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Isolation and characterization of camelid single-domain antibodies against HER2
BMC Research Notes volume 11, Article number: 866 (2018)
To isolate and characterize novel high-affinity llama single-domain antibodies against human HER2.
We immunized a llama with human HER2, constructed a phage-displayed VHH library from the lymphocytes of the animal, and isolated six unique HER2-specific VHHs by panning. All six VHHs were unique at the amino acid level and were clonally unrelated, as reflected by their distinct CDR3 lengths. All six VHHs recognized recombinant human HER2 ectodomain with monovalent affinities ranging from 1 to 51 nM, had comparable affinities for cynomolgus monkey HER2, and bound HER2+ SKOV3 cells by flow cytometry. Three of the VHHs recognized recombinant murine HER2 with no loss of affinity compared with human and cynomolgus monkey HER2. The VHHs recognized three major epitopes on HER2 (including one conserved across the human, simian and murine orthologues), all of which were distinct from that of trastuzumab. These VHHs may be useful in the design of modular cancer immunotherapeutics.
HER2/neu (ERBB2, CD340) is a receptor tyrosine kinase of the epidermal growth factor receptor family that is frequently amplified and/or overexpressed in solid tumors . Antibodies (Abs) and Ab-drug conjugates (ADCs) against HER2, exemplified by trastuzumab (Herceptin), pertuzumab (Perjeta) and trastuzumab emtansine (Kadcyla), play an important role in the diagnosis and treatment of breast cancer . Multiple other anti-HER2 Abs and Ab fragments are in development as naked antibodies, ADCs, bispecific Abs and radioimmunotherapeutics for breast cancer and other indications . A camelid single-domain Ab (sdAb or VHH) against HER2 is currently in clinical trials for breast cancer imaging .
Here, we report the generation and preliminary characterization of a panel of novel llama VHHs directed against human HER2. Several of the VHHs have attractive properties that may make them useful components of modular cancer immunotherapeutics.
We immunized a male llama (Lama glama) with recombinant human HER2 ectodomain (Cat. No. HE2-H5225; ACROBiosystems, Beijing, China) as previously described [5,6,7]. Briefly, the animal was immunized subcutaneously five times with 200 µg of human HER2 (days 0, 21, 28, 35 and 42). The priming immunization was adjuvanted with complete Freund’s adjuvant and boost immunizations were adjuvanted with incomplete Freund’s adjuvant. Blood samples were collected on days 35 and 49, from which serum was obtained after clotting and peripheral blood mononuclear cells were purified by density gradient centrifugation. Interestingly, serum ELISA and western blotting indicated that although HER2 immunization elicited polyclonal Abs against the immunizing antigen, immune sera from unrelated animals showed similar degrees of HER2 reactivity (see Additional file 1). We speculate that serum polyreactivity against this recombinant HER2 ectodomain reflects some degree of unfolding and/or aggregation, although binding by trastuzumab and other antibodies indicated that some proportion was also folded correctly.
We constructed a phage-displayed VHH library from the peripheral blood lymphocytes of the HER2-immunized llama as previously described [5,6,7]. Briefly, total RNA was extracted in 16 replicates from peripheral blood mononuclear cells (eight samples each from the day 35 and 49 bleeds, each containing ~ 1 × 107 cells) using the PureLink™ RNA Mini Kit (Thermo Fisher, Waltham, MA). Approximately 1–2 µg of total RNA was reverse transcribed using qScript® cDNA SuperMix (Quantabio, Beverly, MA) and then rearranged VHH exons were amplified using semi-nested PCR and cloned into the pMED1 phagemid vector. The final library size was ~ 8 × 107 independent transformants with an insert rate of ~ 92%. VHH-displaying phages were rescued from library phagemid-bearing Escherichia coli TG1 cells using M13KO7 helper phage (New England Biolabs, Ipswich, MA), panned for four rounds against human HER2 directly immobilized in wells of microtiter plates, and eluted with triethylamine as previously described [5,6,7]. At the conclusion of four rounds of panning, 96 individual clones (48 each from rounds 3 and 4) were tested for binding to human HER2 by ELISA, yielding six unique and clonally unrelated VHH sequences (Table 1).
