Dynamics inside the cancer cell attractor reveal cell heterogeneity, limits of stability, and escape.

The observed intercellular heterogeneity within a clonal cell population can be mapped as dynamical states clustered around an attractor point in gene expression space, owing to a balance between homeostatic forces and stochastic fluctuations. These dynamics have led to the cancer cell attractor conceptual model, with implications for both carcinogenesis and new therapeutic concepts. Immortalized and malignant EBV-carrying B-cell lines were used to explore this model and characterize the detailed structure of cell attractors. Any subpopulation selected from a population of cells repopulated the whole original basin of attraction within days to weeks. Cells at the basin edges were unstable and prone to apoptosis. Cells continuously changed states within their own attractor, thus driving the repopulation, as shown by fluorescent dye tracing. Perturbations of key regulatory genes induced a jump to a nearby attractor. Using the Fokker-Planck equation, this cell population behavior could be described as two virtual, opposing influences on the cells: one attracting toward the center and the other promoting diffusion in state space (noise). Transcriptome analysis suggests that these forces result from high-dimensional dynamics of the gene regulatory network. We propose that they can be generalized to all cancer cell populations and represent intrinsic behaviors of tumors, offering a previously unidentified characteristic for studying cancer.

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.

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.

Eprenetapopt combined with venetoclax and azacitidine in TP53-mutated acute myeloid leukaemia: a phase 1, dose-finding and expansion study.

BACKGROUND: TP53-mutated acute myeloid leukaemia is associated with poor outcomes. Eprenetapopt (APR-246) is a first-in-class, small-molecule p53 reactivator. We aimed to evaluate the combination of eprenetapopt and venetoclax with or without azacitidine in patients with TP53-mutated acute myeloid leukaemia. METHODS: This phase 1, multicentre, open-label, dose-finding and cohort expansion study was done at eight academic research hospitals in the USA. Inclusion criteria were age of at least 18 years; at least one pathogenic TP53 mutation; treatment-naive acute myeloid leukaemia according to the 2016 WHO classification; an ECOG performance status of 0-2; and a life expectancy of at least 12 weeks. In dose-finding cohort 1 patients received previous therapy with hypomethylating agents for myelodysplastic syndromes. In dose-finding cohort 2, previous use of hypomethylating agents was not permitted. Treatment cycles were 28 days. Patients in cohort 1 received intravenous eprenetapopt 4·5 g/day on days 1-4 and oral venetoclax 400 mg/day on days 1-28; those in cohort 2 also received subcutaneous or intravenous azacitidine 75 mg/m2 on days 1-7. The expansion part of the study proceeded with patients enrolled as in cohort 2. Primary endpoints were safety in all cohorts (assessed in patients receiving at least one dose of assigned treatment) and complete response in the expansion cohort (assessed in patients who completed at least one treatment cycle and had at least one post-treatment clinical response assessment). The trial is registered with ClinicalTrials.gov, NCT04214860, and is complete. FINDINGS: Between Jan 3, 2020, and July 22, 2021, 49 patients were enrolled across all cohorts. Six patients were initially enrolled into each of dose-finding cohorts 1 and 2; after no dose-limiting toxicities were observed, cohort 2 was expanded to enrol an additional 37 patients. The median age was 67 years (IQR 59-73). 24 (49%) of 49 patients were female and 25 (51%) male, and 40 (82%) were White. At data cutoff (Oct 1, 2021), the median length of follow-up was 9·5 months (IQR 6·1-11·5). No dose-limiting toxicities were recorded and the recommended phase 2 dose for eprenetapopt combinations was 4·5 g/day on days 1-4. Across all patients, adverse events of grade 3 or worse occurring in at least 20% of patients were febrile neutropenia (23 [47%] of 49 patients), thrombocytopenia (18 [37%] patients), leukopenia (12 [25%] patients), and anaemia (11 [22%] patients). Treatment-related serious adverse events occurred in 13 (27%) of 49 patients and there was one (2%) treatment-related death (sepsis). 25 (64%, 95% CI 47-79) of 39 patients had an overall response with eprenetapopt and venetoclax with azacytidine; 15 (38%, 23-55) had a complete response. INTERPRETATION: Eprenetapopt and venetoclax with azacitidine had an acceptable safety profile and encouraging activity, supporting further frontline evaluation of this combination in the treatment of TP53-mutated acute myeloid leukaemia. FUNDING: Aprea Therapeutics.

