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Background: BC001 is a novel fully human immunoglobulin G1 monoclonal antibody blocking VEGFR2. This phase I study aimed to assess BC001 alone and plus chemotherapy in solid tumors., Methods: In dose escalation part, BC001 was assessed at dose levels of 2, 4, 8, 12, 16 mg/kg on day 1,15, every 28 days, followed an accelerated titration and "3 + 3" design. BC001 plus paclitaxel was assessed at dose level of 8, 10 mg/kg. The primary endpoints included the DLT, MTD and RDE of BC001. In dose expansion part, it aimed to assess the anti-tumor activity of BC001 alone and plus chemotherapy in gastric cancer as 2nd line treatment., Results: Overall, 53 patients were finally enrolled in this study (phase Ia, n = 28; phase Ib, n = 25). In phase Ia part, 1 DLT (grade 4 neutropenia lasting 4 days) was observed in BC001(8 mg/kg) plus paclitaxel cohort. MTD was not reached. All patients experienced TEAEs of any grade. Twenty-four of them suffered ≥ grade 3 TEAEs. leukopenia (n = 33, 62.3%), hemoglobin decreased (n = 32, 60.4%), neutropenia (n = 29, 54.7%) were commonly observed hematological toxicities. The half-life of 140-240 h. And the PK parameters were not largely influenced by combination with paclitaxel. The serum sVEGFR-1, VEGF-A, sVEGFR-2 could not predict the efficacy. Based on the safety, PK and efficacy data, BC001 of 8 mg/kg was determined as RED. Among the GC patients(n = 21) who receiving BC001 plus paclitaxel as 2nd-line treatment in phase 1b part, 6 patients achieved PR and 10 patients experienced SD. The ORR was 28.6% (95% CI 11.3%, 52.2%) and the DCR was 76.2% (95% CI 52.8%, 91.8%), with the median PFS of 5.4 months (95% CI 1.9, 7.0) and OS of 9.4 months (95% CI 5.4, NA). The median DoR was 5.1 months (95% CI 2.6, NA)., Conclusions: BC001 showed acceptable safety profile and preliminary response in both single-agent and combination with chemotherapy cohorts, especially in GC., (© 2024 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)

Recently, the incidence of malignant tumors is on the rise and searching for new treatments on it has become the research priority. Blocking the vascular endothelial growth factor (VEGF) and its receptor (VEGFR) is one of the treatment strategies that used in the development of specific anti-angiogenic drugs. The deficiencies in tissue penetration and affinity maturation become the weakness of these drugs in anti-tumors applications. The single heavy chain antibody found in Chiloscyllium plagiosum, which has a low molecular weight and superior tissue penetration of variable region (variable new antigen receptor, VNARs), was considered to have the high antigen-binding activity and stability. This type of antibody has a simple structure that can be prokaryoticaly expressed, which makes it easily to produce new antiangiogenic target drugs. Specific anti-IgNAR rabbit multiple antibodies have been used to assess the level of VNARs in sharks and have shown a significant enrichment of IgNAR after triple immunization. An anti-VEGFR2 phage library was used for the targeted VNARs screening, and five candidate VNARs sequences were subsequently obtained by phage screening, followed by combined screening with the transcriptome library, and analysis of conserved regions along with 3D modelling matched the VNAR profile. ELISA and cell-based assays showed that two of the VNARs, VNAR-A6, and VNAR-E3, had a superior antigen affinity and anti-angiogenic activity thereby being able to inhibit human Umbilical Vein Endothelial Cells proliferation and migration. The anti-VEGFR2 VNARs derived from the immunized C. plagiosum and screened by phage library, which provide the new research ideas and specific approaches for the development of new drugs. The anti-VEGFR2 VNARs are capable for blocking the VEGF-VEGFR pathway, which of these may contribute to expanding the use of anti-angiogenic drugs., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Immunology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Transforming growth factor β (TGFβ) is present in blood of patients who do not respond to anti-programmed cell death (ligand) 1 [PD-(L)1] treatment, and through synergy with vascular endothelial growth factor (VEGF), it helps to create an environment that promotes tumor immune evasion and immune tolerance. Therefore, simultaneous inhibition of TGFβ/VEGF is more effective than targeting TGFβ alone. In this study, the dual inhibitory mechanism of TU2218 was identified through in vitro analysis mimicking the tumor microenvironment, and its antitumor effects were analyzed using mouse syngeneic tumor models. TU2218 directly restored the activity of damaged cytotoxic T lymphocytes (CTLs) and natural killer cells inhibited by TGFβ and suppressed the activity and viability of regulatory T cells. The inactivation of endothelial cells induced by VEGF stimulation was completely ameliorated by TU2218, an effect not observed with vactosertib, which inhibits only TGFβ signaling. The combination of TU2218 and anti-PD1 therapy had a significantly greater antitumor effect than either drug alone in the poorly immunogenic B16F10 syngeneic tumor model. The mechanism of tumor reduction was confirmed by flow cytometry, which showed upregulated VCAM-1 expression in vascular cells and increased influx of CD8 + CTLs into the tumor. As another strategy, combination of anti-CTLA4 therapy and TU2218 resulted in high complete regression (CR) rates in CT26 and WEHI-164 tumor models. In particular, immunological memory generated by the combination of anti-CTLA4 and TU2218 in the CT26 model prevented the development of tumors after additional tumor cell transplantation, suggesting that the TU2218-based combination has therapeutic potential in immunotherapy., (© 2024. The Author(s).)

Purpose: The humanized antivascular endothelial growth factor (VEGF) antibody bevacizumab (Bev) is efficacious for the treatment of NF2-related schwannomatosis (NF2), previously known as neurofibromatosis type 2. This study evaluated the safety and efficacy of a VEGF receptor (VEGFR) vaccine containing VEGFR1 and VEGFR2 peptides in patients with NF2 with progressive schwannomas (jRCTs031180184)., Materials and Methods: VEGFR1 and VEGFR2 peptides were injected subcutaneously into infra-axillary and inguinal regions, once a week for 4 weeks and then once a month for 4 months. The primary end point was safety. Secondary end points included tolerability, hearing response, imaging response, and immunologic response., Results: Sixteen patients with NF2 with progressive schwannomas completed treatment and were assessed. No severe vaccine-related adverse events occurred. Among the 13 patients with assessable hearing, word recognition score improved in five patients at 6 months and two at 12 months. Progression of average hearing level of pure tone was 0.168 dB/mo during the year of treatment period, whereas long-term progression was 0.364 dB/mo. Among all 16 patients, a partial response was observed in more than one schwannoma in four (including one in which Bev had not been effective), minor response in 5, and stable disease in 4. Both VEGFR1-specific and VEGFR2-specific cytotoxic T lymphocytes (CTLs) were induced in 11 patients. Two years after vaccination, a radiologic response was achieved in nine of 20 assessable schwannomas., Conclusion: This study demonstrated the safety and preliminary efficacy of VEGFR peptide vaccination in patients with NF2. Memory-induced CTLs after VEGFR vaccination may persistently suppress tumor progression.

Obliterative bronchiolitis (OB) after lung transplantation is a nonreversible, life-threatening complication. Herein, the role of vascular endothelial growth factor receptor (Vegfr)-1 and -2 was investigated in the development of obliterative airway disease (OAD), an experimental model for OB. The nonimmunosuppressed recipients underwent transplantation with fully major histocompatibility complex mismatched heterotopic tracheal allografts and received Vegfr1 and -2-specific monoclonal antibodies either alone or in combination, or rat IgG as a control. The treatment with Vegfr1- or -2-blocking antibody significantly decreased intragraft mRNA expression of natural killer cell activation markers early after transplantation. This was followed by reduced infiltration of Cd11b + cells and Cd4 + T cells as well as down-regulated mRNA expression of proinflammatory chemokines and profibrotic growth factors. However, blocking of both Vegfr1 and -2 was necessary to reduce luminal occlusion. Furthermore, concomitant inhibition of the calcineurin activation pathway almost totally abolished the development of OAD. This study proposes that blocking of Vegf receptors blunted natural killer cell and innate immune responses early after transplantation and attenuated the development of OAD. The results of this study suggest that further studies on the role of Vegfr1 and -2 blocking in development of obliterative airway lesions might be rewarding., (Copyright © 2022 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: As the prognosis of biliary tract cancer (BTC) is extremely poor and treatment options are limited, new treatment modalities are urgently needed. We designed a phase II clinical trial to investigate the immune responses and clinical benefits of OCV-C01, an HLA-A*24:02-restricted three-peptide cancer vaccine targeting VEGFR1, VEGFR2, and KIF20A., Patients and Methods: Participants were patients with advanced BTC who had unresectable tumours and were refractory to standard chemotherapy. OCV-C01 was injected weekly until the discontinuance criteria were met., Results: Six participants, including four patients positive for HLA-A*24:02, were enrolled in this study for assessment of efficacy. Four out of six patients exhibited vaccine-specific T-cell responses to one or more of three antigens. Log-rank tests revealed that vaccine-specific T cell responses contributed significantly to overall survival., Conclusion: The cancer vaccine had positive effects on survival, indicating that this approach warrants further clinical studies., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

