BTN1A1 Antibody

What is BTN1A1 and what is its role in immune regulation?

BTN1A1 (Butyrophilin 1A1) is a 55kDa type I transmembrane glycoprotein belonging to the immunoglobulin superfamily. It consists of an extracellular domain (ECD) (amino acids 27-242), a transmembrane domain, and a cytoplasmic tail (amino acids 270-526) containing the B30.2 domain . The ECD displays two predicted IgV and IgC domains similar to B7 and Skint proteins that interact with other Ig superfamily members .

BTN1A1 functions as an immune checkpoint protein expressed on various immune cells including macrophages, B cells, and activated CD8 T cells, as well as in human tumors . Research demonstrates that BTN1A1 plays an immunomodulatory role by inhibiting T cell activation and proliferation. Specifically, BTN1A1 inhibits the proliferation of T cells activated by anti-CD3 and anti-CD28 antibodies in vitro, and overexpression of BTN1A1 in PC3 cells inhibits T cell-mediated killing of cancer cells .

How does BTN1A1 expression compare to PD-L1 in tumor microenvironments?

Research indicates that BTN1A1 and PD-L1 expression are mutually exclusive in various human solid tumors . This non-overlapping expression pattern is particularly significant because it suggests that BTN1A1 may represent a promising therapeutic target for tumors that are refractory to anti-PD-1/PD-L1 antibody treatments . Furthermore, studies have observed that BTN1A1 had significantly higher detection levels than PD-L1 in cancer patient samples . This distinct expression pattern positions BTN1A1 as a novel cancer immunotherapy target that extends beyond the limitations of low PD-1/PD-L1 expression in tumors .

What are the key binding partners of BTN1A1 and their significance?

Through cell microarray screening, researchers have identified several key binding partners for the extracellular domain of human BTN1A1, including:

• Galectin-1 (Gal1)

• Galectin-9 (Gal9)

• Neuropilin 2 (NRP2)

These binding partners specifically interact with wild-type BTN1A1 but not with the unglycosylated (2NQ) form of the protein . Among these, Galectin-9 (Gal9) exhibited the greatest affinity for human BTN1A1 in immunoprecipitation and Biacore binding assays .

Significantly, Gal9 has been identified as a BTN1A1 receptor that can form a three-protein complex with BTN1A1 and PD-1, thereby suppressing T cell receptor signaling and T cell activation . This BTN1A1-Gal9-PD-1 axis has emerged as a novel therapeutic target for immunotherapeutic drug development, particularly because high Gal9 expression levels correlate with poor prognosis in multiple cancers .

What are the optimal methods for detecting BTN1A1 expression in tissue samples?

For detecting BTN1A1 expression in tissue samples, researchers can employ several validated methods:

Immunohistochemistry (IHC):
Using anti-BTN1A1 antibodies such as Rabbit Anti-Human BTN1A1/Butyrophilin Polyclonal Antibody at an optimized concentration (e.g., 0.3 μg/mL) for 3 hours at room temperature . For visualization, appropriate secondary antibodies like NorthernLights™ 557-conjugated Anti-Rabbit IgG can be used, with DAPI counterstaining to visualize nuclei .

Flow Cytometry:
For cell suspensions, flow cytometry offers quantitative analysis of BTN1A1 expression. HEK293 transfection studies demonstrate that Human BTN1A1 can be detected using Rabbit Anti-Human BTN1A1 Monoclonal Antibody followed by APC-conjugated Goat-anti Rabbit IgG secondary antibody . Quadrant markers should be set based on control antibody staining to ensure specificity.

Fluorescent Immunocytochemistry:
For cultured cells, immersion fixation followed by staining with anti-BTN1A1 antibodies provides localization data. Studies indicate that specific BTN1A1 staining is predominantly localized to the cytoplasm in HEK293 cells .

When comparing expression patterns between BTN1A1 and other immune checkpoints like PD-L1, it's important to note that researchers have observed some inconsistency among existing data when comparing mRNA with protein expression patterns of BTN/BTNL family molecules . Therefore, validating expression using multiple methods is recommended for conclusive results.

