Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity

Significance

Bispecific antibodies expand the function of conventional antibodies. However, therapeutic application of bispecifics is hampered by the reduced physiochemical stability of such molecules. We present a format for bispecific antibodies, fusing two full-sized antibodies via their C termini. This format does not require mutations in the antibody constant domains beyond installation of a five-residue tag, ensuring that the native antibody structure is fully retained in the bispecific product. We have validated the approach by linking two anti-influenza A antibodies, each active against a different subgroup of the virus. The bispecific antibody dimer retains the activity and the stability of the two original antibodies.

Abstract

Bispecific antibodies have therapeutic potential by expanding the functions of conventional antibodies. Many different formats of bispecific antibodies have meanwhile been developed. Most are genetic modifications of the antibody backbone to facilitate incorporation of two different variable domains into a single molecule. Here, we present a bispecific format where we have fused two full-sized IgG antibodies via their C termini using sortase transpeptidation and click chemistry to create a covalently linked IgG antibody heterodimer. By linking two potent anti-influenza A antibodies together, we have generated a full antibody dimer with bispecific activity that retains the activity and stability of the two fusion partners.

With a steady increase of antibodies and antibody derivatives such as antibody drug conjugates and bispecific antibodies entering the clinic, monoclonal human antibodies are now an established source of new therapeutic agents (1, 2). The development of bispecific antibodies has generated particular interest, because it allows expansion of basic antibody functions (3, 4). Through binding two (or more) different targets, a bispecific antibody can simultaneously engage two epitopes of a disease agent, block/activate multiple ligands/receptors at once, or recruit immune effector cells (i.e., T cells or B cells) to a specific (tumor) site (5). There is a growing interest in bispecific antibodies with anticancer properties, which has led to an increase in bispecifics that have entered preclinical testing (5, 6).

Bispecific antibodies with defined functions are generated by means of genetic or biochemical engineering. Many different methods exist to engineer immunoglobulins, with more than 45 bispecific antibody formats at last count (reviewed in ref. 5). These bispecific antibody formats fall into three broad subclasses (5): (i) single-chain double variable domain formats (50–100 kDa) (7–9): Generally these bispecifics consist of multiple variable domains that are connected via peptide linkers. (ii) IgG with multiple variable domains: In this type of bispecific antibody, a second variable domain is genetically linked to any desirable position in the IgG molecule (i.e., the C or N terminus of either the IgG heavy or light chain) (10–12). (iii) Asymmetric IgG molecules: In an asymmetric IgG antibody, two different variable domains are incorporated into a single, asymmetric, antibody molecule via heterodimerization of the constant domains. Heterodimerization may be achieved through engineering the C_H3 domain (13–16) or the hinge region of the antibody (17, 18). Depending on the engineering method, asymmetric IgGs can be made with a common light chain or with two different light chains (19).

Each of these formats has its specific advantages and drawbacks. Most of the limitations arise from the fact that their formats deviate significantly from the natural, highly stable, IgG structure, which compromises stability and ease of manufacture.

Here, we present a bispecific antibody format, in which two antibodies are fused at their C termini, using a combination of sortase transpeptidation and click chemistry (20), to create an IgG heterodimer. This C-C fusion does not require mutations within the antibody constant domains that might interfere with Fc-receptor binding or that would compromise antibody stability. Thus, the native antibody structure is fully retained in our format.

C-to-C fusion is a two-step process (Fig. 1), using a combination of sortase transpeptidation and click chemistry (20). Sortase is a bacterial enzyme that functions to attach cell surface proteins bearing an “LPXTG” motif to the cell wall of Gram-positive bacteria via transacylation (21, 22). Sortase-catalyzed transpeptidation allows for efficient site-specific modifications under physiological conditions, with excellent specificity and near-quantitative yields (23–25). To facilitate site-specific linking of the C termini of two antibodies, the fusion partners are labeled with either an azide or a cyclooctyne (DIBAC) functional group. The modified proteins are then conjugated via a strain-promoted cycloaddition between the azide and the cyclooctyne. This reaction is highly specific and readily proceeds at room temperature in aqueous environments at neutral pH (26), allowing for efficient fusion under mild conditions.

