Efficient production of human interleukin-3 from Escherichia coli using protein disulfide isomerase b'a' domain.

Human interleukin-3 (IL3) is a multifunctional cytokine essential for both clinical and biomedical research endeavors. However, its production in Escherichia coli has historically been challenging due to its aggregation into inclusion bodies, requiring intricate solubilization and refolding procedures. This study introduces an innovative approach employing two chaperone proteins, maltose binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a'), as N-terminal fusion tags. Histidine tag (H) was added at the beginning of each chaperone protein gene for easy purification. This fusion of chaperone proteins significantly improved IL3 solubility across various E. coli strains and temperature conditions, eliminating the need for laborious refolding procedures. Following expression optimization, H-PDIb'a'-IL3 was purified using two chromatographic methods, and the subsequent removal of the H-PDIb'a' tag yielded high-purity IL3. The identity of the purified protein was confirmed through liquid chromatography coupled with tandem mass spectrometry analysis. Biological activity assays using human erythroleukemia TF-1 cells revealed a unique two-step stimulation pattern for both purified IL3 and the H-PDIb'a'-IL3 fusion protein, underscoring the protein's functional integrity and revealing novel insights into its cellular interactions. This study advances the understanding of IL3 expression and activity while introducing novel considerations for protein fusion strategies.

Preclinical Evaluation of HER2-Targeting DARPin G3: Impact of Albumin-Binding Domain (ABD) Fusion.

Designed ankyrin repeat protein (DARPin) G3 is an engineered scaffold protein. This small (14.5 kDa) targeting protein binds with high affinity to human epidermal growth factor receptor 2 (HER2). HER2 is overexpressed in several cancers. The use of the DARPin G3 for radionuclide therapy is complicated by its high renal reabsorption after clearance via the glomeruli. We tested the hypothesis that a fusion of the DARPin G3 with an albumin-binding domain (ABD) would prevent rapid renal excretion and high renal reabsorption resulting in better tumour targeting. Two fusion proteins were produced, one with the ABD at the C-terminus (G3-ABD) and another at the N-terminus (ABD-G3). Both variants were labelled with 177Lu. The binding properties of the novel constructs were evaluated in vitro and their biodistribution was compared in mice with implanted human HER2-expressing tumours. Fusion with the ABD increased the retention time of both constructs in blood compared with the non-ABD-fused control. The effect of fusion with the ABD depended strongly on the order of the domains in the constructs, resulting in appreciably better targeting properties of [177Lu]Lu-G3-ABD. Our data suggest that the order of domains is critical for the design of targeting constructs based on scaffold proteins.

Von-Hippel Lindau protein amyloid formation. The role of GST-tag.

In the last decade, much attention was given to the study of physiological amyloid fibrils. These structures include A-bodies, which are the nucleolar fibrillar formations that appear in the response to acidosis and heat shock, and disassemble after the end of stress. One of the proteins involved in the biogenesis of A-bodies, regardless of the type of stress, is Von-Hippel Lindau protein (VHL). Known also as a tumor suppressor, VHL is capable to form amyloid fibrils both in vitro and in vivo in response to the environment acidification. As with most amyloidogenic proteins fusion with various tags is used to increase the solubility of VHL. Here, we first performed AFM-study of fibrils formed by VHL protein and by VHL fused with GST-tag (GST-VHL) at acidic conditions. It was shown that formed by full-length VHL fibrils are short heterogenic structures with persistent length of 2400 nm and average contour length of 409 nm. GST-tag catalyzes VHL amyloid fibril formation, superimpose chirality, increases length and level of hierarchy, but decreases rigidity of amyloid fibrils. The obtained data indicate that tagging can significantly affect the fibrillogenesis of the target protein.

Targeted Delivery of HSP70 to Tumor Cells via Supramolecular Complex Based on HER2-Specific DARPin9_29 and the Barnase:Barstar Pair.