Eprenetapopt Plus Azacitidine After Allogeneic Hematopoietic Stem-Cell Transplantation for TP53-Mutant Acute Myeloid Leukemia and Myelodysplastic Syndromes.

PURPOSE: Outcomes are poor in TP53-mutant (mTP53) acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), even after allogeneic hematopoietic stem-cell transplant (HCT). Eprenetapopt (APR-246) is a first-in-class, small-molecule p53 reactivator. PATIENTS AND METHODS: We conducted a phase II, multicenter, open-label trial to assess efficacy and safety of eprenetapopt combined with azacitidine as maintenance therapy after HCT (ClinicalTrials.gov identifier: NCT03931291). Patients with mTP53 MDS or AML received up to 12 cycles of eprenetapopt 3.7 g once daily intravenously on days 1-4 and azacitidine 36 mg/m2 once daily intravenously/subcutaneously on days 1-5 in 28-day cycles. The primary outcomes were relapse-free survival (RFS) and safety. RESULTS: Of the 84 patients screened for eligibility before HCT, 55 received a transplant. Thirty-three patients ultimately received maintenance treatment (14 AML and 19 MDS); the median age was 65 (range, 40-74) years. The median number of eprenetapopt cycles was 7 (range, 1-12). With a median follow-up of 14.5 months, the median RFS was 12.5 months (95% CI, 9.6 to not estimable) and the 1-year RFS probability was 59.9% (95% CI, 41 to 74). With a median follow-up of 17.0 months, the median overall survival (OS) was 20.6 months (95% CI, 14.2 to not estimable) and the 1-year OS probability was 78.8% (95% CI, 60.6 to 89.3). Thirty-day and 60-day mortalities from the first dose were 0% and 6% (n = 2), respectively. Acute and chronic (all grade) graft-versus-host disease adverse events were reported in 12% (n = 4) and 33% (n = 11) of patients, respectively. CONCLUSION: In patients with mTP53 AML and MDS, post-HCT maintenance therapy with eprenetapopt combined with azacitidine was well tolerated. RFS and OS outcomes were encouraging in this high-risk population.

Optimal specific radioactivity of anti-HER2 Affibody molecules enables discrimination between xenografts with high and low HER2 expression levels.

PURPOSE: Overexpression of the HER2 receptor is a biomarker for predicting those patients who may benefit from trastuzumab therapy. Radiolabelled Affibody molecules can be used to visualize HER2 expression in tumour xenografts with high sensitivity. However, previous studies demonstrated that the difference in uptake in xenografts with high and low HER2 expression levels is not proportional to the difference in expression levels. We hypothesized that discrimination between tumours with high and low HER2 expression may be improved by increasing the injected dose (reducing the specific activity) of the tracer. METHODS: The influence of injected dose of anti-HER2 (111)In-DOTA-Z(HER2 342) Affibody molecule on uptake in SKOV-3 (high HER2 expression) and LS174T (low expression) xenografts was investigated. The optimal range of injected doses enabling discrimination between xenografts with high and low expression was determined. To verify this, tumour uptake was measured in mice carrying both SKOV-3 and LS174T xenografts after injection of either 1 or 15 µg (111)In-DOTA-Z(HER2:342). RESULTS: An increase in the injected dose caused a linear decrease in the radioactivity accumulation in the LS174T xenografts (low HER2 expression). For SKOV-3 xenografts, the dependence of the tumour uptake on the injected dose was less dramatic. The injection of 10-30 µg (111)In-DOTA-Z(HER2:342) per mouse led to the largest difference in uptake between the two types of tumour. Experiments in mice bearing two xenografts confirmed that the optimized injected dose enabled better discrimination of expression levels. CONCLUSION: Careful optimization of the injected dose of Affibody molecules is required for maximum discrimination between xenografts with high and low levels of HER2 expression. This information has potential relevance for clinical imaging applications.

Imaging of EGFR expression in murine xenografts using site-specifically labelled anti-EGFR 111In-DOTA-Z EGFR:2377 Affibody molecule: aspect of the injected tracer amount.

