Ethical compliance<\/p>\n
Animal studies were performed at the Washington University School of Medicine in compliance with institutional guidelines and protocols approved by the Institutional Animal Care and Use Committee (animal protocol 21-0087, 24-0274; IBC protocol 13652, principal investigator: P.M.R.P.). Radiolabelling and PET\u2013CT imaging experiments were performed using appropriately shielded equipment within a licensed radiation-safety facility by trained personnel under an active licence.<\/p>\n
Preparation of antibody\u2013TCO and antibody\u2013tetrazine conjugates (random and site-specific)Random conjugation<\/p>\n
Antibodies were conjugated at a molar ratio of 15 TCO-PEG4-NHS-ester (TCO; BroadPharm, BP-22418) or tetrazine-PEG5-NHS-ester (tetrazine: BroadPharm, BP-22681) per antibody. Conjugations were performed in PBS (pH\u20098.8\u20139) at 37\u2009\u00b0C, 500\u2009rpm for 1\u2009h. Conjugates were purified using a desalting column (PD-10, GE Healthcare) and concentrated using Amicon filters with a 50\u2009kDa molecular weight cutoff (Millipore, UFC8050) in 1\u00d7 PBS (pH\u20097.4).<\/p>\n
For indocyanine green-Sulfo-Osu (ICG) conjugation, trastuzumab\u2013tetrazine was conjugated with ICG (Fisher Scientific, 501952790) at a molar ratio of 3 ICG per antibody in PBS (pH\u20098.8) at 37\u2009\u00b0C, 500\u2009rpm for 1\u2009h. The fluorescently labelled antibody conjugate was purified using a desalting column (PD-10) and concentrated using 50\u2009kDa molecular weight cutoff Amicon filters in 1\u00d7 PBS (pH\u20097.4).<\/p>\n
Site-specific conjugation<\/p>\n
Antibodies were modified with azide groups through the glycosylation sites in the Fc region (SiteClick Antibody Azido Modification kit, ThermoFisher, S10901). In brief, antibodies (5\u2009mg) were incubated with 100\u2009\u00b5l \u03b2-galactosidase at 37\u2009\u00b0C, 450\u2009rpm for 6\u2009h, followed by overnight incubation at 30\u2009\u00b0C with UDP-GalNAz containing GalT enzyme. Azide-modified antibody was purified and concentrated using Amicon filters with a 50\u2009kDa molecular weight cutoff in 1\u00d7 Tris buffer (pH\u20097.0). Then, DBCO-PEG12-TCO (TCO; BroadPharm, BP-22423) or DBCO-methyl-tetrazine (tetrazine; Vector Laboratories, CCT-1022) at a molar ratio of 15 was conjugated with the azide-modified antibody overnight at room temperature. Site-specific conjugates were purified using a desalting column (PD-10) and concentrated with Amicon filters with a 50\u2009kDa molecular weight cutoff in 1\u00d7 PBS (pH\u20097.4).<\/p>\n
The concentration of the antibody conjugates was determined using a UV-visible spectrophotometer or a Pierce 660 assay (Thermo Fisher Scientific, 22660).<\/p>\n
Cell culture<\/p>\n
The human cancer cell lines NCIN87, A431, BT474, CT26, MDAMB231, MIAPaCa-2 and JIMT1 were purchased from the American Type Culture Collection (ATCC). All cell lines used were tested for bacterial contamination with a PlasmoTest mycoplasma detection kit (InvivoGen, rep-pt1) and authenticated by STR DNA profiling before cell or animal studies. Cells were cultured at 37\u2009\u00b0C in a humidified atmosphere at 5% CO2. All cell culture media were supplemented with 100\u2009units\u2009ml\u22121 penicillin and streptomycin. Details of cell culture media for respective cell lines are detailed in Supplementary Table 3<\/a>. Different cancer cell lines were used for PET and therapeutic studies, as summarized in Supplementary Table 4<\/a>.<\/p>\n Generation of CT26-hHER2 and BT474 trastuzumab-resistant cell lines<\/p>\n We used our previously reported methods to develop the CT26-hHER2 cell line3<\/a>. In brief, we transduced the mouse cancer cell line CT26 using 8\u2009g\u2009ml\u20131 hexadimethrine bromide (Sigma), and the medium was changed 24\u2009h later. Puromycin selection (5\u2009\u03bcg\u2009ml\u20131) was initiated 3\u2009days after transduction and was continued for a minimum of 4\u2009days afterwards. Western blot analysis confirmed human HER2 (hHER2) expression in the CT26 cells (Supplementary Fig. 7<\/a>). HER2 expression in CT26-hHER2 tumours was also confirmed by IHC (Extended Data Fig. 2c<\/a>). PET\u2013CT imaging and ex vivo biodistribution analyses using radiolabelled anti-HER2 trastuzumab confirmed trastuzumab uptake in CT26-hHER2 cells implanted in the mouse (Extended Data Fig. 2a,e<\/a>).<\/p>\n BT474 human breast cancer cells were made resistant to trastuzumab by continually incubating the parental BT474 cancer cells with increasing concentrations (up to 15\u2009\u03bcg\u2009ml\u20131) of the trastuzumab antibody over a period of 9\u2009months. Cell viability studies indicated >90% viability at 48\u2009h after incubation of cells with 20\u2009nM trastuzumab, thereby confirming resistance.