(1) Background: We have previously shown that the use of an artificial supramolecular two-component system based on chimeric recombinant proteins 4D5scFv-barnase and barstar-heat shock protein 70 KDa (HSP70) allows targeted delivery of HSP70 to the surface of tumor cells bearing HER2/neu antigen. In this work, we studied the possibility to using DARPin9_29-barnase as the first targeting module recognizing HER2/neu-antigen in the HSP70 delivery system. (2) Methods: The effect of the developed systems for HSP70 delivery to human carcinomas SK-BR-3 and BT474 cells hyperexpressing HER2/neu on the activation of cytotoxic effectors of the immune cells was studied in vitro. (3) Results: The results obtained by confocal microscopy and cytofluorimetric analysis confirmed the binding of HSP70 or its fragment HSP70-16 on the surface of the treated cells. In response to the delivery of HSP70 to tumor cells, we observed an increase in the cytolytic activity of different cytotoxic effector immune cells from human peripheral blood. (4) Conclusions: Targeted modification of the tumor cell surface with molecular structures recognized by cytotoxic effectors of the immune system is among new promising approaches to antitumor immunotherapy.

Designing Ubiquitin-like protease 1 (Ulp1) based nano biocatalysts: A promising technology for SUMO fusion proteins.

The SUMO proteases (Ulps), a group of cysteine proteases, are well known for their efficient ability to perform structure-based cleavage of SUMO tag from the protein of interest and generation of biotherapeutics with authentic N-terminus. However, the stability of Ulps has remained a challenge for the economical production of difficult-to-produce proteins in E. coli. Therefore, the present study aimed to establish the methodology for developing stable S. pombe Ulp1 preparation using different enzyme immobilization strategies. The whole-cell biocatalyst developed using the Pir1 anchor protein of Pichia cleaved the SUMO tag within 24 h of reaction incubation. The chemical immobilization using commercial epoxy and amino methacrylate beads significantly enhanced the operational reusability of SpUlp1 up to 24 cycles. Silica beads further improved the repetitive usage of the immobilized enzyme for 65 cycles. The SpUlp1 immobilization on laboratory-developed chitosan-coated iron oxide nanoparticles exhibited more than 90 % cleavage of SUMO tag from different substrates even after 100 consecutive reactions. Moreover, an effective SUMO tag removal was observed within 10 min of incubation. The operational stability of the immobilized enzyme was confirmed in a pH range of 5 to 13. The spherical nature of nanoparticles was confirmed by FESEM and TEM results. The successful chitosan coating and subsequent activation with glutaraldehyde were established via FT-IR. Furthermore, HRTEM, SAED, and XRD proved the crystalline nature of nanoparticles, while VSM confirmed the superparamagnetic behavior.

Synthesis of biologically active proteins as L6KD-SUMO fusions forming inclusion bodies in Escherichia coli.

A new platform has been developed to facilitate the production of biologically active proteins and peptides in Escherichia coli. The platform includes an N-terminal self-associating L6 KD peptide fused to the SUMO protein (small ubiquitin-like protein modifier) from the yeast Saccharomyces cerevisiae, which is known for its chaperone activity. The target proteins are fused at the C termini of the L6 KD-SUMO fusions, and the resulting three-component fusion proteins are synthesized and self-assembled in E. coli into so-called active inclusion bodies (AIBs). In vivo, the L6 KD-SUMO platform facilitates the correct folding of the target proteins and directs them into AIBs, greatly simplifying their purification. In vitro, the platform facilitates the effective separation of AIBs by centrifugation and subsequent target protein release using SUMO-specific protease. The properties of the AIBs were determined using five proteins with different sizes, folding efficiencies, quaternary structure, and disulfide modifications. Electron microscopy shows that AIBs are synthesized in the form of complex fibrillar structures resembling "loofah sponges" with unusually thick filaments. The obtained results indicate that the new platform has promising features and could be developed to facilitate the synthesis and purification of target proteins and protein complexes without the use of renaturation.

Theranostic role of 89Zr- and 177Lu-labeled aflibercept in breast cancer.

