Strategies to Obtain Diverse and Specific Human Monoclonal

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Transplantation Publish Ahead of Print DOI: 10.1097/TP.0000000000001702

Antibodies from Transgenic Animals

Marianne Brüggemann, PhD1, Michael J. Osborn, PhD1, Biao Ma, PhD1

and Roland Buelow, PhD2

1 Recombinant Antibody Technology Ltd. Babraham Research Campus, Cambridge

CB22 3AT, UK

2 Ligand Pharmaceuticals, 3911 Sorrento Valley Boulevard, San Diego 92121, CA,

USA

Correspondence: Marianne Brüggemann, PhD, Recombinant Antibody Technology

Ltd., Babraham Research Campus, Cambridge CB22 3AT, UK.

([email protected]).

Authorship:

Marianne Brüggemann: M.B. interpreted the data and wrote the review, MichaelACCEPTED Osborn: M.J.O. corrected and added details, Biao Ma: B.M. participated in critical discussions,

Roland Buelow: R.B. contributed with additions and editing,

Copyright © Wolters Kluwer Health. Unauthorized reproduction of this article is prohibited. The authors declare no conflicts of interest. (or as below: The authors declare no funding or conflicts of interest.)

The authors declare no funding or conflicts of interest.

ACCEPTED

Abstract:

Techniques to obtain large quantities of antigen-specific monoclonal antibodies, mAbs, were first established in the 1970s when Georges Köhler and César Milstein immortalized antibody-producing mouse B-lymphocytes by fusion with myeloma cells

(http://www.whatisbiotechnology.org/exhibitions/milstein). Combined with the expression of human antibodies in transgenic animals, this technique allowed upon immunization the generation of highly specific fully human mAbs for therapeutic applications. Apart from being extremely beneficial, mAbs are a huge success commercially. However, despite cell fusion generating many useful mAbs questions have been asked about which types of cells are prone to fuse and whether other methods may identify a wider range of binders. The discovery that expression libraries, using E.coli or yeast, produced different specificities was intriguing and more recently

Next Generation Sequencing has identified wide-ranging usage with highly diverse and unique repertoires. Another strategy is the combination of flow cytometry sorting of antigen-binding B-lymphocytes and single cell RT-PCR followed by re-expression, which has identified many high affinity mAbs.

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The expression of human Ig loci in transgenic animals was achieved by a combination of DNA transfer and integration strategies, knock-out approaches to silence interfering endogenous genes, and improving cloning, stability and manipulation of large DNA constructs accommodating ~1Mb Ig loci.1 Over 25 years ago it was shown that a germline configured human IgH mini-locus could be stably integrated in the mouse genome, contributing faithfully to immune development by DNA rearrangement and producing human IgM.2 These findings initiated many parallel transgenic approaches of integrating larger and more complete human IgH, Ig and Ig loci, initially in mice and later in cattle, rabbits and rats.3-12 The human IgH variable region, shown in Figure 1, has been as a whole or in large part linked with mouse, rat or human C-genes to generate a diverse human antibody repertoire. Importantly, knock-out approaches secured high expression of these transgenic loci.13-15

Early on it became clear that cell-fusion methods established for immortalizing

B cells from conventional laboratory mouse and rat strains16, 17 were also applicable to generate high levels of specific human mAbs from transgenic lines.2, 6, 7, 18, 19 However, to avoid hybrid cell instability and chromosomal loss it became advisable to identify antibody expression soon after fusion20, which could be done by RT-PCR analysis of individual clones obtained by limiting dilution. Antibody binding using supernatant was tested by ELISA.21 ACCEPTEDIn other early approaches V-domains could be displayed on the surface of filamentous bacteriophage, which allowed the isolation of rare antigen-binders.22

Subsequent improvements in affinities have been obtained by chain shuffling23 and

24, 25 joining of VH and VL domains. Human blood lymphocytes were frequently used to

Copyright © Wolters Kluwer Health. Unauthorized reproduction of this article is prohibited. obtain V-gene libraries and initially there was no need to use transgenic animals. A similar strategy was established by Ribosome display systems.26 Later, surface

