Bispecific Antibody (BsAb) Sequencing

Antibody is a major component in the adaptive immunity and has an essential role in protective and pathogenic immune responses. Recently, the bispecific antibody (BsAb) has been posited as an attractive cancer therapeutic agent for decades. Analysis of bispecific antibody repertoires, contributing to functional immune reactions, provides critical information on protective and pathogenic immunity. In autoimmune diseases, antibody characterization can enable the identification of autoantigens and offer novel insights into the underlying mechanisms of many diseases. Remarkably, Creative Biolabs is pleasure to introduce high-precision bispecific antibody sequencing services for fundamental research, clinical diagnostic, and therapeutic industries. Our sequencing systems are on the basis of database assisted shotgun sequencing (DASS) technology, which depends on next-generation sequencing (NGS) platform.

BsAb

BsAb is an artificial protein that can bind to two different types of antigen at the same time. Its constructs are one of the modalities receiving much attention during recent years. And there are three main dual targeting strategies pursued by BsAb, including, (1) retargeting, (2) simultaneous inhibition of two targets, and (3) increased specificity. As such, BsAb has shown several advantages as follows. First, it can redirect specific immune cells to the tumor cells to facilitate tumor killing. Secondly, it enables the blocking of two different pathways simultaneously that exerts unique or overlapping functions. Thirdly, it may potentially increase binding specificity through interacting with two distinct cell-surface antigens instead of one.

Fig.1 Dual targeting strategies utilizing bispecific antibodies. (Kontermann, 2012)

The development of BsAb has long been restrained by manufacturing problems, for example, low expression yields, product instability, and immunogenicity. The confluence of rapid advancements particularly in recombinant technology and protein engineering has led to the development of the next generation of therapeutic proteins. The constructs of BsAb are a novel approach that is being pursued, combining the ability to simultaneously bind to two different targets as well as the advantages of purpose-designed and optimized antibody-based models. Their pharmacokinetic and pharmacodynamic properties are closely associated with their structural features and capacities to interact with disposition principles of immunoglobulin molecules. Currently, new formats of BsAb that are easier to produce, more stable, and less immunogenic has been made accessible.

BsAb Sequencing

In Creative Biolabs, we make use of a high-field facility (Orbitrap Elite hybrid MS) with 240.000 resolution and <1 ppm mass accuracy to achieve BsAb sequencing services. The available fragmentation techniques contain CID, HCD, and ETD. The combined use of these technologies can ensure the maximum coverage to display high-quality data for our customers.

Fig.2 Sequencing workflow.

• Sequence Validation Guarantee It is known that each project has a tight timeline, and after validation, the sequencing result of BsAb will be guaranteed to be correct in the first attempt.
• Coverage Guarantee If we cannot reach at least 5 folds coverage for the complete V(D)J-region, clients will not be charged and all prepayments are returned. And we also highly recommend that clients could book our 10 folds sequencing services.
• Sequencing of the V and J and C Segments by DASS The V and J and C gene segments of BsAb are available in public databases. But the B cell induces hypermutations into the sequence to optimize the affinity during the BsAb maturation. Our mapping algorithm has error tolerant that could reliably match the “mutated” peptides to the corresponding germline.
• De Novo Sequencing of the CDR3 Region Although the CDR3 of light chain is mostly encoded by germline sequences, its heavy chain is frequently not available in databases. This heavy chain is encoded by D-segments, rest of whom not in matured BsAb is artificial and has to be de-novo sequenced. Our system generates quantities of overlapping peptides during the fragmentation process, enabling to sequence long chains.
• Isobaric Amino Acids Compared with other MS-based methods, most isobaric amino acid combinations can be distinguished by our techniques. For instance, W can be differentiated from GE, AD, and SV by mass accuracy.

Features and Advantages

Antibody sequencing usually has three common traps needed to be avoided that are the impurity of hybridoma cells, mutations introduced by PCR, and misaligned or inaccurate full sequences. We can assist scientists with the sequencing of unknown BsAb antibody using tandem mass spectrometry. Clients can utilize our analysis when there is no record of amino acid sequences of specific antibody in database. Besides, there shows a number of benefits of our BsAb sequencing strategies.

• Superb accuracy: almost no risk to get real sequencing result.
• Faster: 10 working days, all data and analysis will be collected.
• Sensitive: 50-100 μg of a sample, even dilute protein can be performed.
• Powerful: 100% coverage for the VJ and VDJ region is guaranteed.
• High-Throughput: an item is able to be operated with 100, 200, 500 or more BsAb.
• Universality: our techniques can be applied in various BsAb formats from all species (mouse, rat, rabbit, llama, etc.)
• Rich experience: numerous cases have been accomplished successfully.

Sequencing Analysis in Creative Biolabs

Integrative analysis of BsAb development promotes the research in the immune machinery, vaccine design, and therapeutics for autoimmune diseases. The rapid pace of NGS technologies has made it possible to achieve large-scale characterization of functional antibody repertoires. With increasing sequence length and precision, improvement of operation protocols and advancement of bioinformatics analyses, antibody repertoire sequencing would promote studies in antibody-related immune response, and thus expedites vaccine design for clinical diagnosis, infectious diseases, and interference of autoimmune disorders.

Fig.3 Analysis of human antibody repertoires. (Lavinder, 2015)

Creative Biolabs is a professional customized service company with extensive experience in protein engineering as well as a first-class supplier in antibody production markets that has proudly established a series of services for antibody sequencing, especially in BsAbs. By our sequencing platforms, there is a full range of high-quality and cost-efficient sequencing services can be provided. The leading technology allows for multiple forms of purified BsAb to be sequenced within a specific region, leading to a higher degree of accuracy. What's more, we anticipate that antibody sequencing represented by BsAb repertoire will refresh a new generation of biomarkers, diagnostic tools, and therapeutic molecules for a spectrum of diseases, even cancers.

References

1. Kontermann, R.E. Dual targeting strategies with bispecific antibodies. MAbs. 2012, 4(2): 182-197.
2. Lavinder, J.J.; et al. Next-generation sequencing and protein mass spectrometry for the comprehensive analysis of human cellular and serum antibody repertoires. Chem Biol. 2015, 24: 112-120.