Antibody Formats Specification

Overview

The foundry currently supports three main antibody formats/fragments for the binding characterization workflow. Each format features a distinct arrangement of framework and variable regions, which are critical for the binding specificity and affinity toward antigens. Detailed information on the framework, variable regions, and examples of sequences and regions necessary for testing are provided below.

Overview of Antibody Formats - https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.hybrigenics-services.com%2Fvhh-service&psig=AOvVaw2QMiEQMOadEUKztBM91vVJ&ust=1699391268097000&source=images&cd=vfe&opi=89978449&ved=0CBIQjRxqFwoTCNiVmPijsIIDFQAAAAAdAAAAABAZ

Nanobody (VHH)

The Nanobody, also known as a single-domain antibody (sdAb), is a minimalistic antibody fragment comprising a singular variable domain. Originating from Camelidae species, these nanobodies are compact, typically ranging from 12-15 kDa. Their structural similarity to the heavy chain variable domain of conventional antibodies provides them with three complementary determining regions (CDRs), the principal elements dictating their high specificity and affinity for target antigens.

  • Example: Anti-RBD (SARS-CoV-2 WT) Nanobody
  • PDB ID: 7KGK
  • Sequence Length: Approximately 120-130 amino acids
  • Sequence: QVQLVESGGGLVQAGGSLRLSCAASGFPVAYKTMWWYRQAPGKEREWVAAIESYGIKWTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCIVWVGAQYHGQGTQVTVSA
  • Annotated CDR Regions: CDR-H1 (GFPVAYK), CDR-H2 (ESYGIKW), CDR-H3 (WVGAQY) (From left to right, in bold) - CDRs are annotated using Chothia CDR definition.

Single-Chain Variable Fragment (ScFv)

ScFv fragments consist of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins, linked by a short flexible polypeptide linker (Typically -4x(GGGS)-). This format facilitates the formation of a bivalent antigen-binding site that mirrors the natural antigen-recognition properties of a full-size antibody. ScFvs are versatile in their applications due to their reduced size, which is approximately 25-30 kDa, and the ability to retain the specificity of the parental antibody.

  • Example: Anti-Spike S1 (SARS-CoV-2 WT) scFv
  • PDB ID: 7DET
  • Sequence Length: Approximately 250-260 amino acids
  • Sequence: QVQLVQSGAEVKKPGASVKLSCKASGYSFTSYWVNWVRQAPGQGLEWIGMIHPSDSETRLNQKFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCARADGYEWYFDVWGRGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSPGQRATITCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSGTDFTLTINPVEANDVANYYCQQSNEDPWTFGQGTKVEIKAAA
  • Annotated V Regions:
    • VH region (QVQLVQSGAEVKKPGASVKLSCKASGYSFTSYWVNWVRQAPGQGLEWIGMIHPSDSETRLNQKFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCARADGYEWYFDVWGRGTLVTVSS)
    • VL region (DIVLTQSPASLAVSPGQRATITCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSGTDFTLTINPVEANDVANYYCQQSNEDPWTFGQGTKVEIKAAA)
    • Linker (GGGGSGGGGSGGGGS)
  • Annotated CDR Regions: CDR-H1 (GYSFTSY), CDR-H2 (MIHPSDSE), CDR-H3 (RADGYEWYFDV), CDR-L1 (RASESVDSYGNSFMH), CDR-L2 (RASNLE), CDR-L3 (QSNEDPWTF) (From left to right, in bold) - CDRs are annotated using the Chothia CDR definition.

Fragment Antigen-Binding (FAB)

FAB fragments are composed of a constant and variable domain from both the heavy (CH1 and VH) and light chains (CL and VL). This format resembles one arm of a Y-shaped antibody, maintaining the antigen-binding functionality while being smaller than a full antibody, at around 50 kDa. These are expressed as two independent construct which are then incubated together to allow dimerization of the FAB complex.

  • Example: Anti-Spike S1 (SARS-CoV-2 WT) FAB
  • PDB ID: 7KLG
  • Sequence Length: Heavy chain: Approximately 220-250 amino acids; Light chain: Approximately 210-240 amino acids
  • Sequences:
    • Heavy Chain - EVQLVESGGGLVQPGGSLRLSCAASGFDLGGYSMHWVRQAPGKGLEWVAGIYASGGATAYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSYYYGGFGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
    • Light Chain - DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASDLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSYRYPITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
  • Annotated Regions:
    • VH region (EVQLVESGGGLVQPGGSLRLSCAASGFDLGGYSMHWVRQAPGKGLEWVAGIYASGGATAYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSYYYGGFGMDYWGQGTLVTVSS)
    • CH1 domain (ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK)
    • VL region (DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASDLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSYRYPITFGQGTKVEIKRTV)
    • CL domain (AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC).
  • Annotated CDR Regions: CDR-H1 (GFDLGGY), CDR-H2 (IYASGGA), CDR-H3 (SYYYGGFGMDY), CDR-L1 (RASQSVSSAVA), CDR-L2 (SASDLYS), CDR-L3 (QSYRYPIT) (From left to right, in bold) - CDRs are annotated using the Chothia CDR definition.

Additionally, testing other antibody formats is possible, including bispecific and tandem constructs. However, it should be noted that successful expression and proper folding of these molecules cannot be guaranteed.