Full-length Monoclonal Antibodies Research Articles

Production of Full-Length Antibody by Pichia pastoris.

The methylotrophic yeast Pichia pastoris has become an increasingly popular host for recombinant protein expression in recent times. MRL pioneered a glycoengineered humanized P. pastoris expression system that could produce glycoproteins with glycosylation profiles similar to mammalian systems. Therapeutic glycoproteins produced by the humanized P. pastoris platform have shown comparable folding, stability, and in vitro and in vivo efficacies in preclinical models to their counterparts produced from the CHO cells. P. pastoris offers a cost and time efficient alternative platform for therapeutic protein production. This chapter describes a protocol for using P. pastoris to produce full-length monoclonal antibodies. It covers a broad spectrum of antibody expression technologies in P. pastoris, including expression vector construction, yeast transformation, high-throughput strain selection, fermentation, and antibody purification.

Antibody and Antibody Mimetic Immunotherapeutics

Antibody and Antibody Mimetic Immunotherapeutics

Continuous Crystallization of Proteins in a Stirred Classified Product Removal Tank with a Tubular Reactor in Bypass

A lab-scale stirred tank with a cooled tubular reactor in bypass was applied for continuous crystallization of lysozyme and a full-length therapeutic monoclonal antibody. The stirred tank was operated as a mixed suspension classified product removal crystallizer. Lysozyme was crystallized by a combination of cooling crystallization and salting-out. The antibody was crystallized by a combination of cooling crystallization and isoelectric crystallization. It was deduced that nucleation rates were enhanced when the protein solutions passed through the cooled tubular bypass. It was further deduced that crystal growth rates were enhanced in the stirred tank which was operated at a higher temperature compared to the tubular reactor. Classified product removal was possible by controlled sedimentation of protein crystals. The continuous crystallization system allowed a targeted control of crystal morphology and size. No sedimentation occurred in the tubular reactor and precipitation was avoided at all times. High...

Rate of Asparagine Deamidation in a Monoclonal Antibody Correlating with Hydrogen Exchange Rate at Adjacent Downstream Residues.

Antibodies are an important class of drugs, comprising more than half of all new FDA approvals. Therapeutic antibodies must be chemically stable both in storage and in vivo, following administration to patients. Deamidation is a major degradation pathway for all natural and therapeutic proteins circulating in blood. Here, the linkage between deamidation propensity and structural dynamics is investigated by examining two antibodies with differing specificities. While both antibodies share a canonical asparagine-glycine (NG) motif in a structural loop, this is prone to deamidation in one of the antibodies but not the other. We found that the hydrogen-exchange rate at the adjacent two amides, often the autocatalytic nucleophiles in deamidation, correlated with the rate of degradation. This previously unreported observation was confirmed upon mutation to stabilize the deamidation lability via a generally applicable orthogonal engineering strategy presented here. We anticipate that the structural insight into chemical degradation in full-length monoclonal antibodies and the high-resolution hydrogen-exchange methodology used will have broad application across biochemical study and drug discovery and development.

Complete De Novo Assembly of Monoclonal Antibody Sequences.

De novo protein sequencing is one of the key problems in mass spectrometry-based proteomics, especially for novel proteins such as monoclonal antibodies for which genome information is often limited or not available. However, due to limitations in peptides fragmentation and coverage, as well as ambiguities in spectra interpretation, complete de novo assembly of unknown protein sequences still remains challenging. To address this problem, we propose an integrated system, ALPS, which for the first time can automatically assemble full-length monoclonal antibody sequences. Our system integrates de novo sequencing peptides, their quality scores and error-correction information from databases into a weighted de Bruijn graph to assemble protein sequences. We evaluated ALPS performance on two antibody data sets, each including a heavy chain and a light chain. The results show that ALPS was able to assemble three complete monoclonal antibody sequences of length 216–441 AA, at 100% coverage, and 96.64–100% accuracy.

