Progress and challenges in mass spectrometry-based analysis of antibody repertoires
2021; Elsevier BV; Volume: 40; Issue: 4 Linguagem: Inglês
10.1016/j.tibtech.2021.08.006
ISSN0167-9430
AutoresIgor Snapkov, Maria Chernigovskaya, Pavel Sinitcyn, Khang Lê Quý, Tuula A. Nyman, Victor Greiff,
Tópico(s)Mass Spectrometry Techniques and Applications
ResumoRecent advances in high-resolution mass spectrometry (MS) instruments and liquid chromatography (LC)-MS/MS data analysis software have enabled novel insights into the serum and mucosal antibody repertoire.There is a lack of standardization and benchmarking of antibody repertoire proteomics (Ab-seq) in both experimental and analytical pipelines.Knowledge of the sequence composition and dynamics of the antibody repertoire remains limited due to the complexity of antibody biology as well as Ab-seq workflow-related technological, experimental, and computational challenges that hinder the development of vaccines, antibody therapeutics, and immunodiagnostics.Newly developed strategies can improve Ab-seq workflows and technology. Humoral immunity is divided into the cellular B cell and protein-level antibody responses. High-throughput sequencing has advanced our understanding of both these fundamental aspects of B cell immunology as well as aspects pertaining to vaccine and therapeutics biotechnology. Although the protein-level serum and mucosal antibody repertoire make major contributions to humoral protection, the sequence composition and dynamics of antibody repertoires remain underexplored. This limits insight into important immunological and biotechnological parameters such as the number of antigen-specific antibodies, which are for example, relevant for pathogen neutralization, microbiota regulation, severity of autoimmunity, and therapeutic efficacy. High-resolution mass spectrometry (MS) has allowed initial insights into the antibody repertoire. We outline current challenges in MS-based sequence analysis of antibody repertoires and propose strategies for their resolution. Humoral immunity is divided into the cellular B cell and protein-level antibody responses. High-throughput sequencing has advanced our understanding of both these fundamental aspects of B cell immunology as well as aspects pertaining to vaccine and therapeutics biotechnology. Although the protein-level serum and mucosal antibody repertoire make major contributions to humoral protection, the sequence composition and dynamics of antibody repertoires remain underexplored. This limits insight into important immunological and biotechnological parameters such as the number of antigen-specific antibodies, which are for example, relevant for pathogen neutralization, microbiota regulation, severity of autoimmunity, and therapeutic efficacy. High-resolution mass spectrometry (MS) has allowed initial insights into the antibody repertoire. We outline current challenges in MS-based sequence analysis of antibody repertoires and propose strategies for their resolution. The humoral immune response is facilitated via two interdependent arms: cellular humoral immunity (see Glossary) by B cells and their receptors (BCRs), and protein-level humoral immunity facilitated by glycoproteins called antibodies (Figure 1, Key figure). The combined entities of BCRs and antibodies are called immunoglobulins (Igs). BCRs are Igs anchored to the membrane of B cells, whereas antibodies represent the secreted form of Igs (Box 1 and Figure 2 for more information on antibody biology).Box 1The humoral immune response: joint teamwork of B cells and antibodiesThe BCR repertoire is defined as the set of unique BCRs found in the B cell lineage cells in a given individual, from pro-B cell to terminally differentiated plasma cells (B2 cell lineage), or B1 cells (B1 cell lineage). An adult human is estimated to maintain 108–1010 distinct BCRs [113.Glanville J. et al.Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire.Proc. Natl. Acad. Sci. U. S. A. 2009; 06: 20216-20221Google Scholar] at any given time (total number of B cells: 1011–1012 [114.Trepel F. Number and distribution of lymphocytes in man. A critical analysis.J. Mol. Med. 1974; 52: 511-515Google Scholar]). Correspondingly, the antibody repertoire is defined as the entirety of sequence-distinct secreted antibody proteins in a given individual (its size in terms of unique clonal lineage is unknown and is a major focus of discussion). Thus, by definition, the antibody repertoire is a subset of the BCR repertoire and varies as a function of the dynamics of the B cell repertoire and antibody half-life (Table I).ASCs include plasmablasts and plasma cells, which belong to the late stages of B cell ontogeny, as well as 'natural antibody'-producing cells [115.Nutt S.L. et al.The generation of antibody-secreting plasma cells.Nat. Rev. Immunol. 2015; 15: 160-171Google Scholar] (see Figure 2 in the main text). ASCs synthesize and secrete several thousand antibodies per second [116.Helmreich E. et al.The secretion of antibody by isolated lymph node cells.J. Biol. Chem. 1961; 236: 464Google Scholar]. Secretion rates may vary across 3–4 orders of magnitude [117.Eyer K. et al.Single-cell deep phenotyping of IgG-secreting cells for high-resolution immune monitoring.Nat. Biotechnol. 2017; 35: 977-982Google Scholar]. The antibody repertoire (Figure I) is mainly divided into the mucosal compartment (e.g., gut, lung) and the systemic (serum) compartment. In the mucosal compartment, IgA (mostly IgA2) is generally the most abundant antibody isotype. However, IgA is somewhat less abundant than IgG in urine, bile, and in genital and bronchoalveolar secretions. IgD can be detected in nasal, salivary, lacrimal, and bronchoalveolar secretions, whereas IgE is measurable in nasal, bronchoalveolar, and intestinal secretions, at least when allergy is present [118.Cerutti A. et al.Immunoglobulin responses at the mucosal interface.Annu. Rev. Immunol. 2011; 29: 273-293Google Scholar]. The serum of healthy human donors predominantly contains three antibody isotypes: IgG (~85%), IgA (7–15%, mostly IgA1), and IgM (~5%), and IgD and IgE are only present at low concentrations (~1%) [119.Manz R.A. et al.Maintenance of serum antibody levels.Annu. Rev. Immunol. 2005; 23: 367-386Google Scholar]. The half-life of antibody molecules in serum is less than 3 weeks [120.Vieira P. Rajewsky K. The half-lives of serum immunoglobulins in adult mice.Eur. J. Immunol. 1988; 18: 313-316Google Scholar] (Table I).Of note, recent reports suggest that antibody half-life is not only a function of the isotype but is also impacted by non-Fc regions [121.Datta-Mannan A. et al.Balancing charge in the complementarity-determining regions of humanized mAbs without affecting pI reduces non-specific binding and improves the pharmacokinetics.mAbs. 2015; 7: 483-493Google Scholar,122.Schoch A. et al.Charge-mediated influence of the antibody variable domain on FcRn-dependent pharmacokinetics.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 5997-6002Google Scholar].Figure IAntibody structure and function.Show full captionThe combinatorial rearrangement of V, (D), and J gene segments, heavy and light immunoglobulin (Ig) chains (VH, VL), somatic hypermutation, as well as isotype class-switching, can generate a theoretical naïve antibody repertoire of >1014 different Igs (VH/VL, ~110 amino acids in length) (see Figure 2 in the main text) [108.Elhanati Y. et al.Inferring processes underlying B-cell repertoire diversity.Philos. Trans. R. Soc. B. 2015; 370: 20140243Google Scholar,109.Greiff V. et al.Learning the high-dimensional immunogenomic features that predict public and private antibody repertoires.J. Immunol. 2017; 199: 2985-2997Google Scholar]. The region with the highest diversity and variability in an Ig VH and VL sequence is the corresponding complementarity-determining region (CDR) 3 (CDRH3: ~8–20 amino acids). Other regions, such as the framework regions of the Ig molecule, contain large stretches of conserved (very similar) subsequences, which have been recently found to be further diversified by SNPs that have been linked to disease and antigen binding, adding a further level of complexity [110.Collins A.M. et al.Germline immunoglobulin genes: disease susceptibility genes hidden in plain sight?.Curr. Opin. Syst. Biol. 2020; 24: 100-108Google Scholar,111.Watson C.T. et al.The individual and population genetics of antibody immunity.Trends Immunol. 