Site-Specific 68Ga Radiolabeling of Trastuzumab Fab via Methionine for ImmunoPET Imaging

Bioconjugates of antibodies and their derivatives radiolabeled with β+-emitting radionuclides can be utilized for diagnostic PET imaging. Site-specific attachment of radioactive cargo to antibody delivery vectors provides homogeneous, well-defined immunoconjugates. Recent studies have demonstrated the utility of oxaziridine chemistry for site-specific labeling of methionine residues. Herein, we applied this approach to site-specifically radiolabel trastuzumab-derived Fab immunoconjugates with 68Ga, which can be used for in vivo PET imaging of HER2-positive breast cancer tumors. Initially, a reactive azide was introduced to a single solvent-accessible methionine residue in both the wild-type Fab and an engineered derivative containing methionine residue M74, utilizing the principles of oxaziridine chemistry. Subsequently, these conjugates were functionalized with a modified DFO chelator incorporating dibenzocyclooctyne. The resulting DFO-WT and DFO-M74 conjugates were radiolabeled with generator-produced [68Ga]Ga3+, to yield the novel PET radiotracers, [68Ga]Ga-DFO-WT and [68Ga]Ga-DFO-M74. In vitro and in vivo studies demonstrated that [68Ga]Ga-DFO-M74 exhibited a higher affinity for HER2 receptors. Biodistribution studies in mice bearing orthotopic HER2-positive breast tumors revealed a higher uptake of [68Ga]Ga-DFO-M74 in the tumor tissue, accompanied by rapid renal clearance, enabling clear delineation of tumors using PET imaging. Conversely, [68Ga]Ga-DFO-WT exhibited lower uptake and inferior image contrast compared to [68Ga]Ga-DFO-M74. Overall, the results demonstrate that the highly facile methionine-oxaziridine modification approach can be simply applied to the synthesis of stable and site-specifically modified radiolabeled antibody–chelator conjugates with favorable pharmacokinetics for PET imaging.


Materials and Instrumentation
Chemicals were purchased from Merck Chemicals Ltd or Fluorochem Ltd unless otherwise specified and used without further purification.Other solvents were purchased from VWR International and used without further purification.18.2 MΩ water was used to prepare all buffers and aqueous solutions.Oxazridine-N3 1 and DBCO-PEG4-DFO were prepared as previously described. 1,2Trastuzumab was obtained as the biosimilar Herzuma in solution (21 mg/mL) from the Pharmacy Department at Guy's and St. Thomas' NHS Trust, London.Fresh human serum was obtained from a healthy volunteer.PD-10 size exclusion columns were purchased from GE Healthcare UK Ltd.Zeba TM Spin Desalting columns, 7 kDa MWCO, 0.5 mL, 5 mL and 10 mL were purchased from Life Technologies Limited, UK.Amicon Ultra 0.5 mL centrifugal filters (10 kDa MWCO), OverExpress TM C43 (DE3) Chemically Competent Cells and Overnight Express TM Instant TB Medium were purchased from Merck Chemicals Ltd.Instant thin layer chromatography (iTLC-SG) were obtained from Varian Medical Systems UK, Ltd.NuPAGE TM 4 to 12 %, Bis-Tris, 1.0 mm mini protein gels were obtained from Life Technologies Limited, UK.We thank S. Elledge (University of California, San Francisco) for providing the trastuzumab M74 Fab expression vector.Protein A affinity chromatography and size exclusion chromatography were performed on an AKTA pure 25L purification system using an HiTrap Protein A HP 5 mL column (Cytiva) and HiLoad 16/600 Superdex 75 pg column (GE Healthcare), respectively.Protein concentration was determined using a Thermo Scientific Nanodrop One spectrophotometer.High resolution mass spectrometry data and intact protein mass spectrometry were recorded by Mass Spectrometry Service, Imperial College London using a Waters LCT Premier (ES-TOF) spectrometer.HPLC-MS analysis were conducted on a Waters 515 HPLC pump, 2998 photodiode array detector and 3100 mass detector using either a Waters XSelect CSH C18 Column, 130 Å, 5 μm, 4.6 mm x 100 mm column or a Waters XBridge BEH C18 Column, 130 Å, 5 μm, 4.6 mm x 100 mm column.Gamma counting was performed using a Wallac 1282 Compugamma Universal Gamma Counter.Peptide mapping analysis was performed by the BSRC Mass Spectrometry and Proteomics facility at the University of St Andrews.Analytical size exclusion radioHPLC traces were acquired using an Agilent 1260 series HPLC system with an in-line radioactivity detector (LabLogic Systems Limited 1"NaI/PMT Detector).A BioSep TM SEC-s2000 column (5 μM, 145 Å, 300 x 7.8 mm) was used with a mobile phase of PBS supplemented with sodium ethylenediamine tetraacetate (2 mM) at a flow rate of 1 mL/min.iTLC strips were visualised using a Lab Logic Dual Scan-RAM radio-TLC/HPLC Scanner.SDS PAGE gels were visualised using an Invitrogen iBrightFL1000 imaging system (bright view) and GE Healthcare Amersham Typhoon imager (autoradiography).

