Human antibody technology and human antibody responses
Antibodies have emerged as major drugs. They are widely used for treatment of a diversity of conditions in the clinic. In particular the development and use of human, humanised and chimeric antibodies have dramatically improved the management of many disease conditions. My research group have developed and exploited human antibody technology for >30 years for development of human monoclonal antibodies and for studies of human immune responses.
The technology approach
The introduction of gene technology, such as combinatorial antibody library technology, has dramatically expanded our ability to generate and perfect human antibodies for a diversity of purposes, including for use in the treatment of disease. My research group has actively approach this field of research and developed approaches to generate and exploit human antibodies by such means.
- To be well tolerated in therapy, an antibody should not diverge substantially from other human antibodies. For that purpose we developed a human antibody library approach the uses human antibody diversity as generated in vivo. The technology incorporates diversity encoded by human antibody producing B cells into an antibody framework that have biophysically attractive properties. Published in Nature Biotechnology. The advantages of the approach were further discussed in this paper in Nature Biotechnology.
- There is a great need for antibodies against a large number of targets. Antibody library technology is ideally suited to meet this need as it is amenable to automation. We have implemented a high throughput approach to use human antibody library approaches for the large-scale isolation of human antibodies, for instance for analytical application as described in this paper published in Protein Engineering Design and Selection.
- We need to develop antibodies against a great diversity of molecules. These targets differ in size and chemical properties. We reasoned that different antibody populations may target such different molecules with different efficiencies. For that purpose we introduced the concept of designed antibody library diversity for the targeting of molecules of different types, as outlined in this paper in the Journal of Molecular Biology.
- Despite the great power of antibody library technology, initially developed antibodies may be in need for improvement of the binding or biophysical properties. We developed an maturation process based on parallel evolution of multiple antibodies as outlined in this paper in Protein Engineering Design and Selection.
- Human monoclonal antibody technology has a long history. Such studies were indeed conducted long before the introduction of most genetic engineering approaches. My original approach to human antibody technology relied on human hybridoma technology, as summarised in my doctoral thesis, Human monoclonal antibody technology. A tool to investigate human antibody repertoires, defended in 1992.
Beyond our own research we develop and use antibody technology to service the Swedish research Community through SciLifeLab's Drug Discovery and Development Platform's Human Antibody Therapeutics National Facility of which I am Facility Director.
Antibodies in allergic disease
Allergic disease has emerged as an important health problem. Antibodies are critical components of the disease as they, in the form of IgE, initiate the allergic reaction. Antibodies of other types, e.g. IgG, may however also resolve the disease condition. Antibody technology is ideally suited to approach many aspects of the immunology of allergic disease. We have exploited human antibody technology, advances in gene sequencing, protein engineering, X-ray crystallography, immunochemical analysis, and cell biology technology to expand our understanding of allergic disease.
Application in anti-viral antibody research
Antibodies are critically important for our defense against a diversity of viral diseases. We have in particular focused on studies of antibodies against human cytomegalovirus, a common virus that cause substantial morbidity in neonatally infected individuals and in immunocompromised subjects. In particular we have developed and studied antibodies directed against a particular structure of an important envelope glycoprotein (glycoprotein B) of this virus. These studies, that span more the 25 years of research.
Antibody repertoire generation and evolution
The vast diversity of antibodies, critical components of our defense against a hostile environment, are generated from a small number of genes. Gene recombination, combinatorial processes and somatic hypermutation contributes to the vast diversity of antibodies. I have been thoroughly engaged in development of our understanding human antibody repertoires and their evolution.
- Antibody-encoding genes generated through gene rearrangement are further evolved by a process of somatic hypermutation. In humans, this evolution occurs after the stimulation of B cells with an antigen recognised by antibodies displayed on their surface. We have shown in a paper published in Frontiers in Immunology how such evolution occurs through paths directly linked to their germline gene origin. We propose that such germline gene-centric paths must be taken into account during the analysis of antibody evolution.
- Early studies of specific human antibody repertoires largely depended on the the outcomes of human hybridoma-based studies. In an early era of molecular studies of human antibody responses we discussed human antibody diversity as defined by large collections of specific human monoclonal antibodies.