How can researchers effectively evaluate BTN1A1 antibody specificity?

To effectively evaluate BTN1A1 antibody specificity, researchers should implement a multi-faceted approach:

Positive and Negative Controls:
Use HEK293 cells transfected with human BTN1A1 as positive controls and irrelevant transfectants as negative controls . Compare staining patterns between these populations to confirm antibody specificity.

Glycosylation-Dependent Binding Tests:
Since BTN1A1 binding partners interact with the wild-type protein but not with the unglycosylated (2NQ) form, testing antibody binding to both forms can help confirm specificity for properly folded and modified BTN1A1 .

Cross-Reactivity Assessment:
Test the antibody against related butyrophilin family members (e.g., BTN2A1, BTN3A1) to ensure it doesn't cross-react with structurally similar proteins . This is particularly important since butyrophilins share structural features with B7 proteins and may have overlapping functions in immune regulation.

Blocking Experiments:
Pre-incubate the antibody with recombinant BTN1A1 protein before applying to samples. Specific binding should be significantly reduced or eliminated in these conditions.

What protocols are recommended for using BTN1A1 antibodies in functional assays?

For functional assays investigating BTN1A1's immunomodulatory effects, the following protocols are recommended:

T Cell Proliferation Assays:

• Isolate CD4+ T cells from peripheral blood

• Activate cells with anti-CD3 and anti-CD28 antibodies

• Add purified BTN1A1-Ig fusion protein or anti-BTN1A1 antibodies at varying concentrations

• Measure proliferation after 72 hours using standard techniques (e.g., tritiated thymidine incorporation or CFSE dilution)

• Analyze cytokine production (IL-2, IFN-γ) by ELISA or intracellular cytokine staining

Cancer Cell Killing Assays:

• Establish target cells overexpressing BTN1A1 (e.g., transfected PC3 cells)

• Co-culture with activated T cells at various effector:target ratios

• Add anti-BTN1A1 antibodies or control antibodies

• Assess target cell killing using cytotoxicity assays (e.g., LDH release, flow cytometry-based methods)

Receptor Occupancy Studies:
For evaluating therapeutic antibodies, receptor occupancy assays help determine optimal dosing. Studies with humanized STC810 (anti-BTN1A1 antibody) used PK modeling to identify doses needed to achieve tumor concentrations corresponding to different receptor occupancy levels (20%, 80%, or 95%) .

How does the BTN1A1-Gal9-PD-1 axis regulate T cell activation?

The BTN1A1-Gal9-PD-1 axis represents a complex immunoregulatory mechanism with significant implications for cancer immunotherapy. Research has identified Galectin-9 (Gal9) as a BTN1A1 receptor that can form a three-protein complex with BTN1A1 and PD-1 . This molecular complex functions to suppress T cell receptor signaling and inhibit T cell activation through the following mechanisms:

• Complex Formation: Gal9 acts as a bridge between BTN1A1 and PD-1, forming a trimolecular complex that enhances immunosuppressive signaling .

• Signaling Pathway Suppression: This complex inhibits TCR-mediated signaling cascades required for T cell activation, effectively dampening immune responses against tumors expressing BTN1A1 .

• Cell Cycle Regulation: BTN1A1 engagement leads to cell-cycle arrest in activated T cells, similar to the effects observed with related butyrophilin family members like BTNL1 .

• Cytokine Modulation: BTN1A1 signaling reduces expression of cytokines associated with T cell activation, particularly IL-2 and IFN-γ, further suppressing anti-tumor immune responses .

This complex interplay between BTN1A1, Gal9, and PD-1 is particularly significant because high Gal9 expression levels correlate with poor prognosis in multiple cancers . Furthermore, the mutually exclusive expression pattern of BTN1A1 and PD-L1 suggests these checkpoints may operate in different tumor microenvironments, potentially explaining why some patients fail to respond to anti-PD-1/PD-L1 therapies .

What is the role of the B30.2 domain in BTN1A1 function?

