BASS6 Antibody

What is BAG6/BAT3 protein and what cellular functions does it serve?

BAG6/BAT3 functions as an ATP-independent molecular chaperone that prevents the aggregation of misfolded and hydrophobic patches-containing proteins. It serves as part of a cytosolic protein quality control complex (the BAG6/BAT3 complex) that maintains client proteins in a soluble state and participates in their proper delivery to the endoplasmic reticulum .

The protein plays multiple critical roles:

• Post-translational delivery of tail-anchored/type II transmembrane proteins to the ER membrane

• Sorting platform for mislocalized secretory pathway proteins

• Participation in endoplasmic reticulum-associated degradation (ERAD)

• Interaction with ribosomes to facilitate protein trafficking

When client proteins cannot be properly delivered to the endoplasmic reticulum, they are ubiquitinated by RNF126 (an E3 ubiquitin-protein ligase associated with BAG6) and sorted to the proteasome for degradation .

What experimental applications are BAG6/BAT3 antibodies validated for?

BAG6/BAT3 antibodies have been validated for multiple experimental applications according to available product data:

Most commercially available antibodies are tested on human, mouse, and rat samples, with observed molecular weight around 150 kDa, though the calculated molecular weight is approximately 119 kDa .

How should researchers prepare samples for optimal BAG6/BAT3 antibody detection?

For immunohistochemistry applications using paraffin-embedded sections, heat-mediated antigen retrieval in EDTA buffer (pH 8.0) is recommended. Fixation with paraformaldehyde (PFA) is preferred over formalin due to its better tissue penetration ability .

For flow cytometry applications requiring intracellular staining, samples should be:

• Fixed with 4% paraformaldehyde

• Permeabilized with appropriate permeabilization buffer

• Blocked with 10% normal goat serum

• Incubated with antibody at recommended concentration (typically 1-3 μg/1×10^6 cells)

For western blot applications, recommended protocols include:

• Sample loading: 30 μg of sample under reducing conditions

• Blocking: 5% non-fat milk/TBS for 1.5 hours at room temperature

• Primary antibody incubation: 0.5 μg/mL overnight at 4°C

• Secondary antibody: Anti-rabbit IgG conjugated to appropriate detection system (HRP, FITC, etc.)

How do bispecific antibodies (BsAbs) differ from conventional monoclonal antibodies in research applications?

Bispecific antibodies are engineered to bind two distinct targets instead of one, while maintaining the epitope specificity and manufacturability of conventional monoclonal antibodies . This dual-targeting capability offers several research advantages:

• Enhanced avidity effect: According to the "avidity hypothesis," BsAbs may surpass combination therapy by increasing binding strength when two receptors are simultaneously engaged on a target cell .

• Reduced off-target binding: BsAbs are less likely than combination treatments to undergo off-target binding in the presence of decoy cells, potentially improving experimental specificity .

• Novel biological interactions: BsAbs can modulate unexplored biology in ways not possible with single-target mAbs, potentially enabling novel experimental approaches .

• Improved therapeutic window: In therapeutic applications, BsAbs have the theoretical potential to improve safety, efficacy, and selectivity compared to combination therapy approaches .

The actual structure of BsAbs varies widely depending on intended mechanism of action and desired pharmacokinetic/pharmacodynamic properties .

What are the current challenges in developing bioassays for bispecific antibodies?

Developing bioassays for bispecific antibodies presents several unique challenges due to their complex mechanisms of action and dual-target binding capabilities:

• Dual-target characterization requirements: Bioassays must measure two binding events, requiring careful design of assay formats, platforms, and selection of critical reagents .

• Structural complexity: The diverse structural variations of BsAbs necessitate customized approaches for each molecule format .

• Assay selectivity considerations: Assays must differentiate higher-order structure, potency, and efficacy with high precision .

Key aspects requiring characterization include:

Commonly used bi-functional quantitative assay formats include:

• Flow cytometry-based approaches

• Ligand-binding immunoassay setups

• ELISA platforms

• Surface plasmon resonance (SPR)

• Cell-based functional assays (e.g., ADCC)

What are the emerging trends in bispecific antibody research for cancer treatment?

