Impaired proliferation and migration of HUVEC and melanoma cells by human anti-FGF2 mAbs derived from a murine hybridoma by guided selection.

Disadvantages of using murine monoclonal antibodies (mAb) in human therapy, such as immunogenicity response, led to the development of technologies to transform murine antibodies into human antibodies. The murine anti-FGF2 3F12E7 mAb was proposed as a promising agent to treat metastatic melanoma tumors; once it blocks the FGF2, responsible for playing a role in tumor growth, angiogenesis, and metastasis. Considering the therapeutic potential of anti-FGF2 3F12E7 mAb and its limited use in humans due to its origin, we used this antibody as the template for a guided selection humanization technique to obtain human anti-FGF2 mAbs. Three Fab libraries (murine, hybrid, and human) were constructed for humanization. The libraries were phage-displayed, and the panning was performed against recombinant human FGF2 (rFGF2). The selected human variable light and heavy chains were cloned into AbVec vectors for full-length IgG expression into HEK293-F cells. Surface plasmon resonance analyses showed binding to rFGF2 of seven mAbs out of 20 expressed. Assays performed with these mAbs resulted in two that showed proliferation reduction and cell migration attenuation of HUVEC and SK-Mel-28 melanoma cells. In-silico analyses predicted that these two human anti-FGF2 mAbs interact with FGF2 at a similar patch of residues than the chimeric anti-FGF2 antibody, comprehending a region within the heparin-binding domains of FGF2, essential for its function. These results are comparable to those achieved by the murine anti-FGF2 3F12E7 mAb and showed success in the humanization process and selection of two human mAbs with the potential to inhibit undesirable FGF2 roles.

The guided selection humanization process enabled the production of 20 human mAbs anti-FGF2;Seven human anti-FGF2 mAbs showed binding to the rFGF2 antigen in the SPR binding assay;Two human anti-FGF2 mAbs inhibited the proliferation and migration of HUVEC and SK-Mel-28 cells and were predicted to contact the FGF2 at a similar patch of residues than the original mAb.

Potential of an anti-bevacizumab idiotype scFv DNA-based immunization to elicit VEGF-binding antibody response.

Anti-idiotype antibodies have been considered for vaccination approaches against different diseases, including cancers. Based on that, we previously described an anti-bevacizumab idiotype monoclonal antibody, 10.D7, that revealed detectable antitumor effects on a vascular endothelial growth factor (VEGF)-dependent tumor model. Herein, we evaluated the possible applicability of a single-chain variable fragment (scFv) for the 10.D7 antibody in a gene immunization strategy. After checking that mammalian cells transfected to express the 10.D7 scFv are recognized by bevacizumab, it was explored the ability of our scFv construction, in a gene-based scheme, to elicit an immune response containing VEGF-binding antibodies. The results provide evidence that the designed 10.D7 scFv construct maintains the anti-bevacizumab idiotype features and has potential to activate an immune response recognizing VEGF.

Impaired proliferation and migration of HUVEC and melanoma cells by human anti-FGF2 mAbs derived from a murine hybridoma by guided selection

Disadvantages of using murine monoclonal antibodies (mAb) in human therapy, such as immunogenicity response, led to the development of technologies to transform murine antibodies into human antibodies. The murine anti-FGF2 3F12E7 mAb was proposed as a promising agent to treat metastatic melanoma tumors; once it blocks the FGF2, responsible for playing a role in tumor growth, angiogenesis, and metastasis. Considering the therapeutic potential of anti-FGF2 3F12E7 mAb and its limited use in humans due to its origin, we used this antibody as the template for a guided selection humanization technique to obtain human anti-FGF2 mAbs. Three Fab libraries (murine, hybrid, and human) were constructed for humanization. The libraries were phage-displayed, and the panning was performed against recombinant human FGF2 (rFGF2). The selected human variable light and heavy chains were cloned into AbVec vectors for full-length IgG expression into HEK293-F cells. Surface plasmon resonance analyses showed binding to rFGF2 of seven mAbs out of 20 expressed. Assays performed with these mAbs resulted in two that showed proliferation reduction and cell migration attenuation of HUVEC and SK-Mel-28 melanoma cells. In-silico analyses predicted that these two human anti-FGF2 mAbs interact with FGF2 at a similar patch of residues than the chimeric anti-FGF2 antibody, comprehending a region within the heparin-binding domains of FGF2, essential for its function. These results are comparable to those achieved by the murine anti-FGF2 3F12E7 mAb and showed success in the humanization process and selection of two human mAbs with the potential to inhibit undesirable FGF2 roles.