Fig. 1.

Approach for synthesis of C-to-C–fused antibodies. (A) Antibodies are labeled at the C terminus either with an azide (N₃) or DIBAC with a click peptide by using sortase. (B) Click-labeled antibodies are fused via the click reaction.

To test the robustness of this process and determine the features of this bispecific antibody format, we fused two potent anti-influenza antibodies, each active against a different subgroup of the influenza A virus. Based on the hemagglutinin (HA) protein sequence, there are 18 different subtypes of influenza A, divided into two subgroups (27, 28). The HA protein is the common target of almost all neutralizing antibodies, and several antibodies with broadly neutralizing activity between influenza A subtypes in the same group exist (29–33). Combining two such potent subgroup-specific antibodies may result in an IgG heterodimer with even broader anti-influenza A activity. This type of molecule would have therapeutic relevance in a passive immunization setting, because influenza viruses continue to cause significant morbidity and mortality, despite efforts to contain them with seasonal vaccines (34). Because these vaccines are typically only effective against the specific seasonal viral strain, there is an urgent unmet medical need for new treatments active against multiple subtypes of the influenza virus (35).

Results

Isolation and Characterization of Potent Antiinfluenza Antibodies.

To obtain broadly neutralizing influenza A antibodies, we isolated memory B cells from influenza-vaccinated individuals. These B cells were transduced with human Bcl6 and Bcl-xL as described in Kwakkenbos et al. (36, 37) and screened for HA binding. B cells that recognized HA molecules from multiple influenza subtypes were cloned and the antibody derived from them produced in 293T HEK cells. This approach resulted in the identification of two broadly neutralizing antibodies: AT10-002 and AT10-005.

AT10-005 neutralizes two H1N1 and one H5N1 virus (Table 1) and binds four additional group 1 viruses (two H1N1, one H5N1, and one H9N2) (SI Appendix, Table S1). In an ELISA, 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, Table S3) (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.

Table 1.

Neutralizing activity of AT10-002 and AT10-005

Group	Virus	AT10-002	AT10-005
1	H1N1 (A/Hawaii/31/2007)	—	1.29 (± 0.53)
1	H1N1 (A/Neth./602/2009)	—	16.8 (± 1.8)
1	H5N1 (A/Turkey/Turkey/04)	—	8.2 (± 6.1)
2	H3N2 (A/Neth./177/2008)	0.76 (± 0.27)	—
2	H3N2 (A/Swine/St. Oedenrode/1996)	1.79 (± 0.35)	—
2	H7N1 (A/Chicken/Italy/1067/99)	22.3 (± 8.7)	—
2	H7N7 (A/Chicken/Neth./621557/03)	0.78 (± 0.36)	—

Shown are IC₅₀ values in nanomolars. —, no neutralizing activity detected. SI Appendix, Table S1 shows a complete overview of the reactivity of AT10-002 and AT10-005. Data represent mean ± SD of at least two independent experiments.

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 bind to three H3 viruses (SI Appendix, Table S5), and reacted with both the full-length H3 and the HA1 portion of the molecule indicating that the HA head is sufficient for binding (SI Appendix, Table S4). Notably, AT10-003 was unable to block binding of AT10-002 to H3N2-infected cells (SI Appendix, Fig. S2). Therefore, the finding that AT10-002 binding is blocked by CR8020 and not by AT10-003 suggests that the stem region of group 2 HA influenza has the largest contribution to the AT10-002 epitope. Linking the broadly reacting antibodies AT10-005 and AT10-002 would potentially result in a molecule active against a broad spectrum of group 1 and group 2 influenza A viruses.

Synthesis of the C-to-C Fused Bispecific Antibody Dimer.