The B30.2 domain in the cytoplasmic tail of BTN1A1 plays critical roles in its biological functions:

• XOR Binding: The B30.2 domain of BTN1A1 binds to xanthine oxidoreductase (XOR) . This interaction stabilizes the association of XOR with the milk fat globule membrane and appears essential in controlling milk fat globule secretion . This binding property is conserved among BTN1A1 orthologs but is not shared by other butyrophilin family members like BTN2A1 or BTN3A1 .

• Phosphoantigen (pAg) Sensing: The B30.2 domain of butyrophilins functions as a sensor for detecting changes in intracellular phosphoantigen (pAg) concentrations . When the B30.2 domain binds to pAg, it induces a cascade of events leading to the activation of gamma delta T cells .

• Signal Transduction: As part of the BTN1A1 complex that serves as a starting point for signaling pathways, the B30.2 domain likely contributes to the protein's ability to modulate immune responses . This domain helps confer functional specificity to different butyrophilin family members.

Understanding the specific mechanisms by which the B30.2 domain contributes to BTN1A1's immunomodulatory functions remains an active area of research and may provide insights for developing therapies that target this domain specifically.

How do BTN1A1 expression patterns differ across tumor types and what are the implications for immunotherapy?

BTN1A1 expression patterns vary significantly across tumor types, with important implications for immunotherapy:

• Expression Prevalence: BTN1A1 is highly expressed in multiple human tumor types, including urothelial carcinoma and non-small cell lung cancer (NSCLC) . Importantly, studies have shown that BTN1A1 had significantly higher detection levels than PD-L1 in various cancer patient samples .

• Mutually Exclusive Expression with PD-L1: A critical finding is that BTN1A1 and PD-L1 expression are mutually exclusive in various human solid tumors . This non-overlapping pattern suggests distinct immunosuppressive mechanisms operating in different tumor microenvironments.

• Stress-Inducible Expression: BTN1A1 was identified as a potential stress-inducible candidate immune checkpoint using in vivo immune checkpoint target discovery platforms . This suggests that BTN1A1 expression may be dynamic and influenced by conditions in the tumor microenvironment.

Implications for Immunotherapy:

• Complementary Target to PD-1/PD-L1: The mutually exclusive expression pattern positions BTN1A1 as a compelling complementary target to existing PD-1/PD-L1 therapies .

• Potential for Combination Approaches: Preclinical studies demonstrate that anti-BTN1A1 antibodies exhibit antitumor activity both as single agents and in combination with anti-PD-1/PD-L1 or radiation therapy .

• Activity in "Cold" Tumors: Humanized anti-BTN1A1 antibody (hSTC810) was found to suppress tumor growth in A549 CDX humanized murine models, which are considered "immunologically cold" tumors unresponsive to anti-PD-L1 treatment . This suggests potential efficacy in patients who fail to respond to existing checkpoint inhibitors.

• Biomarker Development: The distinct expression pattern of BTN1A1 could serve as a biomarker to guide patient selection for targeted immunotherapies, potentially identifying patients unlikely to respond to PD-1/PD-L1 blockade who might benefit from BTN1A1-targeted approaches.

What is the current status of anti-BTN1A1 antibodies in clinical development?

The clinical development of anti-BTN1A1 antibodies has progressed to early-phase human trials:

• First-in-Human Trial: Humanized STC810 (hSTC810), a monoclonal antibody targeting BTN1A1, is currently being evaluated in a first-in-human Phase I clinical study (NCT05231746) . This trial aims to identify the maximally tolerated dose (MTD) and establish the initial safety profile for patients with advanced solid tumors.

• Trial Design: The Phase I study employs a standard 3+3 escalation design to explore safety, tolerability, dose-limiting toxicities (DLTs), and pharmacokinetics, and to define a recommended phase II dose (RP2D) . The multi-site, non-randomized, open-label study enrolls subjects aged >18 years with various advanced solid tumors.