INTRODUCTION: Overexpression of epidermal growth factor receptor (EGFR) is a prognostic and predictive biomarker in a number of malignant tumours. Radionuclide molecular imaging of EGFR expression in cancer could influence patient management. However, EGFR expression in normal tissues might complicate in vivo imaging. The aim of this study was to evaluate if optimization of the injected protein dose might improve imaging of EGFR expression in tumours using a novel EGFR-targeting protein, the DOTA-Z(EGFR:2377) Affibody molecule. METHODS: An anti-EGFR Affibody molecule, Z(EGFR:2377), was labelled with (111)In via the DOTA chelator site-specifically conjugated to a C-terminal cysteine. The affinity of DOTA-Z(EGFR:2377) for murine and human EGFR was measured by surface plasmon resonance. The cellular processing of (111)In-DOTA-Z(EGFR:2377) was evaluated in vitro. The biodistribution of radiolabelled Affibody molecules injected in a broad range of injected Affibody protein doses was evaluated in mice bearing EGFR-expressing A431 xenografts. RESULTS: Site-specific coupling of DOTA provided a uniform conjugate possessing equal affinity for human and murine EGFR. The internalization of (111)In-DOTA-Z(EGFR:2377) by A431 cells was slow. In vivo, the conjugate accumulated specifically in xenografts and in EGFR-expressing tissues. The curve representing the dependence of tumour uptake on the injected Affibody protein dose was bell-shaped. The highest specific radioactivity (lowest injected protein dose) provided a suboptimal tumour-to-blood ratio. The results of the biodistribution study were confirmed by gamma-camera imaging. CONCLUSION: The (111)In-DOTA-Z(EGFR:2377) Affibody molecule is a promising tracer for radionuclide molecular imaging of EGFR expression in malignant tumours. Careful optimization of protein dose is required for high-contrast imaging of EGFR expression in vivo.

[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.

The influence of Bz-DOTA and CHX-A''-DTPA on the biodistribution of ABD-fused anti-HER2 Affibody molecules: implications for (114m)In-mediated targeting therapy.

PURPOSE: Affibody molecules represent a novel class of high-affinity agents for radionuclide tumour targeting. Fusion of the Affibody molecules with an albumin-binding domain (ABD) enables modification of the blood kinetics of the Affibody molecules and reduction of the renal dose. (177)Lu-CHX-A''-DTPA-ABD-(Z(HER2:342))(2), an anti-HER2 Affibody molecule-ABD fusion protein has earlier demonstrated promising results in treatment of HER2-expressing micro-xenografts in mice. The use of the in vivo generator (114m)In/(114)In as a label for ABD-fused Affibody molecules would create preconditions for efficient treatment of both micrometastases (due to conversion and Auger electrons of (114m)In) and bulky tumours (due to high-energy beta particles from the daughter nuclide (114)In). The goal of this study was to investigate if different chelators influence the biodistribution of ABD-(Z(HER2:342))(2) and to find an optimal chelator for attachment of (114m)In to the Affibody molecule-ABD fusion protein. METHODS: Isothiocyanate derivatives of Bz-DOTA and CHX-A''-DTPA were coupled to ABD-(Z(HER2:342))(2). The cellular processing of both conjugates was studied in vitro. The influence of chelators on the biodistribution was investigated in mice using double isotope ((114m)In and (111)In) labelling. RESULTS: The apparent affinity of CHX-A''-DTPA-ABD-(Z(HER2:342))(2) and Bz-DOTA-ABD-(Z(HER2:342))(2) to the extracellular domain of HER2 was similar, 13.5 and 15.0 pM, respectively. It was found that both conjugates were internalized by SKOV-3 cells. The use of CHX-A''-DTPA provided better cellular retention of the radioactivity, better tumour accumulation of radioactivity and better tumour to organ dose ratios than Bz-DOTA-ABD-(Z(HER2:342))(2). CONCLUSION: CHX-A''-DTPA is more suitable for (114m)In labelling of Affibody molecule-ABD fusion proteins for radionuclide therapy.

Positioning of 99mTc-chelators influences radiolabeling, stability and biodistribution of Affibody molecules.

Affibody molecules represent a novel class of affinity proteins with a high potential as tracers for radionuclide molecular imaging. In this comparative structure-property study, a series of Affibody molecules with the (99m)Tc-chelators maGGG, maSSS, or maESE attached to the epsilon-amine of the internally positioned K49 was prepared by peptide synthesis, for comparison to molecules with similar chelators positioned at the N-terminus. The conjugates were labeled with (99m)Tc and evaluated in vitro and in vivo. It was found that both composition and position of the chelating moiety influence the label stability, biodistribution and targeting properties of HER2-binding Affibody molecules.

