In vivo phage display — A discovery tool in molecular biomedicine

In vivo phage display is a high-throughput method for identifying target ligands specific for different vascular beds. Targeting is possible due to the heterogeneous expression of receptors and other antigens in a particular vascular bed. Such expression is additionally influenced by the physiological or pathological status of the vasculature. In vivo phage display represents a technique that is usable in both, vascular mapping and targeted drug development. In this review, several important methodological aspects of in vivo phage display experiments are discussed. These include choosing an appropriate phage library, an appropriate animal model and the route of phage library administration. In addition, peptides or antibodies identified by in vivo phage display homing to specific types of vascular beds, including the altered vasculature present in several types of diseases are summarized. Still, confirmation in independent experiments and reproduction of identified sequences are needed for enhancing the clinical applicability of in vivo phage display research.     

Phage display screening of therapeutic peptide for cancer targeting and therapy

Recently, phage display technology has been announced as the recipient of Nobel Prize in Chemistry 2018. Phage display technique allows high affinity target-binding peptides to be selected from a complex mixture pool of billions of displayed peptides on phage in a combinatorial library and could be further enriched through the biopanning process; proving to be a powerful technique in the screening of peptide with high affinity and selectivity. In this review, we will first discuss the modifications in phage display techniques used to isolate various cancer-specific ligands by in situ, in vitro, in vivo, and ex vivo screening methods. We will then discuss prominent examples of solid tumor targeting-peptides; namely peptide targeting tumor vasculature, tumor microenvironment (TME) and overexpressed receptors on cancer cells identified through phage display screening. We will also discuss the current challenges and future outlook for targeting peptidebased therapeutics in the clinics.     

Mouse thymus targeted peptide isolated by in vivo phage display can inhibit bioactivity of thymus output in vivo

Heterogeneity of the vasculature in different organs has been well documented by the method of in vivo phage display. Using this technology, several peptide ligands that home to tissue-specific vascular endothelial cell have been isolated. Such peptide ligands directed against specific vascular surface molecules can be used as targeted therapeutic compounds or imaging agents to the vasculature of the specific organ in vivo. In this study, the authors perform in vivo selection in mice using a phage display random peptide library and separated phage peptides homing to mouse thymus by 3 rounds of in vivo panning. Sequence analysis showed that CHAQGSAEC is the dominant peptide sequence. Immunohistochemistry confirmed that the phage peptide CHAQGSAEC can bind specifically to thymus blood vessels in mice. Furthermore, phage peptide CHAQGSAEC and free peptide CHAQGSAEC can inhibit the bioactivity of thymus output in vivo. These results indicate the feasibility of the targeted peptide for possible function as a kind of tool to inhibit thymus bioactivity or as a targeted compound for targeted medicine.     

Angiogenic factors as potential drug target: Efficacy and limitations of anti-angiogenic therapy

Formation of new blood vessels (angiogenesis) has been demonstrated to be a basic prerequisite for sustainable growth and proliferation of tumor. Several growth factors, cytokines, small peptides and enzymes support tumor growth either independently or in synergy. Decoding the crucial mechanisms of angiogenesis in physiological and pathological state has remained a subject of intense interest during the past three decades. Currently, the most widely preferred approach for arresting tumor angiogenesis is the blockade of vascular endothelial growth factor (VEGF) pathway; however, the clinical usage of this modality is still limited by several factors such as adverse effects, toxicity, acquired drug resistance, and non-availability of valid biomarkers. Nevertheless, angiogenesis, being a normal physiological process imposes limitations in maneuvering it as therapeutic target for tumor angiogenesis. The present review offers an updated relevant literature describing the role of well-characterized angiogenic factors, such as VEGF, basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), placenta growth factor (PLGF), hepatocyte growth factor/scatter factor (HGF/SF) and angiopoetins (ANGs) in regulating tumor angiogenesis. We have also attempted to discuss tumor angiogenesis with a perspective of ‘an attractive target with emerging challenges’, along with the limitations and present status of anti-angiogenic therapy in the current state-of-the-art.     

