Site-specific modification of native IgGs with flexible drug-load.

Homogeneous, site-specifically conjugated antibodies have shown to result in antibody-drug conjugates (ADCs) with improved efficacy and tolerability compared to stochastically conjugated ADCs. However, precisely controlling the drug-load as well as attaching multiple payload moieties on the antibody remains challenging. Here, we demonstrate the simple and direct modification of native IgG-antibodies at the residue glutamine 295 (Q295) without the need for any protein engineering at flexible drug-to-antibody ratios of one or multiple payloads. The conjugation is enabled through short, positively charged lysine containing peptides and native, commercially available microbial transglutaminase. In proof-of-concept studies, HER2-targeting ADCs based on trastuzumab were generated with drug-to-antibody ratios (DARs) of 2 and 4 of the same or different payloads using orthogonal conjugation chemistries. Quantitative biodistribution studies performed with 111In-radiolabeled conjugates showed high tumour uptake and low accumulation of radioactivity in non-targeted tissues. A single dose study of trastuzumab conjugated to the highly potent payload α-Amanitin demonstrated complete and long-lasting tumour remissions and was well-tolerated at all dose levels tested.

Cadherin-17 as a target for the immunoPET of adenocarcinoma.

PURPOSE: Cadherin-17 (CDH17) is a calcium-dependent cell adhesion protein that is overexpressed in several adenocarcinomas, including gastric, colorectal, and pancreatic adenocarcinoma. High levels of CDH17 have been linked to metastatic disease and poor prognoses in patients with these malignancies, fueling interest in the protein as a target for diagnostics and therapeutics. Herein, we report the synthesis, in vitro validation, and in vivo evaluation of a CDH17-targeted 89Zr-labeled immunoPET probe. METHODS: The CDH17-targeting mAb D2101 was modified with an isothiocyanate-bearing derivative of desferrioxamine (DFO) to produce a chelator-bearing immunoconjugate - DFO-D2101 - and flow cytometry and surface plasmon resonance (SPR) were used to interrogate its antigen-binding properties. The immunoconjugate was then radiolabeled with zirconium-89 (t1/2 ~ 3.3 days), and the serum stability and immunoreactive fraction of [89Zr]Zr-DFO-D2101 were determined. Finally, [89Zr]Zr-DFO-D2101's performance was evaluated in a trio of murine models of pancreatic ductal adenocarcinoma (PDAC): subcutaneous, orthotopic, and patient-derived xenografts (PDX). PET images were acquired over the course of 5 days, and terminal biodistribution data were collected after the final imaging time point. RESULTS: DFO-D2101 was produced with a degree of labeling of ~ 1.1 DFO/mAb. Flow cytometry with CDH17-expressing AsPC-1 cells demonstrated that the immunoconjugate binds to its target in a manner similar to its parent mAb, while SPR with recombinant CDH17 revealed that D2101 and DFO-D2101 exhibit nearly identical KD values: 8.2 × 10-9 and 6.7 × 10-9 M, respectively. [89Zr]Zr-DFO-D2101 was produced with a specific activity of 185 MBq/mg (5.0 mCi/mg), remained >80% stable in human serum over the course of 5 days, and boasted an immunoreactive fraction of >0.85. In all three murine models of PDAC, the radioimmunoconjugate yielded high contrast images, with high activity concentrations in tumor tissue and low uptake in non-target organs. Tumoral activity concentrations reached as high as >60 %ID/g in two of the cohorts bearing PDXs. CONCLUSION: Taken together, these data underscore that [89Zr]Zr-DFO-D2101 is a highly promising probe for the non-invasive visualization of CDH17 expression in PDAC. We contend that this radioimmunoconjugate could have a significant impact on the clinical management of patients with both PDAC and gastrointestinal adenocarcinoma, most likely as a theranostic imaging tool in support of CDH17-targeted therapies.

Synthesis and Evaluation of a Cathepsin B-Recognizing Trifunctional Chelating Agent to Improve Tumor Retention of Radioimmunoconjugates.