Generation and functional characterization of a single-chain variable fragment (scFv) of the anti-FGF2 3F12E7 monoclonal antibody.

Single-chain variable fragments (scFvs) are small-sized artificial constructs composed of the immunoglobulin heavy and light chain variable regions connected by a peptide linker. We have previously described an anti-fibroblast growth factor 2 (FGF2) immunoglobulin G (IgG) monoclonal antibody (mAb), named 3F12E7, with notable antitumor potential revealed by preclinical assays. FGF2 is a known angiogenesis-associated molecule implicated in tumor progression. In this report, we describe a recombinant scFv format for the 3F12E7 mAb. The results demonstrate that the generated 3F12E7 scFv, although prone to aggregation, comprises an active anti-FGF2 product that contains monomers and small oligomers. Functionally, the 3F12E7 scFv preparations specifically recognize FGF2 and inhibit tumor growth similar to the corresponding full-length IgG counterpart in an experimental model. In silico molecular analysis provided insights into the aggregation propensity and the antigen-recognition by scFv units. Antigen-binding determinants were predicted outside the most aggregation-prone hotspots. Overall, our experimental and prediction dataset describes an scFv scaffold for the 3F12E7 mAb and also provides insights to further engineer non-aggregated anti-FGF2 scFv-based tools for therapeutic and research purposes.

Hybridoma technology: is it still useful?

The isolation of single monoclonal antibodies (mAbs) against a given antigen was only possible with the introduction of the hybridoma technology, which is based on the fusion of specific B lymphocytes with myeloma cells. Since then, several mAbs were described for therapeutic, diagnostic, and research purposes. Despite being an old technique with low complexity, hybridoma-based strategies have limitations that include the low efficiency on B lymphocyte-myeloma cell fusion step, and the need to use experimental animals. In face of that, several methods have been developed to improve mAb generation, ranging from changes in hybridoma technique to the advent of completely new technologies, such as the antibody phage display and the single B cell antibody ones. In this review, we discuss the hybridoma technology along with emerging mAb isolation approaches, taking into account their advantages and limitations. Finally, we explore the usefulness of the hybridoma technology nowadays.

Exploring the Immunological Mechanisms Underlying the Anti-vascular Endothelial Growth Factor Activity in Tumors.

Several studies report the key role of the vascular endothelial growth factor (VEGF) signaling on angiogenesis and on tumor growth. This has led to the development of a number of VEGF-targeted agents to treat cancer patients by disrupting the tumor blood vessel supply. Of them, bevacizumab, an FDA-approved humanized monoclonal antibody against VEGF, is the most promising. Although the use of antibodies targeting the VEGF pathway has shown clinical benefits associated with a reduction in the tumor blood vessel density, the inhibition of VEGF-driven vascular effects is only part of the functional mechanism of these therapeutic agents in the tumor ecosystem. Compelling reports have demonstrated that VEGF confers, in addition to the activation of angiogenesis-related processes, immunosuppressive properties in tumors. It is also known that structural remodeling of the tumor blood vessel bed by anti-VEGF approaches affect the influx and activation of immune cells into tumors, which might influence the therapeutic results. Besides that, part of the therapeutic effects of antiangiogenic antibodies, including their role in the tumor vascular network, might be triggered by Fc receptors in an antigen-independent manner. In this mini-review, we explore the role of VEGF inhibitors in the tumor microenvironment with focus on the immune system, discussing around the functional contribution of both bevacizumab's Fab and Fc domains to the therapeutic results and the combination of bevacizumab therapy with other immune-stimulatory settings, including adjuvant-based vaccine approaches.

Crataeva tapia bark lectin (CrataBL) is a chemoattractant for endothelial cells that targets heparan sulfate and promotes in vitro angiogenesis.

