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Vascular endothelial growth factor-A (VEGF-A) plays a critical role in physiologic and pathologic angiogenesis through its receptors especially through VEGFR2. The lack of cross-reactivity of monoclonal antibodies with human VEGFR2/mouse Flk-1 is a major obstacle in preclinical developments. In this study, using a unique hybridoma technique, we generated a panel of 30 neutralization anti-VEGFR2 rabbit monoclonal antibodies (RabMAbs) either blocking VEGF/VEGFR2 interaction or inhibiting VEGF-stimulated VEGFR2 tyrosine kinase phosphorylation. Among 18 RabMAbs with human/mouse VEGFR2 cross-reactivity, we humanized one lead candidate RabMAb by Mutational Lineage Guided (MLG) method and further demonstrated its potent inhibition of tumor growth in xenograft mouse model. Our study suggests that RabMAbs are highly relevant for therapeutic applications., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Traditional methods to establish animal model of osteonecrosis of the femoral head (ONFH) are not satisfactory because of the undefined underlying mechanism, low rate of the disease, and high incidence of mortality. The present study was to induce ONFH in a rat model through periarticular injection of vascular endothelial growth factor (VEGF) receptor 2 antibody., Materials and Methods: Eighty Sprague-Dawley rats were divided into four groups randomly and equally. VEGF receptor 2 antibody solution with a concentration of 50 μg/mL (group A), 25 μg/mL (group B), and 12.5 μg/mL (group C) was injected periarticularly to the capsular attachment to proximal femur, respectively. The injection lasted 4 continuous days, with a total volume of 2 mL. The rats in the fourth group were treated by saline for control (group D). Two weeks after the first injection, histologic and micro-computed tomographic examinations were used to investigate the presence of ONFH., Results: Histologically, there were 90% of rats that developed ONFH in group A, 60% in group B, and 20% in group C, whereas no ONFH was found in group D. The density of intraosseous vessels was suppressed significantly by the antibody. Radiologically, the features of ONFH including deformation of the femoral head were revealed. There was no occurrence of lethal complications., Conclusions: VEGF receptor 2 antibody can induce ONFH in the rat with a high incidence of the disease. Local blockage of angiogenesis can be used as an effective method to establish animal model of ONFH., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Recent basic and clinical studies have shown that the programmed death ligand (PD-L)/PD-1 pathway has a significant role in tumour immunity, and its blockade has a therapeutic potential against several human cancers. We hypothesized that anti-angiogeneic treatment might augment the efficacy of PD-1 blockade. To this end, we evaluated combining the blockade of PD-1 and vascular endothelial growth factor receptor 2 (VEGFR2) in a murine cancer model using Colon-26 adenocarcinoma. Interestingly, simultaneous treatment with anti-PD-1 and anti-VEGFR2 monoclonal antibodies (mAbs) inhibited tumour growth synergistically in vivo without overt toxicity. Blocking VEGFR2 inhibited tumour neovascularization significantly, as demonstrated by the reduced number of microvessels, while PD-1 blockade had no impact on tumour angiogenesis. PD-1 blockade might promote T cell infiltration into tumours and significantly enhanced local immune activation, as shown by the up-regulation of several proinflammatory cytokine expressions. Importantly, VEGFR2 blockade did not interfere with T cell infiltration and immunological activation induced by PD-1 blockade. In conclusion, simultaneous blockade of PD-1 and VEGFR2 induced a synergistic in-vivo anti-tumour effect, possibly through different mechanisms that might not be mutually exclusive. This unique therapeutic strategy may hold significant promise for future clinical application., (© 2013 British Society for Immunology.)

Ramucirumab (IMC-1121B) is a fully humanized monoclonal antibody that binds to VEGFR2 and can inhibit angiogenesis, a quintessential mechanism for promoting tumor growth and metastasis. Several antiangiogenesis agents are already approved for cancer therapy; however, ramucirumab's selectivity for VEGFR2 makes it interesting. The selectivity of an agent can improve safety and efficacy. This article describes the mechanism of action, pharmacokinetics, safety and clinical trial results of ramucirumab with particular emphasis on gastric cancer.

Most systemic cancer therapies target tumor cells directly, although there is increasing interest in targeting the tumor stroma that can comprise a substantial portion of the tumor mass. We report here a synergy between two T-cell therapies, one directed against the stromal tumor vasculature and the other directed against antigens expressed on the tumor cell. Simultaneous transfer of genetically engineered syngeneic T cells expressing a chimeric antigen receptor targeting the VEGF receptor-2 (VEGFR2; KDR) that is overexpressed on tumor vasculature and T-cells specific for the tumor antigens gp100 (PMEL), TRP-1 (TYRP1), or TRP-2 (DCT) synergistically eradicated established B16 melanoma tumors in mice and dramatically increased the tumor-free survival of mice compared with treatment with either cell type alone or T cells coexpressing these two targeting molecules. Host lymphodepletion before cell transfer was required to mediate the antitumor effect. The synergistic antitumor response was accompanied by a significant increase in the infiltration and expansion and/or persistence of the adoptively transferred tumor antigen-specific T cells in the tumor microenvironment and thus enhanced their antitumor potency. The data presented here emphasize the possible beneficial effects of combining antiangiogenic with tumor-specific immunotherapeutic approaches for the treatment of patients with cancer., (©2013 AACR.)

Although several potential mechanosensors/mechanotransducers have been proposed, the precise mechanisms by which ECs sense and respond to mechanical forces and translate them into biochemical signals remains unclear. Here, we report that two major ligand-dependent tyrosine autophosphorylation sites of VEGFR2, Y1175 and Y1214, are rapidly activated by shear stress in human coronary artery endothelial cells (HCAECs). Neutralizing antibody against VEGFR2 not only abrogates flow-induced phosphorylation of these tyrosine residues, but also has a marked inhibitory effect on downstream eNOS activation. In situ proximity ligation assay revealed that VEGF and VEGFR2 are closely associated in HCAECs, and more importantly, this association is increased with flow. Finally, we show that flow-induced VEGFR2 activation is attenuated in the presence of the broad spectrum matrix metalloproteinase (MMP) inhibitor, GM6001. Taken together, our results suggest that a ligand-dependent mechanism involving the activity of MMPs plays a key role in the early, shear stress-induced activation of VEGFR2., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Monoclonal antibodies (mAbs) that are internalized into cells are a current focus in the development of antibody-drug conjugates (ADCs). We describe a phage display-based high-throughput screening system to rapidly isolate cell-internalizing mAbs. We simultaneously examined the cell-internalizing activities of several hundred independent mAbs and successfully isolated cell-internalizing mAbs against the tumor endothelial markers Roundabout homolog 4 (Robo4) and vascular endothelial growth factor receptor 2 (VEGFR2). Tumor accumulation of mAbs with high cell-internalizing activity was significantly higher than that of mAbs with low cell-internalizing activity. Furthermore, the antitumor effects of ADCs of mAbs with high cell-internalizing activity were significantly stronger than those of mAbs with low cell-internalizing activity. Although anti-VEGFR2 therapy caused a significant loss of body weight, anti-Robo4 therapy did not. These findings indicate that cell-internalizing activity plays an important role in the biodistribution and therapeutic effects of ADCs. Further, Robo4 can be an effective marker for tumor vascular targeting.

