Preclinical development of ZED8, an 89Zr immuno-PET reagent for monitoring tumor CD8 status in patients undergoing cancer immunotherapy.

BACKGROUND: ZED8 is a novel monovalent antibody labeled with zirconium-89 for the molecular imaging of CD8. This work describes nonclinical studies performed in part to provide rationale for and to inform expectations in the early clinical development of ZED8, such as in the studies outlined in clinical trial registry NCT04029181 [1]. METHODS: Surface plasmon resonance, X-ray crystallography, and flow cytometry were used to characterize the ZED8-CD8 binding interaction, its specificity, and its impact on T cell function. Immuno-PET with ZED8 was assessed in huCD8+ tumor-bearing mice and in non-human primates. Plasma antibody levels were measured by ELISA to determine pharmacokinetic parameters, and OLINDA 1.0 was used to estimate radiation dosimetry from image-derived biodistribution data. RESULTS: ZED8 selectively binds to human CD8α at a binding site approximately 9 Å from that of MHCI making mutual interference unlikely. The equilibrium dissociation constant (KD) is 5 nM. ZED8 binds to cynomolgus CD8 with reduced affinity (66 nM) but it has no measurable affinity for rat or mouse CD8. In a series of lymphoma xenografts, ZED8 imaging was able to identify different CD8 levels concordant with flow cytometry. In cynomolgus monkeys with tool compound 89Zr-aCD8v17, lymph nodes were conspicuous by imaging 24 h post-injection, and the pharmacokinetics suggested a flat-fixed first-in-human dose of 4 mg per subject. The whole-body effective dose for an adult human was estimated to be 0.48 mSv/MBq, comparable to existing 89Zr immuno-PET reagents. CONCLUSION: 89Zr immuno-PET with ZED8 appears to be a promising biomarker of tissue CD8 levels suitable for clinical evaluation in cancer patients eligible for immunotherapy.

Development of an 18F-labeled anti-human CD8 VHH for same-day immunoPET imaging.

PURPOSE: Cancer immunotherapies (CITs) have revolutionized the treatment of certain cancers, but many patients fail to respond or relapse from current therapies, prompting the need for new CIT agents. CD8+ T cells play a central role in the activity of many CITs, and thus, the rapid imaging of CD8+ cells could provide a critical biomarker for new CIT agents. However, existing 89Zr-labeled CD8 PET imaging reagents exhibit a long circulatory half-life and high radiation burden that limit potential applications such as same-day and longitudinal imaging. METHODS: To this end, we discovered and developed a 13-kDa single-domain antibody (VHH5v2) against human CD8 to enable high-quality, same-day imaging with a reduced radiation burden. To enable sensitive and rapid imaging, we employed a site-specific conjugation strategy to introduce an 18F radiolabel to the VHH. RESULTS: The anti-CD8 VHH, VHH5v2, demonstrated binding to a membrane distal epitope of human CD8 with a binding affinity (KD) of 500 pM. Subsequent imaging experiments in several xenografts that express varying levels of CD8 demonstrated rapid tumor uptake and fast clearance from the blood. High-quality images were obtained within 1 h post-injection and could quantitatively differentiate the tumor models based on CD8 expression level. CONCLUSION: Our work reveals the potential of this anti-human CD8 VHH [18F]F-VHH5v2 to enable rapid and specific imaging of CD8+ cells in the clinic.

Head-to-head comparison of DFO* and DFO chelators: selection of the best candidate for clinical 89Zr-immuno-PET.

