Affibody PET Imaging of HER2-Expressing Cancers as a Key to Guide HER2-Targeted Therapy.

Human epidermal growth factor receptor 2 (HER2) is a major prognostic and predictive marker overexpressed in 15-20% of breast cancers. The diagnostic reference standard for selecting patients for HER2-targeted therapy is based on the analysis of tumor biopsies. Previously patients were defined as HER2-positive or -negative; however, with the approval of novel treatment options, specifically the antibody-drug conjugate trastuzumab deruxtecan, many breast cancer patients with tumors expressing low levels of HER2 have become eligible for HER2-targeted therapy. Such patients will need to be reliably identified by suitable diagnostic methods. Biopsy-based diagnostics are invasive, and repeat biopsies are not always feasible. They cannot visualize the heterogeneity of HER2 expression, leading to a substantial number of misdiagnosed patients. An alternative and highly accurate diagnostic method is molecular imaging with radiotracers. In the case of HER2, various studies demonstrate the clinical utility and feasibility of such approaches. Radiotracers based on Affibody® molecules, small, engineered affinity proteins with a size of ~6.5 kDa, are clinically validated molecules with favorable characteristics for imaging. In this article, we summarize the HER2-targeted therapeutic landscape, describe our experience with imaging diagnostics for HER2, and review the currently available clinical data on HER2-Affibody-based molecular imaging as a novel diagnostic tool in breast cancer and beyond.

Optimized, automated and cGMP-compliant synthesis of the HER2 targeting [68Ga]Ga-ABY-025 tracer.

BACKGROUND: The Affibody molecule, ABY-025, has demonstrated utility to detect human epidermal growth factor receptor 2 (HER2) in vivo, either radiolabelled with indium-111 (111In) or gallium-68 (68Ga). Using the latter, 68Ga, is preferred due to its use in positron emission tomography with superior resolution and quantifying capabilities in the clinical setting compared to 111In. For an ongoing phase II study (NCT05619016) evaluating ABY-025 for detecting HER2-low lesions and selection of patients for HER2-targeted treatment, the aim was to optimize an automated and cGMP-compliant radiosynthesis of [68Ga]Ga-ABY-025. [68Ga]Ga-ABY-025 was produced on a synthesis module, Modular-Lab PharmTracer (Eckert & Ziegler), commonly used for 68Ga-labelings. The radiotracer has previously been radiolabeled on this module, but to streamline the production, the method was optimized. Steps requiring manual interactions to the radiolabeling procedure were minimized including a convenient and automated pre-concentration of the 68Ga-eluate and a simplified automated final formulation procedure. Every part of the radiopharmaceutical production was carefully developed to gain robustness and to avoid any operator bound variations to the manufacturing. The optimized production method was successfully applied for 68Ga-labeling of another radiotracer, verifying its versatility as a universal and robust method for radiosynthesis of Affibody-based peptides. RESULTS: A simplified and optimized automated cGMP-compliant radiosynthesis method of [68Ga]Ga-ABY-025 was developed. With a decay corrected radiochemical yield of 44 ± 2%, a radiochemical purity (RCP) of 98 ± 1%, and with an RCP stability of 98 ± 1% at 2 h after production, the method was found highly reproducible. The production method also showed comparable results when implemented for radiolabeling another similar peptide. CONCLUSION: The improvements made for the radiosynthesis of [68Ga]Ga-ABY-025, including introducing a pre-concentration of the 68Ga-eluate, aimed to utilize the full potential of the 68Ge/68Ga generator radioactivity output, thereby reducing radioactivity wastage. Furthermore, reducing the number of manually performed preparative steps prior to the radiosynthesis, not only minimized the risk of potential human/operator errors but also enhanced the process' robustness. The successful application of this optimized radiosynthesis method to another similar peptide underscores its versatility, suggesting that our method can be adopted for 68Ga-labeling radiotracers based on Affibody molecules in general. TRIAL REGISTRATION: NCT, NCT05619016, Registered 7 November 2022, https://clinicaltrials.gov/study/NCT05619016?term=HER2&cond=ABY025&rank=1.

Izokibep for the treatment of moderate-to-severe plaque psoriasis: a phase II, randomized, placebo-controlled, double-blind, dose-finding multicentre study including long-term treatment.