Synthetic affibody molecules: a novel class of affinity ligands for molecular imaging of HER2-expressing malignant tumors.

The Affibody molecule Z(HER2:342-pep2), site-specifically and homogeneously conjugated with a 1,4,7,10-tetra-azacylododecane-N,N',N'',N'''-tetraacetic acid (DOTA) chelator, was produced in a single chemical process by peptide synthesis. DOTA-Z(HER2:342-pep2) folds spontaneously and binds HER2 with 65 pmol/L affinity. Efficient radiolabeling with >95% incorporation of (111)In was achieved within 30 min at low (room temperature) and high temperatures (up to 90 degrees C). Tumor uptake of (111)In-DOTA-Z(HER2:342-pep2) was specific for HER2-positive xenografts. A high tumor uptake of 23% injected activity per gram tissue, a tumor-to-blood ratio of >7.5, and high-contrast gamma camera images were obtained already 1 h after injection. Pretreatment with Herceptin did not interfere with tumor targeting, whereas degradation of HER2 using the heat shock protein 90 inhibitor 17-allylamino-geldanamycin before administration of (111)In-DOTA-Z(HER2:342-pep2) obliterated the tumor image. The present results show that radiolabeled synthetic DOTA-Z(HER2:342-pep2) has the potential to become a clinically useful radiopharmaceutical for in vivo molecular imaging of HER2-expressing carcinomas.

Radionuclide therapy of HER2-positive microxenografts using a 177Lu-labeled HER2-specific Affibody molecule.

A radiolabeled anti-HER2 Affibody molecule (Z(HER2:342)) targets HER2-expressing xenografts with high selectivity and gives good imaging contrast. However, the small size (approximately 7 kDa) results in rapid glomerular filtration and high renal accumulation of radiometals, thus excluding targeted therapy. Here, we report that reversible binding to albumin efficiently reduces the renal excretion and uptake, enabling radiometal-based nuclide therapy. The dimeric Affibody molecule (Z(HER2:342))(2) was fused with an albumin-binding domain (ABD) conjugated with the isothiocyanate derivative of CHX-A''-DTPA and labeled with the low-energy beta-emitter (177)Lu. The obtained conjugate [CHX-A''-DTPA-ABD-(Z(HER2:342))(2)] had a dissociation constant of 18 pmol/L to HER2 and 8.2 and 31 nmol/L for human and murine albumin, respectively. The radiolabeled conjugate displayed specific binding to HER2-expressing cells and good cellular retention in vitro. In vivo, fusion with ABD enabled a 25-fold reduction of renal uptake in comparison with the nonfused dimer molecule (Z(HER2:342))(2). Furthermore, the biodistribution showed high and specific uptake of the conjugate in HER2-expressing tumors. Treatment of SKOV-3 microxenografts (high HER2 expression) with 17 or 22 MBq (177)Lu-CHX-A''-DTPA-ABD-(Z(HER2:342))(2) completely prevented formation of tumors, in contrast to mice given PBS or 22 MBq of a radiolabeled non-HER2-binding Affibody molecule. In LS174T xenografts (low HER2 expression), this treatment resulted in a small but significant increase of the survival time. Thus, fusion with ABD improved the in vivo biodistribution, and the results highlight (177)Lu-CHX-A''-DTPA-ABD-(Z(HER2:342))(2) as a candidate for treatment of disseminated tumors with a high level of HER2 expression.

Effects of lysine-containing mercaptoacetyl-based chelators on the biodistribution of 99mTc-labeled anti-HER2 Affibody molecules.