Preparation of Trastuzumab M74 Fab fragment
Trastuzumab Fab plasmid vector containing HC.M107L, LC.T74M mutations was kindly provided by Elledge et al. (University of California, San Francisco).The protein sequences of light chain and heavy chain are shown below.Fab fragment was expressed in OverExpress TM C43 (DE3) chemically competent cells (Merck) on a 2 L scale.C43 (DE3) cells were grown in Overnight Express TM Instant TB Medium (Merck) at 37 o C with shaking at 190 rpm until the optical density at 600 nm reached 0.75 after which the temperature was lowered to 30 o C and further incubated for 16 h.The cells were harvested by centrifugation (5000 g, 20 min), resuspended in 20 mM Phosphate buffer, and lysed by sonication (500 W, 20 % amplitude, 15 s on 45 s off for 10 min).Cell lysate was collected by centrifugation (38758 g, 30 min) and the supernatant was pass through a 0.45 μm syringe filter prior to purification.Purification: 1) Cell lysate was first applied to a 5 mL HiTrap Protein A HP column (binding buffer: Phosphate-buffered saline (PBS), elution buffer: 0.1 M sodium citrate buffer pH 3) on an AKTA pure 25L purification system followed by 2) size exclusion chromatography using a HiLoad 16/600 Superdex 75 pg column (GE Healthcare, UK) (Elution buffer: PBS).Samples for ESI-TOF MS analysis were prepared at 10 μM in 0.1 M ammonium acetate or in H2O (desalted using 0.5 mL Zeba 7-kDa desalting columns, 1500 g, 2 min).Samples were additionally analysed by SDS-PAGE.
Heavy Chain Sequence:

Preparation of Trastuzumab WT Fab fragment
Following a literature procedure. 3Trastuzumab was supplied as the biosimilar Herzuma in solution (21 mg/mL) from the Pharmacy Department at Guy's and St. Thomas' NHS Trust, London.Briefly, Trastuzumab IgG (15 mg, final concentration: 67 μM) was reacted with 1.7 % wt/wt of immobilised papain (250 μg/mL) for 22 h at 37 o C in a buffer containing 20 mM sodium phosphate monobasic, 10 mM disodium EDTA and 80 mM cysteine HCl (pH 7), cysteine HCl was added immediately before digestion.After digestion, Tris.HCl buffer was added (pH 7.5, 2 mL) and the mixture centrifuged (3200 g, 10 min).The supernatant containing the Fab and Fc fragments were collected.Purification was conducted using a 20 mL HIPrep Q HP anion exchange column on an AKTA Pure 25L purification system (binding buffer: 50 mM Tris-HCl pH 8 buffer, elution buffer: 1 M NaCl in 50 mM Tris HCl pH 8 buffer).Samples for ESI-TOF MS analysis were prepared at 10 μM in 0.1 M ammonium acetate or in H2O (desalted using 0.5 mL Zeba 7-kDa desalting columns, 1500 g, 2 min).Samples were additionally analysed by SDS-PAGE.