BACKGROUND: Monoclonal antibodies to interleukin (IL)-17 have shown strong efficacy in patients with psoriasis. Izokibep is a unique IL-17A inhibitor with a small molecular size and favourable distribution to sites of inflammation. OBJECTIVES: To evaluate the dose response, efficacy and safety of izokibep in patients with plaque psoriasis. METHODS: In this double-blind, randomized, phase II dose-finding study (AFFIRM-35) in adults with moderate-to-severe plaque psoriasis and inadequate response to two or more standard therapies, patients were randomized (1:1:1:1:1) to placebo or izokibep 2, 20, 80 or 160 mg every 2 weeks for 12 weeks. During the remainder of the 52-week core study, patients given placebo were switched to izokibep 80 mg, and dosing intervals were adapted based on Psoriasis Area and Severity Index (PASI) scores for all patients. The core study was followed by two optional consecutive 1-year extension periods for a total duration of 3 years. The primary endpoint was a 90% reduction in PASI score (PASI 90) at week 12. Additional efficacy outcomes and adverse event (AE) rates were evaluated. RESULTS: In total, 109 patients were randomized [safety set, n = 108 (one exclusion criteria failure); full analysis set, n = 106]. At week 12, PASI 90 response rates were 0%, 5%, 19%, 71% and 59% for the placebo, 2-, 20-, 80- and 160-mg izokibep groups, respectively. Rapid dose-dependent improvements were also observed across other efficacy outcomes. During the placebo-controlled period, AEs in the izokibep groups were similar to placebo except for mild injection site reactions. AEs were generally mild to moderate and the drug was well tolerated. Izokibep maintained efficacy at the higher dosage groups for up to 3 years, with no new safety signals. CONCLUSIONS: Data from this phase II study indicate that izokibep is well tolerated and efficacious in the treatment of plaque psoriasis. Higher doses or more frequent dosing could be explored to further enhance response rates.

Human Epidermal Growth Factor Receptor 2-Targeting [68Ga]Ga-ABY-025 PET/CT Predicts Early Metabolic Response in Metastatic Breast Cancer.

Imaging using the human epidermal growth factor receptor 2 (HER2)-binding tracer 68Ga-labeled ZHER2:2891-Cys-MMA-DOTA ([68Ga]Ga-ABY-025) was shown to reflect HER2 status determined by immunohistochemistry and in situ hybridization in metastatic breast cancer (MBC). This single-center open-label phase II study investigated how [68Ga]Ga-ABY-025 uptake corresponds to biopsy results and early treatment response in both primary breast cancer (PBC) planned for neoadjuvant chemotherapy and MBC. Methods: Forty patients with known positive HER2 status were included: 19 with PBC and 21 with MBC (median, 3 previous treatments). [68Ga]Ga-ABY-025 PET/CT, [18F]F-FDG PET/CT, and core-needle biopsies from targeted lesions were performed at baseline. [18F]F-FDG PET/CT was repeated after 2 cycles of therapy to calculate the directional change in tumor lesion glycolysis (Δ-TLG). The largest lesions (up to 5) were evaluated in all 3 scans per patient. SUVs from [68Ga]Ga-ABY-025 PET/CT were compared with the biopsied HER2 status and Δ-TLG by receiver operating characteristic analyses. Results: Trial biopsies were HER2-positive in 31 patients, HER2-negative in 6 patients, and borderline HER2-positive in 3 patients. The [68Ga]Ga-ABY-025 PET/CT cutoff SUVmax of 6.0 predicted a Δ-TLG lower than -25% with 86% sensitivity and 67% specificity in soft-tissue lesions (area under the curve, 0.74 [95% CI, 0.67-0.82]; P = 0.01). Compared with the HER2 status, this cutoff resulted in clinically relevant discordant findings in 12 of 40 patients. Metabolic response (Δ-TLG) was more pronounced in PBC (-71% [95% CI, -58% to -83%]; P < 0.0001) than in MBC (-27% [95% CI, -16% to -38%]; P < 0.0001), but [68Ga]Ga-ABY-025 SUVmax was similar in both with a mean SUVmax of 9.8 (95% CI, 6.3-13.3) and 13.9 (95% CI, 10.5-17.2), respectively (P = 0.10). In multivariate analysis, global Δ-TLG was positively associated with the number of previous treatments (P = 0.0004) and negatively associated with [68Ga]Ga-ABY-025 PET/CT SUVmax (P = 0.018) but not with HER2 status (P = 0.09). Conclusion: [68Ga]Ga-ABY-025 PET/CT predicted early metabolic response to HER2-targeted therapy in HER2-positive breast cancer. Metabolic response was attenuated in recurrent disease. [68Ga]Ga-ABY-025 PET/CT appears to provide an estimate of the HER2 expression required to induce tumor metabolic remission by targeted therapies and might be useful as an adjunct diagnostic tool.

Izokibep: Preclinical development and first-in-human study of a novel IL-17A neutralizing Affibody molecule in patients with plaque psoriasis.

