VHH Antibody Discovery

Overview

VHH antibodies, first discovered in 1993, are derived from camelids (such as alpacas, llamas, and camels) heavy chain only antibodies (HCAbs, aka. single-domain antibodies) ,and are the smallest antigen-binding fragments. VHH antibodies possess many unique traits in size, physical-chemical properties, immunogenicity, engineering, multiple-specific antibody construction, and more. These favorable characteristics set them apart from conventional antibodies.

VHH antibodies have proved to have medical values in research, diagnosis industry and therapeutic antibody development. Especially, they can recognize uncommon or concealed epitopes that may be challenging for conventional antibodies, and then effectively bind to cavities or functional sites of targets. In addition, VHH antibodies offer advantages in drug discovery owing to their ease and rapidity of development, along with straightforward manufacturing processes.¹

Leveraging its state-of the-art phage display technology, Biointron has established itself as a leading entity in the arena of VHH antibody discovery, and have proudly offered high-quality antibody discovery services to prestigious institutions and companies globally. In Biointron, your VHH antibody discovery project is in trusted hands.

Highlights

Self-owned Alpaca Breeding Farm

300+ alpacas available
One alpaca just for one project

100+ projects delivered/year

Various immunization/panning/screening methods
Multiple assays for validation (ELISA, SPR, FACS, Internalization, ADCC, etc.)

High Diversity and Large Capacity

Maximizing success with high diversity and large capacity
Library size at least 10⁹

Speak to A Specialist

Biointron Alpaca VHH Affinity Distribution

0%AF-5
4%AF-6
16%AF-7
33%AF-8
32%AF-9
13%AF-10
2%AF-11
0%AF-12
0%AF-13

Data source: 615 VHH antibodies (as part of our developed VHHs)

Calculation example:

Total number of VHH of affnity 10^-9 M from different target

Total number of VHH of from different target (615 unique sequence)

33%

With Biointron VHH discovery platform, VHH hits with affinity ranging from 10^-8 to 10^-11 M have been generated. It should be noted that the presented data represents only a portion of projects from one client and may have sampling bias. The distribution of hits with different affinity may not reflect the variance between different targets. The data is from SPR affinity measurement.

Working Flowchart

Antigen expression or provided by client

Animal immunization

PBMC isolation

mRNA Extraction and Reverse Transcription

Library generation

Library screening and Tailored biopanning ( solid phase panning & liquid phase panning)

Positive Clone Sequencing and Sequence Analysis

VHH expression

VHH Validation

Applications

VHH antibodies have been utilized in a wide variety of settings, such as clinical therapeutics and immunodiagnostics, as well as environmental monitoring, due to their differentiated characteristics.² For example, Caplacizumab, an antitumor therapeutics, is a bivalent VHH antibody treating thrombotic thrombocytopenic purpura and thrombosis.³ VHH antibodies have also been employed in chimeric antigen receptor (CAR) constructs for CAR-T therapy.⁴

VHH Antibody Discovery
Service Details

Service Step	Service Description	Timeline	Deliverables
Phase Ⅰ: Antigen preparation /Antigen validation	Antigen can be prepared by BioIntron or provided by our client	2 weeks for preparation / 2-3 days for validation	1mg purified protein antigen If antigen provided by client, it should be at least 4mg with SDS-PAGE >95%, endotoxin level <1EU/mg
Phase Ⅱ: Alpaca immunization and PBMC isolation /preservation	5ml pre-immune bleed the ELISA titer after 2nd, 3rd and 4th immunization will be monitored and the report will send to client the 50ml PMBC after 2nd, 3rd and 4th immunization will be extracted and stored in Trizol	8-10 weeks	Alpaca's death caused by antigen, there will be setup fee about $5,000 Decision gate: based on the ELISA titer on the 2nd, 3rd and 4th immunization. Our client can decide to use which cell for library generation
Phase Ⅲ: VHH Phage Library Generation	RNA extraction and cDNA preparation VHH amplification Electro competent E. coli Library Diversity and capacity measurement clone NO counting and sequencing to measure the capacity of the library	3-4 weeks	VHH capacity reach to 10⁸ - 10⁹
Milestone Ⅳ: Library Biopanning and Screening	at least 3 round panning (liquid or solid phase panning) ELISA verification, identification and evaluation of positive clones screening positive clone sequencing and sequencing result analysis	3-4 weeks	Guarantee to provide at least 20 unique sequence
VHH-Fc Antibody Production (optional)	Gene synthesis, subcloning, plasmid preparation transient expression and purification QC analysis	2 weeks	Purified antibody (SDS-PAGE >95%, endotoxin level <1EU/mg)
Affinity Ranking (optional)	Affinity measured by Biacore 8K	0.5 weeks

Case Study

Case 1: Anti-X Antigen

After panning and screening, 53 unique sequences were obtained & expressed in HTP CHO expression system.