Formation of new blood vessels from preexisting ones, a process known as angiogenesis, is one of the limiting steps for success in treatment of ischemic disorders. Therefore, efforts to understanding and characterize new agents capable to stimulate neovascularization are a worldwide need. Crataeva tapia bark lectin (CrataBL) has been shown to have chemoattractant properties for endothelial cells through the stimulation of migration and invasiveness of human umbilical vein endothelial cells (HUVEC) because it is a positively charged protein with high affinity to glycosaminoglycan. In addition, CrataBL increased the production of chondroitin and heparan sulfate in endothelial cells. These findings orchestrated specific adhesion on collagen I and phosphorylation of tyrosine kinase receptors, represented by vascular endothelial growth factor receptor-2 (VEGFR-2) and fibroblast growth factor receptor (FGFR), whose downstream pathways trigger the angiogenic cascade increasing cell viability, cytoskeleton rearrangement, cell motility, and tube formation. Moreover, CrataBL inhibited the activity of matrix metalloproteases type 2 (MMP-2), a protein related to tissue remodeling. Likewise, CrataBL improved wound healing and increased the number of follicular structures in lesioned areas produced in the dorsum-cervical region of C57BL/6 mice. These outcomes altogether indicate that CrataBL is a pro-angiogenic and healing agent.

Enkephalin related peptides are released from jejunum wall by orally ingested bromelain.

Stem bromelain [EC 3.4.22.32] is a thiol-endopeptidase and orally recommended in traditional medicine due to its analgesic activity, but the mechanisms are not known. Proenkephalin is expressed in the nervous system, but also in the gastrointestinal tract, where it can be assessed by ingested stem bromelain. Here we demonstrated that stem bromelain hydrolyses synthetic proenkephalin fragments after basic amino acid residues flanking the enkephalin sequences. We also observed with in vivo studies that oral administration of bromelain reduced jejunum proenkephalin levels and increased the serum enkephalin in mice. Effective anti-nociceptive effects in mice were observed 3 h after oral administration of 3 mg/kg stem bromelain by the acetic acid-induced writhing test. However, with higher doses this effect is reduced due to hydrolysis of enkephalin that possibly occurs by the presence of ananain in commercial pineapple stem bromelain preparations, that is also a thiol-protease with broad specificity. The analgesic effects were also evaluated by hot-plate and formalin tests and the obtained results indicated that enkephalin generated in intestine acts in periphery where it also can have anti-inflammatory activity.

Anti-bevacizumab idiotype antibody vaccination is effective in inducing vascular endothelial growth factor-binding response, impairing tumor outgrowth.

Tumors require blood supply and, to overcome this restriction, induce angiogenesis. Vascular endothelial growth factor (VEGF) plays an important role in this process, which explains the great number of antiangiogenic therapies targeting VEGF. The research and development of targeted therapy has led to the approval of bevacizumab, a humanized anti-VEGF monoclonal antibody (mAb), in clinical settings. However, side effects have been reported, usually as a consequence of bolus-dose administration of the antibody. This limitation could be circumvented through the use of anti-idiotype (Id) antibodies. In the present study, we evaluated the efficacy of an active VEGF-binding immune response generated by an anti-bevacizumab idiotype mAb, 10.D7. The 10.D7 anti-Id mAb vaccination led to detectable levels of VEGF-binding anti-anti-Id antibodies. In order to examine whether this humoral immune response could have implications for tumor development, 10.D7-immunized mice were challenged with B16-F10 tumor cells. Mice immunized with 10.D7 anti-Id mAb revealed reduced tumor growth when compared to control groups. Histological analyses of tumor sections from 10.D7-immunized mice showed increased necrotic areas, decreased CD31-positive vascular density and reduced CD68-positive cell infiltration. Our results encourage further therapeutic studies, particularly if one considers that the anti-Id therapeutic vaccination maintains stable levels of VEGF-binding antibodies, which might be useful in the control of tumor relapse.

Blocking FGF2 with a new specific monoclonal antibody impairs angiogenesis and experimental metastatic melanoma, suggesting a potential role in adjuvant settings.