Cathepsin B (CTSB) is a lysosomal protease that is overexpressed in tumor cells. Radioimmunoconjugates (RICs) composed of CTSB-recognizing chelating agents are expected to increase the molecular weights of their radiometabolites by forming conjugates with CTSB in cells, resulting in their improved retention in tumor cells. We designed a novel CTSB-recognizing trifunctional chelating agent, azide-[111In]In-DOTA-CTSB-substrate ([111In]In-ADCS), to synthesize a RIC, trastuzumab-[111In]In-ADCS ([111In]In-TADCS), and evaluated its utility to improve tumor retention of the RIC. [111In]In-ADCS and [111In]In-TADCS were synthesized with satisfactory yield and purity. [111In]In-ADCS was markedly stable in murine plasma until 96 h postincubation. [111In]In-ADCS showed binding to CTSB in vitro, and the conjugation was blocked by the addition of CTSB inhibitor. In the internalization assay, [111In]In-TADCS exhibited high-level retention in SK-OV-3 cells, indicating the in vitro utility of the CTSB-recognizing unit. In the biodistribution assay, [111In]In-TADCS showed high-level tumor accumulation, but the retention was hardly improved. In the first attempt to combine a CTSB-recognizing unit and RIC, these findings show the fundamental properties of the CTSB-recognizing trifunctional chelating agent to improve tumor retention of RICs.

Application of Microfluidic Devices for Automated Two-Step Radiolabeling of Antibodies.

Radioimmunoconjugates (RICs) composed of tumor-targeting monoclonal antibodies and radionuclides have been developed for diagnostic and therapeutic application. A new radiolabeling method using microfluidic devices is expected to facilitate simpler and more rapid synthesis of RICs. In the microfluidic method, microfluidic chips can promote the reaction between reactants by mixing them efficiently, and pumping systems enable automated synthesis. In this study, we synthesized RICs by the pre-labeling method, in which the radiometal is coordinated to the chelator and then the radiolabeled chelator is incorporated into the antibodies, using microfluidic devices for the first time. As a result of examining the reaction parameters including the material of mixing units, reaction temperature, and flow rate, RICs with radiochemical purity (RCP) exceeding 90% were obtained. These high-purity RICs were successfully synthesized without any purification simply by pumping three solutions of a chelating agent, radiometal, and antibody into microfluidic devices. Under the same conditions, the RCP of RICs labeled by conventional methods was below 50%. These findings indicate the utility of microfluidic devices for automatic and rapid synthesis of high-quality RICs.

The Chemistry of Creating Chemically Programmed Antibodies (cPAbs): Site-Specific Bioconjugation of Small Molecules.

Small-molecule drugs have been employed for years as therapeutics in the pharmaceutical industry. However, small-molecule drugs typically have short in vivo half-lives which is one of the largest impediments to the success of many potentially valuable pharmacologically active small molecules. The undesirable pharmacokinetics and pharmacology associated with some small molecules have led to the development of a new class of bioconjugates known as chemically programmed antibodies (cPAbs). cPAbs are bioconjugates in which antibodies are used to augment small molecules with effector functions and prolonged pharmacokinetic profiles, where the pharmacophore of the small molecule is harnessed for target binding and therefore biological targeting. Many different small molecules can be conjugated to large proteins such as full monoclonal antibodies (IgG), fragment crystallizable regions (Fc), or fragment antigen binding regions (Fab). In order to successfully and site-specifically conjugate small molecules to any class of antibodies (IgG, Fc, or Fab), the molecules must be derivatized with a functional group for ease of conjugation without altering the pharmacology of the small molecules. In this Review, we summarize the different synthetic or biological methods that have been employed to produce cPAbs. These unique chemistries have potential to be applied to other fields of antibody modification such as antibody drug conjugates, radioimmunoconjugates, and fluorophore-tagged antibodies.

Anti-HER2 monoclonal antibody based-radioimmunoconjugates: Assessment of the chelating agent influence.

