Site-Specific Photoaffinity Bioconjugation for the Creation of ⁸⁹Zr-Labeled Radioimmunoconjugates

Abstract

Purpose:

Site-specific approaches to bioconjugation produce well-defined and homogeneous immunoconjugates with potential for superior in vivo behavior compared to analogs synthesized using traditional, stochastic methods. The possibility of incorporating photoaffinity chemistry into a site-specific bioconjugation strategy is particularly enticing, as it could simplify and accelerate the preparation of homogeneous immunoconjugates for the clinic. In this investigation, we report the synthesis, in vitro characterization, and in vivo evaluation of a site-specifically modified, ⁸⁹Zr-labeled radioimmunoconjugate created via the reaction between an mAb and an Fc-binding protein bearing a photoactivatable 4-benzoylphenylalanine residue.

Procedures:

A variant of the Fc-binding Z domain of protein A containing a photoactivatable, 4-benzoylphenylalanine residue — Z(35BPA) — was modified with desferrioxamine (DFO), combined with the A33 antigen-targeting mAb huA33, and irradiated with UV light. The resulting immunoconjugate — DFO^Z(35BPA)-huA33 — was purified and characterized via SDS-PAGE, MALDI-ToF mass spectrometry, surface plasmon resonance, and flow cytometry. The radiolabeling of DFO^Z(35BPA)-huA33 was optimized to produce [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33, and the immunoreactivity of the radioimmunoconjugate was determined with SW1222 human colorectal cancer cells. Finally, the in vivo performance of [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 in mice bearing subcutaneous SW1222 xenografts was interrogated via PET imaging and biodistribution experiments and compared to that of a stochastically labeled control radioimmunoconjugate, [⁸⁹Zr]Zr-DFO-huA33.

Results:

HuA33 was site-specifically modified with Z(35BPA)-DFO, producing an immunoconjugate with on average 1 DFO/mAb, high in vitro stability, and high affinity for its target. [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 was synthesized in 95% radiochemical yield and exhibited a specific activity of 2 mCi/mg and an immunoreactive fraction of ~0.85. PET imaging and biodistribution experiments revealed that high concentrations of the radioimmunoconjugate accumulated in tumor tissue (i.e. ~40 %ID/g at 120 h p.i.) but also that the Z(35BPA)-bearing immunoPET probe produced higher uptake in the liver, spleen, and kidneys than its stochastically modified cousin, [⁸⁹Zr]Zr-DFO-huA33.

Conclusions:

Photoaffinity chemistry and an Fc-binding variant of the Z domain were successfully leveraged to create a novel site-specific strategy for the synthesis of radioimmunoconjugates. The probe synthesized using this method — DFO^Z(35BPA)-huA33 — was well-defined and homogeneous, and the resulting radioimmunoconjugate ([⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33) boasted high specific activity, stability, and immunoreactivity. While the site-specifically modified radioimmunoconjugate produced high activity concentrations in tumor tissue, it also yielded higher uptake in healthy organs than a stochastically modified analog, suggesting that optimization of this system is necessary prior to clinical translation.

INTRODUCTION

Radiolabeled antibodies and antibody fragments — radioimmunoconjugates — have proven to be critical tools in diagnostic, theranostic, and therapeutic nuclear medicine [1–2]. Until recently, the overwhelming majority of radioimmunoconjugates were synthesized via stochastic methods centered on the modification of the terminal amine of lysines with chelators or radiolabeled prosthetic groups [3]. While facile, this approach to bioconjugation produces poorly defined and heterogeneous immunoconjugates and can yield products with suboptimal immunoreactivity due to the inadvertent modification of the biomolecule’s complementarity-determining regions [4–5]. To circumvent these issues, a great deal of work has been dedicated to the development of site-specific and site-selective bioconjugation strategies that promise the creation of well-defined and homogeneous immunoconjugates with intact immunoreactivity [6–8]. A variety of effective strategies have been devised, including those focused on cysteine residues, the heavy chain glycans, peptide tags, and unnatural amino acids [9]. Yet each of these strategies come with drawbacks in tow. To wit, the use of peptide tags and unnatural amino acids necessitates genetic engineering; the modification of thiols requires the reduction of disulfide linkages and can still produce heterogeneous mixtures; and the prevailing chemoenzymatic methods for the manipulation of the heavy chain glycans can take days to complete [10]. The exigencies of clinical translation can complicate matters even more. For example, the use of bacterial enzymes (for the modification of the glycans) or sophisticated expression systems (for the use of unnatural amino acids) can be particularly challenging under GMP conditions. In light of this, there is still an unmet need for site-specific bioconjugation strategies that are simultaneously selective, simple, and — critically — clinically translatable.

