Gene Summary

Gene:SCFV; single-chain Fv fragment
Databases:GeneCard, Gene
Source:NCBIAccessed: 11 March, 2017

Cancer Overview

Research Indicators

Publications Per Year (1992-2017)
Graph generated 11 March 2017 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • Molecular Sequence Data
  • Chromosome 14
  • Cancer Gene Expression Regulation
  • gp100 Melanoma Antigen
  • Immunotherapy, Adoptive
  • Transduction
  • Cell Proliferation
  • Antibodies, Neoplasm
  • Genetic Therapy
  • Receptor, erbB-2
  • Monoclonal Antibodies
  • Neuroblastoma
  • Stomach Cancer
  • Ribonucleases
  • Immunoglobulin Fragments
  • Flow Cytometry
  • T-Cell Antigen Receptors
  • Vaccines, DNA
  • Viral Tropism
  • Recombinant Proteins
  • Viral Fusion Proteins
  • Polymerase Chain Reaction
  • Antibody Specificity
  • Tumor Antigens
  • Apoptosis
  • Multiple Myeloma
  • Antibodies
  • Breast Cancer
  • Mice, Inbred BALB C
  • Translocation
  • Cytotoxicity, Immunologic
  • Genetic Vectors
  • Immunotherapy
  • Gene Expression
  • Transfection
  • Immunoglobulin Variable Region
  • Up-Regulation
  • Amino Acid Sequence
  • Recombinant Fusion Proteins
  • Oncogenes
Tag cloud generated 11 March, 2017 using data from PubMed, MeSH and CancerIndex

Specific Cancers (4)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: SCFV (cancer-related)

Mohammadi M, Nejatollahi F, Ghasemi Y, Faraji SN
Anti-Metastatic and Anti-Invasion Effects of a Specific Anti-MUC18 scFv Antibody on Breast Cancer Cells.
Appl Biochem Biotechnol. 2017; 181(1):379-390 [PubMed] Related Publications
Breast cancer is the most common malignancy in women. Altered expression of MUC18, a cell surface receptor, and its interaction with Wnt-5a as its ligand, affects the motility and invasiveness of breast cancer cells. In this study, we explored the Wnt-5a binding site and designed an antigenic epitope on the MUC18 receptor using in silico methods. A specific single-chain variable fragment (scFv) was isolated against the epitope by several panning processes. The binding ability of the scFv to the related epitope was evaluated in ELISA and flow cytometry. The inhibitory effects of the selected scFv on MUC18 positive cell line, MDA-MB231, was assessed by migration and invasion assays. The results demonstrated isolation of specific scFv with frequency of 40 % which showed significant binding with the epitope in both ELISA and fluorescence-activated cell sorting (FACS) analyses. The antibody inhibited the migration (76 %) and invasion (67 %) of MUC18 positive cell line. The results suggest the specific anti-MUC18 scFv as an effective antibody for breast cancer immunotherapy.

Nakazawa Y
Gene-modified T-cell therapy using chimeric antigen receptors for pediatric hematologic malignancies.
Rinsho Ketsueki. 2016; 57(6):701-8 [PubMed] Related Publications
Chimeric antigen receptor (CAR) is the generic name for synthetic T cell receptors redirected to tumor-associated antigens. Most CARs consist of an ectodomain (scFv or ligand), a hinge region, a transmembrane domain, and signaling endodomains derived from one or two co-stimulatory molecules (CD28, 4-1BB, etc) and from a CD3-ζ chain. CD19-targeted CAR T cell therapy has achieved major success in the treatment of B cell malignancies. CD19 CAR-T cells elicited complete remission in 70-90% of adult and pediatric patients with relapsed/refractory acute lymphoblastic leukemia (ALL). CD19 CAR T cell therapy from allogeneic donors including third party donors is a potential option for B-cell malignancies. CAR T cell therapies for myeloma, acute myeloid leukemia, and T-cell leukemia are still under development. Our group is currently preparing a phase I study of CD19 CAR T cell therapy in pediatric and young adult patients with ALL using a non-viral gene transfer method, the piggyBac-transposon system.

Lv X, Zhang J, Xu R, et al.
Gigantoxin-4-4D5 scFv is a novel recombinant immunotoxin with specific toxicity against HER2/neu-positive ovarian carcinoma cells.
Appl Microbiol Biotechnol. 2016; 100(14):6403-13 [PubMed] Related Publications
Immunotoxins are a new class of antibody-targeted therapy in clinical development. Traditional immunotoxins that are constructed from the toxins of plants or bacteria need to be internalized to the cytoplasm and thus have limited antitumor efficacy. In the present study, we combined a recently reported sea anemone cytolysin Gigantoxin-4 with an anti-HER2/neu single-chain variable fragment 4D5 scFv to construct a novel immunotoxin. We fused a SUMO tag to the N-terminus of Gigantoxin-4-4D5 scFv and it was successfully expressed in Escherichia coli strain BL21 (DE3) in a soluble form. After purification, the purity of Gigantoxin-4-4D5 scFv reached 96 % and the yield was 14.3 mg/L. Our results demonstrated that Gigantoxin-4-4D5 scFv exerted a highly cytotoxic effect on the HER2/neu-positive ovarian carcinoma SK-OV-3 cell line. And the hemolytic activity was weaker, making it safe for normal cells. The results of immunofluorescence analysis showed that this novel immunotoxin could specifically bind to SK-OV-3 cells with no recognition of human embryonic kidney 293 cells. Scanning electron microscope observations and extracellular lactate dehydrogenase activity indicated that it could induce necrosis in SK-OV-3 cells by disrupting the cell membrane. Moreover, it could also mediate apoptosis of SK-OV-3 cells.

