Antibody binder sequences and design of pair-FORCE educts
The following binder clones were used in this study to produce antibody derivatives for pair-FORCE. HER2: Trastuzumab, clone 4D5-856, Pertuzumab, clone rhuMAb 2C457. EGFR: Cetuximab, clone C22558, Imgatuzumab, clone GA-20159. Pair-FORCE educts were designed in a similar manner as already described for bsAb FORCE educts29, containing the destabilizing charge mutations E357K and K370E in the CH3 domains of the corresponding dummy chains. Heterodimerization of heavy chains was induced using knob-into-hole mutations in the CH3 domains60,61. The knob mutation used in this study was T366W, and the hole mutations were T366S, L368A, and Y407V. Similar to FORCE dummy chains, pair-FORCE dummy chains do not contain the engineered cysteine residues at positions 354/349 in the CH3 domains, which normally forms an extra disulfide bridge between the CH3 domains29,61. The pair-FORCE products do, however, contain the engineered disulfide bridge in the CH3 domain after chain exchange. The dummy chains were designed with a C-terminal C-tag composing the amino acids EPEA, which allows for selective binding of unreacted educts, dummy-dimers, and aggregates to a CaptureSelect™ C-tagXL affinity column (Thermo Fisher Scientific). For MTG-mediated conjugation, the Q-tag sequence YRYRQ was included in the Fc donor molecule in the positions stated in the text. In the linker-azide molecule, the K-tag sequence was RYESK26,45. For site-specific biotinylation of Fc donor constructs, the BirA recognition sequence GLNDIFEAQKIEWHE was included N-terminal of the hinge. The Lys residue in the recognition sequence is biotinylated by BirA44.
Expression and purification of antibody derivatives
Expression plasmids encoding the respective heavy and light chains of antibody derivatives in this study contained a CMV promoter and an IgG VH signal sequence, which leads to the secretion of the antibodies into the cell culture supernatant29. Recombinant antibody derivatives were expressed in transiently transfected HEK293 cells (Expi293F™, Thermo Fisher Scientific) according to the manufacturer’s instructions, and as previously described29. Expression yields for pair-FORCE Fc donor molecules and binder-acceptor molecules was comparable to standard IgGs, as previously described29. The supernatants were harvested by centrifugation at 3500×g for 45 min, followed by sterile filtration (0.22 µm filter). Antibody derivatives were purified from the supernatant using a HiTrap™ MabSelect SuRe protein A column (Cytiva, 11003494) followed by size exclusion chromatography using a HiLoad® 26/600 Superdex® 200 column (Cytiva, 28989335). Peak fractions were analyzed by reducing and non-reducing SDS-PAGE. Fractions containing the correct chain composition were pooled and concentrated using an Amicon Ultra-15 centrifugal filter with the appropriate molecular weight cut-off (Merck Millipore).
Analytical SEC and capillary SDS electrophoresis (CE-SDS)
To analyze the aggregation and monomeric purity of the purified antibody derivatives, an analytical SEC was performed. For analytical SEC, 40 µg of antibody derivatives were loaded onto a BioSuite 250, 5 µm HR SEC column (7.8 mm × 300 mm, Waters) connected to an UltiMate 3000 UHPLC (Thermo Fisher Scientific), with a running buffer of 200 mM KH2P04, 250 mM KCl at pH 6.2. The data were analyzed with Chromeleon CDS software (Thermo Fisher Scientific). In order to quantify purity and chain composition, as in Fig. 2a, non-reducing and reducing capillary SDS electrophoresis (CE-SDS) was performed. For this purpose, antibody derivatives were analyzed using the LabChip® GXII Touch™ HT Protein Characterization System (Perkin Elmer), according to the manufacturer’s instructions.