PURPOSE: Almost all radiolabellings of antibodies with 89Zr currently employ the hexadentate chelator desferrioxamine (DFO). However, DFO can lead to unwanted uptake of 89Zr in bones due to instability of the resulting metal complex. DFO*-NCS and the squaramide ester of DFO, DFOSq, are novel analogues that gave more stable 89Zr complexes than DFO in pilot experiments. Here, we directly compare these linker-chelator systems to identify optimal immuno-PET reagents. METHODS: Cetuximab, trastuzumab and B12 (non-binding control antibody) were labelled with 89Zr via DFO*-NCS, DFOSq, DFO-NCS or DFO*Sq. Stability in vitro was compared at 37 °C in serum (7 days), in formulation solution (24 h ± chelator challenges) and in vivo with N87 and A431 tumour-bearing mice. Finally, to demonstrate the practical benefit of more stable complexation for the accurate detection of bone metastases, [89Zr]Zr-DFO*-NCS and [89Zr]Zr-DFO-NCS-labelled trastuzumab and B12 were evaluated in a bone metastasis mouse model where BT-474 breast cancer cells were injected intratibially. RESULTS: [89Zr]Zr-DFO*-NCS-trastuzumab and [89Zr]Zr-DFO*Sq-trastuzumab showed excellent stability in vitro, superior to their [89Zr]Zr-DFO counterparts under all conditions. While tumour uptake was similar for all conjugates, bone uptake was lower for DFO* conjugates. Lower bone uptake for DFO* conjugates was confirmed using a second xenograft model: A431 combined with cetuximab. Finally, in the intratibial BT-474 bone metastasis model, the DFO* conjugates provided superior detection of tumour-specific signal over the DFO conjugates. CONCLUSION: DFO*-mAb conjugates provide lower bone uptake than their DFO analogues; thus, DFO* is a superior candidate for preclinical and clinical 89Zr-immuno-PET.

[18F]GTP1 (Genentech Tau Probe 1), a radioligand for detecting neurofibrillary tangle tau pathology in Alzheimer's disease.

OBJECTIVE: Neurofibrillary tangles (NFTs), consisting of intracellular aggregates of the tau protein, are a pathological hallmark of Alzheimer's disease (AD). Here we report the identification and initial characterization of Genentech Tau Probe 1 ([18F]GTP1), a small-molecule PET probe for imaging tau pathology in AD patients. METHODS: Autoradiography using human brain tissues from AD donors and protein binding panels were used to determine [18F]GTP1 binding characteristics. Stability was evaluated in vitro and in vivo in mice and rhesus monkey. In the clinic, whole-body imaging was performed to assess biodistribution and dosimetry. Dynamic [18F]GTP1 brain imaging and input function measurement were performed on two separate days in 5 ß-amyloid plaque positive (Aß+) AD and 5 ß-amyloid plaque negative (Aß-) cognitive normal (CN) participants. Tracer kinetic modeling was applied and reproducibility was evaluated. SUVR was calculated and compared to [18F]GTP1-specific binding parameters derived from the kinetic modeling. [18F]GTP1 performance in a larger cross-sectional group of 60 Aß+ AD participants and ten (Aß- or Aß+) CN was evaluated with images acquired 60 to 90 min post tracer administration. RESULTS: [18F]GTP1 exhibited high affinity and selectivity for tau pathology with no measurable binding to ß-amyloid plaques or MAO-B in AD tissues, or binding to other tested proteins at an affinity predicted to impede image data interpretation. In human, [18F]GTP1 exhibited favorable dosimetry and brain kinetics, and no evidence of defluorination. [18F]GTP1-specific binding was observed in cortical regions of the brain predicted to contain tau pathology in AD and exhibited low (< 4%) test-retest variability. SUVR measured in the 60 to 90-min interval post injection correlated with tracer-specific binding (slope = 1.36, r2 = 0.98). Furthermore, in a cross-sectional population, the degree of [18F]GTP1-specific binding increased with AD severity and could differentiate diagnostic cohorts. CONCLUSIONS: [18F]GTP1 is a promising PET probe for the study of tau pathology in AD.

Impact of drug conjugation on pharmacokinetics and tissue distribution of anti-STEAP1 antibody-drug conjugates in rats.

Antibody-drug conjugates (ADCs) are designed to combine the exquisite specificity of antibodies to target tumor antigens with the cytotoxic potency of chemotherapeutic drugs. In addition to the general chemical stability of the linker, a thorough understanding of the relationship between ADC composition and biological disposition is necessary to ensure that the therapeutic window is not compromised by altered pharmacokinetics (PK), tissue distribution, and/or potential organ toxicity. The six-transmembrane epithelial antigen of prostate 1 (STEAP1) is being pursued as a tumor antigen target. To assess the role of ADC composition in PK, we evaluated plasma and tissue PK profiles in rats, following a single dose, of a humanized anti-STEAP1 IgG1 antibody, a thio-anti-STEAP1 (ThioMab) variant, and two corresponding thioether-linked monomethylauristatin E (MMAE) drug conjugates modified through interchain disulfide cysteine residues (ADC) and engineered cysteines (TDC), respectively. Plasma PK of total antibody measured by enzyme-linked immunosorbent assay (ELISA) revealed ∼45% faster clearance for the ADC relative to the parent antibody, but no apparent difference in clearance between the TDC and unconjugated parent ThioMab. Total antibody clearances of the two unconjugated antibodies were similar, suggesting minimal effects on PK from cysteine mutation. An ELISA specific for MMAE-conjugated antibody indicated that the ADC cleared more rapidly than the TDC, but total antibody ELISA showed comparable clearance for the two drug conjugates. Furthermore, consistent with relative drug load, the ADC had a greater magnitude of drug deconjugation than the TDC in terms of free plasma MMAE levels. Antibody conjugation had a noticeable, albeit minor, impact on tissue distribution with a general trend toward increased hepatic uptake and reduced levels in other highly vascularized organs. Liver uptakes of ADC and TDC at 5 days postinjection were 2-fold and 1.3-fold higher, respectively, relative to the unmodified antibodies. Taken together, these results indicate that the degree of overall structural modification in anti-STEAP1-MMAE conjugates has a corresponding level of impact on both PK and tissue distribution.