2017; 38: 459-470Google Scholar]). Antibody–antigen recognition is mainly mediated by CDRH3 (VH-CDR3) [36.Akbar R. et al.A compact vocabulary of paratope-epitope interactions enables predictability of antibody-antigen binding.Cell Rep. 2021; 34: 108856Google Scholar,112.Xu J.L. Davis M.M. Diversity in the CDR3 region of VH is sufficient for most antibody specificities.Immunity. 2000; 13: 37-45Google Scholar].View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IOverview of antibody abundance in healthy human donorsaAbbreviation: N/A, not available., bReferences for the values presented are provided in the supplemental material online.Ig IsotypeIgMIgDIgGIgAIgERepertoire statisticsSerum (s) abundance (mg ml−1)~0.4–2.3~0.03~7–16~0.7–4~0.0005Gut (g) abundance (mg kg−1 body weight)N/AN/AN/A~40N/ANumber of sAb molecules (ml−1)~2 × 1015 to 9 × 1015~1 × 1014~3 × 1016–6 × 1016~3 × 1015 to 2 × 1016~2 × 1012Number of gAb molecules (kg−1)N/AN/AN/A~2 × 1017N/ASerum antibody half life (days)~6~3~21~6~3a Abbreviation: N/A, not available.b References for the values presented are provided in the supplemental material online. Open table in a new tab Figure 2State-of-the-art knowledge and knowledge gaps in antibody repertoire biology.Show full captionCurrent knowledge gaps in antibody repertoire biology that can be resolved by mass spectrometry (MS) analysis of antibody sequences (Ab-seq) include reliable statistics on clonal diversity and frequency (bulk, VH–VL, antigen-specific) as well as the extent of overlap between genomic and proteomic repertoires.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The BCR repertoire is defined as the set of unique BCRs found in the B cell lineage cells in a given individual, from pro-B cell to terminally differentiated plasma cells (B2 cell lineage), or B1 cells (B1 cell lineage). An adult human is estimated to maintain 108–1010 distinct BCRs [113.Glanville J. et al.Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire.Proc. Natl. Acad. Sci. U. S. A. 2009; 06: 20216-20221Google Scholar] at any given time (total number of B cells: 1011–1012 [114.Trepel F. Number and distribution of lymphocytes in man. A critical analysis.J. Mol. Med. 1974; 52: 511-515Google Scholar]). Correspondingly, the antibody repertoire is defined as the entirety of sequence-distinct secreted antibody proteins in a given individual (its size in terms of unique clonal lineage is unknown and is a major focus of discussion). Thus, by definition, the antibody repertoire is a subset of the BCR repertoire and varies as a function of the dynamics of the B cell repertoire and antibody half-life (Table I). ASCs include plasmablasts and plasma cells, which belong to the late stages of B cell ontogeny, as well as 'natural antibody'-producing cells [115.Nutt S.L. et al.The generation of antibody-secreting plasma cells.Nat. Rev. Immunol. 2015; 15: 160-171Google Scholar] (see Figure 2 in the main text). ASCs synthesize and secrete several thousand antibodies per second [116.Helmreich E. et al.The secretion of antibody by isolated lymph node cells.J. Biol. Chem. 1961; 236: 464Google Scholar]. Secretion rates may vary across 3–4 orders of magnitude [117.Eyer K. et al.Single-cell deep phenotyping of IgG-secreting cells for high-resolution immune monitoring.Nat. Biotechnol. 2017; 35: 977-982Google Scholar]. The antibody repertoire (Figure I) is mainly divided into the mucosal compartment (e.g., gut, lung) and the systemic (serum) compartment. In the mucosal compartment, IgA (mostly IgA2) is generally the most abundant antibody isotype. However, IgA is somewhat less abundant than IgG in urine, bile, and in genital and bronchoalveolar secretions. IgD can be detected in nasal, salivary, lacrimal, and bronchoalveolar secretions, whereas IgE is measurable in nasal, bronchoalveolar, and intestinal secretions, at least when allergy is present [118.Cerutti A. et al.Immunoglobulin responses at the mucosal interface.Annu. Rev. Immunol. 2011; 29: 273-293Google Scholar]. The serum of healthy human donors predominantly contains three antibody isotypes: IgG (~85%), IgA (7–15%, mostly IgA1), and IgM (~5%), and IgD and IgE are only present at low concentrations (~1%) [119.Manz R.A. et al.Maintenance of serum antibody levels.Annu. Rev. Immunol. 