<\/p>\n SDS\u2013PAGE, MALDI, SEC\u2013HPLC and TEM<\/p>\n For antibody click validation, antibody\u2013TCO and antibody\u2013tetrazine were incubated at a 1:1 reaction ratio at 37\u2009\u00b0C, 450\u2009rpm for 90\u2009min. The antibodies (10\u2009\u00b5g) were then mixed with loading buffer (Laemmli buffer), loaded in SDS\u2013PAGE gels (NuPage 4\u201312% Bis-Tris protein gels, Invitrogen) and subjected to standard gel electrophoresis. For reducing conditions, samples were mixed with loading buffer containing reducing agent and boiled at 95\u2009\u00b0C for 10\u2009min. After SDS\u2013PAGE, the gels were rinsed with deionized water, stained using a Pierce Mini Gel Power Staining kit (ThermoFisher, 2284) and scanned on an Odyssey CLx imaging system (LI-COR). No-click reactions (antibody plus antibody\u2013tetrazine), single antibodies and modified antibodies were used as controls.<\/p>\n For ICG studies, trastuzumab\u2013TCO and trastuzumab\u2013tetrazine\u2013ICG were incubated at different ratios (1:1, 1:0.8, 1:0.6, 1:0.4 and 1:0.2) at 37\u2009\u00b0C, 450\u2009rpm for 90\u2009min. Additional experiments were conducted in 1:1 reaction ratios at 37\u2009\u00b0C, 450\u2009rpm, at different incubation times (1, 10, 30 and 90\u2009min). To block the click reactions, 15\u2009\u00b5g trastuzumab\u2013TCO or trastuzumab\u2013tetrazine was pre-incubated with 0.5\u2009\u00b5g unconjugated tetrazine or TCO, respectively. Pre-incubation was performed at 37\u2009\u00b0C, 450\u2009rpm for 90\u2009min. After pre-incubation, the antibody click pairs were reacted in a 1:1 molar ratio for 90\u2009min. Samples were analysed by SDS\u2013PAGE as described above.<\/p>\n Matrix-assisted laser desorption\/ionization-time of flight (MALDI-TOF) mass spectrometry of the antibody conjugates was performed to determine the number of conjugates per antibody at the Alberta Proteomics and Mass Spectrometry Facility at the University of Alberta in Canada.<\/p>\n Size-exclusion chromatography\u2013high performance liquid chromatography (SEC\u2013HPLC) was performed using an Agilent 1260 Infinity\u2009II and a Biozen 3\u2009\u00b5m dSEC-2 column (200\u2009\u00c5, 300\u2009\u00d7\u20097.8\u2009mm, Phenomenex). A flow rate of 1\u2009ml\u2009min\u20131 was used with a mobile phase of PBS buffer for 15\u2009min. Samples were detected at 280\u2009nm. Data collection and analyses were performed using Laura software (LabLogic).<\/p>\n TEM of the antibodies was performed at the Washington University Center for Cellular Imaging. In brief, 20\u2009\u00b5l trastuzumab (single antibody) or click trastuzumab\u2013TCO plus trastuzumab\u2013tetrazine samples (10\u2009\u00b5g\u2009ml\u20131 in mQ water; random and site-specific) were adsorbed for 60\u2009s onto carbon-coated 200\u2009mesh copper grids (01840-F, Ted Pella), which had been glow-discharged for 30\u2009s in a Solarus 950 plasma cleaner (Gatan). After sample adsorption, the grids were washed 5\u2009times with ultrapure water and stained for 2\u2009min with freshly prepared 0.75% uranyl formate. Excess stain was blotted off using filter paper (Whatman no.\u20092, Fisher Scientific) before air drying. Grids were imaged using a JEOL JEM-1400Plus microscope (JEOL) at an operating voltage of 120\u2009kV with a NanoSprint15-MkII 16-megapixel sCMOS camera (Advanced Microscopy Techniques). For 2D classification analysis, images were collected at a nominal magnification of \u00d730,000, which corresponded to a pixel size of a 3.54\u2009\u00c5. Data processing was done with Relion 3.1 (PMID: 30412051). In brief, particles were picked using Laplacian-of-Gaussian blob detection and then extracted with box sizes of 128 or 250\u2009pixels for the single antibody and click samples, respectively. Particles underwent multiple rounds of 2D classification, and the clearest classes were selected for display.<\/p>\n Western blot analyses<\/p>\n Western blot of tumour and cell lysates was performed using our previously reported methods3<\/a>. Primary antibodies included rabbit anti-HER2 (1:800; ab131490, Abcam), rabbit anti-EGFR (1:1,000; ab52894, Abcam), mouse anti-\u03b2-actin (1:10,000; A1978, Sigma), DM1 monoclonal antibody (1:1,000; Invitrogen, MA5-42528) and anti-trastuzumab antibody (2.5\u2009\u00b5g\u2009ml\u20131; Biotechne, MAB95471-SP). After overnight incubation with the primary antibodies at 4\u2009\u00b0C, membranes were washed 3\u2009times with Tris-buffered saline containing Tween-20 buffer (TBS-T) with gentle agitation and then incubated with the following secondary antibodies for 1\u2009h at room temperature: anti-rabbit goat IgG conjugated with Alexa Fluor 680 (1:10,000; Invitrogen, A-21076), anti-mouse goat IgG conjugated with Alexa Fluor 800 (1:10,000; Invitrogen, A32730) and anti-human goat IgG conjugated with Alexa Fluor 680 (1:1,000, ThermoFisher, SA000069). Membranes were washed three times and scanned using an Odyssey CLx imaging system (LI-COR). Western blot source data are provided in Supplementary Fig. 1<\/a>.<\/p>\n IgG\u2013DM1 preparation<\/p>\n Human IgG isotype control (Invitrogen, 31154) was conjugated to SMCC-DM1 (MedChemExpress, HY-101070) at a 1:5 molar ratio in PBS (pH\u20098.7) at 37\u2009\u00b0C, 500\u2009rpm for 1\u2009h. The resulting IgG\u2013DM1 conjugate was purified using a desalting column (PD-10) and concentrated using an Amicon filter with a 50\u2009kDa molecular weight cutoff in 1\u00d7 PBS (pH\u20097.4).