Angiogenesis targeting is an attractive approach for cancer treatment. Vascular endothelial growth factor receptor 2 (VEGFR2) is such an important target that is overexpressed in tumor vasculature compared to the endothelium cells of resting blood vessels and blocking of its signaling inhibits neovascularization and tumor metastasis. Immunotoxins represent a promising group of targeted therapeutics to combat tumors. They consist of an antibody linked to a toxin and are designed to kill specifically the tumor cells. In this study, we fused a VEGFR2-specific Nanobody, the antigen-binding single-domain fragment derived from functional Heavy-chain antibody of Camelidae, to the truncated form of Pseudomonas exotoxin A and evaluated its ability to bind the VEGFR2 molecule on the cell surface. We demonstrate that this immunotoxin inhibits the proliferation of VEGFR2-expressing cells in vitro. This finding is considered to be a significant achievement in tumor therapy and it forms a basis for further studies in animal models., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Objective: To obtain 1-4 IgG-like domains of mouse vascular endothelial growth factor receptor 2 (VEGFR2) fusion protein (mVEGFR2D1-4/GST) and identify its antiginicity and biological activity., Methods: The gene of mVEGFR2D1-4 was amplified by RT-PCR from 14-days embryos of Balb/c mice. The PCR product was cloned into pET-42a prokaryotic expression vector to construct the recombinant plasmid pET-42a-mVEGFR2D1-4, which was transformed into E. coli BL21 (DE3) strain for mVEGFR2D1-4/GST expression. The fusion protein was identified by SDS-PAGE and Western blotting, and the antigenicity of the protein purified by affinity chromatography was characterized by ELISA. The VEGF blocking effect of the purified protein in human umbilical vein endothelial cells (HUVECs) were evaluated in in vitro cell cultures., Results: The mVEGFR2D1-4 gene was obtained, which had an identical sequence to that retrieved in GenBank. The prokaryotic expression vector for mVEGFR2D1-4 was successfully constructed as confirmed by enzyme digestion and DNA sequencing. Both Western blotting and ELISA demonstrated the antigenicity of the purified mVEGFR2D1-4 fusion protein, which obviously blocked the effect of VEGF in promoting HUVEC proliferation in vitro., Conclusion: The mVEGFR2D1-4/GST fusion protein obtained shows a strong antigenicity and biological activity to facilitate further study of active anti-tumor immunotherapy targeting VEGFR2.

Angiogenesis is required for normal physiologic processes, but it is also involved in tumor growth, progression and metastasis. Here, we report the development of an immune-based antiangiogenic strategy based on the generation of T lymphocytes that possess killing specificity for cells expressing vascular endothelial growth factor receptor 2 (VEGFR2). To target VEGFR2-expressing cells, we engineered cytotoxic T lymphocyte (CTL) expressing chimeric T-cell receptors (cTCR-CTL) comprised of a single-chain variable fragment (scFv) against VEGFR2 linked to an intracellular signaling sequence derived from the CD3ζ chain of the TCR and CD28 by retroviral gene transduction methods. The cTCR-CTL exhibited efficient killing specificity against VEGFR2 and a tumor-targeting function in vitro and in vivo. Reflecting such abilities, we confirmed that the cTCR-CTL strongly inhibited the growth of a variety of syngeneic tumors after adoptive transfer into tumor-bearing mice without consequent damage to normal tissue. In addition, CTL expressing both cTCR and tumor-specific TCR induced complete tumor regression due to enhanced tumor infiltration by the CTL and long-term antigen-specific function. These findings provide evidence that the tumor vessel-injuring ability improved the antitumor effect of CTLs in adoptive immunotherapy for a broad range of cancers by inducing immune-mediated destruction of the tumor neovasculature.

The recent approval of a prostate cancer vaccine has renewed hope for anticancer immunotherapies. However, the immunosuppressive tumor microenvironment may limit the effectiveness of current immunotherapies. Antiangiogenic agents have the potential to modulate the tumor microenvironment and improve immunotherapy, but they often are used at high doses in the clinic to prune tumor vessels and paradoxically may compromise various therapies. Here, we demonstrate that targeting tumor vasculature with lower vascular-normalizing doses, but not high antivascular/antiangiogenic doses, of an anti-VEGF receptor 2 (VEGFR2) antibody results in a more homogeneous distribution of functional tumor vessels. Furthermore, lower doses are superior to the high doses in polarizing tumor-associated macrophages from an immune inhibitory M2-like phenotype toward an immune stimulatory M1-like phenotype and in facilitating CD4(+) and CD8(+) T-cell tumor infiltration. Based on this mechanism, scheduling lower-dose anti-VEGFR2 therapy with T-cell activation induced by a whole cancer cell vaccine therapy enhanced anticancer efficacy in a CD8(+) T-cell-dependent manner in both immune-tolerant and immunogenic murine breast cancer models. These findings indicate that vascular-normalizing lower doses of anti-VEGFR2 antibody can reprogram the tumor microenvironment away from immunosuppression toward potentiation of cancer vaccine therapies. Given that the combinations of high doses of bevacizumab with chemotherapy have not improved overall survival of breast cancer patients, our study suggests a strategy to use antiangiogenic agents in breast cancer more effectively with active immunotherapy and potentially other anticancer therapies.

Bispecific antibodies have been exploited as both cancer immunodiagnostics and cancer therapeutics, which have shown promises in clinical trials in cancer imaging and therapy. To improve the anti-tumor effect, an scDb (bispecific single-chain diabody) was constructed from the variable domain genes of two scFvs (single-chain variable fragment antibodies) directed against human EGFR (epidermal growth factor receptor) and VEGFR2 (vascular endothelial growth factor receptor 2) extracellular domains. The anti-EGFR/ anti-KDR scDb was constructed into pHEN2 plasmid and expressed in Escherichia coli HB2151 host. After purification by one-step affinity chromatography of IMAC, scDb protein was characterized by Western blotting. The yield of scDb protein was 570 microg per liter medium. scDb bound to EGFR as efficiently as the parental antibody scFv-E10, while a little bit weaker than the parental antibody scFv-AK404R when bound to KDR. In conclusion, the scDb protein could bind both EGFR and KDR specifically and could be applied for further anti-tumor research.

The aim of this study was to evaluate the safety and efficacy of vaccination with human leukocyte antigen (HLA)-A24-restricted human vascular endothelial growth factor receptor 1 (VEGFR1)-1084 and VEGFR2-169 combined with chemotherapy in patients with advanced gastric cancer. HLA-A 2402-positive patients with advanced or recurrent adenocarcinoma of the stomach were vaccinated with VEGFR1-1084 and VEGFR2-169 combined with S-1 and cisplatin. The study included 22 patients (median age 60.5 years) who received at least one cycle of the combination therapy. No severe adverse effects caused by the vaccine therapy were observed except for an inflammatory reaction at the site of injection in 6 patients. Twelve patients (55%) showed partial response and 10 had stable disease after two cycles of the combination therapy. The disease control rate (partial response and stable disease) was 100% after two cycles. The median time to progression was 9.6 months and median overall survival was 14.2 months. VEGFR1-1084-specific cytotoxic T lymphocyte (CTL) response was induced in 18 (82%) of the 22 patients and VEGFR2-169-specific CTL response was induced in 18 (82%) of the 22 patients. Patients showing CTL response to VEGFR2-169 peptide had significantly better prognosis than those without, as demonstrated by the overall survival (OS) and time to progression (TTP) (OS, p=0.028, TTP, p=0.006). The combination therapy was well tolerated and highly effective in advanced or recurrent gastric cancer. Substantial specific CTL for both peptides was frequently induced even under chemotherapy. Thus, cancer vaccination combined with standard chemotherapy warrants further analysis as a promising strategy for the treatment of advanced cancer.

Abstract: This study is to improve the affinity of scFv-AK404R against VEGFR2. The secondary mutational library was constructed by hydrophilic shuffling in CDR3 region of the heavy chain. VEGFR2-specific screening was performed by phage display technology and the protein of mutants was expressed in periplasm of E.coli HB2151 and purified by affinity chromatography. The affinity constant of scFvs was measured by competitive ELISA, and the structure of scFvs was analyzed by bioinformatics. The result showed that a library with 6.4x10(5) scFv members was established by electro-transformation. Two mutated clones with high absorbance value were isolated after screening. After purification by affinity chromatography, electrophoretically pure scFv proteins were obtained. The competitive ELISA showed that the affinities of WZ01 and WZ02 were three times higher than that of the parental AK404R, and bioinformatics analysis showed that the enlarged contact surface and fitted closely with KDR3 surface may be the reasons for improved affinity. These results suggest that introducing hydrophilic amino acids to the heavy chain CDR3 region is an effective approach to improve the affinity of scFv.