Hinrichs CS
Molecular Pathways: Breaking the Epithelial Cancer Barrier for Chimeric Antigen Receptor and T-cell Receptor Gene Therapy.
Clin Cancer Res. 2016; 22(7):1559-64 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
Adoptive transfer of T cells genetically engineered to express a tumor-targeting chimeric antigen receptor (CAR) or T-cell receptor (TCR) can mediate cancer regression in some patients. CARs are synthetic single-chain proteins that use antibody domains to target cell surface antigens. TCRs are natural heterodimeric proteins that can target intracellular antigens through recognition of peptides bound to human leukocyte antigens. CARs have shown promise in B-cell malignancies and TCRs in melanoma, but neither approach has achieved clear success in an epithelial cancer. Treatment of epithelial cancers may be particularly challenging because of a paucity of target antigens expressed by carcinomas and not by important healthy tissues. In addition, epithelial cancers may be protected by inhibitory ligands and soluble factors in the tumor microenvironment. One strategy to overcome these negative regulators is to modulate expression of T-cell genes to enhance intrinsic T-cell function. Programmable nucleases, which can suppress inhibitory genes, and inducible gene expression systems, which can enhance stimulatory genes, are entering clinical testing. Other work is delineating whether control of genes for immune checkpoint receptors (e.g.,PDCD1, CTLA4) and cytokine and TCR signaling regulators (e.g.,CBLB, CISH, IL12, IL15) can increase the antitumor activity of therapeutic T cells.

Shibata T, Uchida H, Shiroyama T, et al.
Development of an oncolytic HSV vector fully retargeted specifically to cellular EpCAM for virus entry and cell-to-cell spread.
Gene Ther. 2016; 23(6):479-88 [PubMed] Related Publications
Oncolytic herpes simplex virus (HSV) vectors have attracted increasing attention as novel anti-cancer agents. HSV entry is triggered by the binding of glycoprotein D (gD) to its receptors, such as herpesvirus entry mediator or nectin-1. We have recently reported the construction of a fully retargeted HSV platform that incorporates single-chain antibodies (scFv) into gD to mediate entry exclusively via tumor-associated antigens. In this study, we created an scFv directed against epithelial cell adhesion molecule (EpCAM), a recognized carcinoma-associated antigen, and inserted it into the retargeted HSV platform that is ablated for gD recognition of its canonical receptors and contains the entry-enhancing mutations in gB we previously identified. We observed that both initial entry and subsequent cell-to-cell spread of the retargeted virus were stringently dependent on cellular EpCAM expression. Interestingly, the retargeted virus developed larger plaques on some of the human tumor lines tested than the control virus bearing wild-type gD. Intratumoral injection of the retargeted virus revealed antitumor activity in a mouse xenograft model. These observations illustrate the versatility of our retargeted HSV platform as it allows expansion of the oncolytic virus toolbox for the treatment of diverse cancers.

Kim MG, Kim D, Suh SK, et al.
Current status and regulatory perspective of chimeric antigen receptor-modified T cell therapeutics.
Arch Pharm Res. 2016; 39(4):437-52 [PubMed] Related Publications
Chimeric antigen receptor-modified T cells (CAR-T) have emerged as a new modality for cancer immunotherapy due to their potent efficacy against terminal cancers. CAR-Ts are reported to exert higher efficacy than monoclonal antibodies and antibody-drug conjugates, and act via mechanisms distinct from T cell receptor-engineered T cells. These cells are constructed by transducing genes encoding fusion proteins of cancer antigen-recognizing single-chain Fv linked to intracellular signaling domains of T cell receptors. CAR-Ts are classified as first-, second- and third-generation, depending on the intracellular signaling domain number of T cell receptors. This review covers the current status of CAR-T research, including basic proof-of-concept investigations at the cell and animal levels. Currently ongoing clinical trials of CAR-T worldwide are additionally discussed. Owing to the lack of existing approved products, several unresolved concerns remain with regard to safety, efficacy and manufacturing of CAR-T, as well as quality control issues. In particular, the cytokine release syndrome is the major side-effect impeding the successful development of CAR-T in clinical trials. Here, we have addressed the challenges and regulatory perspectives of CAR-T therapy.

Yang JL, Pan XY, Zhao WX, et al.
The antitumor efficacy of a novel adenovirus-mediated anti-p21Ras single chain fragment variable antibody on human cancers in vitro and in vivo.
Int J Oncol. 2016; 48(3):1218-28 [PubMed] Related Publications
Activated ras genes are found in a large number of human tumors, and therefore are one of important targets for cancer therapy. This study investigated the antitumor effects of a novel single chain fragment variable antibody (scFv) against ras protein, p21Ras. The anti-p21Ras scFv gene was constructed by phage display library from hybridoma KGHR1, and then subcloned into replication-defective adenovirus vector to obtain recombinant adenovirus KGHV100. Human tumor cell lines with high expression of p21Ras SW480, MDA-MB‑231, OVCAR-3, BEL-7402, as well as tumor cell line with low expression of p21Ras, SKOV3, were employed to investigate antitumor effects in vitro and in vivo. Fluorescence microscopy demonstrated that KGHV100 was able to express intracellularly anti-p21Ras scFv antibody in cultured tumor cells and in transplantation tumor cells. MTT, Transwell, colony formation, and flow cytometry analysis showed that KGHV100 led to significant growth arrest in tumor cells with high p21Ras expression, and induced G0/G1 cell cycle arrest in the studied tumor cell lines. In vivo, KGHV100 significantly inhibited tumor growth following intratumoral injection, and the survival rates of the mice were higher than the control group. These results indicate that the adenovirus-mediated intracellular expression of the novel anti-p21Ras scFv exerted strong antitumoral effects, and may be a potential method for therapy of cancers with p21Ras overexpression.

Rodgers DT, Mazagova M, Hampton EN, et al.
Switch-mediated activation and retargeting of CAR-T cells for B-cell malignancies.
Proc Natl Acad Sci U S A. 2016; 113(4):E459-68 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
Chimeric antigen receptor T (CAR-T) cell therapy has produced impressive results in clinical trials for B-cell malignancies. However, safety concerns related to the inability to control CAR-T cells once infused into the patient remain a significant challenge. Here we report the engineering of recombinant antibody-based bifunctional switches that consist of a tumor antigen-specific Fab molecule engrafted with a peptide neo-epitope, which is bound exclusively by a peptide-specific switchable CAR-T cell (sCAR-T). The switch redirects the activity of the bio-orthogonal sCAR-T cells through the selective formation of immunological synapses, in which the sCAR-T cell, switch, and target cell interact in a structurally defined and temporally controlled manner. Optimized switches specific for CD19 controlled the activity, tissue-homing, cytokine release, and phenotype of sCAR-T cells in a dose-titratable manner in a Nalm-6 xenograft rodent model of B-cell leukemia. The sCAR-T-cell dosing regimen could be tuned to provide efficacy comparable to the corresponding conventional CART-19, but with lower cytokine levels, thereby offering a method of mitigating cytokine release syndrome in clinical translation. Furthermore, we demonstrate that this methodology is readily adaptable to targeting CD20 on cancer cells using the same sCAR-T cell, suggesting that this approach may be broadly applicable to heterogeneous and resistant tumor populations, as well as other liquid and solid tumor antigens.

