Conventionally, researchers have wrestled to determine just a few antigen-specific B and T cells for weeks and for months, which is insufficient to uncover rare, therapeutically desirable clonotypes. 10x Genomics has made a scientific breakthrough to resolve limitations of former methods when they introduced BEAM (Barcode Enabled Antigen Mapping). 

BEAM lets scientists capture high-resolution cellular profiles of antigen-specific B and T cells with a multiplexed antigen screening workflow built on our Chromium Single Cell Immune Profiling solution. In a mere week’s time, researchers can generate tens to hundreds of high-quality antigen-specific hits, offering unparalleled speed and cellular characterization. 

Unlocking B- and T-cell therapeutic potential 

B and T cells perform a vital function in the adaptive immune system, recognizing molecules the body has determined are foreign, called antigens, and subsequently triggering events that will clear those molecules and the pathogens they’re derived from, such as viruses or bacteria. This function is enacted through antigen recognition by B- and T-cell receptors (BCRs/TCRs).

Antigen recognition by antibodies (BCRs). Retrieved from https://app.biorender.com/biorender-templates/t-5f4fb6cc3b02b700b74df63f-antigen-recognition-by-antibodies. BioRender (2022).

T cells rely on their surface receptors to recognize and bind with foreign and disease-associated antigens—in the form of peptide molecules—presented by innate immune cells, such as dendritic cells or other antigen presenting cells. This is a stylized view of the core components required for TCR-antigen recognition. CREDIT: Leem et al., Nucleic Acids Res. (2018), 46, D406-D412. (BY CC 4.0)

The diversity of the adaptive immune system is determined by BCRs and TCRs repertoire due to recombination of V(D)J gene sequences. Theoretically, there are 10¹⁸ possible BCRs (3) and 10¹⁵ possible TCRs (4), indicating that there are infinite receptors able to bind to and support the clearance of a huge variety of antigens and transform therapeutic research.

Researchers have already discovered receptor–antigen relationships that translated to powerful therapies. The anti-PD1 monoclonal antibody, which binds to PD-1 receptors on T cells to block immune inhibitory signaling and activate T-cell anti-tumor function, is a highly effective therapy for a variety of cancers (5). CAR T-cell therapies are also used to treat some cancers and show promise for treating autoimmune diseases, such as lupus (6), but these treatments only work in limited number of patients due to variation in immunity..

Common hurdles to antigen-specific B- and T-cell discovery

Matching antigens to their corresponding BCRs/TCRs has been historically difficult. Common methods for antibody discovery such as hybridoma technology and phage display are time-consuming, technically daunting, and have low throughput (8).

These methods also limit what samples researchers can use. For example, hybridomas are the product of a fusion of myeloma cells and splenocytes (8), but therapeutically desirable BCRs could be found in blood samples, bone marrow, lymph node aspirates, and more.

Additionally, a common issue that researchers have to confront in antigen-specific T-cell discovery is to develop stable peptide–MHC complexes to tag and subsequently isolate antigen-specific T-cells from a sample. Particularly, MHC molecules are large and tend to collapse, making homebrew stabilization attempts unreliable and painstaking.

MHC I are found on all nucleated body cells, and MHC II are found on macrophages, dendritic cells, and B cells (along with MHC I). The antigen-binding cleft of MHC I is formed by domains α1 and α2. The antigen-binding cleft of MHC II is formed by domains α1 and β1. CREDIT: OpenStax Microbiology.

Besides, T cells with therapeutically relevant antigen specificities tend to occupy a small proportion of the vastly diverse T-cell landscape. Hence, most previous methods tend to overlook rare antigenic T-cell populations. This issue raises researchers’ demands for faster, more robust methods to characterize immune cell phenotypes and antigen specificity and resolve rare clonotypes, not experiments that yield few antigen-specific hits from limited compatible samples.

How BEAM helps to identify antigen-specific clonotypes

BEAM is a multiplexed antigen screening workflow that empowers rapid discovery of antigen-specific B-cell (BEAM-Ab) and T-cell (BEAM-T) clonotypes. It uniquely allows researchers to obtain comprehensive antigen-specific cellular profiles, including full-length paired V(D)J sequences, gene expression, and cell surface proteins from the same single cell.

The BEAM workflow starts with your antigens of interest: researchers can screen binding specificities of up to 15 antigens and a control against hundreds of thousands of B or T cells in a single, one-week experiment. This means you could generate tens to hundreds of antigen-specific hits from the same sample—including compatible samples, such as PBMCs, splenocytes, lymph node aspirates, and enriched B or T cells.

Isolating antigen-specific B and T cells out of a complex sample just got easier too. Researchers can confidently identify rare clonotypes and minimize cell loss since the antigens are coupled to a uniquely barcoded BEAM Conjugate complex that contains a PE fluorescence marker. Flow sorting steps to isolate stained antigen-specific clonotypes simply come down to a check for fluorescence, rather than multicolor analysis with limited parameters.

Having a stable peptide–MHC complex isn’t a problem either. BEAM-T reagent assemblies are powered by kitted, custom loadable MHC monomers, allowing researchers to simply load their antigenic peptide of interest into an empty monomer, then stain T cells. This provides flexibility to design and source peptides from any vendor.