Psoriasis, an immune-mediated inflammatory disease, affects nearly 125 million people globally. The interleukin (IL)-17A homodimer is a key driver of psoriasis and other autoimmune diseases, including psoriatic arthritis, axial spondyloarthritis, hidradenitis suppurativa, and uveitis. Treatment with monoclonal antibodies (mAbs) against IL-17A provides an improvement in the Psoriasis Area and Severity Index compared to conventional systemic agents. In this study, the AffibodyⓇ technology was used to identify and optimize a novel, small, biological molecule comprising three triple helical affinity domains, izokibep (previously ABY-035), for the inhibition of IL-17A signaling. Preclinical studies show that izokibep, a small 18.6 kDa IL-17 ligand trap comprising two IL-17A-specific Affibody domains and one albumin-binding domain, selectively inhibits human IL-17A in vitro and in vivo with superior potency and efficacy relative to anti-IL-17A mAbs. A Phase 1 first-in-human study was conducted to establish the safety, pharmacokinetics, and preliminary efficacy of izokibep, when administered intravenously and subcutaneously as single doses to healthy subjects, and as single intravenous and multiple subcutaneous doses to patients with psoriasis (NCT02690142; EudraCT No: 2015-004531-13). Izokibep was well tolerated with no meaningful safety concerns identified in healthy volunteers and patients with psoriasis. Rapid efficacy was seen in all psoriasis patients after one dose which further improved in patients receiving multiple doses. A therapeutic decrease in joint pain was also observed in a single patient with concurrent psoriatic arthritis. The study suggests that izokibep has the potential to safely treat IL17A-associated diseases such as psoriasis, psoriatic arthritis, axial spondyloarthritis, hidradenitis suppurativa, and uveitis.

Preclinical Evaluation of 99mTc-ZHER2:41071, a Second-Generation Affibody-Based HER2-Visualizing Imaging Probe with a Low Renal Uptake.

Radionuclide imaging of HER2 expression in tumours may enable stratification of patients with breast, ovarian, and gastroesophageal cancers for HER2-targeting therapies. A first-generation HER2-binding affibody molecule [99mTc]Tc-ZHER2:V2 demonstrated favorable imaging properties in preclinical studies. Thereafter, the affibody scaffold has been extensively modified, which increased its melting point, improved storage stability, and increased hydrophilicity of the surface. In this study, a second-generation affibody molecule (designated ZHER2:41071) with a new improved scaffold has been prepared and characterized. HER2-binding, biodistribution, and tumour-targeting properties of [99mTc]Tc-labelled ZHER2:41071 were investigated. These properties were compared with properties of the first-generation affibody molecules, [99mTc]Tc-ZHER2:V2 and [99mTc]Tc-ZHER2:2395. [99mTc]Tc-ZHER2:41071 bound specifically to HER2 expressing cells with an affinity of 58 ± 2 pM. The renal uptake for [99mTc]Tc-ZHER2:41071 and [99mTc]Tc-ZHER2:V2 was 25-30 fold lower when compared with [99mTc]Tc-ZHER2:2395. The uptake in tumour and kidney for [99mTc]Tc-ZHER2:41071 and [99mTc]Tc-ZHER2:V2 in SKOV-3 xenografts was similar. In conclusion, an extensive re-engineering of the scaffold did not compromise imaging properties of the affibody molecule labelled with 99mTc using a GGGC chelator. The new probe, [99mTc]Tc-ZHER2:41071 provided the best tumour-to-blood ratio compared to HER2-imaging probes for single photon emission computed tomography (SPECT) described in the literature so far. [99mTc]Tc-ZHER2:41071 is a promising candidate for further clinical translation studies.

[68Ga]ABY-028: an albumin-binding domain (ABD) protein-based imaging tracer for positron emission tomography (PET) studies of altered vascular permeability and predictions of albumin-drug conjugate transport.

