BASS3 Antibody

What are bispecific antibodies and how do they differ from conventional monoclonal antibodies?

Bispecific antibodies (bsAbs) are engineered proteins capable of binding to two different epitopes, either on the same antigen or on different antigens. Unlike conventional monoclonal antibodies that target a single epitope, bsAbs leverage dual binding activity to enable novel mechanisms of action. The highly modular nature of antibodies allows exogenous antigen-binding domains to be fused within or at the ends of polypeptide chains, creating structurally diverse bsAbs tailored for specific purposes .

These molecules can be constructed in various formats, including:

• Full-length IgG-like structures with two different binding specificities

• Fusion of antigen-binding fragments onto IgG scaffolds

• Direct fusion to Fc domains for smaller constructs that maintain Fc functionality

• Tetra-VH IgGs with distinct binding specificities on each variable domain

The dual-targeting capability makes bsAbs particularly valuable for redirecting immune cells to tumor sites, bridging two different cell types, or simultaneously inhibiting multiple disease pathways.

What is the mechanism of action for T-cell engaging bispecific antibodies?

T-cell engaging bispecific antibodies function by simultaneously binding to CD3 on T cells and a tumor-associated antigen on cancer cells. This dual binding creates a physical bridge that forces T cells into close proximity with target tumor cells, bypassing the need for natural T-cell receptor (TCR) recognition of MHC-presented antigens.

The mechanism follows several steps:

• The antibody binds to both the CD3 complex on T cells and the target antigen on tumor cells

• This engagement activates T cells, as evidenced by upregulation of activation markers like CD25

• Activated T cells initiate cytotoxic responses, releasing granzymes and cytokines

• Target cell killing occurs through both direct cytotoxic activity and inflammatory responses

For example, the BCMAxCD3 bispecific antibody REGN5458 demonstrates this mechanism by binding to B-cell maturation antigen (BCMA) on multiple myeloma cells and CD3 on T cells, effectively inducing polyclonal T-cell killing of both multiple myeloma cell lines and primary human plasma cells .

What are the primary structural configurations for bispecific antibodies?

Bispecific antibodies can be designed with various structural configurations, each offering different advantages depending on the intended application:

• IgG-scaffold based formats: These maintain the basic structure of conventional antibodies while incorporating dual specificity through:

  • Fusion of scFvs or sdAbs to either the N or C terminus of heavy or light chains

  • Incorporation of different binding domains within a single Fab structure (e.g., DutaFab)

• Fragment-based formats: Smaller constructs that lack the Fc region, including:

  • Diabodies

  • Tandem scFvs (BiTEs)

  • Dual-affinity retargeting proteins (DARTs)

• Fc-containing fragments: These retain Fc functionality while being smaller than full IgGs:

  • scFv-Fc fusions

  • Fab-Fc constructs

The choice of configuration significantly impacts properties like size, valency, flexibility, pharmacokinetics, and effector functions. For instance, fragment-based formats typically show better tumor penetration but shorter half-lives, while IgG-based formats exhibit extended circulation time due to FcRn-mediated recycling .

How do the relative binding affinities of the two targeting arms affect bispecific antibody efficacy and safety?

The relative binding affinities between the different antigen-binding arms of a bispecific antibody critically influence both efficacy and safety profiles. This balance is particularly crucial for T-cell engaging bispecific antibodies that target CD3 and a tumor-associated antigen.

Key considerations include:

• Affinity ratio optimization: The relative affinity for tumor antigen versus CD3 influences:

  • On-target efficacy at the tumor site

  • Off-target toxicity in normal tissues with low antigen expression

  • Potential for cytokine release syndrome

• Mechanistic modeling: Research groups have developed computational approaches to understand the affinity interplay for informed bispecific antibody design . These models can predict:

  • Optimal affinity combinations for maximal tumor cell killing

  • Thresholds for minimizing off-target effects

  • Influence of target antigen density on efficacy

• Selectivity enhancement: Higher affinity for tumor-associated antigens compared to CD3 can improve selective killing of cells with high antigen expression while sparing those with low expression .

For example, studies with BCMAxCD3 bispecific antibodies have demonstrated efficient killing of multiple myeloma cell lines expressing varying levels of BCMA, from high-expression NCI-H929 cells (~110,000 surface copies) to low-expression MOLP-8 cells (~4,700 surface copies), showing the importance of tuned affinities for targeting different antigen densities .

