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. 2013 Jan 9;135(1):340-6.
doi: 10.1021/ja309505c. Epub 2012 Dec 21.

Self-assembled antibody multimers through peptide nucleic acid conjugation

Stephanie A Kazane  1 , Jun Y Axup, Chan Hyuk Kim, Mihai Ciobanu, Erik D Wold, Sofia Barluenga, Benjamin A Hutchins, Peter G Schultz, Nicolas Winssinger, Vaughn V Smider
Affiliations

Self-assembled antibody multimers through peptide nucleic acid conjugation

Stephanie A Kazane et al. J Am Chem Soc. .

Abstract

With the recent clinical success of bispecific antibodies, a strategy to rapidly synthesize and evaluate bispecific or higher order multispecific molecules could facilitate the discovery of new therapeutic agents. Here, we show that unnatural amino acids (UAAs) with orthogonal chemical reactivity can be used to generate site-specific antibody-oligonucleotide conjugates. These constructs can then be self-assembled into multimeric complexes with defined composition, valency, and geometry. With this approach, we generated potent bispecific antibodies that recruit cytotoxic T lymphocytes to Her2 and CD20 positive cancer cells, as well as multimeric antibody fragments with enhanced activity. This strategy should accelerate the synthesis and in vitro characterization of antibody constructs with unique specificities and molecular architectures.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

FIGURE 1
FIGURE 1
Construction of self-assembled αHer2 dimers. (A) Fab residues mutated to UAAs for site-specific conjugation (left). These mutations are in the constant region of the light chain (blue) which is paired through a disulfide bond with the heavy chain (red) of the αHer2 Fab (1N8Z). Depiction of oligonucleotide-templated αHer2 homodimer (right, RE=restriction endonuclease site). (B) Site-specific oligonucleotide conjugation and formation of the αHer2 DNA homodimer. An aminooxy-modified single-stranded DNA (ssDNA) was either omitted (lane 1) or coupled to αHer2 K169pAcF Fab (lane 2). The Fabs were analyzed by SDS-PAGE. The conjugate appeared as a lower mobility band (compare lanes 1 and 2) that migrated at a slightly higher molecular weight than the expected molecular weight of the Fab-DNA complex (~60 kDa). Monomeric Fab-ssDNA components with complementary strands were mixed (lane 3) to produce the homodimer which migrates as one band (lane 3) at the calculated molecular weight (~120kD). Treatment with PvuII results in a band with mobility similar to the monomer (lane 4). (C) The remaining five αHer2 heterodimers were created by using different UAA positions (S156, K169 and S202) in the αHer2 Fab. (D) All constructs were tested for phospho-Her2 inhibitory activity on Her2 overexpressing SK-BR-3 breast cancer cells. PBS served as the negative control. RFU indicates relative fluorescence units.
FIGURE 2
FIGURE 2
PNA mediated self-assembly and activity of anti-Her2 bispecific and multimeric antibodies. (A) Structure of the aminooxy-modified PNA linker, where x indicates number of ethylene glycol (EG) units and y indicates number of nucleobases. (B) Schematic depiction of αHer2 PNA dimer (left) and PNA tetramer (right). For the dimer, complementary oligonucleotides A and A’ are coupled to Fabs then mixed to form the dimeric Fab. For the tetramer, complementary oligonucleotides A, B, C, and D allow formation of a cruciform. B. Site-specific conjugation of aminooxy-PNA to αHer2 S202pAcF. The αHer2 S202pAcPhe Fab was left unconjugated (lanes 1 and 3) or conjugated to PNA (lanes 2 and 4) and analyzed by SDS-PAGE, in the absence (lanes 1 and 2) or presence of β-mercaptoethanol (BME) (lanes 3 and 4). C. PNA-mediated αHer2 Fab homodimer and tetramer formation. Each PNA complement was covalently coupled to αHer2 S202pAcF Fab and then hybridized to form either the homodimer (lane 3) or tetramer (lane 4) as seen by SDS-PAGE analysis. D. Phosphorylation inhibition assay of wild-type αHer2 Fab (●), αHer2 IgG (■), αHer2 Fab homodimer (▼), and αHer2 Fab tetramer (▲). ELISA analysis of phosphorylated HER2 levels in SK-BR3 cells using Human Phospho-ErBB2 DuoSet IC kit (R&D Systems).
FIGURE 3
FIGURE 3
Synthesis and activity of αHer2-αCD3 heterodimer. (A) Characterization of the αHer2-αCD3 heterodimer by SDS-PAGE. After purification by size exclusion chromatography, the heterodimer (lane 3) forms one band at the same molecular weight as the αHer2 homodimer (lane 2) on SDS-PAGE. (B) PNA-linked heterodimers mediate T-cell killing of Her2+ tumor cells. Samples were incubated with PBMCs and MDA-MB-435 cells (either Her2+ or Her2-) for 16 hours at 37 °C in RPMI (10% FBS) and cell death measured by LDH release assay. Percent cytotoxicity was determined by maximum killing controls.
FIGURE 4
FIGURE 4
Construction and activity of αCD20 bispecific and tetrameric antibodies. (A) Characterization of the αCD20-αCD3 heterodimer (lane 2) by SDS-PAGE analysis compared to the αCD20 Fab (lane 1). B) PNA-linked heterodimers mediate T-cell killing of CD20+ target cells. The bispecifics were incubated with PBMCs and Ramos cells (10:1) for 16 hours at 37 °C and analyzed by LDH release assay. Percent cytotoxicity was determined by maximum killing controls. (C) Each PNA complement was covalently coupled to αCD20 S202pAcF Fab (lanes 1-4) and hybridized to form the αCD20 tetramer (lane 5). The αCD20 tetramer was purified by size exclusion chromatography and analyzed by SDS-PAGE (lane 6). (D) An αCD20 tetramer induces cell death. The αCD20 tetramer (▲), rituximab IgG (■), or αCD20 Fab (●) were incubated with Ramos cells (105 cells per well) for 48 hours at 37 °C in RPMI (10% FBS) and analyzed by LDH release assay. RAU indicates relative absorbance units.
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