Specific Single-chain Variable Fragment Research Articles

Isolation and Characterization of the Vascular Endothelial Growth Factor Receptor Targeting ScFv Antibody Fragments Derived from Phage Display Technology.

Angiogenesis, as a tumor hallmark, plays an important role in the growth and development of the tumor vasculature system. There is a huge amount of evidence suggesting that the vascular endothelial growth factor receptor (VEGFR-2)/VEGF-A axis is one of the main contributors to tumor angiogenesis and metastasis. Thus, inhibition of the VEGFR-2 signaling pathway by anti-VEGFR-2 mAb can retard tumor growth. In this study, we employ phage display technology and solution-phase biopanning (SPB) to isolate specific single-chain variable fragments (scFvs) against VEGFR-2 and report on the receptor binding characteristics of the candidate scFvs A semisynthetic phage antibody library to isolate anti-VEGFR-2 scFvs through an SPB performed with decreasing concentrations of the VEGFR-2-His tag and VEGFR-2-biotin. After successful expression and purification, the specificity of the selected scFv clones was further analyzed by enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunoblotting. The competition assay was undertaken to identify the VEGFR-2 receptor-blocking properties of the scFvs. Furthermore, the molecular binding characteristics of candidate scFvs were extensively studied by peptide-protein docking. Polyclonal ELISA analysis subsequent to four rounds of biopanning showed a significant enrichment of VEGFR-2-specific phage clones by increasing positive signals from the first round toward the fourth round of selection. The individual VEGFR-2-reactive scFv phage clones were identified by monoclonal phage ELISA. The sequence analysis and complementarity-determining region alignment identified the four unique anti-VEGFR-2-scFv clones. The soluble and purified scFvs displayed binding activity against soluble and cell-associated forms of VEGFR-2 protein in the ELISA and flow cytometry assays. Based on the inference from the molecular docking results, scFvs D3, E1, H1, and E9 recognized domains 2 and 3 on the VEGFR-2 protein and displayed competition with VEGF-A for binding to VEGFR-2. The competition assay confirmed that scFvs H1 and D3 can block the VEGFR-2/VEGF-A interaction. In conclusion, we identified novel VEGFR-2-blocking scFvs that perhaps exhibit the potential for angiogenesis inhibition in VEGFR-2-overexpressed tumor cells.

FGL2-targeting T cells exhibit antitumor effects on glioblastoma and recruit tumor-specific brain-resident memory T cells

Although tissue-resident memory T (TRM) cells specific for previously encountered pathogens have been characterized, the induction and recruitment of brain TRM cells following immune therapy has not been observed in the context of glioblastoma. Here, we show that T cells expressing fibrinogen-like 2 (FGL2)–specific single-chain variable fragments (T-αFGL2) can induce tumor-specific CD8+ TRM cells that prevent glioblastoma recurrence. These CD8+ TRM cells display a highly expanded T cell receptor repertoire distinct from that found in peripheral tissue. When adoptively transferred to the brains of either immunocompetent or T cell-deficient naïve mice, these CD8+ TRM cells reject glioma cells. Mechanistically, T-αFGL2 cell treatment increased the number of CD69+CD8+ brain-resident memory T cells in tumor-bearing mice via a CXCL9/10 and CXCR3 chemokine axis. These findings suggest that tumor-specific brain-resident CD8+ TRM cells may have promising implications for the prevention of brain tumor recurrence.

EGFR as a potent CAR T target in triple negative breast cancer brain metastases.