Moradi-Kalbolandi S, Davani D, Golkar M, et al.
Soluble Expression and Characterization of a New scFv Directed to Human CD123.
Appl Biochem Biotechnol. 2016; 178(7):1390-406 [PubMed] Related Publications
Leukemic cancer stem cells (LSCs), as a unique cell population in acute myeloid leukemia (AML) marked by CD123 overexpression, are thought to play a key role in relapsed AML after chemotherapy. Thus, CD123 is considered as a particularly important target candidate for antibody-derived diagnosis and therapy. In the present work, we constructed an immunized murine antibody phage display library and isolated the functional anti-CD123 Single-chain fragment variable (scFv) clones. We also introduced fusing variable light (VL) and heavy (VH) chains with a new 18-amino acid residue linker as an alternative to conventional linkers. CD123-specific phage clones were progressively enriched through 4 rounds of biopanning, validated by phage ELISA, and anti-CD123 scFv clones with highest affinity were produced in Escherichia coli. The expression and purification of soluble scFv were verified by Western blot, and the results were indicative of the functionality of our proposed linker. The purified scFv specifically recognized CD123 by ELISA and flow cytometry, without any cross-reactivity with other related cell markers. Affinity of anti-CD123 scFv was measured to be 6.9 × 10(-7) M, using the competitive ELISA. Our work, therefore, provides a framework for future studies involving biological functions and applications of our anti-CD123 scFv. It also reveals the feasibility of high throughput methods to isolate biomarker-specific scFvs.

van der Steen SC, van Tilborg AA, Vallen MJ, et al.
Prognostic significance of highly sulfated chondroitin sulfates in ovarian cancer defined by the single chain antibody GD3A11.
Gynecol Oncol. 2016; 140(3):527-36 [PubMed] Related Publications
OBJECTIVE: The extracellular matrix (ECM) of ovarian cancer may provide a number of potential biomarkers. Chondroitin sulfate (CS), a class of sulfated polysaccharides, is abundantly present in the ECM of ovarian cancer. Structural alterations of CS chains (i.e. sulfation pattern) have been demonstrated to play a role in cancer development and progression. In this study we investigate the potential of highly sulfated CS as a biomarker in ovarian cancer using the single chain antibody GD3A11 selected by the phage display technology.
METHODS: The specificity of the antibody was determined by an indirect ELISA. GD3A11 epitope expression was assessed by immunohistochemistry in healthy organs, benign and malignant ovarian tumors (N=359) and correlated to clinical parameters. The CHST15 gene, responsible for the biosynthesis of highly sulfated CS was evaluated for mutation and methylation status.
RESULTS: The GD3A11 epitope was minimally expressed in normal organs. Intense expression was observed in the ECM of different ovarian cancer subtypes, in contrast to benign ovarian tumors. Expression was independent of tumor grade, FIGO stage, and the use chemotherapy. For the aggressive ovarian cancer phenotype, intense expression was identified as an independent predictor for poor prognosis. CHST15 gene analysis showed no mutations nor an altered methylation status.
CONCLUSION: Specific highly sulfated CS motifs expressed in the tumoral ECM hold biomarker potential in ovarian cancer patients. These matrix motifs constitute a novel class of biomarkers with prognostic significance and may be instrumental for innovative diagnostic and therapeutic applications (e.g. targeted therapy) in management of ovarian cancer.

Lu Y, Liu L, Wang Y, et al.
siRNA delivered by EGFR-specific scFv sensitizes EGFR-TKI-resistant human lung cancer cells.
Biomaterials. 2016; 76:196-207 [PubMed] Related Publications
The overexpression of epidermal growth factor receptor (EGFR) is closely associated with a poor outcome in non-small cell lung cancer (NSCLC), and EGFR is an ideal biomarker for the targeted therapy of NSCLC. Although patients with EGFR-activating mutations respond to EGFR tyrosine kinase inhibitors (EGFR-TKIs), they eventually acquire resistance, which typically results from a secondary EGFR mutation or the activation of other signaling pathways. Novel approaches to overcome or prevent EGFR-TKI resistance are clinically important. In this study, we developed an EGFR-scFv-arginine nonamer peptide fusion protein, s-9R, as an siRNA carrier. Here, we show that s-9R effectively and specifically delivers EGFR-siRNAs, KRAS-siRNA and MET-siRNA into NSCLC cells and silences the expression of target genes. The sensitivity of NSCLC cells to gefitinib was restored after treatment with the s-9R/siRNA complex, and the apoptosis rates of the treated cells were significantly higher than those of the control groups. Furthermore, the co-administration of s-9R/siRNA and gefitinib successfully suppressed the progression of H1975 xenograft tumors and extended the life span of tumor-bearing nude mice. Collectively, the results of this study provide not only a new scFv derivative for delivering siRNA into EGFR-overexpressing, TKI-resistant NSCLC cells but also a novel method for overcoming TKI resistance.