Molecular Interaction Characterization Strategies for the Development of New Biotherapeutic Antibody Modalities.

The characterization of target binding interactions is critical at each stage of antibody therapeutic development. During early development, it is important to design fit-for-purpose in vitro molecular interaction characterization (MIC) assays that accurately determine the binding kinetics and the affinity of therapeutic antibodies for their targets. Such information enables PK/PD (pharmacokinetics/pharmacodynamics) modeling, estimation of dosing regimens, and assessment of potency. While binding kinetics and affinities seem to be readily obtained, there is little discussion in the literature on how the information should be generated and used in a systematic manner along with other approaches to enable key drug development decisions. The introduction of new antibody modalities poses unique challenges to the development of MIC assays and further increases the need to discuss the impact of developing context-appropriate MIC assays to enable key decision making for these programs. In this paper, we discuss for the first time the challenges encountered when developing MIC assays supporting new antibody modalities. Additionally, through the presentation of several real case studies, we provide strategies to overcome these challenges to enable investigational new drug (IND) filings.

Total-Body PET and Highly Stable Chelators Together Enable Meaningful 89Zr-Antibody PET Studies up to 30 Days After Injection.

The use of 89Zr-antibody PET imaging to measure antibody biodistribution and tissue pharmacokinetics is well established, but current PET systems lack the sensitivity needed to study 89Zr-labeled antibodies beyond 2-3 isotope half-lives (7-10 d), after which a poor signal-to-noise ratio is problematic. However, studies across many weeks are desirable to better match antibody circulation half-life in human and nonhuman primates. These studies investigated the technical feasibility of using the primate mini-EXPLORER PET scanner, making use of its high sensitivity and 45-cm axial field of view, for total-body imaging of 89Zr-labeled antibodies in rhesus monkeys up to 30 d after injection. Methods: A humanized monoclonal IgG antibody against the herpes simplex viral protein glycoprotein D (gD) was radiolabeled with 89Zr via 1 of 4 chelator-linker combinations (benzyl isothiocyanate-DFO [DFO-Bz-NCS], where DFO is desferrioxamine B; DFO-squaramide; DFO*-Bz-NCS, where DFO* is desferrioxamine*; and DFO*-squaramide). The pharmacokinetics associated with these 4 chelator-linker combinations were compared in 12 healthy young male rhesus monkeys (∼1-2 y old, ∼3 ± 1 kg). Each animal was initially injected intravenously with unlabeled antibody in a peripheral vessel in the right arm (10 mg/kg, providing therapeutic-level antibody concentrations), immediately followed by approximately 40 MBq of one of the 89Zr-labeled antibodies injected intravenously in a peripheral vessel in the left arm. All animals were imaged 6 times over a period of 30 d, with an initial 60-min dynamic scan on day 0 (day of injection) followed by static scans of 30-45 min on approximately days 3, 7, 14, 21, and 30, with all acquired using a single bed position and images reconstructed using time-of-flight list-mode ordered-subsets expectation maximization. Activity concentrations in various organs were extracted from the PET images using manually defined regions of interest. Results: Excellent image quality was obtained, capturing the initial distribution phase in the whole-body scan; later time points showed residual 89Zr mainly in the liver. Even at 30 d after injection, representing approximately 9 half-lives of 89Zr and with a total residual activity of only 20-40 kBq in the animal, the image quality was sufficient to readily identify activity in the liver, kidneys, and upper and lower limb joints. Significant differences were noted in late time point liver uptake, bone uptake, and whole-body clearance between chelator-linker types, whereas little variation (±10%) was observed within each type. Conclusion: These studies demonstrate the ability to image 89Zr-radiolabeled antibodies up to 30 d after injection while maintaining satisfactory image quality, as provided by the primate mini-EXPLORER with high sensitivity and long axial field of view. Quantification demonstrated potentially important differences in the behavior of the 4 chelators. This finding supports further investigation.