Compelling evidence suggests that fibroblast growth factor 2 (FGF2), overexpressed in melanomas, plays an important role in tumor growth, angiogenesis and metastasis. In this study, we evaluated the therapeutic use of a new anti-FGF2 monoclonal antibody (mAb), 3F12E7, using for that the B16-F10 melanoma model. The FGF2 neutralizing effect of this antibody was certified by in vitro assays, which allowed the further track of its possible in vivo application. 3F12E7 mAb could be retained in B16-F10 tumors, as shown by antibody low-pH elution and nuclear medicine studies, and also led to reduction in number and size of metastatic foci in lungs, when treatment starts one day after intravenous injection of B16-F10 cells. Such data were accompanied by decreased CD34(+) tumor vascular density and impaired subcutaneous tumor outgrowth. Treatments starting one week after melanoma cell intravenous injection did not reduce tumor burden, remaining the therapeutic effectiveness restricted to early-adopted regimens. Altogether, the presented anti-FGF2 3F12E7 mAb stands as a promising agent to treat metastatic melanoma tumors in adjuvant settings.

Skin sensitizer identification by IL-8 secretion and CD86 expression on THP-1 cells.

Substantial progress has been made in the development of alternative methods for skin sensitization in the last decade in several countries around the world. Brazil is experiencing an increasing concern about using animals for product development, since the publication of the Law 9605/1998, which prohibits the use of animals when an alternative method is available. In this way, an in vitro test to evaluate allergenic potential is a pressing need.This preliminary study started setting the use of myelomonocytic THP-1 cell line, according to the human cell line activation test (h-CLAT), already under validation process. We found that 48-h chemical exposure was necessary to identify 22 out of 23 sensitizers by the analyses of CD86 expression. In addition, the CD54 expression analyses presented a poor efficiency to discriminate sensitizers from non-sensitizers in our conditions. In view of these results, we looked for changes of pro-inflammatory interleukin profile. The IL-8 secretion analyses after 24-h chemical incubation seemed to be an alternative for CD54 expression assessing.Altogether, our findings showed that the combination of the analyses of CD86 expression and IL-8 secretion allowed predicting allergenicity.

Synergistic effect of vascular endothelial growth factor and granulocyte colony-stimulating factor double gene therapy in mouse limb ischemia.

BACKGROUND: Vascular endothelial growth factor (VEGF) has mostly been tested to treat ischemic diseases, although the outcomes obtained are not satisfactory. Our hypothesis is that the local transient expression of VEGF and stem cell mobilizer granulocyte colony-stimulating factor (G-CSF) genes in ischemic limbs can complement their activities and be more efficient for limb recovery. METHODS: Limb ischemia was surgically induced in mice and 50 microg of VEGF and/or G-CSF genes were locally transferred by electroporation. After 3-4 weeks, evidence of necrosis by visual inspection, capillary density, muscle mass, muscle force and hematopoietic cell mobilization were evaluated. RESULTS: After 4 weeks, 70% and 90% of the animals of the ischemic group (IG) and VEGF-treated group (VG), respectively, presented limb necrosis, in contrast to only 10% observed in the group of mice treated with both VEGF and G-CSF genes (VGG). Recovery of muscle mass and muscle force was higher than 60% in the VGG compared to the non-ischemic group. The mobilization of Sca1+ cells and neutrophils was also higher in the VGG, which may explain the lower level of necrosis observed in this group (22%, in contrast to 70% in the IG). Capillary density and degree of fibrosis were determined in weeks 3 and 4, and also showed a clear benefit as a result of the use of the G-CSF and VEGF genes together. CONCLUSIONS: Gene therapy using VEGF and G-CSF demonstrated a synergistic effect promoting vessel and tissue repair in mouse hind limb ischemia.

Granulocyte-macrophage colony-stimulating factor gene based therapy for acute limb ischemia in a mouse model.