Krug C, Birkholz K, Paulus A, et al.
Stability and activity of MCSP-specific chimeric antigen receptors (CARs) depend on the scFv antigen-binding domain and the protein backbone.
Cancer Immunol Immunother. 2015; 64(12):1623-35 [PubMed] Related Publications
Chimeric antigen receptor (CAR)-modified T cells emerged as effective tools in the immunotherapy of cancer but can produce severe on-target off-tissue toxicities. This risk can conceivably be overcome, at least partially, by transient transfection. The design of CARs, however, has so far not been optimized for use in non-permanent T cell modification. Here we compared the performance of T cells modified with three different first- and second-generation CARs, each specific for MCSP (HMW-MAA) which is commonly expressed by melanoma cells. Upon RNA transfer, the expression of all receptors was limited in time. The second-generation CARs, which combined CD28-CD3ζ signaling, were expressed at higher levels and more prolonged than first-generation CARs with CD3ζ only. The CD28 domain increased the cytokine production, but had only an indirect effect on the lytic capacity, by prolonging the CAR expression. Especially for the second-generation CARs, the scFv clearly impacted the level and duration of CAR expression and the T cell performance. Thus, we identified a CAR high in both expression and anti-tumor cell reactivity. T cells transfected with this CAR increased the mean survival time of mice after challenge with melanoma cells. To facilitate clinical application, this CAR was used to redirect T cells from late-stage melanoma patients by RNA transfection. These T cells mediated effective antigen-specific tumor cell lysis and release of pro-inflammatory cytokines, even after cryoconservation of the transfected T cells. Taken together, the analysis identified a CAR with superior anti-melanoma performance after RNA transfer which is a promising candidate for clinical exploration.

Krenciute G, Krebs S, Torres D, et al.
Characterization and Functional Analysis of scFv-based Chimeric Antigen Receptors to Redirect T Cells to IL13Rα2-positive Glioma.
Mol Ther. 2016; 24(2):354-63 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
Immunotherapy with T cells expressing chimeric antigen receptors (CARs) is an attractive approach to improve outcomes for patients with glioblastoma (GBM). IL13Rα2 is expressed at a high frequency in GBM but not in normal brain, making it a promising CAR T-cell therapy target. IL13Rα2-specific CARs generated up to date contain mutated forms of IL13 as an antigen-binding domain. While these CARs target IL13Rα2, they also recognize IL13Rα1, which is broadly expressed. To overcome this limitation, we constructed a panel of IL13Rα2-specific CARs that contain the IL13Rα2-specific single-chain variable fragment (scFv) 47 as an antigen binding domain, short or long spacer regions, a transmembrane domain, and endodomains derived from costimulatory molecules and CD3.ζ (IL13Rα2-CARs). IL13Rα2-CAR T cells recognized IL13Rα2-positive target cells in coculture and cytotoxicity assays with no cross-reactivity to IL13Rα1. However, only IL13Rα2-CAR T cells with a short spacer region produced IL2 in an antigen-dependent fashion. In vivo, T cells expressing IL13Rα2-CARs with short spacer regions and CD28.ζ, 41BB.ζ, and CD28.OX40.ζ endodomains had potent anti-glioma activity conferring a significant survival advantage in comparison to mice that received control T cells. Thus, IL13Rα2-CAR T cells hold the promise to improve current IL13Rα2-targeted immunotherapy approaches for GBM and other IL13Rα2-positive malignancies.

Deisting W, Raum T, Kufer P, et al.
Impact of Diverse Immune Evasion Mechanisms of Cancer Cells on T Cells Engaged by EpCAM/CD3-Bispecific Antibody Construct AMG 110.
PLoS One. 2015; 10(10):e0141669 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
BACKGROUND: Bispecific T cell engager (BiTE®) are single-chain bispecific antibody constructs with dual specificity for CD3 on T cells and a surface antigen on target cells. They can elicit a polyclonal cytotoxic T cell response that is not restricted by T cell receptor (TCR) specificity, and surface expression of MHC class I/peptide antigen complexes. Using human EpCAM/CD3-bispecific BiTE® antibody construct AMG 110, we here assessed to what extent surface expression of PD-L1, cytoplasmic expression of indoleamine-2,3-deoxygenase type 1, Bcl-2 and serpin PI-9, and the presence of transforming growth factor beta (TGF-β), interleukin-10 (IL-10) and adenosine in culture medium can impact redirected lysis by AMG 110-engaged T cells.
METHODS: The seven factors, which are all involved in inhibiting T cell functions by cancer cells, were tested with human EpCAM-expressing Chinese hamster ovary (CHO) target cells at levels that in most cases exceeded those observed in a number of human cancer cell lines. Co-culture experiments were used to determine the impact of the evasion mechanisms on EC50 values and amplitude of redirected lysis by AMG 110, and on BiTE®-induced proliferation of previously resting human peripheral T cells.
FINDINGS: An inhibitory effect on redirected lysis by AMG 110-engaged T cells was seen upon overexpression of serpin PI-9, Bcl-2, TGF-β and PD-L1. An inhibitory effect on induction of T cell proliferation was only seen with CHO cells overexpressing IDO. In no case, a single evasion mechanism rendered target cells completely resistant to BiTE®-induced lysis, and even various combinations could not.
CONCLUSIONS: Our data suggest that diverse mechanisms employed by cancer cells to fend off T cells cannot inactivate AMG 110-engaged T cells, and that inhibitory effects observed in vitro may be overcome by increased concentrations of the BiTE® antibody construct.

Kuramitsu S, Ohno M, Ohka F, et al.
Lenalidomide enhances the function of chimeric antigen receptor T cells against the epidermal growth factor receptor variant III by enhancing immune synapses.
Cancer Gene Ther. 2015; 22(10):487-95 [PubMed] Related Publications
The epidermal growth factor receptor variant III (EGFRvIII) is exclusively expressed on the cell surface in ~50% of glioblastoma multiforme (GBM). This variant strongly and persistently activates the phosphatidylinositol 3-kinase-Akt signaling pathway in a ligand-independent manner resulting in enhanced tumorigenicity, cellular motility and resistance to chemoradiotherapy. Our group generated a recombinant single-chain variable fragment (scFv) antibody specific to the EGFRvIII, referred to as 3C10-scFv. In the current study, we constructed a lentiviral vector transducing the chimeric antigen receptor (CAR) that consisted of 3C10-scFv, CD3ζ, CD28 and 4-1BB (3C10-CAR). The 3C10-CAR-transduced peripheral blood mononuclear cells (PBMCs) and CD3(+) T cells specifically lysed the glioma cells that express EGFRvIII. Moreover, we demonstrated that CAR CD3(+) T cells migrated to the intracranial xenograft of GBM in the mice treated with 3C10-CAR PBMCs. An important and novel finding of our study was that a thalidomide derivative lenalidomide induced 3C10-CAR PBMC proliferation and enhanced the persistent antitumor effect of the cells in vivo. Lenalidomide also exhibited enhanced immunological synapses between the effector cells and the target cells as determined by CD11a and F-actin polymerization. Collectively, lentiviral-mediated transduction of CAR effectors targeting the EGFRvIII showed specific efficacy, and lenalidomide even intensified CAR cell therapy by enhanced formation of immunological synapses.