Stabilizing the CH2 Domain of an Antibody by Engineering in an Enhanced Aromatic Sequon.

Monoclonal antibodies (mAbs) exhibiting highly selective binding to a protein target constitute a large and growing proportion of the therapeutics market. Aggregation of mAbs results in the loss of their therapeutic efficacy and can result in deleterious immune responses. The CH2 domain comprising part of the Fc portion of Immunoglobulin G (IgG) is typically the least stable domain in IgG-type antibodies and therefore influences their aggregation propensity. We stabilized the CH2 domain by engineering an enhanced aromatic sequon (EAS) into the N-glycosylated C'E loop and observed a 4.8 °C increase in the melting temperature of the purified IgG1 Fc fragment. This EAS-stabilized CH2 domain also conferred enhanced stability against thermal and low pH induced aggregation in the context of a full-length monoclonal IgG1 antibody. The crystal structure of the EAS-stabilized (Q295F/Y296A) IgG1 Fc fragment confirms the design principle, i.e., the importance of the GlcNAc1•F295 interaction, and surprisingly reveals that the core fucose attached to GlcNAc1 also engages in an interaction with F295. Inhibition of core fucosylation confirms the contribution of the fucose-Phe interaction to the stabilization. The Q295F/Y296A mutations also modulate the binding affinity of the full-length antibody to Fc receptors by decreasing the binding to low affinity Fc gamma receptors (FcγRIIa, FcγRIIIa, and FcγRIIIb), while maintaining wild-type binding affinity to FcRn and FcγRI. Our results demonstrate that engineering an EAS into the N-glycosylated reverse turn on the C'E loop leads to stabilizing N-glycan-protein interactions in antibodies and that this modification modulates antibody-Fc receptor binding.

Enhancing full-length antibody production by signal peptide engineering

BackgroundProtein secretion to the periplasm of Escherichia coli offers an attractive route for producing heterologous proteins including antibodies. In this approach, a signal peptide is fused to the N-terminus of the heterologous protein. The signal peptide mediates translocation of the heterologous protein from the cytoplasm to the periplasm and is cleaved during the translocation process. It was previously shown that optimization of the translation initiation region (TIR) which overlaps with the nucleotide sequence of the signal sequence improves the production of heterologous proteins. Despite the progress, there is still room to improve yields using secretion as a means to produce protein complexes such as full-length monoclonal antibodies (mAbs).ResultsIn this study we identified the inefficient secretion of heavy chain as the limitation for full-length mAb accumulation in the periplasm. To improve heavy chain secretion we investigated the effects of various signal peptides at controlled TIR strengths. The signal peptide of disulfide oxidoreductase (DsbA) mediated more efficient secretion of heavy chain than the other signal peptides tested. Mutagenesis studies demonstrated that at controlled translational levels, hydrophobicity of the hydrophobic core (H-region) of the signal peptide is a critical factor for heavy chain secretion and full-length mAb accumulation in the periplasm. Increasing the hydrophobicity of a signal peptide enhanced heavy chain secretion and periplasmic levels of assembled full-length mAbs, while decreasing the hydrophobicity had the opposite effect.ConclusionsThis study demonstrates that under similar translational strengths, the hydrophobicity of the signal peptide plays an important role in heavy chain secretion. Increasing the hydrophobicity of the H-region and controlling TIR strengths can serve as an approach to improve heavy chain secretion and full-length mAb production in E. coli.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0445-3) contains supplementary material, which is available to authorized users.

Conjugation of 10 kDa Linear PEG onto Trastuzumab Fab' Is Sufficient to Significantly Enhance Lymphatic Exposure while Preserving in Vitro Biological Activity.