The Production, Quality Control, and Characterization of ZED8, a CD8-Specific 89Zr-Labeled Immuno-PET Clinical Imaging Agent.

Immuno-PET is a molecular imaging technique utilizing positron emission tomography (PET) to measure the biodistribution of an antibody species labeled with a radioactive isotope. When applied as a clinical imaging technique, an immuno-PET imaging agent must be manufactured with quality standards appropriate for regulatory approval. This paper describes methods relevant to the chemistry, manufacturing, and controls component of an immuno-PET regulatory filing, such as an investigational new drug application. Namely, the production, quality control, and characterization of the immuno-PET clinical imaging agent, ZED8, an 89Zr-labeled CD8-specific monovalent antibody as well as its desferrioxamine-conjugated precursor, CED8, is described and evaluated. PET imaging data in a human CD8-expressing tumor murine model is presented as a proof of concept that the imaging agent exhibits target specificity and comparable biodistribution across a range of desferrioxamine conjugate loads.

SOX1 links the function of neural patterning and Notch signalling in the ventral spinal cord during the neuron-glial fate switch.

During neural development the transition from neurogenesis to gliogenesis, known as the neuron-glial (Nu/G) fate switch, requires the coordinated function of patterning factors, pro-glial factors and Notch signalling. How this process is coordinated in the embryonic spinal cord is poorly understood. Here, we demonstrate that during the N/G fate switch in the ventral spinal cord (vSC) SOX1 links the function of neural patterning and Notch signalling. We show that, SOX1 expression in the vSC is regulated by PAX6, NKX2.2 and Notch signalling in a domain-specific manner. We further show that SOX1 regulates the expression of Hes1 and that loss of Sox1 leads to enhanced production of oligodendrocyte precursors from the pMN. Finally, we show that Notch signalling functions upstream of SOX1 during this fate switch and is independently required for the acquisition of the glial fate perse by regulating Nuclear Factor I A expression in a PAX6/SOX1/HES1/HES5-independent manner. These data integrate functional roles of neural patterning factors, Notch signalling and SOX1 during gliogenesis.

PET of glial metabolism using 2-18F-fluoroacetate.

UNLABELLED: Imaging of the glial activation that occurs in response to central nervous system trauma and inflammation could become a powerful technique for the assessment of several neuropathologies. The selective uptake and metabolism of 2-(18)F-fluoroacetate ((18)F-FAC) in glia may represent an attractive strategy for imaging glial metabolism. METHODS: We have evaluated the use of (18)F-FAC as a specific PET tracer of glial cell metabolism in rodent models of glioblastoma, stroke, and ischemia-hypoxia. RESULTS: Enhanced uptake of (18)F-FAC was observed (6.98 +/- 0.43 percentage injected dose per gram [%ID/g]; tumor-to-normal ratio, 1.40) in orthotopic U87 xenografts, compared with healthy brain tissue. The lesion extent determined by (18)F-FAC PET correlated with that determined by MRI (R(2) = 0.934, P = 0.007). After transient middle cerebral artery occlusion in the rat brain, elevated uptake of (18)F-FAC (1.00 +/- 0.03 %ID/g; lesion-to-normal ratio, 1.90) depicted the ischemic territory and correlated with infarct volumes as determined by 2,3,5-triphenyltetrazolium chloride staining (R(2) = 0.692, P = 0.010) and with the presence of activated astrocytes detected by anti-glial fibrillary acidic protein. Ischemia-hypoxia, induced by permanent ligation of the common carotid artery with transient hypoxia, resulted in persistent elevation of (18)F-FAC uptake within 30 min of the induction of hypoxia. CONCLUSION: Our data support the further evaluation of (18)F-FAC PET for the assessment of glial cell metabolism associated with neuroinflammation.

VCAM-1 Density and Tumor Perfusion Predict T-cell Infiltration and Treatment Response in Preclinical Models.