Background: The investigational oral DNA vaccine VXM01 targets the vascular endothelial growth factor receptor 2 (VEGFR-2) and uses Salmonella typhi Ty21a as a vector. The immune reaction elicited by VXM01 is expected to disrupt the tumor neovasculature and, consequently, inhibit tumor growth. VXM01 potentially combines the advantages of anti-angiogenic therapy and active immunotherapy., Methods/design: This phase I trial examines the safety, tolerability, and immunological and clinical responses to VXM01. The randomized, placebo-controlled, double blind dose-escalation study includes up to 45 patients with locally advanced and stage IV pancreatic cancer. The patients will receive four doses of VXM01 or placebo in addition to gemcitabine as standard of care. Doses from 106 cfu up to 1010 cfu of VXM01 will be evaluated in the study. An independent data safety monitoring board (DSMB) will be involved in the dose-escalation decisions. In addition to safety as primary endpoint, the VXM01-specific immune reaction, as well as clinical response parameters will be evaluated., Discussion: The results of this study shall provide the first data regarding the safety and immunogenicity of the oral anti-VEGFR-2 vaccine VXM01 in cancer patients. They will also define the recommended dose for phase II and provide the basis for further clinical evaluation, which may also include additional cancer indications., Trial Registration: EudraCT No.: 2011-000222-29, NCT01486329, ISRCTN68809279.

Partial response (PR) was obtained in a patient with advanced colon cancer following peptide vaccine therapy. A 61-year-old woman was referred to our hospital for peptide vaccine therapy. She had undergone sigmoidectomy at a nearby hospital and eventually developed multiple metastases to the lung and pelvic lymph nodes with left hydronephrosis. A ureteral stenting catheter had been inserted for left hydronephrosis, and oral opioids had been administered for relief of pain in the left pelvic region. Three tumor-antigen-derived peptides (RNF43, TOMM34, and KOC1) and two human VEGFR-derived peptides (VEGFR1 and VEGFR2) were used as a cocktail. The peptide cocktail was subcutaneously inoculated on days 1, 8, 15, and 22 and repeated at 14-day intervals. The patient's serum level of carcinoembryonic antigen was 28.9 ng/mL (N<5 ng/mL) before treatment, and it decreased promptly after the initiation of therapy to within a normal range. Evaluation of computed tomography images at week 5 revealed PR as determined by the Response Evaluation Criteria in Solid Tumor criteria. After month 3, the oral opioid was discontinued. The PR lasted for 4 months and was followed by stable disease for another 4 months. No particular adverse effects were observed. A cytotoxic T lymphocyte (CTL) response was evaluated by immunosorbent spot assay, and a positive CTL response was recognized against at least one of five peptides at each end of the six courses. Immunotherapy has been proven to slow tumor growth by inducing an active antitumor immune response; and therefore, significant tumor shrinkage is rarely observed. To our knowledge, this is the first case report of PR presented in a patient with advanced colon cancer.

Vascular endothelial growth factor receptor 2 (VEGFR-2) plays a critical role in tumor angiogenesis. None therapeutic antibodies targeting VEGFR-2 are available in clinical use. Herein, we describe the screening of a new single-chain antibody fragment (scFv) targeting extracellular domain 3 of human VEGFR-2 (kinase insert domain-containing receptor [KDR]3) from Griffin phage display scFv library. A comprehensive sequence analysis was performed to assign the framework and complementary-determining regions. The scFv exerted particular binding sites to KDR3 on molecular docking, and the binding affinity was further convinced by binding analysis both in quantitative ELISA and real-time kinetic determination by biosensors (K(D) = 40 nM). Finally, the scFv was revealed to inhibit VEGF-stimulated proliferation of human umbilical vein endothelial cells (HUVECs; IC(50) = 5 nM) and to inhibit HUVEC migration significantly at 17 nM. Taken together, our results indicate that we have successfully isolated a scFv which differentially recognizes KDR3 and has potential clinical applications in the treatment of angiogenesis related diseases., (Copyright © 2012 American Institute of Chemical Engineers (AIChE).)

Background: Endothelial progenitor cells (EPCs) are known to be altered in heart failure (HF), but monocyte-derived EPCs in HF have not been assessed. We aimed to characterize monocyte-derived EPCs in systolic HF., Methods and Results: We recruited 128 subjects with systolic HF: 50 South Asian (SA), 50 white, and 28 African-Caribbean (AC), for interethnic comparisons. Additionally, SAs with HF were compared with 40 SAs with coronary artery disease (CAD) without HF (disease controls [DCs]) and 40 SA healthy controls (HCs). Counts of CD34(+) and kinase domain receptor (KDR)(+) monocytes attributed to specific monocyte subsets (CD14(++) /CD16(-) [Mon1], CD14(++)/CD16(+) [Mon2], and CD14(+)/CD16(++) [Mon3]) and monocyte expression of vascular endothelial growth factor (VEGF) receptor 1 were analyzed by flow cytometry. We also enumerated CD34(+)/KDR(+) EPCs derived from mononuclear cells ('classic' EPC definition)., Results: SAs with HF had significantly reduced counts of CD34(+) monocytes, attributed to the Mon1 and Mon2 subsets. KDR(+) Mon1 counts were 4.5-fold increased in DCs as compared with HCs, but significantly reduced in HF subjects vs. DCs. VEGF receptor type 1 expression on Mon1 and Mon2 cells was significantly reduced in HF patients as compared with DCs. Also, CD34(+)/KDR(+) EPC numbers were reduced in HF subjects. Whites had significantly fewer KDR(+) Mon3 cells than ACs, but significantly more CD34(+) Mon2 cells than SAs and ACs. VEGF receptor type 1 expression by Mon1 cells was predictive for left ventricular ejection fraction after adjustment for ethnicity (β = - 0.25. P = 0.039). CD34(+) Mon2 counts correlated with measures of microvascular endothelial function, and were predictive of the future risk of hospital admission., Conclusions: Circulating counts of monocyte-derived EPCs are significantly altered in HF, with significant ethnic differences in the levels of monocyte-derived EPCs., (© 2012 International Society on Thrombosis and Haemostasis.)

The primary goal of this study was to evaluate the feasibility of using anti-vascular endothelial growth factor receptor 2 (VEGFR2)-conjugated poly(lactic-co-glycolic acid) (PLGA) microspheres as an x-ray phase contrast agent to assess the VEGFR2 expression in cell cultures. The cell lines, mouse LLC (Lewis lung carcinoma) and HUVEC (human umbilical vein endothelial cell), were selected for cell adhesion studies. The bound PLGA microspheres were found to better adhere to LLC cells or HUVECs than unbound ones. Absorption and phase contrast images of PLGA microspheres were acquired and compared in vitro. Phase contrast imaging (PCI) greatly improves the detection of the microspheres as compared to absorption contrast imaging. The cells incubated with PLGA microspheres were imaged by PCI, which provided clear 3D visualization of the beads, indicating the feasibility of using PLGA microspheres as a contrast agent for phase contrast CT. In addition, the microspheres could be clearly distinguished from the wall of the vessel on phase contrast CT images. Therefore, the approach holds promise for assessing the VEGFR2 expression on endothelial cells of tumor-associated vessels. We conclude that PLGA microsphere-based PCI of the VEGFR2 expression might be a novel, promising biomarker for future studies of tumor angiogenesis.

Background: A number of strategies have been used to improve the efficacy of the DNA vaccine for the treatment of tumors. These strategies, ranging from activating CD4+ T cell, manipulating antigen presentation and/or processing to anti-angiogenesis, focus on one certain aspect in the functioning of the vaccine. Therefore, their combination is necessary for rational DNA vaccines design by synergizing different regimens and overcoming the limitations of each strategy., Methods: A DNA fragment (HSV) encoding the C terminal 37 amino acids of human chorionic gonadotropin β chain (hCGβ), 5 different HLA-restricted cytotoxic T lymphocyte epitopes from human survivin and the third and fourth extracellular domains of vascular endothelial growth factor receptor 2 (VEGFR2) was inserted into the sequence between the luminal and transmembrane domain of human lysosome-associated membrane protein-1 cDNA for the construction of a novel DNA vaccine., Results: This novel vaccine, named p-L/HSV, has a potent antitumor effect on the LL/2 lung carcinoma model in syngeneic C57BL/6 mice. The immunologic mechanism involved in the antitumor effect referred to the activation of both cellular and humoral immune response. In addition, the tumor vasculature was abrogated as observed by immunohistochemistry in p-L/HSV immunized mice. Furthermore, the immunized mice received an additional boost with p-L/HSV 6 months later and showed a strong immune recall response., Conclusions: The present study indicates that the strategies of combining antitumor with antiangiogenesis and targeting the tumor antigen to the major histocompatibility complex class II pathway cooperate well. Such a study may shed new light on designing vaccine for cancer in the future., (Copyright © 2012 John Wiley & Sons, Ltd.)