In this investigation, we combine a small Fc domain-binding protein and photochemistry to create an approach to the synthesis of site-specifically modified radioimmunoconjugates. Staphylococcal protein A (SpA) is a well-known immunoglobulin-binding molecule [11]. Engineering two positions in SpA’s B domain creates a new variant: the Z domain. The Z domain is small, stable, and — most importantly for our purposes — has a well characterized binding site for the Fc region of immunoglobulins (Figure 1A) [12]. Our laboratories (and others) have previously created a variant of the Z domain — Z(35BPA) — that contains an unnatural 4-benzoylphenylalanine (BPA) residue (Figure 1B). BPA contains a benzophenone group that forms a diradical upon UV irradiation that can subsequently form covalent crosslinks with neighboring molecules (Figure 1C) [14]. In the case of Z(35BPA), these UV-induced crosslinks facilitate the site-specific bioconjugation of antibodies on the Fc region. This technology has been successfully leveraged for the site-specific modification of antibodies with biotin, fluorophores, DNA oligonucleotides, and peptide nucleic acids, but it has never been employed for the synthesis of radiolabeled mAbs [15–18]. We believe that this technology could provide three key advantages over extant strategies for the construction of site-specifically modified radioimmunoconjugates: (1) it is relatively fast, (2) it eschews the prior manipulation of the antibody, and (3) the resulting immunoconjugate could be purified easily with size exclusion chromatography. Admittedly, however, the use of a large, proteinaceous bioconjugation reagent could increase the odds of immunogenicity.

Figure 1.

(A) Ribbon illustration of a Z domain (gray) binding the Fc region of human IgG₁ (figure prepared based on PDB 5U4Y [13]). (B) The chemical structure of the unnatural amino acid benzoylphenylalanine (BPA). (C) The reaction scheme of the benzophenone group of BPA entering a diradical triplet intermediate state upon irradiation with UV light and cross-linking with a ligand to form a covalent linkage.

Herein, we report the first application of this technology to the synthesis of radioimmunoconjugates. Specifically, we performed the photoaffinity labeling of a colorectal cancer-targeted mAb (huA33) with a desferrioxamine (DFO)-bearing variant of Z(35BPA) [Z(35BPA)-DFO]. The resulting site-specifically modified immunoconjugate — DFO^Z(35BPA)-huA33 — was comprehensively chemically and biologically characterized and radiolabeled with [⁸⁹Zr]Zr⁴⁺ to produce [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33. This final radioimmunoconjugate was then characterized in vitro, and its in vivo performance was compared to that of a stochastically modified analogue in a murine model of colorectal cancer.

MATERIALS AND METHODS

General

All reagents were purchased from Fisher Scientific (Thermo Fisher Scientific; Waltham, MA, USA) unless otherwise noted. The huA33 antibody was produced by the Olivia Newton-John Cancer Research Institute as previously described [19–20]. Protein concentration measurements were performed via UV-Vis spectroscopy with an extinction coefficient of 2.1 × 10⁵ M⁻¹ cm⁻¹ and a molecular weight of 1.5 × 10⁵ Da. All water used was ultrapure (>18.2 MΩcm⁻¹ at 25 °C). p-SCN-Bn-DFO was purchased from Macrocyclics, Inc. (Plano, TX, USA). MALDI-ToF mass spectrometry was performed by the Alberta Proteomics and Mass Spectrometry Facility (University of Alberta; Edmonton, AB, Canada). [⁸⁹Zr]Zr⁴⁺ was provided in 1 M oxalic acid by 3D Imaging, Inc. (Little Rock, AR, USA).

Production of Z(35BPA)-DFO

The unnatural amino acid BPA-containing domain — Z(35BPA) — was recombinantly produced utilizing the Amber suppression system. The Amber codon-containing plasmid pAY430-Z(35BPA) (constructed/used by Stiller [16]) was used as a template for the cloning of the Z(35BPA)-Cys construct with a cysteine at the C-terminus. The DNA sequence coding for Z(35BPA) was PCR-amplified using primers containing NdeI and XhoI restriction sites. The codon for the C-terminal cysteine was inserted into the sequence via the reverse primer during the amplification. The amplified insert was double digested with the enzymes and subcloned into the cut plasmid pET26 containing a His₆-tag for IMAC purification, resulting in the final expression vector pET26-Z(35BPA)-Cys-H₆. After confirming the plasmid sequence by Sanger sequencing, expression and IMAC purification of Z(35BPA)-Cys was performed as previously described [16]. The purity of the final product was analyzed by SDS-PAGE (Thermo Ultimate3000; Thermo Fisher Scientific; Waltham, MA, USA), and electrospray ionization-mass spectroscopy (ESI-MS) (Bruker Impact II; Bruker Daltonics; Billerica, MA, USA) was used to confirm the molecular weight.