2005; 23: 367-386Google Scholar]. The half-life of antibody molecules in serum is less than 3 weeks [120.Vieira P. Rajewsky K. The half-lives of serum immunoglobulins in adult mice.Eur. J. Immunol. 1988; 18: 313-316Google Scholar] (Table I). Of note, recent reports suggest that antibody half-life is not only a function of the isotype but is also impacted by non-Fc regions [121.Datta-Mannan A. et al.Balancing charge in the complementarity-determining regions of humanized mAbs without affecting pI reduces non-specific binding and improves the pharmacokinetics.mAbs. 2015; 7: 483-493Google Scholar,122.Schoch A. et al.Charge-mediated influence of the antibody variable domain on FcRn-dependent pharmacokinetics.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 5997-6002Google Scholar].Table IOverview of antibody abundance in healthy human donorsaAbbreviation: N/A, not available., bReferences for the values presented are provided in the supplemental material online.Ig IsotypeIgMIgDIgGIgAIgERepertoire statisticsSerum (s) abundance (mg ml−1)~0.4–2.3~0.03~7–16~0.7–4~0.0005Gut (g) abundance (mg kg−1 body weight)N/AN/AN/A~40N/ANumber of sAb molecules (ml−1)~2 × 1015 to 9 × 1015~1 × 1014~3 × 1016–6 × 1016~3 × 1015 to 2 × 1016~2 × 1012Number of gAb molecules (kg−1)N/AN/AN/A~2 × 1017N/ASerum antibody half life (days)~6~3~21~6~3a Abbreviation: N/A, not available.b References for the values presented are provided in the supplemental material online. Open table in a new tab Current knowledge gaps in antibody repertoire biology that can be resolved by mass spectrometry (MS) analysis of antibody sequences (Ab-seq) include reliable statistics on clonal diversity and frequency (bulk, VH–VL, antigen-specific) as well as the extent of overlap between genomic and proteomic repertoires. The main function of antibodies in the context of the serum and mucosal humoral immune response is antigen recognition, which also facilitates a variety of effector functions such as opsonization for antibody-dependent cytotoxicity, target neutralization, and gut microbiota regulation [1.Li H. et al.Mucosal or systemic microbiota exposures shape the B cell repertoire.Nature. 2020; 584: 274-278Google Scholar, 2.Forthal D.N. Functions of antibodies.Microbiol. Spectr. 2014; 2: 1-17Google Scholar, 3.Bemark M. Angeletti D. Know your enemy or find your friend? Induction of IgA at mucosal surfaces.Immunol. Rev. 2021; (Published online June 30, 2021)https://doi.org/10.1111/imr.13014Google Scholar]. Together, these functions ensure immunological protection (e.g., vaccine responses), their use in monoclonal antibody (mAb) and serum therapy of infection, cancer, and autoimmunity [4.Carter P.J. Lazar G.A. Next generation antibody drugs: pursuit of the 'high-hanging fruit'.Nat. Rev. Drug Discov. 2017; 17: 197-223Google Scholar, 5.Libster R. et al.Early high-titer plasma therapy to prevent severe Covid-19 in older adults.N. Engl. J. Med. 2021; 384: 610-618Google Scholar, 6.Vaisman-Mentesh A. et al.Molecular landscape of anti-drug antibodies reveals the mechanism of the immune response following treatment with TNFα Antagonists.Front. Immunol. 2019; 10: 2921Google Scholar], as well as the harnessing of antibodies as biosensors for medical diagnostics [e.g., antibody-based tests for HIV and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)] [7.Borrebaeck C.A.K. Antibodies in diagnostics – from immunoassays to protein chips.Immunol. Today. 2000; 21: 379-382Google Scholar,8.Hillman Y. et al.Monoclonal antibody-based biosensor for point-of-care detection of type III secretion system expressing pathogens.Anal. Chem. 2021; 93: 928-935Google Scholar]. .Adaptive immune receptor repertoire (AIRR) [9.Schramm C. AIRR-C Glossary of Terms.zenodo. 2021; https://doi.org/10.5281/zenodo.5095380Google Scholar] DNA/RNA-based sequencing (AIRR-seq) now enables researchers to investigate BCR sequence diversity in great depth (Box 1). These BCR AIRR-seq (hereafter, BCR-seq) studies have led to unprecedented insights into B cell biology in terms of sequence diversity and dynamics across B cell populations and immune states, recapitulating the principles of adaptive immunity as postulated by Burnet half a century ago [10.Wardemann H. Busse C.E. Novel approaches to analyze immunoglobulin repertoires.Trends Immunol. 