text_design_therapeutic_antibodies

 

Design of Therapeutic Antibodies

Antibodies can be dissected into smaller antigen binding fragments, by proteolysis or genetic engineering, to produce mono or multivalent fragments. These are mainly based on either Fab fragments or the single chain Fv (scFv) as building blocks. The Fv portion of an IgG, consisting of the VH and VL domains, is the smallest fragment that maintains the full binding capacity of the intact antibody.

Figure 1. Schematic representation of different antibody classes. (A) IgG antibody with its derivatives Fab- and scFv antibody. (B) Heavy chain IgG antibody subclass lacking the light chain and the VL domain (left), and variable domain of the heavy chain of the heavy chain antibody (VHH) from camelidae. (C) IgNAR antibody from shark with its binding domain VNAR. (Dissertation Alexander Maaß, 2014)
Figure 1. Schematic representation of different antibody classes. (A) IgG antibody with its derivatives Fab- and scFv antibody. (B) Heavy chain IgG antibody subclass lacking the light chain and the VL domain (left), and variable domain of the heavy chain of the heavy chain antibody (VHH) from camelidae. (C) IgNAR antibody from shark with its binding domain VNAR. (Dissertation Alexander Maaß, 2014)
 
 

Antibody engineering using shark vNAR scaffold

Figure 1. Antibody engineering on vNAR scaffold
Figure 1. Antibody engineering on vNAR scaffold

In addition to antibodies with the classical composition of the heavy and the light chains, the adaptive immune repertoire of sharks also includes a heavy-chain only isotype, where antigen binding is mediated exclusively by a small and highly stable domain, referred to as vNAR. In recent years, due to their high affinity and specificity combined with their small size, high physicochemical stability and low-cost production, vNAR fragments have evolved as promising target-binding scaffolds that can be tailor-made for applications in medicine and biotechnology. In our group, we design libraries of these biomolecules to screen for highly potent binders against therapeutically relevant targets, among them the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Therefore we use high throuput screening of yeast cells via FACS (Fluorescent activating cell sorter).

 
 
 
 

Antibody engineering using single-domain camelid antibody scaffold (VHH)

Figure 1. Crystal structure of VHH, modified from PDB file 1YC7
Figure 1. Crystal structure of VHH, modified from PDB file 1YC7

Camelids possess, additionally to conventional heterotetrameric antibodies, unique functional heavy (H)-chain antibodies (HCAbs). The H chain of these homodimeric antibodies consists of one antigen-binding domain, the VHH, and two constant domains. Antigen-specific VHHs and their beneficial properties as size, affinity, specificity, stability, etc. have encouraged antibody engineering of these single-domain antibodies for use as a research tool and in biotechnology and medicine. In our group, we develop binders of tumor-relevant protein targets, namely EphA2, Cadherin, and CD276, based on these challenging biomolecular scaffolds.

 
 

Antibody identification using yeast surface display

Yeast Display

We have established a toolbox of methods to transfer the genetic information for antibodies from B-cells of animals to yeast. Animals are immunized with a target protein of interest and the mRNA is extracted from B-cells, converted into DNA, amplified and transferred into a yeast background. This results in a set of yeast cells each displaying an antibody variant in multiple copies on the cell surface. High throughput fluorescence-activated cell sorting (FACS) is used to identify antibodies with prescribed binding characteristics.

 

Related Publications:

  • Facile generation of antibody heavy and light chain diversities for yeast surface display by Golden Gate Cloning.
    Roth L, Grzeschik J, Hinz SC, Becker S, Toleikis L, Busch M, Kolmar H, Krah S, Zielonka S.
    Biol Chem. 2019 Feb 25;400(3):383-393. doi: 10.1515/hsz-2018-0347.
  • Yeast Surface Display in Combination with Fluorescence-activated Cell Sorting Enables the Rapid Isolation of Antibody Fragments Derived from Immunized Chickens.
    Grzeschik J, Yanakieva D, Roth L, Krah S, Hinz SC, Elter A, Zollmann T, Schwall G, Zielonka S, Kolmar H.
    Biotechnol J. 2019 Apr;14(4):e1800466. doi: 10.1002/biot.201800466.
  • A Streamlined Approach for the Construction of Large Yeast Surface Display Fab Antibody Libraries.
    Krah S, Grzeschik J, Rosowski S, Gaa R, Willenbuecher I, Demir D, Toleikis L, Kolmar H, Becker S, Zielonka S.
    Methods Mol Biol. 2018;1827:145-161. doi: 10.1007/978-1-4939-8648-4_8.
  • Construction of Histidine-Enriched Shark IgNAR Variable Domain Antibody Libraries for the Isolation of pH-Sensitive vNAR Fragments.
    Könning D, Hinz S, Grzeschik J, Schröter C, Krah S, Zielonka S, Kolmar H.
    Methods Mol Biol. 2018;1827:109-127. doi: 10.1007/978-1-4939-8648-4_6.