Conjugation of the desferrioxamine chelator to Z(35BPA)-Cys was achieved via maleimidethiol coupling through the reaction between the thiol side chain of the C-terminal cysteine residue and a maleimide-functionalized variant of desferrioxamine (DFO-mal) (Macrocyclics, Inc.). Z(35BPA)-Cys dissolved in Chelex-treated PBS (pH 7.4) was reduced with 10 mM TCEP (tris(2-carboxyethyl)phosphine) before conjugation to reduce dimer formation. The conjugation mixture containing Z(35BPA)-Cys (~1.5 mg/mL) and 4 molar equivalents of DFO-mal was incubated for 1–2 hours at room temperature. The conjugation reaction was monitored with MALDI-ToF (MALDI ToF/ToF Analyzer; SCIEX Applied Biosystems; Foster City, CA, USA), and the product was purified with size exclusion chromatography (PD-10 Column; GE Healthcare; Chicago, IL, USA). The final Z(35BPA)-DFO product was lyophilized from 10 mM ammonium acetate (pH 4.5) and stored at −20 °C.

Bioconjugation of DFO^Z(35BPA)-huA33

HuA33 in Chelex-treated PBS (pH 7.4) was diluted to a final concentration of 1.0 mg/mL. Z(35BPA)-DFO (5.0 equiv.) was resuspended in Chelex-treated PBS (pH 7.4) and added to the solution of huA33. The mixture was placed on ice and irradiated with a lamp producing a narrow distribution of UV light centered on 370 nm for 2 h (Kessil PR160L, Kessil Lighting; Richmond, CA, USA).The ice was replaced every 30 min to dissipate the heat generated from the lamp and to cool the reaction. After 2 h, the reaction was quenched with 100 mM glycine-HCl (pH 2.5), washed with PBS, and concentrated with a 50 μL Amicon filter with a 50 kDa molecular weight cut-off (MilliporeSigma).

Bioconjugation of DFO-huA33

HuA33 antibody in Chelex-treated PBS (pH 7.4) was diluted to a final concentration of 1.0 mg/mL. The pH of the solution was increased to 8.9 with 0.1 M Na₂CO₃. p-SCN-Bn-DFO in DMSO (12.5 mg/mL, 20 equiv.) was slowly added to the solution of antibody and thoroughly mixed. The mixture was incubated for 1 h on an agitating ThermoMixer at 37 °C. After 1 h, the reaction was purified with size exclusion chromatography (PD-10 Column), and the final product was concentrated using a 2 mL Amicon Ultra centrifugal filter with a 50 kDa molecular weight cut-off (MilliporeSigma) [21].

Gel Electrophoresis

HuA33, DFO-huA33, and DFO^Z(35BPA)-huA33 were prepared for SDS-PAGE according to the manufacturer’s instructions (NuPAGE^™, ThermoFisher). In brief, 2 μg of each immunoglobulin were reduced with dithiothreitol and lithium dodecyl sulfate loading buffer and diluted to 20 μL using HPLC-grade water (MilliporeSigma). The samples were then denatured with heat on a ThermoMixer set to 80 °C and mixed for 15 min at 300 rpm. Subsequently, the samples were loaded to the wells of a NuPAGE^™ 4–12% Bis-Tris Protein Gel, and the gel box was filled with diluted NuPAGE^™ MOPS SDS Running Buffer. 10 μL of a Novex^™ Sharp Pre-Stained Protein Ladder was loaded to the outermost wells. The gel was allowed to run at 70 V until the lowest molecular weight ladder band reached the bottom of the gel. The gel was washed 3× with MilliQ H₂O, stained with SimplyBlue^™ SafeStain (ThermoFisher) for 90 min, and washed 3× more with MilliQ H₂O. The gel was imaged using a LI-COR Odyssey^® CLx instrument and analyzed using Image Studio^™ Acquisition Software.

Cell Culture

The human colorectal cancer cell line SW1222 was obtained from Sigma-Aldrich and maintained under sterile conditions in Iscove’s Modified Dulbecco’s Medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 units/mL penicillin, and 100 units/mL streptomycin. The cells were allowed to grow in a 37 °C environment with 5% CO₂ and were passaged every 7 days or upon reaching 80% confluency with 0.25% trypsin / 0.53 mM EDTA in Hank’s Balanced Salt Solution without calcium and magnesium. Cells were not used for in vitro or in vivo experiments beyond the fifteenth passage, and routine mycoplasma tests revealed no contamination. All media were purchased from the Media Preparation Core at Memorial Sloan Kettering Cancer Center.

