Antibody sequencing provides a new avenue for antibody therapy research

In the face of various pathogens from the outside world, a wide variety of natural functional antibodies in our body form a strong immune defense line to protect our health
.
Today, with the development of immunology and molecular biology technology, researchers have been able to isolate, purify and prepare antibodies in vitro, and directly promote the birth
of antibody drugs.

Antibody drugs

Since the first therapeutic antibody muromonomab-CD3 (Muromonab-CD3) entered clinical application in 1986, more than 100 therapeutic antibody drugs have entered clinical application, and new antibody drugs are in the process
of research and development and marketing every year.

Figure 1.
Common antibody drugs
.
Includes CD3 monoclonal antibodies, which are used to treat a variety of cancers; Infliximab, used to treat immune system disorders such as Crohn's disease; adalimumab, used to treat rheumatoid arthritis and ankylosing spondylitis; Etanerceptimab (Etanercept), used to treat rheumatoid arthritis [1].

With the development of technology, common techniques for antibody screening and discovery include phage display technology, transgenic murine hybridoma technology and single B-cell technology
.
Phage display technology realizes the unification of protein/peptide genotype and phenotype, and the existing gene fragments can be quickly screened to obtain the required antibodies, but its dependence on the library is relatively high, and it cannot cope with the diversity and complexity
of antibody types.
Transgenic murine hybridoma technology does not need to go through the traditional molecular biology step of expressing recombinant antibodies, and only needs to go through animal immunity, cell culture and cell fusion, mouse ascites preparation and monoclonal antibody purification to complete the preparation of target antibodies, but it still has the disadvantages
of complex operation, difficulty and low success rate.
Single B-cell technology has the advantages of
high efficiency, complete humanization, and rich genetic diversity.
However, to date, the application potential of single B-cell antibody preparation technology has not been realized
.
The preparation technology of single B-cell antibody is still limited by some practical conditions, such as imperfect high-throughput PCR antibody gene amplification technology, difficulty in the preparation of some target antigens, and large demand for suitable human donors
.
The antibody discovery technology that relies on de novo sequencing of proteins is simple and fast to operate, does not rely on any antibody library, and can cope with a variety of antibody detection, so it is more and more concerned and favored
by antibody drug discovery and developers.

Fig.
2.
(left) Phage display technology screening monoclonal antibody [2], which can quickly screen to obtain target antibodies, but its dependence on the library is relatively high, and it cannot cope with the diversity and complexity of antibody types; (Right) Transgenic murine hybridoma technology does not need to go through the traditional molecular biology step of expressing recombinant antibodies, but it still has the disadvantages
of complex operation, difficulty and low success rate.

Protein sequencing

The discovery of antibody sequences relies on gene sequencing and protein sequencing
.
Compared to gene sequencing, protein sequencing produces much fewer random matches and is more conducive to the discovery
of new antibody sequences.
Among the protein sequencing methods, there are currently three commonly used methods: sequencing based on the Edman degradation method, peptide profiling and De novo sequencing
.
Among them, de novo sequencing of proteins has the advantage of not relying on any protein database and can sequence the full length of any unknown protein, making it the preferred technology
for obtaining protein sequences.

Figure 3.
Comparison of three protein sequencing methods[3].

Edman degradation method can only determine dozens of amino acid sequences at the N-terminus, peptide profiling needs to rely on existing protein databases, while De novo sequencing can de-novo
sequence unknown proteins without relying on any protein database.

At present, antibody sequencing technology based on de novo sequencing of proteins has gradually attracted the attention
of basic research and antibody drug developers.
Fast's antibody discovery platform can directly and efficiently discover natural antibodies with high affinity that play a role in the immune system, facilitating the production of recombinant antibodies and providing a new way
for antibody research and development.

Figure 4.
Fast sequence antibody discovery platform

The Fast Order Biological Antibody Discovery Platform requires only 1 mg of antigen-affinity purification of polyclonal antibodies, or 3-5 mL of blood sample volume, and then hydrolyzes protein samples containing multiple antibodies into short peptides using a variety of enzymes, which are further detected using a high-precision mass spectrometer (Thermo Orbitrap Fusion), and a de novo sequencing algorithm to change the tandem mass spectrometry data into short peptide sequences, and finally assemble the short peptide sequences back into a complete high-quality antibody protein sequence
.

The de novo sequencing technology of protein independently developed by Fast Sequence Biology breaks through the limitation of samples, can obtain protein sequences in complex samples, breaks the database dependence of traditional proteomics, and truly realizes de novo sequencing
of proteins.
Among them, Shanghai Fast Biotechnology Co.
, Ltd.
has continuously optimized and upgraded antibody sequencing technology based on its more than 20 years of technology accumulation, and developed a simpler, faster and more accurate antibody sequencing platform
.
The technical advantages of the protein de novo sequencing technology platform of Fast Sequence Bio are as follows:

1.
Simpler, break through the limitation of samples

Using our unique de novo sequencing technology to obtain protein sequences in complex samples, breaking the database dependency of traditional proteomics and truly realizing de novo sequencing of
proteins.

2.
Obtain information more quickly and with high-throughput sequencing

Obtain information faster on the job with high-throughput sequencing and identify special, high-quality molecules that cannot be found by other methods, greatly reducing the time
to obtain protein sequencing results.