BACKGROUND: Albumin is commonly used as a carrier platform for drugs to extend their circulatory half-lives and influence their uptake into tissues that have altered permeability to the plasma protein. The albumin-binding domain (ABD) protein, which binds in vivo to serum albumin with high affinity, has proven to be a versatile scaffold for engineering biopharmaceuticals with a range of binding capabilities. In this study, the ABD protein equipped with a mal-DOTA chelator (denoted ABY-028) was radiolabeled with gallium-68 (68Ga). This novel radiotracer was then used together with positron emission tomography (PET) imaging to examine variations in the uptake of the ABD-albumin conjugate with variations in endothelial permeability. RESULTS: ABY-028, produced by peptide synthesis in excellent purity and stored at - 20 °C, was stable for 24 months (end of study). [68Ga]ABY-028 could be obtained with labeling yields of > 80% and approximately 95% radiochemical purity. [68Ga]ABY-028 distributed in vivo with the plasma pool, with highest radioactivity in the heart ventricles and major vessels of the body, a gradual transport over time from the circulatory system into tissues and elimination via the kidneys. Early [68Ga]ABY-028 uptake differed in xenografts with different vascular properties: mean standard uptake values (SUVmean) were initially 5 times larger in FaDu than in A431 xenografts, but the difference decreased to 3 after 1 h. Cutaneously administered, vasoactive nitroglycerin increased radioactivity in the A431 xenografts. Heterogeneity in the levels and rates of increases of radioactivity uptake was observed in sub-regions of individual MMTV-PyMT mammary tumors and in FaDu xenografts. Higher uptake early after tracer administration could be observed in lower metabolic regions. Fluctuations in the increased permeability for the tracer across the blood-brain-barrier (BBB) direct after experimentally induced stroke were monitored by PET and the increased uptake was confirmed by ex vivo phosphorimaging. CONCLUSIONS: [68Ga]ABY-028 is a promising new tracer for visualization of changes in albumin uptake due to disease- and pharmacologically altered vascular permeability and their potential effects on the passive uptake of targeting therapeutics based on the ABD protein technology.

Kinetic analysis of HER2-binding ABY-025 Affibody molecule using dynamic PET in patients with metastatic breast cancer.

BACKGROUND: High expression of human epidermal growth factor receptor type 2 (HER2) represents an aggressive subtype of breast cancer. Anti-HER2 treatment requires a theragnostic approach wherein sufficiently high receptor expression in biopsy material is mandatory. Heterogeneity and discordance of HER2 expression between primary tumour and metastases, as well as within a lesion, present a complication for the treatment and require multiple biopsies. Molecular imaging using the HER2-targeting Affibody peptide ABY-025 radiolabelled with 68Ga-gallium for PET/CT is currently under investigation as a non-invasive tool for whole-body evaluation of metastatic HER2 expression. Initial studies demonstrated a high correlation between 68Ga-ABY-025 standardized uptake values (SUVs) and histopathology. However, detecting small liver lesions might be compromised by high background uptake. This study aimed to explore the applicability of kinetic modelling and parametric image analysis for absolute quantification of 68Ga-ABY-025 uptake and HER2-receptor expression and how that relates to static SUVs. METHODS: Dynamic 68Ga-ABY-025 PET of the upper abdomen was performed 0-45 min post-injection in 16 patients with metastatic breast cancer. Five patients underwent two examinations to test reproducibility. Parametric images of tracer delivery (K1) and irreversible binding (Ki) were created with an irreversible two-tissue compartment model and Patlak graphical analysis using an image-derived input function from the descending aorta. A volume of interest (VOI)-based analysis was performed to validate parametric images. SUVs were calculated from 2 h and 4 h post-injection static whole-body images and compared to Ki. RESULTS: Characterization of HER2 expression in smaller liver metastases was improved using parametric images. Ki values from parametric images agreed very well with VOI-based gold standard (R2 > 0.99, p < 0.001). SUVs of metastases at 2 h and 4 h post-injection were highly correlated with Ki values from both the two-tissue compartment model and Patlak method (R2 = 0.87 and 0.95, both p < 0.001). 68Ga-ABY-025 PET yielded high test-retest reliability (relative repeatability coefficient for Patlak 30% and for the two-tissue compartment model 47%). CONCLUSION: 68Ga-ABY-025 binding in HER2-positive metastases was well characterized by irreversible two-tissue compartment model wherein Ki highly correlated with SUVs at 2 and 4 h. Dynamic scanning with parametric image formation can be used to evaluate metastatic HER2 expression accurately.

Perspectives on the Phase 0 clinical trial of microdose administration of ABY-029 for fluorescence guided surgery: Stability testing.

ABY-029, an anti-epidermal growth factor receptor (EGFR) Affibody molecule labeled with IRDye 800CW, has been used in three Phase 0 microdosing clinical trials for fluorescence guided surgery. In May of 2019, the clinical trials were put on hold because the ABY-029 produced under Good Manufacturing Practices (GMP) for human administration had come to the end of term in which the drug product was known to be stable. Stability testing was halted due to limitations in supply of a suitable reference standard and a required test product being discontinued from commercial sale. In order to complete the remaining patients in the three clinical trials, new stability tests were developed and the GMP batch of ABY-029 drug product tested under the new protocols. The GMP batch of ABY-029 passed all stability tests under the new protocols and the Federal Drug Administration (FDA) has given permission to complete the remaining patients with stability testing of ABY-029 performed for each patient. The tests developed and used to test ABY-029 drug product stability are described here.

Measuring microdose ABY-029 fluorescence signal in a primary human soft-tissue sarcoma resection.