Purpose: We investigated the feasibility of delivering the proinflammatory cytokine interleukin (IL)-12 into tumor using T cells genetically engineered to express a chimeric antigen receptor (CAR) against the VEGF receptor-2 (VEGFR-2)., Experimental Design: Two different strains of mice bearing five different established subcutaneous tumors were treated with syngeneic T cells cotransduced with an anti-VEGFR-2 CAR and a constitutively expressed single-chain murine IL-12 or an inducible IL-12 gene after host lymphodepletion. Tumor regression, survival of mice, and persistence of the transferred cells were evaluated., Results: Adoptive transfer of syngeneic T cells cotransduced with an anti-VEGFR-2 CAR and a constitutively expressing single-chain IL-12 resulted in the regression of five different established tumors of different histologies without the need for IL-2 administration. T cells transduced with either anti-VEGFR-2 CAR or single-chain IL-12 alone did not alter the tumor growth indicating that both of them had to be expressed in the same cell to mediate tumor regression. Anti-VEGFR-2 CAR and IL-12-cotransduced T cells infiltrated the tumors, expanded, and persisted for prolonged periods. The antitumor effect did not require the presence of host T and B cells but was dependent on host IL-12R-expressing cells. The anti-VEGFR-2 CAR changed the immunosuppressive tumor environment by altering/reducing both the systemic and the intratumoral CD11b(+)Gr1(+) myeloid suppressor cell subsets that expressed VEGFR-2., Conclusions: These results suggest that targeted delivery of IL-12 into the tumor environment with T cells redirected against VEGFR-2 is a promising approach for treating patients with a variety of solid tumor types.

Intact immunoglobulin G antibody has a relatively large molecule size of approximately 150 kDa that remains in the bloodstream for many weeks, which is a considerable disadvantage when it is used to carry radioactive materials for imaging. To lower background activity and increase the contrast of images, we investigated antivascular endothelial growth factor (VEGF) receptor 2 antibody (DC101) conjugated dextran for VEGF receptor 2 imaging in tumor xenografted mice. DTPA-conjugated aminodextran was synthesized, reacted with sulfo-LC-SPDP, and then reacted with DC101. The binding affinity of DTPA-dextran-DC101 to Flk-1 was measured. The gamma imaging and biodistributions of (99m)Tc-DTPA-dextran-DC101, (99m)Tc-DTPA-DC101, and (125)I-DC101 were studied in B16F10 melanoma xenografted mice. The dissociation values for DC101, DTPA-DC101, and DTPA-dextran-DC101 were 22.48, 3.05, and 14.74 pM, respectively. In gamma images, (99m)Tc-DTPA-dextran-DC101 showed weak liver uptake and rapid kidney elimination. In biodistribution results, the liver uptake of (99m)Tc-DTPA-dextran-DC101 was similar with that of (99m)Tc-DTPA-DC101 at each time point. However, the blood activity of (99m)Tc-DTPA-dextran-DC101 has shown significant differences, compared with (99m)Tc-DTPA-DC101 at all time points. The tumor accumulation of dextran-conjugated antibody was increased with time, whereas that of dextran nonconjugated antibody decreased. In particular, the pattern of tumor uptake of (99m)Tc-DTPA-dextran-DC101 was similar to that of (125)I-DC101, so this was thought to reflect the kinetics of DC101, unlike the nonconjugated form. The results of this study suggested that introduction of dextran moiety to make (99m)Tc-radiolabeled DC101 imaging agent could provide better images with the impaired background and the steady increasing binding to the receptor. However, further studies are necessary to improve clinical pharmacokinetics, such as enhancement of tumor uptake and impaired renal uptake.

Arterial dysfunction has been documented in patients with beta-thalassaemia major. This study aimed to determine the quantity and proliferative capacity of circulating CD133(+)VEGFR2(+) and CD34(+)VEGFR2(+) cells in patients with beta-thalassaemia major and those after haematopoietic stem cell transplantation (HSCT), and their relationships with arterial function. Brachial arterial flow-mediated dilation (FMD), carotid arterial stiffness, the quantity of these circulating cells and their number of colony-forming units (CFUs) were determined in 17 transfusion-dependent thalassaemia patients, 14 patients after HSCT and 11 controls. Compared with controls, both patient groups had significantly lower FMD and greater arterial stiffness. Despite having increased CD133(+)VEGFR2(+) and CD34(+)VEGFR2(+) cells, transfusion-dependent patients had significantly reduced CFUs compared with controls (p = 0.002). There was a trend of increasing CFUs across the three groups with decreasing iron load (p = 0.011). The CFUs correlated with brachial FMD (p = 0.029) and arterial stiffness (p = 0.02), but not with serum ferritin level. Multiple linear regression showed that CFU was a significant determinant of FMD (p = 0.043) and arterial stiffness (p = 0.02) after adjustment of age, sex, body mass index, blood pressure and serum ferritin level. In conclusion, arterial dysfunction found in patients with beta-thalassaemia major before and after HSCT may be related to impaired proliferation of CD133(+)VEGFR2(+) and CD34(+)VEGFR2(+) cells.

Vascular endothelial growth factor receptor-2 (VEGFR2) is an important tumor-associated receptor and blockade of the VEGF receptor signaling can lead to the inhibition of neovascularization and tumor metastasis. Nanobodies are the smallest intact antigen binding fragments derived from heavy chain-only antibodies occurring in camelids. Here, we describe the identification of a VEGFR2-specific Nanobody, named 3VGR19, from dromedaries immunized with a cell line expressing high levels of VEGFR2. We demonstrate by FACS, that 3VGR19 Nanobody specifically binds VEGFR2 on the surface of 293KDR and HUVECs cells. Furthermore, the 3VGR19 Nanobody potently inhibits formation of capillary-like structures. These data show the potential of Nanobodies for the blockade of VEGFR2 signaling and provide a basis for the development of novel cancer therapeutics., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Purpose: Angiogenesis is a key process in tumor progression. By binding VEGF, VEGF receptor-2 (VEGFR2) is a main signaling transducer in tumor-associated angiogenesis. Accordingly, therapeutic approaches against the VEGF/VEGFR2 signaling axis have been designed. However, an efficient and specific chemotherapeutic targeting of tumor-associated endothelial cells has not yet been achieved., Experimental Design: We have employed anti-VEGFR2 antibodies covalently linked to pegylated liposomal doxorubicin (PLD) to specifically ablate tumor-associated endothelial cells in the Rip1Tag2 mouse model of insulinoma, in the MMTV-PyMT mouse model of breast cancer, and in the HT-29 human colon cancer xenograft transplantation model., Results: In each model, anti-VEGFR2-targeted immunoliposomes (ILs) loaded with doxorubicin (anti-VEGFR2-ILs-dox) were superior in therapeutic efficacy to empty liposomes, empty anti-VEGFR2-ILs, antibodies alone, and PLD. Efficacy was similar to that of the oral VEGFR1, -2, and -3 inhibitor PTK787. Detailed histopathologic and molecular analysis revealed a strong antiangiogenic effect of anti-VEGFR2-ILs-dox, and the observed antiangiogenic therapy was significantly more efficient in reducing tumor burden in well-vascularized transgenic mouse models as compared with the less-vascularized xenograft model., Conclusions: Anti-VEGFR2 ILs provide a highly efficient approach to selectively deplete VEGFR2-expressing tumor vasculature. They offer a novel and promising anticancer strategy., (©2011 AACR.)

