Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity Koen Wagnera,1, Mark J. Kwakkenbosa,1, Yvonne B. Claassena, Kelly Maijoora, Martino Böhnea, Koenraad F. van der Sluijsb, Martin D. Wittec,2, Diana J. van Zoelend, Lisette A. Cornelissend, Tim Beaumonta, Arjen Q. Bakkera, Hidde L. Ploeghc, and Hergen Spitsa,3 aAIMM Therapeutics, 1105 BA Amsterdam, The Netherlands; bLaboratory of Experimental Intensive Care and Anesthesiology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands; cWhitehead Institute for Biomedical Research, Cambridge, MA 02142; and dDepartment of Virology, Central Veterinary Institute, Wageningen University and Research Centre, 8200 AB Lelystad, The Netherlands Edited by K. Christopher Garcia, Stanford University, Stanford, CA, and approved October 21, 2014 (received for review May 9, 2014) Bispecific antibodies have therapeutic potential by expanding the Here, we present a bispecific antibody format, in which two functions of conventional antibodies. Many different formats of antibodies are fused at their C termini, using a combination of bispecific antibodies have meanwhile been developed. Most are sortase transpeptidation and click chemistry (20), to create an IgG genetic modifications of the antibody backbone to facilitate heterodimer. This C-C fusion does not require mutations within the incorporation of two different variable domains into a single antibody constant domains that might interfere with Fc-receptor molecule. Here, we present a bispecific format where we have binding or that would compromise antibody stability. Thus, the fused two full-sized IgG antibodies via their C termini using sor- native antibody structure is fully retained in our format. tase transpeptidation and click chemistry to create a covalently C-to-C fusion is a two-step process (Fig. 1), using a combina- linked IgG antibody heterodimer. By linking two potent anti- tion of sortase transpeptidation and click chemistry (20). Sortase influenza A antibodies together, we have generated a full anti- is a bacterial enzyme that functions to attach cell surface proteins body dimer with bispecific activity that retains the activity and bearing an “LPXTG” motif to the cell wall of Gram-positive bac- stability of the two fusion partners. teria via transacylation (21, 22). Sortase-catalyzed transpeptidation allows for efficient site-specific modifications under physiological antibody engineering | immunotherapy | influenza conditions, with excellent specificity and near-quantitative yields INFLAMMATION IMMUNOLOGY AND (23–25). To facilitate site-specific linking of the C termini of two ith a steady increase of antibodies and antibody derivatives antibodies, the fusion partners are labeled with either an azide or Wsuch as antibody drug conjugates and bispecific antibodies a cyclooctyne (DIBAC) functional group. The modified proteins entering the clinic, monoclonal human antibodies are now an are then conjugated via a strain-promoted cycloaddition between established source of new therapeutic agents (1, 2). The development the azide and the cyclooctyne. This reaction is highly specific and of bispecific antibodies has generated particular interest, because readily proceeds at room temperature in aqueous environments at it allows expansion of basic antibody functions (3, 4). Through neutral pH (26), allowing for efficient fusion under mild conditions. binding two (or more) different targets, a bispecific antibody can To test the robustness of this process and determine the features simultaneously engage two epitopes of a disease agent, block/acti- of this bispecific antibody format, we fused two potent anti- influenza antibodies, each active against a different subgroup of vate multiple ligands/receptors at once, or recruit immune effector the influenza A virus. Based on the hemagglutinin (HA) protein cells (i.e., T cells or B cells) to a specific (tumor) site (5). There is a growing interest in bispecific antibodies with anticancer proper- ties, which has led to an increase in bispecifics that have entered Significance preclinical testing (5, 6). Bispecific antibodies with defined functions are generated by Bispecific antibodies expand the function of conventional anti- means of genetic or biochemical engineering. Many different bodies. However, therapeutic application of bispecifics is ham- methods exist to engineer immunoglobulins, with more than 45 pered by the reduced physiochemical stability of such molecules. bispecific antibody formats at last count (reviewed in ref. 5). We present a format for bispecific antibodies, fusing two full- These bispecific antibody formats fall into three broad subclasses sized antibodies via their C termini. This format does not require mutations in the antibody constant domains beyond installation (5): (i) single-chain double variable domain formats (50–100 of a five-residue tag, ensuring that the native antibody structure kDa) (7–9): Generally these bispecifics consist of multiple vari- ii is fully retained in the