguish between direct and indirect effects of anti-PDPN antibodies in complex tumor models?

Distinguishing between direct and indirect effects of anti-PDPN antibodies in complex tumor models requires sophisticated experimental designs and analyses. Researchers should develop parallel models with selective PDPN knockdown or knockout in either tumor cells or stromal components (particularly CAFs) to decouple their respective contributions to antibody effects. Sequential depletion studies—where specific immune cell populations (NK cells, macrophages, T cells) are selectively eliminated before antibody administration—can isolate the contribution of each effector mechanism. Time-course analyses are critical, as direct cytotoxic effects via ADCC/CDC typically occur rapidly (hours to days), while indirect effects through microenvironment modulation may require longer timeframes (days to weeks). Multiplexed imaging approaches combining antibody tracking with simultaneous visualization of target engagement, immune cell recruitment, and downstream signaling events can provide spatial and temporal resolution of different mechanisms. Additionally, single-cell RNA sequencing of tumors at various timepoints post-treatment can reveal transcriptional changes across different cell populations, helping differentiate primary from secondary effects. Finally, comparing the effects of Fc-functional antibodies with F(ab')2 fragments or Fc-mutated versions that retain binding but lack effector functions can isolate direct signaling effects from immune-mediated mechanisms .