ResourcesBlogAntibody Basics: Part 2 - Antibody formats: VHHs
Antibody Basics: Part 2 - Antibody formats: VHHs
Biointron2024-02-02Read time: 10 mins
Welcome to Antibody Basics by Biointron, Part 2. Here we’ll give an introduction on the smallest antibody format, VHH. We've mentioned full length antibodies in our previous video, with IgG, IgM, IgD, IgE, and IgA. However, several other engineered formats have been developed:
Monovalent antibody fragments such as single-chain fragment variable (scFv) and fragment antigen-binding (Fab), as well as Fragment with two Fab regions, lacking the fragment crystallizable (Fc) region, and scFv-Fc (single-chain variable fragment - Fc)
Multimeric formats such as diabodies (dimeric scFvs) or triabodies (trimeric scFvs).
A VHH antibody is the antigen-binding fragment of heavy chain-only antibodies
Derived from heavy-chain only IgG antibodies found in the Camelidae family
Beta-sandwich fold supports presentation of 3 CDRs to bind antigens
Convex-shaped paratope formed allows grant to small clefts on the antigen
History of VHHs
Late 20th Century: Discovery of camelid antibodies and initial recognition of their potential.
1990s: First VHH sequences published in 1996, and cloning and expression took place.
Early 2000s: VHH antibodies gain popularity in research, diagnostics, and therapeutics due to their small size and unique binding properties.
Mid-2000s: Investment in VHH antibodies for therapeutic use increases, leading to preclinical and clinical trials.
2010s to Present: Diversification: Ongoing advancements in VHH antibody engineering and applications contribute to their adoption in various fields.
VHHs are ideal for a wide range of applications, including diagnostic tests, therapeutics, and research tools.
VHH antibody immune libraries are produced by phage display:
Antigen Expression
Animal Immunization
PBMC Isolation
mRNA Extraction and
Reverse Transcription
Library Generation
Library Screening
and Biopanning
Positive Clone Sequencing
and Sequence Analysis
VHH Expression
There are currently only four approved VHH-based antibody therapeutics.
Caplacizumab: FDA in 2019. Bivalent, anti-VWF factor, humanized antibody to treat acquired thrombotic thrombocytopenic purpura.
Envafolimab: Approved in 2022 in China. Anti-PD-L1 antibody to treat various solid tumours and chronic hepatitis B; soft tissue sarcomas and biliary tract cancer.
Ozoralizumab: Approved in Japan in 2022. Trivalent, bispecific, humanized anti-TNFα antibody to treat rheumatoid arthritis. Binds to two subunits of TNFα to potently neutralize its action.
Ciltacabtagene autoleucel (cilta-cel): Approved by FDA in 2022. VHH-based, BCMA-directed genetically modified autologous CAR T-cell therapy to treat relapsed/refractory multiple myeloma.
Several VHH-based drugs are in various stages of clinical trials.
VHH antibodies are particularly useful as diagnostics.
Lateral flow immunoassays: Rapid diagnostic test that specifically detects the presence of an antigen of interest within a mixture. VHH antibodies are highly stable, possess a large paratope repertoire, prevent cross-reactions with host, and can be adsorbed onto gold nanoparticles.
Enzyme-linked immunoassay (ELISA): Competitive ELISA: a labeled antigen competes with target antigen for binding to immobilized antibodies. Sandwich ELISA: two VHH antibodies sandwich the target antigen, with one immobilized on a solid support and the other labeled with an enzyme.
Biosensors: A device with a bioreceptor specific to a target antigen affixed to a semiconductor, and binding causes an electric potential change. VHH antibodies can withstand high temperatures and pH changes allows for storage in less-than-ideal environments.
In vivo diagnostic imaging: Medical/nuclear optical imaging techniques, PET, and SPECT use fluorescently or radioactively labeled VHH antibodies. The small size of VHH allows for rapid tissue penetration, ease of modification/conjugation, and rapid renal clearance.
VHH antibodies can thus be applied to disease diagnosis, pharmaceutical quality control, food and environmental analysis, and disease imaging and progression monitoring.
Antibodies are versatile molecules that perform a range of effector functions, many of which engage different arms of the immune system. Their modes of action extend beyond simple antigen binding, enabling the activation of various immune mechanisms that lead to pathogen neutralization and clearance. These functions include blocking molecular interactions, activating the complement system, and linking the humoral immune response to cellular immune responses via Fc receptor engagement.
In today’s competitive biotech landscape, intellectual property (IP) protection has become an essential pillar in fostering innovation and collaboration across drug discovery and development. By offering clear IP terms and no royalty fees,pharmaceutical companies and research institutes
In addition to isotypes and subtypes, antibodies exhibit genetic variation known as allotypes, which are polymorphic epitopes on immunoglobulins. These allotypic differences arise from allelic variations in immunoglobulin genes, causing certain antibody subtypes to differ between individuals or ethnic groups. The presence of these polymorphic forms can influence immune responses, particularly when an individual is exposed to a non-self allotype, potentially triggering an anti-allotype immune reaction.
In mammals, antibodies are classified into five major isotypes: IgA, IgD, IgE, IgG, and IgM. Each isotype is defined by the heavy chain it contains: alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM). These structural differences in the heavy chains determine the antibody's function, tissue localization, and role in the immune response. Furthermore, antibody light chains fall into two classes—kappa and lambda—with kappa being more common, though both exhibit similar functions despite differences in sequence.