- Despite the vast diversity generated in the antibody population, many antibody responses are limited to certain niches of this diversity. We have described such restricted diversities in the case of responses against an important target on a envelope glycoprotein of human cytomegalovirus and an important grass pollen allergen. We have also discussed potential uses of the latter restricted niches in the studies of allergic immune responses.
- Antibody evolution is usually considered to involve point mutations of antibody-encoding genes. We however have identified processes that, in addition to single base mutations, introduced insertions and deletions into antibody-encoding sequences during the process of somatic hypermutation. We recently demonstrated the different modes whereby antibodies of different germline gene origins are targeted by such evolution.
- We are committed to the FAIR principles of data sharing. NGS data set have thus been made available for research purposes through SRA/European Nucleotide Archive.
Antibody germline repertoires - a best practice approach to germline gene inference
A detailed understanding of germline genes available in study subjects are critical for properrepertoire analysis. Germline gene databases are known to contain alleles of such genes that have been incorrectly identified. Furthermore, some common alleles are missing from these databases, and most genes in databases will not be relevant in the context of a single subject as it is not present in his/her genotype. These problems result in many incorrect assignments of germline gene origin of individual sequences, a fact that affect downstream interpretation of antibody responses, the role of somatic hypermutation etc.
As part of my interest in antibody repertoires I have been engaged in the Adaptive Immune Receptor Repertoire Community, more specifically in its Germline Database Working Group, Software Working Group, and its Inferred Allele Review Committee (IARC). My aim in this context is to develop valid, best-practice procedures for computational inference of immunoglobulin germline genes using information obtained from studies employing high throughput sequencing of immunoglobulin-encoding transcriptomes, and to support the incorporation of such information into existing germline gene databases.
As outlined above, studies of antibody repertoires are critically dependent on an understanding of the genes available to each study subject for the generation of antibody diversity. Unfortunately, sequencing of human immunoglobulin germline gene loci is well beyond the capabilities of virtually every antibody research project. Consequently, other approaches, in particular gene inference based on analysis of antibody-encoding transcripts using next generation sequencing, have become important for our understanding of individual subjects' immunoglobulin repertoires and their development. We have described:
- approaches that enhance validity of such inference in particular using haplotype-based analysis and analysis of the quality of individual sequence reads. This study was published in Molecular Immunology.
- the impact of the V-DJ rearrangement process of our ability to infer the 3'-end of IGHV genes, a study that was also published in Molecular Immunology. We concluded that inference of the last base of an IGHV gene cannot be made with confidence as the most likely germline-encoded gene may not even be the dominant base in rearrangements. We postulated that factors related to antibody structure may limit the ability of the last base of the germline gene to be incorporated during productive recombination.
- the difficulties associated with inference of the final bases of novel alleles of IGHV genes as illustrated by the difficult to achieve inference of allelic variant IGHV3-7*02 A318G, an allele that seems to be present in a substantial number of subjects. We proposed that inference of IGHV alleles should currently be supported by a detailed analysis of the final bases of the reads supporting a specific inference in order to validate the germline gene inferences. This study was published in Molecular Immunology.
- a number of alleles of IGHV genes that are considered as being functional by standard criteria but that are poorly expressed. We hypothesized that this might be a consequence of uncommon residues in the germline gene-encoded product, residues that might prevent production of a functional product from these genes. This study was published in Frontiers in Immunology.
- a curated set of leader sequences and upstream regions of human heavy chain variable domain germline genes. We used this set to build hypothesis regarding the development and organisation of the immunoglobulin heavy chain locus. This study was published in Frontiers in Immunology.
I am also engaged, through my involvement in IARC, in establishment of principles for discovery and documentations of immunoglobulin germline gene alleles through the use of NGS data and inference technology, alleles that are not recognised by standard immunoglobulin gene databases. For more information on these processes, please review the Open Germline Receptor Database (OGRDB) web page. Major results include for instance:
- Approaches to curation and affirmation of novel alleles of immunoglobulin genes identified by computational inference using repertoire NGS data. This study was published in Frontiers in Immunology.