The fibronectin type III (FN3) monobody domain is a promising non-antibody scaffold, which features a less complex architecture than an antibody while maintaining analogous binding loops. We previously developed FN3Con, a hyperstable monobody derivative with diagnostic and therapeutic potential. Prestabilization of the scaffold mitigates the stability-function trade-off commonly associated with evolving a protein domain toward biological activity. Here, we aimed to examine if the FN3Con monobody could take on antibody-like binding to therapeutic targets, while retaining its extreme stability. We targeted the first of the Adnectin derivative of monobodies to reach clinical trials, which was engineered by directed evolution for binding to the therapeutic target VEGFR2; however, this function was gained at the expense of large losses in thermostability and increased oligomerization. In order to mitigate these losses, we grafted the binding loops from Adnectin-anti-VEGFR2 (CT-322) onto the prestabilized FN3Con scaffold to produce a domain that successfully bound with high affinity to the therapeutic target VEGFR2. This FN3Con-anti-VEGFR2 construct also maintains high thermostability, including remarkable long-term stability, retaining binding activity after 2 years of storage at 36 °C. Further investigations into buffer excipients doubled the presence of monomeric monobody in accelerated stability trials. These data suggest that loop grafting onto a prestabilized scaffold is a viable strategy for the development of monobody domains with desirable biophysical characteristics and that FN3Con is therefore well-suited to applications such as the evolution of multiple paratopes or shelf-stable diagnostics and therapeutics., Competing Interests: Conflict of interest The authors declare no competing financial interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: T cells engineered to express chimeric antigen receptors (CARs) are a novel modality to treat refractory cancers. The development of CAR T cells against Ewing sarcoma (EwS) is limited by a lack of targetable surface antigens. We investigated vascular endothelial growth factor receptor 2 (VEGFR2) expressed on tumor-associated blood vessels as potential CAR target in this cancer., Methods: Expression of VEGFR2 was studied by immunohistochemistry in human EwS biopsies and in murine xenografts and by flow cytometry in EwS cell lines. CARs with short, medium, and long hinge domains against either human or murine VEGFR2 were generated and expressed in human T cells by retroviral gene transfer. The capacity of the individual CARs to activate T cells in response to VEGFR2-expressing cells was compared in vitro., Results: Tumor-associated endothelial cells in human EwS biopsies and in xenografts expressed VEGFR2. Tumor cells in the majority of EwS biopsies were also VEGFR2-positive. Following modification with anti-mouse or anti-human VEGFR2-specific CAR genes, T cells specifically lysed VEGFR2-expressing target cells of the respective species. CAR T cells with short-length or medium-length hinge domains were functionally superior over those with the long hinge region by in vitro parameters, including antigen-specific degranulation responses, lysis of tumor spheroids, tumor necrosis factor α secretion, sequential killing, and proliferation., Conclusions: VEGFR2 is consistently expressed on endothelial cells of the tumor stroma in EwS and thus is a candidate target for CAR T cells in this cancer. Among various VEGFR2-specific CARs, a construct with a short hinge domain was chosen to be further developed toward clinical translation., (© 2020 Wiley Periodicals, Inc.)

The immunosuppressive properties of vascular endothelial growth factors (VEGFs) suggest a new role of angiogenic factors in T cell modulation in cancer and pregnancy. Most of VEGF effects on T cells are mediated through the VEGF receptor type 2 (VEGFR-2). This study aims to investigate the role of placental growth factor (PlGF) as a selective VEGFR-1 ligand in the modulation of human T cells functions. For this, PBMCs from healthy donors were stimulated with anti-CD3 mAbs (a-CD3) or Concanavalin A (ConA) in the absence or presence of PlGF and assessed for T cell proliferation, IL-10 production, programmed cell death, and the expression of inhibitory receptors (PD-1, CTLA-4, TIM-3) using radiometric ( 3 H-thymidine incorporation) and FACS analysis. We showed that most T cells in freshly isolated PBMCs lacked VEGFR-1. However, activation with a-CD3 or ConA strongly increased the percentages of VEGFR-1 expressing CD4 + and CD8 + T cells. PlGF in a wide dose range suppressed PBMC cell proliferation, inhibiting both CD4 + and CD8 + T cells. Blockade of VEGFR-1, but not VEGFR-2 with neutralizing Abs completely abolished the suppressive effect of PlGF. Furthermore, we found that treatment with PlGF up-regulated IL-10 production in CD4 + and CD8 + T cells, promoted CD8 + T cells apoptosis and enhanced the expression of inhibitory receptors (PD-1 and TIM-3) on activated T cells. Our in vitro findings suggest the involvement of PlGF/VEGFR-1 signaling in the modulation of T cell responses in a-CD3-stimulated PBMCs., (©2020 Society for Leukocyte Biology.)

Bisphosphonate-related osteonecrosis of the jaw (ONJ) is a major oral complication; however, its pathogenesis remains unclear. Impairment of osteoclast differentiation by bisphosphonates may be associated with the pathogenesis of ONJ. In our previous study, we reported that the expression of the gene encoding nuclear factor of activated T cells c1 (NFATc1), a known osteoclast differentiation marker, was significantly silenced by zoledronate, a bisphosphonate, in mouse osteoclast precursor cells (mOCPCs) using cDNA microarray. In the present study, the expression value of the NFATc1 gene was regarded as a cut-off and genes whose expression value was significantly decreased compared with that of the NFATc1 gene were extracted in mOCPCs. For validation, CD11b-positive (CD11b+) cells were used, which were purified from human peripheral blood mononuclear cells as human OCPCs. A total of 19 genes were identified; sequential expression analysis revealed that the gene encoding vascular endothelial growth factor receptor 2 (VEGFR2) was frequently silenced by zoledronate in CD11b+ cells. Furthermore, the number of tartrate-resistant acid phosphatase-positive multinucleated cells was decreased by VEGFR2 suppression using a VEGFR2 neutralizing antibody. Zoledronate inhibits human osteoclast differentiation via suppressing VEGFR2 expression. These results suggest that low expression of VEGFR2 in OCPCs may be involved in the pathogenesis of zoledronate-induced ONJ. The understanding of the role of VEGFR2 on bone remodeling is important to elucidate the pathogenesis of bisphosphonate-related ONJ.

Background and Aims: Activation of the antitumor immune response using programmed death receptor-1 (PD-1) blockade showed benefit only in a fraction of patients with hepatocellular carcinoma (HCC). Combining PD-1 blockade with antiangiogenesis has shown promise in substantially increasing the fraction of patients with HCC who respond to treatment, but the mechanism of this interaction is unknown., Approach and Results: We recapitulated these clinical outcomes using orthotopic-grafted or induced-murine models of HCC. Specific blockade of vascular endothelial receptor 2 (VEGFR-2) using a murine antibody significantly delayed primary tumor growth but failed to prolong survival, while anti-PD-1 antibody treatment alone conferred a minor survival advantage in one model. However, dual anti-PD-1/VEGFR-2 therapy significantly inhibited primary tumor growth and doubled survival in both models. Combination therapy reprogrammed the immune microenvironment by increasing cluster of differentiation 8-positive (CD8 + ) cytotoxic T cell infiltration and activation, shifting the M1/M2 ratio of tumor-associated macrophages and reducing T regulatory cell (Treg) and chemokine (C-C motif) receptor 2-positive monocyte infiltration in HCC tissue. In these models, VEGFR-2 was selectively expressed in tumor endothelial cells. Using spheroid cultures of HCC tissue, we found that PD-ligand 1 expression in HCC cells was induced in a paracrine manner upon anti-VEGFR-2 blockade in endothelial cells in part through interferon-gamma expression. Moreover, we found that VEGFR-2 blockade increased PD-1 expression in tumor-infiltrating CD4 + cells. We also found that under anti-PD-1 therapy, CD4 + cells promote normalized vessel formation in the face of antiangiogenic therapy with anti-VEGFR-2 antibody., Conclusions: We show that dual anti-PD-1/VEGFR-2 therapy has a durable vessel fortification effect in HCC and can overcome treatment resistance to either treatment alone and increase overall survival in both anti-PD-1 therapy-resistant and anti-PD-1 therapy-responsive HCC models., (© 2019 by the American Association for the Study of Liver Diseases.)