In the present work, the radioimmunoconjugates 111In-DTPA-trastuzumab and 177Lu-DOTA-trastuzumab were evaluated regarding the influence of the chelating agents on the physical-chemical parameters and human epidermal growth factor receptor 2 (HER2) tumor cell binding. Data showed that both chelating agents, at predetermined molar ratios (antibody:chelator - 1:10 and 1:20), did not influence the immunoconjugates integrity, the radiolabeling process and the radiolabeled antibodies stability. However, differences were observed in the lipophilic feature between DOTA and DTPA radioimmunoconjugates and in the specific binding to SK-BR-3 tumor cells (HER2 positive). Therefore, this study showed the importance of assessing the influence of chelating agents and their molar ratios in the development process of radioimmunoconjugates.

Re-Evaluations of Zr-DFO Complex Coordination Chemistry for the Estimation of Radiochemical Yields and Chelator-to-Antibody Ratios of 89Zr Immune-PET Tracers.

(1) Background: Deferoxamine B (DFO) is the most widely used chelator for labeling of zirconium-89 (89Zr) to monoclonal antibody (mAb). Despite the remarkable developments of the clinical 89Zr-immuno-PET, chemical species and stability constants of the Zr-DFO complexes remain controversial. The aim of this study was to re-evaluate their stability constants by identifying species of Zr-DFO complexes and demonstrate that the stability constants can estimate radiochemical yield (RCY) and chelator-to-antibody ratio (CAR). (2) Methods: Zr-DFO species were determined by UV and ESI-MS spectroscopy. Stability constants and speciation of the Zr-DFO complex were redetermined by potentiometric titration. Complexation inhibition of Zr-DFO by residual impurities was investigated by competition titration. (3) Results: Unknown species, ZrHqDFO2, were successfully detected by nano-ESI-Q-MS analysis. We revealed that a dominant specie under radiolabeling condition (pH 7) was ZrHDFO, and its stability constant (logß111) was 49.1 ± 0.3. Competition titration revealed that residual oxalate inhibits Zr-DFO complex formation. RCYs in different oxalate concentration (0.1 and 0.04 mol/L) were estimated to be 86% and >99%, which was in good agreement with reported results (87%, 97%). (4) Conclusion: This study succeeded in obtaining accurate stability constants of Zr-DFO complexes and estimating RCY and CAR from accurate stability constants established in this study.

Evaluating 225Ac and 177Lu Radioimmunoconjugates against Antibody-Drug Conjugates for Small-Cell Lung Cancer.

Interest in the use of 225Ac for targeted alpha therapies has increased dramatically over the past few years, resulting in a multitude of new isotope production and translational research efforts. However, 225Ac radioimmunoconjugate (RIC) research is still in its infancy, with most prior experience in hematologic malignancies and only one reported preclinical solid tumor study using 225Ac RICs. In an effort to compare 225Ac RICs to other current antibody conjugates, a variety of RICs are tested against intractable small-cell lung cancer (SCLC). We directly compare, in vitro and in vivo, two promising candidates of each α or ß- category, 225Ac and 177Lu, versus pyrrolobenzodiazepine (PBD) nonradioactive benchmarks. The monoclonal antibody constructs are targeted to either delta like 3 protein (DLL3), a recently discovered SCLC target, or CD46 as a positive control. An immunocompromised maximum tolerated dose assay is performed on NOD SCID mice, along with tumor efficacy proof-of-concept studies in vivo. We overview the conjugation techniques required to create serum-stable RICs and characterize and compare in vitro cell killing with RICs conjugated to nonspecific antibodies (huIgG1) with either native or site-specific thiol loci against tumor antigen DLL3-expressing and nonexpressing cell lines. Using patient-derived xenografts of SCLC onto NOD SCID mice, solid tumor growth was controlled throughout 3 weeks before growth appeared, in comparison to PBD conjugate controls. NOD SCID mice showed lengthened survival using 225Ac compared to 177Lu RICs, and PBD dimers showed full tumor suppression with nine out of ten mice. The exploration of RICs on a variety of antibody-antigen systems is necessary to direct efforts in cancer research toward promising candidates. However, the anti-DLL3-RIC system with 225Ac and 177Lu appears to be not as effective as the anti-DLL3-PBD counterpart in SCLC therapy with matched antibodies and portrays the challenges in both SCLC therapy as well as the specialized utility of RICs in cancer treatment.