- The development of a database (OGRDB) for open documentation of adaptive immune receptor repertoire germline gene alleles. This study was published in Nucleic Acids Research.
- Development of a forward looking approach to naming of adaptive immune receptor repertoire alleles (e.g. those alleles that encode antibodies), an approach that allows for refinement and consolidation into genes over time as more detailed knowledge of the relevant loci emerges. This study was published in Immunoinformatics.
- Definition of transcribed immunoglobulin alleles of mouse Balb/c mice. The germline repertoire of this mouse strains is almost completely different from that of the previously characterized C57BL/6 strain. Extreme caution must be observed when analysing antibody repertoires derived from different mouse strains. This study was published in Frontiers in Immunology.
- Description of the caution that must be observed when identifiying novel immunoglobulin germline alleles based on short read sequencing data. This study was published in Genes & Immunity.
The technology approach
- Ljungars A, Schiött T, Mattson U, Steppa J, Hambe B, Semmrich M, Ohlin M, Tornberg UC, Mattsson M (2020) A bispecific IgG format containing four independent antigen binding sites. Sci Rep 10, 1546. (Abstract at publisher's website)
- Ljungars A, Svensson C, Carlsson A, Birgersson E, Tornberg U-C, Frendéus B, Ohlin M, Mattsson M (2019) Deep mining of complex antibody phage pools generated by cell panning enables discovery of rare antibodies binding new targets and epitopes. Front Pharmacol 10, 847 (Abstract at publisher's website)
- Ljungars A, Mårtensson L, Mattsson J, Kovacek M, Sundberg A, Tornberg UC, Jansson B, Persson N, Kuci Emruli V, Ek S, Jerkeman M, Hansson M, Juliusson G, Ohlin M, Frendéus B, Teige I, Mattsson M (2018) Phenotypic discovery of therapeutic antibodies and targets for cancer treatment. NPJ Precis Oncol 2, 18. (Abstract at publisher's website)
- Persson H, Kirik U, Thörnqvist L, Greiff L, Levander F, Ohlin M (2018) In vitro evolution of antibodies inspired by in vivo evolution. Front Immunol 9, 1391. (Abstract at publisher's website)
- Säll A, Walle M, Wingren C, Müller S, Nyman T, Vala A, Ohlin M, Borrebaeck CAK, Persson H (2016) Generation and analyses of human synthetic antibody libraries and their application for protein microarrays. Prot Eng Des Select 29, 427-437. (Abstract at publisher's website)
- Säll A, Persson H, Ohlin M, Borrebaeck CAK, Wingren C (2016) Advancing the Global Proteome Survey platform by using an oriented single chain antibody fragment immobilization approach. New Biotechnol 33, 503-513. (Abstract at publisher's website)
- Persson H, Wallmark H, Ljungars A, Hallborn J and Ohlin M (2008) In vitro evolution of an antibody fragment population to find high affinity hapten binders. Protein Eng Des Sel 21, 485-493. (Abstract)
- Persson H and Ohlin M (2007) Exploring central and peripheral diversity in antibody evolution. Mol Immunol 44, 2729-2736. (Abstract at publisher's website)
- Persson H, Lantto J and Ohlin M (2006) Creating a focused antibody library for improved hapten recognition. J Mol Biol 357, 607-620. (Abstract at publisher's website)
- Borrebaeck CAK and Ohlin M (2002) Antibody evolution beyong Nature. Nat. Biotechnol. 20, 1189-1190. (Abstract at publisher's website)
- Söderlind E, Strandberg L, Jirholt P, Kobayashi N, Alexeiva V, Åberg AM, Nilsson A, Jansson B, Ohlin M, Wingren C, Danielsson L, Carlsson R and Borrebaeck CAK (2000) Recombining germline-derived CDR-sequences for creating diverse single-framework antibody libraries. Nat. Biotechnol. 18, 852-856. (Abstract at publisher's website)
Antibodies in allergic disease
Anti-viral antibody research