There is currently no curative treatment for patients diagnosed with triple-negative breast cancer brain metastases (TNBC-BM). CAR T cells hold potential for curative treatment given they retain the cytolytic activity of a T cell combined with the specificity of an antibody. In this proposal we evaluated the potential of EGFR re-directed CAR T cells as a therapeutic treatment against TNBC cells in vitro and in vivo. We leveraged a TNBC-BM tissue microarray and a large panel of TNBC cell lines and identified elevated epidermal growth factor receptor (EGFR) expression. Next, we designed a second-generation anti-EGFR CAR T construct incorporating a clinically relevant mAb806 tumor specific single-chain variable fragment (scFv) and intracellular 4-1BB costimulatory domain and CD3ζ using a lentivirus system and evaluated in vitro and in vivo anti-tumor activity. We demonstrate EGFR is enriched in TNBC-BM patient tissue after neurosurgical resection, with six of 13 brain metastases demonstrating both membranous and cytoplasmic EGFR. Eleven of 13 TNBC cell lines have EGFR surface expression ≥ 85% by flow cytometry. EGFR806 CAR T treated mice effectively eradicated TNBC-BM and enhanced mouse survival (log rank p < 0.004). Our results demonstrates anti-tumor activity of EGFR806 CAR T cells against TNBC cells in vitro and in vivo. Given EGFR806 CAR T cells are currently undergoing clinical trials in primary brain tumor patients without obvious toxicity, our results are immediately actionable against the TNBC-BM patient population.

Preparation and application of a specific single-chain variable fragment against artemether

Artemether, an artemisinin derivative, is a component of the commonly used artemisinin-based combination therapy, artemether-lumefantrine. In this study, we cloned the VH and VL genes of a cell line (mAb 2G12E1) producing a monoclonal antibody specific to artemether, and used to construct a recombinant DNA of single-chain variable fragment (scFv). The scFv was constructed into prokaryotic expression vectors pET32a (+), pET22b (+), pGEX-2T, and pMAL-p5x, respectively. However, only the pMAL-p5x/scFv could be induced to express soluble scFv with comparable sensitivity and specificity to that of mAb 2G12E1. Based on the anti-artemether scFv, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed. The 50% of inhibition concentration (IC50) value and the working range based on IC20 to IC80 were 4.33 ng mL−1 and 1.05–22.65 ng mL−1, respectively. The artemether content in different drugs were determined by the developed icELISA, and the results were consistent to those determined by ultra performance liquid chromatography (UPLC). The anti-artemether scFv prepared in the current study could be a valuable genetically engineered antibody applied for artemether monitoring and specific binding mechanism studying.

The Effects of Chimeric Antigen Receptor (CAR) Hinge Domain Post-Translational Modifications on CAR-T Cell Activity.

To improve the efficacy and safety of chimeric antigen receptor (CAR)-expressing T cell therapeutics through enhanced CAR design, we analysed CAR structural factors that affect CAR-T cell function. We studied the effects of disulphide bonding at cysteine residues and glycosylation in the HD on CAR-T function. We used first-generation CAR[V/28/28/3z] and CAR[V/8a/8a/3z], consisting of a mouse vascular endothelial growth factor receptor 2 (VEGFR2)-specific single-chain variable fragment tandemly linked to CD28- or CD8α-derived HD, transmembrane domain (TMD) and a CD3ζ-derived signal transduction domain (STD). We constructed structural variants by substituting cysteine with alanine and asparagine (putative N-linked glycosylation sites) with aspartate. CAR[V/28/28/3z] and CAR[V/8a/8a/3z] formed homodimers, the former through a single HD cysteine residue and the latter through the more TMD-proximal of the two cysteine residues. The absence of disulphide bonds did not affect membrane CAR expression but reduced antigen-specific cytokine production and cytotoxic activity. CAR[V/28/28/3z] and CAR[V/8a/8a/3z] harboured one N-linked glycosylation site, and CAR[V/8a/8a/3z] underwent considerable O-linked glycosylation at an unknown site. Thus, N-linked glycosylation of CAR[V/28/28/3z] promotes stable membrane CAR expression, while having no effect on the expression or CAR-T cell activity of CAR[V/8a/8a/3z]. Our findings demonstrate that post-translational modifications of the CAR HD influence CAR-T cell activity, establishing a basis for future CAR design.