To enable the C-to-C protein fusion, we modified the C termini of the heavy chains of both antiinfluenza antibodies with a small tag, consisting of a GGGGS (G4S) linker sequence, followed by the sortase recognition site LPETGG and a His6 tag. The His tag is used to monitor the sortase labeling reaction, because it will be removed upon sortase-catalyzed transpeptidation. Triglycine peptides containing either an azide or a DIBAC moiety were synthesized as described (20). We chose to label AT10-002 with DIBAC and AT10-005 with azide. The extent of sortase labeling was monitored by using a fluorescent azide-containing nucleophile (GGG-TAMRA-azide): We observed excellent labeling with the azide-containing probe after 4 h at 41 °C in 25 mM Tris buffer, pH 8.0 and 150 mM NaCl (Fig. 2A). For the TAMRA-DIBAC-modified peptide, sortase labeling is somewhat less efficient (Fig. 2B). We attribute this difference to the nonspecific thiol-yne coupling reaction between DIBAC and free thiol groups (41), such as the unpaired cysteine residues in the antibody or in sortase (Fig. 2A). Because the active site of sortase contains a free thiol (42), sortase itself would be particularly vulnerable to the thiol-yne reaction (43). Nonetheless, we observed efficient installation of DIBAC after 4-h incubation at 33 °C, in 25 mM Tris buffer, pH 6.8 and 150 mM NaCl, demonstrating that a lower incubation temperature and pH reduces thiol-yne coupling.

Fig. 2.

Preparation of BiFlu. (A) Determining optimal reaction conditions for sortase labeling. ST-tagged antibody AT10-005-ST (1.0 μM) is mixed with sortase (2.0 μM) and GGG-TAMRA-azide or GGG-TAMRA-DIBAC (125 μM), in labeling buffer (25 mM Tris⋅HCl, 150 mM NaCl, and 10 mM CaCl₂) and incubated for 6 h at the indicated pH and temperature. C, control reaction (incubation with GGG-TAMRA-DIBAC at 37 °C, pH 7.5). After incubation, the reaction mixture is analyzed with reducing fluorescent SDS/PAGE (λ_ex, 532 nm; λ_em, 580 nm). (B) Quantification of sortase-labeling results. Fluorescence is measured as relative to labeling with GGG-TAMRA-azide at 37 °C, pH 7.5 (set at 1.00 RU). (C) Gel filtration chromatogram of click-reaction products. AT10-002-DIBAC (5.0 μM) is mixed with AT10-005-aizde (5.0 μM). After the indicated time, a sample is analyzed with gel filtration chromatography (column volume: 120 mL). (D) Coomassie-stained SDS/PAGE gel of purified BiFlu (1.5 μg). (E) Anti-His Western blot of purified BiFlu (0.5 μg). HC, antibody heavy chain; LC, antibody light chain; M, Dual Color Protein Standard (Bio-Rad); S, sortase. Products of thiol-yne coupling are indicated with asterisks.

Following sortase labeling, the antibodies were separated from sortase, excess triglycine peptide, and other reaction products by size exclusion chromatography. AT10-002–DIBAC was readily coupled to AT10-005–azide at 20 °C. Antibody dimers were resolved from any remaining antibody monomers by size exclusion chromatography (Fig. 2C). After prolonged incubation of click-labeled antibodies, a second peak was visible in the elution profile (Fig. 2C). This second peak consists of higher order antibody oligomers that may form when the two click-linked C termini of one antibody connect with two different antibodies (as shown in Fig. 2D). Formation of higher-order complexes also occurred when the click reactions were performed at higher temperatures or at a higher antibody concentration, suggesting that this reaction requires careful control by choice of temperature and antibody concentration, to obtain the optimal yield of the click-linked antibody dimer.