The lymphatic system is a major conduit by which many diseases spread and proliferate. There is therefore increasing interest in promoting better lymphatic drug targeting. Further, antibody fragments such as Fabs have several advantages over full length monoclonal antibodies but are subject to rapid plasma clearance, which can limit the lymphatic exposure and activity of Fabs against lymph-resident diseases. This study therefore explored ideal PEGylation strategies to maximize biological activity and lymphatic exposure using trastuzumab Fab' as a model. Specifically, the Fab' was conjugated with single linear 10 or 40 kDa PEG chains at the hinge region. PEGylation led to a 3-4-fold reduction in binding affinity to HER2, but antiproliferative activity against HER2-expressing BT474 cells was preserved. Lymphatic pharmacokinetics were then examined in thoracic lymph duct cannulated rats after intravenous and subcutaneous dosing at 2 mg/kg, and the data were evaluated via population pharmacokinetic modeling. The Fab' displayed limited lymphatic exposure, but conjugation of 10 kDa PEG improved exposure by approximately 11- and 5-fold after intravenous (15% dose collected in thoracic lymph over 30 h) and subcutaneous (9%) administration, respectively. Increasing the molecular weight of the PEG to 40 kDa, however, had no significant impact on lymphatic exposure after intravenous (14%) administration and only doubled lymphatic exposure after subcutaneous administration (18%) when compared to 10 kDa PEG-Fab'. The data therefore suggests that minimal PEGylation has the potential to enhance the exposure and activity of Fab's against lymph-resident diseases, while no significant benefit is achieved with very large PEGs.

In vivo optical imaging of early osteoarthritis using an antibody specific to damaged arthritic cartilage.

BackgroundThe lack of specific and sensitive serum and radiographic biomarkers for early diagnosis of osteoarthritis (OA) as well as for monitoring subtle changes in disease activity in clinical trials has hampered the development of treatments for OA. We previously showed that 1-11E, a human single chain fragment variable (scFv) specific to collagen type II that has been post-translationally modified by reactive oxidants (ROS-CII), binds exclusively to arthritic cartilage. Here we test the validity of 1-11E as a radiographic biomarker for early disease in experimental OA.MethodsMurine OA was induced by destabilisation of the medial meniscus (DMM) in adult male mice. Immunohistochemistry of destabilised or sham-operated knees was performed from 2 to 8 weeks post-surgery with Cy5.5-labelled 1-11E and negative control scFv, C7. Prospective in vivo optical images were taken 4 and 8 weeks post-DMM following intra-articular injection of Cy5.5-labelled scFvs, or intravenous injection of Cy5.5-labelled full length monoclonal antibodies (mAbs).ResultsSpecific cartilage staining with 1-11E was apparent as early as 4 weeks post-DMM at the time of earlier cartilage degradation assessed by histology. Prospective in vivo optical images taken 4 and 8 weeks post-DMM following local intra-articular injection of Cy5.5-labelled scFv (n = 7) showed specific in vivo retention of Cys5.5-1-11E scFv following local administration into the knee joint (tissue half-life >78 hours, n = 7, signal to noise ratio (SNR) > 2.1). Specific localization of Cys-5.5-1-11E-mAb to DMM knees (SNR >1.65) was also observed (p < 0.01, n = 8, SNR >1.65). In both cases the SNR increased with time post-DMM.Conclusions1-11E binds specifically to early osteoarthritic cartilage and can be used as a radiographic biomarker following local or systemic delivery to facilitate early diagnosis and monitor disease progression in OA.

Structural Characterization of Nanoyeast Single-Chain Fragment Variable Affinity Reagents

Nanoyeast single-chain variable fragments (nanoyeast-scFv) are a new class of low-cost and stable protein capture agents developed as alternatives to full length monoclonal antibodies for use in immunosensors. Physical characteristics that impart nanoyeast-scFv with these advantages have yet to be investigated. We investigate the structure, size and surface loading of nanoyeast-scFv to better understand its ability to specifically and sensitively capture proteins of interest while retaining activity in solution. Nanoyeast-scFv fragments were found to be globular in structure and heterogeneous in size, typically <100 nm, and were found to aggregate in solution. Optimal fragment size was determined to be <220 nm, with larger fragments interfering with the electrochemical biosensor. We hypothesize that the stability of nanoyeast-scFv is conferred in part by components of the yeast cell wall fragments, which provide stability to the associated scFv both during selection, lyophilization, and rehydration.

