【Literature Sharing】When SMARTer RACE and In-Fusion are used to prepare mAbs

Monoclonal antibodies are highly homogeneous antibodies produced by a single B cell clone that are directed against only a specific epitope
.
Since the advent of hybridoma monoclonal antibody technology in 1975, monoclonal antibodies have rapidly been widely used in the field of life science research and in clinical detection and diagnosis, making great contributions to research in many fields and the diagnosis and treatment of diseases
.
The current monoclonal antibody preparation technologies include hybridoma technology, EBV transformed B lymphocyte technology, phage display technology, transgenic mouse technology and single B cell antibody preparation technology [1]
.

Among them, the single B cell antibody preparation technology is a newly developed technology in recent years, which is independent of hybridoma technology and phage display technology to rapidly prepare monoclonal antibodies
.
This technology starts with B cells and utilizes the fact that each B cell contains only one functional light chain and heavy chain variable region DNA sequence, and produces only one specific antibody, and can be directly expanded from a single B cell.
Increase antibody genes to obtain antigen-specific antibodies expressed by single B cells
.
This method ensures the natural pairing of the variable regions of the light and heavy chains of antibodies, and has the advantages of high throughput, high efficiency, all-natural origin, and richer gene diversity.
It is currently an important strategy for rapid development of antibodies against viral infectious diseases.
, has a wider application prospect
.

The technical process of single B cell antibody preparation includes: identification and isolation of single B cells; amplification and cloning of antibody genes; expression, screening and identification of antigen-specific antibodies and other steps
.
Among them, when amplifying unknown antibody genes, it is necessary to design primers with strong versatility, high sensitivity and good specificity for nested or semi-nested RT-PCR in order to amplify the complete antibody gene sequence and avoid non-specific amplification.
The requirements for primer design are very high
.
However, in practical applications, limited by the diversity of variable region sequences, it is difficult to design universal primers to cover all sorted B cell functional antibody genes
.
In addition, if high-throughput antibody screening is performed, a large number of amplified antibody genes need to be cloned into expression vectors, but using traditional enzyme digestion and ligation methods requires a lot of time, cost and effort
.

Below are two articles to understand how SMARTer RACE technology and In-Fusion seamless cloning can help the production of single B cell antibodies
.

Literature 1: "Sequence analysis of feline immunoglobulin mRNAs and the develop- ment of a felinized monoclonal antibody specific to feline panleukopenia virus"

Summary

The use of antibody-based immunotherapy in cats is limited by the lack of species-specific intact sequences in mRNAs encoding rearranged heavy and light chain immunoglobulins in B cells
.
To overcome this problem, we isolated mRNAs from feline peripheral blood mononuclear cells (PBMCs) and used existing immunoglobulin sequences and 5′ and 3′ RACE to decloned and sequenced heavy and light chain immunoglobulins mRNAs
.
Using these sequences, two bicistronic vectors were prepared for the expression of mammalian representative cat heavy chains (IGHG1a) and light chains (lambda or kappa)
.
The article reports novel feline immunoglobulin sequences, techniques for expressing antigen-specific feline monoclonal antibodies, and preliminary characterization of functional feline monoclonal antibodies against feline panleukopenia virus
.

RACE library construction

The principle of SMARTer® RACE is that when SMARTScribe reverse transcriptase (RT) reaches the end of the mRNA template, it will automatically add several non-template bases.
At this time, the preset SMARTer II A Oligonucleotide in the kit has a modified base.
The base sequence can be annealed to the end of the cDNA and used as the extension template of SMARTScribe reverse transcriptase.
The reverse transcriptase will automatically convert the template and continue to extend the cDNA until the end of SMART Oligo without additional adapters or tails.
Generate a complete cDNA copy with SMARTer sequence at the end and corresponding to the original RNA, which is especially suitable for high-throughput research of antibody variable region extraction, without the need to design primers for variable regions
.

Figure 1.
Mechanism of SMARTer cDNA synthesis
.

The author of this paper used the preset primers (UPM) in the kit with the gene-specific primers designed by himself to carry out PCR amplification of different Ig fragments (IGHV, IGHC, IGLV, IGLC and full-length IGK), and the gene-specific primers were designed.
The idea and sequence are as follows:

Figure 2.
Schematic diagram of feline Ig heavy and light chain structures
.
The positions of primers used to generate 5' or 3' RACE libraries are shown
.
FR, framework region; CDR, complementarity determining region
.

Table 1.
Primers used in the RACE library

result

A total of 71 unique IGH variable region (IGHV) and 96 IGH constant region (IGHC) RACE sequences, 50 unique IGLV sequences, 5 IGLC genes, 37 unique sequences of full-length IGK transcripts were obtained after sequencing, The new full-length IGHA, IGLV, IGLC and IGK sequences were successfully identified through alignment and confirmation with existing database information
.
The representative full-length sequences of the identified cat IgG heavy and light chains (lambda, IgL and kappa, IgK) were cloned into the vector and the bicistronic vector containing cat IgG IgL or IgG IgK was transfected into HEK It is expressed in cells, and after testing, the secreted antibody chain has been successfully assembled into a complete antibody molecule
.
As a proof of principle, a Felizized monoclonal antibody against CPV and FPV was generated by exchanging the CDRs of the rat anti-CPV monoclonal antibody into the backbone of the expression vector.
After testing, it was proved that the modified monoclonal antibody was fully functional.
and maintain its antigen recognition ability
.
The constructed feline expression vector will facilitate the development of innovative antibody therapeutics in cats
.