Flow Cytometry

2.0 × 10⁶ A33-expressing SW1222 cells were aliquoted per sample and washed 3× with ice-cold PBS. 50 μL of huA33, DFO-huA33, or DFO^Z(35BPA)-huA33 (12 μg/mL) were added to the cells, and the resultant suspensions were incubated on ice for 30 min. After 30 min, the cells were washed 3× with ice-cold PBS. Then, 50 μL of goat anti-human IgG Alexa-488 secondary antibody (12 μg/mL) were added to the cells, and the resultant suspensions were incubated on ice for 30 min in the dark. After 30 min, the cells were washed 3× with ice-cold PBS. Following the washes, the cell pellets were resuspended in FACS buffer (PBS + 0.05% FCS + 2 mM EDTA). The samples were analyzed using a Becton-Dickinson Biosciences FACSCalibur Flow Cytometer, and the data were analyzed using FlowJo^™ Software. All suspensions were washed by centrifuging at 650 rcf for 2.5 min.

Surface Plasmon Resonance

HuA33, DFO-huA33, and DFO^Z(35BPA)-huA33 were analyzed for their affinities for their target — the A33 antigen — using surface plasmon resonance (Nicoya OpenSPR). Per the manufacturer’s instructions, protein A was immobilized onto an activated carboxyl sensor using a Nicoya OpenSPR kit. The antibodies were diluted in running buffer (Cytiva HBS-P Buffer + PBS-BSA) and captured on the protein A surface. Then, a multicycle kinetics experiment was performed by flowing 1.23 nM, 3.4 nM, 11 nM, 33 nM, and 100 nM solutions of the A33 antigen in running buffer over the sensor for 300 s. 10 mM glycine-HCl (pH 1.5) was used as a regeneration solution between each antigen injection to strip the mAb from the protein A prior to the subsequent injection of antigen. Blank buffer runs were subtracted from the results and the kinetics parameters were determined using TraceDrawer. The A33 antigen was purchased from Sino Biological.

Radiolabeling

DFO^Z(35BPA)-huA33 and DFO-huA33 (0.2 mg) were diluted in Chelex-treated PBS (pH 7.4). [⁸⁹Zr]Zr⁴⁺ [74.0 – 185.0 MBq (2.0 – 5.0 mCi)] in 1.0 M oxalic acid was diluted with Chelex-treated PBS, and the pH of the solution was adjusted to 7.4 with 1.0 M Na₂CO₃. The mAb solutions were combined with pH-adjusted zirconium-89, mixed thoroughly, and incubated on a ThermoMixer for 1 h at 37 °C and 500 rpm. The reaction was monitored using glass fiber silica-impregnated instant thin-layer chromatography (iTLC) paper (Pall Corp.; East Hills, NY, USA) using an EDTA solution as the eluent (50 mM, pH 5.5). The iTLC plates were analyzed on an AR-2000 radioi-TLC plate reader with WinScan Software (Bioscan, Inc.; Washington, DC, USA). Following the reaction, the radioimmunoconjugates were purified using size exclusion chromatography (PD-10 Column). Radiochemical purities were assayed using radio-iTLC with EDTA as the eluent (50 mM, pH 5.5).

Immunoreactivity

The immunoreactivities of the radioimmunoconjugates were interrogated using a saturation binding assay. 2 × 10⁷ A33-expressing SW1222 cells were washed with ice-cold media and resuspended in fresh media. 5 ng of either [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 or [⁸⁹Zr]Zr-DFO-huA33 were administered to the cells, and the resultant suspensions were allowed to incubate on ice for 1 h (the tubes containing the cells and the radioimmunoconjugates were mixed every 15 min). In additional cohorts, the A33 antigens were also blocked with cold immunoconjugates in PBS-BSA. After incubation, the supernatant was collected, and the cells were washed 3× with ice-cold PBS and subsequently analyzed on a ⁸⁹Zr-calibrated gamma counter. The activities (counts/minute) were background- and decay-corrected to the start of the run, and the immunoreactivities were expressed as a percentage by comparing the activity remaining in the cells to those of the supernatant and washes.

Animal Models and Subcutaneous Xenografts

Five-to-seven week old athymic nude mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and allowed to acclimatize for one week prior to inoculation. The animals were housed in ventilated cages and given food and water ad libitum. The mice were anesthetized by inhalation of 2% isoflurane/oxygen gas mixture (Baxter Healthcare; Deerfield, IL, USA). The injection site was sanitized with ethanol and tumors were induced by the subcutaneous injection of 5 × 10⁶ SW1222 cells in a 1:1 mixture of media:MatriGel in the right shoulder (Corning Life Sciences; Corning, NY, USA). To ensure homogenous tumors, the implantation suspension was thoroughly mixed prior to each inoculation. Tumors reached ideal size for PET imaging and biodistribution studies (~100 mm³) after 2 weeks. All animal work was approved by the IACUCs of Weill Cornell Medicine, Hunter College, and Memorial Sloan Kettering Cancer Center.