Targeting B-cell malignancies with the beta-emitting anti-CD37 radioimmunoconjugate 177Lu-NNV003.

PURPOSE: The aim of this study was to explore the ß-emitting lutetium-177 labelled anti-CD37 antibody NNV003 (177Lu-NNV003, Humalutin®) for the treatment of non-Hodgkin's lymphoma in in vitro studies and in animal models. METHODS: Cytotoxicity of 177Lu-NNV003 was measured in REC-1 (mantle cell lymphoma) and DOHH-2 (diffuse large B cell lymphoma) cell lines. Biodistribution was studied in mice bearing subcutaneous DOHH-2 or MEC-2 (chronic lymphocytic leukaemia) xenografts. The therapeutic effect of a single injection of 177Lu-NNV003 was measured in mice intravenously or subcutaneously injected with REC-1 cells. Haematological and histopathological assessments were used to evaluate the toxic effect of 177Lu-NNV003. The immunotherapeutic effect of NNV003 was assessed by measuring binding to Fcγ receptors, activation of ADCC and ADCP. NNV003's immunogenicity potential was assessed using in silico immunogenicity prediction tools. RESULTS: 177Lu-NNV003 showed an activity dependent antiproliferative effect in all cell lines. Maximum tumour uptake in vivo was 45% of injected activity/g in MEC-2 tumours and 15% injected activity/g in DOHH-2 tumours. In mice injected intravenously with REC-1 cells, 177Lu-NNV003 (50-100 MBq/kg) improved survival compared to control groups (p < 0.02). In mice with subcutaneous REC-1 xenografts, 500 MBq/kg 177Lu-NNV003 extended survival compared to the control treatments (p < 0.005). Transient haematological toxicity was observed in all mice treated with radioactivity. NNV003 induced ADCC and ADCP and was predicted to have a lower immunogenicity potential than its murine counterpart. CONCLUSION: 177Lu-NNV003 had a significant anti-tumour effect and a favourable toxicity profile. These results warrant further clinical testing in patients with CD37-expressing B cell malignancies.

MIB Guides: Measuring the Immunoreactivity of Radioimmunoconjugates.

Immunoglobulins, both full-length antibodies and smaller antibody fragments, have long been regarded as effective platforms for diagnostic and therapeutic radiopharmaceuticals. The construction of radiolabeled immunoglobulins (i.e., radioimmunoconjugates) requires the manipulation of the biomolecule through the attachment of a radiohalogen or the bioconjugation of a chelator that is subsequently used to coordinate a radiometal. Both synthetic approaches have historically relied upon the stochastic modification of amino acids within the immunoglobulin, a process which poses a risk to the structural and functional integrity of the biomolecule itself. Not surprisingly, radioimmunoconjugates with impaired antigen binding capacity will inevitably exhibit suboptimal in vivo performance. As a result, the biological characterization of any newly synthesized radioimmunoconjugate must include an assessment of whether it has retained its ability to bind its antigen. Herein, we provide straightforward and concise protocols for three assays that can be used to determine the immunoreactivity of a radioimmunoconjugate: (1) a cell-based linear extrapolation assay; (2) a cell-based antigen saturation assay; and (3) a resin- or bead-based assay. In addition, we will provide a critical analysis of the relative merits of each assay, an examination of the inherent limitations of immunoreactivity assays in general, and a discussion of other approaches that may be used to interrogate the biological behavior of radioimmunoconjugates.

Regulatory considerations in the design, development and quality of monoclonal antibodies and related products for the diagnosis and treatment of cancer.

Over 160 therapeutic and in vivo diagnostic monoclonal antibodies have been approved by the US FDA since the first monoclonal antibody, muromonab, was approved in 1986. Approximately 42% of these approvals were for the treatment or in vivo diagnosis of oncology indications, although some products are no longer marketed. This review will look at the history of monoclonal antibody development and approvals, discuss current antibody-based modalities, regulatory considerations for engineering approaches, critical quality attributes for different modalities, immunogenicity of mAbs across oncology products, and the future directions for development of therapeutic and diagnostic monoclonal antibody-based products.

Methods for the Production of Radiolabeled Bioagents for ImmunoPET.