The Salmonella type III secretion system (T3SS) efficiently translocates heterologous proteins into the cytosol of eukaryotic cells. This leads to an antigen-specific CD8 T-cell induction in mice orally immunized with recombinant Salmonella. Recently, we have used Salmonella's T3SS as a prophylactic and therapeutic intervention against a murine fibrosarcoma. In this study, we constructed a recombinant Salmonella strain translocating the immunogenic H-2D(b)-specific CD8 T-cell epitope VILTNPISM (KDR2) from the murine vascular endothelial growth factor receptor 2 (VEGFR2). VEGFR2 is a member of the tyrosine protein kinase family and is upregulated on proliferating endothelial cells of the tumor vasculature. After single orogastric vaccination, we detected significant numbers of KDR2-tetramer-positive CD8 T cells in the spleens of immunized mice. The efficacy of these cytotoxic T cells was evaluated in a prophylactic setting to protect mice from challenges with B16F10 melanoma cells in a flank tumor model, and to reduce dissemination of spontaneous pulmonary melanoma metastases. Vaccinated mice revealed a reduction of angiogenesis by 62% in the solid tumor and consequently a significant decrease of tumor growth as compared to non-immunized mice. Moreover, in the lung metastasis model, immunization with recombinant Salmonella resulted in a reduction of the metastatic melanoma burden by approximately 60%.

Vascular endothelial growth factor (VEGF) is an angiogenic factor that also functions as an autocrine growth factor for VEGF receptor (VEGFR)-2(+) melanomas. In multiple studies, VEGFR-2 was detected by immunostaining in 78-89% of human melanoma cells, suggesting that most patients with melanoma would benefit from anti-VEGF therapy. Here, we evaluated 167 human melanoma specimens in a tissue microarray to verify the presence of VEGFR-2, but found disparities in staining with commercial antibodies A-3 and 55B11. Antibody A-3 stained melanoma cells in 79% of specimens, consistent with published results; however, we noted extensive nonspecific staining of other cells such as smooth muscle and histiocytes. In contrast, antibody 55B11 stained melanoma cells in only 7% (95% confidence interval: 3.3-11.5) of specimens. As an internal positive control for VEGFR-2 detection, vascular endothelial cells were stained with antibody 55B11 in all specimens. We compared VEGFR-2(+) and VEGFR-2(-) melanoma cell lines by immunoblotting and immunohistochemistry after small interfering RNA (siRNA) knockdown and transient overexpression of VEGFR-2 to validate antibody specificity. Immunoblotting revealed that A-3 primarily cross-reacted with several proteins in both cell lines and these were unaffected by siRNA knockdown of VEGFR-2. In contrast, 55B11 staining of VEGFR-2(+) cells was mostly eliminated by siRNA knockdown of VEGFR-2 and increased in VEGFR-2(-) melanoma cell lines following transfection to express ectopic VEGFR-2. Our results show that relatively few melanoma cells (<10%) express detectable levels of VEGFR-2, and therefore, the majority of patients with melanoma are unlikely to benefit from antiproliferative effects of anti-VEGF therapy., (Copyright © 2011 UICC.)

Purpose: The purpose of this study was to quantify and model the longitudinal intra-tumor physiological response to a single dose of a monoclonal antibody specific to the VEGFR2 using dynamic contrast-enhanced CT., Material and Methods: Dynamic contrast-enhanced CT imaging was performed on athymic nude mice bearing xenograft VEGF-transfected MCF-7 tumors (MCF7(VEGF)) to quantify intra-tumor physiology pre- and post-injection (days 2, 7, and 14) of a nonspecific (IgG1, controls) and specific (DC101, treated) monoclonal antibody targeting VEGFR2. Parametrical maps of tumor physiology-perfusion (F), permeability surface area (PS), fractional plasma (f(p)), and interstitial space (f (is))-were obtained at four time points over a 2-week period., Results: A temporal multistage recovery process whereby a decoupling of the fractional change in physiological parameters (f (p), F) was observed when comparing treated to control tumors: f (p) and perfusion decreased by a combined 27% (P < 0.01) and 65% (P < 0.01) on day 2, while only perfusion remained reduced by 46% (P < 0.01) on day 7. Intra-tumor heterogeneity defined by the change in variance of perfusion decreased on days 2 and 7; no change in the variance of f(p) was observed. Analysis based on a mathematical model linking perfusion and vascular morphology indicates that a decrease in f(p) and perfusion was consistent with a reduction in blood vessel radius, followed by an increase in the vascular radius and tortuosity resulting in the decoupling of f(p) and perfusion before returning to control levels., Conclusion: Inhibiting VEGFR2 activity results in a temporal decoupling of physiological parameters, which can be explained by a combination of morphological changes influencing perfusion. Such a decoupling has the potential to significantly impact the delivery of pharmaceuticals and oxygen within solid tumors, critical factors in combined anti-angiogenic and radio- and chemotherapies.

Vascular endothelial growth factor receptor type 2 (VEGFR2)-targeted tumor treatment is an antiangiogenic therapeutic strategy. The human sodium iodide symporter (hNIS) gene is a useful reporter gene for tumor imaging and radiotherapy. In this study, we investigated the evaluation of therapeutic efficacy in hNIS gene-transfected tumor xenografts using a gamma imaging system after treatment with an anti-VEGFR2 antibody. Human breast cancer MDA-MB-231 cells transfected with the hNIS gene were injected subcutaneously into the right flanks of BALB/c nude mice. Therapy was initiated when the tumor volume reached approximately 130-180 mm(3). The animals were intravenously injected with 50, 100, or 150 μg of antibody every 3 days for 16 days. Gamma imaging was performed 1 and 2 weeks after the first injection to monitor the effects of tumor therapy. Mice were sacrificed 2 weeks after the first injection of antibody and the tumors were removed for CD31 staining and reverse transcription-polymerase chain reaction (RT-PCR) assay. All groups of mice that were treated with anti-hVEGFR2 antibody showed markedly reduced tumor growth compared to control mice. In vivo gamma imaging results showed that, at 1 week after the first injection of the anti-hVEGFR2 antibody, (125)I uptake of a tumor treated with 150 μg of antibody was 24.5% lower than that in the controls. At 2 weeks, (125)I uptake in the tumor treated with 150 μg of antibody was as low as 44.3% of that in the controls. CD31 staining and RT-PCR assays showed that blood vessel formation and expression of the hNIS gene were reduced with increased treatment doses. This study demonstrated the feasibility of molecular imaging and the therapeutic efficacy of developing therapeutic antibody anti-hVEGFR2 using a gamma imaging system in hNIS gene-transfected tumor xenograft mice.

Human vascular endothelial growth factor (VEGF) and its receptor (VEGFR-2/kinase domain receptor [KDR]) play a crucial role in angiogenesis, which makes the VEGFR-2 signaling pathway a major target for therapeutic applications. In this study, a single-chain antibody phage display library was constructed from spleen cells of mice immunized with recombinant human soluble extracellular VEGFR-2/KDR consisting of all seven extracellular domains (sKDR D1-7) to obtain antibodies that block VEGF binding to VEGFR-2. Two specific single-chain antibodies (KDR1.3 and KDR2.6) that recognized human VEGFR-2 were selected; diversity analysis of the clones was performed by BstNI fingerprinting and nucleotide sequencing. The single-chain variable fragments (scFvs) were expressed in soluble form and specificity of interactions between affinity purified scFvs and VEGFR-2 was confirmed by ELISA. Binding of the recombinant antibodies for VEGFR-2 receptors was investigated by surface plasmon resonance spectroscopy. In vitro cell culture assays showed that KDR1.3 and KDR2.6 scFvs significantly suppressed the mitogenic response of human umbilical vein endothelial cells to recombinant human VEGF(165) in a dose-dependent manner, and reduced VEGF-dependent cell proliferation by 60% and 40%, respectively. In vivo analysis of these recombinant antibodies in a rat cornea angiogenesis model revealed that both antibodies suppressed the development of new corneal vessels (p < 0.05). Overall, in vitro and in vivo results disclose strong interactions of KDR1.3 and KDR2.6 scFvs with VEGFR-2. These findings indicate that KDR1.3 and KDR2.6 scFvs are promising antiangiogenic therapeutic agents., (Copyright © 2011 International Union of Biochemistry and Molecular Biology, Inc.)