PET Imaging

PET images were obtained using a microPET Focus 120 small animal scanner (Siemens Medical Solutions; Malvern, PA, USA). Once the SW1222 tumors reached an appropriate size for experiments (~100 mm³), [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 or [⁸⁹Zr]Zr-DFO-huA33 were administered to the mice via intravenous tail vein injection. All mice underwent static scans every 24 h until 120 h post-injection for scan times ranging from 10–30 minutes. The counting rates in the reconstructed images were converted to activity concentrations (percent injected dose per gram of tissue [%ID/g]) using a system calibration factor derived from the imaging of a mouse-sized water-equivalent phantom containing ⁸⁹Zr. Maximum intensity projection (MIP) images were generated from 3-dimensional ordered subset expectation maximum reconstruction (3D-OSEM). The images were analyzed with ASIPro VM^™ (Concorde Microsystems; Knoxville, TN, USA).

Biodistribution Experiments

After the final PET imaging timepoint, the animals were euthanized by CO_2(g) asphyxiation followed by cervical dislocation. The most relevant organs were harvested, rinsed in water, dried, weighed, and quantified on a ⁸⁹Zr-calibrated PerkinElmer Wizard² γ-counter. The counts/minute in each tissue was background- and decay-corrected to the start of the activity measurement. The %ID/g for each sample was calculated by normalization to the total injected activity.

RESULTS

Model System

The model system for this study is centered on huA33, a humanized mAb that targets the A33 antigen, a transmembrane glycoprotein that is expressed in more than 95% of human colorectal cancers [22]. We chose to radiolabel the antibody with ⁸⁹Zr, the current radionuclide-of-choice for immunoPET due to the alignment of its physical half-life (t_1/2 ~ 3.3 d) with the multi-day biological half-life of an mAb [23]. Accordingly, we employed the chelator desferrioxamine (DFO), an acyclic linear siderophore that provides the radiometal with six oxygen donor atoms and has been used in the overwhelming majority of preclinical and clinical studies with ⁸⁹Zr-labeled probes. Importantly, ⁸⁹Zr-labeled variants of huA33 have already been employed for immunoPET, which we viewed as advantageous as it would enable comparisons with previously obtained data; after all, the focus of this investigation was the bioconjugation strategy rather than the radioimmunoconjugate itself [24–26]. Finally, we chose SW1222 human colorectal cancer cells for in vitro and in vivo studies, as they are well known to overexpress our target: the A33 antigen [27–28].

Synthesis of Z(35BPA)-DFO

A Z domain containing the photoactivatable amino acid BPA was used for site-specific photoaffinity labelling of the huA33 antibody. For efficient UV crosslinking to a human IgG₁ antibody, BPA is introduced in position 35 of the Z domain. Z(35BPA) was modified with a C-terminal cysteine for functionalization with a DFO chelator for radiolabeling of the final DFO^Z(35BPA)-mAb complex. The production of Z(35BPA)-Cys was performed recombinantly using the Amber suppression system allowing for incorporation of the unnatural amino acid BPA. Following expression and IMAC purification, the resulting final product displayed high purity and the correct molecular weight when analyzed with SDS-PAGE and MALDI-ToF (Figures S1–S2).

The desferrioxamine chelator was coupled to the Z domain via maleimide chemistry. The reaction mixture containing an excess of maleimide-modified DFO was incubated with Z(35BPA)-Cys at room temperature. Samples taken after 1 and 2 h of incubation were analyzed by MALDI-ToF and showed that the reaction is complete after 2 h with no detectable unreacted starting material (Z(35BPA)-Cys) left in the reaction mixture (Figure S3). Unreacted DFO-mal was removed by gel filtration using a PD-10 Column, and the molecular weight of the final product was confirmed with MALDI-ToF.

Photoaffinity Bioconjugation of huA33 with Z(35BPA)-DFO

The photoaffinity labeling of huA33 was achieved via a facile one-pot reaction in which the mAb was incubated with Z(35BPA)-DFO and irradiated with UV light at 370 nm (Figure 2A). An experiment in which the mAb was irradiated in the presence of Z(35BPA)-DFO for 5 min, 10 min, 15 min, 1 h, 2 h, and 4 h suggests that the reaction is complete after around ~20 min (Figure S4). Furthermore, additional optimization experiments using different molar ratios of Z(35BPA)-DFO to huA33 confirm that a ratio of 5:1 was sufficient for reaching a 1:1 conjugation yield. With these data, an irradiation time of 2 h and a molar ratio of 5:1 Z(35BPA)-DFO to huA33 were chosen for the production of the immunoconjugate at the center of this investigation. After the irradiation, the reaction was quenched with 100 mM glycine-HCl (pH 2.5) and purified using centrifugal filtration, ultimately yielding the modified mAb — DFO^Z(35BPA)-huA33 — in >80% yield. As a control, a second immunoconjugate of huA33 — DFO-huA33 — was synthesized via traditional stochastic methods using p-SCN-Bn-DFO (Figure 2B). Both immunoconjugates were purified via centrifugal filtration or size exclusion chromatography prior to chemical and biological characterization.