Immunoglobulin-based positron emission tomography (ImmunoPET) is making increasingly significant contributions to the nuclear imaging toolbox. The exquisite specificity of antibodies combined with the high-resolution imaging of PET enables clinicians and researchers to localize diseases, especially cancer, with a high degree of spatial certainty. This review focuses on the radiopharmaceutical preparation necessary to obtain those images-the work behind the scenes, which occurs even before the patient or animal is injected with the radioimmunoconjugate. The focus of this methods review will be the chelation of four radioisotopes to their most common and clinically relevant chelators.

Development of 177Lu-Cetuximab-PAMAM dendrimeric nanosystem: a novel theranostic radioimmunoconjugate.

PURPOSE: Epidermal growth factor receptors (EGFRs) are overexpressed in a wide range of tumors and are attractive candidates to target in targeted therapies. This study aimed to introduce a novel radiolabeled compound, 177Lu-cetuximab-PAMAM G4, for the treatment of EGFR-expressing tumors. METHODS: In this study, the cetuximab mAb was bound to PAMAM G4 and labeled with 177Lu via DTPA-CHX chelator. The synthesized nanosystem was confirmed by different analyses such as DLS, FT-IR, TEM, and RT-LC. Cell viability of the radioimmunoconjugate was assessed over the EGFR-expressing cell line of SW480. The biodistribution of 177Lu-Cetuximab-PAMAMG4 was determined in different intervals after injection of the radiolabeled compound in normal and tumoral nude mice via scarification and SPECT images. RESULTS: The average size of PAMAM G4 and PAMAM-Cetuximab-DTPA-CHX nanoparticles were 2 and 70 nm, respectively. 177Lu-Cetuximab-PAMAMG4 was prepared with radiochemical purity of more than 98%. The survival rates of SW480 cells at 24, 48, and 72 h post-treatment with177Lu-Cetuximab-PAMAMG4 (500 nM) were 18%, 15%, and 14%, respectively. The biodistribution studies showed a significant accumulation of 177Lu-Cetuximab-PAMAM in the EGFR-expressing tumor. CONCLUSION: According to the results, this new agent can be considered as an efficient therapeutic complex for tumors expressing EGFR receptors.

Application of Magnetic Particles for Fast Determination of Immunoreactive Fraction of 68Ga-Labelled VHH Antibodies to PD-L1.

Quantification of the immunoreactive fraction (IRF) of radioactive isotope-labeled antibodies or their fragments is necessary to assess the specific activity of radiopharmaceuticals. Traditionally, cells expressing the target molecules on their surface are used to determine IRF, but such analysis is time-consuming and has difficulties with standardization. The aim of the study was to develop a fast and reliable method for quantitative determination of IRF by 68Ga-labeled VHH antibodies to PD-L1 based on the use of magnetic particles coated with antigen molecules. Materials and Methods: Commercially available magnetic particles coated with protein A have been used in our study. The antigen conjugated with the Fc fragment (PD-L1-Fc) was immobilized on the particles. The IRF value of 68Ga radionuclide-labeled nanobodies (VHH) against PD-L1 (68Ga-VHH-PD-L1) was determined using magnetic particles coated with antigen molecules and cells expressing the antigen on their surface. When VHH antibodies were conjugated to 68Ga radionuclide, protein molecules were modified using bifunctional chelating agents: tetraazacyclododecanetetraacetic acid (DOTA) or deferoxamine (DFO). The magnitude of IRF was defined as the ratio of radioactivity specifically bound to particles or cells to the total radioactivity added to the sample. Results: The specificity of the 68Ga-VHH-PD-L1 radioimmunoconjugate binding to the antigen-coated magnetic particles has been proved. Some special aspects, which should be taken into consideration when using this method, have been established. The comparison of the IRF estimates using the antigen-expressing cells and magnetic particles has not revealed any significant differences in the results obtained in our study. Nevertheless, the presented method based on magnetic particles with immobilized antigen molecules requires only 15 min to determine the radioimmunoconjugate IRF, which is of fundamental importance for the routine assessment of the specificity of radiopharmaceuticals containing short-lived isotopes.