DAR determination of conjugated Fc donor molecules and pair-FORCE products by mass spectrometry
In order to calculate the DAR and to check the integrity of the conjugated Fc donor molecules and pair-FORCE products, mass spectrometry (MS) was performed. Before measurement, the samples were deglycosylated by adding N-glycosidase F (Roche Diagnostics, Penzberg, Germany). The deglycosylation was performed in 0.1 M sodium phosphate buffer at pH 7.1, at a ratio of 0.14 U/µg antibody. The reaction was incubated for 16 h at 37 °C, and samples were subsequently separated by reverse-phase chromatography. This was performed using a PLRP-S column (Agilent, Waldbronn, Germany) with mobile phase A containing 0.1% (v/v) formic acid in UPLC grade water, and mobile phase B containing acetonitrile (Fisher Chemical, Schwerte, Germany). The column temperature was 75 °C, and a gradient of 25% to 40% mobile phase B was used for separation. MS spectra were acquired using a MaXis Q-TOF instrument (Bruker Daltonics, Bremen, Germany) controlled by Compass 6.2 software. A total of 49 samples were analyzed, with one technical replicate each. One control sample (two technical replicates), consisting of a mixture of two different bispecific antibodies in different formats and one standard IgG, was run within the sequence to check LC separation and MS data quality. For data evaluation, in-house-developed software was used. The data annotation was performed using m/z spectra, and a deviation of a maximum of 75 ppm between theoretical and experimental mass was used to confirm the identity of the species. For DAR estimation, the average intensity ratio of each suitable charge state was used. Detailed MS acquisition settings were as follows: ESI Apollo source parameters: Capillary: 5000 V, Nebulizer: 1.6 Bar, Dry Gas 9 l/min, and 230 °C dry temp. Further details are provided in Table 2.
Payload conjugation to exchange-enabled Fc donor molecules
Fc donor molecules were conjugated with payloads to generate Fc donor-payload entities for chain exchange reactions. Enhanced GFP (EGFP) was genetically fused to the C-terminus of the Fc donor with a Gly4Ser (G4S) linker. Biotin was conjugated in a site-directed manner via AviTag/BirA technology using the BirA Bulk Kit (Avidity LLC), as per the manufacturer’s instructions. Conjugation of reactive amines on Fc donor molecules with the NHS-coupled pH-sensitive fluorescent dye pHAb was performed using the Promega pHAb Amine Reactive Dye Kit (Promega, G9845), according to the manufacturer’s instructions (including the DAR calculation). Labeling of reactive amines with horseradish peroxidase (HRP) was performed using the EZ-Link® Activated Peroxidase Antibody Labeling Kit (Thermo Fisher Scientific, 31497), as per the manufacturer’s instructions. Site-directed MTG-mediated conjugation of K-tag containing azide-linker adapters to Q-tags on Fc donor molecules is described below.
Two-step, site-specific Fc donor conjugation with MMAE and fluorescent dyes
In order to conjugate MMAE and AF488 onto Fc donor molecules for ADC matrices, a two-step conjugation method was performed in a similar manner as previously described28,45. In the first step, a linker-azide moiety was conjugated onto a Q-tag or multiple Q-tags on the Fc donor molecule using a specialized MTG enzyme26. A tenfold molar excess of linker-azide was mixed with the antibody, along with the MTG enzyme at a 1:50 molar ratio of enzyme to antibody. The reaction mixture was incubated for 3 h at 37 °C and was terminated by adding excess ammonium sulfate. The linker-azide-conjugated Fc donor molecule was then purified by SEC. In the second step, the linker-azide-conjugated Fc donor was coupled with MMAE or AF488 using strain-promoted azide-alkyne cycloaddition (SPAAC) (Cu-free click chemistry reaction). For this purpose, the linker-azide-conjugated Fc donor was mixed with a fivefold molar excess of DBCO-(PEG)3-VC-PAB-MMAE (MedChem-Express, Cat. No. HY-111012) or DBCO-AF488 (Jena Bioscience, Cat. No. CLK-1278-1). The reaction was incubated overnight at 25°C, and the final conjugated Fc donor molecule was purified by SEC. Conjugation efficiency was analyzed by HIC after each step.