Figure 2.

(A) The site-specific bioconjugation and radiosynthesis of [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 (top) and (B) the random lysine bioconjugation and radiosynthesis of [⁸⁹Zr]Zr-DFO-huA33.

Structural and Biological Characterization

The next step of the investigation was the comprehensive chemical and biological characterization of the immunoconjugates. SDS-PAGE was performed to interrogate the site-specificity of the photoaffinity ligation. To this end, huA33, DFO-huA33, and DFO^Z(35BPA)-huA33 were analyzed on a denaturing gel (Figure 3A). The attachment of the DFO-bearing Z domain clearly splits the heavy chain band of the immunoconjugate into two bands of equal intensity: one with the same molecular weight as that of parental huA33 and another ~10 kDa heavier. The binding site for the Z domain is located in the Fc region of the antibody. Upon irradiation, the photoreaction creates two distinct types of heavy chains: one with and one without Z(35BPA)-DFO attached. Critically, no corresponding shift is observed in the light chain of DFO^Z(35BPA)-huA33 compared to huA33, demonstrating the selectivity of the reaction. Furthermore, subtle shifts in the molecular weights of both the light and heavy chains can be observed between huA33 and DFO-huA33, underscoring the random nature of the stochastic lysine conjugation. Quantitative colorimetric analysis of the gel determined a degree-of-labeling (DOL) of ~1 Z domain/mAb (and thus 1 DFO/mAb) for DFO^Z(35BPA)-huA33, while MALDI-ToF mass spectrometry revealed a DOL of ~2.1 DFO/mAb for DFO-huA33 (Figure S5).

Figure 3.

Structural, biological, and radiochemical evaluation of the mAbs. (A) SDS-PAGE analyzing the composition of huA33, DFO-huA33, and DFO^Z(35BPA)-huA33. (B) Fluorescence-associated cell sorting of huA33, huA33, DFO-huA33, and DFO^Z(35BPA)-huA33 with A33-expressing SW1222 cells and an AlexaFlour^™ 488 secondary antibody. (C) Longitudinal stability study of [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 in PBS over 5 days. (D) Cell-based immunoreactivity assay comparing [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 and [⁸⁹Zr]Zr-DFO-huA33 with A33-expressing SW1222 cells.

Flow cytometry and surface plasmon resonance were used to assess the binding of DFO^Z(35BPA)-huA33 and DFO-huA33 to their target antigen. Flow cytometry was first performed with A33 antigen-expressing SW1222 cells. The data revealed that huA33, DFO-huA33, and DFO^Z(35BPA)-huA33 all bound A33 in a similar manner, suggesting that the modification of the latter pair did not significantly impede their abilities to target membrane-bound antigen (Figure 3B). Surface plasmon resonance data reinforced these findings (Figure S6). The K_D values obtained for the three immunoglobulins were strikingly similar: 3.0 × 10⁻⁹ M (huA33), 5.3 × 10⁻⁹ M (DFO-huA33), and 7.3 × 10⁻⁹ M (DFO^Z(35BPA)-huA33). Similar antigen binding on- (k_a) and off- (k_d) rates further illustrate that the attachment of Z(35BPA)-DFO — and p-SCN-Bn-DFO, for that matter — does not perturb the biological activity of the antibody (Table 1).

Table 1.

Surface plasmon resonance binding parameters of huA33, DFO-huA33, and DFO^Z(35BPA)- huA33 for A33

Immunoconjugate	ka (M−1s−1)	kd (s−1)	KD (M)
huA33	9.1 × 104 (± 2.9 × 101)	2.7 × 10−4 (± 1.8 × 10−5)	3.0 × 10−9 (± 2.0 × 10−10)
DFO-huA33	1.2 × 105 (± 1.1 × 102)	6.5 × 10−4 (± 1.5 × 10−6)	5.3 × 10−9 (± 1.7 × 10−11)
DFOZ(35BPA)-huA33	1.3 × 105 (± 9.2 × 101)	9.4 × 10−4 (± 2.0 × 10−6)	7.3 × 10−9 (± 2.1 × 10−11)

