Comparative Preclinical Evaluation of HER2-Targeting ABD-Fused Affibody® Molecules 177Lu-ABY-271 and 177Lu-ABY-027: Impact of DOTA Position on ABD Domain.

Radiolabeled Affibody-based targeting agent 177Lu-ABY-027, a fusion of an anti-HER2 Affibody molecule with albumin binding domain (ABD) site-specifically labeled at the C-terminus, has demonstrated a promising biodistribution profile in mice; binding of the construct to albumin prevents glomerular filtration and significantly reduces renal uptake. In this study, we tested the hypothesis that site-specific positioning of the chelator at helix 1 of ABD, at a maximum distance from the albumin binding site, would further increase the strength of binding to albumin and decrease the renal uptake. The new construct, ABY-271 with DOTA conjugated at the back of ABD, has been labelled with 177Lu. Targeting properties of 177Lu-ABY-271 and 177Lu-ABY-027 were compared directly. 177Lu-ABY-271 specifically accumulated in SKOV-3 xenografts in mice. The tumor uptake of 177Lu-ABY-271 exceeded uptake in any other organ 24 h and later after injection. However, the renal uptake of 177Lu-ABY-271 was two-fold higher than the uptake of 177Lu-ABY-027. Thus, the placement of chelator on helix 1 of ABD does not provide desirable reduction of renal uptake. To conclude, minimal modification of the design of Affibody molecules has a strong effect on biodistribution, which cannot be predicted a priori. This necessitates extensive structure-properties relationship studies to find an optimal design of Affibody-based targeting agents for therapy.

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.

Evaluating the Therapeutic Efficacy of Mono- and Bivalent Affibody-Based Fusion Proteins Targeting HER3 in a Pancreatic Cancer Xenograft Model.

Human epidermal growth factor receptor 3 (HER3) has been increasingly scrutinized as a potential drug target since the elucidation of its role in mediating tumor growth and acquired therapy resistance. Affibody molecules are so-called scaffold proteins with favorable biophysical properties, such as a small size for improved tissue penetration and extravasation, thermal and chemical stability, and a high tolerance to modifications. Additionally, affibody molecules are efficiently produced in prokaryotic hosts or by chemical peptide synthesis. We have previously evaluated the biodistribution profiles of five mono- and bivalent anti-HER3 affibody molecules (designated as 3) fused to an albumin-binding domain (designated as A), 3A, 33A, 3A3, A33, and A3, that inhibit ligand-dependent phosphorylation. In the present study, we examined the therapeutic efficacy of the three most promising variants, 3A, 33A, and 3A3, in a direct comparison with the HER3-targeting monoclonal antibody seribantumab (MM-121) in a preclinical BxPC-3 pancreatic cancer model. Xenografted mice were treated with either an affibody construct or MM-121 and the tumor growth was compared to a vehicle group. Receptor occupancy was estimated by positron emission tomography/computed tomography (PET/CT) imaging using a HER3-targeting affibody imaging agent [68Ga]Ga-(HE)3-Z08698-NODAGA. The affibody molecules could inhibit ligand-dependent phosphorylation and cell proliferation in vitro and demonstrated tumor growth inhibition in vivo comparable to that of MM-121. PET/CT imaging showed full receptor occupancy for all tested drug candidates. Treatment with 3A and 3A3 affibody constructs was more efficient than with 33A and similar to the anti-HER3 antibody seribantumab, showing that the molecular design of affibody-based therapeutics targeting HER3 in terms of the relative position of functional domains and valency has an impact on therapeutic effect.

Computed tomography and clinical outcome in patients with severe traumatic brain injury.

OBJECTIVE: To study: (i) acute computed tomography (CT) characteristics and clinical outcome; (ii) clinical course and (iii) Corticosteroid Randomisation after Significant Head Injury acute calculator protocol (CRASH) model and clinical outcome in patients with severe traumatic brain injury (sTBI). METHODS: Initial CT (CTi) and CT 24 hours post-trauma (CT24) were evaluated according to Marshall and Rotterdam classifications. Rancho Los Amigos Cognitive Scale-Revised (RLAS-R) and Glasgow Outcome Scale Extended (GOSE) were assessed at three months and one year post-trauma. The prognostic value of the CRASH model was evaluated. RESULTS: Thirty-seven patients were included. Marshall CTi and CT24 were significantly correlated with RLAS-R at three months. Rotterdam CT24 was significantly correlated with GOSE at three months. RLAS-R and the GOSE improved significantly from three months to one year. CRASH predicted unfavourable outcome at six months for 81% of patients with bad outcome and for 85% of patients with favourable outcome according to GOSE at one year. CONCLUSION: Neither CT nor CRASH yielded clinically useful predictions of outcome at one year post-injury. The study showed encouragingly many instances of significant recovery in this population of sTBI. The combination of lack of reliable prognostic indicators and favourable outcomes supports the case for intensive acute management and rehabilitation as the default protocol in the cases of sTBI.