Microdose administration of ABY-029, an anti-epidermal growth factor receptor Affibody molecule conjugated to IRDye 800CW, is being studied in a Phase 0 trial for resection of soft-tissue sarcomas. The excised tissue of a single patient in the microdose administration group was imaged with both a wide-field fluorescence surgical system and a flat-bed scanning fluorescence imaging system. Here the resultant fluorescence from a breadloaf section of the primary tumor specimen and six region-specific tissue samples collected from that breadloaf are compared using these two imaging systems - a flatbed, black-box, fluorescence scanning system, the Odyssey CLx, and a open-air, wide-field, pre-clinical surgical imaging system, the Solaris. Florescence signal is compared using a variety of methods including: mean, standard deviation, variance, tumor-to-background ratio, biological-variance ratio, and contrast-to-noise ratio. The images produced from the Odyssey scanner have higher signal variance but more accurately represent the EGFR expression in small tissue sections. The Solaris system has higher depth sensitivity and volume averaging, and as such has lower signal variation and higher contrast-to-noise ratio.

Preclinical imaging of epidermal growth factor receptor with ABY-029 in soft-tissue sarcoma for fluorescence-guided surgery and tumor detection.

BACKGROUND AND OBJECTIVES: Fluorescence-guided surgery using epidermal growth factor receptor (EGFR) targeting has been performed successfully in clinical trials using a variety of fluorescent agents. We investigate ABY-029 (anti-EGFR Affibody® molecule labeled with IRDye 800CW) compared with a small-molecule perfusion agent, IRDye 700DX carboxylate, in a panel of soft-tissue sarcomas with varying levels of EGFR expression and vascularization. METHODS: Five xenograft soft-tissue sarcoma cell lines were implanted into immunosuppressed mice. ABY-029 and IRDye 700DX were each administered at 4.98 µM. Fluorescence from in vivo and ex vivo (fresh and formalin-fixed) fixed tissues were compared. The performance of three fluorescence imaging systems was assessed for ex vivo tissues. RESULTS: ABY-029 is retained longer within tumor tissue and achieves higher tumor-to-background ratios both in vivo and ex vivo than IRDye 700DX. ABY-029 fluorescence is less susceptible to formalin fixation than IRDye 700DX, but both agents have disproportional signal loss in a variety of tissues. The Pearl Impulse provides the highest contrast-to-noise ratio, but all systems have individual advantages. CONCLUSIONS: ABY-029 demonstrates promise to assist in wide local excision of soft-tissue sarcomas. Further clinical evaluation of in situ or freshly excised ex vivo tissues using fluorescence imaging systems is warranted.

Diagnostic HER2-binding radiopharmaceutical, [68Ga]Ga-ABY-025, for routine clinical use in breast cancer patients.

[68Ga]Ga-ABY-025/PET-CT targeting human epidermal growth factor receptor type 2 (HER2) has demonstrated its potential clinical value for the detection and quantification of HER2 in a phase I clinical study with breast cancer patients. Previously, the radiopharmaceutical was prepared manually, however larger scale of multicenter clinical trials and routine healthcare requires automation of the production process to limit the operator radiation dose, improve tracer manufacturing robustness, and provide on-line documentation for good manufacturing practice (GMP) compliance. The production of [68Ga]Ga-ABY-025 was implemented on the Modular-Lab PharmTrace synthesis platform (Eckert & Ziegler) and disposable cassettes were developed. Pharmaceutical grade 68Ge/68Ga generator (GalliaPharm®) was used in the study. The active pharmaceutical ingredient starting material ABY-025 (GMP grade) was provided by Affibody AB. The patient examinations were conducted using a Discovery MI PET/CT scanner (20 cm FOV, GE Healthcare). Reproducible and GMP compliant fully automated production of [68Ga]Ga-ABY-025 was developed. The radiochemical purity of the product was 98.7 ± 0.6% with total peptide content of 315 ± 15 µg (n = 3). Radionuclidic purity, sterility, endotoxin content, residual solvent content, and sterile filter integrity were controlled and met acceptance criteria. The product was stable at ambient temperature for at least 2 h. The primary tumor and metastasis were detected with SUVmax values of 8.3 and 16.0, respectively. Automated production of [68Ga]Ga-ABY-025 was established and the process was validated enabling standardized multicenter phase II and III clinical trials and routine clinical use. Patient examinations conformed to the radiopharmaceutical biodistribution observed in the previous phase I study.

Application of Fluorescence-Guided Surgery to Subsurface Cancers Requiring Wide Local Excision: Literature Review and Novel Developments Toward Indirect Visualization.