Hydrophobic interaction chromatography (HIC)
In order to assess the conjugation efficiency of the two-step, site-specific Fc donor conjugation, analytical HIC was performed. Conjugation of Fc donor educts with the azide-linker adapter and subsequent conjugation of DBCO-containing payloads with Cu-free click chemistry increases the hydrophobicity of the molecule, which can be analyzed by HIC. Briefly, 40 µg of each sample was applied to a TSKgel Butyl-NPR HPLC column (2.5 μm, 4.6 mm × 35 mm; Tosoh Bioscience, 0014947) using an UltiMate 3000 UHPLC (Thermo Fisher Scientific). Binding was performed in HIC buffer A (20 mM Na2HPO4, 1.5 M (NH4)2SO4, pH 7.0), with 5% of HIC buffer B (20 mM Na2HPO4, 25 % (v/v) isopropanol, pH 7). A gradient of 5–100% HIC buffer B was applied during the run.
Transfer of payloads from Fc donor-payload educts to binder-acceptor educts by chain exchange
The chain-exchange reaction was performed essentially as previously described29, with some minor modifications. Briefly, Fc donor-payload and binder-acceptor educts were combined in equimolar amounts at a concentration of 1 mg/ml in PBS, pH 7.4. In order to reduce the disulfide bridges in the hinge region, a 20-fold molar excess of TCEP containing 0.05% Tween 20 was added, which initiates the chain-exchange reaction. The mixture was incubated for 3 h at 37 °C while shaking at 300 rpm. The chain-exchange products were purified by application of the reaction mixture onto a CaptureSelect C-tagXL column (Thermo Fisher Scientific, 494307205), which binds to C-tag-containing aggregates, unreacted educts, and dummy-dimers. The flow-through contains the pair-FORCE product. Pair-FORCE products were incubated for 5 days at 4°C to allow for reoxidation of disulfide bridges. The products were further analyzed by analytical SEC and CE-SDS, as described above.
Cell culture
The cell lines SK-BR-3 (ATCC, HTB-30), MCF-7 (ATCC, HTB-22), A431 (ATCC, CRL-1555), SK-OV-3 (ATCC, HTB-77), MDA-MB-453 (ATCC, HTB-131), NCI-H1650 (ATCC, CRL-5883), and MDA-MB-468 (ATCC, HTB-132) were cultivated in RPMI-1640 media supplemented with 10% FCS and 2 mM glutamine. For A431 cultivation, the media was supplemented with 1 mM sodium pyruvate. The cell lines were cultured at 37 °C with 5% CO2 and 80% humidity. For sub-culturing, the cells were detached with Accutase (PAN Biotech) and were counted using a Vi-CELL XR cell counter (Beckman Coulter).
Immunoprecipitation and Western blot
In Supplementary Fig. 4, immunoprecipitation of EGFR from whole cell lysate was performed using a biotinylated EGFR-binding antibody derivative generated by pair-FORCE. A total of 3 × 106 A431 cells were resuspended on ice in 1 ml RIPA lysis and extraction buffer (Thermo Fisher Scientific, 89900). Cell debris was removed by centrifugation at 15,000×g for 15 min, and the supernatant was collected. Immunoprecipitation with the biotinylated C225 anti-EGFR antibody derivative was performed with Pierce™ Streptavidin Magnetic Beads (Thermo Fisher Scientific, 88816), according to the manufacturer’s instructions. Briefly, the biotinylated C225 antibody was incubated with the A431 lysate for 2 h at room temperature. The antibody-lysate mixture was added to streptavidin magnetic beads pre-washed with PBS, and the mixture was incubated for 1 h at room temperature with rotation. The beads were washed three times with PBS, and elution was performed with 50 µl of NuPAGE™ LDS Sample Buffer (Thermo Fisher Scientific, NP0007), with heating at 95 °C for 5 min. After the separation of the magnetic beads, 20 µl of eluate was loaded onto a 4–12% Bis-Tris gel (Invitrogen, NP0322). Blotting was performed using Trans-Blot Turbo Mini 0.2 µm PVDF Transfer Packs (Bio-Rad, 1704156) with the Trans-Blot Turbo Transfer System (Bio-Rad), according to the manufacturer’s instructions. The membrane was blocked for 30 min at room temperature in a blocking buffer (TBS with 5% skim milk and 0.05% Tween 20). The membrane was then incubated overnight in a blocking buffer containing an anti-EGFR primary antibody (Abcam, ab264540, 1:1000 dilution). On the following day, the membrane was washed three times with TBS-T (TBS with 0.05% Tween 20) and was incubated with the secondary antibody (Polyclonal Goat anti-Mouse Immunoglobulins/HRP; Agilent Dako, P044701-2, 1:1500 dilution) in blocking buffer for 1 h at RT. After three rounds of washing with TBS-T, chemiluminescence was detected using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, 34579) and the Gel Doc XR+ Gel Documentation System (Bio-Rad). For testing the functionality of the pair-FORCE-generated C225-HRP antibody derivative (Supplementary Fig. 2), the western blot procedure was performed in a similar manner. Briefly, A431 whole cell lysate was loaded onto the gel, and blotting was performed as above. C225-HRP was applied as the primary antibody at a concentration of 0.24 µg/ml in blocking buffer, and the detection protocol was performed as above, but with SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34095).