Effect of Intravitreal Bevacizumab Injection on Iris and Iridocorneal Angle Neovascularization in Neovascular Glaucoma

Neovascular glaucoma is a serious complication associated with retinal ischemic changes, which increase the production of vascular endothelial growth factor. Vascular endothelial growth factor has been implicated as a key molecule in the development of newly formed vessels and neovascular glaucoma. Intravitreal injection of bevacizumab, a full-length humanized anti-vascular endothelial growth factor monoclonal antibody, leads to a dramatic regression of the new iris and iridocorneal angle vessels on slitlamp examination. However, anterior segment angiography reveals that bevacizumab does not cause a regression of the neovascular vessels themselves but reduces vascular permeability while newly formed vessels are still present in the iris and iridocorneal angle. This review focuses on the pathology and diagnosis of neovascula glaucoma and the effect of intravitreal bevacizumab on the iris and iridocorneal angle neovascularization.

A noncanonical function of sortase enables site-specific conjugation of small molecules to lysine residues in proteins.

We provide the first demonstration that isopeptide ligation, a noncanonical activity of the enzyme sortase A, can be used to modify recombinant proteins. This reaction was used in vitro to conjugate small molecules to a peptide, an engineered targeting protein, and a full-length monoclonal antibody with an exquisite level of control over the site of conjugation. Attachment to the protein substrate occurred exclusively through isopeptide bonds at a lysine ε-amino group within a specific amino acid sequence. This reaction allows more than one molecule to be site-specifically conjugated to a protein at internal sites, thereby overcoming significant limitations of the canonical native peptide ligation reaction catalyzed by sortase A. Our method provides a unique chemical ligation procedure that is orthogonal to existing methods, supplying a new method to site-specifically modify lysine residues that will be a valuable addition to the protein conjugation toolbox.

Functional production of a soluble and secreted single-chain antibody by a bacterial secretion system.

Single-chain variable fragments (scFvs) serve as an alternative to full-length monoclonal antibodies used in research and therapeutic and diagnostic applications. However, when recombinant scFvs are overexpressed in bacteria, they often form inclusion bodies and exhibit loss of function. To overcome this problem, we developed an scFv secretion system in which scFv was fused with osmotically inducible protein Y (osmY), a bacterial secretory carrier protein, for efficient protein secretion. Anti-EGFR scFv (αEGFR) was fused with osmY (N- and C-termini) and periplasmic leader sequence (pelB) to generate αEGFR-osmY, osmY-αEGFR, and pelB-αEGFR (control), respectively. In comparison with the control, both the osmY-fused αEGFR scFvs were soluble and secreted into the LB medium. Furthermore, the yield of soluble αEGFR-osmY was 20-fold higher, and the amount of secreted protein was 250-fold higher than that of osmY-αEGFR. In addition, the antigen-binding activity of both the osmY-fused αEGFRs was 2-fold higher than that of the refolded pelB-αEGFR from inclusion bodies. Similar results were observed with αTAG72-osmY and αHer2-osmY. These results suggest that the N-terminus of osmY fused with scFv produces a high yield of soluble, functional, and secreted scFv, and the osmY-based bacterial secretion system may be used for the large-scale industrial production of low-cost αEGFR protein.