Efficient selection of DARPins with sub-nanomolar affinities using SRP phage display

There is an ever-increasing demand to select specific, high-affinity binding molecules against targets of biomedical interest. The success of such selections depends strongly on the design and functional diversity of the library of binding molecules employed, and on the performance of the selection strategy. We recently developed SRP phage display that employs the cotranslational signal recognition particle (SRP) pathway for the translocation of proteins to the periplasm. This system allows efficient filamentous phage display of highly stable and fast-folding proteins, such as designed ankyrin repeat proteins (DARPins) that are virtually refractory to conventional phage display employing the post-translational Sec pathway. DARPins comprise a novel class of binding molecules suitable to complement or even replace antibodies in many biotechnological or biomedical applications. So far, all DARPins have been selected by ribosome display. Here, we harnessed SRP phage display to generate a phage DARPin library containing more than 10¹⁰ individual members. We were able to select well behaved and highly specific DARPins against a broad range of target proteins having affinities as low as 100 pM directly from this library, without affinity maturation. We describe efficient selection on the Fc domain of human IgG, TNFα, ErbB1 (EGFR), ErbB2 (HER2) and ErbB4 (HER4) as examples. Thus, SRP phage display makes filamentous phage display accessible for DARPins, allowing, for example, selection under harsh conditions or on whole cells. We envision that the use of SRP phage display will be beneficial for other libraries of stable and fast-folding proteins.     

血管内皮细胞生长因子受体Flt-1结合小肽的融合表达及活性鉴定

血管内皮细胞生长因子 (VEGF)通过结合其酪氨酸激酶受体KDR、fms样酪氨酸激酶 1(Flt 1)调节新生血管形成 ;筛选能封闭VEGF结合Flt 1的小肽 ,可以通过阻断肿瘤血管形成 ,抑制实体瘤生长 .将从噬菌体 12肽库中筛选获得的 2个能与Flt 1结合的阳性噬菌体克隆 (F5 6和F90 )十二肽DNA(36bp)克隆到表达载体pQE4 2中 ,在大肠杆菌M15中稳定表达二氢叶酸还原酶融合蛋白(DHFR F5 6 F90 ) ,经变性、复性后得到纯度达 90 %的可溶性蛋白 .ELISA检测表明 ,DHFR F5 6 F90能结合可溶性受体sFlt 1和血管内皮细胞 ;12 5I VEGF竞争抑制实验显示 ,DHFR F5 6能竞争抑制VEGF同可溶性受体sFlt 1结合 .结果提示 ,F5 6可能是VEGF受体Flt 1的有效拮抗剂 ,具有抗肿瘤新生血管形成的潜在应用前景     

Molecular addresses in blood vessels as targets for therapy

We have isolated several organ- and tumor-homing peptides by using in vivo phage display. This technology involves the screening of peptide libraries in a living animal. The peptides that result from such a selection home to specific organs or tissues because they recognize molecular 'addresses', receptors that are differentially expressed in vascular beds. Targeted delivery of chemotherapeutics, pro-apoptotic peptides and cytokines to tumors using these peptides improved therapeutic efficacy in animal models. Translation of this technology into clinical applications will form the basis for targeting therapeutic and imaging agents in the context of cancer and other diseases.     

In vivo phage display and vascular heterogeneity: implications for targeted medicine

The vascular endothelium expresses differential receptors depending on the functional state and tissue localization of its cells. A method to characterize this receptor heterogeneity with phage display random peptide libraries has been developed. Using this technology, several peptide ligands have been isolated that home to tissue-specific endothelial cell receptors following intravenous administration. Such peptide ligands, or antibodies directed against specific vascular receptors, can be used to target therapeutic compounds or imaging agents to endothelial cells in vitro and in vivo. Recent advances in the field include identification of novel endothelial receptors expressed differentially in normal and pathological conditions and the isolation of peptides or antibody ligands to such receptors in in vitro assays, in animal models and in a human patient. These milestones, which extend the 'functional map' of the vasculature, should lead to clinical applications in diseases such as cancer and other conditions that exhibit distinct vascular characteristics.     