We obtained pure and fully intact click-linked antibody dimers as judged from analysis by SDS/PAGE (Fig. 2D). The presence of a ∼100-kDa polypeptide (100 kDa equals twice the size of the antibody heavy chain) in the reduced dimer sample (Fig. 2D) confirms covalent fusion at the C termini of the heavy chains. The reduced dimer samples also showed some monomeric heavy chains (Fig. 2D). The presence of these monomeric heavy chains upon SDS/PAGE of the dimer sample demonstrates that not all antibody heavy chains have fused. This finding is not unexpected, because a single covalent HC-HC link suffices to generate an antibody dimer. This single HC-HC linkage is not due to incomplete sortase labeling; anti-His Western blotting demonstrates a near-complete loss of the His tag in the purified dimer (Fig. 2E). Apparently, after the first HC-HC linkage, a second click coupling between the two remaining modified HC C termini is disfavored, presumably due to steric hindrance.

BiFlu Retains Structural Stability and Fc-Receptor Binding.

To ensure that the C-to-C fusion of two antibodies does not compromise the stability of the resulting structure, we assessed the stability of our antibody dimer (BiFlu) by means of dynamic scanning fluorescence (DSF).

The DSF curves (Fig. 3A) showed that the melting properties of BiFlu are adequately described by summation of the curves of the two individual antibodies. Thermal stability of the two antibodies is therefore unchanged when they are linked via their C termini. In view of its small size (24 aa + the click product) relative to the mass of BiFlu (±320 kDa), the linker sequence that connects the two antibodies contributes little if at all to the DSF curve of BiFlu.

Fig. 3.

Stability of the BiFlu antibody dimer. (A) DSF curves of BiFlu and monomeric IgG (25 μg/mL) in PBS buffer. (B) Long-term stability test of BiFlu and monomeric IgG in PBS buffer. BiFlu or monomeric IgG (AT10-002 or AT10-005) is diluted into PBS buffer (final concentration: 250 μg/mL) and incubated at 37 °C. After the indicated number of days, 2.5 μg of antibody is heated for 10 min at 55 °C (the lower temperature is used to minimize antibody breakdown) and analyzed with SDS/PAGE. (C) Long-term stability test of BiFlu and monomeric IgG in IgG-depleted human serum. BiFlu or monomeric IgG (mixture of AT10-002 and AT10-005) is diluted into IgG-depleted human serum (final concentration: 250 μg/mL) and incubated at 37 °C. After the indicated time, 0.25 μg of antibody (+ serum) is heated for 10 min at 55 °C and analyzed with anti-IgG HC Western blotting. In B and C, BiFlu and monomeric IgG are not fully denatured; therefore, they run at a lower molecular mass than expected (IgG molecular mass: 160 kDa).

We further assessed the stability of the antibody dimer over a more extended observation window. After 3 wk of incubation at 37 °C in PBS (Fig. 3B) or in PBS plus (antibody-depleted) human serum (Fig. 3C), the majority of BiFlu remains intact, suggesting that the antibody dimer would also be stable at physiological conditions. Only a small amount of BiFlu (<< 10%) has decomposed into the separate IgG monomers. The covalent link between the two C termini in the antibody dimer is stable under these conditions.

In this bispecific full antibody dimer format, the two antibodies are linked via the C termini of the heavy chain. Because the Fc portion contains the binding sites for the IgG Fc receptors (FcγRs) and the neonatal Fc receptor (FcRn) (44), we verified that such a fusion did not impair interaction of the antibody dimer with Fc receptors. We measured binding of BiFlu to soluble Fc receptors by ELISA and found that BiFlu binds FcRn and all three Fcγ receptors with similar affinity as the parental IgGs (SI Appendix, Fig. S3). We also tested binding of BiFlu to THP-1 cells, which express Fc receptors on their cell surface (45), and observed equivalent binding of BiFlu and the parental antibodies (SI Appendix, Fig. S3).

BiFlu retains FcR-binding activity, implying that it should be capable of engaging FcR effector functions. If in vivo neutralization of virus by the single antibodies were to somehow involve engagement with Fc receptors, then the bispecific antibody retains functionality for this parameter as well.