BACKGROUND: Granulocyte-colony-stimulating factor (GM-CSF) is a pleiotropic factor for hematopoiesis that stimulates myeloblasts, monoblasts and mobilization of bone marrow stem cells. Therefore, the GM-CSF gene is a potential candidate for vessel formation and tissue remodeling in the treatment of ischemic diseases. METHODS: A new mouse limb ischemia was established by surgery and gene transfer was performed by injection of 100 microg of a plasmid carrying GM-CSF. Muscle force and weight, histology, capillary density, circulating stem cells and monocytes were determined after 3-4 weeks. RESULTS: More than 60% of nontreated ischemic animals showed gangrene below the heel after 4 weeks, whereas the GM-CSF gene-treated animals showed only darkening of nails or toes. These animals demonstrated a full recovery of the affected muscles in terms of weight, force and muscle fiber structure, but the muscles of nontreated ischemic animals lost approximately 50% weight, 86% force and their regular structure. When the GM-CSF gene was injected into the contralateral limb, only partial loss was observed, demonstrating a distant effect of GM-CSF. The capillary density in the GM-CSF-treated group was 52% higher in relation to the nontreated group. Blood analysis by flow cytometry showed that the GM-CSF-treated group had 10-20% higher levels of circulating monocytes and Sca-1(+). CONCLUSIONS: We conclude that the direct administration of GM-CSF gene in limb ischemia had a strong therapeutic effect because it promoted the recovery of muscle mass, force and structure by mobilizing therapeutic cells and augmenting the number of vessels.

Humoral immune response after genetic immunization is consistently improved by electroporation.

Aiming to evaluate some parameters to influence the immune response to DNA vaccination, we compare three protocols of DNA immunization (i.m. injections, i.m. injections followed by electroporation, and the effect of i.p. injection of stably antigen-transfected cells before DNA administration), using three different antigens. Statistical analyses showed that electroporation after intramuscular injections provided an immune response comparable to that obtained by pre-treatment with antigen-transfected cells and similar to that obtained by protein immunization. The results allowed us selecting a protocol that worked well for all three antigens and reinforced the idea that high level of gene expression is essential to get good immunization.

Carcinoembryonic antigen (CEA) mimicry by an anti-idiotypic scFv isolated from anti-Id 6.C4 hybridoma.

Since carcinoembryonic antigen (CEA) is expressed during embryonic life, it is not immunogenic in humans. The use of anti-idiotypic (Id) antibodies as a surrogate of antigen in the immunization has been considered a promising strategy for breaking tolerance to some tumor associated antigens. We have described an anti-Id monoclonal antibody (MAb), designated 6.C4, which is able to mimic CEA functionally. The anti-Id MAb 6.C4 was shown to elicit antibodies that recognized CEA in vitro and in vivo. In the present study, we sought to verify whether a single chain (scFv) antibody obtained, the scFv 6.C4, would retain the ability to mimic CEA. Two scFv containing the variable heavy and light chain domains of 6.C4 were constructed with a 15-amino acid linker: one with and another without signal peptide. DNA immunization of mice with both forms of scFv individually elicited antibodies able to recognize CEA.

Análise quantitativa e qualitativa do conteúdo proteíco do humor aquoso de portadores de síndrome de exfoliaçäo / Quantitative and qualitative analysis of the protein content of the aqueous humor in patients with exfoliation syndrome

Com o objetivo de analisar, qualitativa e quantitativamente, o conteúdo protéico do humor aquoso de portadores de síndrome de exfoliaçäo (EXF) e compará-lo com o de näo portadores da síndrome, foram selecionados 40 indivíduos (22 portadores e 18 näo portadores de EXF, com indicaçäo de tratamento cirúrgico de cataratae/ou glaucoma em pelo menos um dos olhos) que tiveram seu humor aquoso colhido , por ocasiäo no ato operatório. Utilizando o método de BRADFORD para quantificaçäo de proteínas totais no humor aquoso de portadores de EXF foi maior que nos dos näo portadores da síndrome (p<0,005). Observou-se também que a concentraçäo média de proteínas totais, nos portadores de EXF associada a glaucoma, foi maior que nos portadores de glaucoma primário de ângulo aberto (p<0,005). O mesmo ocorreu com relaçäo à concentraçäo média de proteínas totais do humor aquoso de portadores de EXF associada a catarata em comparaçäo com a dos portadores de catarata (p<0,025). Utilizando a técnica de eletroforese de proteínas em gel de poliacrilamida , com gradiente de 8 a 25 por cento na presença de SDS, a revelaçäo pela prata com as unidades de eletroforese e de revelaçäo do PhastSystem, respectivamente, observou-se, em 100 por cento dos membros de ambos os grupos estudados, a presença de uma única banda, bem definida e situada na regiäo correspondente 67 quilodaltons, a albumina. Após a separaçäo parcial da albumina com Affie-Gel blue, nenhuma outra proteína foi identificada em nenhum das amostras de humor aquoso de ambos de ambos os grupos.