PURPOSE: Triple-negative breast cancer (TNBC) has a poor prognosis due to the absence of effective therapeutic targets. Vascular endothelial growth factor (VEGF) family are expressed in 30-60% of TNBC, therefore providing potential therapeutic targets for TNBC. Aflibercept (Abe), a humanized recombinant fusion protein specifically bound to VEGF-A, B and placental growth factor (PIGF), has proven to be effective in the treatment in some cancers. Therefore, 89Zr/177Lu-labeled Abe was investigated for its theranostic role in TNBC. METHODS: Abe was radiolabeled with 89Zr and 177Lu via the conjugation of chelators. Flow cytometry and cell immunofluorescent staining were performed to evaluate the binding affinity of Abe. Sequential PET imaging and fluorescent imaging were conducted in TNBC tumor bearing mice following the injection of 89Zr-labeled Abe and Cy5.5-labeled Abe. Treatment study was performed after the administration of 177Lu-labeled Abe. Tumor volume and survival were monitored and SPECT imaging and biodistribution studies were conducted. Safety evaluation was performed including body weight, blood cell measurement, and hematoxylin-eosin (H&E) staining of major organs. Expression of VEGF and CD31 was tested by immunohistochemical staining. Dosimetry was estimated using the OLINDA software. RESULTS: FITC-labeled Abe showed a strong binding affinity to VEGF in TNBC 4T1 cells and HUVECs by flow cytometry and cell immunofluorescence. Tumor uptake of 89Zr-labeled Abe peaked at 120 h (SUVmax = 3.2 ± 0.64) and persisted before 168 h (SUVmax = 2.54 ± 0.42). The fluorescence intensity of the Cy5.5-labeled Abe group surpassed that of the Cy5.5-labeled IgG group, implying that Cy5.5-labeled Abe is a viable candidate monitoring in vivo tumor targeting and localization. 177Lu-labeled Abe (11.1 MBq) served well as the therapeutic component to suppress tumor growth with standardized tumor volume at 16 days, significantly smaller than PBS group (about 815.66 ± 3.58% vs 3646.52 ± 11.10%, n = 5, P < 0.01). Moreover, SPECT images confirmed high contrast between tumors and normal organs, indicating selective tumor uptake of 177Lu-labeled Abe. No discernible abnormalities in blood cells, and no evident histopathological abnormality observed in liver, spleen, and kidney. Immunohistochemical staining showed that 177Lu-labeled Abe effectively inhibited the expression of VEGF and CD31 of tumor, suggesting that angiogenesis may be suppressed by 177Lu-labeled Abe. The whole-body effective dose for an adult human was estimated to be 0.16 mSv/MBq. CONCLUSION: 89Zr/177Lu-labeled Abe could be a TNBC-specific marker with diagnostic value and provide insights into targeted therapy in the treatment of TNBC. Further clinical evaluation and translation may be of high significance for TNBC.

Novel Anti-Mesothelin Nanobodies and Recombinant Immunotoxins with Pseudomonas Exotoxin Catalytic Domain for Cancer Therapeutics.

Recombinant immunotoxins (RITs) are fusion proteins consisting of a targeting domain linked to a toxin, offering a highly specific therapeutic strategy for cancer treatment. In this study, we engineered and characterized RITs aimed at mesothelin, a cell surface glycoprotein overexpressed in various malignancies. Through an extensive screening of a large nanobody library, four mesothelin-specific nanobodies were selected and genetically fused to a truncated Pseudomonas exotoxin (PE24B). Various optimizations, including the incorporation of furin cleavage sites, maltose-binding protein tags, and tobacco etch virus protease cleavage sites, were implemented to improve protein expression, solubility, and purification. The RITs were successfully overexpressed in Escherichia coli, achieving high solubility and purity post-purification. In vitro cytotoxicity assays on gastric carcinoma cell lines NCI-N87 and AGS revealed that Meso(Nb2)-PE24B demonstrated the highest cytotoxic efficacy, warranting further characterization. This RIT also displayed selective binding to human and monkey mesothelins but not to mouse mesothelin. The competitive binding assays between different RIT constructs revealed significant alterations in IC50 values, emphasizing the importance of nanobody specificity. Finally, a modification in the endoplasmic reticulum retention signal at the C-terminus further augmented its cytotoxic activity. Our findings offer valuable insights into the design and optimization of RITs, showcasing the potential of Meso(Nb2)-PE24B as a promising therapeutic candidate for targeted cancer treatment.

Use of the selected metal-dependent enzymes for exploring applicability of human annexin A1 as a purification tag.

Several fusion tags have been developed for non-chromatographic fusion protein purification. Previously, we identified that human annexin A1 as a novel N-terminal purification tag was used for purifying the fusion proteins produced in Escherichia coli through precipitation in 10 mM Ca2+ buffer, and redissolution of the precipitate in 15 mM EDTA buffer. In this work, we selected four metal-dependent enzymes including E. coli 5-aminolevulinate dehydratase, yeast 3-hydroxyanthranilate 3,4-dioxygenase, maize serine racemase and copper amine oxidase for investigating the annexin A1 tag applicability. Fusion of the His6-tag or the enzyme changed the behavior of precipitation-redissolution. The relatively high recovery yields of three tagged enzymes with the improved purities were obtained through two rounds of purification, whereas low recovery yield of the annexin A1 tagged maize amine oxidase was prepared. The added EDTA displayed different abilities to redissolve the fusion proteins precipitates in two precipitation-redissolution cycles. It inactivated three enzymes and obviously inhibited the activity of the fused maize serine racemase. Based on current findings, we believe that four enzymes could be applied for evaluating applicability of the proteins or peptides as affinity tags for chromatographic purification in a calcium dependent manner.