<\/p>\n To validate the conjugation of IgG to DM1, IgG and IgG\u2013DM1 were run on a SDS\u2013PAGE gel (Invitrogen) and then transferred to a polyvinylidene difluoride membrane (iBlot, Invitrogen) using an iBlot 2 Gel transfer system (Invitrogen). The membrane was washed and stained with an anti-DM1 monoclonal antibody. Before analysis, the gel was stained using a Pierce Mini Gel Power Staining kit. Protein bands were imaged using an Odyssey CLx imaging system (LI-COR). Western blot source data are provided in Supplementary Fig. 1<\/a>.<\/p>\n For the generation of IgG\u2013DM1\u2013tetrazine, IgG\u2013DM1 was conjugated to tetrazine\u2013PEG5-NHS-ester as described for the antibody\u2013tetrazine random conjugation procedure.<\/p>\n In-cell western assay<\/p>\n NCIN87 cancer cells (24,000 cells per well) were plated in a 96-well plate and incubated at 37\u2009\u00b0C in 5% CO2. After 24\u2009h, the cells were fixed with 4% (v\/v) paraformaldehyde (PFA) at room temperature for 20\u2009min, washed with PBS and permeabilized with 0.25% Triton X-100 prepared in PBS containing 0.02% BSA and 0.02% NaN3. The NCIN87 cells were blocked with 1% BSA prepared in PBS containing 0.02% BSA and 0.02% NaN3 at room temperature for 30\u2009min and incubated at 4\u2009\u00b0C overnight with trastuzumab, trastuzumab\u2013tetrazine, trastuzumab\u2013ss-tetrazine, panitumumab, panitumumab\u2013TCO or panitumumab\u2013ss-TCO. All antibodies were prepared at 100\u2009nM in 1% BSA in PBS. Cells were washed with PBS, followed by incubation with anti-human goat IgG conjugated with AlexaFluor 680 (1:1,000; ThermoFisher, SA000069) at room temperature for 1\u2009h. The plates were scanned using an Odyssey CLx imaging system (LI-COR), and quantifications were performed using Empiria Studio software (v.3.2).<\/p>\n In vitro serum reactivity of antibody click pairs<\/p>\n Panitumumab\u2013TCO or trastuzumab\u2013tetrazine was incubated in mouse serum (Sigma-Aldrich, M5905) at a final concentration of 5\u2009mg\u2009ml\u20131 in a 1:1 mixture (PBS to mouse serum) at 37\u2009\u00b0C, 500\u2009rpm for 0, 4 or 24\u2009h. At each time point, 5\u2009\u00b5g of the corresponding antibody was collected and reacted with its complementary click pair in PBS (20\u2009\u00b5l) at 37\u2009\u00b0C, 500\u2009rpm for 90\u2009min. For the no-click group, unmodified panitumumab was used.<\/p>\n To evaluate antibody click reactivity following incubation in mouse serum, click and no-click reaction samples were analysed by SDS\u2013PAGE and transferred onto polyvinylidene difluoride membranes as described above (see the section \u2018IgG\u2013DM1 preparation\u2019). Membranes were washed in TBS-T and stained with revert 700 total protein (LI-COR, 92611016) according to the manufacturer\u2019s instructions. Membranes were then incubated with anti-human goat IgG conjugated with Alexa Fluor 680 (1:5,000) in 5% BSA in TBS-T at room temperature for 1\u2009h. Following washing, images were acquired using an Odyssey CLx imaging system (LI-COR). Western blot source data are provided in Supplementary Fig. 1<\/a>.<\/p>\n Immunofluorescence<\/p>\n For immunofluorescence assays, TCO-conjugated or tetrazine-conjugated antibodies were labelled with Alexa Fluor 488 NHS ester (Thermo Fisher Scientific, A20000) or Alexa Fluor 594 NHS ester (Thermo Fisher Scientific, A20004) at a molar ratio of 4 fluorophores per antibody at 37\u2009\u00b0C, 450\u2009rpm for 1\u2009h in PBS (pH\u20098.8). The fluorescently labelled antibody conjugates were purified via desalting chromatography (PD-10) and concentrated using 50\u2009kDa molecular weight cutoff Amicon filters in 1\u00d7 PBS (pH\u20097.4). For control studies, unconjugated antibodies were labelled with Alexa Fluor 488 NHS ester.<\/p>\n Confocal microscopy<\/p>\n NCIN87 and MIAPaCa-2 cancer cells (1\u2009million cells) were grown on coverslips of 1.5\u2009mm thickness (ThermoFisher Scientific, 12-542B) pretreated with poly-l-lysine (Sigma-Aldrich) for 24\u2009h. Cells were then first incubated with 100\u2009nM panitumumab (no click) or panitumumab\u2013TCO (click) conjugated with Alexa Fluor 488 in cell culture medium at 4\u2009\u00b0C for 30\u2009min. Next, the cells were washed to remove unbound antibodies with PBS (containing Ca2+ and Mg2+, DPBS) and incubated with 100\u2009nM trastuzumab\u2013tetrazine conjugated with Alexa Fluor 594 in cell culture medium at 37\u2009\u00b0C in 5% CO2 for 3 or 24\u2009h. Cells were then washed with PBS and fixed with 4% PFA for 20\u2009min at room temperature before incubation with 4\u2032,6-diamidino-2-phenylindole (DAPI; 1:5,000, Santa Cruz Biotechnology) for 5\u2009min. Fluorescence images were acquired using a \u00d763 oil-immersion objective using a Zeiss 980 microscope (excitation at 488, 561 and 405\u2009nm, and emission at 499\u2013570, 577\u2013642 and 420\u2013478\u2009nm) at the Washington University Center for Cellular Imaging.