Radiochemistry and the Characterization of the Radioimmunoconjugates

The pair of immunoconjugates were radiolabeled with [⁸⁹Zr]Zr⁴⁺ according to published protocols [29]. In brief, DFO^Z(35BPA)-huA33 and DFO-huA33 were incubated with [⁸⁹Zr]Zr⁴⁺ at a neutral pH on an agitating ThermoMixer at 37 °C for 1 h and subsequently purified via size exclusion chromatography. Radio-instant thin layer chromatography (radio-iTLC) and radio-size exclusion chromatography (radio-SEC) revealed that both radioimmunoconjugates were synthesized in high radiochemical yield (>95%) and high radiochemical purity (>99%) with specific activities ranging from 2–5 mCi/mg (Figure S7). The unconjugated chelator-bearing Z domain was radiolabeled as a control, yielding [⁸⁹Zr]Zr-DFO-Z(35BPA) in >95% yield and purity. Next, [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33, [⁸⁹Zr]Zr-DFO-huA33, and [⁸⁹Zr]Zr-DFO-Z(35BPA) were analyzed via radioactive SDS-PAGE and autoradiography to further evaluate the site-specificity of the bioconjugation reactions. [⁸⁹Zr]Zr-DFO-Z(35BPA) displayed a radioactive band at 9–10 kDa, while [⁸⁹Zr]Zr-DFO-huA33 had radioactive signal associated with both the heavy and light chains. [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33, in contrast, was only radiolabeled on the heavy chain, reinforcing the site-selectivity of the strategy (Figure S8). Importantly, both [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 and [⁸⁹Zr]Zr-DFO-Z(35BPA) displayed excellent stability in PBS: radio-iTLC and radio-SEC measurements displayed that the two radiolabeled constructs remained >92% and >89% stable to demetallation and aggregation, respectively, after 5 d at 37 °C (Figure 3C, Figure S9). Both [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 and [⁸⁹Zr]Zr-DFO-huA33 were determined to have high immunoreactive fractions — >0.85 ± 0.01 and >0.88 ± 0.03, respectively — with A33 antigen-expressing SW1222 human colorectal cells (Figure 3D). Finally, in an effort to probe the potential effects of UV irradiation on the reactivity of huA33, DFO-huA33 was irradiated for 2 h in a manner analogous to DFO^Z(35BPA)-huA33. Subsequently, the immunoconjugate was radiolabeled with [⁸⁹Zr]Zr⁴⁺, and the immunoreactive fraction of the [⁸⁹Zr]Zr-DFO-huA33 product was measured with SW1222 cells. Its immunoreactive fraction, ~0.85 ± 0.02, mirrors that of both [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 and unirradiated [⁸⁹Zr]Zr-DFO-huA33.

In Vivo Validation

The culmination of this investigation was the evaluation of the in vivo behavior of [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 in a murine model of human colorectal cancer. To this end, female athymic nude mice (n = 5 per group) were inoculated subcutaneously with 5 × 10⁶ A33 antigen-expressing SW1222 cells on the right shoulder. After the xenografts had reached a volume of ~100 mm³ (ca. ~14 d), the mice were intravenously administered 80 μCi of either [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 or — for comparison — [⁸⁹Zr]Zr-DFO-huA33 via tail vein injection. PET scans were acquired every 24 h over the next 5 d, after which the mice were euthanized and dissected to determine the acute biodistribution of the radioimmunoconjugates at 120 h p.i (Figure 4A–C). PET imaging revealed the specific accumulation of [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 in the tumor tissue as early as 24 h as well as its retention in the xenograft over the course of the 120 h experiment. The tumor is easily the tissue with the highest activity concentration, with the liver and kidneys the healthy organs with the highest uptake (as shown in the maximum intensity projections). The biodistribution data confirm these observations: at 120 h, the tumor boasts the highest concentration of [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 (39.6 ± 1.8 %ID/g), while the liver and spleen lie quite a bit lower (19.3 ± 3.4 %ID/g and 5.7 ± 2.9 %ID/g, respectively) (Figure 4D). While these data are promising, the in vivo performance of [⁸⁹Zr]Zr-DFO-huA33 clearly exceeded that of [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33. Both the PET and biodistribution data showed that the randomly labeled radioimmunoconjugate produced higher tumor uptake and lower activity concentrations in healthy tissues compared to the site-specifically modified variant. To wit, the tumoral activity concentrations of the two radioimmunoconjugates were 56.2 ± 5.1 %ID/g ([⁸⁹Zr]Zr-DFO-huA33) and 39.6 ± 1.8 %ID/g ([⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33) at 120 h p.i. while those in the liver at the same timepoint were 2.2 ± 0.4 %ID/g ([⁸⁹Zr]Zr-DFO-huA33) and 19.3 ± 3.4 %ID/g ([⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33) (Table S1). In the end, while the chemical, biological, and radiochemical characterization data for [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 were excellent, its suboptimal in vivo performance compared to [⁸⁹Zr]Zr-DFO-huA33 suggests that the system must be developed further prior to clinical translation.

Figure 4.