Schutsky K, Song DG, Lynn R, et al.
Rigorous optimization and validation of potent RNA CAR T cell therapy for the treatment of common epithelial cancers expressing folate receptor.
Oncotarget. 2015; 6(30):28911-28 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
Using lentiviral technology, we recently demonstrated that incorporation of CD27 costimulation into CARs greatly improves antitumor activity and T cell persistence. Still, virus-mediated gene transfer is expensive, laborious and enables long-term persistence, creating therapies which cannot be easily discontinued if toxic. To address these concerns, we utilized a non-integrating RNA platform to engineer human T cells to express FRα-specific, CD27 CARs and tested their capacity to eliminate human FRα(+) cancer. Novel CARs comprised of human components were constructed, C4-27z and C4opt-27z, a codon-optimized variant created for efficient expression. Following RNA electroporation, C4-27z and C4opt-27z CAR expression is initially ubiquitous but progressively declines across T cell populations. In addition, C4-27z and C4opt-27z RNA CAR T cells secrete high levels of Th-1 cytokines and display strong cytolytic function against human FRα(+) cancers in a time- and antigen-dependent manner. Further, C4-27z and C4opt-27z CAR T cells exhibit significant proliferation in vivo, facilitate the complete regression of fully disseminated human ovarian cancer xenografts in mice and reduce the progression of solid ovarian cancer. These results advocate for rapid progression of C4opt-27z RNA CAR to the clinic and establish a new paradigm for preclinical optimization and validation of RNA CAR candidates destined for clinical translation.

Zhang M, Chakraborty SK, Sampath P, et al.
Fluoromodule-based reporter/probes designed for in vivo fluorescence imaging.
J Clin Invest. 2015; 125(10):3915-27 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
Optical imaging of whole, living animals has proven to be a powerful tool in multiple areas of preclinical research and has allowed noninvasive monitoring of immune responses, tumor and pathogen growth, and treatment responses in longitudinal studies. However, fluorescence-based studies in animals are challenging because tissue absorbs and autofluoresces strongly in the visible light spectrum. These optical properties drive development and use of fluorescent labels that absorb and emit at longer wavelengths. Here, we present a far-red absorbing fluoromodule-based reporter/probe system and show that this system can be used for imaging in living mice. The probe we developed is a fluorogenic dye called SC1 that is dark in solution but highly fluorescent when bound to its cognate reporter, Mars1. The reporter/probe complex, or fluoromodule, produced peak emission near 730 nm. Mars1 was able to bind a variety of structurally similar probes that differ in color and membrane permeability. We demonstrated that a tool kit of multiple probes can be used to label extracellular and intracellular reporter-tagged receptor pools with 2 colors. Imaging studies may benefit from this far-red excited reporter/probe system, which features tight coupling between probe fluorescence and reporter binding and offers the option of using an expandable family of fluorogenic probes with a single reporter gene.

Xi JM, Pang H, Hu XL, Wang ZJ
Construction of a natural phage antibody library of human anaplastic thyroid carcinoma.
Genet Mol Res. 2015; 14(3):8397-406 [PubMed] Related Publications
The objective of this study was to identify and construct a human natural phage single-chain antibody (scFv) library of human anaplastic thyroid carcinoma (ATC) using phage display technology. Total RNA was extracted from lymphatic tissue near an ATC and used to amplify variable heavy chain (VH) and variable light chain (VL) fragments with added linker sequences using reverse transcription-polymerase chain reaction (RT-PCR). After purification, the VH and VL amplicons were used to produce scFv fragments with added SfiI and NotI restriction enzyme recognition sites using splicing-overlap-extension PCR. Following digestion, the scFv gene was cloned in the pCANTAB-5E plasmid, and the recombinant phagemids were transformed into the susceptible Escherichia coli TG1 strain. After infection by the helper phage M13K07, a human ATC phage antibody library was successfully constructed. Clear 28 S and 18 S bands could be seen in the total RNA from the library, and the sizes of the VH, VL, and scFv genes contained therein were approximately 370, 350, and 750 bp, respectively. In addition, the conversion efficiency as measured by the pUC19 standard plasmid was 10(8) CFU/μg, and the positive insert ratio was 86.4% (19/22). These results demonstrated the successful construction of a human ATC scFv antibody gene library, and might provide the experimental basis for the further screening and identification of a phage single-chain antibody with ATC cell-specificity.

Liu X, Jiang S, Fang C, et al.
Affinity-Tuned ErbB2 or EGFR Chimeric Antigen Receptor T Cells Exhibit an Increased Therapeutic Index against Tumors in Mice.
Cancer Res. 2015; 75(17):3596-607 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
Target-mediated toxicity is a major limitation in the development of chimeric antigen T-cell receptors (CAR) for adoptive cell therapy of solid tumors. In this study, we developed a strategy to adjust the affinities of the scFv component of CAR to discriminate tumors overexpressing the target from normal tissues that express it at physiologic levels. A CAR-expressing T-cell panel was generated with target antigen affinities varying over three orders of magnitude. High-affinity cells recognized target expressed at any level, including at levels in normal cells that were undetectable by flow cytometry. Affinity-tuned cells exhibited robust antitumor efficacy similar to high-affinity cells, but spared normal cells expressing physiologic target levels. The use of affinity-tuned scFvs offers a strategy to empower wider use of CAR T cells against validated targets widely overexpressed on solid tumors, including those considered undruggable by this approach.