27, 28 expression of ScFv by random combination of VH and VL in, both, phage and yeast29, 30 permitted selection of high-affinity antigen binders directly and became an important tool to select human mAbs from transgenic animals.31, 32 In other general selection approaches to establish single copy expression in mammalian cells virus- mediated display33 and, separately, homologous integration were successful.34

Specific screening or enrichment of single B-lymphocytes from bone marrow, spleen or lymph nodes has also been used to identify antigen-specific binders.35, 36 This technique often combines flow cytometry with single cell VH and VL cloning by RT-

PCR.37 Variations using magnetic beads for enrichment have also been described.38

Another successful strategy, next generation sequencing, identified binders by sequence

39, 40 occurrence. For example, when the most abundant VH and VL sequences from bone marrow plasma cells of immunized mice were paired a large number of antigen-specific binders, some with nanomolar affinity, could be obtained.41

Here a concise overview is presented of how high affinity mAbs can be isolated using cell fusion, display libraries, FACS and NGS approaches. Table 1 presents a summary of the technologies.

Hybridoma mAbs

GeneratingACCEPTED mAbs by hybridoma cell-fusion using spleen or lymph node cells is the most successful approach of deriving antigen-specific human antibodies from transgenic rodents.1, 6, 8, 9 Previously, this generated fully human Abs4, 42 whilst more recently chimeric products with human variable region but endogenous mouse or rat

Copyright © Wolters Kluwer Health. Unauthorized reproduction of this article is prohibited. constant (C) region were obtained.6, 8, 9 Expression of human V(D)Js linked to mouse or rat C has also increased antibody expression to near normal levels. Optimisation of the fusion process by using a range of myeloma partners has made this process highly successful and rapid immunization schemes using transgenic rats produced diverse binders easily by this technology.6, 19.

The great manufacturability of animal derived antibodies has resulted in more

FDA approvals compared to display antibodies selected from human cells (Table 2).43

Moreover, the development of commercial late-stage antibody therapeutics has exceeded all expectations44; with a recent increase of fully human mAbs from transgenic animals to >25% and a decrease of all other animal mAbs, fully mouse, chimeric or humanised, to <75%. Interestingly, the latest fully human therapeutic antibodies were derived by cell fusion and genetic engineering (CH replacement) from recently produced transgenic animals carrying larger and near complete human V(D)J regions. For these novel transgenic lines it was important, as a proof of principle, to show that many diverse mAbs could be easily produced after immunization. This was achieved and amongst many targets, identified blockers of receptor-ligand interaction, addressed neurodegeneration and provided toxin neutralisers.6, 8, 9, 45

Surface expression technologies:

Phage, yeast and mammalian display libraries Phage displayACCEPTED and selection has been extensively described27, 46 and a comparison of this technology with generating antigen-specific hybridomas revealed that quite different and few overlapping or competing specificities could be obtained.45 One reason may be that random combinations of VH and VL sequences are expressed on the

VHVL combination when fused. Nevertheless, a considerable number of display-derived antibodies are in clinical development and phage libraries have been described as goldmines for therapeutic mAbs.31, 47

A variety of yeast strains have also been employed as expression host to generate antibody fragments with Saccharomyces cerevisiae being extensively utilized.48 This single cell organism provides some of the advantages of a eukaryotic expression system with posttranscriptional modification, such as glycosylation or disulphide linkage, and secretory expression. Readily available expression plasmids allow single copy surface display as a fusion protein with Saccharomyces’ Aga2 adhesion receptor. Cloning and selection strategies have been extensively described49, 50 and tackling infectious disease by isolation of anti-bacterial and anti-viral mAbs has been successful.51 Enrichment of antigen-specific yeast cells by flow cytometry or with magnetic beads is well established49, 52 but there are drawbacks because of a reduced library size, ~108 for yeast compared to ~1010 for phage libraries51, and the slower growth of yeast cells compared to bacteria.