Chimeric anti-IL-17 full-length monoclonal antibody is a novel potential candidate for the treatment of rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease, primarily manifesting as inflammatory arthritis. It is associated with chronic inflammation of the synovial joints, mostly in the hands and feet, as well as with systemic extra-articular inflammation. The chimeric anti-interleukin (IL)-17 full-length monoclonal antibody (CMa17Aab) targets IL-17A, which is an important cytokine in the pathogenesis of RA and other inflammatory disorders. In this study, we investigated whether CMa17Aab exerts therapeutic effects in a mouse model of type II collagen-induced arthritis (CIA). Mice with CIA were subcutaneously injected with the humanized CMa17Aab antibody. The effects of treatment were assessed by estimating the arthritis severity score, the extent of histological damage and bone destruction, the autoreactive humoral and cellular immune responses and the production of cytokines. Treatment with CMa17Aab exerted beneficial effects in the mice with CIA as regards clinical and histological parameters. Compared with the controls, treatment with CMa17Aab resulted in a significant alleviation of the severity of the symptoms of arthritis, by preventing bone damage and cartilage destruction, reducing humoral and cellular immune responses, and downregulating the expression of IL-6, IL-8, matrix metalloproteinase (MMP)-3, IL-17, IL-1β, tumor necrosis factor (TNF)-α, receptor activator for nuclear factor-κB ligand (RANKL) and interferon (IFN)-γ in inflamed tissues. In conclusion, our study demonstrates that treatment with CMa17Aab exerts beneficial effects in mice with CIA, by preventing joint inflammation, cartilage destruction and bone damage. These preliminary results suggest that CMa17Aab is an important regulator in RA, and that it may represent a novel therapeutic agent that may prove useful in the treatment of this disease.

Abstract B247: Integration of a modularized protein engineering technology and the RheoSwitch Therapeutic System® platform to develop high affinity trastuzumab single chain variable fragment-Fc proteins for gene therapy applications.

Abstract While monoclonal antibodies remain highly effective anticancer agents, their complex post-translational processing preclude their use in multi-effector gene therapy platforms. Breast and ovarian cancers often overexpress HER family receptors and consequently the receptors are targets for effective therapeutics such as the monoclonal antibodies trastuzumab and cetuximab. To address the need for new immune modulating formats that can be encoded in a single therapy, we have designed a platform for rapid conversion and assay of monoclonal antibodies into functional single chain variable fragment-Fc fusion proteins. Here we demonstrate results with trastuzumab and cetuximab as examples of the platform scFv-Fc conversion. The scFv-Fc trastuzumab displays efficient expression from transfected cell lines and high-affinity, high specificity binding to HER2 in plate-based screens with measured KD of 0.15nM compared to commercial trastuzumab's KD of 0.08nM in the same assay. scFv-Fc trastuzumab was shown to bind the HER2+ breast cancer cell lines BT-474 and SKBR3 in FACS-based assays while showing no affinity towards HER2- control cells SHP-77. Antibody-dependent cell-mediated cytotoxicity assays (ADCC) were performed to evaluate ADCC potency of the scFv-Fc trastuzumab. Using NK92/CD16A(158V/V) cells as effectors and BT474 cells as targets, the EC50 of the scFv-Fc trastuzumab was 3.6ng/ml, compared to the EC50 of monoclonal format trastuzumab at 0.94ng/ml. The data provide a direct demonstration of the scFv-Fc format as a viable alternative for achieving molecular targeting in a single chain entity at a level commensurate with KD values of full-length monoclonal antibodies while maintaining ADCC activity. In addition, functional scFv-Fc trastuzumab was expressed in combination with scFv-Fc cetuximab from a single vector. scFv-Fc molecules generated using this modular protein engineering platform were cloned into plasmid DNA vectors controlled by the RheoSwitch Therapeutic System®. Transient transfections into differentiated C2C12 myocytes demonstrated the production of scFv-Fc proteins from muscle cells under control of the RheoSwitch® Activator Ligand. The integrated scFv-Fc optimization platform and inducible multigenic systems described here provides support for future work for the treatment of cancer using multigenic therapeutic antibodies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B247. Citation Format: Charles Reed, Vernon Dailey, Anissa Elayadi, Lindsay Williams, Dina Schneider, Thomas Reed, Jonathan Lewis, Richard Einstein, Samuel Broder. Integration of a modularized protein engineering technology and the RheoSwitch Therapeutic System® platform to develop high affinity trastuzumab single chain variable fragment-Fc proteins for gene therapy applications. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B247.