Construction of a Collagen-like Protein Based on Elastin-like Polypeptide Fusion and Evaluation of Its Performance in Promoting Wound Healing.

In the healing of wounds, human-like collagen (hCol) is essential. However, collagen-based composite dressings have poor stability in vivo, which severely limits their current therapeutic potential. Based on the above, we have developed a recombinant fusion protein named hCol-ELP, which consists of hCol and an elastin-like peptide (ELP). Then, we examined the physicochemical and biological properties of hCol-ELP. The results indicated that the stability of the hCol-ELP fusion protein exhibited a more compact and homogeneous lamellar microstructure along with collagen properties, it was found to be significantly superior to the stability of free hCol. The compound hCol-ELP demonstrated a remarkable capacity to induce the proliferation and migration of mouse embryo fibroblast cells (NIH/3T3), as well as enhance collagen synthesis in human skin fibroblasts (HSF) when tested in vitro. In vivo, hCol-ELP demonstrated significant enhancements in healing rate and a reduction in the time required for scab removal, thereby exhibiting a scar-free healing effect. The findings provide a crucial theoretical foundation for the implementation of an hCol-ELP protein dressing in fields associated with the healing of traumatic injuries.

sTREM2 is a plasma biomarker for human NASH and promotes hepatocyte lipid accumulation.

BACKGROUND: Pathogenetic mechanisms of the progression of NAFL to advanced NASH coupled with potential noninvasive biomarkers and novel therapeutic targets are active areas of investigation. The recent finding that increased plasma levels of a protein shed by myeloid cells -soluble Triggering Receptor Expressed on Myeloid cells 2 (sTREM2) -may be a biomarker for NASH has received much interest. We aimed to test sTREM2 as a biomarker for human NASH and investigate the role of sTREM2 in the pathogenesis of NASH. METHODS: We conducted studies in both humans (comparing patients with NASH vs. NAFL) and in mice (comparing different mouse models of NASH) involving measurements of TREM2 gene and protein expression levels in the liver as well as circulating sTREM2 levels in plasma. We investigated the pathogenetic role of sTREM2 in hepatic steatosis using primary hepatocytes and bone marrow derived macrophages. RESULTS: RNA sequencing analysis of livers from patients with NASH or NAFL as well as livers from 2 mouse models of NASH revealed elevated TREM2 expression in patients/mice with NASH as compared with NAFL. Plasma levels of sTREM2 were significantly higher in a well-characterized cohort of patients with biopsy-proven NASH versus NAFL (area under receiver-operating curve 0.807). Mechanistic studies revealed that cocultures of primary hepatocytes and macrophages with an impaired ability to shed sTREM2 resulted in reduced hepatocyte lipid droplet formation on palmitate stimulation, an effect that was counteracted by the addition of exogenous sTREM2 chimeric protein. Conversely, exogenous sTREM2 chimeric protein increased lipid droplet formation, triglyceride content, and expression of the lipid transporter CD36 in hepatocytes. Furthermore, inhibition of CD36 markedly attenuated sTREM2-induced lipid droplet formation in mouse primary hepatocytes. CONCLUSIONS: Elevated levels of sTREM2 due to TREM2 shedding may directly contribute to the pathogenesis of NAFLD by promoting hepatocyte lipid accumulation, as well as serving as a biomarker for distinguishing patients with NASH versus NAFL. Further investigation of sTREM2 as a clinically useful diagnostic biomarker and of the therapeutic effects of targeting sTREM2 in NASH is warranted.

Molecular reshaping of phage-displayed Interleukin-2 at beta chain receptor interface to obtain potent super-agonists with improved developability profiles.

Interleukin-2 (IL-2) engineered versions, with biased immunological functions, have emerged from yeast display and rational design. Here we reshaped the human IL-2 interface with the IL-2 receptor beta chain through the screening of phage-displayed libraries. Multiple beta super-binders were obtained, having increased receptor binding ability and improved developability profiles. Selected variants exhibit an accumulation of negatively charged residues at the interface, which provides a better electrostatic complementarity to the beta chain, and faster association kinetics. These findings point to mechanistic differences with the already reported superkines, characterized by a conformational switch due to the rearrangement of the hydrophobic core. The molecular bases of the favourable developability profile were tracked to a single residue: L92. Recombinant Fc-fusion proteins including our variants are superior to those based on H9 superkine in terms of expression levels in mammalian cells, aggregation resistance, stability, in vivo enhancement of immune effector responses, and anti-tumour effect.