He H, Tu X, Zhang J, et al.
A novel antibody targeting CD24 and hepatocellular carcinoma in vivo by near-infrared fluorescence imaging.
Immunobiology. 2015; 220(12):1328-36 [PubMed] Related Publications
Liver cancer is one of the most common malignant cancers worldwide. The poor response of liver cancer to chemotherapy has whipped up the interest in targeted therapy with monoclonal antibodies because of its potential efficiency. One promising target is cluster of differentiation 24 (CD24), which is known to beover-expressed on hepatocellular carcinoma (HCC), providing prospect for HCC targeted diagnosis and therapy. In this study we developed a novel CD24 targeted monoclonal antibody G7mAb based on hybridoma technology and then generated a single-chain antibodyfragment (scFv) G7S. Firstly, ELISA, western blot, and flow cytometry assays demonstrated specific binding of CD24 by G7mAb and G7S. Further, G7mAb was demonstrated to have similar binding capacity as ML5 (a commercial Anti-CD24 Mouse Antibody) inimmunohistochemical assay. Further more, a near-infrared fluorescent dye multiplex probe amplification (MPA) was conjugated to G7mAb and G7S to form G7mAb-MPA and G7S-MPA. The near-infrared fluorescence imaging revealed that G7mAb and G7S aggregate in CD24+Huh7 hepatocellular carcinoma xenograft tissuevia specific binding to CD24 in vivo. In conclussion, G7mAb and G7S were tumor targeted therapeutic and diagnostic potentials in vitro and in vivo as anticipated.

Khan I, Zakaria MK, Kumar M, et al.
A novel placental like alkaline phosphatase promoter driven transcriptional silencing combined with single chain variable fragment antibody based virosomal delivery for neoplastic cell targeting [corrected].
J Transl Med. 2015; 13:254 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
BACKGROUND: Placental like alkaline phosphatase (PLAP), an oncofetal antigen, is highly expressed in germ cell, cervical, ovarian and several other tumour types but minimally in normal tissues [corrected]. The expression of a PLAP promoter based transcriptional unit following antigen mediated cell specific delivery is a possible approach for tumour targeting.
METHODS: PLAP promoter alone or in combination with NFκB DNA response elements was used for expressing shRNA targeting the long control region (LCR) of human papillomavirus (HPV)-16 oncogenes E6 and E7 via transcriptional gene silencing in PLAP expressing cervical cancer cell lines, SiHa and CaSki. This was packaged in a Sendai virus envelope incorporating a single chain variable fragment antibody (scFv) for antibody mediated targeting. Specificity and efficacy of the shRNA was assessed by studying the heterochromatization, down regulation of the HPV-16 E6/E7 genes and subsequent effects on their targets and cell growth properties.
RESULTS: Reduction of HPV-16 E6 and E7 expression by TGS led to the activation of the previously suppressed target genes of p53 (PUMA and NOXA) and Rb (cyclins A2 and E). Cell death was seen only in PLAP expressing HPV-16 infected SiHa and CaSki cells but not in the HPV-18 integrated HeLa and non-PLAP CHO cells. There was reduction in the enhancer associated transcripts of the long control region (LCR) of HPV-16 E6/E7 genes. Also, an increase in the enrichment of dimethylated histone three lysine nine (H3K9Me2) and trimethylated histone three lysine twenty-seven (H3K27Me3) was observed by ChIP assay, which decreased upon trichostatin A treatment, indicating a possible mechanism for the heterochromatization of the target LCR region.
CONCLUSION: A combination of novel PLAP promoter and antibody based specificities has the potential for being developed as a possible therapeutic strategy for PLAP positive neoplasia.

Chen F, Fan C, Gu X, et al.
Construction of Anti-CD20 Single-Chain Antibody-CD28-CD137-TCRζ Recombinant Genetic Modified T Cells and its Treatment Effect on B Cell Lymphoma.
Med Sci Monit. 2015; 21:2110-5 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
BACKGROUND: Immunotherapy has been explored as a new therapy for B cell lymphoma, which is a non-Hodgkin's lymphoma. Because CD20 is a B lymphocyte-specific marker, anti-CD20 single chain-tagged T lymphocytes have already begun to be experimentally used in B cell lymphoma treatment, but its use is still limited because of its unspecific targeting. T cells transfected with CD28 and CD137 can significantly improve the ability of cytokines secretion and anti-tumor effect, as well as extending T cell survival time and improving their proliferation ability.
MATERIAL AND METHODS: Genes containing anti-CD20-CD28-CD137-TCRζ were constructed. After cloning and sequencing, the plasmid was constructed and packaged by lentivirus. It was transfected to the peripheral blood T lymphocyte after identification transfection to induce the fusion protein expression. The cells were incubated with Raji cells and the LDH test was performed to detect the cytotoxic effect of CAR-T cells; the tumor volume and survival rate were measured to observe its inhibitory effect on B cell lymphoma in nude mice.
RESULTS: Gene with anti-CD20-CD28-CD137-TCRζ was successfully constructed and transfected to the T cell surface. LDH assay revealed that CAR-T cells can kill the Raji cells with a killing rate of 32.89±6.26%. It can significantly inhibit B cell lymphoma growth in nude mice.
CONCLUSIONS: T lymphocytes transfected with anti-CD20-CD28-CD137-TCRζ fusion gene can kill B cell lymphoma, which could provide a new strategy for tumor treatment.

Chan G, Jordaan G, Nishimura RN, Weisbart RH
Combining intracellular antibodies to restore function of mutated p53 in cancer.
Int J Cancer. 2016; 138(1):182-6 [PubMed] Related Publications
TP53 is a tumor suppressor gene that is mutated in 50% of cancers, and its function is tightly regulated by the E3 ligase, Mdm2. Both p53 and Mdm2 are localized in the cell nucleus, a site that is impervious to therapeutic regulation by most antibodies. We identified a cell-penetrating lupus monoclonal anti-DNA antibody, mAb 3E10, that targets the nucleus, and we engineered mAb 3E10 to function as an intranuclear transport system to deliver therapeutic antibodies into the nucleus as bispecific single chain Fv (scFv) fragments. Bispecific scFvs composed of 3E10 include PAb421 (3E10-PAb421) that binds p53 and restores the function of mutated p53, and 3G5 (3E10-3G5) that binds Mdm2 and prevents destruction of p53 by Mdm2. We documented the therapeutic efficacy of these bispecific scFvs separately in previous studies. In this study, we show that combination therapy with 3E10-PAb421 and 3E10-3G5 augments growth inhibition of cells with p53 mutations compared to the effect of either antibody alone. By contrast, no enhanced response was observed in cells with wild-type p53 or in cells homozygous null for p53.