• Dosing Schedule: Eligible patients receive hSTC810 intravenously at doses ranging from 0.3mg/kg to 15 mg/kg once every 2 weeks as a single agent until disease progression or lack of tolerability .

• Current Status: As of the most recent data available, 13 patients had been enrolled, and 2 dose levels had been completed with no dose-limiting toxicities observed . Enrollment was continuing at the time of reporting.

• Future Plans: Dose expansion cohorts are planned at the recommended Phase II dose to further assess safety, pharmacokinetics, pharmacodynamics, and immunohistochemistry in pre- and post-treatment tumor tissue samples .

The development of hSTC810 follows promising preclinical studies that demonstrated antitumor activity in A549 CDX humanized murine models, which are typically unresponsive to anti-PD-L1 treatment . This suggests potential efficacy in patients with tumors that are refractory to existing immune checkpoint inhibitors.

How do preclinical models inform the therapeutic potential of anti-BTN1A1 antibodies?

Preclinical models have provided crucial insights into the therapeutic potential of anti-BTN1A1 antibodies:

• Efficacy in Monotherapy: Mouse studies demonstrated that anti-BTN1A1 antibodies exhibited antitumor activity as single agents . This establishes BTN1A1 as a valid standalone therapeutic target.

• Combination Therapy Potential: These antibodies also showed enhanced efficacy when combined with anti-PD-1/PD-L1 antibodies or radiation therapy in preclinical models . This supports the potential for combination approaches in clinical settings.

• Activity in "Cold" Tumor Models: Particularly significant was the finding that humanized anti-BTN1A1 antibody (hSTC810) suppressed tumor growth in A549 CDX humanized murine models, which are considered immunologically "cold" tumors unresponsive to anti-PD-L1 treatment . This suggests potential efficacy in patients who fail to respond to existing checkpoint inhibitors.

• Safety Profile: Preclinical studies indicated that hSTC810-treated mice exhibited significantly reduced tumor volumes compared to hIgG4-treated control animals without any treatment-related toxicity . This favorable safety profile supported advancement to human trials.

• Mechanistic Insights: Studies identified Galectin 9 (Gal9) as a BTN1A1 receptor that can form a three-protein complex with BTN1A1 and PD-1, thereby suppressing T cell receptor signaling and T cell activation . This mechanistic understanding helps explain the therapeutic activity and supports rational combination strategies.

• Pharmacokinetic Modeling: PK modeling including allometric scaling was performed to conduct simulations predicting the PK profile of hSTC810 after IV administration in humans. This informed the dosing strategies for clinical trials, identifying doses needed to achieve tumor concentrations corresponding to different receptor occupancy levels (20%, 80%, or 95%) .

What biomarkers might predict response to anti-BTN1A1 therapy?

Based on current research, several potential biomarkers might predict response to anti-BTN1A1 therapy:

• BTN1A1 Expression Levels: The presence and level of BTN1A1 expression in tumor tissues would be a primary biomarker for patient selection . Immunohistochemical staining using validated anti-BTN1A1 antibodies could identify patients whose tumors express this checkpoint.

• PD-L1 Expression Status: Given the mutually exclusive expression pattern of BTN1A1 and PD-L1, the absence or low levels of PD-L1 expression might identify patients more likely to respond to anti-BTN1A1 therapy . This could be particularly relevant for patients who have failed prior PD-1/PD-L1 inhibitor therapy.

• Galectin-9 Expression: Since Galectin-9 functions as a BTN1A1 receptor and forms a three-protein complex with BTN1A1 and PD-1, Gal9 expression levels might predict response to anti-BTN1A1 antibodies . High Gal9 expression correlates with poor prognosis in multiple cancers, potentially identifying patients who might benefit from disrupting this axis.

• Tumor Immune Microenvironment Characteristics: Assessment of the tumor immune microenvironment, including the presence and activity state of T cells, could help identify patients more likely to respond. "Cold" tumors with suppressed immune infiltration but BTN1A1 expression might be particularly suitable candidates .

• Stress-Related Markers: Since BTN1A1 was identified as a potential stress-inducible candidate immune checkpoint, markers of cellular stress in the tumor microenvironment might correlate with BTN1A1 expression and predict response to therapy .