Peptide mapping analysis
Proteins were diluted to 10 μM with ammonium bicarbonate and digested with trypsin.The samples were analysed by nanoLCMSMS on a Sciex 5600 QTof mass spectrometer coupled with Sciex Eksigent 425 nanoLC.The LC was configured in trap elute format, with Waters acuity UPLC M class symmetry trap column 100 Å 5 μm 2 G 180 μm x 20 mm trap and Waters acquity UPLC Mclass HSS1 1.8 μm 75 μm x 150 mm column, both Waters.5μL of sample was injected onto the trap in 15 μL/min of loading buffer (0.05 % Trifluoroacetic acid in water), and run for 5 min.The trap was switched in line with the analytical column and the sample eluted at a gradient over 35 min (A = 100 % water with 0.1 % formic acid, B = 20 % water 80 % acetonitrile, 0.1 % formic acid, 2 % A to 1 min, linear to 40% A over 25 min, linear to 95 % A over 4 min, hold for 1 min, linear back to 2 % A, and re-equilibrate for 4 min).The flow from the column was sprayed directly into the nanospray orifice at a voltage of 2300 V positive ionisation.Mass spectrometry data was collected from 350 to 1250 m/z for the survey scan with the top 20 most intense peaks selected by Data Dependant Acquisition conditions for MSMS with collision induced dissociation (CID) fragmentation.Raw data was exported and extracted using ms convert (ProteoWizard).The data searched using Mascot search engine (MatrixScience) against an internally generated database of 6700 protein sequences.Settings were 20 ppm on the MS and 0.1Da on the MSMS data, with variable oxidation of methionine.For the identification of the Trastuzumab modification (C(6) H(9) N(5) O 167.1686 Da) was set as a variable modification on methionine residues.

Cell culture for HER2 in vitro binding studies
The human breast cancer cell lines HCC1954 (cultured in RPMI-1640) and MDA-MB-231 (cultured in DMEM low glucose (1 g/L)) were purchased from American Type Culture Collection.Growth media were supplemented with 10 % foetal bovine serum (FBS), 100 units/mL penicillin, 100 µg/mL streptomycin, and 2 mM ʟ-glutamine.Cell lines were harvested twice weekly using a formulation of 0.25 % trypsin/0.53mM EDTA in Phosphate Buffered Salt (PBS) Solution without calcium and magnesium, which was then neutralised with the appropriate medium containing FBS.Cells were maintained in a humidified chamber containing 5 % CO2 at 37 o C.

FigureFigure
Figure S7 ESI-deconvoluted mass spectrum of the reaction between trastuzumab N3-WT and DBCO-PEG4-DFO, to yield a mixture containing unmodified WT Fab and DFO-WT.

FigureFigureFigure
Figure S9 ESI-deconvoluted mass spectra of the reaction between trastuzumab WT Fab and 20 eq. of Ox-N3 1. Signals observed at 47566 m/z (labelled red) corresponds to unmodified WT Fab and 47732 m/z (labelled blue) corresponds to singly modified WT Fab conjugate and 47898 m/z (labelled green) corresponds to dual modified WT Fab conjugate.

Figure
Figure S12 SDS PAGE analysis (Left: bright view image, Right: autoradiography) of [ 68 Ga]Ga-DFO-M74 and [ 68 Ga]Ga-DFO-WT from crude labelling reactions (1 and 2) or after Zeba spin purification (3 and 4).Unchelated [ 68 Ga]Ga migrates to bottom of SDS-gel.The radioactivity signal from the radiolabelled conjugates were coincident with the stained protein bands corresponding to the M74 and WT Fab fragments.