Probing the structural and molecular diversity of tumor vasculature

The molecular diversity of the vasculature provides a rational basis for developing targeted diagnostics and therapeutics for cancer. Targeted imaging agents would offer better localization of primary tumors and metastases, and targeted therapies would improve efficacy and reduce side effects. The development of targeted pharmaceuticals requires the identification of specific ligand-receptor pairs, and knowledge of their cellular distribution and accessibility. Using in vivo phage display, a technique by which we can identify organ-specific and disease-specific proteins expressed on the endothelial surface, it is now possible to decipher the molecular signature of blood vessels in normal and diseased tissues. These studies have already led to the identification of peptides that target the normal vasculature of the brain, kidney, pancreas, lung and skin, as well as the abnormal vasculature of tumors, arthritis and atherosclerosis. Membrane dipeptidase in the lungs, interleukin-11 receptor in the prostate, and aminopeptidase N in tumors are examples of molecular targets on blood vessels. Corresponding confocal-microscopic imaging and ultrastructural studies are providing a more complete understanding of the cellular abnormalities of tumor blood vessels, and the distribution and accessibility of potential targets. The combined approach offers a strategy for creating a ligand-receptor map of the human vasculature, and forms a foundation for the development and application of targeted therapies in cancer and other diseases.     

Inhibition of FGF‐FGFR and VEGF‐VEGFR signalling in cancer treatment

The sites of targeted therapy are limited and need to be expanded. The FGF‐FGFR signalling plays pivotal roles in the oncogenic process, and FGF/FGFR inhibitors are a promising method to treat FGFR‐altered tumours. The VEGF‐VEGFR signalling is the most crucial pathway to induce angiogenesis, and inhibiting this cascade has already got success in treating tumours. While both their efficacy and antitumour spectrum are limited, combining FGF/FGFR inhibitors with VEGF/VEGFR inhibitors are an excellent way to optimize the curative effect and expand the antitumour range because their combination can target both tumour cells and the tumour microenvironment. In addition, biomarkers need to be developed to predict the efficacy, and combination with immune checkpoint inhibitors is a promising direction in the future. The article will discuss the FGF‐FGFR signalling pathway, the VEGF‐VEGFR signalling pathway, the rationale of combining these two signalling pathways and recent small‐molecule FGFR/VEGFR inhibitors based on clinical trials.     

Hypoxia-inducible factor 1-alpha and vascular endothelial growth factor in cartilage tumors

Neoangiogenesis has been demonstrated in chondrosarcoma. Anti-angiogenic therapies are being tested in clinical trials for chondrosarcomas. Studies of the underlying mechanisms have been performed almost exclusively in cell lines. We immunostained 20 samples of chondrosarcoma and 20 samples of enchondromas with antibodies against hypoxia-inducible factor 1-alpha (HIF-1-alpha) and vascular endothelial growth factor (VEGF). The immunohistochemical staining of HIF-1-alpha and VEGF were highly correlated. Enchondromas were HIF-1-alpha and VEGF negative, whereas all chondrosarcoma exhibited HIF-1-alpha and VEGF immunostaining. HIF-1-alpha/VEGF double positive cases were almost exclusively chondrosarcomas with a high tumor grade. We suggest that HIF-1-alpha is a marker of malignancy in chondrosarcomas that correlates with tumor neo-angiogenesis. Our findings also suggest that a HIF-1-alpha/VEGF angiogenic pathway may exist in chondrosarcoma in vivo as in other malignant tumors. The inclusion of novel inhibitors to HIF-1-alpha and other factors may optimize anti-angiogenic interventions in chondrosarcoma.     

Biomarkers predicting tumor response and evasion to anti-angiogenic therapy

No fully validated biological markers currently exist to predict responsiveness to or the development of evasion to anti-angiogenic therapy of cancer. The identification of such biomarkers is vital to move these therapies forward, as failure to respond to these treatments is often associated with rapid tumor progression that could have been averted had the intrinsic or acquired evasion to anti-angiogenic therapy been identified in a timely fashion. Furthermore, the high cost of antiangiogenic therapies makes it important to avoid utilizing them in the setting of lack of response or developing evasion, making the identification of biomarkers even more important. A number of potential physiologic, circulating, tissue, and imaging biomarkers have emerged from recently completed preclinical animal studies and clinical trials. In this review, we define 5 different types of biomarkers (physiologic, circulating, intratumoral, genetic polymorphisms, and radiographic); discuss the challenges in establishing biomarkers of antiangiogenic therapy in animal models and in clinical trials; and discuss future strategies to identify and validate biomarkers of anti-angiogenic therapy.     

