CD276 Antibody

What is CD276 and what role does it play in immune regulation?

CD276 (B7-H3) is a member of the B7 family of immune checkpoint molecules. It is a type I transmembrane protein with a molecular weight of approximately 57.2 kDa and length of 534 amino acids in humans . Although initially described as a co-stimulatory molecule for T cell activation, subsequent research revealed that CD276 actually has strong immunosuppressive effects, inhibiting T cell proliferation and reducing the secretion of IFN-γ, tumor necrosis factor-alpha (TNF-α), and other cytokines .

CD276 may participate in the regulation of T-cell-mediated immune responses, play a protective role in tumor cells by inhibiting natural-killer mediated cell lysis, and serve as a marker for neuroblastoma cell detection . It may also be involved in transplant rejection and regulation of lymphocytic activity at mucosal surfaces. Additionally, CD276 could play a key role in providing the placenta and fetus with a suitable immunological environment during pregnancy .

Both isoforms of CD276 (isoform 1 and isoform 2) appear to modulate CD4 T-cell responses, with isoform 2 shown to enhance cytotoxic T-cell induction and selectively stimulate interferon gamma production when T-cell receptor signaling is present .

How is CD276 expression characterized across different cancer types?

CD276 is frequently overexpressed in malignant tissues, with more than 70% of colorectal, prostate, ovarian, pancreatic, and breast cancer specimens showing CD276 expression . High CD276 expression correlates with poor clinical prognosis across multiple cancer types .

Interestingly, despite similar CD276 expression levels, different cancer cell types show varying sensitivity to CD276-targeted therapies. For example, CD276-positive glioblastomas tend to be resistant to CD276-targeted antibody-drug conjugates (ADCs), while neuroblastomas, breast cancers, colon cancers, lung cancers, and Ewing's sarcomas show high sensitivity . This suggests that factors beyond simple expression levels, such as cellular context and downstream signaling pathways, influence the response to CD276-targeted interventions.

In the tumor microenvironment, CD276 is expressed not only on cancer cells but also on tumor-associated vasculature, which allows CD276-targeted therapies to potentially disrupt tumor blood supply .

What experimental methods are recommended for detecting CD276 expression?

Several validated methods can be employed for reliable CD276 detection:

When selecting anti-CD276 antibodies, consider the specific epitope targeted. Antibodies against amino acids 1-150 (N-terminal/extracellular region) are particularly suitable for applications requiring native protein recognition, while those targeting the C-terminal region (amino acids 450 to C-terminus) may work better for detecting denatured proteins .

For validating antibody specificity, CRISPR-Cas9-engineered CD276 knockout cell lines serve as excellent negative controls. Multiple antibodies recognizing different epitopes should be tested when establishing new detection protocols to ensure result reliability .

What are common applications for anti-CD276 antibodies in cancer research?

Anti-CD276 antibodies serve multiple purposes in cancer research, with applications extending beyond simple detection:

For basic research, these antibodies enable characterization of CD276 expression patterns across normal and malignant tissues, exploration of CD276's role in immune evasion mechanisms, and investigation of its functions in promoting tumor growth, invasion, and metastasis .

In mechanistic studies, anti-CD276 antibodies help elucidate CD276's involvement in the tumor microenvironment, particularly regarding macrophage recruitment and interactions with extracellular matrix components . They also facilitate the study of metabolic reprogramming in tumor cells, as CD276 increases glucose uptake and promotes the Warburg effect through regulation of HIF-1α and glycolytic enzymes .

For translational research, anti-CD276 antibodies are crucial for developing targeted therapies including blocking monoclonal antibodies, antibody-drug conjugates (ADCs), bispecific antibodies, and chimeric antigen receptor (CAR) T-cells . They also help evaluate CD276 as a biomarker for cancer diagnosis, prognosis, and treatment response prediction .

When designing experiments with anti-CD276 antibodies, researchers should consider cross-reactivity with CD276 orthologs in other species (e.g., mouse, rat, bovine) when planning for translational studies, as this can facilitate more seamless bench-to-bedside progression .

How does CD276 contribute to tumor immune evasion?

CD276 employs multiple mechanisms to facilitate tumor immune evasion:

CD276 inhibits T cell proliferation and activity, reducing the secretion of critical anti-tumor cytokines including IFN-γ and TNF-α . This dampens the adaptive immune response against tumor cells and creates an immunosuppressive microenvironment.