The excision of tumors by wide local excision is challenging because the mass must be removed entirely without ever viewing it directly. Positive margin rates in sarcoma resection remain in the range of 20% to 35% and are associated with increased recurrence and decreased survival. Fluorescence-guided surgery (FGS) may improve surgical accuracy and has been utilized in other surgical specialties. ABY-029, an anti-epidermal growth factor receptor Affibody molecule covalently bound to the near-infrared fluorophore IRDye 800CW, is an excellent candidate for future FGS applications in sarcoma resection; however, conventional methods with direct surface tumor visualization are not immediately applicable. A novel technique involving imaging through a margin of normal tissue is needed. We review the past and present applications of FGS and present a novel concept of indirect FGS for visualizing tumor through a margin of normal tissue and aiding in excising the entire lesion as a single, complete mass with tumor-free margins.

Intra-image referencing for simplified assessment of HER2-expression in breast cancer metastases using the Affibody molecule ABY-025 with PET and SPECT.

PURPOSE: In phase I/II-studies radiolabelled ABY-025 Affibody molecules identified human epidermal growth factor receptor 2 (HER2) expression in breast cancer metastases using PET and SPECT imaging. Here, we wanted to investigate the utility of a simple intra-image normalization using tumour-to-reference tissue-ratio (T/R) as a HER2 status discrimination strategy to overcome potential issues related to cross-calibration of scanning devices. METHODS: Twenty-three women with pre-diagnosed HER2-positive/negative metastasized breast cancer were scanned with [111In]-ABY-025 SPECT/CT (n = 7) or [68Ga]-ABY-025 PET/CT (n = 16). Uptake was measured in all metastases and in normal spleen, lung, liver, muscle, and blood pool. Normal tissue uptake variation and T/R-ratios were established for various time points and for two different doses of injected peptide from a total of 94 whole-body image acquisitions. Immunohistochemistry (IHC) was used to verify HER2 expression in 28 biopsied metastases. T/R-ratios were compared to IHC findings to establish the best reference tissue for each modality and each imaging time-point. The impact of shed HER2 in serum was investigated. RESULTS: Spleen was the best reference tissue across modalities, followed by blood pool and lung. Spleen-T/R was highly correlated to PET SUV in metastases after 2 h (r = 0.96, P < 0.001) and reached an accuracy of 100% for discriminating IHC HER2-positive and negative metastases at 4 h (PET) and 24 h (SPECT) after injection. In a single case, shed HER2 resulted in intense tracer retention in blood. In the remaining patients shed HER2 was elevated, but without significant impact on ABY-025 biodistribution. CONCLUSION: T/R-ratios using spleen as reference tissue accurately quantify HER2 expression with radiolabelled ABY-025 imaging in breast cancer metastases with SPECT and PET. Tracer binding to shed HER2 in serum might affect quantification in the extreme case.

Fluorescent Affibody Molecule Administered In Vivo at a Microdose Level Labels EGFR Expressing Glioma Tumor Regions.

PURPOSE: Fluorescence guidance in surgical oncology provides the potential to realize enhanced molecular tumor contrast with dedicated targeted tracers, potentially with a microdose injection level. For most glioma tumors, the blood brain barrier is compromised allowing some exogenous drug/molecule delivery and accumulation for imaging. The aberrant overexpression and/or activation of epidermal growth factor receptor (EGFR) is associated with many types of cancers, including glioblastoma, and so the use of a near-infrared (NIR) fluorescent molecule targeted to the EGFR receptor provides the potential for improving tumor contrast during surgery. Fluorescently labeled affibody molecule (ABY-029) has high EGFR affinity and high potential specificity with reasonably fast plasma clearance. In this study, ABY-29 was evaluated in glioma versus normal brain uptake from intravenous injection at a range of doses, down to a microdose injection level. PROCEDURE: Nude rats were inoculated with the U251 human glioma cell line in the brain. Tumors were allowed to grow for 3-4 weeks. ABY-029 fluorescence ex vivo imaging of brain slices was acquired at different time points (1-48 h) and varying injection doses from 25 to 122 µg/kg (from human protein microdose equivalent to five times microdose levels). RESULTS: The tumor was most clearly visualized at 1-h post-injection with 8- to 16-fold average contrast relative to normal brain. However, the tumor still could be identified after 48 h. In all cases, the ABY-029 fluorescence appeared to localize preferentially in EGFR-positive regions. Increasing the injected dose from a microdose level to five times, a microdose level increased the signal by 10-fold, and the contrast was from 8 to 16, showing that there was value in doses slightly higher than the microdose restriction. Normal tissue uptake was found to be affected by the tumor size, indicating that edema was a likely factor affecting the expected tumor to normal tissue contrast. CONCLUSION: These results suggest that the NIR-labeled affibody molecules provide an excellent potential to increase surgical visualization of EGFR-positive tumor regions.