- Bahnan W, Wrighton S, Sundwall M, Bläckberg A, Höglund U, Larsson O, Khakzad H, Godzwon M, Walle M, Elder E, Söderlund Strand A Happonen L , André O, Kumra Ahnlide J, Hellmark T, Wendel-Hansen V, Wallin RPA, Malmström J, Malmström L, Ohlin M, Rasmussen M, Nordenfelt P (2022) Opsonization by non-neutralizing antibodies can confer protection to SARS-CoV-2 despite spike-dependent modulation of phagocytosis. Front Immunol (in press)
- Devito C, Ellegård R, Falkeborn T, Svensson L, Ohlin M, Larsson M, Broliden K, Hinkula J (2018) Human IgM monoclonal antibodies block HIV-transmission to immune cells in cervico-vaginal tissues and across polarized epithelial cells in vitro. Sci Rep 8, 10180. (Abstract at publisher's website)
- Ohlin M, Söderberg-Nauclér C (2015) Human antibody technology and the development of antibodies against cytomegalovirus. Mol Immunol 67, 153-170. (Abstract at publisher's website)
- Ohlin M (2014) A new look at a poorly immunogenic neutralization epitope on cytomegalovirus glycoprotein B. Is there cause for antigen redesign? Mol Immunol 60, 95-102. (Abstract at publisher's website)
- Axelsson F, Persson J, Moreau E, Côté MH, Lamarre A and Ohlin M (2009) Novel antibody specificities targeting glycoprotein B of cytomegalovirus identified by molecular library technology. N Biotechnol 25, 429-436. (Abstract at publisher's website)
- Barrios Y, Knör S, Lantto J, Mach M and Ohlin M (2007) Clonal repertoire diversification of a neutralizing cytomegalovirus glycoprotein B-specific antibody results in variants with diverse anti-viral properties. Mol Immunol 44, 680-690. [Epub ahead of print July 7, 2006] (Abstract at publisher's website)
- Gicklhorn D, Eickmann M, Meyer G, Ohlin M and Radsak K (2003) Differential effects of glycoprotein B epitope specific antibodies on human cytomegalovirus induced cell-cell fusion. J. Gen. Virol. 84, 1859-1862. (Abstract at publisher's website)
- Lantto J, Fletcher J and Ohlin M (2003) Binding characteristics determine the neutralizing potential of antibody fragments specific for antigenic domain 2 on glycoprotein B of human cytomegalovirus. Virology 305, 201-209. (Abstract at publisher's website)
- Lantto J, Fletcher J and Ohlin M (2002) A divalent antibody format is required for neutralisation of human cytomegalovirus via antigenic domain 2 on glycoprotein B. J. Gen. Virol. 83, 2001-2005. (Abstract at publisher's website)
- Furebring C, Speckner A, Mach M, Sandlie I, Norderhaug L, Borrebaeck CAK, Turesson H and Ohlin M (2001) Antibody-mediated neutralization of cytomegalovirus: Modulation of efficacy induced through the IgG constant region. Mol. Immunol. 38, 833-840. (Abstract at publisher's website)
- Speckner A, Glykofrydes D, Ohlin M and Mach M (1999) Antigenic domain 1 of human cytomegalovirus glycoprotein B induces a multitude of different antibodies which, when combined, results in incomplete virus neutralization. J. Gen. Virol. 80, 2183-2191. (Abstract at publisher's website)
- Eickmann M, Lange R, Ohlin M, Reschke M, Radsak K (1998) Effect of cysteine substitutions on dimerization and interfragment linkage of human cytomegalovirus glycoprotein B (gpUL55). Arch. Virol. 143, 1865-1880. (Abstract at publisher's website)
- Ohlin M, Silvestri M, Sundqvist V-A, Borrebaeck CAK (1997) Cytomegalovirus glycoprotein B-specific antibody analysis using electrochemiluminescence detection-based techniques. Clin. Diagn. Lab. Immunol. 4, 107-111. (Abstract at publisher's website)
- Bold S, Ohlin M, Garten W, Radsak K (1996) Structural domains involved in human cytomegalovirus glycoprotein B mediated cell-cell fusion. J. Gen. Virol. 77, 2297-2302. (Abstract at publisher's website)
- Ohlin M, Owman H, Mach M, Borrebaeck CAK (1996) Light chain shuffling of a high affinity antibody results in a drift in epitope recognition. Mol. Immunol. 33, 47-56.
