Technical Service

Antibody Discovery Platform for Phage Display Technology

Introduction

Phage display antibody technology unifies genotype and phenotype, and combines selectivity with amplification. With powerful screening capabilities, it can mimic in vitro the antibody generation process in vivo, and can prepare and produce monoclonal antibodies without hybridoma route or even immunization.

Phage display technology provides a simple and fast route for the preparation of genetically engineered antibodies for fully human recombinant antibodies, which is an important turning point in the development history of fully human recombinant antibody preparation.

In recent years, single domain antibodies (SdAb) have been increasingly concerned. They are special antibodies consisting of only two heavy chains naturally found in camelids and cartilaginous fish, containing only one variable domain of heavy chain antibodies (VHH) and two conventional CH2 and CH3 regions. Single domain antibodies bind antigen through a variable region of the heavy chain (VHH), which can be stable in vitro on its own, called camel-like single domain antibodies (SdAb) or nanobodies. Nanobody crystals are 2.5 nm wide and 4 nm long, and their molecular weighs only 1/10 of the traditional intact antibodies (about 15 kD), but they still have intact antigen recognition ability. Thanks to their tiny structure, intact antigen recognition ability, and phage screening technology, nanobodies feature with high affinity, high specificity, strong penetration, as well as easy modification and expression. Moreover, because the complete sequence of antibodies can be obtained, it allows stable production by in vitro recombinant expression and effectively avoids the batch-to-batch variation problem of conventional antibodies.


Nanobody Features

1. Longer CDR3 region (nanobody CDR3 is usually composed of 16-24 amino acids, while the traditional monoclonal antibody CDR3 has only 7-12 amino acids), stronger antigen-specific binding ability

2. Simple but stable structure

3. Easy to produce from small to industrial scale-ups

4. Easy to humanize


Classification of Phage Antibody Libraries

Antibody libraries are huge collections (usually greater than 1010) of antibody genes encoding antibodies of unknown properties. Antibody libraries are an important source of antibody discovery through phage display and other in vitro selection techniques, whose design is key to successful antibody discovery.

Random Peptide Libraries: Chemically synthesized random oligonucleotide sequences are fused to phage surface protein genes to express short random sequence peptides of various amino acid combinations on the phage surface. Using phage to display random peptide libraries, it is possible to screen antigenic epitopes of various antigens in high throughput.

Immunobanks: Immunobanks are constructed from antibody genes of donor B-lymphocytes after immunization (including vaccination, microbial infection, autoimmune diseases, tumors, etc.). The efficiency of antibody selection for specific immune antigens is high, but it is generally only applicable to the selection of one specific antibody, and the library capacity is not required to be high, and the library capacity of 106-108 can meet the needs in general.

Naïve Library: B cells of peripheral blood lymphocytes, bone marrow, and spleen cells of human or animal origin that are not immunized are used as materials for gene amplification, but the affinity is usually low. All antibody genes within and outside the bone marrow are included, and the antibody libraries constructed are suitable for the selection of antibodies corresponding to all antigens. The library capacity is required to be high, with a minimum of 109, because the current estimate of antigenic species in nature is on this order of magnitude.

  


Principle of Phage Display Technology

Comparison of Phage Display Technology for Monoclonal Antibody Development with Conventional Antibody Technology

Advantages

1. Experienced in phage antibody technology, over 5 years development in phage antibody technology, hundreds of phage display antibody development projects delivered.

2. Full human recombinant antibody library with library capacity up to 100 billion (1011 pfu), two-step library construction method, ensuring the diversity of libraries and high correct insertion rate of antibody library fragments; Get the full human recombinant antibody in two weeks.

3. Antibody libraries of multiple species (human, mouse, rabbit, alpaca, etc.) can be constructed.

4. Variety of antibody libraries (scfv, Fab, VHH, etc.) can be constructed.



Workflow

Contents

Types

Projects

Time Frame

Deliverables

Panning Directly

Recombinant Human scFV Phage Antibody Library Panning

   2-4 weeks

   1-3 positive strains

1-3 sequences

Service report

Rabbit Natural scFV Phage Antibody Library Panning

Alpaca nanobody natural VHH library panning

7-peptide library and 12-peptide phage library panning

Canine scFV phage antibody library search

Human scFV phage antibody library panning (COVID-19)

Library Construction + Panning

Phage library construction and panning: human, rabbit, mouse, alpaca, dog, and cat, etc.

12-14 weeks

Immune library

    5-10 positive strains

 5-10 sequences

Service report


Case Study

Based on the analysis of hundreds of random antibody sequences in the library, CDR3 lengths basically conformed to a normal distribution, being consistent with the distribution of CDR3 lengths of natural VHH antibodies reported in the literature.



The target A protein was rapidly screened with a natural nanobody library, and after four rounds of panning, 56 out of 96 selected monoclones were positive (difference values were all greater than 0.5), with a positivity rate of 58%.



The optimal 9 antibody sequences were expressed to soluble VHH antibody and verified again :