In vivo evaluation of the radioimmunoconjugates. Representative coronal PET images acquired 24, 48, 72, 96, and 120 h after the administration of (A) [⁸⁹Zr]Zr-DFO-Z(35BPA), (B) [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33, or (C) [⁸⁹Zr]Zr-DFO-huA33 to athymic nude mice bearing subcutaneous SW1222 colorectal cancer xenografts. (D) Ex vivo distribution data collected 120 h after the administration of [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 (red) and [⁸⁹Zr]Zr-DFO-huA33 (blue) to athymic nude mice bearing subcutaneous SW1222 xenografts in the right shoulder. Statistical significance was determined via a two-tailed t test with a Welch’s correction using GraphPad Prism: * = p <0.05; *** = p <0.001.

DISCUSSION

To the best of our knowledge, this is the first time that photoaffinity labeling has been harnessed for the preparation of site-specifically modified radioimmunoconjugates. This approach could offer several key advantages over extant methods. Unlike chemoenzymatic approaches, it is rapid. Unlike strategies predicated on peptide tags and unnatural amino acids, it employs native mAbs. And unlike thiol-targeted methodologies, it eschews the reduction of the immunoglobulin. Furthermore, the chelator-modified immunoconjugates could be separated simply and easily from excess Z(35BPA)-DFO via affinity chromatography. That said, it is important to note that we did have some concerns regarding the Z domain itself given that it is a relatively large (which could theoretically impact the pharmacokinetic profile of the immunoconjugate) and unnatural (which could theoretically lead to immunogenicity) modification.

From a chemical and in vitro biological perspective, the modification strategy worked extremely well, producing a well-defined and homogeneous immunoconjugate with unperturbed antigen binding ability relative to its parent mAb. Likewise, [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 was produced in high yield and specific activity while exhibiting high immunoreactivity and stability. Indeed, problems only emerged upon in vivo validation, in which [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 produced lower tumoral activity concentrations and higher background uptake in healthy organs (e.g. the liver and spleen) than [⁸⁹Zr]Zr-DFO-huA33. These data were especially surprising because this phenomenon — i.e. attenuated tumor uptake and heightened healthy organ retention — has not previously been observed with any other immunoconjugates created using this technology [17, 30]. Our earliest hypothesis was that free [⁸⁹Zr]Zr-DFO-Z(35BPA) could be responsible for the background uptake, but PET imaging experiments clearly demonstrated that the ⁸⁹Zr-labeled Z domain accumulated only in its excretory organs: the kidneys. Next, we hypothesized that the background uptake was caused by aggregates of [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33, but SE-HPLC measurements revealed that the radioimmunoconjugate did not aggregate to a significant degree (Figure S10). Finally, we theorized that increased affinity for FcγRI could be responsible for the enhanced uptake in the liver and spleen, as tissue-resident macrophages in both organs are known to express high levels of the receptor [31–33]. However, a subsequent ELISA illustrated that DFO^Z(35BPA)-huA33 has an affinity for the Fc receptor that is similar to that of DFO-huA33 and native huA33 (Figure S11). While other FcγR could be at the root of this problem, it is unlikely, as only FcγRI binds monomeric immunoglobulins. Ultimately, the explanation for this phenomenon remains elusive, though the entire body of work on this technology suggests that it may be unique to this antibody, cell line, or mouse model. Moving forward, we are interested in using smaller, truncated variants of Z(35BPA) that may simultaneously offer better affinity for the Fc domain and attenuate the influence of the conjugation method on the pharmacokinetic profile of the radioimmunoconjugate [34–36].

CONCLUSION

This investigation clearly demonstrates that photoaffinity labeling with Z(35BPA) is an effective route for the site-specific bioconjugation of homogeneous radioimmunoconjugates with high immunoreactivity and stability. However, our in vivo validation data suggest that optimization of the system — at least in the context of the huA33 mAb — is necessary before this platform can be considered for clinical translation.

ABBREVIATIONS

DFO

desferrioxamine

BPA

4-benzoylphenylalanine

Z(35BPA)

Z domain of protein A with 4-benzoylphenylalanine

ADC

antibody-drug conjugate

PET

positron emission tomography

DOL

degree of labeling

IgG

immunoglobulin

mAb

monoclonal antibody

SDS-PAGE

sodium dodecyl sulfate polyacrylamide gel electrophoresis

IMAC

immobilized metal affinity chromatography

MALDI-ToF

matrix-assisted laser desorption / time-of-flight mass spectrometry

IACUC

Institutional Animal Care and Use Committee

%ID/g

% of injected dose per gram

PBS

phosphate-buffered saline

DMSO

dimethyl sulfoxide

RPM

revolutions per minute

RCF

relative centrifugal force

EDTA

ethylenediaminetetreaacetic acid

iTLC

instant-thin layer chromatography