Using mammalian cell display is a very efficient strategy to generate libraries expressing correctly folded single chain antibody fragments.53, 54 A CMV expression plasmid with a trans-membrane sequence and a tag allowed cloning of single VH or VH

55 linked to VL and transfection into human embryonic kidney cells (HEK-293T).

Library sizes are relatively small, with between 103-106 clones, but flow sorting ensuredACCEPTED rapid isolation of specific binders. Expression in mammalian cells may also avoid incompatibility issues and codon optimization of human sequences, which may be required for efficient antibody fragment expression in yeast56 or E.coli57. Despite the method’s success, one concern was that multiple antibody sequences with different

Copyright © Wolters Kluwer Health. Unauthorized reproduction of this article is prohibited. specificities could be expressed on the cell surface. This was overcome by site-specific integration of one copy using the Flp-InTM system (Invitrogen), where a gene of interest is integrated after a Flp recombination target site is inserted into a cell line of choice such as HEK or CHO.34

Enrichment and analysis of single cells

Selection of antigen-specific B cells by FACS, immunomagnetic sorting or panning combined with single cell VH and VL RT-PCR is a swift and effective way to obtain high affinity mAbs58-61. However, in transgenic lines lower B cell numbers and reduced antibody levels compared to normal animals had been reported.1 Fortunately, the analysis of recently derived transgenic animals revealed a comprehensive level of antigen-specific B cells, which could be visualized and separated by flow cytometry.

For example, successful enrichments of antigen-specific single spleen or lymph node cells was achieved by staining with anti-IgG and antigen, which had been conjugated with different fluorophores. Interestingly, selection of high affinity binders was promptly achieved by including an irrelevant antigen in the staining process for the exclusion of non-specific cells.62

Generally applicable RT-PCR methods with extensive lists of primer sequences and mixes to retrieve most human V(D)J products are available; either by single reaction or using a nested, perhaps more sensitive, amplification.63, 64 Essentially fine- tuning isACCEPTED required when transcription levels are reduced in, for example, long-lived or memory B cells compared to plasmablasts.64, 65 Amplification where VH and VL products can be obtained jointly has also been described; the aim was to speed up the process and include cloning and re-expression as Fab.66 In this VH-VL linkage-cloning,

Copyright © Wolters Kluwer Health. Unauthorized reproduction of this article is prohibited. termed Symplex PCR, FACS sorted individual human plasma cells produced near 1% antigen-specific binders and with adaptation this method could be used to amplify sorted B cells from transgenic rodents.

Next generation sequencing and identification of binders

Major improvements in sequence processing in the last few years has established a very efficient high-throughput technology termed Next Generation Sequencing (NGS). This has allowed the discovery of near-complete immune repertoires.67 68 As explained in detail39, the success of V-gene reads or sequence recovery depends on comprehensive library preparation methods and the use of highly efficient sequencing platforms eg,

Illumina HiSeq sequencing by synthesis. An equally important consideration for increased data processing was the addition of adapters essential for reaction priming of flow cell attached sequences (http://nextgen.mgh.harvard.edu/Illumina

Chemistry.html). A comparison of V-gene recovery and expediency using NGS strategies to identify extensive near-complete antibody repertoires, revealed that primer extensions are most suitable for small RNA samples and that sequence additions or a ligation approach may be less efficacious but possibly easier to perform.39, 69

To overcome the problem with correct VH-VL pairing of NGS sequences 2 strategies have been tried; both finding antigen-specific antibodies with nanomolar affinity. In one approach single-cell emulsion droplets provided natively paired and subsequentlyACCEPTED correctly linked VH and VL sequences and in the other strategy abundant VH and VL sequences were paired based on their frequencies revealed by NGS.41, 70 In both approaches distinct human or mouse B cell populations were enriched by FACS.

Nevertheless, avoiding selection or enrichment procedures prior to NGS is also possible

Copyright © Wolters Kluwer Health. Unauthorized reproduction of this article is prohibited. and this was demonstrated by identifying antigen-specific domain antibodies from a blood lymphocyte library of an immunized Llama.71 However, comparison with sequences from a phage library generated from the same material helped to select high affinity binders. Encouraging results have been obtained by deriving NGS libraries from transgenic rodents expressing human antibody repertoires with a fixed light chain as presented in the following talk

(http://content.stockpr.com/omniab/db/252/746/file/OmniAb.pdf) and by 2 companies

(http://www.omtinc.net; http://omttherapeutics.com).