In the innate immune compartment, CD276 protects tumor cells from natural killer (NK) cell-mediated lysis, preventing this critical first-line defense against malignant cells . This protection appears to be dependent on direct CD276-mediated signaling rather than indirect effects through other pathways.

CD276 also synergizes with other immune checkpoints, particularly the PD-1/PD-L1 pathway. PD-1 (Programmed Cell Death-1) is another immunosuppressive receptor that interacts with PD-L1 (B7-H1) and PD-L2 (B7-DC) to down-regulate effector T cell activity in both normal tissues and tumors . The combined effect of CD276 and PD-1/PD-L1 signaling creates a powerful immunosuppressive network that effectively shields tumors from immune surveillance.

Research has also revealed that CD276 affects the recruitment and function of macrophages in the tumor microenvironment, potentially skewing them toward a tumor-promoting phenotype . Together, these mechanisms contribute to a comprehensive immune evasion strategy that promotes tumor progression.

What are the current known binding partners or receptors for CD276?

The binding partners and receptors for CD276 have remained elusive for many years, hampering full understanding of its signaling mechanisms. This represents a significant knowledge gap in CD276 biology, as receptor identification is crucial for understanding downstream signaling pathways.

A breakthrough came in 2019 when Husain et al. implemented a new platform called "conditioned medium AlphaScreen" to sensitively detect receptor-ligand interactions at high throughput . Using this technology, they identified interleukin receptor IL20RA as a potential receptor for CD276. This discovery provided the first concrete lead for a CD276 receptor, though further confirmatory studies are still needed to fully validate this interaction .

Understanding CD276's binding partners is essential for developing effective blocking strategies and for predicting potential off-target effects of CD276-targeted therapies. The elucidation of CD276-receptor interactions will likely open new avenues for therapeutic intervention and may explain some of the contradictory results observed in early CD276 functional studies .

Researchers should design experiments that can directly test the interaction between CD276 and IL20RA, as well as continue screening for additional potential binding partners that may contribute to CD276's diverse effects on immune function and tumor progression.

How can researchers effectively validate CD276 antibody specificity?

Validating CD276 antibody specificity is crucial for ensuring reliable experimental results. Researchers should implement a comprehensive validation strategy:

Genetic validation approaches:

• Use CRISPR-Cas9-engineered CD276 knockout cell lines as negative controls to definitively confirm signal specificity

• Compare signal intensity with CD276 expression levels determined by orthogonal methods (e.g., mRNA quantification, proteomics)

• Test multiple cell lines with varying CD276 expression levels to establish signal correlation with expected expression patterns

Biochemical validation methods:

• Perform blocking experiments with recombinant CD276 protein to confirm binding specificity

• Test multiple anti-CD276 antibodies targeting different epitopes to verify consistent detection patterns

• Include appropriate isotype controls matched to the primary antibody class and concentration

Application-specific validation:

• For Western blot: Verify proper molecular weight and band pattern, with CD276 expected at approximately 57.2 kDa (though glycosylation can increase apparent molecular weight)

• For IHC/ICC: Compare staining patterns across multiple tissues with known CD276 expression profiles

• For flow cytometry: Correlate surface expression with total protein levels by comparing permeabilized versus non-permeabilized staining

Through these rigorous validation steps, researchers can ensure that their observations are genuinely attributable to CD276 rather than non-specific binding or technical artifacts.

What molecular mechanisms underlie CD276's promotion of tumor cell proliferation and invasion?

CD276 drives tumor progression through multiple interconnected molecular pathways:

Metabolic reprogramming: CD276 enhances the Warburg effect (aerobic glycolysis), promoting glucose uptake by tumor cells even in oxygen-rich environments . It increases HIF-1α expression and activates downstream molecules including key glycolytic enzymes LDHA and PDK1. Metabolic imaging experiments confirmed that CD276 enhances glucose uptake by tumor cells, thereby promoting tumor growth in mouse breast cancer xenograft models .

Oxidative stress regulation: CD276 promotes HIF-1α stability by inhibiting the stress-activated transcriptional factor Nrf2 and its antioxidant targets (SOD1, SOD2, and PRX3) . This redox signaling modulation creates conditions favorable for tumor cell proliferation.

JAK/STAT signaling activation: CD276 activates the JAK3/STAT3/SLUG signaling pathway in liver cancer, which regulates MMP2 expression and drives epithelial-mesenchymal transition (EMT) . In melanoma cells, CD276 regulates metastasis-related proteins including MMP-2, tissue inhibitors of metalloproteinase (TIMP1 and TIMP2), STAT3, and IL-8 .