Developing of specific monoclonal recombinant antibody fused to alkaline phosphatase (AP) for one-step detection of fig mosaic virus.

Present study was performed to develop a fusion recombinant monoclonal antibody for one-step and accurate detection of FMV with a specific single-chain variable fragment (scFv) fused to alkaline phosphatase (AP) named as scFv(FMV-NP)-AP. The gene encoding-specific scFv recombinant antibody binding to nucleocapsid protein of Fig Mosaic Virus (FMV-NP) was fused to upstream of AP gene and integrated in pET26b bacterial expression vector. As vector contain pelB signal peptide, the expressed protein is secreted into periplasmic compartment. Recombinant fusion protein was produced in transformed E. coli following induction by IPTG. Extraction and purification of fusion protein was performed under denatured condition. The results of SDS-PAGE and western blot analysis indicated high integrity and purity with a single band protein with expected size of 72kDa. The total yield of purified scFv(FMV-NP)-AP fusion protein estimated around 0.5-1mg/l cultured medium. Subsequent colorimetric analysis confirmed presence of alkaline phosphatase activity in prepared scFv-AP fusion protein. Specificity of generated recombinant fusion antibody against cognate antigen and the native virus presented in infected plant extracts was assessed by ELISA, western blot and dot blot assays. Results revealed that scFv(FMV-NP)-AP is able to detect the presence of FMV in infected fig plants. The novel approach, implementing specific recombinant fusion antibody developed in this research, leads to one-step detection of FMV in plants by avoiding the use of chemical enzyme-labeled secondary antibodies.

Sustainable Efficacy and Safety Results from Lummicar Study 1: A Phase 1/2 Study of Fully Human B-Cell Maturation Antigen-Specific CAR T Cells (CT053) in Chinese Subjects with Relapsed and/or Refractory Multiple Myeloma