<\/p>\n For studies in which pertuzumab was used as the first pair of antibody click, the experimental procedure was performed in NCIN87 cancer cells as described above, but replacing panitumumab or panitumumab\u2013TCO with pertuzumab or pertuzumab\u2013TCO conjugated with Alexa Fluor 488, respectively.<\/p>\n Experiments of LAMP1 staining were performed after 3\u2009h of incubation with trastuzumab\u2013tetrazine conjugated with Alexa Fluor 594, as described above. Then, cells were fixed with 4% PFA for 20\u2009min at room temperature, permeabilized for 5\u2009min with 0.2% Triton X-100 prepared in PBS containing 0.02% BSA and 0.02% NaN3 and blocked with 1% BSA in TBS-T for 30\u2009min at room temperature. Cells were incubated with CoraLite Plus 647 anti-human CD107a\/LAMP1 (1:400; Proteintech, CL647-65051) for 1\u2009h at room temperature. Fluorescence images were acquired using a \u00d763 oil-immersion objective using a Zeiss 980 microscope (excitation 639\u2009nm, and emission 660\u2013750\u2009nm) at the Washington University Center for Cellular Imaging.<\/p>\n Immunofluorescence with site-specific conjugations was performed in MIAPaCa-2 cancer cells as described above, but incubating trastuzumab\u2013ss-tetrazine for 1\u2009h at 37\u2009\u00b0C in 5% CO2. For the pre-click condition, panitumumab\u2013ss-TCO conjugated with Alexa Fluor 488 and trastuzumab\u2013ss-tetrazine conjugated with Alexa Fluor 594 were reacted for 90\u2009min at 37\u2009\u00b0C at a 1:1 molar ratio before incubation with cells. Fluorescence images were acquired using a \u00d760 oil-immersion objective using an EVOS M5000 imaging system (excitation at 482 and 585\u2009nm, and emission at 524 and 628\u2009nm).<\/p>\n Fluorescence quantification<\/p>\n NCIN87 (20,000 cells) and MIAPaCa-2 (15,000 cells) cancer cells were plated in a 96-well plate and incubated at 37\u2009\u00b0C in 5% CO2. After 24\u2009h, the cells were first incubated with 100\u2009nM panitumumab (no click) or panitumumab\u2013TCO (click) conjugated with Alexa Fluor 488 in culture medium at 4\u2009\u00b0C for 30\u2009min. Next, the cells were washed with PBS (containing Ca2+ and Mg2+, DPBS) and incubated with 100\u2009nM trastuzumab\u2013tetrazine conjugated with Alexa Fluor 594 in culture medium at 37\u2009\u00b0C in 5% CO2 for 3\u2009h. Cells were then washed with PBS, and fluorescence intensity (excitation at 488 and585\u2009nm, emission at 530 and 626\u2009nm) was measured using a BioTek Synergy H1 microplate reader (Agilent). Protein quantification was performed by lysing the cells in PBS containing 1% (m\/v) SDS and measured by BCA assay.<\/p>\n pHrodo internalization assay<\/p>\n Trastuzumab\u2013tetrazine was labelled with the amine-reactive pHrodo Red (ThermoFisher, P36600) according to the manufacturer\u2019s instructions at a molar ratio of 19:1 (dye to antibody). In brief, trastuzumab\u2013tetrazine in 0.1\u2009M NaHCO3 (pH\u20098.3) was incubated with pHrodo red succinimidyl ester at room temperature for 1\u2009h. The resulting conjugate was purified using Zeba spin desalting column (7\u2009K MWCO, 0.5\u2009ml, ThermoFisher, 89882) in 1\u00d7 PBS pH\u20097.4. For control, IgG\u2013tetrazine was conjugated to pHrodo Red succinimidyl ester.<\/p>\n NCIN87 (24,000 cells), MIAPaCa-2 (15,000 cells), MDA-MB-231 (15,000 cells), CT26 (15,000 cells) and CT26-hHER2 (15,000 cells) cancer cells were plated in a 96-well plate and incubated at 37\u2009\u00b0C in 5% CO2. After 24\u2009h, the cells were first incubated with 5\u2009\u00b5g\u2009ml\u20131 panitumumab (no click) or panitumumab\u2013TCO (click) in cell culture medium at 4\u2009\u00b0C for 30\u2009min. Next, the cells were washed with PBS (containing Ca2+ and Mg2+, DPBS) and incubated with 5\u2009\u00b5g\u2009ml\u20131 trastuzumab\u2013tetrazine conjugated with pHrodo. Live-cell imaging was performed on an IncuCyte S3 (excitation at 565\u2013605\u2009nm, emission at 625\u2013705\u2009nm) under a \u00d710 objective at an interval of 1\u2009h at 37\u2009\u00b0C at the Siteman Flow Cytometry Core. For pHrodo assays with site-specific conjugates, the procedure was performed as described above, using 10\u2009\u00b5g\u2009ml\u20131 of the corresponding antibodies. Data were collected at an interval of 2\u2009h at 37\u2009\u00b0C.<\/p>\n IHC and co-registration<\/p>\n IHC staining of HER2 and EGFR was performed on formalin-fixed, paraffin-embedded sections (4\u2009\u00b5m) of NCIN87, A431,\u00a0MIAPaCa-2, admix model and CT26-hHER2 subcutaneous tumours. For paraffin-embedding and sectioning, tumours were submitted to the Pulmonary Morphology Core at Washington University School of Medicine. For NCIN87, A431 and\u00a0MIAPaCa-2 tumours, sections were submitted to HistoWiz for HER2 and EGFR staining. For the admixed tumours, EGFR IHC was performed by HistoWiz, and HER2 by the Anatomic and Molecular Pathology core laboratory. For CT26-hHER2, sections were submitted to the Laboratory of Comparative Pathology at Memorial Sloan Kettering Cancer Center for HER2 staining.