Hyperprogressive disease (HPD), an unexpected acceleration of tumor growth kinetics, is described in cancer patients treated with anti-PD-1/anti-PD-L1 agents. Here, our aim was to take into consideration the host and explore whether single nucleotide polymorphisms (SNPs) in key genes involved in immune response might predispose to HPD. DNA was extracted from blood-samples from 98 patients treated under CPI monotherapy. Four candidate genes (PD-1, PD-L1, IDO1 and VEGFR2) and 15 potential SNPs were selected. The TGK R (ratio of the slope of tumor growth before treatment and the slope of tumor growth on treatment) was calculated. Hyperprogression was defined as a TGK R ≥2. TGK R calculation was feasible for 80 patients (82%). HPD was observed for 11 patients (14%) and was associated with shorter overall survival (P = 0.003). In univariate analysis, HPD was significantly associated with age ≥70 y (P = 0.025), immune-related toxicity (P = 0.016), VEGFR2 rs1870377 A/T or A/A (P = 0.005), PD-L1 rs2282055 G/T or G/G (P = 0.024) and PD-L1 rs2227981 G/A or A/A (P = 0.024). Multivariate analysis confirmed the correlation between HPD and age ≥70 y (P = 0.006), VEGFR2 rs1870377 A/T or A/A (P = 0.007) and PD-L1 rs2282055 G/T or G/G (P = 0.018). Immunogenetics could become integral predictive factors for CPI-based immunotherapy.

Hypothyroidism is a well-established toxicity of small-molecule anti-vascular endothelial growth factor (VEGF) tyrosine kinase inhibitors. However, its association with anti-VEGF biologics is uncertain. The aim of this study was to investigate the incidence, time course, clinical features, and severity of thyroid dysfunction in patients receiving ramucirumab (an antiangiogenic VEGF receptor 2-binding monoclonal antibody). After retrospectively reviewing electronic medical records from September 2015 to December 2018 at Kyoto-Katsura Hospital, we identified 38 patients who received ramucirumab and had thyroid function testing available to review (case series). We also evaluated the change of thyroid-stimulating hormone (TSH) level during ramucirumab chemotherapy in 16 out of 38 patients who were regularly confirmed TSH (descriptive study). A total of 14 (36.8%) patients developed thyroid dysfunction (TSH >10 mU/L) after ramucirumab chemotherapy. Thyroid autoantibodies were detected in one of the 10 patients (10.0%) who were tested for thyroid autoantibodies. The median time to onset of thyroid dysfunction after ramucirumab initiation was 275 (range, 63-553) days. Levothyroxine replacement was needed in 10 (71.4%) patients. Sixteen patients had thyroid function regularly monitored; the mean TSH level was significantly increased after ramucirumab chemotherapy compared with that at baseline (10.7 ± 10.0 mU/L vs. 4.1 ± 2.8 mU/L; p < 0.01). Our findings indicate that ramucirumab can result in thyroid dysfunction. We propose that thyroid function testing should be performed regularly to detect hypothyroidism and guide its management in patients receiving ramucirumab chemotherapy.

The anti-VEGF antibody bevacizumab has shown efficacy for the treatment of neurofibromatosis type 2 (NF2). Theoretically, vascular endothelial growth factor receptors (VEGFRs)-specific cytotoxic T lymphocytes (CTLs) can kill both tumor vessel cells and tumor cells expressing VEGFRs. Here we show an exploratory clinical study of VEGFRs peptide vaccine in seven patients with progressive NF2-derived schwannomas. Hearing improves in 2/5 assessable patients (40%) as determined by international guidelines, with increases in word recognition scores. Tumor volume reductions of ≥20% are observed in two patients, including one in which bevacizumab had not been effective. There are no severe adverse events related to the vaccine. Both VEGFR1-specific and VEGFR2-specific CTLs are induced in six patients. Surgery is performed after vaccination in two patients, and significant reductions in the expression of VEGFRs in schwannomas are observed. Therefore, this clinical immunotherapy study demonstrates the safety and preliminary efficacy of VEGFRs peptide vaccination in patients with NF2.

Objective: To improve the production yield of N-glycosylated anti-VEGFR2 (vascular endothelial growth factor receptor 2) monobody (FN3 VEGFR2 -Gly) in lpp knockout Escherichia coli cells harboring Campylobacter jejuni N-glycosylation pathway., Results: The leaky CLM37-Δlpp strain efficiently secreted FN3 VEGFR2 -Gly into culture medium. The extracellular levels of glycosylated FN3 VEGFR2 -Gly in CLM37-Δlpp culture medium were approximately 11 and 15 times higher compared to those in CLM37 cells via IPTG and auto-induction, respectively. In addition, the highest level of total glycosylated FN3 VEGFR2 -Gly (70 ± 3.4 mg/L) was found in culture medium via auto-induction. Furthermore, glycosylated FN3 VEGFR2 -Gly was more stable than unglycosylated FN3 VEGFR2 -Gly in this expression system, but their bioactivities were relatively similar., Conclusions: Lpp knockout leaky E. coli strain combined with auto-induction method can enhance the extracellular production of homogenous N-glycosylated FN3 VEGFR2 -Gly, and facilitate the downstream protein purification. The findings of this study may provide practical implications for the large-scale production and cost-effective harvesting of N-glycosylation proteins.

When targeted by the tumor-promoting enzyme heparanase, cleaved and shed syndecan-1 (Sdc1) then couples VEGFR2 (also known as KDR) to VLA-4, activating VEGFR2 and the directed migration of myeloma cells. But how VEGFR2 activates VLA-4-mediated motility has remained unknown. We now report that VEGFR2 causes PKA-mediated phosphorylation of VLA-4 on S988, an event known to stimulate tumor metastasis while suppressing cytotoxic immune cells. A key partner in this mechanism is the chemokine receptor CXCR4, a well-known mediator of cell motility in response to gradients of the chemokine SDF-1 (also known as CXCL12). The entire machinery necessary to phosphorylate VLA-4, consisting of CXCR4, AC7 (also known as ADCY7) and PKA, is constitutively associated with VEGFR2 and is localized to the integrin by Sdc1. VEGFR2 carries out the novel phosphorylation of Y135 within the DRY microswitch of CXCR4, sequentially activating Gα i βγ, AC7 and PKA, which phosphorylates S988 on the integrin. This mechanism is blocked by a syndecan-mimetic peptide (SSTN VEGFR2 ), which, by preventing VEGFR2 linkage to VLA-4, arrests tumor cell migration that depends on VLA-4 phosphorylation and stimulates the LFA-1-mediated migration of cytotoxic leukocytes., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Solid tumors that are responsible for more than 85% of cancer death cases need angiogenesis for their growth and metastasis. Among antiangiogenic therapies, targeting the vascular endothelial growth factor receptor 2 (VEGFR2) that is over-expressed on tumor vasculatures has been a promising strategy. In this study, we developed a second generation nanobody (VHH)-based CAR T cell targeting VEGFR2-expressing tumor cells. The CAR T cell was developed by linking the anti-VEGFR2 VHH to a spacer, and signaling domains of CD28 and CD3 ζ. The T cells were activated with anti-CD3 plus rIL-2 and electroporated with a plasmid encoding the CAR construct. The expression of activation markers, CD69 and CD25, on CAR T cells upon coculturing with VEGFR2-expressing cells were 41% and 48%, and the IL-2 and IFN-γ production were 470 pg/mL and 360 pg/mL, respectively. The expression of degranulation marker, CD107a, was 30% and the cytotoxic activity of the CAR T cells reached to more than 30% with E:T ratio of 9:1. The anti-VEGFR2 CAR but not mock T cells mediated specific lysis of 293-KDR cells expressing human VEGFR2 and might be considered as a candidate for adoptive T-cell immunotherapy of solid tumors. © 2019 IUBMB Life, 71(9):1259-1267, 2019., (© 2019 International Union of Biochemistry and Molecular Biology.)

