B-BODY™ MULTISPECIFIC PLATFORMS

 

Bispecific antibodies represent an emerging class of drug molecules that enable unique mechanisms of action relative to their monoclonal counterparts. Although two bispecific antibody molecules have gained approval and numerous others have reached the clinic, challenges remain for the creation of highly developable, human IgG-like bispecific scaffolds. Here, we describe the generation of a robust multispecific platform exhibiting biophysical properties comparable to the parent monoclonal IgGs. Through a novel domain-substitution strategy, we have enabled highly orthogonal heavy chain-light chain pairing to ensure the formation of properly paired, four-chain and five-chain multispecific molecules. The B-Body™ retains high stability and possesses transient expression titers comparable to mAbs. Furthermore, the B-Body™ is compatible with Invenra’s proprietary antibody libraries to afford a truly “plug-and-play” platform for efficient high-throughput bispecific discovery.  We have successfully exploited the high-throughput potential of the B-Body platform to create a series of bispecific molecules with potent biological activity acting through multiple mechanisms of action. 

Design Strategy

First Fab Arm

  • Wild type Fab architecture

  • Compatible with all variable domain frameworks

CH1/CL Domain Substitution

  • Substituted domain derived from another human antibody domain

  • Substitution enables highly specific LC/HC pairing

  • Minimal mutagenesis to domain enables Trispecific antibody design

  • Domain substituted Fab has thermostability comparable to the native Fab

  • Easy separation of initial by-products after Protein A

Knob-in-Hole-like Mutation

  • Enables robust HC/HC pairing

  • Mutation set is clinically validated

Design Output
Bispecific 1x1 B-Body
Bispecific 2x1 B-Body
Trispecific 2x1 B-Body
Binding Specificity #1
Binding Specificity #2
Binding Specificity #3

The B-Body™ platform enables high throughput functional discovery in the final therapeutic format. In-format discovery avoids missing rare combinations of affinity, epitope and architecture required for first-in-class therapeutics. B-Bodies™ are designed for compatibility with standard manufacturing processes required for therapeutic development.

Design Validation
The B-Body Possesses Superior Developability Characteristics
Parameter
Trispecific 1x2
B-Body
Bispecific 2x1
B-Body
Bispecific 1x1
B-Body
Unit
Purification Yield After
Protein A Purification
mg/L
300
150
100
Homogeneity After
Affinity Purification
%SEC
Area
98
98
85
Fab Arm
Denaturation Temp ( T
m)
C
o
77
77
70 & 77

Expression, purification and stability of the B-Body platform. Small-scale expression yields are derived from the Expi293 expression system. Thermostability of the Fab arm was determined by differential scanning fluorimetry.  

The B-Body Yields Highly Pure and Stable Bispecifics
Size exclusion chromatography of the 1x1 and 2x1 B-Body™ yield monodisperse samples after single-step purification.
SDS-PAGE gel of variants of the B-Body™ platform yield highly pure protein after purification. Proper chain assembly was confirmed by LC-MS.
Representative accelerated stability study of the B-Body™ at 8.6 mg/ml at 40ºC in PBS validates the design principles of the platform.

Technology Applications

 

The compatibility of the B-Body™ platform with true plug-n-play high throughput discovery enables a new approach to in-format identification and validation of multispecific drug candidates.  Invenra’s multispecific discovery campaigns begin with the selection of new fab domains to each target that cover a broad range of affinities and epitopes.  Invenra has demonstrated that identification of multispecific molecules with activities beyond target binding requires empirical testing of combinations of affinities, epitopes, and platform architectures.  A typical campaign can test 1,000 to 10,000 combinations to identify molecules with new biological activities.  Invenra has used our B-Body™ Platform for the following indications:

 

SNIPER™ ADCs - Unique target combinations are selected to specifically target and eliminate new classes of cells without traditionally clean ADC targets.  SNIPERs™ are optimized to maximize the difference between the monovalent binding affinity to each target and the bivalent avidity to the target combination.

ClustR Multispecific Agonist Discovery - Receptor clustering on the cell membrane is critical in the signaling of many immune receptors.  By targeting multiple constrained epitopes, the ClustR™ technology forces high-order receptor clustering. ClustR™ agonist discovery often involves rapid empirical testing to define the appropriate target epitopes, affinities, and B-Body™ geometries that lead to receptor clustering and amplified cell signaling.   

T-cell Redirecting B-Bodies™ -  Combinations of tumor target epitopes and platform geometry are empirically screened for impact on T-cell activation and killing.  The final format is selected by determining the impact on target affinities, potency and developability.

For easy navigation, select the button below to walk through all three applications in further detail, or select an application from the list above to jump to.

POSTERS & PRESENTATIONS

 

AACR 2019

Abstract 563: A Biparatopic Agonist Antibody for OX40 That Exhibits Superior Activity Without Secondary Crosslinking

presented by Bonnie J Hammer

Cancer Immunotherapy: Executive Summit 2019

Modulating Anti-Tumor Immune Responses with B-Body™ Bispecific Antibodies (Invenra’s OX40 Agonist)

presented by Bonnie J Hammer

PEGS Europe 2018

Multispecific and Multivalent Antibodies as OX40 Agonists

presented by Bryan Glaser

Immuno Oncology Summit 2018

Using B-Body™ Bispecific/Multispecific Antibodies to Modulate Anti-tumor Immune Responses

presented by Bonnie J Hammer

PEGS Boston 2018

Multispecific and Multivalent Antibodies as OX40 Agonists

presented by Francis Qufei Li

PEGS Boston 2018

Multispecific and Multivalent Antibodies as OX40 Agonists

presented by Francis Qufei Li

PEGS Boston 2018

A Bispecific Approach for the Selective Elimination of Tumor Tregs

presented by Lucas Bailey

PepTalk: The Protein Science Week 2018

Multispecific and Multivalent Antibodies as OX40 Agonists

presented by Francis Qufei Li

World Bispecific 2017

A Highly Developable Bispecific Antibody Platform Enables High-Throughput Bispecific Discovery

presented by Lucas Bailey

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