<\/p>\n To assess how EGFR and HER2 proteins are distributed across the same tumour tissue, we used automated image analysis58<\/a>. First, we digitally aligned (co-registered) the stained tissue images by enhancing contrast, reducing background noise and matching key tissue structures between the two. This process ensured that the same regions of tissue could be directly compared. Next, we segmented each image to isolate the areas with the highest levels of protein staining. This step involved identifying and highlighting distinct regions corresponding to high EGFR or HER2 signals. Together, these steps enabled us to quantify and visualize the extent to which the two proteins overlapped in each tumour via DSC. Additional details involving the co-registration methods are provided in Supplementary Information<\/a>.<\/p>\n Antibody radiolabelling<\/p>\n Antibodies used in this work were radiolabelled with 89Zr or 64Cu according to published methods38<\/a>,59<\/a>. 89Zr and 64Cu were purchased from the WUSTL Cyclotron and Nuclear Pharmacy. In brief, antibodies were first conjugated with the chelators p-isothiocyanatobenzyl-desferrioxamine (DFO-Bz-NCS; Macrocyclics) or 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA, Macrocyclics) and then radiolabelled with 89Zr or 64Cu, respectively. Antibodies were purified after conjugation and radiolabelling using desalting columns (PD-10) and concentrated using Amicon filters with a 50\u2009kDa molecular weight cutoff. Radiolabelled antibodies used in the study had a radiochemical yield and purity above around 95%.<\/p>\n For bispecific antibody radiolabelling, BSCFV-155 (Creative Biolabs; anti-EGFR based on clone C225 and anti-HER2 based on clone 4D5) was conjugated with p-SCN-Bn-NOTA and radiolabelled with 64Cu as previously described59<\/a>. Serum stability was performed following a previously described method59<\/a>. In brief, 10\u2009\u00b5Ci (0.37\u2009MBq) of the purified radiolabelled antibody was incubated with human serum at 37\u2009\u00b0C for 48 or 72\u2009h.<\/p>\n PET\u2013CT imaging and biodistribution studies<\/p>\n Female nu\/nu mice (6\u20138\u2009weeks old) or female BALB\/c mice (4\u20136\u2009weeks old), purchased from Charles River Laboratories, were used for imaging studies. Tumour implantation and tumour models used are summarized in Supplementary Table 4<\/a>. The tumour volume was estimated by external vernier caliper measurements of the longest axis, \u03b1 (in mm), and the axis perpendicular to the longest axis, b (in mm). The tumours were assumed to be spheroidal, and the volume was calculated in accordance with the equation V\u2009=\u2009(4\u03c0\/3)\u2009\u00d7\u2009(\u03b1\/2)2\u2009\u00d7\u2009(b\/2). PET\u2013CT imaging was performed when tumour volumes reached approximately 150\u2013200\u2009mm3.<\/p>\n For random conjugates, mice were first injected with TCO-conjugated antibodies (50\u2009\u00b5g). At 24 or 48\u2009h after injection, mice were injected with [89Zr]Zr-DFO-trastuzumab\u2013tetrazine or [64Cu]Cu-NOTA-trastuzumab\u2013tetrazine (7.4\u2009MBq, 50\u2009\u00b5g) and used for both biodistribution and PET\u2013CT imaging studies. Control groups included 64Cu\/89Zr-labelled trastuzumab (single antibody) or panitumumab\u2013TCO plus [64Cu]Cu-NOTA-IgG\u2013tetrazine (control IgG click). Acute biodistribution and PET\u2013CT imaging studies were performed at 48\u2009h or 24\u2009h after tail vein injection of the tetrazine-conjugated radiolabelled antibodies.<\/p>\n For site-specific conjugations, mice were first injected with panitumumab\u2013ss-TCO (50\u2009\u00b5g). At 24\u2009h after injection, mice were injected with [89Zr]Zr-DFO-trastuzumab\u2013ss-tetrazine or [64Cu]Cu-NOTA-trastuzumab\u2013ss-tetrazine (7.4\u2009MBq, 50\u2009\u00b5g). PET\u2013CT images were acquired at 24\u2009h for [64Cu]Cu-NOTA-trastuzumab\u2013ss-tetrazine or at 24, 48, 72, 96 and 120\u2009h for [89Zr]Zr-DFO-trastuzumab\u2013ss-tetrazine. Acute biodistribution studies were performed at the last point of the PET\u2013CT imaging study. Control groups included IgG\u2013ss-TCO plus [89Zr]Zr-DFO\u2013trastuzumab\u2013ss-tetrazine (control IgG click) or antibodies pre-clicked (panitumumab\u2013ss-TCO pre-clicked with [89Zr]Zr-DFO-trastuzumab\u2013ss-tetrazine) before being injected.<\/p>\n Mice used for biodistribution studies were euthanized by controlled carbon dioxide overdose followed by cervical dislocation, and organs were collected and weighed. Radioactivity associated with each organ was assessed using a gamma counter (2480 Wizard, PerkinElmer) and quantified as a percentage of the injected dose per gram of the organ (%ID\u2009g\u20131).