Cancer immunotherapies have demonstrated durable responses in a range of different cancers. However, only a subset of patients responds to these therapies. We set out to test if non-invasive imaging of tumor perfusion and vascular inflammation may be able to explain differences in T-cell infiltration in pre-clinical tumor models, relevant for treatment outcomes. Tumor perfusion and vascular cell adhesion molecule (VCAM-1) density were quantified using magnetic resonance imaging (MRI) and correlated with infiltration of adoptively transferred and endogenous T-cells. MRI biomarkers were evaluated for their ability to detect tumor rejection 3 days after T-cell transfer. Baseline levels of these markers were used to assess their ability to predict PD-L1 treatment response. We found correlations between MRI-derived VCAM-1 density and infiltration of endogenous or adoptively transferred T-cells in some preclinical tumor models. Blocking T-cell binding to endothelial cell adhesion molecules (VCAM-1/ICAM) prevented T-cell mediated tumor rejection. Tumor rejection could be detected 3 days after adoptive T-cell transfer prior to tumor volume changes by monitoring the extracellular extravascular volume fraction. Imaging tumor perfusion and VCAM-1 density before treatment initiation was able to predict the response of MC38 tumors to PD-L1 blockade. These results indicate that MRI based assessment of tumor perfusion and VCAM-1 density can inform about the permissibility of the tumor vasculature for T-cell infiltration which may explain some of the observed variance in treatment response for cancer immunotherapies.

Preparation and evaluation of L- and D-5-[18F]fluorotryptophan as PET imaging probes for indoleamine and tryptophan 2,3-dioxygenases.

Indoleamine and tryptophan 2,3-dioxygenases (IDO1 and TDO2) are pyrrolases catalyzing the oxidative cleavage of the 2,3-double bond of L-tryptophan in kynurenine pathway. In the tumor microenvironment, their increased activity prevents normal immune function, i.e. tumor cell recognition and elimination by cytotoxic T-cells. Consequently, inhibition of the kynurenine pathway may enhance the activity of cancer immunotherapeutics by reversing immune dysfunction. We sought to investigate the properties of radiolabeled 5-[18F]fluorotryptophan with respect to its ability for measuring IDO1 and TDO2 activity by positron emission tomography (PET). RESULTS: L-5-[18F]fluorotryptophan and D-5-[18F]fluorotryptophan were synthesized by Cu(I) catalyzed [18F]fluorodeboronylation of Boc/tBu protected precursors in moderate yields (1.5±0.6%) sufficient for pre-clinical studies. The specific activity of the product was 407-740GBq/µmol, radiochemical purity >99% and enantiomeric excess 90-99%. Enzymatic assay confirmed that L-5-fluorotryptophan is an IDO1 and TDO2 substrate whereas the D-isomer is not. In-vitro cell uptake experiments using CT26 cells with doxycycline-induced overexpression of human-IDO1 and human-TDO2 revealed an elevated cell uptake of L-5-[18F]fluorotryptophan upon induction of IDO1 or TDO2 enzymes compared to baseline; however, the uptake was observed only in the presence of low L-tryptophan levels in media. PET imaging experiments performed using tumor bearing mouse models expressing IDO1 at various levels (CT26, CT26-hIDO1, 17082A, 17095A) showed tumor uptake of the tracer elevated up to 8%ID/g; however, the observed tumor uptake could not be attributed to IDO1 activity in the tumor tissue. The metabolism of L- and D- isomers was markedly different in vivo, the D-isomer was excreted by a combination of hepatobiliary and renal routes, the L-isomer underwent extensive metabolism to [18F]fluoride. CONCLUSION: The observed in vivo tumor uptake of the tracer could not be attributed to IDO1 or TDO2 enzyme activity in the tumor, presumably due to competition with endogenous tryptophan as well as rapid tracer metabolism.

Evaluation of a 3-hydroxypyridin-2-one (2,3-HOPO) Based Macrocyclic Chelator for (89)Zr(4+) and Its Use for ImmunoPET Imaging of HER2 Positive Model of Ovarian Carcinoma in Mice.