MHC class I-related chain A (MICA) is one of the major ligands for natural killer group 2 member D (NKG2D), which is an activating NK receptor. MICA is expressed on the surface of human epithelial tumor cells, and its shedding from tumor cells leads to immunosuppression. To activate immune response in the tumor microenvironment, we designed an anti-VEGFR2-MICA bispecific antibody (JZC01), consisting of MICA and an anti-VEGFR2 single chain antibody fragment (JZC00) and explored its potential anti-tumor activity. JZC01 targeted vascular endothelial growth factor receptor 2 (VEGFR2) and inhibited tumorigenesis by blocking the VEGFR2 signaling pathway. Additionally, JZC01 promoted NK and CD8 + T cells to release IFN-γ and engaged activated lymphocytes to lysis of VEGFR2-expressing tumor cells. The in vivo anti-tumor activity of JZC01 was investigated by establishing a Lewis lung cancer cell-transplanted mouse model. It effectively reduced the tumor vascular density and increased the infiltration and activation of NK and CD8 + T cells in the tumor microenvironment. Thus, JZC01 functions in anti-tumor angiogenesis and anti-tumor immune activation, and showed improved anti-tumor efficacy combined with docetaxel, which provides a new insight into anti-tumor therapy.

Gastric cancer is an important cancer type worldwide, the anti‑angiogenic agent BC001 can target the vascular endothelial growth factor receptor 2 (VEGFR2), and significantly suppresses the growth of gastric cancer BGC823 cells in vitro and in vivo. However, numerous results indicated that antiangiogenic drugs could induce autophagy, and the inhibition of autophagy enhanced the anticancer effects of antiangiogenic agents. In the present study, hydroxychloroquine (HCQ), an inhibitor of autophagy, enhanced the antiproliferative and proapoptotic effects of BC001 in vitro. Furthermore, HCQ enhanced the antitumor effects of BC001 on BGC823 xenograft tumors in vivo. Of note, BC001 neither induced nor inhibited autophagy. RNA‑sequencing results revealed that HCQ regulated autophagy or lysosomal‑associated genes, such as tumor protein p53‑inducible nuclear protein 1, interleukin (IL)1B, tumor necrosis factor (TNF), Mediterranean fever, ubiquitin specific peptidase 36, IL6, neuraminidase (NEU)1, ATP‑binding cassette subfamily A member 1, proprotein convertase subtilisin/kexin type 9, myelin basic protein and NEU3. Importantly, HCQ was determined to affect multiple pathways, including 'negative regulation of endothelial cell proliferation', 'blood vessel remodeling', 'cell surface receptor signaling pathways' and 'notch receptor processing' associated with 'signal transduction', 'cancers' and 'immune system', through regulating C‑X‑C motif chemokine ligand 8, TNF, IL6, intercellular adhesion molecule 1 and FOS genes. In summary, HCQ was proposed to enhance the anticancer effects of BC001 in gastric cancer via complex mechanisms.

Dendritic cells (DCs) are potent and specialized antigen presenting cells, which play a crucial role in initiating and amplifying both the innate and adaptive immune responses against cancer. Tumor cells can escape from immune attack by secreting suppressive cytokines that solely or cooperatively impair the immune function of DCs. However, the underlying mechanisms are not fully defined. Vascular endothelial growth factor (VEGF) has been identified as a major cytokine in the tumor microenvironment. To elucidate the effects of VEGF on the motility and immune function of mature DCs (mDCs), the cells were treated with 50 ng/mL VEGF and investigated by proteomics and molecular biological technologies. The results showed that VEGF can impair the migration capacity and immune function of mDCs through the RhoA-cofilin1 pathway mediated by the VEGF receptor 2, suggesting impaired motility of mDCs by VEGF is one of the aspects of immune escape mechanisms of tumors. It is clinically important to understand the biological behavior of DCs and the immune escape mechanisms of tumor as well as how to improve the efficiency of antitumor therapy based on DCs., (© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

The stimulator of interferon genes (STING) signaling pathway is a critical link between innate and adaptive immunity, and induces anti-tumor immune responses. STING is expressed in vasculatures, but its role in tumor angiogenesis has not been elucidated. Here we investigated STING-induced tumor vascular remodeling and the potential of STING-based combination immunotherapy. Endothelial STING expression was correlated with enhanced T-cell infiltration and prolonged survival in human colon and breast cancer. Intratumoral STING activation with STING agonists (cGAMP or RR-CDA) normalized tumor vasculatures in implanted and spontaneous cancers, but not in STING-deficient mice. These were mediated by upregulation of type I/II interferon genes and vascular stabilizing genes (e.g., Angpt1, Pdgfrb, and Col4a). STING in non-hematopoietic cells is as important as STING in hematopoietic cells to induce a maximal therapeutic efficacy of exogenous STING agonist. Vascular normalizing effects of STING agonists were dependent on type I interferon signaling and CD8+ T cells. Notably, STING-based immunotherapy was maximally effective when combined with VEGFR2 blockade and/or immune checkpoint blockade (αPD-1 or αCTLA-4), leading to complete regression of immunotherapy-resistant tumors. Our data show that intratumoral STING activation can normalize tumor vasculature and the tumor microenvironment, providing a rationale for combining STING-based immunotherapy and anti-angiogenic therapy.

VEGF-A/VEGFR2 complex is the major signaling pathway involved in angiogenesis and the inhibition of this axis retards tumor growth and inflammatory disorders progression, reducing vessel sprouting. Signaling by VEGFR2 requires receptor dimerization and a well-defined orientation of monomers in the active dimer. The extracellular portion of receptor is composed of seven Ig-like domains, of which D2-3 are the ligand binding domains, while D4 and D7, establishing homotypic contacts, allosterically regulate receptor activity. The allosteric targeting of VEGFR2 represents a promising alternative to study neovascular disorders overcoming drawbacks related to competition with VEGF. In this work, we expressed in bacterial host domain 4 of VEGFR2 (VEGFR2D4). After protein refolding, we characterized the purified domain and administered it in mice for monoclonal antibodies production. One of them, mAbD4, was tested in ELISA assays, showing a nanomolar affinity for VEGFR2D4. Finally, the methodology here described could contribute to the development of antibodies which can allosterically bind VEGFR2 and therefore to be used for imaging purposes or to modulate receptor signaling.

Clinical and biomedical research seeks single-cell quantification to better understand their roles in a complex, multicell environment. Recently, quantification of vascular endothelial growth factor receptors (VEGFRs) provided important insights into endothelial cell characteristics and response in tumor microenvironments. However, existing technologies for quantifying plasma membrane receptor tyrosine kinases (RTKs) lack multiplexing capabilities, limiting detailed characterization. Here, we use the unique spectral properties of quantum dots (Qdots) to optimize and dually quantify VEGFR1 and VEGFR2 on human umbilical vein endothelial cells (HUVECs). To enable this quantification, we reduce nonspecific binding between Qdot-conjugated antibodies and cells via buffer optimization. Second, we identify optimal labeling conditions by examining Qdot-conjugated antibody binding to five receptors: VEGFRs (VEGFR1 and VEGFR2), their coreceptor neuropilin1 (NRP1), and platelet-derived growth factor receptor (PDGFRα and PDGFRβ). We establish that 800-20 000 is the dynamic range where accurate Qdot-enabled quantification can be achieved. Through these optimizations, we demonstrate measurement of 1 100 VEGFR1 and 6 900 VEGFR2 per HUVEC. We induce ∼90% upregulation of VEGFR1 and ∼30% downregulation of VEGFR2 concentration via 24 h VEGF-A 165 treatment. We observe no change in VEGFR1 or VEGFR2 concentration with 24 h VEGF-B 167 treatment. We further apply Qdots to analyze HUVEC heterogeneity and observe that 24 h VEGF-A 165 treatment induces a ∼15% decrease in VEGFR2 heterogeneity, but little to no change in VEGFR1 heterogeneity. We observe that VEGF-B 167 induces little to no change in either VEGFR1 or VEGFR2 heterogeneity. Overall, we demonstrate experimental and analytical strategies for quantifying two or more RTKs at single-level using Qdots, which will help provide new insights into biological systems.

High intensity focused ultrasound (HIFU) is a noninvasive thermal ablation technique for the treatment of benign and malignant solid masses. To improve the efficacy of HIFU ablation, we developed poly (lactide-co-glycolide) (PLGA) nanoparticles encapsulating perfluoropentane (PFP) and hematoporphyrin monomethyl ether (HMME) as synergistic agents (HMME+PFP/PLGA). Two-step biotin-avidin pre-targeting technique was applied for the HIFU ablation. We further modified the nanoparticles with streptavidin (HMME+PFP/PLGA-SA). HMME+PFP/PLGA-SA were highly dispersed with spherical morphology (477.8 ± 81.8 nm in diameter). The encapsulation efficiency of HMME and PFP were 46.6 ± 3.3% and 40.1 ± 2.6%, respectively. The binding efficiency of nanoparticles to streptavidin was 95.5 ± 2.5%. The targeting ability of the HMME+PFP/PLGA-SA nanoparticles was tested by parallel plate flow chamber in vitro. In the pre-targeting group (HMME+PFP/PLGA-SA), a large number of nanoparticles bound to the peripheral and surface of the cell. In the HIFU ablation experiment in vivo, compared with the other groups, the largest gray-scale changes and coagulation necrosis areas were observed in the pre-targeting (HMME+PFP/PLGA-SA) group, with the lowest energy efficiency factor value. Moreover, the microvessel density and proliferation index declined, while the apoptotic index increased, in the tumor tissue surrounding the coagulation necrosis area in the pre-targeting group. Meanwhile, the survival time of the tumor-bearing nude mice in the pre-targeting group was significantly longer than that in the HIFU treatment group. These results suggest that HMME+PFP/PLGA-SA have high potential to act as synergistic agents in HIFU ablation.