Successful treatment of a ruptured aneurysm at the vertebral artery-posterior inferior cerebellar artery junction and simultaneous treatment of the stenotic vertebral artery with a single flow-diverting stent: a case report.

INTRODUCTION: This is the first report on the simultaneous successful treatment of a large ruptured saccular aneurysm and stenotic parent artery with a single flow-diverting stent. CASE PRESENTATION: We report the case of a 68-year-old Caucasian man with occlusion of the right vertebral artery and a ruptured aneurysm at the junction of the left posterior inferior cerebellar artery-left vertebral artery that was successfully treated by the deployment of a single flow-diverting stent in the stenotic left vertebral artery. Stent deployment was complicated by thrombotic occlusion of the basilar artery, which was successfully reopened. The patient recovered completely, and follow-up angiography at 4 months and 1 year showed patent vertebral artery with gradual shrinkage of the aneurysm. CONCLUSIONS: This report contributes to the literature on treatment of large ruptured aneurysms localized in stenotic arteries and in areas of the endocranium where a mass of embolic material in the aneurysm (coils) might compromise the circulation in the parent blood vessel or compress vital brain structures.

Imaging of platelet-derived growth factor receptor ß expression in glioblastoma xenografts using affibody molecule 111In-DOTA-Z09591.

UNLABELLED: The overexpression and excessive signaling of platelet-derived growth factor receptor ß (PDGFRß) has been detected in cancers, atherosclerosis, and a variety of fibrotic diseases. Radionuclide in vivo visualization of PDGFRß expression might help to select PDGFRß targeting treatment for these diseases. The goal of this study was to evaluate the feasibility of in vivo radionuclide imaging of PDGFRß expression using an Affibody molecule, a small nonimmunoglobulin affinity protein. METHODS: The PDGFRß-binding Z09591 Affibody molecule was site-specifically conjugated with a maleimido derivative of DOTA and labeled with (111)In. Targeting of the PDGFRß-expressing U-87 MG glioblastoma cell line using (111)In-DOTA-Z09591 was evaluated in vitro and in vivo. RESULTS: DOTA-Z09591 was stably labeled with (111)In with preserved specific binding to PDGFRß-expressing cells in vitro. The dissociation constant for (111)In-DOTA-Z09591 binding to U-87 MG cells was determined to be 92 ± 10 pM. In mice bearing U-87 MG xenografts, the tumor uptake of (111)In-DOTA-Z09591 was 7.2 ± 2.4 percentage injected dose per gram and the tumor-to-blood ratio was 28 ± 14 at 2 h after injection. In vivo receptor saturation experiments demonstrated that targeting of U-87 MG xenografts in mice was PDGFRß-specific. U-87 MG xenografts were clearly visualized using small-animal SPECT/CT at 3 h after injection. CONCLUSION: This study demonstrates the feasibility of in vivo visualization of PDGFRß-expressing xenografts using an Affibody molecule. Further development of radiolabeled Affibody molecules might provide a useful clinical imaging tool for PDGFRß expression during various pathologic conditions.

Long-term follow-up of intracranial aneurysms treated with endovascular coiling: experience from one institution.

BACKGROUND: Our aim was to evaluate the long-term treatment results in patients with intracranial aneurysms treated with endovascular techniques. METHODS: Forty-four patients treated due to intracranial aneurysms between 1996 and 2002 were investigated with a time-of-flight sequence magnetic resonance angiography (TOF MRA). RESULTS: Depending on the assessment, 47% to 51% of the treated aneurysms had a residual neck at the last digital subtraction angiography follow-up. There was filling of the aneurysm base (2%) in only 1 patient, whereas the remaining aneurysms were totally occluded. A TOF MRA performed 6 to 14 (mean 9.68) years after the last procedure showed a stable result in 93.9% of the treated aneurysms. There were no de novo aneurysms and previously untreated aneurysms were unchanged in size. CONCLUSION: Our long-term follow-up showed a stable result in previously coiled intracranial aneurysms.