DTPA(DOTA)-Nimotuzumab Radiolabeling with Generator-produced Thorium for Radioimmunotherapy of EGFR-overexpressing Carcinomas.

INTRODUCTION: The feasibility of preparing the "in-house" generators and the Th- DTPA(DOTA)-Nimotuzumab radioimmunoconjugate was evaluated. 226Th is perspective for TAT, however, due to short half-life it is preferable to apply this radionuclide for readily available epithelial malignancies. Nimotuzumab being specific for EGFR expressing cells as a targeting moiety is considered to be suitable for thorium delivery. METHODS: TEVA extraction chromatographic resin and anion exchange resin AG 1x8 were used as sorbents for 226Th generator. In order to determine features of labeling by Th4+ we applied 234Th as a longer-lived analog of short-lived 226Th and the immunoconjugates DTPA(DOTA)-Nimotuzumab were used for radiolabeling. RESULTS: The generator on the base of TEVA resin has shown higher volume activity of the product compared to the AG 1x8. The 226Th volume concentration was up to 80%/mL. The radiolabeling of BFCA by thorium radioisotopes reached 95% at the MR(Th:p-SCN-Bn-DTPA) = 1:100 and 86% for MR(Th:p-SCN-Bn-DOTA) = 1:5000 at 90°C. The procedure of Nimotuzumab labeling with Th4+ for radiotherapy of EGFR-overexpressing carcinomas was established. The overall labeling yield in both radioimmunoconjugates - DTPA and DOTA functionalized - was in the range of 45-50%. The immunoconjugate Nimotuzumab-p-SCN-Bn-DTPA was obtained with a molar ratio 1:25 (Nimotuzumab: BFCA), within 1 hour of conjugation at 25°C and labelled via postconjugation approach. Whereas Nimotuzumab-p-SCN-Bn-DOTA was obtained at the same conditions, but radiolabeled by the method of pre-conjugation. CONCLUSION: Thorium-234 incorporation into both radioimmunoconjugates reached 45-50%. It has been shown that Th-DTPA-Nimotuzumab radioimmunoconjugate specifically bound with EGFR overexpressing epidermoid carcinoma A431 cells.

Antibody based conditioning for allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapeutic option for many patients with hematological malignancies and nonmalignant hematopoietic disorders. To achieve stable engraftment of donor hematopoietic stem cells (HSCs), recipient HSC deletion is needed to create space for incoming donor HSCs and donor HSCs must escape immune rejection by the recipient. Conventional allo-HSCT requires high dose of irradiation and/or chemotherapy to produce sufficient host stem cell and immune system ablation to permit donor HSC engraftment. However, these procedures also result in nonspecific tissue injury that can cause short- and long-term adverse effects as well as incite and amplify graft-versus-host-disease (GVHD). The delivery of targeted radiotherapy to hematopoietic tissues with the use of a radioimmunoconjugate (ROIC) as a part of transplant preparative regimen has shown clinical benefits. ROIC clinical data provide evidence for decreased relapse without increased transplant-related mortality by delivering higher targeted radiation to sites of malignancy than when given in a nontargeted fashion. An alternative approach to allo-HSCT has been developed and tested in preclinical mouse models in which nonmyeloablative preconditioning with low dose of the alkylating agent (busulfan) or lower systemic dose of irradiation combined with co-stimulatory pathway blockade (CTLA4-Ig, anti-CD40L monoclonal antibody) and/or immunosuppressive drugs have been used. Under these conditions, mixed chimerism and transplantation tolerance to fully MHC mismatched donor marrow was observed. Recently, several novel proof-of-concept antibody-mediated preconditioning methods have been developed that can selectively target hematopoietic stem and immune cells with minimal overall toxicity. Antibody-drug-conjugate (ADC) combined with reduced intensity conditioning or high dose ADC as single dose monotherapy have shown promise for allo-HSCT in preclinical models. The purpose of the current review is to discuss the literature exploring antibody-based conditioning that includes native antibody, radiolabeled antibody conjugates, and ADC for allo-HSCT.

Radioimmunotheranostic Pair Based on the Anti-HER2 Monoclonal Antibody: Influence of Chelating Agents and Radionuclides on Biological Properties.