Robust production of active Ulp1 (SUMO protease) from inclusion bodies.

High yield purification of Ulp1 is required during the isolation and purification of SUMO-tagged recombinant proteins. However, when expressed as a soluble protein, Ulp1 is toxic to E. coli host cells and most of the protein forms inclusion bodies. The extraction of insoluble Ulp1 followed by its purification and refolding into its active form is a lengthy and costly procedure. In our present study, we developed a simple, cost effective procedure for the large scale production of active Ulp1 that can be used for industrial scale requirements.

Inhibition of complement C3 signaling ameliorates locomotor and visual dysfunction in autoimmune inflammatory diseases.

Neuromyelitis optica (NMO) is an autoimmune inflammatory disease of the central nervous system (CNS) characterized by transverse myelitis and optic neuritis. The pathogenic serum IgG antibody against the aquaporin-4 (AQP4) on astrocytes triggers the activation of the complement cascade, causing astrocyte injury, followed by oligodendrocyte injury, demyelination, and neuronal loss. Complement C3 is positioned as a central player that relays upstream initiation signals to activate downstream effectors, potentially stimulating and amplifying host immune and inflammatory responses. However, whether targeting the inhibition of C3 signaling could ameliorate tissue injury, locomotor defects, and visual impairments in NMO remains to be investigated. In this study, using the targeted C3 inhibitor CR2-Crry led to a significant decrease in complement deposition and demyelination in both slice cultures and focal intracerebral injection models. Moreover, the treatment downregulated the expression of inflammatory cytokines and improved motor dysfunction in a systemic NMO mouse model. Similarly, employing serotype 2/9 adeno-associated virus (AAV2/9) to induce permanent expression of CR2-Crry resulted in a reduction in visual dysfunction by attenuating NMO-like lesions. Our findings reveal the therapeutic value of inhibiting the complement C3 signaling pathway in NMO.

Detection of Protein-Protein Interactions Using Glutathione-S-Transferase (GST) Pull-Down Assay Technique.

Pull-down assay is a technique to analyze direct protein-protein interaction under in vitro condition. Also, this technique is appropriate for investigating the direct interaction between two purified proteins. Glutathione-s-transferase (GST) protein is a widely used affinity tag for affinity purification. In this chapter, we explain the widely used GST pull-down assay to identify the protein-protein interaction between purified proteins.

Production of recombinant His-tagged triple-FLAG peptide in Brevibacillus choshinensis and its utilization as an easy-to-remove affinity peptide.

Triple-FLAG (3 × FLAG)-tagged proteins can be affinity purified through binding to an anti-FLAG antibody and competitive elution with excess free 3 × FLAG peptide. To expand the availability of the 3 × FLAG purification system, we produced a recombinant His-tagged 3 × FLAG peptide in Brevibacillus choshinensis. The screening of connecting linkers between His-tag and the 3 × FLAG peptide, culture containers, and culture media showed that the His-tagged 3 × FLAG peptide with an LA linker was most expressed in 2SY medium using a baffled shake flask. The peptide was affinity-purified to give a yield of about 25 mg/L of culture. The peptide was effective for eluting 3 × FLAG-tagged α-amylase from anti-FLAG magnetic beads. Finally, the peptide remaining in the amylase fraction was removed by His-tag affinity purification. These results show that the recombinant His-tagged 3 × FLAG peptide can function as an easy-to-remove affinity peptide in the 3 × FLAG purification system.

Protein Transduction System Based on Tryptophan-zipper against Intracellular Infections via Inhibiting Ferroptosis of Macrophages.