Toxicity and Pharmacokinetic Profile for Single-Dose Injection of ABY-029: a Fluorescent Anti-EGFR Synthetic Affibody Molecule for Human Use.

PURPOSE: ABY-029, a synthetic Affibody peptide, Z03115-Cys, labeled with a near-infrared fluorophore, IRDye® 800CW, targeting epidermal growth factor receptor (EGFR) has been produced under good manufacturing practices for a US Food and Drug Administration-approved first-in-use human study during surgical resection of glioma, as well as other tumors. Here, the pharmacology, phototoxicity, receptor activity, and biodistribution studies of ABY-029 were completed in rats, prior to the intended human use. PROCEDURES: Male and female Sprague Dawley rats were administered a single intravenous dose of varying concentrations (0, 245, 2449, and 24,490 µg/kg corresponding to 10×, 100×, and 1000× an equivalent human microdose level) of ABY-029 and observed for up to 14 days. Histopathological assessment of organs and tissues, clinical chemistry, and hematology were performed. In addition, pharmacokinetic clearance and biodistribution of ABY-029 were studied in subgroups of the animals. Phototoxicity and ABY-029 binding to human and rat EGFR were assessed in cell culture and on immobilized receptors, respectively. RESULTS: Histopathological assessment and hematological and clinical chemistry analysis demonstrated that single-dose ABY-029 produced no pathological evidence of toxicity at any dose level. No phototoxicity was observed in EGFR-positive and EGFR-negative glioma cell lines. Binding strength and pharmacokinetics of the anti-EGFR Affibody molecules were retained after labeling with the dye. CONCLUSION: Based on the successful safety profile of ABY-029, the 1000× human microdose 24.5 mg/kg was identified as the no observed adverse effect level following intravenous administration. Conserved binding strength and no observed light toxicity also demonstrated ABY-029 safety for human use.

Simultaneous In Vivo Fluorescent Markers for Perfusion, Protoporphyrin Metabolism, and EGFR Expression for Optically Guided Identification of Orthotopic Glioma.

Purpose: While extent of tumor resection is an important predictor of outcome in glioma, margin delineation remains challenging due to lack of inherent contrast between tumor and normal parenchyma. Fluorescence-guided surgery is promising for its ability to enhance contrast through exogenous fluorophores; however, the specificity and sensitivity of the underlying contrast mechanism and tumor delivery and uptake vary widely across approved and emerging agents.Experimental Design: Rats with orthotopic F98 wild-type and F98 EGFR-positive (EGFR+) gliomas received in vivo administration of IRDye680RD, 5-aminioleuvulinic acid, and ABY-029-markers of perfusion, protoporphyrin metabolism, and EGFR expression, respectively. Ex vivo imaging demonstrates the contrast mechanism-dependent spatial heterogeneity and enables within-animal comparisons of tumor-to-background ratio (TBR).Results: Generally, ABY-029 outperformed PpIX in F98EGFR orthotopic tumor margins and core (50% and 60% higher TBR, respectively). PpIX outperformed ABY-029 in F98wt margins by 60% but provided equivalent contrast in the bulk tumor. IRDye680RD provided little contrast, having an average TBR of 1.7 ± 0.2. The unique spatial patterns of each agent were combined into a single metric, the multimechanistic fluorescence-contrast index (MFCI). ABY-029 performed best in EGFR+ tumors (91% accuracy), while PpIX performed best in wild-type tumors (87% accuracy). Across all groups, ABY-029 and PpIX performed similarly (80% and 84%, respectively) but MFCI was 91% accurate, supporting multiagent imaging when tumor genotype was unknown.Conclusions: Human use of ABY-029 for glioma resection should enhance excision of EGFR+ tumors and could be incorporated into current PpIX strategies to further enhance treatment in the general glioma case. Clin Cancer Res; 23(9); 2203-12. ©2016 AACR.

Vision 20/20: Molecular-guided surgical oncology based upon tumor metabolism or immunologic phenotype: Technological pathways for point of care imaging and intervention.