Flow cytometry: binding and internalization assays
In Supplementary Fig. 3, the binding of an EGFP-conjugated HER2 pair-FORCE product to target cells was assessed by flow cytometry. For this purpose, a total of 2 × 105 A431 cells were incubated with 200 nM of antibody derivatives in FACS buffer (PBS with 2% FCS) for 1 h on ice. Cells were then washed twice with cold PBS, and fluorescence was measured in the FITC channel of a FACS Canto II instrument (BD Biosciences). FlowJo software (BD Biosciences) was used for data analysis and visualization. Gating for single viable cells was performed for all flow cytometry analyses using standard forward-scatter and side-scatter analysis. For epitope binning assays (Supplementary Fig. 3), flow cytometry analyses were performed as above, with additional pre-incubation of A431 target cells with 200 nM of different anti-EGFR antibodies (P2X, mab806, and P1X), which bind to known epitopes on EGFR domains I, II, and III, respectively62,63. FITC intensity was measured using a FACS Canto II instrument as above. To assess internalization of HER2-targeting, pair-FORCE-generated molecules (Figs. 4, 5e), 0.75 × 105 SK-BR-3 cells were seeded out in flat-bottom 96-well plates and treated with various concentrations of HER2 binders conjugated with pHAb by pair-FORCE (500 nM, 50 nM, 5 nM, 0.5 nM, 0.05 nM, and 0 nM final antibody concentration), in a total volume of 200 µl. The cells were incubated for 24 h at 37 °C in a humidified 5% CO2 atmosphere. The cells were then detached with 100 µl Accutase (Pan Biotech, P10-21100), washed twice with PBS, and analyzed via flow cytometry in the PE channel as described above. In order to calculate the relative internalization efficacy of pair-FORCE-generated HER2 binders (defined as absolute internalization divided by absolute binding), binding studies were performed with pair-FORCE-generated HER2 binders labeled with AF488. Briefly, 2 × 105 SK-BR-3 cells were incubated with 200 nM of AF488-labeled HER2 binders in FACS buffer for 30 min on ice. The cells were subsequently washed twice with cold PBS, and fluorescence was measured by flow cytometry in the FITC channel, as described above. Data were collected for 20,000 cells per condition. Absolute internalization and absolute binding were defined as the geometric mean of fluorescence histograms from flow cytometry internalization (pHAb) and binding (AF488) experiments, respectively, and were calculated using FlowJo software. For visualization of bar graphs, GraphPad Prism 9 software was used (GraphPad Software, San Diego, CA, USA).
HER2 binding kinetics of pair-FORCE ADCs and HER2 binder-acceptor modules
For the assessment of the HER2 binding kinetics of HER2-targeting pair-FORCE ADCs and HER2 binder-acceptor modules, a Biacore 8 K+ (GE Healthcare) SPR system was used. First, an anti-human Fab capture ligand was immobilized onto a Series S CM4 (Cytiva 29104989) sensor chip via amine coupling, according to the manufacturer’s instructions (Human Fab Capture Kit, Cytiva, 28958325). Pair-FORCE antibodies were captured on the chip at a flow rate of 10 µl/min for 180 s, and capture ranged between 360–600 RU. An Fc-fused HER2 extracellular domain (ECD) was flowed as an analyte at a rate of 30 µl/min for 120 s, followed by dissociation for 600 s at the same flow rate. The analyte concentrations tested were 0, 5, 25, and 125 nM. Regeneration was performed using regeneration buffer (10 mM glycine, pH 2.0) for 60 s at a flow rate of 30 µl/min at the end of each SPR cycle. The equilibrium constant (KD) and kinetic rate constants were determined by fitting the data to a 1:1 Langmuir interaction model using Biacore™ Insight software (Cytiva).