• Glycosylation Status: Since BTN1A1 binding partners interact with the wild-type protein but not with the unglycosylated form, assessment of BTN1A1 glycosylation status might predict functional activity and response to antibody therapy .

What are common challenges in detecting BTN1A1 expression and how can they be overcome?

Researchers face several challenges when detecting BTN1A1 expression:

• Inconsistency Between mRNA and Protein Expression: There is documented inconsistency among existing data when comparing mRNA with protein expression patterns of BTN/BTNL family molecules . To overcome this:

  • Use multiple detection methods (qPCR, Western blot, IHC) to validate expression

  • Employ antibodies targeting different epitopes of BTN1A1

  • Include appropriate positive and negative controls in each experiment

• Specificity Issues: Given the structural similarity between butyrophilin family members, ensuring antibody specificity is crucial . Strategies include:

  • Validate antibody specificity using cells transfected with BTN1A1 versus irrelevant transfectants

  • Perform blocking experiments with recombinant BTN1A1 protein

  • Test for cross-reactivity with related butyrophilin family members (BTN2A1, BTN3A1)

• Optimal Dilution Determination: Finding the optimal antibody concentration is essential for specific staining. Recommendations:

  • Perform titration experiments to determine optimal antibody dilutions

  • Follow manufacturer guidelines that optimal dilutions should be determined by each laboratory for each application

  • Include both positive and negative controls at each dilution

• Fixation Effects: The detection of membrane proteins can be affected by fixation methods. To address this:

  • Compare different fixation protocols (formaldehyde, methanol, acetone)

  • For immunocytochemistry, use methods validated for BTN1A1 detection (e.g., immersion fixation followed by staining at 0.3 μg/mL for 3 hours at room temperature)

  • Consider live-cell staining approaches for flow cytometry applications

• Subcellular Localization: While BTN1A1 is a transmembrane protein, specific staining has been localized to the cytoplasm in some cell types . To clarify localization:

  • Use confocal microscopy with membrane and organelle markers

  • Compare surface staining (non-permeabilized cells) with total staining (permeabilized cells)

  • Consider subcellular fractionation followed by Western blotting

How can researchers optimize BTN1A1 antibodies for therapeutic applications?

For researchers developing therapeutic BTN1A1 antibodies, several optimization strategies are critical:

• Antibody Format Selection:

  • Consider different antibody formats (full IgG, Fab, scFv) based on desired tissue penetration and effector functions

  • Evaluate various isotypes (IgG1, IgG4) depending on whether effector functions are desired

  • The humanized anti-BTN1A1 antibody hSTC810 has shown promise in preclinical models

• Epitope Targeting:

  • Target functional epitopes involved in BTN1A1 interactions with its binding partners

  • Focus on domains that interact with Galectin-9, as this interaction forms part of the BTN1A1-Gal9-PD-1 immunosuppressive axis

  • Consider epitope mapping studies to identify optimal binding regions

• Affinity Optimization:

  • Perform affinity maturation to enhance binding while maintaining specificity

  • Balance high affinity with potential for optimal tissue penetration

  • Use techniques like phage display or yeast display for affinity optimization

• Pharmacokinetic Optimization:

  • Implement PK modeling including allometric scaling to predict antibody behavior in humans

  • Identify doses needed to achieve different receptor occupancy levels (20%, 80%, or 95%)

  • Use the partition coefficient to predict concentrations at tumor sites based on serum concentrations

• Combination Strategies:

  • Evaluate synergy with other immunotherapeutic agents, particularly anti-PD-1/PD-L1 antibodies

  • Consider combinations with radiation therapy, which has shown enhanced efficacy in preclinical models

  • Develop rational combination approaches based on understanding of the BTN1A1-Gal9-PD-1 axis

• Humanization and Immunogenicity Reduction:

  • Employ state-of-the-art humanization techniques to minimize immunogenicity

  • Test for anti-drug antibody responses in preclinical models

  • Consider the use of human antibody discovery platforms (e.g., transgenic mice, phage display libraries)

What are key considerations for developing and validating BTN1A1 knockout/knockdown models?