Background: CT053 is a fully human autologous chimeric antigen receptor (CAR) T-cell therapy comprising a B-cell maturation antigen (BCMA)-specific single-chain variable fragment (25C2). A clinical trial of CT053 is ongoing in LUMMICAR STUDY 1 (NCT03975907) for patients with relapsed and refractory multiple myeloma (RRMM) in China. The pivotal Phase 2 of LUMMICAR STUDY 1 is actively enrolling patients. Here, we report clinical data from Phase 1 with 12 months of follow-up.Methods: Subjects with RRMM who had received ≥3 prior therapies, including at least one proteasome inhibitor and one immunomodulatory drug were enrolled in Phase 1 study. Adverse events (AEs) were graded according to CTCAE, v5.0; Cytokine release syndrome (CRS) and neurotoxicity were graded according to ASTCT CRS consensus grading system (Lee DW et al, 2019). Response was assessed per IMWG 2016 criteria. Minimal residual disease (MRD) was tested by next-generation flow cytometry on bone marrow aspirates by the EuroFlow assay with a minimum sensitivity of 1 in 10⁵ nucleated cells or higher, and CAR copies in the subject peripheral blood were monitored by quantitative real-time polymerase chain reaction (qPCR).Results: Fourteen subjects with a median age of 54 years (range 34-62) received CT053 infusion. Three subjects received 1.0×10 8 CAR+ T cells and eleven subjects received 1.5×10 8 CAR+ T cells. As of July 8, 2021, 14 subjects with a median follow-up of 13.6 months since infusion. Of the 14 subjects, 11 (78.6%) had received autologous stem cell transplantation, 2 (14.2%) had extramedullary disease at baseline, 2 (14.3%) had ISS stage III, and 5 (35.7%) had high-risk cytogenetics. The median prior therapies was 6 (range 3-7) and no subject received bridging therapy.The most common AEs were expected hematological toxicities. All subjects (100%) experienced ≥ grade 3 neutropenia, 91.7% experienced ≥ grade 3 thrombocytopenia, and most recovered to ≤ grade 2 within 2 weeks. No dose limiting toxicity or treatment-related death was reported. Additionally, no ≥ grade 3 CRS or neurotoxicity was observed, 92.9% subjects (13/14) experienced grade 1 or 2 CRS (9 grade 1, 4 grade 2). CRS occurred at a median of 6 days (range 2-12) post-infusion with a median duration of 7 days. No severe infections were reported except one case of grade 3 lung infection.A 100% overall response rate was achieved in 14 subjects, with 11 stringent complete responses (sCR, 78.6%), 2 very good partial responses (VGPR), and 1 partial response, with a ≥VGPR rate of 92.9%. Four subjects achieved sCR at week 52. As of July 8, 2021, 12 subjects with at least 12 months of efficacy assessment were still in the study, and the 12-month progression-free survival (PFS) rate was 85.7%. With a median follow-up of 13.6 months, the median duration of response and the median PFS had not been reached. All 11 subjects with sCR were MRD-negative, and 9 subjects reached sustained CR/sCR for more than 12 months (Figure 1). Three subjects had disease progressed including two subjects with extramedullary disease progressed at week 16. Interestingly, the CR/sCR rate for the subjects without extramedullary disease is 91.7% (11/12). The 1-year PFS rate for subjects without extramedullary disease reached 100%。CT053 cells expanded and persisted well. No immunogenicity was detected.Conclusion: These results demonstrate that CT053 CAR T cells at a dose of 1.0-1.5×10 8 cells achieve a deep and durable response, including a high MRD-negative sCR rate, with an acceptable safety profile in subjects with heavily pretreated RRMM. [Display omitted] DisclosuresLi: CARsgen: Current Employment, Current equity holder in publicly-traded company. Wang: CARsgen Therapeutics Corp: Current Employment. Xiao: CARsgen Therapeutics Corp: Current Employment. Wang: CARsgen Therapeutics Co. LtD: Current Employment, Current equity holder in publicly-traded company. Tang: CARsgen Therapeutics Corp: Current Employment.

Isolation of single-chain variable fragment (scFv) antibodies for detection of Chickpea chlorotic dwarf virus (CpCDV) by phage display.

Chickpea chlorotic dwarf virus (CpCDV, genus Mastrevirus), has a wide host range and geographic distribution in many parts of the world, and it is one of the most important legume-infecting viruses. Detection of CpCDV-infected plants in the field and evaluation of viral resistance of plant cultivars are possible by conducting serological assays. Here, development and characterization of a specific recombinant monoclonal antibody for CpCDV as a diagnostic tool are described. For this purpose, the coat protein of CpCDV was expressed in Escherichia coli strain Rosetta (DE3) and used to screen a Tomlinson phage display antibody library to select a specific single-chain variable fragment (scFv). In each round of biopanning, the affinity of the phage for CpCDV-CP was tested by enzyme-linked immunosorbent assay (ELISA). The results showed that the specificity of the eluted phages increased after each round of panning. Testing of individual clones by ELISA showed that five clones of the monoclonal phage were more strongly reactive against CpCDV than the other clones. All selected positive clones contained the same sequence. The phage-displayed scFv antibody, which was named CpCDV-scFvB9, did not bind to other tested plant pathogens and showed high sensitivity in the detection of CpCDV. A Western blot assay demonstrated that CpCDV-scFvB9 reacted with the recombinant coat protein of CpCDV. Finally, the interaction CpCDV-scFvB9 and CpCDV-CP was analyzed in a molecular docking experiment. This is the first report on production of an scFv antibody against CpCDV, which could be useful for immunological detection of the virus.