Summary

In conclusion, numerous antibody screening libraries in different formats have been instrumental in identifying antigen-specific mAbs. Important technical advances have improved the success rate, increasing the number of specific sequences found (Figure

2). For conventional antibodies in (H-L)n format, single cell strategies facilitated correct pairing while high-throughput sequencing and bioinformatics analyses can predict correct VH-VL combinations. These strategies identified many immensely beneficial high affinity human anti-human mAbs from transgenic rodents; some are already in the clinic others in late phase trials1, 44, with the majority for cancer treatment, followed by treatment of autoimmunity, musculoskeletal problems and infectious diseases. Table 2 provides a comprehensive list of therapeutic mAbs in clinical use but it comes at a high price.72 ACCEPTEDRecently in the UK the use of Nivolumab, trade name Opdivo, received extensive attention when described as a life-extending drug that was too expensive

(http://news.sky.com/story/aa-gills-last-article-reveals-life-extending-drug-was-too- expensive-10692057).

Copyright © Wolters Kluwer Health. Unauthorized reproduction of this article is prohibited. As significant improvements have been made recently with the derivation of rats and mice expressing high levels of human antibodies in human V(D)J rodent C chimeric form, cost reductions are highly likely. It is also interesting to speculate how high affinity mAbs may be isolated in the next few years. A clue may be provided by an increased use of sophisticated computation models so that in future it may be possible to identify specific binders directly from NGS libraries by occurrence and

CDR pattern.73, 74 Thus, to rapidly obtain specific human antibodies from transgenic rodents a combination of the above described strategies may be advantageous; first enrichment of antigen-binding B cells which is then followed by high-throughput sequencing and usage of analyses programmes to identify high affinity binders.

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Figure 1. The human IgH V, D, J region linked with C-region genes or locus and expressed in transgenic animals.

Figure 2. Strategies to isolate antigen-specific human mAbs. Human peripheral blood lymphocytes can be obtained from healthy volunteers while transgenic rodents provide lymphocyte tissue, such as spleen or lymph nodes, after immunization. B cells from these tissues can be used directly for immortalization such as cell fusion. Display libraries using phage, yeast or mammalian expression vectors are generated by RT-PCR cloning. Extensive transcript analysis can also be obtained immediately by high- throughput Next Generation sequencing. Improvements in mRNA processing facilitated the retrieval of H- and L-chain sequences from single cells while enrichment of antigen-binding cells by, for example, Flow Cytometry improved the recovery efficiency significantly. At the final stage re-expression and specificity analysis of the obtained sequences may include data analysis using Bioinformatic tools.

ACCEPTED

ACCEPTED22

ACCEPTED23

Methodologies to obtain mAbs from lymphocyte tissues of immunized transgenic animals

Method Description Advantages Problems

immortalization of antibody- Ig secretion, good stability, hybridoma cell fusion limited repertoire producing cells ready for testing

phage surface display of RT- large libraries, binders can be correct combination of VH and VL, phage libraries PCR products selected expression bias

yeast surface display of RT-PCR original folding maintained, correct combination of VH and VL, yeast libraries products binders can be selected medium size library, expression bias

mammalian cell expression of authentic products, easy correct combination of VH and VL, mammalian cell libraries RT-PCR products screening small library size

chip attachment and reads of extensive near complete correct combination of VH and VL, Next Generation Sequencing single (RT-)PCR products sequence info re-expression

ACCEPTED24

Copyright © Wolters Kluwer Health. Unauthorized reproduction of this article is prohibited. authentic VH and VL Single cell analysis PCR analysis of 1 cell / well analysis of larger numbers combinations

microtiter well collection and antigen-specific, authentic VH Flow cytometry retrieving and analysing larger numbers PCR of deflected single cells and VL combinations