Introduction: Colorectal cancer (CRC) is one of the main causes of cancer-related morbidity and mortality worldwide. Mortality is most often attributable to metastatic disease. Despite the progress achieved so far, life expectancy continues to be limited in most patients. Ramucirumab, a most recent antiangiogenic drug, is vying in the race to metastatic CRC (mCRC) treatment since its approval by the Food and Drug Administration (FDA), based on the results of the RAISE study., Areas Covered: This article reviews the role of ramucirumab in mCRC, including clinical indication, safety issues, and future perspectives., Expert Opinion: The use of Ramucirumab in clinical practice is still limited, probably due to economic burden and the lack of specific biomarkers. Future efforts will be addressed to improve our knowledge in the use of this drug and better guide us in patients' care.

Background: Even with drug-eluting stents, the risk of in-stent restenosis (ISR) remains high. The goal of this study was to investigate the use of an endothelial progenitor cell (EPC) capture stent plus regional EPC transplantation to reduce the ISR rate., Methods: Endothelial progenitor cell capture stents were fabricated using fibrin gel and anti-CD34 plus anti-VEGFR-2 dual antibodies. Twenty male New Zealand white rabbits established as an atherosclerotic model were randomly divided into two groups: group 1 (n = 10), in which EPC capture stents were deployed into the right iliac artery; and group 2 (n = 10), in which sirolimus-eluting stents were placed. In both groups, EPCs were transplanted into target vessels beyond the stents, with outflow blocked. Radiologic-pathologic correlation outcomes were reviewed after 2 months., Results: The technical success rate of EPC capture stent placement plus EPC transplantation was 100%. The ISR rate in group 1 was lower than in group 2 (1/10 vs. 4/10; p > 0.05). Minimal luminal diameters were larger in group 1 than in group 2 (computed tomographic angiography, 1.85 ± 0.15 mm vs. 1.50 ± 0.20 mm; duplex ultrasound, 1.90 ± 0.10 mm vs. 1.70 ± 0.30 mm; p > 0.05). Transplanted EPCs were tracked positively only in group 1. Pathologic analysis demonstrated neointimal hyperplasia thickness of 0.21 ± 0.09 mm in group 1 vs. 0.11 ± 0.07 mm in group 2 (p < 0.05)., Conclusion: Endothelial progenitor cell capture stent placement plus local EPC transplant decreases the ISR rate through thrombosis reduction rather than through neointimal hyperplasia inhibition.

Purpose: Tumor vessels influence the growth and response of tumors to therapy. Imaging vascular changes in vivo using dynamic contrast-enhanced MRI (DCE-MRI) has shown potential to guide clinical decision making for treatment. However, quantitative MR imaging biomarkers of vascular function have not been widely adopted, partly because their relationship to structural changes in vessels remains unclear. We aimed to elucidate the relationships between vessel function and morphology in vivo Experimental Design: Untreated preclinical tumors with different levels of vascularization were imaged sequentially using DCE-MRI and CT. Relationships between functional parameters from MR ( i AUC, K trans , and BAT frac ) and structural parameters from CT (vessel volume, radius, and tortuosity) were assessed using linear models. Tumors treated with anti-VEGFR2 antibody were then imaged to determine whether antiangiogenic therapy altered these relationships. Finally, functional-structural relationships were measured in 10 patients with liver metastases from colorectal cancer. Results: Functional parameters i AUC and K trans primarily reflected vessel volume in untreated preclinical tumors. The relationships varied spatially and with tumor vascularity, and were altered by antiangiogenic treatment. In human liver metastases, all three structural parameters were linearly correlated with i AUC and K trans For i AUC, structural parameters also modified each other's effect. Conclusions: Our findings suggest that MR imaging biomarkers of vascular function are linked to structural changes in tumor vessels and that antiangiogenic therapy can affect this link. Our work also demonstrates the feasibility of three-dimensional functional-structural validation of MR biomarkers in vivo to improve their biological interpretation and clinical utility. Clin Cancer Res; 24(19); 4694-704. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Increased interest and insights gained by researchers on the roles of endothelial cells in the pathophysiology of cancer, inflammatory, and cardiovascular diseases have led to the design of pharmacological interventions aimed at the endothelium lining in the diseased sites. Toward this end, we used established brain microvascular endothelial cell lines mouse (bEND3), human (hCMEC/D3), and Toggle Cell-SELEX to identify a species cross-reactive, endothelial cell-internalizing aptamer R11-3. This 2'F-modified RNA aptamer is specific for endothelial cells as no internalization was seen with cells of nonendothelial origin. R11-3 was truncated in size, and its potential in endothelial targeted therapeutics was established using VEGFR2 targeting long interfering RNA (liRNA) aptamer chimera. Due to its specificity for both mouse and human endothelial cells, we believe that this aptamer not only fits for development of endothelial targeted drug development for human diseases but is also suitable for preclinical evaluation in mice.

Key Message: This research has shown, for the first time, that plant chloroplasts are a suitable compartment for synthesizing recombinant immunotoxins and the transgenic immunotoxin efficiently causes the inhibition of VEGFR2 overexpression, cell growth and proliferation. Angiogenesis refers to the formation of new blood vessels, which resulted in the growth, invasion and metastasis of cancer. The vascular endothelial growth factor receptor 2 (VEGFR2) plays a major role in angiogenesis and blocking of its signaling inhibits neovascularization and tumor metastasis. Immunotoxins are promising therapeutics for targeted cancer therapy. They consist of an antibody linked to a protein toxin and are designed to specifically kill the tumor cells. In our previous study, VGRNb-PE immunotoxin protein containing anti-VEGFR2 nanobody fused to the truncated form of Pseudomonas exotoxin A has been established. Here, we expressed this immunotoxin in lettuce chloroplasts. Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, multigene engineering in a single transformation event and maternal inheritance of the transgenes. Site specific integration of transgene into chloroplast genomes, and homoplasmy were confirmed. Immunotoxin levels reached up to 1.1% of total soluble protein or 33.7 µg per 100 mg of leaf tissue (fresh weight). We demonstrated that transgenic immunotoxin efficiently causes the inhibition of VEGFR2 overexpression, cell growth and proliferation. These results indicate that plant chloroplasts are a suitable compartment for synthesizing recombinant immunotoxins.

Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2) is the main mediator of angiogenic signaling in endothelial cells and a primary responder to VEGF. VEGF dependent VEGFR-2 activation regulates endothelial cell migration and proliferation, as well as vessel permeability. VEGF is presented as an antiparallel homodimer, and its binding to VEGFR-2 brings two receptors in close proximity. Downstream signaling is triggered by receptor dimerization, kinase activation, and receptor internalization. Our aim was to further investigate allosteric inhibition using binders targeting extracellular subdomains 4⁻7 of VEGFR-2 as an alternative to existing anti-angiogenic therapies, which rely on neutralizing VEGF or blocking of the ligand-binding site on the receptor. We applied phage display technology to produce single chain antibody fragments (scFvs) targeting VEGFR-2. Selected antibody fragments were characterized using biophysical and biological assays. We characterized several antibody fragments, which exert their inhibitory effect of VEGFR-2 independent of ligand binding. These reagents led to rapid clearance of VEGFR-2 from the cell surface without kinase activation, followed by an increase in intracellular receptor-positive vesicles, suggesting receptor internalization. Our highly specific VEGFR-2 binders thus represent novel tools for anti-angiogenic therapy and diagnostic applications.