Design of an optimized scaffold for affibody molecules.

Affibody molecules are non-immunoglobulin-derived affinity proteins based on a three-helical bundle protein domain. Here, we describe the design process of an optimized Affibody molecule scaffold with improved properties and a surface distinctly different from that of the parental scaffold. The improvement was achieved by applying an iterative process of amino acid substitutions in the context of the human epidermal growth factor receptor 2 (HER2)-specific Affibody molecule Z(HER2:342). Replacements in the N-terminal region, loop 1, helix 2 and helix 3 were guided by extensive structural modeling using the available structures of the parent Z domain and Affibody molecules. The effect of several single substitutions was analyzed followed by combination of up to 11 different substitutions. The two amino acid substitutions N23T and S33K accounted for the most dramatic improvements, including increased thermal stability with elevated melting temperatures of up to +12 degrees C. The optimized scaffold contains 11 amino acid substitutions in the nonbinding surface and is characterized by improved thermal and chemical stability, as well as increased hydrophilicity, and enables generation of identical Affibody molecules both by chemical peptide synthesis and by recombinant bacterial expression. A HER2-specific Affibody tracer, [MMA-DOTA-Cys61]-Z(HER2:2891)-Cys (ABY-025), was produced by conjugating MMA-DOTA (maleimide-monoamide-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to the peptide produced either chemically or in Escherichia coli. ABY-025 showed high affinity and specificity for HER2 (equilibrium dissociation constant, K(D), of 76 pM) and detected HER2 in tissue sections of SKOV-3 xenograft and human breast tumors. The HER2-binding capacity was fully retained after three cycles of heating to 90 degrees C followed by cooling to room temperature. Furthermore, the binding surfaces of five Affibody molecules targeting other proteins (tumor necrosis factor alpha, insulin, Taq polymerase, epidermal growth factor receptor or platelet-derived growth factor receptor beta) were grafted onto the optimized scaffold, resulting in molecules with improved thermal stability and a more hydrophilic nonbinding surface.

Coil embolization of anterior circulation aneurysms supported by the Solitaire AB Neurovascular Remodeling Device.

INTRODUCTION: The purpose of the study is to evaluate patients with wide-necked or complex aneurysms of the anterior circulation who underwent Solitaire AB Neurovascular Remodeling Device-assisted coil embolization. METHODS: From February 2008 to March 2009, consecutive data were collected from 45 patients with anterior circulation aneurysms. Eighteen of the patients presented with acute subarachnoid hemorrhage. Forty-six aneurysms were treated with the aid of different applications (n = 49) of the Solitaire AB Remodeling Device followed by standard coiling procedure (n = 43) using bioactive coils or/and bare coils. RESULTS: Successful positioning of the remodeling device was obtained in 95.9% of the cases. There were two thromboembolic complications (4.1%) and one severe vasospasm requiring retrieval of the device. Permanent procedural morbidity was observed in one patient (2%). The proportion of patients in whom Raymond class 1 occlusion was obtained was 53.5% (n = 23). Raymond class 2 occlusion was achieved in 42% (n = 18) and Raymond class 3 occlusion in 4.7% (n = 2). Thirty-nine patients left the hospital with a good clinical status. CONCLUSION: The initial technical and clinical results of Solitaire AB device-assisted coiling of aneurysms in the anterior circulation are highly encouraging. This technique may enhance the possibilities of the endovascular treatment of these aneurysms in clinical routine.

Spontaneous resorption of intradural lumbar disc fragments.

BACKGROUND CONTEXT: Intradural disc herniation is relatively rare complication of the spinal degenerative process that occurs most frequently in the lumbar part of the spine. Both myelographic and magnetic resonance features of this entity have been described, and the mechanism of intradural herniation has already been proposed and generally accepted. In this article, we present a case of spontaneous resorption of an intradural, fragmented intervertebral disc. Spontaneous resorption of intradural disc fragments has not been previously reported. PURPOSE: To discuss a possible mechanism of spontaneous resorption of the subdural disc fragments. STUDY DESIGN: Case report and literature review. METHODS: Radiological follow-up of a 46-year-old man with the intradural herniation of disc fragments. CONCLUSION: The reaction generated by the meninges might lead to the complete resorption of intrathecally localized disc fragments.