体内筛选技术在靶向治疗中的新进展

用噬菌体展示技术进行体内筛选可以更好地模拟靶抗原的天然环境 ,以筛选到与活体内某些器官或组织有特异结合活性的肽或抗体。近年来利用该技术在动物体内的研究已取得了可喜的进展。综述了体内筛选技术在器官和组织血管靶向载体的筛选、基因治疗及绘制人类血管分子图谱方面的应用 ,并对其今后的研究发展方向进行了阐述。     

Exploitation of phage display for the development of anti-cancer agents targeting fibroblast growth factor signaling pathways: New strategies to tackle an old challenge

A tumor is defined as a group of cancer cells and ‘surrounding’ stromal bio-entities. Alongside the extracellular matrix (ECM) in the tumor microenvironment (TME), the stromal cells play key roles in cancer affliction and progression. Carcinoma-associated fibroblasts (CAFs) in the area of the tumor, whether activated or not, dictate the future of tumor cells. The CAFs and corresponding secreted growth factors (GFs), which mediate the crosstalk within the TME, can be targeted in therapies directed at the stroma. The impact of the fibroblast growth factor-fibroblast growth factor receptor (FGF-FGFR) signaling pathway in different kinds of tumors has been explored. Several tyrosine kinase inhibitors (TKIs), monoclonal antibodies (mAbs), and ligand traps targeting the formation of FGF-FGFR complex are in preclinical or early development phases. Moreover, there are numerous studies in the literature reporting the application of phage display technology for the development of peptides and proteins capable of functioning as FGF mimetics or traps, which are able to modulate FGF-related signaling pathways. In this review, prominent research in relation to phage display-assisted ligand identification for the FGF/FGFR system is discussed.     

KRN633, an Inhibitor of Vascular Endothelial Growth Factor Receptor Tyrosine Kinase,Induces Intrauterine Growth Restriction in Mice

We previously reported that treatment with KRN633, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, during mid‐pregnancy caused intrauterine growth restriction resulting from impairment of blood vessel growth in the labyrinthine zone of the placenta and fetal organs. However, the relative sensitivities of blood vessels in the placenta and fetal organs to vascular endothelial growth factor (VEGF) inhibitors have not been determined. In this study, we aimed to examine the effects of KRN633 on the vasculatures of organs in mother mice and their newborn pups by immunohistochemical analysis. Pregnant mice were treated daily with KRN633 (5 mg/kg) either from embryonic day 13.5 (E13.5) to E17.5 or from E13.5 to the day of delivery. The weights of the pups of KRN633‐treated mice were lower than those of the pups of vehicle‐treated mothers. However, no significant difference in body weight was observed between the vehicle‐ and KRN633‐treated mice. The vascular development in the organs (the pancreas, kidney, and intestine) and intestinal lymphatic formation of the pups of KRN633‐treated mothers was markedly impaired. In contrast, the KRN633 treatment showed no significant effect on the vascular beds in the organs, including the labyrinthine zone of the placenta, of the mother mice. These results suggest that blood vessels in fetal organs are likely to be more sensitive to reduced VEGF signaling than those in the mother. A partial loss of VEGF function during pregnancy could suppress vascular growth in the fetus without affecting the vasculature in the mother mouse, thereby increasing the risk of intrauterine growth restriction.     

Purpurin inhibits adipocyte-derived leucine aminopeptidase and angiogenesis in a zebrafish model

Adipocyte-derived leucine aminopeptidase (A-LAP) is a novel member of the M1 family of zinc metallopeptidases, which has been reported to play a crucial role in angiogenesis. In the present study, we conducted a target-based screening of natural products and synthetic chemical libraries using the purified enzyme to search novel inhibitors of A-LAP. Amongst several hits isolated, a natural product purpurin was identified as one of the most potent inhibitors of A-LAP from the screening. In vitro enzymatic analyses demonstrated that purpurin inhibited A-LAP activity in a non-competitive manner with a K_i value of 20 M. In addition, purpurin showed a strong selectivity toward A-LAP versus another member of M1 family of zinc metallopeptidase, aminopeptidase N (APN). In angiogenesis assays, purpurin inhibited the vascular endothelial growth factor (VEGF)-induced invasion and tube formation of human umbilical vein endothelial cells (HUVEC). Moreover, purpurin inhibited in vivo angiogenesis in zebrafish embryo without toxicity. These data demonstrate that purpurin is a novel specific inhibitor of A-LAP and could be developed as a new anti-angiogenic agent.     