Cancer vaccines have been developed as a new therapeutic approach, however, their clinical benefit remains limited. We previously performed a phase II study for advanced colorectal cancer (CRC) using five human leukocyte antigen (HLA-A*24:02)-restricted peptides derived from kinase of the outer chloroplast membrane 1, translocase of outer mitochondrial membrane 34 (TOMM34), ring finger protein 43 (RNF43), vascular endothelial growth factor receptor 1 (VEGFR1) and VEGFR2. In the present study the relationship between overall survival (OS) and several biomarkers, including cytotoxic T lymphocyte (CTL) and immunoglobulin G (IgG) responses to these five peptides, was investigated. In 89 advanced CRC patients treated with a combination therapy consisting of these five peptides and oxaliplatin-based chemotherapy, plasma was collected before and after 3 months of vaccine administration. IgGs reactive to each of the five peptides were assessed using the multiplex bead suspension Luminex system. Antigen-specific T-cell responses were estimated by enzyme-linked immunoSpot assay. Plasma levels of TOMM34 IgG (P<0.001), RNF43 IgG (P<0.001) and VEGFR2 IgG (P<0.001) were significantly increased after vaccination and stronger VEGFR2 IgG responses correlated significantly with OS in HLA-matched patients (P=0.034). CTL responses to VEGFR1 and VEGFR2 were also significantly increased in the HLA-matched group (P=0.049 and P<0.001, respectively). However, increased CTL response did not correlate with OS. Multivariate analysis indicated that IgG responses to VEGFR2 were the most significant predictor for OS in the HLA-A*24:02-matched group (P=0.04). Our findings indicated that VEGFR2 IgG responses may be an important immunological biomarker in the early course of treatment for CRC patients treated with therapeutic epitope peptides.

Background Tanibirumab is a fully human monoclonal antibody to vascular endothelial growth factor receptor 2 (VEGFR-2). We conducted a first-in-human phase I study of tanibirumab in patients with solid tumors refractory to standard chemotherapy. Primary endpoints were evaluating safety, pharmacokinetics (PKs), estimating maximum-tolerated dose (MTD) and recommended phase II dose (RP2D). Methods We designed our study to escalate tanibirumab at 9 different dose levels with a 3 + 3 method and tanibirumab (1-28 mg/kg) was administered intravenously on D1, 8, 15 in 28-day courses. Dose limiting toxicities (DLTs) were only assessed during the first cycle of treatment and response evaluation was performed every 2 cycles. The effects of tanibirumab on several angiogenic factors were analyzed. Results From October 2011 to September 2013, a total of 26 patients with refractory solid tumors were enrolled. The median age was 58 years (range, 27-75) and 20 patients were male. The most common tumor type was colorectal cancer (N = 19) and seven patients had a history of previous bevacizumab treatment. As hemangioma continued to occur, the final dose level, 28 mg/kg, was not performed. DLTs were not found, and the MTD was confirmed to be 24 mg/kg. Hemangioma was observed in 16 patients (61.5%), but all were grade 1-2 and disappeared after discontinuation of the study drug. Among the 18 patients in the efficacy set, no objective response was observed, but 11 patients showed stable disease. PKs were characterized by dose-dependent linear exposure and the mean trough concentrations exceeded biologically relevant target levels at 12 mg/kg and above. Serum VEGF, soluble VEGFR-2, and PlGF increased at the 4 mg/kg dose level and above. Conclusions Treatment with tanibirumab showed a tolerable toxicity profile and modest clinical efficacy in patients with refractory solid tumors. A phase II trial of tanibirumab is ongoing now.

Background: Ramucirumab, the human immunoglobulin G1 monoclonal antibody receptor antagonist of vascular endothelial growth factor receptor 2, has been approved for treating gastric/gastroesophageal junction, non-small-cell lung, and metastatic colorectal cancers. With the completion of six global, randomized, double-blind, placebo-controlled, phase III trials across multiple tumor types, an opportunity now exists to further establish the safety parameters of ramucirumab across a large patient population., Materials and Methods: An individual patient meta-analysis across the six completed phase III trials was conducted and the relative risk (RR) and associated 95% confidence intervals (CIs) were derived using fixed-effects or mixed-effects models for all-grade and high-grade adverse events (AEs) possibly related to vascular endothelial growth factor pathway inhibition. The number needed to harm was also calculable due to the placebo-controlled nature of all six registration standard trials., Results: A total of 4996 treated patients (N = 2748 in the ramucirumab arm and N = 2248 in the control, placebo arm) were included in this meta-analysis. Arterial thromboembolic events [ATE; all-grade, RR: 0.8, 95% CI 0.5-1.3; high-grade (grade ≥3), RR: 0.9, 95% CI 0.5-1.7], venous thromboembolic events (VTE; all-grade, RR: 0.7, 95% CI 0.5-1.1; high-grade, RR: 0.7, 95% CI 0.4-1.2), high-grade bleeding (RR: 1.1, 95% CI 0.8-1.5), and high-grade gastrointestinal (GI) bleeding (RR: 1.1, 95% CI 0.7-1.7) did not demonstrate a definite increased risk with ramucirumab. A higher percentage of hypertension, proteinuria, low-grade (grade 1-2) bleeding, GI perforation, infusion-related reaction, and wound-healing complications were observed in the ramucirumab arm compared with the control arm., Conclusions: Ramucirumab may be distinct among antiangiogenic agents in terms of ATE, VTE, high-grade bleeding, or high-grade GI bleeding by showing no clear evidence for an increased risk of these AEs in this meta-analysis of a large and diverse patient population. Ramucirumab is consistent with other angiogenic inhibitors in the risk of developing certain AEs. Clinical Trial Numbers: NCT00917384 (REGARD), NCT01170663 (RAINBOW), NCT01168973 (REVEL), NCT01183780 (RAISE), NCT01140347 (REACH), and NCT00703326 (ROSE)., (© The Author 2017. Published by Oxford University Press on behalf of the European Society for Medical Oncology.)

In this study, a xenogeneic DNA vaccine encoding for human vascular endothelial growth factor receptor-2 (hVEGFR-2) was evaluated in two murine tumor models, the B16-F10 melanoma and the EO771 breast carcinoma model. The vaccine was administered by intradermal injection followed by electroporation. The immunogenicity and the biological efficacy of the vaccine were tested in (1) a prophylactic setting, (2) a therapeutic setting, and (3) a therapeutic setting combined with surgical removal of the primary tumor. The tumor growth, survival, and development of an immune response were followed. The cellular immune response was measured by a bioluminescence-based cytotoxicity assay with vascular endothelial growth factor-2 (VEGFR-2)-expressing target cells. Humoral immune responses were quantified by enzyme-linked immunosorbent assay (ELISA). Ex vivo bioluminescence imaging and immunohistological observation of organs were used to detect (micro)metastases. A cellular and humoral immune response was present in prophylactically and therapeutically vaccinated mice, in both tumor models. Nevertheless, survival in prophylactically vaccinated mice was only moderately increased, and no beneficial effect on survival in therapeutically vaccinated mice could be demonstrated. An influx of CD3+ cells and a slight decrease in VEGFR-2 were noticed in the tumors of vaccinated mice. Unexpectedly, the vaccine caused an increased quantity of early micrometastases in the liver. Lung metastases were not increased by the vaccine. These early liver micrometastases did however not grow into macroscopic metastases in either control or vaccinated mice when allowed to develop further after surgical removal of the primary tumor.

Malaria is a severe disease and kills over 400,000 people each year. Malarial complications are the main cause of death and include cerebral malaria and malaria-associated acute respiratory distress syndrome (MA-ARDS). Despite antimalarial treatment, lethality rates of MA-ARDS are still between 20 and 80%. Patients develop pulmonary edema with hemorrhages and leukocyte extravasation in the lungs. The vascular endothelial growth factor-A (VEGF-A) and the placental growth factor (PlGF) are vascular permeability factors and may be involved in the disruption of the alveolar-capillary membrane, leading to alveolar edema. We demonstrated increased pulmonary VEGF-A and PlGF levels in lungs of mice with experimental MA-ARDS. Depletion of pathogenic CD8 + T cells blocked pulmonary edema and abolished the increase of VEGF-A and PlGF. However, neutralization of VEGF receptor-2 (VEGFR-2) with the monoclonal antibody clone DC101 did not decrease pulmonary pathology. The broader spectrum receptor tyrosine kinase inhibitor sunitinib even increased lung pathology. These data suggest that the increase in alveolar VEGF-A and PlGF is not a cause but rather a consequence of the pulmonary pathology in experimental MA-ARDS and that therapeutic inhibition of VEGF receptors is not effective and even contra-indicated.