The DNA sequences encoding the six VHHs were cloned into the pSJF2H expression vector . C-terminally c-Myc- and His6-tagged VHHs were expressed in 200 mL overnight cultures of E. coli TG1 under IPTG induction and purified by Ni2+ affinity chromatography as previously described [5,6,7]. All six VHHs were primarily monomeric by size exclusion chromatography, although trace aggregates or impurities were observed for NRC-sdAb035 and NRC-sdAb037 (Fig. 1a). We immobilized human (ACROBiosystems HE2-H5225), cynomolgus (ACROBiosystems HE2-C52Hb) and murine HER2 (ACROBiosystems ER2-M5220) ectodomains on adjacent flow cells of a CM5 Series S sensor chip (GE Healthcare, Piscataway, NJ) by amine coupling and analyzed binding of the VHHs to each surface using single-cycle kinetics on a Biacore T200 surface plasmon resonance (SPR) instrument (GE Healthcare). All six VHHs showed high-affinity binding to HER2 (KD range 1–51 nM), with nearly equivalent kinetic and affinity parameters observed for human and cynomolgus HER2 (Fig. 1b, Table 1 and Additional file 2); moreover, three of the VHHs also cross-reacted with murine HER2 with no apparent loss of binding affinity. All six VHHs bound to HER2+ SKOV3 cells by flow cytometry, although staining by NRC-sdAb034 was weak (Fig. 1c). Epitope binning experiments indicated that despite their unique amino acid sequences, all three cross-reactive VHHs (NRC-sdAb034, NRC-sdAb036 and NRC-sdAb038) targeted a nearly identical epitope (Fig. 1d and Additional file 3); the epitopes of NRC-sdAb037 and NRC-sdAb039 also showed a high degree of overlap, while NRC-sdAb035’s epitope was distinct. The epitopes of all six VHHs were distinct from the trastuzumab epitope.
In summary, we have reported the isolation and preliminary characterization of six novel anti-HER2 llama VHHs. Interestingly, despite their unique and clonally unrelated sequences, the VHHs targeted only three major epitopes on HER2, including an apparently immunodominant epitope conserved across the human, simian and murine orthologues. The nearly identical binding of the VHHs to cynomolgus HER2 would permit toxicity assessment and, for mouse cross-reactive VHHs, evaluation in syngeneic tumor models in combination with immunomodulatory agents. Several other groups have described anti-HER2 VHHs and characterized them in some detail , but in most cases cross-reactivity with murine HER2 was not assessed. Moreover, targeting of a non-trastuzumab epitope is clearly an advantage for imaging of patients being dosed with Herceptin/Kadcyla . The other major potential advantage of these VHHs in comparison with conventional antibodies is their modularity, which would permit facile incorporation into multifunctional biologics.
At the current time, we have not been able to explore whether these VHHs have anti-cancer activity. We do not know: (i) the precise locations of their epitopes on HER2, (ii) whether any of the VHHs inhibit HER2 signaling or inhibit receptor dimerization, permitting their development as naked Ab therapeutics, (iii) whether any of the VHHs internalize into HER2+ tumor cells, permitting their development as ADCs or radioimmunotherapeutics, or (iv) whether their sequences can be humanized without loss of stability or binding affinity. Moreover, we are unable to disclose the amino acid sequences of these VHHs for intellectual property reasons. In future studies, we hope to comprehensively investigate the roles of molecular size, valency and serum half-life on tumor uptake using anti-HER2 VHH-based biologics as a model system.
- sdAb or VHH:
surface plasmon resonance
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KAH designed the study, KAH and MJL isolated the VHHs, SR and GH performed and analyzed SPR experiments. All authors read and approved the final manuscript.
We gratefully acknowledge Qingling Yang for technical assistance.
The authors declare that have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its additional files.
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Experiments involving animals were conducted using protocols approved by the National Research Council Canada Animal Care Committee and in accordance with the guidelines set out in the OMAFRA Animals for Research Act, R.S.O. 1990, c. A.22.
This work was funded by the National Research Council Canada.
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Hussack, G., Raphael, S., Lowden, M.J. et al. Isolation and characterization of camelid single-domain antibodies against HER2. BMC Res Notes 11, 866 (2018) doi:10.1186/s13104-018-3955-8
- Single-domain antibody