What are the primary developability challenges specific to bispecific antibodies and how can they be addressed?

Bispecific antibodies face unique developability challenges beyond those of conventional monoclonal antibodies. These challenges and their solutions include:

Importantly, developability properties of the complete bispecific construct cannot be reliably predicted from analysis of individual building blocks alone. Recent research has shown that fusion of single-domain antibodies (sdAbs) onto IgG scaffolds causes changes in expression yields and biophysical stability that depend on the molecular geometry, fusion site, and number of domains . Therefore, comprehensive evaluation of the complete bispecific molecule is essential.

How does the molecular geometry of bispecific antibodies impact their functional activity?

The molecular geometry of bispecific antibodies significantly influences their functional activity through several mechanisms:

For BCMAxCD3 bispecific antibodies specifically, the molecular geometry must allow efficient engagement of both BCMA on multiple myeloma cells and CD3 on T cells to facilitate effective T-cell activation and tumor cell killing, as demonstrated in both in vitro cytotoxicity assays and in vivo tumor models .

What are the key considerations when designing in vitro assays to evaluate bispecific antibody efficacy?

When designing in vitro assays to evaluate bispecific antibody efficacy, researchers should consider several critical factors:

• Cell line selection:

  • Target antigen expression levels should reflect clinical reality

  • For example, MOLP-8 multiple myeloma cell line (~4,700 BCMA copies per cell) may better represent patient samples (median 3,155 copies) than high-expressing lines like NCI-H929 (~110,000 copies)

  • Include cell lines with varying levels of target expression to assess specificity and potency across expression ranges

• Readout selection:

  • Multiple complementary assays should be employed:

    • Cytotoxicity measurements (e.g., LDH release, live/dead staining)

    • T-cell activation markers (e.g., CD25 upregulation)

    • Cytokine and granzyme production

    • Signal transduction (e.g., NFAT activation for T-cell engaging bispecifics)

• Effector cell considerations:

  • Source variability (donor-to-donor variation in PBMCs)

  • Effector:Target ratios optimization

  • T-cell population (bulk T cells vs CD4+ or CD8+ enriched populations)

• Kinetic measurements:

  • Time-course experiments to capture both early and late effects

  • Differential kinetics between cytotoxicity and cytokine production

For BCMAxCD3 bispecific antibodies specifically, researchers observed that in vitro cytotoxicity occurs at lower concentrations than required for detectable cytokine production, suggesting either decoupled mechanisms or different signaling thresholds for these two responses . This highlights the importance of multiple readouts and time points when evaluating bispecific antibody function.

How should researchers approach combinatorial testing of bispecific antibodies with other immunotherapies?

Combinatorial testing of bispecific antibodies with other immunotherapies requires a systematic approach to maximize therapeutic potential while minimizing toxicity. Based on current research, the following strategy is recommended:

• Rationale-driven combination selection:

  • Identify complementary mechanisms of action

  • For T-cell engaging bispecifics, combinations with checkpoint inhibitors are particularly promising

  • Research has demonstrated that BCMAxCD3 bispecific antibodies show potent combinatorial efficacy with PD-1 blockade in preclinical models

• Model selection for combination studies:

  • Humanized models with intact immune components are preferred

  • CD3-humanized immunocompetent mice allow assessment of both bispecific activity and checkpoint inhibition

  • Models should reflect the tumor microenvironment when possible

• Mechanistic evaluation of combinatorial effects:

  • BCMAxCD3 and PD-1 blockade combinations likely work through:

    • Enhanced bispecific-mediated tumor killing by effector T cells due to augmented T-cell activation

    • Boosting of endogenous immune responses against tumor antigens released upon tumor cell killing

• Sequential vs. concurrent administration:

  • Timing of administration may impact efficacy and toxicity

  • Consider whether priming with one agent before adding the second provides advantages

• Toxicity monitoring:

  • Potential for enhanced cytokine release with combinations

  • Monitoring inflammatory markers (e.g., C-reactive protein, serum cytokines)

Research on BCMAxCD3 bispecific antibodies suggests that "combining BCMAxCD3 bsAb with BCMA CAR T-cell therapy may provide even further enhancement of early and durable disease control," highlighting the potential for novel combination approaches beyond traditional checkpoint inhibitors .