A novel octadentate 3-hydroxypyridin-2-one (2,3-HOPO) based di-macrocyclic ligand was evaluated for chelation of (89)Zr; subsequently, it was used as a bi-functional chelator for preparation of (89)Zr-labeled antibodies. Quantitative chelation of (89)Zr(4+) with the octadentate ligand forming (89)ZrL complex was achieved under mild conditions within 15 minutes. The (89)Zr-complex was stable in vitro in presence of DTPA, but a slow degradation was observed in serum. In vivo, the hydrophilic (89)Zr-complex showed prevalently renal excretion; and an elevated bone uptake of radioactivity suggested a partial release of (89)Zr(4+) from the complex. The 2,3-HOPO based ligand was conjugated to the monoclonal antibodies, HER2-specific trastuzumab and an isotypic anti-gD antibody, using a p-phenylene bis-isothiocyanate linker to yield products with an average loading of less than 2 chelates per antibody. Conjugated antibodies were labeled with (89)Zr under mild conditions providing the PET tracers in 60-69% yield. Despite the limited stability in mouse serum; the PET tracers performed very well in vivo. The PET imaging in mouse model of HER2 positive ovarian carcinoma showed tumor uptake of (89)Zr-trastuzumab (29.2 ± 12.9 %ID/g) indistinguishable (p = 0.488) from the uptake of positive control (89)Zr-DFO-trastuzumab (26.1 ± 3.3 %ID/g). In conclusion, the newly developed 3-hydroxypyridin-2-one based di-macrocyclic chelator provides a viable alternative to DFO-based heterobifunctional ligands for preparation of (89)Zr-labeled monoclonal antibodies for immunoPET studies.

ImmunoPET helps predicting the efficacy of antibody-drug conjugates targeting TENB2 and STEAP1.

The efficacy of antibody-drug conjugates (ADCs) targeted to solid tumors depends on biological processes that are hard to monitor in vivo. 89Zr-immunoPET of the ADC antibodies could help understand the performance of ADCs in the clinic by confirming the necessary penetration, binding, and internalization. This work studied monomethyl auristatin E (MMAE) ADCs against two targets in metastatic castration-resistant prostate cancer, TENB2 and STEAP1, in four patient-derived tumor models (LuCaP35V, LuCaP70, LuCaP77, LuCaP96.1). Three aspects of ADC biology were measured and compared: efficacy was measured in tumor growth inhibition studies; target expression was measured by immunohistochemistry and flow cytometry; and tumor antibody uptake was measured with 111In-mAbs and gamma counting or with 89Zr-immunoPET. Within each model, the mAb with the highest tumor uptake showed the greatest potency as an ADC. Sensitivity between models varied, with the LuCaP77 model showing weak efficacy despite high target expression and high antibody uptake. Ex vivo analysis confirmed the in vivo results, showing a correlation between expression, uptake and ADC efficacy. We conclude that 89Zr-immunoPET data can demonstrate which ADC candidates achieve the penetration, binding, and internalization necessary for efficacy in tumors sensitive to the toxic payload.

ImmunoPET imaging of phosphatidylserine in pro-apoptotic therapy treated tumor models.

UNLABELLED: An immunoPET imaging probe for the detection of phosphatidylserine was developed and tested in animal models of human cancer treated with pro-apoptotic therapy. We hypothesized that the relatively long plasma half-life of a probe based on a full-length antibody coupled with a residualizing radionuclide would be able to catch the wave of drug-induced apoptosis and lead to a specific accumulation in apoptotic tumor tissue. METHODS: The imaging probe is based on a 89Zr-labeled monoclonal antibody PGN635 targeting phosphatidylserine. The probe was evaluated pre-clinically in four tumor xenograft models: one studied treatment with paclitaxel to trigger the intrinsic apoptotic pathway, and three others interrogated treatment with an agonistic death-receptor monoclonal antibody to engage the extrinsic apoptotic pathway. RESULTS: High accumulation of 89Zr-PGN635 was observed in treated tumors undergoing apoptosis reaching 30 %ID/g and tumor-to-blood ratios up to 13. The tumor uptake in control groups treated with vehicle or imaged with a non-binding antibody probe was significantly lower. CONCLUSIONS: The results demonstrate the ability of 89Zr-PGN635 to image drug-induced apoptosis in animal models and corroborate our hypothesis that radiolabeled antibodies binding to intracellular targets transiently exposed on the cell surface during apoptosis can be employed for detection of tumor response to therapy.

An integrated approach to identify normal tissue expression of targets for antibody-drug conjugates: case study of TENB2.