The oncogene HER2 is an important molecular target in oncology because it is associated with aggressive disease and the worst prognosis. The development of non-invasive imaging techniques and target therapies using monoclonal antibodies is a rapidly developing field. Thus, this work proposes the study of the radioimmunotheranostic pair, [111In]In-DTPA-trastuzumab and [177Lu]Lu-DOTA-trastuzumab, evaluating the influence of the chelating agents and radionuclides on the biological properties of the radioimmunoconjugates (RICs). The trastuzumab was immunoconjugated with the chelators DTPA and DOTA and radiolabeled with [111In]InCl3 and [177Lu]LuCl3, respectively. The stability of the RICs was evaluated in serum, and the immunoreactive and internalization fractions were determined in SK-BR-3 breast cancer cells. The in vivo pharmacokinetics and dosimetry quantification and the ex vivo biodistribution were performed in normal and SK-BR-3 tumor-bearing mice. The data showed that there was no influence of the chelating agents and radionuclides on the immunoreactive and internalization fractions of RICs. In contrast, they influenced the stability of RICs in serum, as well as the pharmacokinetics, dosimetry and biodistribution profiles. Therefore, the results showed that the nature of the chelating agent and radionuclide could influence the biological properties of the radioimmunotheranostic pair.

The Influence of Glycans-Specific Bioconjugation on the FcγRI Binding and In vivo Performance of 89Zr-DFO-Pertuzumab.

Rationale: The overwhelming majority of radioimmunoconjugates are produced via random conjugation methods predicated on attaching bifunctional chelators to the lysines of antibodies. However, this approach inevitably produces poorly defined and heterogeneous immunoconjugates because antibodies have several lysines distributed throughout their structure. To circumvent this issue, we have previously developed a chemoenzymatic bioconjugation strategy that site-specifically appends cargoes to the biantennary heavy chain glycans attached to CH2 domains of the immunoglobulin's Fc region. In the study at hand, we explore the effects of this approach to site-specific bioconjugation on the Fc receptor binding and in vivo behavior of radioimmunoconjugates. Methods: We synthesized three desferrioxamine (DFO)-labeled immunoconjugates based on the HER2-targeting antibody pertuzumab: one using random bioconjugation methods (DFO-nsspertuzumab) and two using variants of our chemoenzymatic protocol (DFO-sspertuzumab-EndoS and DFO-sspertuzumab-ßGal). Subsequently, we characterized these constructs and evaluated their ability to bind HER2, human FcγRI (huFcγRI), and mouse FcγRI (muFcγRI). After radiolabeling the immunoconjugates with zirconium-89, we conducted PET imaging and biodistribution studies in two different mouse models of HER2-expressing breast cancer. Results: MALDI-ToF and SDS-PAGE analysis confirmed the site-specific nature of the bioconjugation, and flow cytometry and surface plasmon resonance (SPR) revealed that all three immunoconjugates bind HER2 as effectively as native pertuzumab. Critically, however, SPR experiments also illuminated that DFO-sspertuzumab-EndoS possesses an attenuated binding affinity for huFcγRI (17.4 ± 0.3 nM) compared to native pertuzumab (4.7 ± 0.2 nM), DFO-nsspertuzumab (4.1 ± 0.1 nM), and DFO-sspertuzumab-ßGal (4.7 ± 0.2 nM). ImmunoPET and biodistribution experiments in athymic nude mice bearing HER2-expressing BT474 human breast cancer xenografts yielded no significant differences in the in vivo behavior of the radioimmunoconjugates. Yet experiments in tumor-bearing humanized NSG mice revealed that 89Zr-DFO-sspertuzumab-EndoS produces higher activity concentrations in the tumor (111.8 ± 39.9 %ID/g) and lower activity concentrations in the liver and spleen (4.7 ± 0.8 %ID/g and 13.1 ± 4.0 %ID/g, respectively) than its non-site-specifically labeled cousin, a phenomenon we believe stems from the altered binding of the former to huFcγRI. Conclusion: These data underscore that this approach to site-specific bioconjugation not only produces more homogeneous and well-defined radioimmunoconjugates than traditional methods but may also improve their in vivo performance in mouse models by reducing binding to FcγRI.