BiFlu Retains Functional Activity and Neutralizes Influenza A.

Having determined stability and receptor binding of BiFlu, we measured its functional activity. The binding of BiFlu to HA proteins was tested with capture surface plasmon resonance (SPR), using either heavy or light chain-specific anti-IgG antibodies, followed by subsequent injections with H1 or H3 HA protein. H1 HA represents group 1 Influenza A viruses and should be detected by BiFlu component AT10-005; H3 is a representative of group 2 Influenza A and should be recognized by BiFlu component AT10-002 (Fig. 4A).

Fig. 4.

Capture SPR analysis of HA binding. (A) HA binding of BiFlu, AT10-002, and AT10-005, immobilized on an anti-HC–coated spot. (B) HA binding of BiFlu, AT10-002, and AT10-005, immobilized on an anti-lambda light-chain coated spot. First, antibody is injected, followed by HA antigens (H3, then H1), and then anti-light chain antibody (either anti-kappa or anti-lambda). Because BiFlu is twice as large as IgG, BiFlu gives a greater capture response when binding to the capture antibody (anti-HC or anti-lambda). Kinetic constants are shown in Table 2.

When immobilized via a heavy chain-specific antibody, BiFlu bound both H1 and H3 HA with similar, low picomolar (K_D = ± 15 pM for both antigens) affinity, as did the parental antibodies (Fig. 4A and Table 2). In the same setup, we determined binding to H7 and H9 HA protein, finding that BiFlu binds these antigens with an affinity similar to that of the parental antibodies (Table 2).

Table 2.

Kinetic constants for HA binding

Antibody	ka	kd	KD
H1 (A/New Caledonia/20/1999)
AT10-002	—	—	—
AT10-005	19.5 (± 1.8)	0.29 (± 0.10)	15.0 (± 5.8)
BiFlu	21.4 (± 2.8)	0.34 (± 0.17)	16.2 (± 8.4)
H3 (A/Wyoming/03/2003)
AT10-002	19.2 (± 1.5)	0.23 (± 0.08)	11.8 (± 5.9)
AT10-005	—	—	—
BiFlu	22.2 (± 2.5)	0.33 (± 0.16)	14.7 (± 9.2)
H7 (A/Netherlands/219/03)
AT10-002	0.59 (± 0.15)	0.25 (± 0.05)	441 (± 109)
AT10-005	—	—	—
BiFlu	0.57 (± 0.12)	0.21 (± 0.01)	375 (± 88)
H9 (A/Hong Kong/1073/1999)
AT10-002	—	—	—
AT10-005	0.81 (± 0.06)	21.6 (± 0.1)	27,000 (± 2,000)
BiFlu	0.94 (± 0.02)	27.1 (± 0.2)	29,000 (± 1,000)

k_a in 10⁴ sec⁻¹⋅M⁻¹, k_d in 10⁻⁵ sec⁻¹, K_D in picomolars; —, no binding detected. Data represent mean ± SD of at least two independent experiments. SPR curves and fits are shown in SI Appendix, Figs. S4 (H1 + H3 binding), S5 (H7), and S6 (H9).

When using anti-light chain antibodies for immobilization, BiFlu could be captured on both anti-kappa and anti-lambda, because AT10-002 and AT10-005 have different light chains (lambda and kappa, respectively). BiFlu captured via its light chains binds both H1 and H3 HA (Fig. 4B), demonstrating that BiFlu is a bivalent heterodimer. The fact that light chain-captured BiFlu binds both antigens with similar kinetics as the single antibodies (here: AT10-002 + H3) indicates that, in the BiFlu sample, the two antibodies are linked in a 1:1 ratio and that homodimers must be absent. We estimate that BiFlu contains > 95% heterodimers.