Dangaj D, Scholler N
Isolation and Validation of Anti-B7-H4 scFvs from an Ovarian Cancer scFv Yeast-Display Library.
Methods Mol Biol. 2015; 1319:37-49 [PubMed] Related Publications
B7-H4 (VTCN1, B7x, B7s) is an inhibitory modulator of T-cell response implicated in antigen tolerization. As such, B7-H4 is an immune checkpoint of potential therapeutic interest. To generate anti-B7-H4 targeting reagents, we isolated antibodies by differential cell screening of a yeast-display library of recombinant antibodies (scFvs) derived from ovarian cancer patients and we screened for functional scFvs capable to interfere with B7-H4-mediated inhibition of antitumor responses. We found one antibody binding to B7-H4 that could restore antitumor T cell responses. This chapter gives an overview of the methods we developed to isolate a functional anti-B7-H4 antibody fragment.

Kato T, Yui M, Deo VK, Park EY
Development of Rous sarcoma Virus-like Particles Displaying hCC49 scFv for Specific Targeted Drug Delivery to Human Colon Carcinoma Cells.
Pharm Res. 2015; 32(11):3699-707 [PubMed] Related Publications
PURPOSE: Virus-like particles (VLPs) have been used as drug carriers for drug delivery systems. In this study, hCC49 single chain fragment variable (scFv)-displaying Rous sarcoma virus-like particles (RSV VLPs) were produced in silkworm larvae to be a specific carrier of an anti-cancer drug.
METHOD: RSV VLPs displaying hCC49 scFv were created by the fusion of the transmembrane and cytoplasmic domains of hemagglutinin from influenza A (H1N1) virus and produced in silkworm larvae. The display of hCC49 scFv on the surface of RSV VLPs was confirmed by enzyme-linked immunosorbent assay using tumor-associated glycoprotein-72 (TAG-72), fluorescent microscopy, and immunoelectron microscopy. Fluorescein isothiocyanate (FITC) or doxorubicin (DOX) was incorporated into hCC49 scFv-displaying RSV VLPs by electroporation and specific targeting of these VLPs was investigated by fluorescent microscopy and cytotoxicity assay using LS174T cells.
RESULTS: FITC was delivered to LS174T human colon adenocarcinoma cells by hCC49 scFv-displaying RSV VLPs, but not by RSV VLPs. This indicated that hCC49 scFv allowed FITC-loaded RSV VLPs to be delivered to LS174T cells. DOX, which is an anti-cancer drug with intrinsic red fluorescence, was also loaded into hCC49 scFv-displaying RSV VLPs by electroporation; the DOX-loaded hCC49 scFv-displaying RSV VLPs killed LS174T cells via the specific delivery of DOX that was mediated by hCC49 scFv. HEK293 cells were alive even though in the presence of DOX-loaded hCC49 scFv-displaying RSV VLPs.
CONCLUSION: These results showed that hCC49 scFv-displaying RSV VLPs from silkworm larvae offered specific drug delivery to colon carcinoma cells in vitro. This scFv-displaying enveloped VLP system could be applied to drug and gene delivery to other target cells.

Jiang K, Li J, Yin J, et al.
Targeted delivery of CXCR4-siRNA by scFv for HER2(+) breast cancer therapy.
Biomaterials. 2015; 59:77-87 [PubMed] Related Publications
Therapeutics based on short interfering RNAs (siRNAs) have great potential to treat human diseases. However, the clinical application of siRNAs has been limited by their poor intracellular uptake, low serum stability, and inability to target specific cells. In this study, we addressed this lack of specificity by synthesizing a molecularly targeted CXCR4-siRNA (CXCR4si) for the treatment of HER2(+) breast cancers using a HER2-scFv-arginine nonamer peptide fusion protein (e23sFv-9R) as an siRNA carrier. The e23sFv-9R binding siRNA is able to specifically deliver the siRNA to HER2(+) breast cancer cells and concentrate and persist in orthotopic HER2(+) breast cancer xenografts for at least 36 h. CXCR4si delivered by e23sFv-9R inhibited CXCR4 gene expression, reduced proliferation and metastasis and induced apoptosis in the HER2(+) breast cancer BT-474 cell line in vitro. Moreover, the systemic delivery of CXCR4si by e23sFv-9R is able to suppress tumor growth, reduce metastasis and prolong survival in mice bearing HER2(+) xenografts. This approach causes no systemic toxicity and does not activate the innate immune response, suggesting that a fusion protein carrying CXCR4si shows promise in the treatment of HER2-overexpressing breast cancer.

Patil SS, Railkar R, Swain M, et al.
Novel anti IGFBP2 single chain variable fragment inhibits glioma cell migration and invasion.
J Neurooncol. 2015; 123(2):225-35 [PubMed] Related Publications
Insulin like growth factor binding protein 2 (IGFBP2) is highly up regulated in glioblastoma (GBM) tissues and has been one of the prognostic indicators. There are compelling evidences suggesting important roles for IGFBP2 in glioma cell proliferation, migration and invasion. Extracellular IGFBP2 through its carboxy terminal arginine glycine aspartate (RGD) motif can bind to cell surface α5β1 integrins and activate pathways downstream to integrin signaling. This IGFBP2 activated integrin signaling is known to play a crucial role in IGFBP2 mediated invasion of glioma cells. Hence a molecular inhibitor of carboxy terminal domain of IGFBP2 which can inhibit IGFBP2-cell surface interaction is of great therapeutic importance. In an attempt to develop molecular inhibitors of IGFBP2, we screened single chain variable fragment (scFv) phage display libraries, Tomlinson I (Library size 1.47 × 10(8)) and Tomlinson J (Library size 1.37 × 10(8)) using human recombinant IGFBP2. After screening we obtained three IGFBP2 specific binders out of which one scFv B7J showed better binding to IGFBP2 at its carboxy terminal domain, blocked IGFBP2-cell surface association, reduced activity of matrix metalloprotease 2 in the conditioned medium of glioma cells and inhibited IGFBP2 induced migration and invasion of glioma cells. We demonstrate for the first time that in vitro inhibition of extracellular IGFBP2 activity by using human scFv results in significant reduction of glioma cell migration and invasion. Therefore, the inhibition of IGFBP2 can serve as a potential therapeutic strategy in the management of GBM.