Production of monoclonal camelid antibodies by means of hybridoma technology (P3376)

Abstract Here we describe the first monoclonal immunoglobulins produced by camelid hybridomas. Hybridomas were generated by using the CBF7 heteromyeloma line to fuse peripheral blood lymphocytes from New World camels (llamas, alpacas) and Old World camels (Bactrian camels). The hybridomas produced immunoglobulins of IgG1, IgG2 and IgG3 subclasses as defined by both enzyme immunoassays and PCR and subsequent gene sequencing. The subclass-specific murine monoclonal antibodies were also produced in the experiments described here. They could be used to detect conventional four-chain camelid antibodies (of subclass IgG1) and heavy chain-only antibodies (both of subclasses IgG2 and IgG3). The different monoclonal heavy chain-only immunoglobulins and the producing hybridomas are of special interest since they can be used to produce further secondary reagents (murine monoclonal antibodies) for the characterization of different camelid antibodies and to solve some as yet unresolved problems in the molecular biology of heavy chain antibody-producing cells. Most importantly, the technique described here can be the basis for the production of full-length heavy chain-only monoclonal antibodies of camelids directed against specific antigens.

Aminoglycoside phosphotransferase II gene as primary selection marker for Pichia pastoris producing full-length monoclonal antibody

Historically aminoglycoside phosphotransferase gene I (APH-I) has been used as a selection marker in Pichia pastoris (P. pastoris) whereas APH-II widely employed for generating stable mammalian cell lines. These drug resistance genes emerge from two separate transposons; their prevalence/regulations/substrate affinities among bacterial species are significantly different and vary with reaction conditions. Whether APH-II could function as a selection marker in P. pastoris for expressing heterologous proteins was unknown, and whether direct selection by antibiotic agent G418 could be used for the production of a full-length monoclonal antibody was yet to be established. In this study, these two possibilities were tested in wild-type P. pastoris using a single dual-expression vector, in which both of antibody light and heavy chain genes were constructed and controlled by two separate GAP promoters. It was demonstrated for the first time that APH-II in combination with G418 could directly select the transformants for simultaneous production of functional antibody light chain and heavy chain. The broad titer range obtained from G418-selected clones may provide a good opportunity to generate high producers.

Engineering Aggregation-Resistant Antibodies

The ability of antibodies to bind to target molecules with high affinity and specificity has led to their widespread use in diagnostic and therapeutic applications. Nevertheless, a limitation of antibodies is their propensity to self-associate and aggregate at high concentrations and elevated temperatures. The large size and multidomain architecture of full-length monoclonal antibodies have frustrated systematic analysis of how antibody sequence and structure regulate antibody solubility. In contrast, analysis of single and multidomain antibody fragments that retain the binding activity of mono-clonal antibodies has provided valuable insights into the determinants of antibody aggregation. Here we review advances in engineering antibody frameworks, domain interfaces, and antigen-binding loops to prevent aggregation of natively and nonnatively folded antibody fragments. We also highlight advances and unmet challenges in developing robust strategies for engineering large, multidomain antibodies to resist aggregation.