Validation of VHHs Binding to X (by ELISA)

49 of 53 VHH Antibodies bound to X (ELISA)
Validation of VHHs affinity with X (by SPR)
5Not Binding

9E-7

21E-8

16E-9

2E-10
Affinity range 5.36x10^-7 M to 7.66x10^-10 M.
48 of 53 VHH Antibodies bound to X (SPR)
Validation of Cell-based VHHs Binding to X (FACS)

43 of 53 VHH Antibodies Bound against CHO-Kl/X Cell

VHHs Agonist Activity Validation

31 of 53 antibodies showed significant agonist activity

VHHs Validation (Blocking assay)

18 of 53 antibodies showed significant blocking activity.

Case 2: VHH Antibody Discovery (Target: Claudin 18.2)

Alpacas were immunized with Claudin 18.2-encoding DNAs, followed by immunizations with Claudin 18.2-overexpressing cells. Alpaca serum taken after immunization manifested specific binding to Claudin 18.2- overexpressing cells (but not to parental ones).

Phage Library construction from PBMC

Library size is ~1.1x10E10

Insert rate >95%

Antigen	Immunization
DNA encoding Target A	1&2
CHO-K1-Target A	3, 4, 5

Cell panning	Input (CFU/ml)	Output	Output/Input
1	2.00E+12	9.00E+07	4.50E-05
2	1.33E+12	2.10E+08	2.10E+08
3	2.10E+08	2.10E+08	2.10E+08

	Total clone	OD450 (293T-Target A) >0.5	OD450 (293T-blank) >0.1	Combined with Target A clone number	Targeted Clone %	Unique
0635-Human Target A	88	2	0	2	2.27%	1
0635-Human Target A	88	18	12	6	6.82%	2
0635-Human Target A	88	55	41	14	15.91%	10

Four VHHs obtained specifically bound to human Claudin 18.2, but not to Claudin 18.1.

“As a dedicated scientist in the forefront of VHH antibody discovery, I am proud to contribute to Biointron’s commitment to advancing healthcare through cutting-edge biotechnology. Our VHH antibodies epitomize precision, innovation, and the relentless pursuit of scientific excellence.”

Yang Xiang

Antibody Discovery Team

FAQs

What are VHH antibodies?

A VHH antibody is the antigen binding fragment of heavy chain only antibodies. They are derived from the immune system of camelids such as alpacas. As they are highly stable and much smaller than traditional antibodies, VHH antibodies are ideal for a wide range of applications, including diagnostic tests, therapeutics, and research tools.
What are recombinant antibodies?

Recombinant antibodies are monoclonal antibodies produced in vitro through synthetic genes and antibody fragments, instead of using hybridomas. They can take several different formats, such as full-length immunoglobulins (Ig), monovalent antibody fragments such as single-chain fragment variable (scFv) and fragment antigen-binding (Fab), and multimeric diabodies (dimeric scFvs) or triabodies (trimeric scFvs).⁵
How are VHHs isolated from camelids?

There are several ways to isolate VHHs from camelids against a target of interest and to build a library for screening, such as immunized, naïve, and synthetic/semi-synthetic libraries. The most common approach is by immunizing camelids and building a library based on the repertoire of heavy-chain immunoglobulins.⁶

Resources

References

Atarhouch, T., Muyldermans, S., Robinson, G., Hammers, C., Songa, E. B., Bendahman, N., & Hammers, R. (1993). Naturally occurring antibodies devoid of light chains. Nature, 363(6428), 446-448. https://doi.org/10.1038/363446a0
Bever, C. S., Dong, X., Vasylieva, N., Barnych, B., Cui, Y., Xu, L., Hammock, B. D., & Gee, S. J. (2016). VHH antibodies: Emerging reagents for the analysis of environmental chemicals. Analytical and Bioanalytical Chemistry, 408(22), 5985. https://doi.org/10.1007/s00216-016-9585-x
Bannas, P., & Hambach, J. (2017). Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics. Frontiers in Immunology, 8, 309808. https://doi.org/10.3389/fimmu.2017.01603
Bao, C., Gao, Q., Li, L., Han, L., Zhang, B., Ding, Y., Song, Z., Zhang, R., Zhang, J., & Wu, X. (2021). The Application of Nanobody in CAR-T Therapy. Biomolecules, 11(2), 238. https://doi.org/10.3390/biom11020238
Muyldermans, S. (2021). A guide to: Generation and design of nanobodies. The Febs Journal, 288(7), 2084-2102. https://doi.org/10.1111/febs.15515
Arbabi-Ghahroudi, M. (2022). Camelid Single-Domain Antibodies: Promises and Challenges as Lifesaving Treatments. International Journal of Molecular Sciences, 23(9). https://doi.org/10.3390/ijms23095009

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VHH Antibody Discovery

Overview