- Ohlin M, Plachter B, Sundqvist V-A, Middeldorp JM, Steenbakkers PGA, Borrebaeck CAK (1995) Human antibody reactivity against the lower matrix protein (pp65) produced by cytomegalovirus. Clin. Diagn. Lab. Immunol. 2, 325-329.
- Rioux J, Ohlin M, Borrebaeck CAK, Newkirk MM (1995) Molecular characterization of human monoclonal antibodies specific for the human cytomegalovirus: relationship of variable region sequence to antigen specificity and rheumatoid factor associated idiotype expression. Immunol. Infect. Dis. 5, 43-52.
- Ohlin M, Owman H, Rioux JD, Newkirk MM, Borrebaeck CAK (1994) Restricted variable region gene usage and possible rheumatoid factor relationship among human monoclonal antibodies specific for the AD-1 epitope on cytomegalovirus glycoprotein B. Mol. Immunol. 31, 983-991.
- Ohlin M, Sundqvist V-A, Mach M, Wahren B, Borrebaeck CAK (1993) Fine specificity of the human immune response to the major neutralization epitopes expressed on cytomegalovirus gp58/116 (gB) studied by human monoclonal antibodies. J. Virol. 67, 703-710.
- Ohlin M, Hinkula J, Broliden P-A, Grunow R, Borrebaeck CAK, Wahren B (1992) Human monoclonal antibodies produced from normal, HIV-1 seronegative donors and specific for glycoprotein gp120 of HIV-1 envelope. Clin. Exp. Immunol. 89, 290-295.
- Ohlin M, Sundqvist V-A, Wahren B, Gilliam G, Rudèn U, Gombert F, Borrebaeck CAK (1991) Characterization of human monoclonal antibodies directed against the pp65 kD matrix antigen of human cytomegalovirus. Clin. Exp. Immunol. 84, 508-514.
- Ohlin M, Broliden P-A, Danielsson L, Wahren B, Rosen J, Jondal M, Borrebaeck CAK (1989) Human monoclonal antibodies against a recombinant HIV envelope-antigen produced by primary in vitro immunization. Characterization and epitope mapping. Immunology 68, 325-331.
Antibody repertoire generation, evolution, and analysis
- Lees WD, Christley S, Jackson KJL, Peres A, Kos JT, Corrie B, Ralph D, Breden F, Matsen E, Cowell LG, Yaari G, Corcoran M, Karlsson Hedestam G, Ohlin M, Collins AM, Watson CT, Busse CE, The AIRR Community (2023) AIRR community curation and standardised representation for immunoglobulin and T cell receptor germline sets. Immunoinformatics (in press)
- Jackson KJL, Kos JT, Lees WD, Gibson WS, Laird Smith M, Peres A, Yaari G, Corcoran MM, Busse CE, Ohlin M, Watson CT, Collins AM (2022) A BALB/c IGHV reference set, defined by haplotype analysis of long-read VDJ-C sequences from F1 (BALB/c / C57BL/6) mice. Front Immunol 13, 888555 (Link to full text at publisher's web site)
- Babrak L, Marquez S, Busse CE, Lees WD, Miho E, Ohlin M, Rosenfeld AM, Stervbo U, Watson CT, Schramm CA (2022) Adaptive Immune Receptor Repertoire (AIRR) Community guide to TR and IG gene annotation. In: Anton W. Langerak AW (ed.), Immunogenetics: Methods and Protocols. Methods Mol Biol 2453 (in press) (Link to full text at publisher's web site)
- Collins AM, Peres A, Corcoran MM, Watson CT, Yaari G, Lees WD, Ohlin M (2021) Commentary on Population matched (pm) germline allelic variants of immunoglobulin (IG) loci: Relevance in infectious diseases and vaccination studies in human populations. Genes Immun 22, 335-338. (Link to full text at publisher's web site)
- Huang Y, Thörnqvist L, Ohlin M (2021) Computational inference, validation, and analysis of 5'UTR-leader sequences of alleles of immunoglobulin heavy chain variable genes. Front Immunol 12, 730105. (Abstract at publisher's web site)