Affibody-mediated transferrin depletion for proteomics applications.

An Affibody (Affibody) ligand with specific binding to human transferrin was selected by phage display technology from a combinatorial protein library based on the staphylococcal protein A (SpA)-derived Z domain. Strong and selective binding of the selected Affibody ligand to transferrin was demonstrated using biosensor technology and dot blot analysis. Impressive specificity was demonstrated as transferrin was the only protein recovered by affinity chromatography from human plasma. Efficient Affibody-mediated capture of transferrin, combined with IgG- and HSA-depletion, was demonstrated for human plasma and cerebrospinal fluid (CSF). For plasma, 85% of the total transferrin content in the samples was depleted after only two cycles of transferrin removal, and for CSF, 78% efficiency was obtained in single-step depletion. These results clearly suggest a potential for the development of Affibody-based resins for the removal of abundant proteins in proteomics analyses.

Hypofractionated conformal stereotactic radiotherapy for arteriovenous malformations.

OBJECTIVE: Arteriovenous malformations (AVMs) are congenital vascular lesions that are associated with high morbidity and mortality if left untreated. There are several options for treatment, including radiotherapy. Safe and effective single-fraction radiotherapy for patients with large AVMs has been considered difficult. METHODS: Between December 1986 and June 2001, 36 patients with cerebral AVMs were treated with hypofractionated conformal stereotactic radiotherapy at Umeå University Hospital. Twenty-nine patients have been followed angiographically to date and are reported in this study. RESULTS: Twenty-four (83%) of 29 patients (mean AVM volume, 11.5 cm(3)) underwent complete obliteration of their AVMs. The rates of angiographically verified total obliteration at 2 years after treatment were 56% for AVMs 4 to 10 cm(3) and 50% for AVMs larger than 10 cm(3). The obliteration rate increased considerably with extended follow-up. Five years after treatment, the obliteration rates were 81% for AVMs 4 to 10 cm(3) and 70% for AVMs larger than 10 cm(3). CONCLUSION: Hypofractionated conformal stereotactic radiotherapy may be an important alternative to single-fraction radiotherapy in patients with large AVMs or AVMs located in eloquent areas, because it allows the administration of a higher radiation dose than is possible to deliver in single-fraction radiosurgery. With our technique of hypofractionated conformal stereotactic radiotherapy, the rate of obliterating AVMs was comparable to that of single-dose radiosurgery, although the volumes of the irradiated AVMs in our study were larger than those reported previously.

Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli.

Genetic strategies have been used for more than two decades to improve bacterial bioprocesses and to simplify recovery procedures. Such strategies include the design of efficient expression vectors and the improvement of bacterial production strains in different ways, e.g. by deletion of protease genes or engineering for overexpression of rare-codon tRNAs, foldases or chaperones. Gene multimerization is another such principle that has proved beneficial to improve production yields. Genetic strategies have furthermore been exploited to facilitate recovery processes by adapting the product for a particular purification principle. In this area, affinity fusions have been commonly used, but other principles, such as modified isoelectric point (pI) or hydrophobic properties have also been successfully investigated. A recent drastic step forward in the use of gene technology to improve recovery processes for recombinant proteins is the introduction of combinatorial protein engineering to generate tailor-made product-specific affinity ligands. This strategy, which allows efficient recovery of a recombinant protein in its native form, is likely to be increasingly used also in industrial-scale bioprocesses, since novel protein ligands have been described that can be sanitized using common industrial cleaning-in-place procedures. The examples presented in this review make it evident that genetic strategies will be of utmost importance in the future for facilitating production and recovery of recombinant proteins.

Structure, specificity, and mode of interaction for bacterial albumin-binding modules.

We have determined the solution structure of an albumin binding domain of protein G, a surface protein of group C and G streptococci. We find that it folds into a left handed three-helix bundle similar to the albumin binding domain of protein PAB from Peptostreptococcus magnus. The two domains share 59% sequence identity, are thermally very stable, and bind to the same site on human serum albumin. The albumin binding site, the first determined for this structural motif known as the GA module, comprises residues spanning the first loop to the beginning of the third helix and includes the most conserved region of GA modules. The two GA modules have different affinities for albumin from different species, and their albumin binding patterns correspond directly to the host specificity of C/G streptococci and P. magnus, respectively. These studies of the evolution, structure, and binding properties of the GA module emphasize the power of bacterial adaptation and underline ecological and medical problems connected with the use of antibiotics.