The effects of polar (mercaptoacetyl-triseryl) and negatively charged (mercaptoacetyl-triglumatyl) chelators on the biodistribution of 99mTc-labeled anti-HER2 Affibody molecules were previously investigated. With glycine, serine, and glutamate, we demonstrated that substitution with a single amino acid in the chelator can significantly influence the biodistribution properties and the excretion pathways. Here, we have taken this investigation further, by analyzing the effects of introduction of a positive amino acid residue on the in vivo properties of the 99mTc-labeled Affibody molecule. The Affibody molecules with mercaptoacetyl-seryl-lysyl-seryl (maSKS) and mercaptoacetyl-trilysyl (maKKK) extensions were produced by peptide synthesis and labeled with 99mTc in alkaline conditions. A comparative biodistribution was performed in normal mice to evaluate the excretion pathway. A shift toward renal excretion was obtained when serine was substituted with lysine in the chelating sequence. The radioactivity in the gastrointestinal tract was reduced 3-fold for the 99mTc-maSKS-Z(HER2:342) and 99mTc-maKKK-Z(HER2: 342) in comparison with the 99mTc-maSSS-Z(HER2:342) conjugate 4 h post injection (p.i.). The radioactivity in the liver was elevated when a triple substitution of positively charged lysine was used. The tumor targeting properties of 99mTc-maSKS-Z(HER2:342) were further investigated in SKOV-3 xenografts. The tumor uptake of 99mTc-maSKS-Z(HER2: 342) was 17+/-7% IA/g 4 h p.i. Tumor xenografts were well-visualized by gamma scintigraphy. In conclusion, the substitution with one single lysine in the chelator results in better tumor imaging properties of the Affibody molecule Z(HER2:342) and is favorable for imaging of tumors and metastases in the abdominal area. Multiple lysine residues in the chelator are, however, undesirable due to elevated uptake both in the liver and kidneys.

Development and preclinical characterisation of 99mTc-labelled Affibody molecules with reduced renal uptake.

PURPOSE: Affibody molecules are low molecular weight proteins (7 kDa), which can be selected to bind to tumour-associated target proteins with subnanomolar affinity. Because of rapid tumour localisation and clearance from nonspecific compartments, Affibody molecules are promising tracers for molecular imaging. Earlier, (99m)Tc-labelled Affibody molecules demonstrated specific targeting of tumour xenografts. However, the biodistribution was suboptimal either because of hepatobiliary excretion or high renal uptake of the radioactivity. The goal of this study was to optimise the biodistribution of Affibody molecules by chelator engineering. MATERIALS AND METHODS: Anti-HER2 Z(HER2:342) Affibody molecules, carrying the mercaptoacetyl-glutamyl-seryl-glutamyl (maESE), mercaptoacetyl-glutamyl-glutamyl-seryl (maEES) and mercaptoacetyl-seryl-glutamyl-glutamyl (maSEE) chelators, were prepared by peptide synthesis and labelled with (99m)Tc. The tumour-targeting capacity of these conjugates was compared with each other and with the best previously available conjugate, (99m)Tc-maEEE-Z(HER2:342,) in nude mice bearing SKOV-3 xenografts. The tumour-targeting capacity of the most promising conjugate, (99m)Tc-maESE-Z(HER2:342,) was compared with radioiodinated Z(HER2:342). RESULTS: All novel conjugates demonstrated successful tumour targeting and a low degree of hepatobiliary excretion. The renal uptakes of serine-containing conjugates, 33 +/- 5, 68 +/- 21 and 71 +/- 10%IA/g, for(99m)Tc-maESE-Z(HER2:342), (99m)Tc-maEES-Z(HER2:342) and (99m)Tc-maSEE-Z(HER2:342), respectively, were significantly reduced in comparison with (99m)Tc-maEEE-Z(HER2:342) (102 +/- 13%IA/g). For (99m)Tc-maESE-Z(HER2:342), a tumour uptake of 9.6 +/- 1.8%IA/g and a tumour-to-blood ratio of 58 +/- 6 were reached at 4 h p.i. CONCLUSIONS: A combination of serine and glutamic acid residues in the chelator sequence confers increased renal excretion and relatively low renal uptake of (99m)Tc-labelled Affibody molecules. In combination with preserved targeting capacity, this improved imaging of targets in abdominal area.

111In-benzyl-DTPA-ZHER2:342, an affibody-based conjugate for in vivo imaging of HER2 expression in malignant tumors.