Thermal stability measurements
Thermal stability measurements of MMAE-conjugated Fc donor molecules, HER2-targeting binder-acceptor molecules, and pair-FORCE-generated HER2-targeting MMAE ADCs were performed using an Uncle instrument (Unchained Labs). Briefly, 9 µl of each sample was loaded in duplicates into the capillaries of the Uncle Uni. As a standard, an in-house reference antibody was used at a concentration of 1 mg/ml. The Unis were loaded into the Uncle instrument and a thermal ramp from 30–90 °C was performed with a ramp rate of 0.1 °C/min. Uncle software was used to calculate the Tm of each sample using the first derivative of the barycentric mean (BCM) of intrinsic fluorescence intensity. The Tagg of each sample was calculated according to the standard Uncle algorithm using the intensity of scattered light at 266 nm.
Cell proliferation assays for assessment of ADC cytotoxicity
The cytotoxicity of pair-FORCE-generated ADCs was assessed using the bromodeoxyuridine (BrdU) colorimetric cell proliferation assay (Roche, Cat. No. 11647229001). Cells were seeded into 96-well flat-bottom tissue culture-treated microplates at a density of 7500 cells per well for MCF-7 and A431 cells, and 10,000 cells per well for SK-BR-3 cells (to account for the slower growth rate of SK-BR-3 cells). The cells were incubated for 24 h at 37 °C, 5% CO2, and 80% humidity, and were subsequently treated with serial dilutions of pair-FORCE-generated ADCs (500, 50, 5, 0.5, and 0.05 nM) for 72 h. After 72 h, the cells were incubated with BrdU for 3 h, fixed using FixDenat for 30 min, and incubated with Anti-BrdU POD for 90 min. The cells were washed with washing solution and the substrate solution was added and incubated for 3 min. Subsequently, cell proliferation was recorded by measuring absorbance at 370 nm using a plate reader (Tecan Group AG). The cytotoxic activity of ADCs was calculated from the BrdU assay, in which the measured signal represents DNA synthesis in viable cells. The percentage of viable cells depicted on the Y axis of cytotoxicity assays was calculated as the percentage of BrdU signal reduction relative to untreated cells. The IC50 value was determined by fitting the data with the equation y = 100/(1 + x/IC50) in GraphPad Prism 9, where x corresponds to the ADC concentration in nM, and y corresponds to the percentage of cell viability. Triplicates were performed for each data point.
Quantification of HER2 receptors on different cell lines
Quantification of HER2 receptors on different cell lines was performed using the QIFIKIT® (Agilent, K0078), according to the manufacturer’s instructions. Briefly, 100 µl of setup and calibration beads were washed three times with 1 ml of FACS buffer (PBS + 2% FCS). The beads were then resuspended in 98 µl of FACS buffer. A total of 3 × 105 cells (SK-OV-3, SK-BR-3, MDA-MB-453, NCI-H1650, MCF-7, MDA-MB-468) were incubated with 3.75 µg of anti-human CD340 (Clone 24D2, BioLegend) or isotype control (Mouse IgG1 clone MOPC-21, BioLegend) in FACS buffer for 45 min on ice. The cells were then washed three times with FACS buffer and resuspended in a total volume of 97 µl. Subsequently, 3 µl of FITC-conjugated anti-mouse antibody (QIFIKIT®) were added to both the cells and the beads, followed by another 45 min incubation on ice. After three additional washes, the cells and beads were resuspended in FACS buffer in a total volume of 125 µl (cells) and 200 µl (beads). Measurement and data acquisition of the beads and cells were performed according to the manufacturer’s instructions (QIFIKIT®), using a FACS Canto II instrument (BD Biosciences). Ten thousand cells were measured per condition. Generation of the calibration curve and HER2 quantification were carried out according to the manufacturer’s instructions (QIFIKIT®), using FlowJo software (BD Biosciences) and GraphPad Prism 9.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
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