EMT promotion: CD276 plays a crucial role in EMT, which enables metastasis. In CD276-knockout liver cancer cells, expression of the epithelial marker E-cadherin is significantly increased, indicating that CD276 normally suppresses this anti-metastatic protein .

Extracellular matrix modulation: CD276 regulates the extracellular matrix modulator PAI-1, affecting tumor stiffness and invasiveness . This modulation likely contributes to creating a physical environment conducive to tumor cell invasion and metastasis.

These mechanistic insights reveal CD276 as a multifunctional oncoprotein that simultaneously affects metabolism, signaling, and extracellular interactions to promote tumor growth and spread.

How does CD276 influence the tumor microenvironment, particularly regarding macrophage recruitment?

CD276 exerts profound effects on the tumor microenvironment through multiple mechanisms:

Macrophage recruitment and polarization: Studies using 3D spheroid coculture systems with human cells have demonstrated that tumor-expressed CD276 plays a significant role in macrophage recruitment into tumor spheroids . Tumor-associated macrophages (TAMs) can comprise up to 50% of tumor mass and typically support tumor growth through various mechanisms. CD276 may influence not only the recruitment but also the polarization of these macrophages toward a tumor-promoting phenotype.

Extracellular matrix remodeling: CD276 regulates the extracellular matrix modulator PAI-1 (Plasminogen Activator Inhibitor-1) . PAI-1 inhibits tissue plasminogen activator and urokinase, enzymes that convert plasminogen to plasmin and degrade fibrin. By modulating PAI-1, CD276 may alter the architecture and stiffness of the tumor stroma, affecting both cancer cell invasion and immune cell infiltration.

Immune cell exclusion: High CD276 expression correlates with lower tumor-infiltrating leukocytes . This reduction in immune cell infiltration contributes to immune evasion and may protect tumors from immune surveillance and attack.

Vascular effects: CD276 is expressed not only on cancer cells but also on tumor-associated vasculature . This expression pattern may affect blood vessel formation, integrity, and permeability within the tumor, influencing both nutrient delivery to tumor cells and immune cell trafficking into the tumor site.

These interactions collectively create an immunosuppressive and tumor-promoting microenvironment that supports cancer progression and resistance to therapy. Understanding these effects is essential for developing combination therapies that can address both the tumor cells and their supportive microenvironment.

What are the current strategies for developing CD276-targeted immunotherapy?

Multiple CD276-targeted immunotherapy approaches are in development, each with unique mechanisms and potential advantages:

Blocking monoclonal antibodies:
These aim to inhibit CD276's immunosuppressive functions by preventing its interaction with receptors on immune cells. Early antibodies like mAb 376.96 have shown promise, particularly when combined with tyrosine kinase inhibitors like sunitinib .

Antibody-drug conjugates (ADCs):
These deliver cytotoxic payloads specifically to CD276-expressing cells. Several iterations have been developed:

• m276-PBD dimer: Shows extensive tumor killing and anti-metastasis activity in vivo

• m276-SL-PBD: An engineered ADC with improved therapeutic index that can eradicate large triple-negative breast cancer xenografts at doses 10-40 fold lower than maximum tolerated dose

• MGC018: Demonstrates antitumor activity across various human tumor xenografts

Bispecific antibodies:
B7-H3Bi-Ab targets both T cell receptors and CD276, enhancing production of IFN-γ, TNF-α, and IL-2. When bound to activated T-cells, it significantly inhibits tumor growth and prolongs survival in animal models .

CAR-T cell therapy:
CD276 CAR-T cells show promising results against solid tumors including osteosarcoma, medulloblastoma, Ewing's sarcoma, and atypical teratoid/rhabdoid tumors . Their efficacy depends on CD276 antigen density on tumor surfaces .

Dual-payload ADCs:
Recent innovations include anti-CD276 monoclonal antibodies conjugated with both cytotoxic drugs and immune-boosting reagents. A DualADC targeting triple-negative breast cancer effectively killed multiple TNBC subtypes, enhanced immune functions in the tumor microenvironment, and reduced tumor burden by 90-100% in animal studies .

Combination approaches:
CD276-targeted therapies may be combined with other modalities, such as PD-1/PD-L1 inhibitors. CD276 CAR-T cells show stronger anti-tumor activity when targeted at tumor cells expressing PD-L1, suggesting potential synergy .