Surgical guidance with fluorescence has been demonstrated in individual clinical trials for decades, but the scientific and commercial conditions exist today for a dramatic increase in clinical value. In the past decade, increased use of indocyanine green based visualization of vascular flow, biliary function, and tissue perfusion has spawned a robust growth in commercial systems that have near-infrared emission imaging and video display capabilities. This recent history combined with major preclinical innovations in fluorescent-labeled molecular probes, has the potential for a shift in surgical practice toward resection guidance based upon molecular information in addition to conventional visual and palpable cues. Most surgical subspecialties already have treatment management decisions partially based upon the immunohistochemical phenotype of the cancer, as assessed from molecular pathology of the biopsy tissue. This phenotyping can inform the surgical resection process by spatial mapping of these features. Further integration of the diagnostic and therapeutic value of tumor metabolism sensing molecules or immune binding agents directly into the surgical process can help this field mature. Maximal value to the patient would come from identifying the spatial patterns of molecular expression in vivo that are well known to exist. However, as each molecular agent is advanced into trials, the performance of the imaging system can have a critical impact on the success. For example, use of pre-existing commercial imaging systems are not well suited to image receptor targeted fluorophores because of the lower concentrations expected, requiring orders of magnitude more sensitivity. Additionally the imaging system needs the appropriate dynamic range and image processing features to view molecular probes or therapeutics that may have nonspecific uptake or pharmacokinetic issues which lead to limitations in contrast. Imaging systems need to be chosen based upon objective performance criteria, and issues around calibration, validation, and interpretation need to be established before a clinical trial starts. Finally, as early phase trials become more established, the costs associated with failures can be crippling to the field, and so judicious use of phase 0 trials with microdose levels of agents is one viable paradigm to help the field advance, but this places high sensitivity requirements on the imaging systems used. Molecular-guided surgery has truly transformative potential, and several key challenges are outlined here with the goal of seeing efficient advancement with ideal choices. The focus of this vision 20/20 paper is on the technological aspects that are needed to be paired with these agents.

Good manufacturing practice production of [(68)Ga]Ga-ABY-025 for HER2 specific breast cancer imaging.

Therapies targeting human epidermal growth factor receptor type 2 (HER2) have revolutionized breast cancer treatment, but require invasive biopsies and rigorous histopathology for optimal patient stratification. A non-invasive and quantitative diagnostic method such as positron emission tomography (PET) for the pre-therapeutic determination of the presence and density of the HER2 would significantly improve patient management efficacy and treatment cost. The essential part of the PET methodology is the production of the radiopharmaceutical in compliance with good manufacturing practice (GMP). The use of generator produced positron emitting (68)Ga radionuclide would provide worldwide accessibility of the agent. GMP compliant, reliable and highly reproducible production of [(68)Ga]Ga-ABY-025 with control over the product peptide concentration and amount of radioactivity was accomplished within one hour. Two radiopharmaceuticals were developed differing in the total peptide content and were validated independently. The specific radioactivity could be kept similar throughout the study, and it was 6-fold higher for the low peptide content radiopharmaceutical. Intrapatient comparison of the two peptide doses allowed imaging optimization. The high peptide content decreased the uptake in healthy tissue, in particular liver, improving image contrast. The later imaging time points enhanced the contrast. The combination of high peptide content radiopharmaceutical and whole-body imaging at 2 hours post injection appeared to be optimal for routine clinical use.

Measuring HER2-Receptor Expression In Metastatic Breast Cancer Using [68Ga]ABY-025 Affibody PET/CT.

PURPOSE: Positron Emission Tomography (PET) imaging of HER2 expression could potentially be used to select patients for HER2-targed therapy, predict response based on uptake and be used for monitoring. In this phase I/II study the HER2-binding Affibody molecule ABY-025 was labeled with (68)Ga-gallium ([(68)Ga]ABY-025) for PET to study effect of peptide mass, test-retest variability and correlation of quantified uptake in tumors to histopathology. EXPERIMENTAL DESIGN: Sixteen women with known metastatic breast cancer and on-going treatment were included and underwent FDG PET/CT to identify viable metastases. After iv injection of 212±46 MBq [(68)Ga]ABY-025 whole-body PET was performed at 1, 2 and 4 h. In the first 10 patients (6 with HER2-positive and 4 with HER2-negative primary tumors), [(68)Ga]ABY-025 PET/CT with two different doses of injected peptide was performed one week apart. In the last six patients (5 HER2-positive and 1 HER2-negative primary tumors), repeated [(68)Ga]ABY-025 PET were performed one week apart as a test-retest of uptake in individual lesions. Biopsies from 16 metastases in 12 patients were collected for verification of HER2 expression by immunohistochemistry and in-situ hybridization. RESULTS: Imaging 4h after injection with high peptide content discriminated HER2-positive metastases best (p<0.01). PET SUV correlated with biopsy HER2-scores (r=0.91, p<0.001). Uptake was five times higher in HER2-positive than in HER2-negative lesions with no overlap (p=0.005). The test-retest intra-class correlation was r=0.996. [(68)Ga]ABY-025 PET correctly identified conversion and mixed expression of HER2 and targeted treatment was changed in 3 of the 16 patients. CONCLUSION: [(68)Ga]ABY-025 PET accurately quantifies whole-body HER2-receptor status in metastatic breast cancer.