We report the synthesis and characterization of two amine reactive fluorescent dyes with efficient two-photon absorption (2PA) properties and high fluorescence quantum yields. Bioconjugation of these dyes with the DC-101 antibody proved to be useful for selectively imaging the vascular endothelial growth factor receptor 2 (VEGFR-2) in cells expressing this receptor in vitro and in "whole" mounted excised tumors (ex vivo) by two-photon fluorescence microscopy (2PFM). The penetration depths reached within the tumors by 2PFM was over 800 μm. In addition, the concentration of dye required for incubation of these bioconjugates was in the picomolar domain, the probes possessed very good photostability, and the 2PFM setup did not require any additional means of increasing the collection efficiencies of fluorescent photons to achieve the relatively deep tissue imaging that was realized, due, in large part, to the favorable photophysical properties of the new probes.

Many recombinant murine monoclonal antibodies (mAbs) were studied under pre-clinical or clinical development and became one of the most prolific drug classes in oncology. Vascular endothelial growth factors receptor 2 (VEGFR2) has been implicated to play an important role in tumors. We have established a murine anti-VEGFR2 mAb. To reduce the shortcoming of the mAb, a murine-human chimeric Fab (cFab) named FA8H1 was constructed with gene engineering techniques and expressed as a soluble and functional protein in Escherichia coli Top10F'. Several immunological methods were used to characterize the cFab, including ELISA, affinity and kinetics assay, IP, IF, FACS, and IHC. The results illuminated that cFab maintained the specificity for the VEGFR2 antigen. The active cFab also effectively identified VEGFR2 over-expressing cells in a number of archived human cancer tissues, compared to its parental antibody. The FA8H1 provided the basis for potential therapy research against over-expressing VEGFR2 human solid tumors.

It has been shown that antibodies to donor CD34+/VEGFR-2+ stem cells or antibodies against mismatched HLA are associated with graft rejection after hematopoietic stem cell transplantation (HSCT). CD34/VEGFR-2 positive stem cells have been implicated to play a major role in engraftment after HSCT. In this study we treated four patients with an imminent risk of antibody-mediated rejection with immune modulation, i.e. plasma exchange, intravenous immunoglobulin (IVIG), and rituximab before HSCT. Three of the patients had been previously transplanted and rejected their initial grafts after 12 months, 1 month, and less than 1 month, respectively. The fourth patient was not transplanted previously but had HLA directed antibodies present against the graft. During the immune modulatory treatment we followed the pattern of antibodies in sera using FACS and microcytotoxicity assay. We could show that two patients had antibodies against donor CD34+/VEGFR-2+ cells while the other two had antibodies directed against HLA. All four patients tolerated the immune modulatory regimen without any side effects. In this preliminary study we show that immune modulatory treatment may be used to reduce antibody levels and to prevent rejection after HSCT. In two of the three patients which experienced previous rejections and had detectable anti-HLA or anti-CD34+/VEGFR-2+ antibodies, immune modulation resulted in engraftment. In the fourth patient with known anti-HLA-class I antibodies, the treatment also resulted in engraftment. Our results encourage further studies regarding this treatment regimen., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Vascular endothelial growth factor (VEGF) has been known as a potential vasculogenic and angiogenic factor and its receptor (VEGFR2) is a major receptor to response to the angiogenic activity of VEGF. The technique that to break the immune tolerance of "self-antigens" associated with angiogenesis is an attractive approach for cancer therapy with T4 phage display system. In this experiment, mouse VEGFR2 was constructed on T4 phage nanometer-particle surface as a recombinant vaccine. T4-mVEGFR2 recombinant vaccine was identified by PCR and western blot assay. Immunotherapy with T4-mVEGFR2 was confirmed by protective immunity against Lewis lung carcinoma (LLC) in mice. The antibody against mVEGFR2 was detected by ELISPOT, ELISA and Dot ELISA. The inhibitive effects against angiogenesis were studied using CD31 and CD105 via histological analysis. VEGF-mediated endothelial cells proliferation and tube formation were inhibited in vitro by immunoglobulin induced by T4-mVEGFR2. The antitumor activity was substantiated from the adoptive transfer of the purified immunoglobulin. Antitumor activity and autoantibody production of mVEGFR2 could be neutralized by the depletion of CD4+T lymphocytes. These studies strongly suggest that T4-mVEGFR2 recombinant vaccine might be a promising antitumor approach., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Using an orthotopic intracerebral model from our established HM55-BGIV-101 tumor line, we investigated the antitumor effect on the angiogenesis and growth of human glioblastoma after treatment with monoclonal antibody DC101 against the vascular endothelial growth factor receptor-2 and monoclonal antibody C225 against the epidermal growth factor receptor. Nude mice bearing intracerebral glioblastoma xenografts were treated intraperitoneally with DC101 and C225 either alone or in combination. Histopathological analysis of solid tumor volume, satellite tumor number, microvessel density, tumor cell proliferation, and apoptosis was performed. In the DC101-treated group, solid tumor volume and microvessel density were reduced by 59.7 and 64%, respectively; tumor cell proliferative activity was reduced by 53.2% and the apoptotic index (AI) was increased by 66.7%; satellite tumor number was enhanced by 84.4%. C225 alone reduced satellite tumor number by 43.3%, but had no effect on solid tumor volume, microvessel density, tumor cell proliferation, and apoptosis. C225 combined with DC101 not only reduced solid tumor volume, microvessel density, tumor cell proliferative activity, and increased AI, but also reduced satellite tumor number. Inhibition of angiogenesis achieved by DC101 can cause increased tumor cell invasiveness. In our studies this increased tumor cell invasiveness was inhibited simultaneously by C225, which provides a theoretical basis for treatment of glioblastoma by the method of combining drugs with different pharmacological activity.

Aim: To construct and express an anti-VEGFR2/anti-CD3 bispecific single-chain antibody (bscVEGFR2×CD3)and to identify its binding specificities to CD3 and VEGFR2., Methods: The gene encoding anti-VEGFR2/anti-CD3 bispecific single-chain antibody was designed and synthesized. Bispecific single-chain antibody (bsc-Ab) DNA was subcloned into a eukaryotic expression vector pcDNA3.1(+), then transfected into Chinese hamster ovary (CHO) cells and stable expression cell lines were selected. Expressed Bsc-Ab was purified by His-tag affinity chromatography and confirmed by 120 g/L SDS-PAGE and Western blotting. Antigen binding activity of the bsc-Ab was analyzed by FACS., Results: The plasmid DNA containing bispecific single-chain fragments were confirmed. BscVEGFR2×CD3 was secreted by CHO into the supernatant. Six stable expression cell lines were established. The molecular weight of bsc-Ab was correct indicated by SDS-PAGE and Western blotting. The bsc-Ab could specifically bind to CD3(+); jurkat cells and VEGFR2(+); A375 cells., Conclusion: An anti-VEGFR2/anti-CD3 bispecific single-chain antibody is successfully constructed and expressed, and the antibody has specific binding capacity to CD3 and VEGFR2.

To determine the adeno-associated virus (AAV) serotype that most efficiently mediates muscle expression of antiangiogenic proteins, we injected four different serotype (1, 2, 7, and 8) AAV vectors encoding mouse endostatin (mEnd) or human soluble FLK-1 (hsFLK-1) into a quadriceps muscle of C57BL/6 mice. The highest plasma levels of therapeutic protein were observed in AAV8-injected mice (8 > 7 > 1 > 2). Sustained expression of mEnd was detected for 6 months, whereas concentrations of hsFLK-1 declined to the background level within 2 weeks caused by neutralizing anti-hsFLK-1 antibody. These data demonstrate that AAV8 (mEnd) serotype is the most efficient mediator for protein expression.

Purpose: Lymphangiogenesis (LG) accompanies many corneal diseases after inflammatory, infectious, or chemical insults and is a primary mediator of transplant rejection. The purpose of this study was to investigate whether there is a time window for therapeutic intervention of corneal LG and whether a combined blockade of VEGFR-2 and VEGFR-3 effectively suppresses early-, middle-, or late-stage LG., Methods: Corneal inflammatory neovascularization was induced by a standard suture placement model in mice. Neutralizing antibodies against VEGFR-3 and/or VEGFR-2 were administrated systemically with the treatment started at postoperative day 0, day 7, or day 14. Whole mount corneas were sampled for immunofluorescence microscopic studies using LYVE-1 (a lymphatic marker) antibodies. Digital images were analyzed by software., Results: Both VEGFR-3 and VEGFR-2 were involved in corneal suture-induced inflammatory LG. Their combined blockade led to a significant inhibition of both early- and middle-stage LG while demonstrating no effect on late-stage LG., Conclusions: Corneal inflammatory LG has a discrete time window for intervention therapy. Although it is important to start the treatment as soon as possible, interventions initiated in the middle of the LG process are still effective. These novel findings will shed some light on our understanding of inflammatory LG and the development of new therapeutic protocols for LG-related diseases at different stages.