bispecific product. We have validated the able domains that are connected via peptide linkers. ( ) IgG with approach by linking two anti-influenza A antibodies, each active multiple variable domains: In this type of bispecific antibody, against a different subgroup of the virus. The bispecific antibody a second variable domain is genetically linked to any desirable dimer retains the activity and the stability of the two original position in the IgG molecule (i.e., the C or N terminus of either antibodies. the IgG heavy or light chain) (10–12). (iii) Asymmetric IgG molecules: In an asymmetric IgG antibody, two different vari- Author contributions: K.W., M.J.K., T.B., H.L.P., and H.S. designed research; K.W., M.J.K., able domains are incorporated into a single, asymmetric, anti- Y.B.C., K.M., M.B., and A.Q.B. performed research; K.F.v.d.S., M.D.W., D.J.v.Z., and L.A.C. contributed new reagents/analytic tools; K.W., M.J.K., and H.S. analyzed data; and K.W., body molecule via heterodimerization of the constant domains. M.J.K., H.L.P., and H.S. wrote the paper. Heterodimerization may be achieved through engineering the – Conflict of interest statement: K.W., M.J.K., Y.B.C., K.M., M.B., T.B., A.Q.B., and H.S. are CH3domain(13 16) or the hinge region of the antibody (17, employees of AIMM Therapeutics. 18). Depending on the engineering method, asymmetric IgGs This article is a PNAS Direct Submission. can be made with a common light chain or with two different 1K.W. and M.J.K. contributed equally to this work. light chains (19). 2Present address: Bio-Organic Chemistry, Stratingh Institute for Chemistry, University of Each of these formats has its specific advantages and draw- Groningen, 9747 AG Groningen, The Netherlands. backs. Most of the limitations arise from the fact that their for- 3To whom correspondence should be addressed. Email: [email protected]. mats deviate significantly from the natural, highly stable, IgG This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. structure, which compromises stability and ease of manufacture. 1073/pnas.1408605111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1408605111 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 this antibody binds full-length H5 HA protein and the HA2 subunit. The subunits of HA are described in SI Appendix, Table S2. AT10-005 contains the IGHV1-69 gene segment and harbors the hydrophobic signature commonly found in group 1-specific antibodies (SI Appendix,TableS3) (38, 39). Antibody competition using AT10-005 and the stem-binding antibody CR6261 (40) (SI Appendix,Fig.S1) for H1 binding on H1N1 (A/Hawaii/31/2007)- infected cells confirms that both antibodies bind similar regions. AT10-002 is specific for HA proteins of group 2 viruses (SI Appendix, Table S1) and shows neutralizing activity against four group 2 viruses (two H3N2, one H7N1, and one H7N7 virus) (Table 1). The antibody binds full-length H3 but not the separate HA1 portion (SI Appendix, Table S4). In addition, AT10-002 competes with the group 2 HA stem-specific antibody CR8020 (31) for binding to H3N2-infected cells (SI Appendix, Fig. S2). To further analyze the binding site of AT10-002, we have isolated a third HA-specific antibody: AT10-003. AT10-003 was found to – Fig. 1. Approach for synthesis of C-to-C fused antibodies. (A) Antibodies bind to three H3 viruses (SI Appendix, Table S5), and reacted are labeled at the C terminus either with an azide (N ) or DIBAC with a click 3 with both the full-length H3 and the HA1 portion of the mole- peptide by using sortase. (B) Click-labeled antibodies are fused via the click SI reaction. cule indicating that the HA head is sufficient for binding ( Appendix, Table S4). Notably, AT10-003 was unable to block binding of AT10-002 to H3N2-infected cells (SI Appendix, Fig. sequence, there are 18 different subtypes of influenza A, divided S2). Therefore, the finding that AT10-002 binding is blocked by into two subgroups (27, 28). The HA protein is the common target CR8020 and not by AT10-003 suggests that the stem region of of almost all neutralizing antibodies, and several antibodies with group 2 HA influenza has the largest contribution to the AT10- broadly neutralizing activity between influenza A subtypes in the 002 epitope. Linking the broadly reacting antibodies AT10-005 same group exist (29–33). Combining two such potent subgroup- and AT10-002 would potentially result in a molecule active specific antibodies may result in an IgG heterodimer with even against a broad spectrum of group 1 and group 2 influenza A broader anti-influenza A activity. This type of molecule would viruses. have therapeutic relevance in a passive immunization setting, Synthesis of the C-to-C Fused Bispecific Antibody Dimer. To enable because influenza viruses continue to cause significant morbidity the C-to-C protein fusion, we modified the C termini of the and mortality, despite efforts to contain them with seasonal heavy chains of both antiinfluenza antibodies with a small tag, vaccines (34).