Liu F, Si Y, Liu G, et al.
The tetravalent anti-DR5 antibody without cross-linking direct induces apoptosis of cancer cells.
Biomed Pharmacother. 2015; 70:41-5 [PubMed] Related Publications
Tumor necrosis factor-related apoptosis-inducing ligand can induce apoptosis in many tumor cell lines. This apoptotic effect is mediated by interaction of TRAIL and its receptors, which include Death Receptor 4 (DR4) and Death Receptor 5 (DR5). Some antibodies to DR4 or DR5 do not have anti-tumor ability without cross-linking but exhibit anti-tumor ability in the presence of a cross-linking reagent. Here, we suggest that the tetravalent anti-DR5 antibody can induce apoptosis of cancer cells independent of cross-linking reagent. The single-chain variable fragment of the anti-DR5 antibody, HSA (human serum albumin) - p53 gene, comprising residues 490-513 of HSA and the tetramerization domain of human p53 were assembled into the tetravalent antibody by an overlapping PCR. Results of size exclusion HPLC indicated that the purified protein exhibited a major peak (tetramer) and a minor peak (dimer). MTT assay demonstrated the tetravalent antibody without cross-linking could inhibit survival of Jurkat and EC9706 cells in a dose-dependent manner while the monovalent antibody could not inhibit survival of Jurkat and EC9706 cells. IC50 of Jurkat cell was 3.2 mg/L and IC50 of EC9706 cell was 3.9 mg/L. Furthermore, the Annexin V/PI assay and the Hoechst 33258 staining showed that the tetravalent antibody could efficiently induce apoptosis of Jurkat and EC9706 cells. Therefore, the tetravalent anti-DR5 antibody can act as a direct agonistic antibody, and initiate efficient apoptotic independent of cross-linking reagent. Thus, the tetravalent anti-DR5 antibody will be a new kind of candidate for potential cancer therapeutics.

Guerzoni C, Fiori V, Terracciano M, et al.
CD99 triggering in Ewing sarcoma delivers a lethal signal through p53 pathway reactivation and cooperates with doxorubicin.
Clin Cancer Res. 2015; 21(1):146-56 [PubMed] Related Publications
PURPOSE: The paucity of new drugs for the treatment of Ewing sarcoma (EWS) limits the cure of these patients. CD99 has a strong membranous expression in EWS cells and, being also necessary for tumor survival, is a suitable target to aim at. In this article, we described a novel human monospecific bivalent single-chain fragment variable diabody (dAbd C7) directed against CD99 of potential clinical application.
EXPERIMENTAL DESIGN: In vitro and in vivo evaluation of cell death and of the molecular mechanisms triggered by anti-CD99 agents were performed alone or in combination with doxorubicin to demonstrate efficacy and selectivity of the new dAbd C7.
RESULTS: The dAbd C7 induced rapid and massive EWS cell death through Mdm2 degradation and p53 reactivation. Mdm2 overexpression as well as silencing of p53 in p53wt EWS cells decreased CD99-induced EWS cell death, whereas treatment with nutlin-3 enhanced it. Furthermore, cell death was associated with induction of p21, bax, and mitochondrial depolarization together with substantial inhibition of tumor cell proliferation. Combined treatment of anti-CD99 dAbd C7 with doxorubicin was additive both in vitro and in vivo against EWS xenografts. Normal mesenchymal stem cells showed no p53 activation and were resistant to cell death, unless transformed by EWS-FLI, the oncogenic driver of EWS.
CONCLUSIONS: These results indicate that dAbd C7 is a suitable candidate tool to target CD99 in patients with EWS able to spare normal stem cells from death as it needs an aberrant genetic context for the efficient delivery of CD99-triggered cell death.

Oberst MD, Fuhrmann S, Mulgrew K, et al.
CEA/CD3 bispecific antibody MEDI-565/AMG 211 activation of T cells and subsequent killing of human tumors is independent of mutations commonly found in colorectal adenocarcinomas.
MAbs. 2014; 6(6):1571-84 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
Individual or combinations of somatic mutations found in genes from colorectal cancers can redirect the effects of chemotherapy and targeted agents on cancer cell survival and, consequently, on clinical outcome. Novel therapeutics with mechanisms of action that are independent of mutational status would therefore fulfill a current unmet clinical need. Here the CEA and CD3 bispecific single-chain antibody MEDI-565 (also known as MT111 and AMG 211) was evaluated for its ability to activate T cells both in vitro and in vivo and to kill human tumor cell lines harboring various somatic mutations commonly found in colorectal cancers. MEDI-565 specifically bound to normal and malignant tissues in a CEA-specific manner, and only killed CEA positive cells. The BiTE® antibody construct mediated T cell-directed killing of CEA positive tumor cells within 6 hours, at low effector-to-target ratios which were independent of high concentrations of soluble CEA. The potency of in vitro lysis was dependent on CEA antigen density but independent of the mutational status in cancer cell lines. Importantly, individual or combinations of mutated KRAS and BRAF oncogenes, activating PI3KCA mutations, loss of PTEN expression, and loss-of-function mutations in TP53 did not reduce the activity in vitro. MEDI-565 also prevented growth of human xenograft tumors which harbored various mutations. These findings suggest that MEDI-565 represents a potential treatment option for patients with CEA positive tumors of diverse origin, including those with individual or combinations of somatic mutations that may be less responsive to chemotherapy and other targeted agents.

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