Angiogenesis, a well-recognized characteristic of malignancy, has been exploited more than any other pathway targeted by biologic anti-neoplastic therapies. Vascular endothelial growth factor receptor-2 (VEGFR-2) is the critical receptor involved in malignant angiogenesis with its activation inducing a number of other cellular modifications resulting in tumor growth and metastases. Ramucirumab (IMC-1121B; ImClone Systems Corporation, Branchburg, NJ) is a fully human monoclonal antibody developed to specifically inhibit VEGFR-2. Ramucirumab is currently being investigated in multiple clinical trials across a variety of tumor types. Herein, angiogenesis inhibition in cancer is reviewed and up-to-date information on the clinical development of ramucirumab is presented.

Background: Vascular endothelial growth factor receptor 2 (VEGFR2) has been suggested to play an important role in solid tumours. Although several reports have shown the relationship between VEGFR2 expression and hepatocellular carcinoma (HCC), the expression pattern of VEGFR2 in HCC parenchyma or stroma, as well as the relationship between VEGFR2 expression and clinicopathological characteristics in HCC, are yet to be satisfactorily defined., Methods: One-step real-time PCR, western blotting and immunohistochemistry were used to characterise the expression of VEGFR2 in HCC using a self-made anti-VEGFR2 monoclonal antibody (A8H1)., Results: Expression of VEGFR2 in HCC cells was higher than in hepatic cells (p<0.001). Comparison of clinicopathological characteristics and immunohistochemistry by χ(2) test analysis showed that the high expression of VEGFR2 in HCC was related to large tumour diameter (p=0.012), poor differentiation (p=0.007), high serum α-fetoprotein (p=0.029), multifocal gross classification (p=0.007), and less than 5 years' survival (p=0.029). Kaplan-Meier survival and Cox regression analyses showed that high VEGFR2 expression (p=0.009) and stage grouping with TNM classification (p=0.004) were independent prognotic factors., Conclusions: The efficacy of A8H1 in immunohistochemistry using HCC tissues was confirmed. There was a correlation of high VEGFR2 expression with prognostic significance in HCC. Additionally, the self-made anti-VEGFR2 monoclonal antibody could be used for future anti-HCC-targeted therapy research.

Circulating endothelial progenitor cells (EPCs) in the peripheral blood of adults represent an auspicious cell source for tissue engineering of an autologous endothelium on blood-contacting implants. Novel materials biofunctionalised with EPC-specific capture molecules represent an intriguing strategy for induction of selective homing of progenitor cells. The trapped EPCs can differentiate into endothelial cells and generate a non-thrombogenic surface on artificial materials. However, the success of this process mainly depends on the use of optimised capture molecules with a high selectivity and affinity. In recent years, various biomedical engineering strategies have emerged for in situ immobilisation of patient's own stem cells on blood contacting materials. The realisation of this in vivo tissue engineering concept and generation of an endothelium on artificial surfaces could exceedingly enhance the performance of not only small calibre vascular grafts and stents, but also, in general all blood-contacting medical devices, such as heart valves, artificial lungs, hearts, kidneys, and ventricular assist devices.

The benefits of inhibiting vascular endothelial growth factor (VEGF) signaling in cancer patients are predominantly attributed to effects on tumor endothelial cells. Targeting non-endothelial stromal cells to further impact tumor cell growth and survival is being pursued through the inhibition of additional growth factor pathways important for the survival and/or proliferation of these cells. However, recent data suggest that VEGF receptor (VEGFR)-specific inhibitors may target lymphatic vessels and pericytes in addition to blood vessels. Here, in fact, we demonstrate that DC101 (40 mg/kg, thrice a week), an antibody specific to murine VEGFR2, significantly reduces all three of these stromal components in subcutaneous (SKRC-29) and orthotopic (786-O-LP) models of renal cell carcinoma (RCC) established in nu/nu athymic mice. Sunitinib (40 mg/kg, once daily), a receptor tyrosine kinase inhibitor of VEGFR2 and other growth factor receptors, also caused significant loss of tumor blood vessels in RCC models but had weaker effects than DC101 on pericytes and lymphatic vessels. In combination, sunitinib did not significantly add to the effects of DC101 on tumor blood vessels, lymphatic vessels, or pericytes. Nevertheless, sunitinib increased the effect of DC101 on tumor burden in the SKRC-29 model, perhaps related to its broader specificity. Our data have important implications for combination therapy design, supporting the conclusion that targeting VEGFR2 alone in RCC has the potential to have pleiotropic effects on tumor stroma.

Purpose: Vascular endothelial growth factor receptor 2-blocking antibody (DC101) has inhibitory effects on tumor growth and angiogenesis in vivo. The human sodium/iodide symporter (hNIS) gene has been shown to be a useful molecular imaging reporter gene. Here, we investigated the evaluation of therapeutic efficacy by molecular imaging in reporter gene transfected tumor xenografts using a gamma imaging system., Methods: The hNIS gene was transfected into MDA-MB-231 cells using Lipofectamine. The correlation between the number of MDA-MB-231-hNIS cells and the uptake of (99m)Tc-pertechnetate or (125)I was investigated in vitro by gamma imaging and counting. MDA-MB-231-hNIS cells were injected subcutaneously into mice. When the tumor volume reached 180-200 mm(3), we randomly assigned five animals to each of three groups representing different tumor therapies; no DC101 (control), 100 μg, or 150 μg DC101/mouse. One week and 2 weeks after the first injection of DC101, gamma imaging was performed. Mice were sacrificed 2 weeks after the first injection of DC101. The tumor tissues were used for reverse transcriptase-polymerase chain reaction (RT-PCR) and CD31 staining., Results: Uptake of (125)I and (99m)Tc-pertechnetate into MDA-MB-231-hNIS cells in vitro showed correlation with the number of cells. In DC101 treatment groups, the mean tumor volume was smaller than that of the control mice. Furthermore, tumor uptake of (125)I was lower than in the controls. The CD31 staining and RT-PCR assay results showed that vessel formation and expression of the hNIS gene were significantly reduced in the tumor tissues of treatment groups., Conclusion: This study demonstrated the power of molecular imaging using a gamma imaging system for evaluating the therapeutic efficacy of an antitumor treatment. Molecular imaging systems may be useful in evaluation and development of effective diagnostic and/or therapeutic antibodies for specific target molecules., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Use of adequate adjuvant is necessary for induction of effective antitumor immune responses. To develop an effective adjuvant for cancer immunotherapy, we selected formalin-inactivated (f)-HSV as an adjuvant component, and analyzed the mechanisms underlying its adjuvant effects. First, we found that f-HSV can induce the tumor antigen-specific CTLs by enhancing antigen cross-presentation by dendritic cells (DCs), mainly through TLR2, but not TLR9. Next, f-HSV was also found to prevent the accumulation of myeloid-derived suppressor cells (MDSCs). We demonstrated that the expansion of MDSCs in the blood and spleen during tumor progression required B cells producing the inflammatory angiogenesis factors, vascular endothelial growth factor (VEGF)-A and neuropilin-1 (NRP-1), a co-receptor for VEGF receptor-2 (VEGFR-2). Interestingly, the transmembrane-type NRP-1 on B cells changed to soluble-type NRP-1 (sNRP-1) by f-HSV treatment. We further showed that the sNRP-1 and VEGF-A secreted from B cells by f-HSV treatment could abrogate the immunosuppressive ability of MDSCs. These results suggest that f-HSV can enhance antitumor immune responses as an adjuvant, not only through activation of DCs, but also inactivation of MDSCs via B cells., (Copyright © 2010 UICC.)

