Understanding Trimerbody™

The Trimerbody concept
Trimerbody is a novel class of proprietary multivalent and multispecific therapeutic proteins based on specific antigen-binding domains and the unique structural and functional properties of naturally occurring collagen trimerization domains. LeadArtis’s proprietary platform allows rapid generation and production of novel biological therapeutics with potential in a wide range of human diseases.
The technology was originally developed following the discovery that fully functional multivalent and multispecific antibodies could be generated by employing certain engineered collagen trimerization domains and target antigen binding domains consisting of conventional single-chain antibody fragments (scFv), single-domain antibodies or antibody mimetics.  As a result, the recombinant Trimerbodies display enhanced antigen-binding capacity and are very stable.
Trimerbodies, with their unique structural and functional properties, form the basis of a new generation of therapeutic molecules, which due to their small size and unique structure are ideal for the generation of novel biological drugs with multiple competitive advantages over other therapeutic molecules.
LeadArtis has developed different Trimerbody configurations, each of which with distinctive properties:

Multichain Trimerbody (MTs) made by fusing the target antigen-binding domain/s [single-chain fragment variable (scFv), single-domain antibody (sdAb) or antibody mimetic] to the N- and/or C-terminus of a collagen TrImErization (TIE) domain flanked by short flexible linkers (Figure 1). Due to their multivalence, MTs demonstrated specific antigen binding, increased avidity, slow off-rates in vitro and improved tumor-targeting efficacy in vivo in several mouse models of cancer. Additionally, these molecules are efficiently produced as soluble functional proteins by standard manufacturing methods including bacteria (E. coli), yeast (P. pastoris), insect and mammalian cells (CHO, 293, etc).

Tandem Trimerbodies (TT)
This strategy allows the generation of mono- or multi-specific antibodies within a single polypeptide chain (Tandem Trimerbody or TTs). These molecules are generated by fusing single-domain antibodies (sdAb) from camelid heavy-chain-only immunoglobulins (VHHs) or antibody mimetics to the N-terminus of the TIE domains that are connected with additional glycine-serine based linkers (Figure 2). TTs are soluble, exhibit excellent antigen binding capacity to their cognate antigen/s with high affinity and specificity, and are efficiently produced as soluble functional proteins by standard manufacturing methods including mammalian, yeast, insect, and bacterial cells.

The TT strategy allows the efficient generation of multispecific molecules from pre-existent single-domain antibodies. As this new format targets up to three antigens per molecule it might have therapeutic potential and conceivable diagnostic or imaging use in many diseases, as simultaneously inhibiting different pathways involved in pathogenesis can prevent resistance from being established. 

T cell-recruiting Trimerbodies: ATTACK
The ATTACK (Asymmetric Tandem Trimerbody for T Cell Activation and Cancer Killing) is a novel concept in antibody engineering created by fusing an anti-CD3 scFv to the C-terminus of a TT (Figure 3). The product is efficiently secreted as soluble protein by transfected mammalian cells, easily purified using standard chromatographic methods and very efficient at recognizing the cognate antigens. 
The ATTACK molecules have been designed to specifically activate T cells to kill cancer cells overcoming some of the limitations of earlier T cell bispecifics. For instance, the absence of the Fc domain precludes the unwanted unspecific T cell activation; additionally, the molecules can be designed with one, two or three TAA specificities to fine tune cancer cell killing and reduce on-target off-tumor toxicity.

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