Several significant trends are emerging in bispecific antibody research for cancer therapeutics:

• T-cell engagement approaches: T-cell-engaging BsAbs like mosunetuzumab, epcoritamab, and glofitamab (targeting CD20×CD3) have shown promising clinical activity in B-cell non-Hodgkin lymphoma by redirecting T-cell cytotoxicity toward tumor cells .

• Target diversification: While international research has traditionally focused on CD3-based (n=63) bispecific antibodies, Chinese research enterprises are increasingly developing PD-1/PD-L1 axis compounds .

• Novel structural designs: Advanced engineering approaches include:

  • Bivalent binding configurations that increase target affinity

  • "Head-to-tail" orientation designs that enhance cytolytic activity

  • Multivalent antibodies targeting multiple tumor-associated antigens

• Expansion to solid tumors: The scope of BsAbs now extends beyond hematologic malignancies to solid tumors, representing a significant advancement .

• Safety optimization: Next-generation BsAbs are being designed with controlled Fc regions to modulate immune responses and reduce cytokine release syndrome (CRS) risk .

• Combination approaches: Researchers are exploring combinations of BsAbs with other modalities such as immune checkpoint inhibitors and traditional chemotherapy to enhance efficacy and potentially reduce adverse events .

How can researchers address and mitigate cytokine release syndrome in bispecific antibody research?

Cytokine release syndrome (CRS) represents one of the primary challenges in bispecific antibody research and therapeutic application. Several strategies are being explored to address this concern:

• Antibody engineering approaches:

  • Development of BsAbs with controlled Fc regions to modulate immune response

  • Creation of low-affinity CD3-binding moieties to reduce cytokine secretion

  • Structural modifications to optimize binding kinetics

• Dosing strategy optimization:

  • Implementation of two-dose step-up regimens

  • Controlled dose escalation protocols

  • Clinical trials have shown regimens with step-up dosing can significantly reduce CRS rates

• Biomarker identification:

  • Development of predictive biomarkers for CRS susceptibility

  • Tailoring treatment approaches based on patient-specific risk factors

  • Integration of biomarker-driven strategies into clinical trial design

• Prophylactic measures:

  • Pre-medication protocols to mitigate initial immune activation

  • Close monitoring during administration periods

  • Standardized management algorithms for early intervention

Case Study: AZD0486, a CD19×CD3 BsAb incorporating a low-affinity CD3-binding moiety, demonstrated significantly reduced CRS in phase 1 studies - only 22% of patients experienced grade 1-2 CRS with no grade ≥3 events observed when using a two-dose step-up regimen .

What methodological considerations are important when validating BAG6/BAT3 antibodies for specific research applications?

Comprehensive antibody validation requires multiple complementary approaches to ensure specificity, sensitivity, and reproducibility:

• Positive control selection:
  For BAG6/BAT3 antibodies, validated positive controls include:

  • WB: Human Hela, U87, SH-SY5Y whole cell lysates; rat brain tissue; mouse brain tissue

  • IHC: Human lung cancer tissue, human ovarian serous adenocarcinoma tissue

  • ICC/IF: PC3 cells

  • Flow cytometry: SH-SY5Y cells

• Western blot validation:

  • Expected molecular weight: Approximately 150 kDa (observed) vs. 119 kDa (calculated)

  • Sample preparation: 30 μg under reducing conditions

  • Detection system selection impacts sensitivity and specificity

• Cross-reactivity assessment:

  • Test across multiple species (human, mouse, rat) to confirm specificity

  • Negative controls to rule out non-specific binding

  • Competition assays with immunizing peptides to verify epitope specificity

• Application-specific optimization:

  • For IHC: Test multiple antigen retrieval methods (EDTA buffer pH 8.0 recommended)

  • For ICC/IF: Optimize fixation and permeabilization conditions

  • For flow cytometry: Carefully titrate antibody concentrations

• Isotype control usage:

  • Always include appropriate isotype controls (e.g., rabbit IgG for rabbit-derived antibodies)

  • Use at equivalent concentrations to test antibody

  • Critical for flow cytometry and immunofluorescence applications

Implementing these validation strategies ensures experimental reproducibility and data reliability across different research applications.