3.
More rigorous, 100% ensure sequence accuracy

This core technology ensures the correctness of each amino acid and the sequence accuracy of the whole antibody, and the antibody sequencing technology can ensure that the amino acids at each site of the sequence are supported by more than ten different peptides, and quality control is carried out at the amino acid level to ensure that each amino acid has strong evidence of ion fragment peaks in the mass spectrometry, which can accurately distinguish Leu/Ile residues in antibody sequencing
.

Fast sequence antibody discovery platform application scenarios

Based on the above characteristics and advantages, the antibody discovery service of Fast Bio has been fully applied
in many fields.
These practical applications further highlight the expertise and industry leadership of Fast Bio in antibody discovery
.

1.
New natural antibody discovery in tumors: Targeted therapy has been applied in many tumor clinical treatments, and the basis of targeted therapy is the discovery
of various natural antibodies in tumors.
A 2022 study published in Cell titled "Tumor-reactive antibodies evolve from non-binding and autoreactive precursors" [4] proposed a new way
to treat tumors using natural antibodies in patients.
This method overcomes the shortcomings of relying on existing protein sequences to develop targeted drugs, and provides technical support
for the development of new targeted drugs.
Using Fast-Sequence's protein discovery platform, researchers can quickly identify and develop different natural antibodies in the body, thereby accelerating the drug development process
.

2.
As mentioned earlier, antibody drugs have become an important part
of biomedicine.
In the future-oriented research and development of antibody drugs, the preparation of antibody drugs using natural antibodies derived from patients has become an important research direction
.
In a 2022 research paper titled "Rapid, site-specific labeling of "off-the-shelf" and native serum autoantibodies with T cell–redirecting domains" published in the top international journal Science Advances, researchers developed a way to convert T A technique
in which cell redirection domains are specifically covalently attached to any off-the-shelf human immunoglobulin G (IgG) or isolated native IgG in serum, generating bispecific antibodies in just a few hours.
The technology can use cancer patients' own antibodies to find and treat their own cancer, enabling precision medicine and personalized treatment
.
Using the antibody discovery platform of Fast Sequence Biologics, we can quickly and accurately sequence and identify new antibodies in organisms, so as to quickly advance the above laboratory results to the clinic and accelerate the development of new antibody drugs [5].

3.
Research and development of therapeutic antibodies for autoimmune diseases: The onset of many autoimmune diseases is due to the production of pathogenic immunoglobulin G (IgG) antibodies, and the subsequent formation of immune complexes that poison and attack normal cells and tissues, endangering human health
.
Through the antibody discovery platform of Fast-Sequence, which does not rely on the existing protein database, the antibodies in the patient's blood can be quickly isolated and detected, so as to develop targeted therapeutic antibodies, so as to develop targeted clinical treatment plans
.

4.
Identification of immune response after intervention: Through the antibody sequencing platform of Rapid Sequencers, the level of natural antibodies produced in the immune response can be rapidly evaluated and intervened
.

5.
Conversion of multiple antibodies into monoclonal antibodies or monoclonal antibody mixtures: According to whether the number of antigen epitopes recognized by the antibody is single, the antibody is divided into monoclonal antibody and polyantibody, and the targeted therapeutic effect of monoclonal antibody is better than that of polyantibody, which is the first choice
for clinical development of antibody drugs.
However, the development process of monoclonal antibodies is time-consuming, labor-intensive, and technically difficult
.
With the support of the rapid biological antibody discovery platform, the convenient preparation of multiple antibodies can be quickly analyzed and detected, so as to convert them into specific monoclonal antibodies or monoclonal antibody mixtures, accelerating the preparation process
of monoclonal antibodies.

summary

With the continuous increase of human medical and health level, antibodies have played an increasingly important role in clinical diagnosis and treatment, and the research and development of various antibody biological products is also an important part of
the field of biomedicine.
However, time-consuming, labor-intensive and technical difficulties limit the preparation and development
of antibody products.
Based on this, the antibody discovery platform created by Fast Bio not only breaks through the limitations of sample types and quantities, but also improves speed and accuracy, greatly reducing the research and development cycle
of antibody products.

Choose the fast-order biological antibody discovery platform, so that antibody research and development is no longer "stuck in the neck"!

References:

[1] Smith, Susan L.
"Ten years of Orthoclone OKT3 (muromonab-CD3): a review.
" Journal of Transplant Coordination 6.
3 (1996): 109-121.

[2] Diebolder, P.
, & Krawczyk, A.
(2017).
Detailed Protocols for the Selection of Antiviral Human Antibodies from Combinatorial Immune Phage Display Libraries.
In (Ed.
), Antibody Engineering.
IntechOpen

[3] Janin-Bussat, Marie-Claire, et al.
"Characterization of antibody drug conjugate positional isomers at cysteine residues by peptide mapping LC–MS analysis.
" Journal of Chromatography B 981 (2015): 9-13.

[4] Roei D.
Mazor et al, Tumor-reactive antibodies evolve from non-binding and autoreactive precursors, Cell (2022).

[5] Zappala F, Higbee-Dempsey E, Jang B, et al.
Rapid, site-specific labeling of "off-the-shelf" and native serum autoantibodies with T cell-redirecting domains.
Sci Adv.
2022.

[6] Muñoz-Durango N, Pizarro-Ortega MS, Rey-Jurado E, Díaz FE, Bueno SM, Kalergis AM.
Patterns of antibody response during natural hRSV infection: insights for the development of new antibody-based therapies.
Expert Opin Investig Drugs.
2018.