When developing BTN1A1 knockout/knockdown models for research purposes, researchers should consider several critical factors:

• Selection of Appropriate Model Systems:

  • Choose cell lines that naturally express BTN1A1 or can be induced to express it

  • Consider both cancer cell lines and immune cell models to study different aspects of BTN1A1 biology

  • For in vivo studies, consider both syngeneic mouse models and humanized mouse models for more translational relevance

• Knockout Strategy Design:

  • For CRISPR-Cas9 approaches, design multiple guide RNAs targeting different exons

  • Consider targeting functional domains like the extracellular IgV/IgC domains or the B30.2 domain

  • For conditional knockouts, implement inducible systems to study temporal aspects of BTN1A1 function

• Knockdown Alternatives:

  • Use siRNA or shRNA approaches for temporary silencing to study acute effects

  • Implement doxycycline-inducible shRNA systems for controllable knockdown

  • Consider antisense oligonucleotides as an alternative approach

• Comprehensive Validation:

  • Validate knockout/knockdown at both mRNA level (qPCR) and protein level (Western blot, flow cytometry)

  • Use multiple antibodies targeting different epitopes to confirm complete loss of expression

  • Perform functional validation to confirm loss of BTN1A1-mediated immunomodulatory effects

• Control Considerations:

  • Include appropriate controls (wild-type, scrambled shRNA, non-targeting gRNA)

  • Generate rescue models re-expressing BTN1A1 to confirm phenotype specificity

  • Consider knockout of related butyrophilin family members for comparative studies

• Phenotypic Characterization:

  • Assess effects on T cell activation and proliferation in co-culture systems

  • Evaluate changes in cytokine production patterns

  • For in vivo models, examine tumor growth kinetics and immune infiltration

  • Investigate the formation of the BTN1A1-Gal9-PD-1 complex in the presence/absence of BTN1A1

• Off-Target Effect Assessment:

  • Perform whole-genome or targeted sequencing to identify potential off-target mutations

  • Validate key findings using multiple independent knockout/knockdown clones

  • Consider using different knockout technologies to confirm results

By carefully addressing these considerations, researchers can develop robust BTN1A1 knockout/knockdown models that advance understanding of this novel immune checkpoint's biology and therapeutic potential.

What are the most promising future directions for BTN1A1 antibody research?

The field of BTN1A1 antibody research presents several promising future directions:

• Expanding Clinical Trials: Building on the current Phase I trial of hSTC810 , future studies should evaluate anti-BTN1A1 antibodies in larger patient populations, particularly focusing on tumors with low PD-L1 expression that might be unresponsive to existing checkpoint inhibitors.

• Combination Immunotherapy Approaches: Given the mutually exclusive expression pattern of BTN1A1 and PD-L1 , rational combination strategies targeting both pathways could potentially address resistance mechanisms and improve response rates.

• Biomarker Development: Refining predictive biomarkers based on BTN1A1 expression, Galectin-9 levels, and immune microenvironment characteristics will be crucial for patient selection and treatment personalization.

• Novel Antibody Formats: Exploring bispecific antibodies targeting BTN1A1 and other immune checkpoints, or antibody-drug conjugates that selectively deliver cytotoxic payloads to BTN1A1-expressing cells, could enhance therapeutic efficacy.

• Targeting the BTN1A1-Gal9-PD-1 Axis: Developing therapeutic approaches that specifically disrupt the formation or function of this three-protein complex could provide more selective immune modulation than targeting individual components .

• Understanding Resistance Mechanisms: As anti-BTN1A1 therapies advance clinically, investigating mechanisms of primary and acquired resistance will become increasingly important for developing next-generation approaches.

• Applications Beyond Oncology: Given BTN1A1's protective effect against experimental autoimmune encephalomyelitis (EAE) , exploring its potential in autoimmune and inflammatory diseases represents an exciting frontier.