How might bispecific antibody technology evolve to address current limitations in cancer therapy?

The future of bispecific antibody technology is likely to evolve along several promising paths to address current limitations:

• Next-generation designs:

  • BsAbs with extended half-lives and improved pharmacokinetic profiles

  • Enhanced stability and prolonged circulation to enable less frequent dosing

  • Incorporation of bispecific T-cell engagers and dual-affinity retargeting platforms

• Trispecific antibody development:

  • Targeting two different cancer-associated antigens simultaneously

  • Providing additional co-stimulatory signals to prevent T-cell exhaustion

  • Particularly relevant for multiple myeloma treatment approaches

• Tumor microenvironment focus:

  • Improved understanding of the dynamic interplay between immune cells, stromal components, and tumor cells

  • Design of therapeutic strategies that can navigate the complexities of the tumor microenvironment

  • BsAbs targeting both tumor cells and immunosuppressive elements

• Addressing resistance mechanisms:

  • Development of sequential therapy strategies as resistance to BsAbs is anticipated

  • Optimization of BsAb positioning within current treatment landscapes

  • Combined approaches targeting multiple resistance pathways

• Expanded therapeutic applications:

  • Growth beyond oncology into autoimmune, inflammatory, and infectious disease areas

  • Novel target combinations beyond currently explored pathways

  • Application in diseases with complex pathogenic mechanisms

What methodological innovations are enhancing bispecific antibody manufacturing efficiency?

Several technological advancements have significantly improved the manufacturing efficiency of bispecific antibodies:

• "Knobs-into-holes" technology:

  • Mutations altering the physical structure of CH3 domains on heavy chains

  • Creates "knobs" on one heavy chain that fit into "holes" on the other

  • Facilitates desired heterodimerization and improves assembly efficiency

• CrossMab technology:

  • Induces crossover of heavy chain and light chain domains within one Fab region

  • Forces specific structural orientation during antibody assembly

  • Significantly improves correct pairing efficiency

• Multi-Fab configuration innovations:

  • Engineering antibodies containing more than two Fab regions

  • Creating bivalent binding to increase affinity (e.g., glofitamab with 2:1 structure)

  • Optimizing spatial orientation through "head-to-tail" configurations using flexible linkers

• Production platform advancements:

  • Single-cell systems programmed to generate specific heavy and light chain combinations

  • Controlled expression systems to regulate chain production ratios

  • Advanced purification methods to isolate correctly assembled BsAbs

These manufacturing innovations have been critical in enabling the clinical development of bispecific antibodies, with ongoing research focused on further improving design efficiency and production yields.

What is the global landscape of bispecific antibody clinical research and development?

The global landscape of bispecific antibody research reveals distinct regional trends and research priorities:

• Geographic distribution:

  • 272 clinical trials regarding BsAbs research were conducted from 1997 to 2020

  • United States leads in BsAbs clinical research

  • China ranks second, contributing 29% of studies worldwide

• Clinical trial phases:

  • Global BsAbs trials concentrated in Phase I (n=161) and early Phase I/II studies

  • Phase III trials remain relatively rare

  • International pharmaceutical development accelerated after 2014

  • Chinese research surge observed since 2017

• Mechanism of action focus:

  • Global and international companies primarily focus on T-cell recruitment approaches

  • Chinese research concentrated on double immune checkpoint blocking strategies

  • Research on tumor-targeted immunomodulators is comparatively rare

• Target preferences by region:

  • International R&D companies: CD3-based (n=63) bispecific antibodies

  • Chinese enterprises: PD-1 (n=9)/PD-L1 (n=7) axis compounds

  • Regional differences reflect distinct research priorities and strategies

• Cancer type focus:

  • International studies: Nearly equal distribution between solid tumors (58%) and hematological tumors (42%)

  • Chinese studies: Predominantly solid tumors (91%) with hematological tumors underrepresented (9%)

  • Significant regional variation in specific cancer subtypes investigated