Cells penetrating molecules in living systems hold promise of capturing and eliminating threats and damage that can plan intracellular fate promptly. However, it remains challenging to construct cell penetration systems that are physiologically stable with predictable self-assembly behavior and well-defined mechanisms. In this study, we develop a core-shell nanoparticle using a hyaluronic acid (HA)-coated protein transduction domain (PTD) derived from the human immunodeficiency virus (HIV). This nanoparticle can encapsulate pathogens, transporting the PTD into macrophages via lipid rafts. PTD forms hydrogen bonds with the components of the membrane through TAT, which has a high density of positive charges and reduces the degree of membrane order through Tryptophan (Trp)-zipper binding to the acyl tails of phospholipid molecules. HA-encapsulated PTD increases the resistance to trypsin and proteinase K, thereby penetrating macrophages and eliminating intracellular infections. Interestingly, the nonagglutination mechanism of PTD against pathogens ensures the safe operation of the cellular system. Importantly, PTD can activate the critical pathway of antiferroptosis in macrophages against pathogen infection. The nanoparticles developed in this study demonstrate safety and efficacy against Gram-negative and Gram-positive pathogens in three animal models. Overall, this work highlights the effectiveness of the PTD nanoparticle in encapsulating pathogens and provides a paradigm for transduction systems-anti-intracellular infection therapy.

E6 Tagged Protein Production, Extraction, and Purification from Escherichia coli Lysate.

Cervical cancer has been extensively associated with human papillomavirus (HPV) due to the expression of oncoproteins such as E6. This protein can interfere with p53 tumor suppressor activity, blocking apoptosis of abnormal cells. The functional inhibition of E6 protein is a promising therapeutic strategy for HPV-induced cancers. Conducting biointeraction and characterization studies between E6 protein and potential anti-HPV drugs is necessary to obtain large quantities of high-purity and soluble E6 protein. The recombinant production of E6 protein is particularly challenging because it tends to aggregate. One way to circumvent this problem is to use a dual MBP-His6 tag that can facilitate the expression, proper folding, and solubility of the E6 protein. This chapter outlines effective methods for expressing and obtaining E6 protein with a dual affinity tag by combining different chromatographic methods.

A Simple Purification Method for Heat-Stable Recombinant Low Molecular Weight Proteins and Peptides Via GST-Fusion Products.

Here, we describe a simple, rapid, cost-effective, and efficient novel one-step purification method for GST-tagged peptides and small proteins. This novel technique applies to proteins and peptides that are known to be thermally stable at 60 °C and do not have elaborate structure(s) and whose heat-induced unfolding is reversible. This method takes advantage of glutathione S-transferase from Schistosoma japonicum (sj26GST) precipitating when heated at 60 °C. Purified GST-fusion products are subjected to enzymatic cleavage to separate the GST tag from the target peptide or small proteins. In our proposed method, the cleavage products are heated at 60 °C for 20 min which results in the precipitation of the GST tag. Subsequently, the GST tag is separated from the target peptide or small protein by high-speed centrifugation. Biophysical experiments such as SDS-PAGE, circular dichroism, isothermal titration calorimetry, mass spectroscopy, and multidimensional NMR spectroscopy confirm that the target peptides and small proteins are purified to more than 95% homogeneity, intact native conformation, and no significant change in the binding affinity of heat-treated purified product to the interacting partners.

Duration of Vascular Endothelial Growth Factor Suppression After Intravitreal Injection of Brolucizumab and Aflibercept in Macaque Eyes.

Purpose: To compare the duration of vascular endothelial growth factor (VEGF) suppression in the aqueous humor of macaque eyes after intravitreal injection of brolucizumab and aflibercept. Methods: Clinical dose of intravitreal brolucizumab (IVBr; 6.0 mg/50 µL) or intravitreal aflibercept (IVA; 2 mg/50 µL) was injected into the right eye of each of 8 macaques. Aqueous humor samples (150 µL) from both eyes were obtained just before injection and on days 1, 3, 7, 14, 21, 28, 42, 56, 84, and 112 after IVBr injection or IVA injection. VEGF concentrations were measured using enzyme-linked immunosorbent assays. Results: In the injected eyes, the mean VEGF suppression durations (range) were 4.9 (3-8) weeks for IVBr injection and 6.8 (6-8) weeks for IVA injection (P = 0.04). The VEGF concentrations returned to the preinjection level in the aqueous humor at 12 weeks both after IVBr and IVA injection. In the noninjected fellow eyes, the aqueous VEGF concentrations had decreased least at 1 day after IVBr injection and at 3 days after IVA injection, but were still detectable. The VEGF concentrations in the fellow eyes returned to the preinjection level in the aqueous humor at 1 week after IVBr injection and at 2 weeks after IVA injection. Conclusions: The duration of VEGF suppression in the aqueous humor after IVBr injection may be shorter than that after IVA injection, which may be related with clinical usage.