<\/p>\n PET\u2013CT imaging was conducted in a Mediso nanoScan PET\u2013CT scanner at 24\u2013120\u2009h after injection of the radiolabelled antibodies modified with or without tetrazine. The mice were anaesthetized by inhalation of 2% isoflurane (Baxter Healthcare) in an oxygen gas mixture 5\u2009min before the PET\u2013CT experiments. CT scanning was recorded for 5\u2009min to obtain anatomical information, followed by a static PET scan for 20\u2009min. PET\u2013CT images were analysed using Imalytics Preclinical software (v.3.1, Gremse-IT). PET\u2013CT images were calibrated as a percentage of injected dose per millilitre (%ID\u2009ml\u20131). Regions of interest were delineated in the tumour, and activity values were obtained as mean %ID\u2009ml\u20131.<\/p>\n In vivo pharmacokinetic studies<\/p>\n Female and male nu\/nu mice (4\u20135\u2009weeks old; n\u2009=\u200912) or female BALB\/c mice (4\u20135\u2009weeks old; n\u2009=\u20097) were obtained from Charles River Laboratories. Mice were intravenously injected with 5\u2009mg\u2009kg\u20131 panitumumab\u2013TCO (nu\/nu mice) or pertuzumab\u2013TCO (BALB\/c mice). At 24\u2009h after injection, the mice received a second intravenous injection of 5\u2009mg\u2009kg\u20131 trastuzumab\u2013tetrazine. Blood samples were collected by cardiac puncture at 5\u2009min, 30\u2009min, 4\u2009h, 16\u2009h and 24\u2009h after administration of trastuzumab\u2013tetrazine, after euthanizing the mice. Blood was coagulated at room temperature for 30\u2009min, and serum was obtained after centrifugation at 2,000g at 4\u2009\u00b0C for 15\u2009min. Then, 1\u2009\u00b5l serum was diluted with 14\u2009\u00b5l PBS and 5\u2009\u00b5l loading buffer (Laemmli buffer), subjected to SDS\u2013PAGE electrophoresis and western blot analyses. Membranes were stained with revert 700 total protein, anti-human goat IgG conjugated with AlexaFluor 680 (1:5,000) and anti-trastuzumab antibody (2.5\u2009\u00b5g\u2009ml\u20131, Biotechne, MAB95471-SP) in 5% BSA in TBS-T. Protein bands were visualized using an Odyssey CLx imaging system. Western blot source data are provided in Supplementary Fig. 1<\/a>.<\/p>\n Antibody\u2013ADC click therapeutic efficacy studies (random and site-specific)<\/p>\n Female and male nu\/nu mice (6\u20138\u2009weeks old) or female BALB\/c mice (4\u20136\u2009weeks old) were obtained from Charles River Laboratories. Information regarding tumour models is provided in Supplementary Table 4<\/a>. Tumour volumes were measured twice a week by external caliper measurements of the longest axis, \u03b1 (in mm), and the axis perpendicular to the longest axis, b (in mm). The tumours were assumed to be spheroidal, and the volume was calculated in accordance with the equation V\u2009=\u2009(4\u03c0\/3)\u2009\u00d7\u2009(\u03b1\/2)2\u2009\u00d7\u2009(b\/2). Once tumour volumes reached 100\u2013250\u2009mm3, ADC treatments were initiated, and doses were selected on the basis of commonly used preclinical dosing ranges for ADC studies, including those reported for T-DXd in mouse tumour models4<\/a>,5<\/a>,60<\/a>,61<\/a>. Humane end points were defined as tumour volumes >1,500\u2009mm3, body-weight loss of >20%, tumour ulceration and other clinical symptoms of acute toxicity. At the terminal stage, selected tumours were collected for western blot analyses.\u00a0Stratified random sampling was used to assign the mice randomly\u00a0to experimental or control groups to ensure comparable tumour size\u00a0at baseline. The personnel involved in tumour measurements and mouse weight were blinded to the treatment groups.<\/p>\n Gastric cancer model (NCIN87)<\/p>\n Female or male nu\/nu mice inoculated with NCIN87 xenografts were stratified into the following two groups: no click (panitumumab plus T-DXd; n\u2009=\u20096) or click (panitumumab\u2013TCO plus T-DXd\u2013tetrazine; n\u2009=\u20096). Panitumumab or panitumumab\u2013TCO was administered at 10\u2009mg\u2009kg\u20131 via the tail vein. At 24\u2009h after injection, the mice received an intravenous injection of T-DXd or T-DXd\u2013tetrazine (10\u2009mg\u2009kg\u20131).<\/p>\n Bilateral tumour model<\/p>\n Female and male nu\/nu mice were subcutaneously injected with NCIN87 and A431 cancer cells inoculated in the right and left flanks, respectively. Mice were randomized into three groups: saline (n\u2009=\u20095), no click (panitumumab plus T-DXd, n\u2009=\u20099) or click (panitumumab\u2013TCO plus T-DXd\u2013tetrazine, n\u2009=\u20099). Panitumumab or panitumumab\u2013TCO was administered at 5\u2009mg\u2009kg\u22121 via the tail vein. At 24\u2009h after injection, mice received an intravenous injection of T-DXd or T-DXd\u2013tetrazine (5\u2009mg\u2009kg\u20131).