Each approach has specific advantages and challenges that researchers must consider when designing CD276-targeted immunotherapies.

How can researchers optimize antibody-drug conjugates targeting CD276?

Optimizing CD276-targeted ADCs involves several critical strategies across antibody engineering, linker design, payload selection, and manufacturing:

Antibody engineering:

• Fc domain modification: Engineering specific mutations (L234A, L235A, P329G - collectively known as LALAPG) blocks Fcγ receptor binding, reducing off-target toxicity without altering antibody stability or immunogenicity .

• Site-specific conjugation: Introducing a free cysteine at position S239C enables site-specific maleimide-mediated drug conjugation, increasing ADC solubility and reducing aggregation . This approach partly buries the hydrophobic drug in a pocket of the Fc domain and shields it with the nearby hydrophilic carbohydrate side chain at N297.

Linker optimization:

• Stability enhancement: Site-specific conjugation at S239C protects the payload from premature release through retro-Michael reactions and prevents cleavage of the valine-alanine drug linker by circulating enzymes .

• Hydrophilicity: Using more hydrophilic linkers reduces non-specific binding to cells of the reticuloendothelial system and prevents aggregation .

Purification strategies:

• Drug-to-antibody ratio (DAR) optimization: Hydrophobic interaction chromatography (HIC) can be used to enrich for optimal DAR fractions. Studies showed 2.9-3.6-fold higher killing activity in HIC DAR2-enriched fractions compared to unfractionated samples .

Cancer sensitivity screening:

• In vitro testing across multiple cancer types helps identify those most sensitive to the ADC, aiding in personalized therapy approaches and ensuring optimal therapeutic windows .

These approaches have led to significant improvements in CD276 ADCs, creating therapeutics with substantially wider therapeutic windows and greater efficacy against large established tumors .

What challenges must be addressed in developing effective CD276 CAR-T cell therapies?

Developing effective CD276 CAR-T cell therapies presents several significant challenges:

Target density dependence:
The efficacy of CD276 CAR-T cells depends critically on the density of CD276 antigens on tumor surfaces. Studies have demonstrated that activity against cells expressing low levels of CD276 is significantly reduced . This could lead to heterogeneous responses within tumors with variable CD276 expression and potential for antigen-loss escape variants.

Solid tumor barriers:
Unlike hematological malignancies where CAR-T therapy has shown great success, solid tumors present physical and biochemical barriers:

• Dense extracellular matrix limiting CAR-T cell infiltration

• Immunosuppressive microenvironment inhibiting CAR-T function

• Hypoxic conditions affecting CAR-T cell metabolism and persistence

Specificity considerations:
While CD276 is overexpressed in tumors compared to normal tissues, it is not tumor-specific. This raises concerns about potential on-target, off-tumor toxicity affecting normal tissues expressing lower levels of CD276. Careful antibody selection and affinity tuning may be necessary to achieve a therapeutic window.

Manufacturing optimization:
Producing consistent, high-quality CD276 CAR-T cells for clinical use requires standardized protocols addressing:

• Optimal CAR design (costimulatory domains, hinge regions)

• T cell selection and activation methods

• Expansion protocols maintaining CAR-T functionality

• Cryopreservation and shipping considerations

Despite these challenges, CD276 CAR-T cells have shown promise against several solid tumor types, including osteosarcoma, medulloblastoma, Ewing's sarcoma, and atypical teratoid/rhabdoid tumors . Recent studies have demonstrated that CD276 CAR-T cells can control tumor growth both in vitro and in vivo, with significant inhibition of tumor growth and no obvious toxicity or side effects in preclinical models .

How do different cancer types vary in their sensitivity to CD276-targeted therapies despite similar expression levels?

An intriguing paradox has emerged in CD276-targeted therapy development: despite similar CD276 expression levels, different cancer types show remarkably variable sensitivity to CD276-targeted interventions:

Observed sensitivity patterns:

• Highly sensitive: Neuroblastomas, breast cancers, colon cancers, lung cancers, and Ewing's sarcomas show high sensitivity to CD276-targeted ADCs, with IC50 values generally much less than 1 nM .

• Relatively resistant: All tested glioblastomas were highly resistant to CD276-targeted ADCs, with IC50 values greater than 1 nM, despite high CD276 expression . Similarly, pancreatic cancer cell lines demonstrated relative resistance.