PET imaging of HER-2-positive tumours

Human epidermal growth factor receptor 2 (HER-2; neu, cerb-B2) is a transmembrane protein and a member of the erbB family of receptor tyrosine kinase proteins that amplifies the signal provided by other members of the HER family (HER-1, HER-3, and HER-4) by forming heterodimers with them. Overexpression of HER-2 increases tumorigenicity, production of vascular endothelial growth factor, angiogenesis and metastatic potential, and it also confers a survival advantage on cancer cells by making them resistant to apoptosis induced by certain proapoptotic stimuli. HER-2 is amplified and overexpressed in 20–30% of breast cancers, and is also overexpressed in other tumours, including ovarian, lung, gastric and oral cancers. Several studies have found that breast cancers that overexpress HER-2 have a more aggressive course and are associated with a higher relapse rate and mortality rate [1]. Therefore HER-2 has great value as a prognostic indicator of patient survival, as a molecular target for therapeutic intervention and as a predictive marker of response to antineoplastic drugs. Indeed, previous studies have shown that agents that target HER-2 are remarkably effective both in the metastatic and adjuvant settings in breast cancer. Trastuzumab, a humanized monoclonal antibody, improves response rates, time to progression and even survival when used alone or added to chemotherapy in patients with stage IV disease. Moreover, in recent years several targeted agents have become available including lapatinib, a selective, reversible dual inhibitor of the epidermal growth factor receptor (EGFR; HER-1) and HER-2 [2]. However, HER-2 tumour expression can vary during treatment and can differ across metastatic lesions, and also between primary tumour and distant localization within a patient. This is well documented in breast cancer, where a change in HER-2 status has been reported in up to 38% of patients, raising questions about the validity of making decisions concerning the use of trastuzumab based on the HER-2 status of the primary tumour alone [3–5]. Furthermore, the assessment of HER-2 status is still plagued by a high rate of false-positive and false-negative results, leading to the administration of unnecessary therapies (that could be associated with side effects, such as serious cardiac toxicity), and to the termination of effective treatments, respectively [6, 7]. Therefore there is the need for methods that are able to assess the HER-2 status repeatedly, preferably in all lesions and noninvasively. HER-2 imaging through radioisotopelabelled trastuzumab-like molecules could be very useful in this setting. Currently available HER-2 targeted ligands include full-length monoclonal antibodies, Fab fragments, F(ab’)2 fragments, diabodies, minibodies, Affibodies and peptides. When radiolabelling these HER-2-targeted molecules, the physical half-life of the radioisotope ideally should suit the biological half-life of the HER-2-targeting molecules to allow imaging at the optimal time-point. Thus fulllength monoclonal antibodies are mostly radiolabelled with long-lived isotopes, while the smaller HER-2-targeting molecules, which have a more rapid clearance, are radiolabelledwith shorter-lived isotopes. In, I and Tc have been used to label full-length HER-2 monoclonal antibodies and smaller HER-2-targeting antibody fragments, proteins and peptides for SPECT/gamma camera imaging. Moreover, full-length monoclonal antibodies have been labelled with This Editorial Commentary refers to the articles http://dx.doi.org/10.1007/ s00259-011-1810-4 and http://dx.doi.org/10.1007/s00259-011-1879-9

Utilisation du bévacizumab (Avastin®) en injection intravitréenne avant vitrectomie pour rétinopathie diabétique sévère, à propos de six cas

Bevacizumab (Avastin(®), Roche) is a full-length humanized monoclonal antibody applicable to all subtypes of vascular endothelial growth factor (VEGF). The purpose of this study was to report the results of its use as a surgical additive in severe cases of proliferative diabetic retinopathy (PDR). This retrospective study focused on six eyes of six patients with complicated diabetic retinopathy. A vitrectomy was performed within 13.6 days after an intravitreal bevacizumab injection of 0.1 mL (2.5mg), with dissection of the fibrovascular proliferation using a mono- or bimanual delamination technique. The mean follow-up after intravitreal injection was 13.3 months. The mean surgery time was 64 minutes. The bimanual technique was not necessary. Only one iatrogenic retinal tear was repaired. The intraoperative bleeding was negligible. No adverse events resulting from the drug nor recurrence were observed throughout the follow-up period. Intravitreal bevacizumab is useful as a surgical additive in severe cases of PDR, significantly improving surgical conditions. Nevertheless, its use beyond approved indications should be reserved for complex surgical cases.