How should researchers interpret differences in kinetics between bispecific antibodies and CAR T-cell therapies?

When analyzing data comparing bispecific antibodies and CAR T-cell therapies, researchers should carefully consider the distinct kinetic profiles of each approach:

• Temporal differences in tumor clearance:

  • BCMAxCD3 bispecific antibodies rapidly clear established systemic multiple myeloma tumors

  • BCMA CAR T cells clear tumors with slower kinetics despite similar in vitro potency

• Mechanistic basis for kinetic differences:

  • Bispecific antibodies immediately engage T cells already present at the tumor site

  • CAR T cells require time to traffic to the tumor site, activate, and numerically expand before exerting their effects

  • These fundamental differences persist even when using the same BCMA-binding domain in both therapeutic approaches

• Implications for therapeutic design:

  • Rapid action of bispecific antibodies may be advantageous for immediate disease control

  • Potentially prolonged persistence of CAR T cells may provide longer-term surveillance

  • Combining approaches might leverage complementary kinetic profiles

• Analytical considerations:

  • Different measurement timepoints may be needed to fully capture effects

  • Area under the curve analysis rather than single timepoint comparisons

  • Consideration of tumor burden rebound rates after treatment cessation

When evaluating efficacy data, researchers should recognize that "using the same BCMA-binding domain, these results suggest that BCMAxCD3 bsAb rapidly exerts its therapeutic effects by engaging T cells already in place at the tumor site, whereas anti-BCMA CAR T cells require time to traffic to the tumor site, activate, and numerically expand before exerting antitumor effects" .

What metrics should be used to evaluate the developability profile of bispecific antibodies?

Comprehensive evaluation of bispecific antibody developability requires assessment across multiple parameters:

• Expression and production metrics:

  • Yield from transient and stable expression systems

  • Correct assembly percentage

  • Purification efficiency and recovery

  • Scale-up consistency

• Biophysical stability parameters:

  • Thermal stability (Tm, Tagg)

  • Conformational stability

  • Resistance to aggregation under stress conditions

  • Long-term stability in formulation

• Chemical stability indicators:

  • Susceptibility to oxidation, deamidation, isomerization

  • pH sensitivity

  • Photostability

  • Freeze-thaw stability

• Binding functionality:

  • Retention of dual binding capacity after stress

  • Maintenance of affinity and specificity

  • Functional activity preservation

Recent research emphasizes that "bsAb developability profile cannot be ascertained from analysis of the individual building blocks or the parental antibodies alone," highlighting the importance of evaluating the complete construct . High-throughput screening approaches can efficiently assess developability profiles early in the development process, including:

• In silico predictive tools for sequence-based liability identification

• Bioconjugation approaches to generate combinatorial panels without individual expression

• Inclusion of developability pressure during the discovery process

The goal should be to identify bispecific antibodies with developability profiles that "align with, or even surpass, those of conventional monospecific antibodies" .

How can researchers address apparent contradictions in bispecific antibody efficacy data between different model systems?

When faced with contradictory efficacy data between different model systems, researchers should implement a systematic approach to reconciliation and interpretation:

• Identify system-specific variables:

  • Target antigen density differences between models

  • T-cell functionality and activation state variations

  • Microenvironmental factors that may influence efficacy

  • Different pharmacokinetic properties across systems

• Conduct bridging studies:

  • Use identical readouts across systems where possible

  • Implement standardized controls

  • Perform dose-response analyses in each system

  • Consider sequential testing in multiple models

• Mechanistic investigations:

  • Analyze T-cell phenotype and functional state ex vivo from different models

  • Assess effector mechanisms (cytotoxicity vs. cytokine production)

  • Evaluate pharmacodynamic markers across systems

• Consider limitations of each model:

  • As noted for BCMAxCD3 studies, "unappreciated differences that could affect the outcomes of in vivo studies may exist, such as T-cell metabolic states"

  • Xenograft models lack "an intact and endogenous immune system"

  • Syngeneic models may not "recapitulate the BM TME [bone marrow tumor microenvironment]"

• Triangulate with clinical data when available:

  • "Accumulating clinical data with bsAbs and CAR T cells may shed light on the differential mechanisms underpinning efficacy or lack thereof"

When interpreting contradictory results, researchers should remember that different model systems are optimized to answer specific questions, and comprehensive understanding may require integration of data across multiple complementary models.