Hemangioma is a common benign tumor affecting infants. In this study, we prepared sodium morrhuate immunoliposomes through encapsulation of sodium morrhuate with liposomes coupled with an anti-VEGFR2/KDR antibody and examined its effect on the biology of human hemangioma endothelial cells (HECs). It was found that compared to the liposomal sodium morrhuate group, treatment with sodium morrhuate immunoliposomes facilitated cell detachment and apoptotic death. Confocal microscopy analysis revealed that sodium morrhuate immunoliposomes had a higher binding activity to HECs than liposomal sodium morrhuate. Apoptosis analysis further demonstrated that treatment with liposomal sodium morrhuate or sodium morrhuate immunoliposomes significantly induced apoptosis in HECs, compared to the control group. Western blot analysis revealed an induction of caspase-3 and caspase-9 levels and reduction of caspase-8 and Bcl-2 levels in HECs treated with liposomal sodium morrhuate or sodium morrhuate immunoliposomes. Taken together, these results indicate that sodium morrhuate immunoliposomes have an increased capacity to target HECs and promote mitochondrial apoptosis. Therefore, sodium morrhuate immunoliposomes may represent a promising agent in the treatment of hemangiomas., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Microbubbles (MB) are routinely used as contrast agents for ultrasound (US) imaging. We describe different types of targeted and drug-loaded poly(n-butyl cyanoacrylate) (PBCA) MB, and demonstrate their suitability for multiple biomedical applications, including molecular US imaging and US-mediated drug delivery. Molecular imaging of angiogenic tumor blood vessels and inflamed atherosclerotic endothelium is performed by modifying the surface of PBCA MB with peptides and antibodies recognizing E-selectin and VCAM-1. Stable and inertial cavitation of PBCA MB enables sonoporation and permeabilization of blood vessels in tumors and in the brain, which can be employed for direct and indirect drug delivery. Direct drug delivery is based on US-induced release of (model) drug molecules from the MB shell. Indirect drug delivery refers to US- and MB-mediated enhancement of extravasation and penetration of co-administered drugs and drug delivery systems. These findings are in line with recently reported pioneering proof-of-principle studies showing the usefulness of (phospholipid) MB for molecular US imaging and sonoporation-enhanced drug delivery in patients. They aim to exemplify the potential and the broad applicability of combining MB with US to improve disease diagnosis and therapy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Elevated vascular endothelial growth factor (VEGF) and complement activation are implicated in the pathogenesis of different ocular diseases. The objective of this study was to investigate the hypothesis that dual inhibition of both VEGF and complement activation would confer better protection against ocular inflammation and neovascularization. In this study, we engineered a secreted chimeric VEGF inhibitor domain (VID), a complement inhibitor domain (CID) and a dual inhibitor (ACVP1). Vectors expressing these three inhibitors were constructed and packaged into AAV2 (sextY-F) particles. The expression and secretion of the proteins were validated by Western blot. The effects of these inhibitors expressed from AAV2 vectors were examined in endotoxin-induced uveitis (EIU), experimental autoimmune uveoretinitis (EAU) and choroidal neovascularization (CNV) mouse models. The AAV2 vectors expressing the CID- and ACVP1-attenuated inflammation in EIU and EAU model, whereas the vector expressing VID showed improved retinal structure damaged by EAU, but not affect the infiltration of inflammatory cells in EAU or EIU eyes. Both VID and CID vectors improved laser-induced retinal and choroid/RPE injuries and CNV, whereas ACVP1 vector provided significantly better protection. Our results suggest that gene therapy targeting VEGF and complement components could provide an innovative and long-term strategy for ocular inflammatory and neovascular diseases., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

We investigated peptide cocktail vaccine OCV-C01 containing epitope peptides derived from KIF20A, vascular endothelial growth factor receptor (VEGFR)1 and VEGFR2 combined with gemcitabine in the adjuvant treatment for resected pancreatic cancer patients. A single-arm multicenter phase II study was performed on 30 patients with pancreatic ductal carcinoma who underwent pancreatectomy. At each 28-day treatment cycle, patients received weekly subcutaneous injection of OCV-C01 for 48 weeks and gemcitabine was administered intravenously at 1,000 mg/m 2 on days 1, 8 and 15 for 24 weeks. Patients were followed for 18 months. The primary endpoint was disease-free survival (DFS) and secondary endpoints included safety, overall survival (OS) and immunological assays on peptide-specific cytotoxic T lymphocyte (CTL) activity and KIF20A expression in resected pancreatic cancer. The median DFS was 15.8 months [95% confidence interval (CI), 11.1-20.6] and the DFS rate at 18 months was 34.6% (95% CI, 18.3-51.6). The median OS was not reached and the OS rate at 18 months was 69.0% (95% CI, 48.8-82.5). The administration of OCV-C01 was well tolerated. In the per protocol set, there were significant differences in DFS between patients with KIF20A-specific CTL responses and without (p = 0.027), and between patients with KIF20A expression and without (p = 0.014). In addition, all four patients who underwent R0 resection with KIF20A expression had no recurrence of pancreatic cancer with KIF20A-specific CTL responses. OCV-C01 combined with gemcitabine was tolerable with a median DFS of 15.8 months, which was favorable compared with previous data for resected pancreatic cancer., (© 2016 UICC.)

Objectives: To investigate the effect of pazopanib on different CTCs subpopulations in patients with recurrent SCLC and evaluate their clinical relevance., Methods: Peripheral blood was obtained before the administration of pazopanib (n=56 patients), after the first cycle (n=35 patients) and at disease progression (n=45 patients). CTCs were detected by CellSearch and double immunofluorescent staining using antibodies against the cytokeratins (CK), TTF-1, CD56 and VEGFR2., Results: Before treatment, CTCs could be detected in 50% of patients by CellSearch; phenotypic characterization of CTCs demonstrated that 50%, 46.6% and 27.6% of patients had CD45 - /TTF1 + , CD45 - /CD56 + and TTF-1 + /CD56 + CTCs, respectively. Additionally, 59% of CTCs were TTF-1 + /VEGFR2 + and 53% CK + /VEGFR2 + . One pazopanib cycle resulted to a significant decrease of the number of CTCs (CellSearch: p=0.043) and CK + /VEGFR2 + cells (p=0.027). At the time of PD, both the total number of CTCs (p=0.027) and the number of the different subpopulations were significantly increased compared to post-1st cycle values; this increased CTCs number was associated with a significant increase of TTF-1 + /VEGFR2 + (p=0.028) and CK + /VEGFR2 + CTCs (p=0.018). In multivariate analysis, only the number of CTCs as assessed by CellSearch after one treatment cycle was significantly associated with OS (HR: 0.21; p=0.005)., Conclusions: Pazopanib has a significant effect on different subpopulations of CTCs in patients with relapsed SCLC; the detection of VEGFR2 + CTCs during treatment could be a surrogate marker associated with resistance to pazopanib., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

In order to achieve a synergistic effect on anti-tumour and anti-angiogenesis activity, we designed and constructed a DNA vaccine that expresses MUC1and VEGFR2 in the same reading frame. The aim of this study was to investigate the anti-tumour activity of this DNA vaccine. Furthermore, we also investigated the enhanced synergistic anti-Lewis lung carcinoma effect of this DNA vaccine by using GM-CSF as an adjuvant. A series of DNA plasmids encoding MUC1, VEGFR2, GM-CSF, and their conjugates were constructed and injected into mice intramuscularly (i.m.) followed by an electric pulse. The humoral and cellular immune responses after immunization were detected by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot (ELISPOT), respectively. To evaluate the anti-tumour efficacy of these plasmids, murine models with MUC1-expressing tumours were generated. After injection into the tumour-bearing mouse model, the plasmid carrying the fusion gene of MUC1 and VEGFR2 showed stronger inhibition of tumour growth than the plasmid expressing MUC1 or VEGFR2 alone, which indicated that MUC1 and VEGFR2 could exert a synergistic anti-tumour effect. Furthermore, mice vaccinated with the combination of the GM-CSF expressing plasmid and the plasmid carrying the fusion gene of MUC1 and VEGFR2 showed an increased inhibition in the growth of MUC1-expressing tumours and prolonged mouse survival. These observations emphasize the potential of the synergistic anti-tumour and anti-angiogenesis strategy used in DNA vaccines, and the potential of the GM-CSF gene as an adjuvant for DNA vaccines, which could represent a promising approach for tumour immunotherapy., (© 2016 John Wiley & Sons Australia, Ltd.)