<\/p>\n NCIN87 gastric cancer model of acquired resistance to T-DXd<\/p>\n Male and female nu\/nu mice inoculated with NCIN87 xenografts (n\u2009=\u200916) were treated with T-DXd monotherapy intravenously (5\u2009mg\u2009kg\u20131). Nearly 1\u2009month after therapy initiation, mice were stratified into responders versus non-responders on the basis of tumour volume measurements. Tumours from responder and non-responder tumours were subjected to western blot analyses and HER2-targeting PET imaging. Responder tumours were defined as having a fold change in tumour volume lower than or equal to 0 and a decrease in HER2 by PET. A fold change in tumour volume for non-responders was close to 1, and demonstrated an increase in EGFR protein levels as detected by western blotting. HER2-targeting immuno-PET was performed at 24\u2009h after intravenous injection of [64Cu]Cu-NOTA-trastuzumab (50\u2009\u00b5g, 7.4\u2009MBq). The mice that initially did not respond to T-DXd therapy (non-responder) were treated intravenously with panitumumab\u2013TCO (5\u2009mg\u2009kg\u20131) on day\u20091 and with T-DXd\u2013tetrazine on day\u20092 (5\u2009mg\u2009kg\u20131).<\/p>\n BT474 trastuzumab-resistant model<\/p>\n Female nu\/nu mice were subcutaneously implanted with oestrogen-receptor-positive BT474 trastuzumab-resistant cells (n\u2009=\u200910). Drinking water of mice was supplemented with 0.67\u2009\u03bcg\u2009ml\u20131 \u03b2-oestradiol (Sigma) from 1\u2009week in advance of tumour inoculation and continued until mice were killed. Fresh oestradiol-supplemented water was provided twice a week. T-DXd monotherapy (5\u2009mg\u2009kg\u20131) was initiated when the tumour volume reached 200\u2013500\u2009mm3. Nearly 1\u2009month after therapy initiation, mice were stratified into responders versus non-responders on the basis of tumour volume measurements. Non-responder mice were treated with pertuzumab\u2013TCO (5\u2009mg\u2009kg\u20131) on day\u20091. Mice were then injected with T-DXd\u2013tetrazine (5\u2009mg\u2009kg\u20131) on day\u20092, and tumour volumes were measured twice a week.<\/p>\n Immunocompetent CT26-hHER2 model<\/p>\n Female BALB\/c mice were subcutaneously injected with CT26-hHER2 cancer cells. Mice were randomized into five cohorts: saline (n\u2009=\u20097), pertuzumab (n\u2009=\u20099), T-DXd (n\u2009=\u20099), no click (pertuzumab plus T-DXd, n\u2009=\u200914) or click (pertuzumab\u2013TCO plus T-DXd\u2013tetrazine, n\u2009=\u200914). Pertuzumab or pertuzumab\u2013TCO was administered at 5\u2009mg\u2009kg\u20131 via the tail vein. At 24\u2009h after injection, mice received an intravenous injection of T-DXd or T-DXd\u2013tetrazine (5\u2009mg\u2009kg\u20131). For individual antibodies (pertuzumab or T-DXd), animals received a single dose at 5\u2009mg\u2009kg\u20131.<\/p>\n Admixed breast tumour model (random and site-specific)<\/p>\n Female mice\u00a0(nu\/nu) inoculated with admixed breast tumour (MDA-MB-231 plus JIMT1) xenografts were divided into nine groups: saline (n\u2009=\u200910), panitumumab (n\u2009=\u200910), T-DXd (n\u2009=\u200910), T-DM1 (n\u2009=\u200910), T-DXd no click (panitumumab plus T-DXd, n\u2009=\u200910), T-DM1 no click (panitumumab plus T-DM1, n\u2009=\u200910), T-DXd click random (panitumumab\u2013TCO plus T-DXd\u2013tetrazine, n\u2009=\u200914), T-DXd click site-specific (panitumumab\u2013ss-TCO plus T-DXd\u2013ss-tetrazine, n\u2009=\u200910) or T-DM1 click random (panitumumab\u2013TCO plus T-DM1\u2013tetrazine, n\u2009=\u20096). The admixed breast tumour model was developed by co-injecting HER2+ JIMT1 and HER2\u2013 MDA-MB-231 breast cancer cells at a 4:1 ratio at the time of implantation. Panitumumab or panitumumab\u2013TCO (random or site-specific) was administered at 5\u2009mg\u2009kg\u20131 via the tail vein. At 24\u2009h after injection, mice received an intravenous injection of 5\u2009mg\u2009kg\u20131 T-DXd, T-DM1, T-DM1\u2013tetrazine or T-DXd\u2013tetrazine (random or site-specific). For individual antibodies (panitumumab, T-DM1 or T-DXd), animals received a single dose at 5\u2009mg\u2009kg\u20131.<\/p>\n Pancreatic tumour model (random and site-specific)<\/p>\n Female or male mice\u00a0(nu\/nu) inoculated with MiaPaCa-2 pancreatic xenografts were divided into 13 groups: saline (n\u2009=\u20098), panitumumab (n\u2009=\u200910), T-DXd (n\u2009=\u200910), T-DM1 (n\u2009=\u200910), T-DXd no click (panitumumab plus T-DXd, n\u2009=\u200910), T-DM1 no click (panitumumab plus T-DM1, n\u2009=\u200910), T-DXd click random (panitumumab\u2013TCO plus T-DXd\u2013tetrazine, n\u2009=\u200910), T-DM1 click random (panitumumab\u2013TCO plus T-DM1\u2013tetrazine, n\u2009=\u200910), IgG\u2013DM1 (n\u2009=\u20098), IgG\u2013DM1 no click (panitumumab plus IgG\u2013DM1, n\u2009=\u20096), IgG\u2013DM1 click random (panitumumab\u2013TCO plus IgG\u2013DM1\u2013tetrazine, n\u2009=\u20096), T-DXd click site-specific (panitumumab\u2013ss-TCO plus T-DXd\u2013ss-tetrazine, n\u2009=\u200910) or T-DXd click random in the presence of excess tetrazine (panitumumab\u2013TCO plus T-DXd\u2013tetrazine in the presence of 5\u2009mg\u2009kg\u20131 NHS-PEG5-tetrazine). Panitumumab or panitumumab\u2013TCO (random or site-specific) was administered at 5\u2009mg\u2009kg\u20131 via the tail vein. At 24\u2009h after injection, mice received an intravenous injection of 5\u2009mg\u2009kg\u20131 T-DXd, T-DM1, T-DM1\u2013tetrazine or T-DXd\u2013tetrazine (random or site-specific). For individual antibodies (panitumumab, T-DM1, IgG\u2013DM1 or T-DXd), animals received a single dose at 5\u2009mg\u2009kg\u20131.