2017; 38: 471-482Google Scholar,11.Greiff V. et al.Bioinformatic and statistical analysis of adaptive immune repertoires.Trends Immunol. 2015; 36: 738-749Google Scholar]. BCR-seq has also fueled advances in therapeutic antibody and vaccine design [12.Robinson W.H. Sequencing the functional antibody repertoire – diagnostic and therapeutic discovery.Nat. Rev. Rheumatol. 2014; 11: 171-182Google Scholar, 13.Mason D.M. et al.Optimization of therapeutic antibodies by predicting antigen specificity from antibody sequence via deep learning.Nat. Biomed. Eng. 2021; 5: 600-612Google Scholar, 14.Galson J.D. et al.Studying the antibody repertoire after vaccination: practical applications.Trends Immunol. 2014; 35: 319-331Google Scholar]. Although advances in the genomic analysis of BCR diversity have been numerous, the sequence diversity and antigen-specific dynamics of the mucosal and serological antibody repertoire remain underexplored. Indeed, although the antigen- and epitope-binding patterns of antibody repertoires can be assessed in a fairly straightforward fashion via peptide array proteomics and microfluidics, these technologies do not resolve the amino acid sequence of the binders and are thus not suitable for profiling the (antigen-specific) sequence diversity of antibody repertoires (Box 2). Without a detailed description of the sequence identities and relative amounts of the antibodies that comprise the antibody repertoire, establishing precisely how the B cell developmental program shapes protective immunity remains unfeasible, hindering the conception and testing of precise, personalized, and targeted novel therapeutics, vaccines, and diagnostics.Box 2MS-independent antibody repertoire binding profilingAlthough MS-based analysis of antibody repertoires has remained challenging, there are numerous orthogonal proteomics approaches that do not aim to analyze antibody repertoire sequence diversity but instead focus on antibody binding repertoire. The main approaches for profiling the antibody binding repertoire are peptide/protein display and microfluidics.Peptide and protein displayBriefly, display-based methods encompass approaches such as peptide and protein arrays as well as other related technologies such as PhIP-seq and VIRscan [123.Sikorski K. et al.A high-throughput pipeline for validation of antibodies.Nat. Methods. 2018; 15: 909Google Scholar,124.Larman H.B. et al.Autoantigen discovery with a synthetic human peptidome.Nat. Biotechnol. 2011; 29: 535-541Google Scholar]. These technologies allow profiling of the antibody reactivity of hundreds of thousands of peptides or proteins. For all these approaches, the serum is incubated and binding is registered. The binding is a function of the distribution of the antibodies in the serum (which is still largely unknown as explained in the main text). Although studies using these technologies have elucidated some aspects of humoral immunity in health and disease, the insight afforded by these technologies into humoral immunity is ultimately relatively restricted as it remains unclear how many and which antibodies bind to each peptide and protein. Nota bene: many reports describing these technologies claim to perform epitope mapping. This is incorrect. Any stretch of the surface of a given protein may be an epitope [125.Kunik V. Ofran Y. The indistinguishability of epitopes from protein surface is explained by the distinct binding preferences of each of the six antigen-binding loops.Protein Eng. Des. Sel. 2013; 26: 599-609Google Scholar]. To define an epitope, the corresponding antibody is required. Indeed, for proper biophysical and functional characterization, antibodies are preferably assayed as monoclonal species [56.Greiff V. et al.A minimal model of peptide binding predicts ensemble properties of serum antibodies.BMC Genomics. 2012; 13: 79Google Scholar,126.Georgiev I.S. et al.Delineating antibody recognition in polyclonal sera from patterns of HIV-1 isolate neutralization.Science. 