How might epitope prediction tools enhance bispecific antibody development?

Epitope prediction tools represent a significant opportunity to accelerate and optimize bispecific antibody development through several key applications:

• Target selection and validation:

  • Identification of optimal epitopes on target antigens

  • Prediction of shared epitopes across species for translational development

  • Visualization tools like Venn diagrams and UpSet plots to compare predicted epitopes across proteins

• Cross-reactivity assessment:

  • Prediction of potential off-target binding

  • Identification of epitope conservation across family members

  • Early flagging of potential safety concerns

• Optimization of dual-targeting strategies:

  • Epitope mapping to ensure non-overlapping binding sites

  • Design of complementary epitope pairs for optimal target engagement

  • Structure-guided epitope selection for optimal molecular geometry

• Immunogenicity risk reduction:

  • Identification of potential T-cell epitopes within the bispecific construct

  • De-immunization strategies based on epitope prediction

  • Assessment of epitope novelty in engineered interfaces and linkers

Interactive web applications for epitope prediction allow researchers to analyze multiple proteins simultaneously, visualize results through various plots and tables, and implement these analyses within individualized pipelines . These computational approaches can significantly reduce experimental burden during early-stage bispecific antibody development and guide rational design decisions.

What innovative engineering approaches are emerging to address current limitations of bispecific antibodies?

The field of bispecific antibody engineering is rapidly evolving with innovative approaches addressing current limitations:

• Novel molecular architectures:

  • Tetra-VH IgGs: Replacing traditional VH/VL pairs with independent single-domain antibodies on each arm

  • DutaFab technology: Spatially segregating CDRs of a single Fab domain into a VH paratope and a VL paratope to create bispecific Fab domains

  • Rational fusion site selection based on structural and functional considerations

• Affinity and selectivity engineering:

  • Mechanistic modeling to optimize relative binding affinities

  • Computational approaches for understanding affinity interplay to allow informed design

  • Targeted affinity tuning for enhanced safety and efficacy

• Developability-focused design:

  • High-throughput screening pipelines for developability assessment

  • Bioconjugation approaches to generate combinatorial panels without individual expression

  • Inclusion of selective pressure for drug-like qualities during discovery

• Combinatorial therapy optimization:

  • Rational design of bispecific antibodies specifically for combination with checkpoint inhibitors

  • Engineering for enhanced penetration of immunosuppressive tumor microenvironments

  • Exploration of novel combinations like bispecific antibodies with CAR T-cell therapy

These innovative approaches aim to create bispecific antibodies with "dual binding activity, while concurrently addressing the imperative need for developability profiles that align with, or even surpass, those of conventional monospecific antibodies" .

What are the key considerations when translating preclinical bispecific antibody findings to first-in-human trials?

Successful translation of bispecific antibodies from preclinical studies to clinical trials requires careful consideration of several critical factors:

• Dose selection and escalation strategy:

  • Integration of preclinical pharmacokinetic/pharmacodynamic data

  • Consideration of potential cytokine release syndrome

  • Selection of starting dose with appropriate safety margin

  • Identification of biomarkers for dose optimization

• Patient population selection:

  • Target antigen expression screening strategies

  • Disease stage considerations (refractory vs. earlier intervention)

  • For BCMAxCD3 bispecific antibodies, selection of relapsed/refractory multiple myeloma patients

• Safety monitoring plan:

  • Cytokine release monitoring

  • On-target, off-tumor toxicity assessment

  • Mitigation strategies for anticipated adverse events

  • In non-human primate studies, BCMAxCD3 bispecific antibody administration was "well tolerated, resulting in the depletion of BCMA+ cells and mild inflammatory responses characterized by transient increases in C-reactive protein and serum cytokines"

• Biomarker strategy:

  • Target engagement confirmation

  • T-cell activation assessment

  • Pharmacodynamic marker selection

  • Resistance mechanism monitoring

• Combination strategy planning:

  • Sequential vs. concurrent administration with other agents

  • Dosing adjustments for combinations

  • Separate safety run-in for combination cohorts

The comprehensive preclinical evaluation of BCMAxCD3 bispecific antibodies provided "strong rationale for clinical testing of BCMAxCD3 bsAb in patients with MM," leading to the initiation of a phase 1 clinical trial (NCT03761108) evaluating REGN5458 in patients with relapsed/refractory multiple myeloma .