ACCEPTED25

Derivation and targets of therapeutic fully human mAbs*

Name Trade name System/Source Target Use

transgenic

Actoxumab mouse Clostridium difficile Clostridium difficile colitis

Rheumatoid/Psoriatic/Juvenile Idiopathic Arthritis, Crohn's

Disease, Plaque Psoriasis, Ankylosing Spondylitis, Hemolytic

Adalimumab Humira phage display TNF-α disease of the newborn

Adecatumumab MT201 phage display EpCAM (CD326) prostate and breast cancer, colorectal liver metastases

transgenic

Alirocumab Praluent mouse PCSK9 hypercholesterolemia

Anetumab

ravtansine BAY 94-9343 phage display MSLN, mesothelin cancer, Mesothelioma

transgenic

Anifrolumab mouse interferon α/β receptor Systemic Lupus Erythematosus

ACCEPTED26

Atinumab mouse RTN4, Anti-IFNAR1 Systemic Lupus Erythematosus

Avelumab MSB0010718C phage display CD274, PD-L1 mesothelioma, Merkel Cell Carcinoma

Benlysta,

Belimumab LymphoStat-B phage display BAFF non-Hodgkin lymphoma etc., systemic lupus

Bertilimumab iCo phage display CCL11 (eotaxin-1) severe allergic disorders, ulcerative colitis

transgenic

Bezlotoxumab mouse Clostridium difficile Clostridium difficile colitis

Bimagrumab BYM338 phage display ACVR2B myostatin inhibitor

transgenic

Brazikumab mouse IL23 Crohn's disease

psoriasis, rheumatoid arthritis, inflammatory bowel diseases,

Briakinumab phage display IL-12, IL-23 multiple sclerosis

transgenic

Brodalumab mouse IL-17 inflammatory diseases

ACCEPTED27

Canakinumab Ilaris mouse IL-1? rheumatoid arthritis

Carlumab CNTO 888 phage display MCP-1 oncology/immune indications, prostate cancer

IGF-1 receptor

Cixutumumab IMC-A12 phage display (CD221) solid tumors, melanoma of the eye, liver

transgenic CD38 (cyclic ADP

Daratumumab DARZALEX® mouse ribose hydrolase) cancer, multiple myeloma

transgenic

Denosumab Prolia mouse RANKL osteoporosis, bone metastases etc.

Drozitumab phage display DR5 cancer

transgenic

Dupilumab mouse IL4 atopic diseases

transgenic interferon gamma-

Eldelumab mouse induced protein Crohn's disease, ulcerative colitis

Elgemtumab phage display ERBB3 (HER3) cancer: carcinoma, breast, gastric

ACCEPTED28

Enoticumab mouse DLL4 solid tumors

transgenic

Evinacumab REGN1500 mouse angiopoietin 3 dyslipidemia

transgenic

Evolocumab mouse PCSK9 hypercholesterolemia

transgenic hepatitis B surface

Exbivirumab mouse antigen hepatitis B

transgenic

Fasinumab mouse HNGF acute sciatic pain, pain due to osteoarthritis, chronic back pain

rabies virus

Foravirumab CL-184 phage display glycoprotein rabies (prophylaxis)

idiopathic pulmonary fibrosis, focal segmental

Fresolimumab GC-1008 phage display TGF-β glomerulosclerosis, cancer, primary brain tumors

Fulranumab transgenic NGF pain

ACCEPTED29

IGF-1 receptor

Ganitumab AMG 479 phage display (CD221) cancer (pancreatic, colorectal, breast)

Gantenerumab phage display beta amyloid Alzheimer's disease

Simponi, transgenic

Golimumab CNTO 148 mouse TNF-α rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis

Guselkumab Stelara phage display IL23 psoriasis

transgenic

Icrucumab IMC-18F1 mouse VEGFR-1 cancer, solid tumors

Imalumab BAX69 phage display MIF colorectal cancer, solid tumors

transgenic

Inclacumab mouse selectin P cardiovascular disease

transgenic

Intetumumab CNTO 95 mouse CD51 solid tumors (prostate cancer, melanoma)