<\/p>\n Toxicology studies<\/p>\n To assess the potential for antigen-independent toxicity, female BALB\/c mice (n\u2009=\u200924) were purchased from Charles River Laboratories. For serum chemistry analyses, mice were randomized into four groups: saline (n\u2009=\u20097), no click (panitumumab plus T-DXd, n\u2009=\u20094), random click (panitumumab\u2013TCO plus T-DXd\u2013tetrazine, n\u2009=\u20093) or site-specific click (panitumumab\u2013ss-TCO plus T-DXd\u2013ss-tetrazine, n\u2009=\u20094). Panitumumab, panitumumab\u2013TCO or panitumumab\u2013ss-TCO was intravenously injected at 20\u2009mg\u2009kg\u20131. At 24\u2009h after injection, mice received an intravenous administration of T-DXd, T-DXd\u2013tetrazine or T-DXd\u2013ss-tetrazine (20\u2009mg\u2009kg\u20131). Blood samples were analysed 15\u2009days after administration of antibodies to measure the levels of enzymes associated with liver function, specifically AST, ALT and ALP.<\/p>\n For random and site-specific click groups (n\u2009=\u20093 per group), selected organs (lungs and liver) were collected and formaldehyde-fixed for histopathology 7\u2009days after administration of antibodies. Liver and lung tissue slices were stained with haematoxylin and eosin. Damage was evaluated by microscopy by a board-certified pathologist at the Division of Comparative Medicine at Washington University as summarized in Supplementary Table 2<\/a>.<\/p>\n Statistical analyses<\/p>\n Fluorescence quantification and biodistribution data were analysed using ANOVA followed by Student\u2019s t-tests. These analyses were conducted using GraphPad Prism (v.9; www.graphpad.com<\/a>) and R statistical (v.4.4.0) software.<\/p>\n Longitudinal PET image quantification data were analysed using a linear regression model with group, time and their interaction term. For tumour uptake (Extended Data Fig. 5b<\/a>), for which the groups showed diverging accumulation rates over time, we used the emtrends function from the emmeans package to estimate the slope for each group and to perform pairwise contrasts of slopes with Tukey\u2019s correction for multiple comparisons. This tests whether the temporal rate of antibody accumulation differs between groups. For liver uptake (Extended Data Fig. 5c<\/a>), for which accumulation trajectories were roughly parallel across groups, we first confirmed that the group \u00d7 time interaction term was not significant (F-test, P\u2009=\u20090.46) and then fit a no-interaction model. Group mean differences were estimated and compared using Tukey-adjusted pairwise contrasts from this model. For point comparisons at the 120\u2009h end point cited in the main text, we fit a one-way linear model restricted to 120\u2009h observations and obtained pairwise contrasts with Tukey\u2019s correction (tumour) or a two-sample equal-variance t-test (liver, click versus pre-click).<\/p>\n For tumour volume comparison, the longitudinal data were analysed using a linear mixed-effects model with a first-order autoregressive (AR(1)) correlation structure to account for correlations among repeated measures from the same mouse. We included all time points at which every treatment group had data to perform balanced comparisons across groups. The mixed model included the treatment group, time points and the interaction term between group and time points. The P\u2009value of the interaction term from the type\u2009III (marginal) analysis was used to assess whether tumour volume fold change trajectories differed between treatment groups across time points. To quantify effect sizes, pairwise contrasts between each treatment group and the click group were estimated at the final common time point using the emmeans package (v.1.10.7), with Dunnett\u2019s correction for multiple comparisons. Dunnett\u2019s test was chosen over Bonferroni because it accounts for the positive correlation among contrasts that share a common reference group. As a pre-specified primary comparison, we also fit a separate model restricted to the click and no-click groups. This model used all time points at which both groups have data, extending the observation window beyond the final time point available in the full five-group model (which is constrained by the earlier dropout of control groups). Differences are reported on the fold change scale (relative to day\u20090 tumour volume) with 95% confidence intervals. Models were fit using restricted maximum likelihood via the nlme package (v.3.1-166) in R (v.4.4.0).<\/p>\n Survival was compared across treatment groups using the log-rank test. Pairwise comparisons between all group pairs were performed with Bonferroni correction for multiple testing. Hazard ratios with 95% confidence intervals were estimated using Cox proportional hazards regression. Kaplan\u2013Meier survival curves were generated for visualization. All survival analyses were conducted using the survival package (v.3.5-8) at the two-sided 5% significance level.<\/p>\n Reporting summary<\/p>\n