The BEAM workflow also includes easy-to-use data analysis and visualization software to explore and interpret antigen–clonotype relationships. Antigen specificity scores are calculated to identify antigen-specific clonotypes by comparing UMI counts associated with antigens of interest against UMI counts associated with the control antigen.

The BEAM workflow starts with user-supplied antigens, which are barcoded using 10x Genomics BEAM reagents. Barcoded antigens are then used to stain B or T cells prior to flow sorting for enrichment. Normal library preparation and sequencing steps are then carried out on the sample. (Note: BEAM-Ab and BEAM-T are performed in separate workflows with unique reagents.)

Some of the ways BEAM will advance science

How can multiplexing and screening up to 15 antigens in a single experiment help your research? What is the benefit of capturing a comprehensive cellular profile, including gene expression and full-length, paired V(D)J sequences, alongside antigen specificity? We have some ideas.

Antibody discovery

Hunting for potent antibodies against a known antigen target, such as a viral peptide or a surface receptor on cancer cells has always drawn infectious disease researcher attention. These antigen targets not only have different epitopes with varying affinity to antibodies, but they also often mutate or recombine as viruses or cancer cells evolve, which can alter antibody affinity as well. Screening B cells against a more comprehensive antigen panel increases your chances of finding the most potent neutralizing antibodies.

In a recent internal study, 10x Genomics scientists validated this approach using BEAM-Ab to identify antigen-specific immune cells against the SARS-CoV-2 spike protein. They developed a panel of three spike protein antigens, and a human control protein, then assayed PBMCs from a convalescent COVID-19 survivor. In a one-week workflow, they identified hundreds of antigen-specific B-cell clonotypes. They also obtained full-length, paired V(D)J sequences for specific BCRs, which can be used to fast-track production of recombinant antibodies for downstream validation. 

Cancer immunotherapeutics

BEAM promises to accelerate crucial translational oncology research to enhance efficiency of immunotherapies to treat cancer. Specifically, when the researcher aims to determine patient T cells specific to cancer neoantigens—antigens derived from a patient’s cancer cells, there could be dozens of candidates for the ideal neoantigen target from a single patient. It suggests the value of multiplexed antigen screening by BEAM-T to reveal the full breadth of antigen-specific T-cell clonotypes in the patient sample.

Furthermore, BEAM-T can be used to better understand how antigen–receptor interactions influence effector functions, and ultimately guide development of personalized cancer vaccines. And access to full-length, paired TCR sequences forges a path to investigate the potential of receptors for adoptive cell therapy, such as CAR T-cell therapy.

Vaccine development 

Unraveling the cellular and molecular immune response to pathogens as well as to vaccines plays an essential role in infectious disease. One of the most common questions usually centers around which antigenic peptides produce the most robust antiviral immune response in the body to inform vaccine development. With BEAM-T, you could screen against multiple viral peptides, including antigens from different strains of the same virus, in one experiment to track their effects on the T-cell immune response, and thus identify which antigens drive a unique immune response and, ultimately, inform development of a multivalent vaccine that ensures the broadest protection against all viral strains.

BEAM is uniquely positioned to offer rapid insights into a previously unobtainable diversity of antigen-specific BCR/TCR repertoire data at an unprecedented scale. This data could serve as the springboard for faster vaccine development, tracking the immune response in autoimmune and infectious disease, and identifying novel therapeutics against cancer.

Summary

BEAM is a transformative approach to the search for therapeutically relevant antigen-specific B- and T-cell clonotypes by screening multiple antigens simultaneously, driving innovation in therapies and cures against immune-related diseases such as cancer, autoimmune disorders and infectious diseases.

References:

1. Dr. Biology. “B-cells”. ASU – Ask A Biologist. 16 February, 2011. https://askabiologist.asu.edu/b-cell
2. Dr. Biology. “T-cells”. ASU – Ask A Biologist. 16 February, 2011. https://askabiologist.asu.edu/t-cell
3. Hoehn K, et al. The diversity and molecular evolution of B-cell receptors during infection. Mol Biol Evol 33: 1147–1157 (2016). doi: 10.1093/molbev/msw015
4. Lythe G, et al. How many TCR clonotypes does a body maintain? J Theor Biol 389: 214–24 (2016). doi: 10.1016/j.jtbi.2015.10.016
5. https://www.cancer.gov/publications/dictionaries/cancer-drug/def/anti-pd-1-monoclonal-antibody-medi0680
6. Mackensen A, et al. Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus. Nat Med 28: 2124–2132 (2022). doi: 10.1038/s41591-022-02017-5
7. DeMaio A, et al. The role of the adaptive immune system and T cell dysfunction in neurodegenerative diseases. J Neuroinflammation 19: 251 (2022). doi: 10.1186/s12974-022-02605-9
8. Mitra S and Tomar PC. Hybridoma technology; advancements, clinical significance, and future aspects. J Genet Eng Biotechnol 19: 159 (2021). doi: 10.1186/s43141-021-00264-6

Source: Introducing BEAM (Barcode Enabled Antigen Mapping): Benefits of rapid, antigen-specific B- and T-cell discovery