Ipilimumab Yervoy, MDX- transgenic CD152 melanoma

ACCEPTED30

transgenic

Iratumumab MDX-060 mouse CD30 (TNFRSF8) Hodgkin's lymphoma

Lanadelumab DX2930 phage display kallikrein angioedema

Lerdelimumab phage display TGF beta 2 reduction of scarring after glaucoma surgery

Lexatumumab phage display TRAIL-R2 cancer

transgenic

Lirilumab 1-7F9 mouse KIR2D solid and hematological cancers

transgenic multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's

Lucatumumab HCD122 mouse CD40 lymphoma

Mapatumumab phage display TRAIL-R1 cancer, non-Hodgkin lymphoma, liver, cervical

GMCSF receptor α-

Mavrilimumab CAM-3001 phage display chain rheumatoid arthritis

Metelimumab phage display TGF beta 1 systemic scleroderma

Namilumab MT203 phage display CSF2 Granulocyte macrophage-colony stim. factor receptor

ACCEPTED31

Necitumumab Portrazza phage display EGFR nonsmall cell lung carcinoma

transgenic

Nesvacumab REGN910-3 mouse angiopoietin 2 cancer

transgenic

Nivolumab Opdivo mouse PD-1 nonsmall cell, nonsquamous lung cancer

transgenic

Ofatumumab Arzerra mouse CD20 chronic lymphocytic leukemia, lymphomas

Opicinumab BIIB033 phage display LINGO-1 multiple sclerosis

BI-204/

Orticumab MLDL1278A phage display oxLDL Artherosclerotic vascular disease

transgenic

Panitumumab Vectibix mouse EGFR colorectal cancer

transgenic

Patritumab mouse ERBB3 (HER3) cancer, immunomodulator

ACCEPTED32

Radretumab phage display domain-B Hodgkin lymphoma

Cyramza,

Ramucirumab IMC-1121B phage display VEGFR2 solid tumors, breast, metastatic gastric adenocarcinoma

anthrax toxin,

Raxibacumab Abthrax phage display protective antigen anthrax (prophylaxis and treatment)

transgenic

Rilotumumab mouse HGF solid tumors

platelet-derived

transgenic growth factor receptor

Rinucumab REGN2176-3 mouse beta neovascular age-related macular degeneration

transgenic

Sarilumab REGN88 mouse IL6 rheumatoid arthritis, ankylosing spondylitis

transgenic

Secukinumab Cosentyx mouse IL 17A uveitis, rheumatoid arthritis psoriasis

ACCEPTED33

Tanibirumab TTAC-0001 phage display VEGFR-2 advanced cancer

Tarextumab phage display Notch 2/3 receptor pancreatic cancer

transgenic IGF-1 receptor

Teprotumumab mouse (CD221) hematologic tumors

Tesidolumab phage display complement C5 Geographic atropy

Ticilimumab (= transgenic

tremelimumab) CP-675,206 mouse CTLA-4 non small lung cancer

HuMax-TF- transgenic

Tisotumab vedotin ADC) mouse coagulation factor III tumor signaling and angiogenesis

Tralokinumab CAT-354 phage display IL-13 asthma etc., colitis

transgenic growth differentiation

Trevogrumab mouse factor 8 muscle atrophy due to orthopedic disuse and sarcopenia

transgenic

Ulocuplumab mouse CXCR4 (CD184) hematologic malignancies

ACCEPTED34

Ustekinumab Stelara mouse IL-12, IL-23 multiple sclerosis, psoriasis, psoriatic arthritis

Utomilumab phage display 4-1BB (CD137) cancer, solid tumors

Vantictumab OMT- 18R5 phage display Frizzled receptor cancer solid tumors, breast cancer

transgenic

Zalutumumab HuMax-EGFr mouse EGFR squamous cell carcinoma of the head and neck

transgenic

Zanolimumab HuMax-CD4 mouse CD4 rheumatoid arthritis, psoriasis, T cell lymphoma

* https://en.wikipedia.org/wiki/List_of_therapeutic_monoclonal_antibodies

ACCEPTED35