We previously conducted a phase I clinical trial combining the HLA-A*2402-restricted KIF20A-derived peptide vaccine with gemcitabine for advanced pancreatic cancer (PC) and confirmed its safety and immunogenicity in cancer patients. In this study, we conducted a multicenter, single-armed, phase II trial using two antiangiogenic cancer vaccines targeting VEGFR1 and VEGFR2 in addition to the KIF20A peptide. We attempted to evaluate the clinical benefit of the cancer vaccination in combination with gemcitabine. Chemotherapy naïve PC patients were enrolled to evaluate primarily the 1-year survival rate, and secondarily overall survival (OS), progression free survival (PFS), response rate (RR), disease control rate (DCR) and the peptide-specific immune responses. All enrolled patients received therapy without the HLA-A information, and the HLA genotypes were used for classification of the patients. Between June 2012 and May 2013, a total of 68 patients were enrolled. No severe systemic adverse effects of Grade 3 or higher related to these three peptides were observed. The 1-year survival rates between the HLA-A*2402-matched and -unmatched groups were not significantly different. In the HLA-A*2402 matched group, patients showing peptide-specific CTL induction for KIF20A or VEGFR1 showed a better prognosis compared to those without such induction (P = 0.023, P = 0.009, respectively). In the HLA-A*2402-matched group, the patients who showed a strong injection site reaction had a better survival rate (P = 0.017) compared to those with a weak or no injection site reaction. This phase II study demonstrated that this therapeutic peptide cocktail might be effective in patients who demonstrate peptide-specific immune reactions although predictive biomarkers are needed for patient selection in its further clinical application., (© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Targeted delivery of anticancer drugs to brain tumors, especially glioblastoma multiforme, which is the most frequent and aggressive type, is one of the important objectives in nanomedicine. Vascular endothelial growth factor (VEGF) and its receptor type II (VEGFR2) are promising targets because they are overexpressed by not only core tumor cells but also by migrated glioma cells, which are responsible for resistance and rapid progression of brain tumors. The purpose of the present study was to develop the liposomal drug delivery system combining enhanced loading capacity of cisplatin and high binding affinity to glioma cells. This was achieved by using of highly soluble cisplatin analogue, cis-diamminedinitratoplatinum(II), and antibodies against the native form of VEGF or VEGFR2 conjugated to liposome surface. The developed drug delivery system revealed sustained drug release profile, high affinity to antigens, and increased uptake by glioma C6 and U-87 MG cells. Pharmacokinetic study on glioma C6-bearing rats revealed prolonged blood circulation time of the liposomal formulation. The above features enabled the present drug delivery system to overcome both poor pharmacokinetics typical for platinum formulations and low loading capacity typical for conventional liposomal cisplatin formulations.

Introduction of selectivity/specificity into viral-based gene delivery systems, such as lentiviral vectors (LVs), is crucial in their systemic administration for cancer gene therapy. The pivotal role of tumor-associated endothelial cells (TAECs) in tumor angiogenesis and overexpression of vascular endothelial growth factor receptor-2 (VEGFR2 or KDR) in TAECs makes them a potent target in cancer treatment. Herein, we report the development of VEGFR2-targeted LVs pseudotyped with chimeric sindbis virus E2 glycoprotein (cSVE2s). For this purpose, either sequence of a VEGFR2-specific nanobody or its natural ligand (VEGF 121 ) was inserted into the binding site of sindbis virus E2 glycoprotein. In silico modeling data suggested that the inserted targeting motifs were exposed in the context of cSVE2s. Western blot analysis of LVs indicated the incorporation of cSVE2s into viral particles. Capture ELISA demonstrated the specificity/functionality of the incorporated cSVE2s. Transduction of 293/KDR (expressing VEGFR2) or 293T cells (negative control) by constructed LVs followed by fluorescent microscopy and flow cytometric analyses indicated selective transduction of 293/KDR cells (30 %) by both targeting motifs compared to 293T control cells (1-2 %). These results implied similar targeting properties of VEGFR2-specific nanobody compared to the VEGF 121 and indicated the potential for transductional targeting of tumor vasculature by the nanobody displaying LVs.

Background: Only a small proportion of patients respond to anti-VEGF therapy, pressing the need for a reliable biomarker that can identify patients who will benefit. We studied the biological activity of anti-VEGF antibodies in patients' blood during anti-VEGF therapy by using the Ba/F3-VEGFR2 cell line, which is dependent on VEGF for its growth., Methods: Serum samples from 22 patients with cancer before and during treatment with bevacizumab were tested for their effect on proliferation of Ba/F3-VEGFR2 cells. Vascular endothelial growth factor as well as bevacizumab concentrations in serum samples from these patients were determined by enzyme linked immunosorbent assay (ELISA)., Results: The hVEGF-driven cell proliferation was effectively blocked by bevacizumab (IC 50 3.7 μg ml -1 ; 95% CI 1.7-8.3 μg ml -1 ). Cell proliferation was significantly reduced when patients' serum during treatment with bevacizumab was added (22-103% inhibition compared with pre-treatment). Although bevacizumab levels were not related, on-treatment serum VEGF levels were correlated with Ba/F3-VEGFR2 cell proliferation., Conclusions: We found that the neutralising effect of anti-VEGF antibody therapy on the biological activity of circulating VEGF can be accurately determined with a Ba/F3-VEGFR2 bioassay. The value of this bioassay to predict clinical benefit of anti-VEGF antibody therapy needs further clinical evaluation in a larger randomised cohort.

We obtained the morphologically, cytofluorometrically, and functionally mature dendritic cells from rats that were pulsed with antigens of the C6 glioma tissue extract. The concentrations of angiogenesis antigens (VEGF, VEGFR-1, and VEGFR-2) and periglioma zone proteins (GFAP, connexin 43, and BSAT1) in the pulsing extract were measured by ELISA. Our results drove us to a conclusion that despite mature phenotype of pulsed dendritic cell, the antigenic composition of glioma tissue extracts should be modified.

Cutaneous leishmaniasis causes a spectrum of diseases from self-healing to severe nonhealing lesions. Defining the factors contributing to lesion resolution may help in developing new therapies for those patients with chronic disease. We found that infection with Leishmania major increases the expression of vascular endothelial growth factor-A and vascular endothelial growth factor receptor (VEGFR)-2 and is associated with significant changes in the blood and lymphatic vasculature at the site of infection. Ab blockade of VEGFR-2 during infection led to a reduction in lymphatic endothelial cell proliferation and simultaneously increased lesion size without altering the parasite burden. These data show that L. major infection initiates enhanced vascular endothelial growth factor-A/VEGFR-2 signaling and suggest that VEGFR-2-dependent lymphangiogenesis is a mechanism that restricts tissue inflammation in leishmaniasis., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Human papillomavirus (HPV) has been identified as the primary etiological factor in cervical cancer as well as in subsets of anogenital and oropharyngeal cancers. The two HPV viral oncoproteins, E6 and E7, are uniquely and consistently expressed in all HPV-infected cells and are therefore promising targets for therapeutic vaccination. In order to achieve a synergistic antitumor and anti-angiogenesis effect, we designed and constructed a novel DNA vaccine that can express the HPV 16 E6E7 fusion protein and VEGFR2 in the same reading frame. A series of DNA plasmids encoding E6E7, VEGFR2 and their conjugates were constructed and injected into mice. The resultant humoral and cellular immune responses were detected by ELISA and enzyme-linked immunospot (ELISPOT), respectively. To evaluate the antitumor efficacy of these plasmids, tumor-bearing mice expressing the E6E7 fusion protein were constructed. After injection into the tumor-bearing mouse model, the plasmid harboring the E6E7 fusion gene and VEGFR2 showed stronger inhibition of tumor growth than the plasmid expressing E6E7 or VEGFR2 alone, which indicated that the combination of E6E7 and VEGFR2 could exert a synergistic antitumor effect. These observations emphasize the potential of a synergistic antitumor and anti-angiogenesis strategy using a DNA vaccine, which could be a promising approach for tumor immunotherapy., (© 2016 The Societies and John Wiley & Sons Australia, Ltd.)

MHC class I polypeptide-related sequence A (MICA), which is normally expressed on cancer cells, activates NK cells via NK group 2-member D pathway. However, some cancer cells escape NK-mediated immune surveillance by shedding membrane MICA causing immune suppression. To address this issue, we designed an antibody-MICA fusion targeting tumor-specific antigen (vascular endothelial growth factor receptor 2, VEGFR2) based on our patented antibody (mAb04) against VEGFR2. In vitro results demonstrate that the fusion antibody retains both the antineoplastic and the immunomodulatory activity of mAb04. Further, we revealed that it enhanced NK-mediated immunosurveillance against K562 cells through increasing degranulation and cytokine production of NK cells. The overall data suggest our new fusion protein provides a promising approach for cancer-targeted immunotherapy and has prospects for potential application of chronic myeloid leukemia.