Potential mechanisms for differential sensitivity:
Research suggests these sensitivity differences are not attributable to CD276 expression levels alone, as all tested cancer cell types displayed similar CD276 mRNA levels in the Cancer Dependency Map (DepMap) dataset and comparable cell surface protein levels as assessed by flow cytometry .

Instead, sensitivity variations may be attributed to cancer-type-specific factors:

• Driver mutations affecting downstream signaling pathways

• Variations in mitotic index and proliferation rates

• Differences in intrinsic apoptotic sensitivity

• Variable DNA repair capacity affecting response to DNA-damaging payloads

• Cancer-specific drug trafficking, metabolism, or efflux mechanisms

Interestingly, sensitivity patterns to free payload (e.g., SGD-1882 PBD) often mirror sensitivity to the complete ADC, suggesting that intrinsic cellular responses to the payload rather than antibody binding or internalization may be the predominant determinant of efficacy .

These findings highlight the importance of in vitro sensitivity screening as a valuable tool for identifying cancer types intrinsically hypersensitive to specific CD276-targeted approaches, potentially guiding patient selection and helping ensure optimal therapeutic windows.

What methodological considerations are important when studying CD276 in 3D tumor models versus 2D cultures?

When investigating CD276 biology and therapeutics, the choice between 2D and 3D culture systems significantly impacts experimental outcomes:

Expression and localization differences:

• 3D models often show altered CD276 expression patterns compared to 2D cultures

• Polarized expression may occur in 3D structures, affecting accessibility to antibodies

• 3D tumor spheroids better recapitulate the gradient of hypoxia seen in tumors, which may influence CD276 expression via HIF-1α regulation

Functional assessments:

• 3D spheroid coculture systems with human cells provide more physiologically relevant insights into CD276's role in macrophage recruitment

• These systems enable studies of CD276's influence on extracellular matrix components and tumor-stroma interactions that are absent in 2D models

Drug delivery considerations:

• ADC penetration differs substantially between 2D and 3D models

• Binding site barrier effects (where antibodies bind primarily to the outer layer of cells) are only observable in 3D models

• Drug efficacy typically decreases in 3D models due to limited penetration, more accurately predicting in vivo challenges

Experimental setup:
When creating 3D models to study CD276:

• Consider co-culture systems incorporating relevant stromal and immune components

• Validate CD276 expression and accessibility using imaging techniques like confocal microscopy

• Optimize dissociation protocols for flow cytometry that maintain CD276 epitopes

• Establish image analysis workflows for quantifying CD276 expression in different regions of spheroids

The 3D spheroid model is particularly valuable for studying CD276's role in the tumor microenvironment, as demonstrated by research showing tumor-expressed CD276's influence on macrophage recruitment into tumor spheroids . This approach provides insights that would be impossible to obtain using traditional 2D culture methods.

How can researchers design effective combination therapies targeting CD276 alongside other immune checkpoint modulators?

Designing effective combination therapies targeting CD276 requires systematic consideration of multiple factors:

Mechanistic rationale:
CD276 synergizes with other immune checkpoints, particularly the PD-1/PD-L1 pathway. Understanding these interactions helps identify non-redundant pathways for simultaneous targeting. CD276 CAR-T cells have shown stronger anti-tumor activity against tumor cells expressing PD-L1, indicating potential synergy between these pathways . Combination therapies should target complementary aspects of tumor immunity.

Strategic combination partners:
Several promising combination approaches have emerged:

Dual-function approaches:
Recent innovations include dual-payload antibody-drug conjugates targeting CD276, which combine cytotoxic drugs with immune-boosting reagents. These DualADCs have effectively killed multiple triple-negative breast cancer subtypes while enhancing immune functions in the tumor microenvironment, reducing tumor burden by up to 90-100% in animal studies .

Biomarker-guided patient selection:
Single-cell RNA sequencing, multiplex cytokine analysis, and histological approaches can help characterize treatment effects on tumor cells and the immune landscape . These methods can identify predictive biomarkers for response to combination therapy and guide patient selection.

Optimizing drug delivery:
Nanoparticle-based delivery systems can co-deliver CD276-targeted agents with other immunomodulators, potentially enhancing efficacy while reducing systemic toxicity. Consideration should be given to the pharmacokinetics of each agent to ensure optimal exposure at the tumor site.

By taking these factors into account, researchers can design rational combination approaches that overcome resistance to single-agent immunotherapies and achieve more durable responses in cancer patients.