We then tested the in vitro neutralization activity of BiFlu against H3N2 and H1N1. BiFlu neutralized both strains efficiently (Fig. 5) with an IC₅₀ value of ∼1.0 nM, similar to the two single antibodies tested separately (Table 3). We then tested the in vivo activity of our antibodies in a murine H1N1 (A/PR/8/1934) challenge model. Prophylactic administration of 1 mg/kg AT10-005 protected the mice against lethal infection, all mice pretreated with AT10-002 or rituximab lost more then 25% of their body weight and were killed by day 8 (SI Appendix, Fig. S7). We examined the in vivo activity of BiFlu in the same challenge model, injecting mice with either BiFlu (2 mg/kg) or a mixture of AT10-005 and AT10-002 (1 mg/kg each). All BiFlu-treated mice were protected against H1N1 challenge, similar to the AT10-002 + AT10-005–treated mice, whereas 50% of the mice treated with rituximab failed to recover from the infection (Fig. 6A) [the control group survival is different from the first experiment (SI Appendix, Fig. S7); we attribute this result to variation in the model]. Mice injected with BiFlu or AT10-002 + AT10-005 also showed a significantly lower body weight loss compared with the control group (Fig. 6B).

Fig. 5.

Virus neutralization assays. (A) Neutralization of influenza A H1N1 virus (strain: A/Hawaii/31/2007). (B) Neutralization of influenza A H3N2 virus (strain: A/Netherlands/177/2008). Data represent mean ± SD of at least two independent experiments.

Table 3.

IC₅₀ values for influenza neutralization

Virus	AT10-002	AT10-005	BiFlu
H1N1 (A/Hawaii/31/2007)	—	1.29 (± 0.53)	0.36 (± 0.25)
H3N2 (A/Neth./177/2008)	0.76 (± 0.27)	—	1.37 (± 0.41)

IC₅₀ values in nanomolars. —, no neutralizing activity detected. Data represent mean ± SD of at least two independent experiments.

Fig. 6.

In vivo protective activity of BiFlu. Kaplan–Meier survival curves (A) and body weight loss (mean ± SD) of C57BL/6J mice (B) that were i.v. injected with either BiFlu (2 mg/kg), AT10-002 + AT10-005 (1 mg/kg each), or rituximab (1 mg/kg). Twenty-four hours later, the mice were challenged intranasally with 50 μL of a 10^4.5 TCID₅₀ H1N1 (A/PR/8/1934) preparation. Compared with mice treated with control antibody (rituximab), the survival and body weight loss of mice treated with BiFlu or the AT10-002 + AT10-005 antibody mixture was significantly improved (survival: P < 0.05; body weight BiFlu P < 0.01 from day 6, AT10-002 + AT10-005 P < 0.01 from day 3). No significant difference was found between BiFlu- and AT10-002 + AT10-005–treated mice. (C) Integrity of BiFlu in mouse plasma, 24 h after injection. Samples containing 0.25 μg of antibody are heated for 10 min at 55 °C and analyzed with anti-IgG HC Western blotting. (D) Antibody concentration in mouse plasma, 24 h after injection. IgG concentration of all mice in each group (n = 6) is determined by ELISA.

To test the integrity of the BiFlu molecule at the time of viral infection, we performed an anti-human IgG HC Western blot (Fig. 6C), demonstrating that BiFlu remained intact as a dimer. The human IgG concentration in the mice, 24 h after injection, was determined with ELISA (Fig. 6D). For both the BiFlu and the antibody mixture group, we found approximately 9.5 μg of human IgG/mL, indicating that BiFlu remained in the circulation at similar levels as the single antibodies.

BiFlu has the ability to bind H1, H3, H7, and H9 HAs, and exhibits neutralization potency against both H1 and H3. The activity of BiFlu is consistent with the combined activities of the individual parental antibodies, which neutralize a wide range of influenza subtypes. Thus, we have created a bispecific antibody dimer capable of broad HA binding and potentially broad neutralization potency.