2013; 340: 751-756Google Scholar] since incubating uncharacterized mixtures with peptide/protein arrays only enables limited, if any, insight into epitope mapping [56.Greiff V. et al.A minimal model of peptide binding predicts ensemble properties of serum antibodies.BMC Genomics. 2012; 13: 79Google Scholar].MicrofluidicsMicrofluidic technology has recently enabled the profiling of adaptive immunity at single-cell resolution. Eyer and colleagues developed the DropMap technology which enables profiling of ASC secretion rate and affinity at the single-cell level with high-throughput and an extensive dynamic range (102 to 105 or more cells [117.Eyer K. et al.Single-cell deep phenotyping of IgG-secreting cells for high-resolution immune monitoring.Nat. Biotechnol. 2017; 35: 977-982Google Scholar,127.Eyer K. One by one – insights into complex immune responses through functional single-cell analysis.Chim. Int. J. Chem. 2020; 74: 716-723Google Scholar,128.Kräutler N.J. et al.Quantitative and qualitative analysis of humoral immunity reveals continued and personalized evolution in chronic viral infection.Cell Rep. 2020; 30: 997-1012Google Scholar]). Although the technology is high-throughput on the plasma cell side, it is so far relatively low-throughput on the antigen side, and is therefore not suitable for exploring the repertoire of serum antibodies that bind to large-scale protein landscapes. In addition, it also does not allow concurrent sequencing of the profiled antibody-secreting cells. However, recent advances have started to address this shortcoming [129.Gérard A. et al.High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics.Nat. Biotechnol. 2020; 38: 715-721Google Scholar].A future aim must be to enable sequence determination and antibody binding affinity measurements at the single-molecule level and repertoire scale (discussed in the concluding section of the main text). Although MS-based analysis of antibody repertoires has remained challenging, there are numerous orthogonal proteomics approaches that do not aim to analyze antibody repertoire sequence diversity but instead focus on antibody binding repertoire. The main approaches for profiling the antibody binding repertoire are peptide/protein display and microfluidics. Peptide and protein display Briefly, display-based methods encompass approaches such as peptide and protein arrays as well as other related technologies such as PhIP-seq and VIRscan [123.Sikorski K. et al.A high-throughput pipeline for validation of antibodies.Nat. Methods. 2018; 15: 909Google Scholar,124.Larman H.B. et al.Autoantigen discovery with a synthetic human peptidome.Nat. Biotechnol. 2011; 29: 535-541Google Scholar]. These technologies allow profiling of the antibody reactivity of hundreds of thousands of peptides or proteins. For all these approaches, the serum is incubated and binding is registered. The binding is a function of the distribution of the antibodies in the serum (which is still largely unknown as explained in the main text). Although studies using these technologies have elucidated some aspects of humoral immunity in health and disease, the insight afforded by these technologies into humoral immunity is ultimately relatively restricted as it remains unclear how many and which antibodies bind to each peptide and protein. Nota bene: many reports describing these technologies claim to perform epitope mapping. This is incorrect. Any stretch of the surface of a given protein may be an epitope [125.Kunik V. Ofran Y. The indistinguishability of epitopes from protein surface is explained by the distinct binding preferences of each of the six antigen-binding loops.Protein Eng. Des. Sel. 2013; 26: 599-609Google Scholar]. To define an epitope, the corresponding antibody is required. Indeed, for proper biophysical and functional characterization, antibodies are preferably assayed as monoclonal species [56.Greiff V. et al.A minimal model of peptide binding predicts ensemble properties of serum antibodies.BMC Genomics. 2012; 13: 79Google Scholar,126.Georgiev I.S. et al.Delineating antibody recognition in polyclonal sera from patterns of HIV-1 isolate neutralization.Science. 2013; 340: 751-756Google Scholar] since incubating uncharacterized mixtures with peptide/protein arrays only enables limited, if any, insight into epitope mapping [56.Greiff V. et al.A minimal model of peptide binding predicts ensemble properties of serum antibodies.BMC Genomics. 