Objective: To explore the specific cellular and humoral immunity induced by dendritic cells (DC) vaccine loading allogenic microvascular endothelial cell bEnd.3 antigen against U14 cervical cancer cell of mice., Methods: Mouse brain microvascular endothelial cell bEnd.3 was cultured and identified for preparation endothelial cell bEnd.3 antigen. The level of mRNA expression of vascular endothelial growth factor receptor 2 (VEGF-R₂) and integrin αV was detected by reverse transcription (RT)-PCR. The BALB/c mice were immuned with DC loading bEnd.3 antigen 4 times in 4 weeks (bEnd.3-DC group), while the mice only were immuned with DC or injected with phosphate buffer saline (PBS group) as control group. One week after last vaccination, U14 cervical cancer cells were injected subcutaneously into the mice. The tumor size, cytotoxic T lymphocyte (CTL) response of spleen lymphocytes in vitro, the percentage of CD₃+CD₈+ surface markers of spleen lymphocytes, and the titer of serum antibody were detected. The specific immunity was examined by immunocytochemistry and western blot., Results: The expression of VEGF-R₂ and integrin αV gene in bEnd.3 cells were expressed highly. After the vaccine was injected, the tumors of mice in PBS group grew faster than those in other groups, while the tumors in bEnd.3-DC group grew slowly and disappeared after 2 weeks. The volume of tumors in DC group grew slower than those in PBS group [(0.11 ± 0.13) cm³ versus (3.38 ± 0.34) cm³]. The CTL response of spleen lymphocytes in vitro showed that bEnd.3-DC cells could kill bEnd.3 cells, the special lysis rate was more than 60%. The percentage of CD₃+CD₈+ spleen lymphocytes in bEnd.3-DC group [(38.6 ± 0.7)%] was higher than those in other groups (P < 0.05). The titer of serum antibody of bEnd.3-DC group was 1:3200, while it was 1:800 in DC group and there were not any in PBS group. Immunocytochemistry analysis indicated there were specific antigen-antibody reaction to bEnd.3 cell in bEnd.3-DC group. Western blot analysis revealed that there were specific bands at 220,000 (VEGF-R₂)., Conclusions: bEnd.3-DC vaccine can inhibit the tumor growth of U14 cervical cancer cell of mice, which indicates that the special cellular and humoral immunity are induced by bEnd.3-DC antigen which maybe have some antigens in bEnd.3 cells that reacts with endothelial cell proliferation-related antigens.

Tumors induce new blood vessel growth primarily from host organ microvascular endothelial cells (EC), and microvasculature differs significantly between the lung and liver. Vascular endothelial growth factor (VEGF or VEGF-A) promotion of tumor angiogenesis is thought to be mediated primarily by VEGF receptor-2 (VEGFR-2). In this study, VEGFR-2 antibody (DC101) inhibited growth of RenCa renal cell carcinoma lung metastases by 26%, whereas VEGFR-1 antibody (MF-1) had no effect. However, VEGFR-2 neutralization had no effect on RenCa liver metastases, whereas VEGFR-1 neutralization decreased RenCa liver metastases by 31%. For CT26 colon carcinoma liver metastases, inhibition of both VEGFR-1 and VEGFR-2 was required to induce growth delay. VEGFR-1 or VEGFR-2 inhibition decreased tumor burden not by preventing the establishment of micrometastases but rather by preventing vascularization and growth of micrometastases by 55% and 43%, respectively. VEGF induced greater phosphorylation of VEGFR-2 in lung ECs and of VEGFR-1 in liver ECs. EC proliferation, migration, and capillary tube formation in vitro were suppressed more by VEGFR-2 inhibition for lung EC and more by VEGFR-1 inhibition for liver EC. Collectively, our results indicate that liver metastases are more reliant on VEGFR-1 than lung metastases to mediate angiogenesis due to differential activity of VEGFRs on liver EC versus lung EC. Thus, therapies inhibiting specific VEGFRs should consider the targeted sites of metastatic disease., (©2010 AACR.)

Vascular endothelial growth factor (VEGF) is a key mediator in the development of airway immune dysfunction to inhaled allergens. However, the exact role of its receptors-mediated signaling is controversial. In this study, we evaluated the role of VEGF receptor (VEGFR)-1- and VEGFR-2-mediated signaling in T cell priming and polarization in the context of inhalation of LPS-containing allergens. A murine asthma model of mixed Th1 and Th17 cell responses was generated using intranasal sensitization with LPS-containing allergens. Pharmacologic intervention was performed during sensitization. In vivo production of VEGF and Th1- and Th17-polarizing cytokines (IL-12p70 and IL-6, respectively) were upregulated by airway exposure to LPS. Pharmacological intervention with a VEGFR-2-neutralizing Ab (anti-Flk1 mAb) abolished the production of IL-6 (but not IL-12p70) and the subsequent development of allergen-specific Th17 cell response. On the other hand, blocking VEGFR-1 signaling with a VEGFR-1 antagonist (anti-Flt1 hexapeptide) did not affect the production of IL-12p70 and IL-6. However, blocking VEGFR-1 signaling resulted in T cell tolerance rather than priming, mainly by inhibiting the maturation of lung dendritic cells, and their migration into lung-draining lymph nodes. These results suggest that T cell priming to LPS-containing allergens depends on VEGFR-1-mediated signaling, and the subsequent Th17 polarization depends on VEGFR-2 signaling.

Although the critical role of complement component C3d as a molecular adjuvant in preventing virus infection is well established, its role in cancer therapies is unclear. In this study, we have engineered a DNA vaccine that expresses extracellular region of murine VEGFR-2 (FLK1(265-2493)) and 3 copies of C3d (C3d3), a component of complement as a molecular adjuvant, designed to increase antitumor immunity. VEGFR-2 has a more restricted expression on endothelial cells and is upregulated once these cells proliferate during angiogenesis in the tumor vasculature. Immunization of mice with vector encoding FLK1(265-2493) alone generated only background levels of anti-VEGFR-2 antibodies and slight inhibitory effect on tumor growth. However, the addition of C3d3 to the vaccine construct significantly augmented the anti-VEGFR-2 humoral immune response and inhibited the tumor growth. The antitumor activity induced by vaccination with vector encoding FLK1(265-2493)-C3d3 fusion protein was also demonstrated via growth inhibition of established tumors following passive transfer of immune serum from vaccinated mice. Our results suggest that vaccination with vector encoding FLK1(265-2493) with C3d3 as a molecular adjuvant induces adaptive humoral activity, which is directed against the murine VEGFR-2 and can significantly inhibit tumor growth, and that administration of C3d as a molecular adjuvant to increase antibodies levels to VEGFR-2 may provide an alternative treatment modality for cancer therapies., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The role of lymphangiogenesis in inflammation has remained unclear. To investigate the role of lymphatic versus blood vasculature in chronic skin inflammation, we inhibited vascular endothelial growth factor (VEGF) receptor (VEGFR) signaling by function-blocking antibodies in the established keratin 14 (K14)-VEGF-A transgenic (Tg) mouse model of chronic cutaneous inflammation. Although treatment with an anti-VEGFR-2 antibody inhibited skin inflammation, epidermal hyperplasia, inflammatory infiltration, and angiogenesis, systemic inhibition of VEGFR-3, surprisingly, increased inflammatory edema formation and inflammatory cell accumulation despite inhibition of lymphangiogenesis. Importantly, chronic Tg delivery of the lymphangiogenic factor VEGF-C to the skin of K14-VEGF-A mice completely inhibited development of chronic skin inflammation, epidermal hyperplasia and abnormal differentiation, and accumulation of CD8 T cells. Similar results were found after Tg delivery of mouse VEGF-D that only activates VEGFR-3 but not VEGFR-2. Moreover, intracutaneous injection of recombinant VEGF-C156S, which only activates VEGFR-3, significantly reduced inflammation. Although lymphatic drainage was inhibited in chronic skin inflammation, it was enhanced by Tg VEGF-C delivery. Together, these results reveal an unanticipated active role of lymphatic vessels in controlling chronic inflammation. Stimulation of functional lymphangiogenesis via VEGFR-3, in addition to antiangiogenic therapy, might therefore serve as a novel strategy to treat chronic inflammatory disorders of the skin and possibly also other organs.