Document 2: "Rapid high-throughput cloning and stable expression of antibodies in HEK293 cells"

Summary

Single cell-based antibody variable region amplification is a rapid and efficient technique for cloning antigen-specific monoclonal antibodies, from the initial screening process (including small numbers of hundreds or thousands of mAbs) to in vitro characterization, to For subsequent in vivo experiments (requiring only small amounts of mAb), it is critical to have a robust mAb production system from clone to stable cell line preparation
.
This paper develops a robust high-throughput monoclonal antibody discovery and production system that integrates the advantages of efficient cloning and stable pool selection methods, reducing the time and effort required to clone and express large numbers of variable region genes, ranging from 4 to 6 months reduced to 4 to 6 weeks
.

efficient cloning

During the cloning of individual HCs and LCs, the biggest bottleneck is the selection of recombinant transformants on antibiotic agar plates and subsequent colony screening, which is time-consuming
.
These steps can be omitted using the highly efficient ligase-independent In-Fusion seamless cloning kit, and there is no background caused by vector self-ligation
.
In-Fusion Cloning is a ligase-free, high-efficiency seamless cloning technology.
Based on the principle of homologous recombination between the vector and the insert sequence, it helps to quickly and accurately clone any PCR fragment into any destination vector.
, the background is low, the clone is guaranteed to be directional, and the success rate of single-segment cloning exceeds 95%
.
The speed and ease of use of In-Fusion cloning, as well as the accuracy of results, make the system easy to handle high-throughput workflows
.

experimental method

The V region cDNA insert was cloned into its C region linearized vector using In-Fusion HD cloning
.
After transformation, 50 μl of each transformed product was added to a 96-well deep-well plate containing 1 ml of TB and incubated overnight with shaking
.
Small-scale plasmid extraction was performed on the culture, and HC and LC plasmids were co-transfected into HEK293 cells for 8-10 days of culture.

Figure 3.
Flow chart for high-throughput cloning, transfection, and stable cell line selection
.

To validate and quantify this cloning method, 44 VH and 51 VL genes were RT-PCR amplified from cloned B cell populations, in addition to sequencing the DNA of the half clones to confirm the presence of the desired insert, and cloned into the C region vector
.
Mini-preparation of the half-clonal DNA pool with restriction enzymes showed that the vast majority of LC plasmids (98%) and HC plasmids (95.
4%) contained the expected insert.
PCR screening to determine the presence of inserts, further quantification of the percentage of plasmids with inserts in each transformation, the average percentage of HC plasmids was 71.
6% and the average percentage of LC plasmids was 81.
1% (table below).
Subsequent preparation of plasmids without enzyme digestion has been greatly improved (presumably due to incomplete digestion by restriction enzymes)
.

result

Amplify the VH and VL regions from a single B cell and insert them into a bicistronic constant region vector, respectively.
The transformation product omits the step of plating to isolate a single colony, which can shorten the cloning process from 1 to 2 weeks to 2 to 4 weeks.
days
.
Since the entire system links the expression of HC and LC with the expression of antibiotic resistance genes, theoretically all antibiotic-resistant cells express mAbs, and there is no need for cell sorting and enrichment or subcloning of high-producing cells, so that the lead mAb Rapid transfer to large-scale production, combined with subsequent optimization of co-transfected cell culture and selection process, can save an additional 7 to 8 weeks
.
Overall, the semi-clonal approach developed here reduces the time from cloning to stable cell line development by 3 to 5 months
.

The author and his colleagues have also evaluated other cloning methods.
The main reasons for choosing In-Fusion are its ease of use, no self-association background, and high positive clone rate, all of which omit the step of screening single clones by plating A necessary prerequisite for conducting high-throughput experiments
.

Through the sharing of the above two literatures, it is not difficult to see the promoting role of SMARTer RACE and In-Fusion seamless cloning technology in the preparation of single B cell antibodies.
SMARTer RACE technology is an effective method for cloning new genes.
When amplifying genes with high variability such as antibody variable regions, the operation is simple and the success rate is high, and the cloned gene information is comprehensive, which ensures the activity and function of the antibody
.
With the high accuracy rate of In-Fusion and the characteristics of no vector self-ligation background interference, the operation process can be further simplified, the screening step of plating plate can be omitted, and the transformed product can be directly cultivated to extract plasmids for subsequent co-transfection.
The process is greatly shortened, and it is especially suitable for large-scale high-throughput screening in the process of mAb development
.
I look forward to further exploration and attempts by researchers, and I hope to see the combination of the above two methods and further expansion and optimization, which will bring more possibilities for single B cell antibody preparation technology in the process of drug development
.

If you are eager to try it after reading the literature, you might as well buy it and go back and try it!

Promotion time: 2022.

references

[1] Wang SX.

[2] Lv Xinping, Wu Jing, Chen Jingtao.