Discussion

We have presented a bispecific antibody format, in which two full-length IgG antibodies are joined at their C termini. One advantage of this format is the stability of the C-C–linked IgG heterodimer, produced with minimal modification of the native IgG structure. The chemical structure of the C-C linkage includes the sortase recognition site, plus a triazole moiety resulting from the click reaction and eventual linker peptides. This product, like any other nonnative protein modification, could be immunogenic, a notion that would require testing in a human host.

The C-C–linked IgG heterodimer stands out from other IgG-scFv formats; the latter bispecifics, in which the extra domains are genetically fused to the antibody backbone, are often unstable and aggregation-prone (46, 47). Several formats for asymmetric bispecific IgG antibody formats now exist. Antibody asymmetry is facilitated through engineering of the CH₃ domain (13–16) or the hinge region of the antibody (17, 18), promoting heterodimerization of the constant domains. Some of the constant domain mutations required to enable IgG heterodimerization compromise stability and may affect binding to Fc receptors as well (48). Solving these issues requires extensive antibody engineering (48). Also, an asymmetric IgG binds monovalently to its target because it contains only one copy of each variable domain, which may affect its activity. Production of these asymmetric IgG antibodies requires either a mild-reduction step to convert homodimers into heterodimers (17, 18) or coexpression of two different antibodies (13–16), adding further complication.

In contrast, the preparation of the C-C–fused IgG heterodimers lends itself to large-scale manufacturing without loss of product quality or the need for elaborate optimization. The antibodies to be joined can be expressed separately as full-length antibodies, and the coupling reactions occur under physiological conditions. Likewise, sortase-catalyzed reactions enable large-scale preparation of modified biomolecules (49) and immunotoxins (50). A panel of sortase-modified antibodies equipped with click handles far more readily facilitates combinatorial exploration of many different combinations of C-C–linked bispecific antibodies than genetic fusions would allow.

Antibodies against the stem region of Influenza virus HA antigens provide protection against virus in a prophylactic setting in animal models (30, 51) and based on this property, they are being tested in clinical trials. BiFlu combines the activities of two broadly neutralizing antibodies into a single unit. From a developmental and regulatory perspective, combining two antibodies into a single drug makes development less complex and more cost-effective, because preclinical and clinical testing will be reduced to a single molecule.

Materials and Methods

Isolation and Selection of Antiinfluenza Antibodies from Human B Cells.

Immortalization of human memory B cells was performed as described (36, 37). Human memory B cells were isolated using FACS, out of peripheral blood mononucleated cells (PBMCs) from an influenza vaccinated donor, and immortalized through retroviral transduction with a bicistronic construct coding for Bcl6 and Bcl-xL. The use of human PBMCs was approved by the Medical Ethical Committee of the Academic Medical Center and was contingent on informed consent. B cells with reactivity to more than one HA type were characterized for HA recognition by ELISA and binding to HA-expressing cells.

Preparation of SDS-PAGE and Western Blot Samples.

Unless indicated otherwise, samples are prepared in 1x XT-sample buffer (BIORAD) and heated for 10 min at 95 °C. If indicated, samples are reduced by adding 10 mM DTT. Mini-Protean TGX precast 4-20% gradient gels (BIORAD) were used for electrophoresis.

SPR.

SPR is performed on an IBIS Mx96 instrument (IBIS Technologies). Anti-human IgG heavy and anti-human light chain antibodies are immobilized on an amine-specific E2S gold-film SPR chip (Ssens Technologies) using a CFM microfluidics spotter device (Wasatch Microfluidics). Antibodies and full-length HA-antigens are injected over the chip surface in cycles of concatenated injections. Data is processed with SprintX software (IBIS Technologies).

Virus Neutralization Assay.

MDCK-SIAT cells are incubated with virus and antibody. Cells are fixed 24 h after infection. The amount of infected cells is detected with FITC-labeled antiinfluenza nuclear protein (NP) antibody; total cell count is measured with DAPI staining.

Additional detailed information is described in SI Appendix, SI Materials and Methods.