The Impact of FcγRI Binding on Immuno-PET.

Antibodies are promising vectors for PET imaging. However, the high uptake of radioimmunoconjugates in nontarget tissues such as the liver and spleen hampers their performance as radiotracers. This off-target uptake can lead to suboptimal tumor-to-background activity concentration ratios, decreasing the contrast of images and reducing their diagnostic utility. A possible cause of this uptake is the sequestration of radioimmunoconjugates by immune cells bearing Fc-γ-receptors (FcγR) that bind to the Fc regions of antibodies. Methods: Since the heavy chain glycans influence the affinity of FcγR for the Fc domain, we set out to investigate whether radioimmunoconjugates with truncated glycans would exhibit altered binding to FcγRI and, in turn, improved in vivo performance. Using the HER2-targeting antibody trastuzumab, we synthesized a series of desferrioxamine-bearing immunoconjugates with differing glycosylation states and interrogated their FcγRI binding via surface plasmon resonance, enzyme-linked immunosorbent assay, and flow cytometry. Furthermore, we labeled these immunoconjugates with 89Zr and explored their biodistribution in athymic nude, NSG, and humanized NSG mice bearing human epidermal growth factor receptor 2-expressing human breast cancer xenografts. Results: We observed a strong correlation between the impaired in vitro FcγRI binding of deglycosylated immunoconjugates and significant decreases in the in vivo off-target uptake of the corresponding 89Zr-labeled radioimmunoconjugates (i.e., liver activity concentrations are reduced by ∼3.5-fold in humanized NSG mice). These reductions in off-target uptake were accompanied by concomitant increases in the tumoral activity concentrations of the glycoengineered radioimmunoconjugates, ultimately yielding improved tumor-to-healthy organ contrast and higher quality PET images. Conclusion: Our findings suggest that the deglycosylation of antibodies represents a facile strategy for improving the quality of immuno-PET in animal models as well as in certain patient populations.

Clinical Results in Medullary Thyroid Carcinoma Suggest High Potential of Pretargeted Immuno-PET for Tumor Imaging and Theranostic Approaches.

Monoclonal antibody (mAb)-based therapies have experienced considerable growth in cancer management. When labeled with radionuclides, mAbs also represent promising probes for imaging or theranostic approaches. Initially, mAbs have been radiolabeled with single-photon emitters, such as 131I, 99mTc, or 111In, for diagnostic purposes or to improve radioimmunotherapy (RIT) using dosimetry estimations. Today, more accurate imaging is achieved using positron- emission tomography (PET). Thanks to the important technical advances in the production of PET emitters and their related radiolabeling methods, the last decade has witnessed the development of a broad range of new probes for specific PET imaging. Immuno-PET, which combines the high sensitivity and resolution of a PET camera with the specificity of a monoclonal antibody, is fully in line with this approach. As RIT, immuno-PET can be performed using directly radiolabeled mAbs or using pretargeting to improve imaging contrast. Pretargeted immuno-PET has been developed against different antigens, and promising results have been reported in tumor expressing carcinoembryonic antigen (CEA; CEACAM5) using a bispecific mAb (BsmAb) and a radiolabeled peptide. Medullary thyroid carcinoma (MTC) is an uncommon thyroid cancer subtype which accounts for <10% of all thyroid neoplasms. Characterized by an intense expression of CEA, MTC represents a relevant tumor model for immuno-PET. High sensitivity of pretargeted immunoscintigraphy using murine or chimeric anti-CEA BsMAb and pretargeted haptens-peptides labeled with 111In or 131I were reported in metastatic MTC patients 20 years ago. Recently, an innovative clinical study reported high tumor uptake and contrast using pretargeted anti-CEA immuno-PET in relapsed MTC patients. This review focuses on MTC as an example, but the same pretargeting technique has been applied with success for clinical PET imaging of other CEA-expressing tumors and other pretargeting systems. In particular, those exploiting bioorthogonal chemistry also appear interesting in preclinical animal models, suggesting the high potential of pretargeting for diagnostic and theranostic applications.