2012; 13: 79Google Scholar]. Microfluidics Microfluidic technology has recently enabled the profiling of adaptive immunity at single-cell resolution. Eyer and colleagues developed the DropMap technology which enables profiling of ASC secretion rate and affinity at the single-cell level with high-throughput and an extensive dynamic range (102 to 105 or more cells [117.Eyer K. et al.Single-cell deep phenotyping of IgG-secreting cells for high-resolution immune monitoring.Nat. Biotechnol. 2017; 35: 977-982Google Scholar,127.Eyer K. One by one – insights into complex immune responses through functional single-cell analysis.Chim. Int. J. Chem. 2020; 74: 716-723Google Scholar,128.Kräutler N.J. et al.Quantitative and qualitative analysis of humoral immunity reveals continued and personalized evolution in chronic viral infection.Cell Rep. 2020; 30: 997-1012Google Scholar]). Although the technology is high-throughput on the plasma cell side, it is so far relatively low-throughput on the antigen side, and is therefore not suitable for exploring the repertoire of serum antibodies that bind to large-scale protein landscapes. In addition, it also does not allow concurrent sequencing of the profiled antibody-secreting cells. However, recent advances have started to address this shortcoming [129.Gérard A. et al.High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics.Nat. Biotechnol. 2020; 38: 715-721Google Scholar]. A future aim must be to enable sequence determination and antibody binding affinity measurements at the single-molecule level and repertoire scale (discussed in the concluding section of the main text). Since antibodies are proteins, they are, as opposed to B cells, not amenable to BCR-seq [15.Miho E. et al.Computational strategies for dissecting the high-dimensional complexity of adaptive immune repertoires.Front. Immunol. 2018; 9: 224Google Scholar,16.Yaari G. Kleinstein S.H. Practical guidelines for B-cell receptor repertoire sequencing analysis.Genome Med. 2015; 7: 121Google Scholar]. To analyze the sequence diversity of complex antibody mixtures, mass spectrometry (MS) is required – hereafter referred to as Ab-seq (Figure 1). First demonstrated by Sato and colleagues [17.Sato S. et al.Proteomics-directed cloning of circulating antiviral human monoclonal antibodies.Nat. Biotechnol. 2012; 30: 1039-1043Google Scholar] and Cheung and colleagues [18.Cheung W.C. et al.A proteomics approach for the identification and cloning of monoclonal antibodies from serum.Nat. Biotechnol. 2012; 30: 447-452Google Scholar] on serological antibody repertoires for antibody discovery, Ab-seq was subsequently applied to a variety of model organisms and immunological and biotechnological research questions (Table 1). We review here the use, progress, and challenges of Ab-seq and its constituent technologies (Figure 1). Specifically, we provide an overview of Ab-seq workflow, concepts and methods, state-of-the-art understanding, and knowledge gaps in our conception of antibody repertoire sequence and dynamics as obtained by Ab-seq. We then outline current challenges in Ab-seq as imposed by antibody repertoire biology, sample preparation, MS-based technical constraints, and computational analysis. Finally, we discuss future necessary short- and long-term developments in the Ab-seq field that will be necessary for its large-scale implementation in the holistic analysis of humoral immunity and the design of next-generation biologic drugs.Table 1Bottom-up proteomics antibody repertoire studiesFinding, antibody repertoire subset, speciesNext-generation sequencing (NGS) library preparation and sequencing platformAntibody isolation and fractionationAntibody proteolysis/digestion strategyLC-MS/MSComputational pipelineRefsIdentification of antigen-specific serum antibodies from immunized animals, IgG, rabbit, mouseMultiplex PCR (MTPX); 454 Life SciencesProtein A sepharose beads; Protein G magnetic beadsNo Fab-Fc fragmentation.Either chymotrypsin, elastase, pepsin, or trypsinLTQ Orbitrap Velos in parallel processing mode when possibleSEQUE
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