Multiple Novel Classes of APRIL-specific Receptor-blocking Peptides Isolated by Phage Display

A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor (TNF) ligand superfamily and has a proliferative effect on both normal and tumor cells. The TNF family receptors (B-cell maturation antigen (BCMA), transmembrane activator and CAML-interactor (TACI), and BAFF receptor-3 (BR3)) for APRIL and the closely related ligand, B-cell activating factor of the TNF family (BAFF), bind these ligands through a highly conserved six residue DXL motif ((F/Y/W)-D-X-L-(V/T)-(R/G)). Panning peptide phage display libraries led to the identification of several novel classes of APRIL-binding peptides, which could be grouped by their common sequence motifs. Interestingly, only one of these ten classes consisted of peptides containing the DXL motif. Nevertheless, all classes of peptides prevented APRIL, but not BAFF, from binding BCMA, their shared receptor. Synthetic peptides based on selected sequences inhibited APRIL binding to BCMA with IC₅₀ values of 0.49-27 μM. An X-ray crystallographic structure of APRIL bound to one of the phage-derived peptides showed that the peptide, lacking the DXL motif, was nevertheless bound in the DXL pocket on APRIL. Our results demonstrate that even though a focused, highly conserved motif is required for APRIL-receptor interaction, remarkably, many novel and distinct classes of peptides are also capable of binding APRIL at the ligand receptor interface.     

Therapeutic angiogenesis for revascularization in peripheral artery disease

Therapeutic angiogenesis for peripheral artery disease (PAD), achieved by gene and cell therapy, has recently raised a great deal of hope for patients who cannot undergo standard revascularizing treatment. Although pre-clinical studies gave very promising data, still clinical trials of gene therapy have not provided satisfactory results. On the other hand, cell therapy approach, despite several limitations, demonstrated more beneficial effects but initial clinical studies must be constantly validated by larger randomized, multi-center, double-blinded, placebo-controlled trials. This review focuses on previous and recent gene and cell therapy studies for limb ischemia, including both experimental and clinical research, and summarizes some important papers published in this field. Moreover, it provides a short comment on combined gene and cell therapy approach on the example of heme oxygenase-1 overexpressing cells with therapeutic properties.     

Function blocking antibodies to neuropilin-1 generated from a designed human synthetic antibody phage library

Non-immune (na?ve) antibody phage libraries have become an important source of human antibodies. The synthetic phage antibody library described here utilizes a single human framework with a template containing human consensus complementarity-determining regions (CDRs). Diversity of the libraries was introduced at select CDR positions using tailored degenerate and trinucleotide codons that mimic natural human antibodies. Neuropilin-1 (NRP1), a cell-surface receptor for both vascular endothelial growth factor (VEGF) and class 3 semaphorins, is expressed on endothelial cells and neurons. NRP1 is required for vascular development and is expressed widely in the developing vasculature. To investigate the possibility of function blocking antibodies to NRP1 as potential therapeutics, and study the consequence of targeting NRP1 in murine tumor models, panels of antibodies that cross-react with human and murine NRP1 were generated from a designed antibody phage library. Antibody (YW64.3) binds to the CUB domains (a1a2) of NRP1 and completely blocks Sema3A induced neuron collapse; antibody (YW107.4.87) binds to the coagulation factor V/VIII domains (b1b2) of NRP1 and blocks VEGF binding and VEGF induced cell migration. YW107.4.87 inhibits tumor growth in animal xenograft models. These antibodies have provided valuable tools to study the roles of NRP1 in vascular and tumor biology.     

VEGF受体功能研究进展

血管内皮生长因子受体（VEGFR）调控心血管系统的发育。VEGFR1对于造血祖细胞的招募及单核巨噬细胞的迁移是必需的;VEGFR2、VEGFR3在调控血管及淋巴管内皮细胞的功能时发挥重要作用,而现在很多研究都聚焦于阻断VEGFR信号通路以达到阻断肿瘤血管生长的目的。