- Ohlin M (2020) Poorly expressed alleles of several human immunoglobulin heavy chain variable (IGHV) genes are common in the human population. Front Immunol, 11:603980. (Abstract at publisher's website)
- Lees W, Busse CE, Corcoran M, Ohlin M, Scheepers C, Matsen FA IV, Yaari G, Watson CT, The AIRR Community, Collins A, Shepherd A (2020) OGRDB – a reference database of inferred immune receptor genes. Nucleic Acids Res 48, D964–D970. (Abstract at publisher's website)
- Smakaj E, Babrak L, Ohlin M, Shugay M, Briney B, Tosoni D, Galli C, Grobelsek V, D’Angelo I, Olson B, Watson C, Reddy S, Greiff V, Trück J, Marquez S, Lees W, Miho E (2019) Benchmarking immunoinformatic tools for the analysis of antibody repertoire sequences. Bioinformatics (in press) (Abstract at publisher's website).
- Ohlin M, Scheepers C, Corcoran M, Lees WD, Busse CE, Bagnara D, Thörnqvist L, Bürckert LP, Jackson KJL, Ralph DK, Schramm CA, Marthandan N, Breden F, Scott JK, Matsen IV FA, Greiff V, Yaari G, Kleinstein SH, Christley S, Sherkow JS, Kossida S, Lefranc MP, van Zelm MC, Watson CT, Collins AM (2019) Inferred allelic variants of immunoglobulin receptor genes: a system for their evaluation, documentation and naming. Front Immunol 10, 435. (Abstract at publisher's website)
- Thörnqvist L, Ohlin M (2018) Critical steps for computational inference of the 3’-end of novel alleles of immunoglobulin heavy chain variable genes – illustrated by an allele of IGHV3-7. Mol Immunol 103, 1-6. (Abstract at publisher's website)
- Thörnqvist L, Ohlin M (2018) The functional 3´-end of immunoglobulin heavy chain variable (IGHV) genes. Mol Immunol 96, 61-68. (Abstract at publisher's website)
- Thörnqvist L, Ohlin M (2018) Data on the nucleotide composition of the first codons encoding the complementary determining region 3 (CDR3) in immunoglobulin heavy chains. Data Brief 19, 337-352. (Abstract at publisher's website)
- Kirik U, Persson H, Levander F, Greiff L, Ohlin M (2017) Antibody heavy chain variable domains of different germline gene origins diversify through different paths. Front Immunol 8, 1433. (Abstract at publisher's website)
- Kirik U, Greiff L, Levander F, Ohlin M (2017) Parallel antibody germline gene and haplotype analyses support the validity of immunoglobulin germline gene inference and discovery. Mol Immunol 87, 12-22. (Abstract at publisher's website)
- Kirik U, Greiff L, Levander F, Ohlin M (2017) Data on haplotype-supported immunoglobulin germline gene inference. Data Brief 13, 620-640. (Abstract at publisher's website)
- Lantto J, Ohlin M (2002) Functional consequences of insertions and deletions in the complementarity determining regions of human antibodies. J. Biol. Chem. 277, 45108-45114. (Abstract at publisher's website)
- Lantto J, Ohlin M (2002) Uneven distribution of repetitive sequence motifs in immunoglobulin heavy variable genes. J. Mol. Evol. 54, 346-353. (Abstract at publisher's website)
- Ohlin M, Borrebaeck CAK (1998) Insertions and deletions in hypervariable loops of antibody heavy chains contribute to molecular diversity. Mol. Immunol. 35, 233-238. (Abstract at publisher's website)
- Ohlin M, Borrebaeck CAK (1996) Characteristics of human antibody repertoires following active immune responses in vivo. Mol. Immunol. 33, 583-592. (Abstract at publisher's website)