BACKGROUND AND PURPOSE: The success of antibody-drug conjugates (ADCs) depends on the therapeutic window rendered by the differential expression between normal and pathological tissues. The ability to identify and visualize target expression in normal tissues could reveal causes for target-mediated clearance observed in pharmacokinetic characterization. TENB2 is a prostate cancer target associated with the progression of poorly differentiated and androgen-independent tumour types, and ADCs specific for TENB2 are candidate therapeutics. The objective of this study was to locate antigen expression of TENB2 in normal tissues, thereby elucidating the underlying causes of target-mediated clearance. EXPERIMENTAL APPROACH: A series of pharmacokinetics, tissue distribution and mass balance studies were conducted in mice using a radiolabelled anti-TENB2 ADC. These data were complemented by non-invasive single photon emission computed tomography - X-ray computed tomography imaging and immunohistochemistry. KEY RESULTS: The intestines were identified as a saturable and specific antigen sink that contributes, at least in part, to the rapid target-mediated clearance of the anti-TENB2 antibody and its drug conjugate in rodents. As a proof of concept, we also demonstrated the selective disposition of the ADC in a tumoural environment in vivo using the LuCaP 77 transplant mouse model. High tumour uptake was observed despite the presence of the antigen sink, and antigen specificity was confirmed by antigen blockade. CONCLUSIONS AND IMPLICATIONS: Our findings provide the anatomical location and biological interpretation of target-mediated clearance of anti-TENB2 antibodies and corresponding drug conjugates. Further investigations may be beneficial in addressing the relative contributions to ADC disposition from antigen expression in both normal and pathological tissues.

Enhanced tumor retention of a radiohalogen label for site-specific modification of antibodies.

A known limitation of iodine radionuclides for labeling and biological tracking of receptor targeted proteins is the tendency of iodotyrosine to rapidly diffuse from cells following endocytosis and lysosomal degradation. In contrast, radiometal-chelate complexes such as indium-111-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (In-111-DOTA) accumulate within target cells due to the residualizing properties of the polar, charged metal-chelate-amino acid adduct. Iodine radionuclides boast a diversity of nuclear properties and chemical means for incorporation, prompting efforts to covalently link radioiodine with residualizing molecules. Herein, we describe the Ugi-assisted synthesis of [I-125]HIP-DOTA, a 4-hydroxy-3-iodophenyl (HIP) derivative of DOTA, and demonstration of its residualizing properties in a murine xenograft model. Overall, this study displays the power of multicomponent synthesis to yield a versatile radioactive probe for antibodies across multiple therapeutic areas with potential applications in both preclinical biodistribution studies and clinical radioimmunotherapies.

Preparation of 18F-labeled peptides using the copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition.

An optimized procedure for preparing fluorine-18 ((18)F)-labeled peptides by the copper-catalyzed azide-alkyne 1,3-dipolar cyloaddition (CuAAC) is presented here. The two-step radiosynthesis begins with the microwave-assisted nucleophilic (18)F-fluorination of a precursor containing a terminal p-toluenesulfonyl, terminal azide and polyethylene glycol backbone. The resulting (18)F-fluorinated azide-containing building block is coupled to an alkyne-decorated peptide by the CuAAC. The reaction is accelerated by the copper(I)-stabilizing ligand bathophenanthroline disulfonate and can be performed in either reducing or nonreducing conditions (e.g., to preserve disulfide bonds). After an HPLC purification, (18)F-labeled peptide can be obtained with a 31 ± 6% radiochemical yield (n = 4, decay-corrected from (18)F-fluoride elution) and a specific activity of 39.0 ± 12.4 Ci µmol(-1) within 77 ± 4 min.

The development of peptide-based tools for the analysis of angiogenesis.

Limitations to the application of molecularly targeted cancer therapies are the inability to accurately match patient with effective treatment and the absence of a prompt readout of posttreatment response. Noninvasive agents that rapidly report vascular endothelial growth factor (VEGF) levels using positron emission tomography (PET) have the potential to enhance anti-angiogenesis therapies. Using phage display, two distinct classes of peptides were identified that bind to VEGF with nanomolar affinity and high selectivity. Co-crystal structures of these different peptide classes demonstrate that both bind to the receptor-binding region of VEGF. (18)F-radiolabelling of these peptides facilitated the acquisition of PET images of tumor VEGF levels in a HM7 xenograph model. The images obtained from one 59-residue probe, (18)F-Z-3B, 2 hr postinjection are comparable to those obtained with anti-VEGF antibody B20 72 hr postinjection. Furthermore, VEGF levels in growing SKOV3 tumors were followed using (18)F-Z-3B as a PET probe with VEGF levels increasing with tumor size.