UNLABELLED: Data on expression of the HER2 (erbB-2) receptor in breast carcinoma make it possible to select the most efficient treatment. There are strong indications that HER2 expression possesses prognostic and predictive values in ovarian, prostate, and lung carcinomas as well. Visualization of HER2 expression using radionuclide targeting can provide important diagnostic information. The Affibody Z(HER2:342) is a short (approximately 7 kDa) phage-display-selected protein that binds HER2 with an affinity of 22 pmol/L. The goal of this study was to evaluate whether (111)In-labeled HER2:342 can be used for imaging of HER2 overexpression in vivo. METHODS: Z(HER2:342) was labeled with (111)In via isothiocyanate-benzyl-DTPA (DTPA is diethylenetriaminepentaacetic acid) and the conjugate was characterized in vitro and in vivo. RESULTS: (111)In-Benzyl-DTPA-Z(HER2:342) preserved the capacity to bind living HER2-expressing cells specifically. The affinity of In-benzyl-DTPA-Z(HER2:342) to HER2 was 21 pmol/L according to surface plasmon resonance measurements. In nude mice bearing HER2-expressing SKOV-3 xenografts, a tumor uptake of 12% +/- 3% injected activity per gram and a tumor-to-blood ratio of about 100 were obtained 4 h after injection. Tumor uptake in vivo was receptor specific, as it could be blocked with an excess of nonlabeled Z(HER2:342). HER2-expressing xenografts were clearly imaged 4 h after injection using a gamma-camera. CONCLUSION: (111)In-Benzyl-DTPA-Z(HER2:342) is a promising candidate for visualization of HER2 expression in carcinomas, using the single-photon detection technique.

Transcript identification by analysis of short sequence tags--influence of tag length, restriction site and transcript database.

There exist a number of gene expression profiling techniques that utilize restriction enzymes for generation of short expressed sequence tags. We have studied how the choice of restriction enzyme influences various characteristics of tags generated in an experiment. We have also investigated various aspects of in silico transcript identification that these profiling methods rely on. First, analysis of 14 248 mRNA sequences derived from the RefSeq transcript database showed that 1-30% of the sequences lack a given restriction enzyme recognition site. Moreover, 1-5% of the transcripts have recognition sites located less than 10 bases from the poly(A) tail. The uniqueness of 10 bp tags lies in the range 90-95%, which increases only slightly with longer tags, due to the existence of closely related transcripts. Furthermore, 3-30% of upstream 10 bp tags are identical to 3' tags, introducing a risk of misclassification if upstream tags are present in a sample. Second, we found that a sequence length of 16-17 bp, including the recognition site, is sufficient for unique transcript identification by BLAST based sequence alignment to the UniGene Human non-redundant database. Third, we constructed a tag-to-gene mapping for UniGene and compared it to an existing mapping database. The mappings agreed to 79-83%, where the selection of representative sequences in the UniGene clusters is the main cause of the disagreement. The results of this study may serve to improve the interpretation of sequence-based expression studies and the design of hybridization arrays, by identifying short tags that have a high reliability and separating them from tags that carry an inherent ambiguity in their capacity to discriminate between genes. To this end, supplementary information in the form of a web companion to this paper is located at http:// biobase.biotech.kth.se/tagseq.

Identification of progression markers in B-CLL by gene expression profiling.

OBJECTIVE: B-cell chronic lymphocytic leukemia is a heterogeneous disease with a pronounced variation in the clinical course. With the purpose of identifying genes that could be related to disease progression, we have performed gene expression profiling on B-CLL patients with an indolent disease and patients with a progressive disease with need for therapy. MATERIALS AND METHODS: we applied the Affymetrix GeneChip technique to 11 B-CLL patients with stable and 10 patients with clinically progressive disease. Supervised and unsupervised clustering methods with different algorithms were used to identify genes that tend to give a distinction between stable and progressive disease. RESULTS: The supervised learning procedures identified groups of genes with a combined power to discriminate samples from progressive and stable disease with 70-90% accuracy. The gene for protein phosphatase 2 regulatory subunit B' (B56) gamma isoform (PPP2R5C) and the gene for retinoblastoma-like 2 (p130) (RBL2) were included among the best discriminators; both genes were downregulated in progressive as compared to stable B-CLL. In a hierarchical clustering analysis based on gene expression pattern three clinical subcategories could be identified: one with a more severe clinical outcome, a second one with good prognosis, and a third one that was intermediate between the other two groups. CONCLUSIONS: Our application of microarray analysis on a clinically well defined material has identified a number of genes with combined expression patterns related to stable or progressive disease in general. Unsupervised clustering suggested the existence of subclasses of samples in the progressive group that may be identifiable through gene expression patterns.

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.