Generation and molecular docking analysis of specific single-chain variable fragments selected by phage display against the recombinant nucleocapsid protein of fig mosaic virus

The mosaic disease caused by fig mosaic virus (FMV) is considered the plague of fig worldwide. A naïve phage display library, raised against the recombinant nucleocapsid protein of FMV (FMV-Np) was screened to obtain specific monoclonal recombinant antibodies in the form of single chain variable fragments (scFvs). After three rounds of biopanning, the bacterially expressed FMV-Np was used as an antigen for selecting specific phages for the production of specific soluble scFvs to be used in immunological assays. The binding specificity of scFvs against FMV-infected fig samples was evaluated by immunoblotting and Plate trapped antigen-ELISA (PTA-ELISA), which revealed efficient of the resultant scFvs to the target antigen. Silico homology-modelling and molecular docking analysis confirmed the scFv and FMV-Np interactions with the anti-FMV-Np scFv through an estimated binding energy of -650 kj mol−1; considered to be generated from the interactions between 13 amino acids residues predicted as putative epitopes in the interface pocket of FMV-Np and scFv antibody. This high affinity was further confirmed in the specificity of ELISA and immunoblotting assays. This is the first report on the application of phage display technology to generate specific recombinant scFvs against FMV that can be applied in development of antibody-mediated protection strategy to control the fig mosaic disease.

Novel On-Target Restoration of a Split T Cell-Engaging Antibody for Precision Immunotherapy of Multiple Myeloma

T cell-engaging immunotherapies are changing the therapeutic landscape of cancer. However, suitable target antigens are scarce, restricting these strategies to very few tumor types. To overcome this limitation, we developed a T cell-engaging antibody derivative, which comes in two complementary halves and addresses antigen combinations instead of single molecules. Each half, originally coined hemibody, contains an antigen specific single-chain variable fragment fused to either the variable light (VL) or variable heavy (VH) chain domain of an anti-CD3 antibody.

Preparation of a highly specific single chain variable fragment antibody targeting miroestrol and its application in quality control of Pueraria candollei by enzyme-linked immunosorbent assay.

Miroestrol is the potent phytoestrogen isolated from White Kwao Krua (Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham, a Thai traditional medicinal plant. Nowadays, various health supplementary products featuring White Kwao Krua are available worldwide. A sensitive and rapid analytical method for quantification of miroestrol is necessary for quality control of these products. To prepare a single-chain variable fragment (scFv) antibody specific to miroestrol and develop a scFv-based enzyme-linked immunosorbent assay (ELISA) for quantitative analysis of miroestrol in plant materials and health supplementary products. A gene encoding anti-miroestrol scFv antibody was constructed and expressed in Escherichia coli SHuffle T7 strain. Anti-miroestrol scFv antibody was characterised and applied to ELISA. The developed scFv-based ELISA method was validated for its sensitivity, specificity, accuracy and precision. Anti-miroestrol scFv antibody was highly specific to miroestrol. The scFv-based ELISA was applied to determine miroestrol in the range 0.06-7.81μg/mL, with the limit of quantification of 0.06μg/mL miroestrol. The accuracy of the assay was validated by its 95.08-103.99% recovery from the spiked miroestrol recovery experiment and in good correlation with the results from the monoclonal antibody-based ELISA. The relative standard deviation of the intra- and inter-assay were less than 6.0%. The developed scFv-based ELISA was sensitive, specific, accurate, and precise for determination of miroestrol and useful for quality control of P. candollei plant raw materials and supplementary products.

Preparation of single-chain antibody against VP3 protein of duck hepatitis virus type 1 by phage display technology

To construct phage antibody library for VP3 protein of duck hepatitis virus type 1(DHAV-1) and pan the specific single-chain variable fragment antibody (scFv), total RNA was extracted from the protein VP3- immunized mice spleen., vp3 gene encoding VP3 protein was amplified from the genome of DHAV-1 by RT-PCR method for the following recombinant pET-VP3 construction, immunogenic VP3 expression and purification, and combined with SOE-PCR method to complete the assembly of scFv. The scFv gene was cloned into pCANTAB5E vector for phage antibody library construction. Finally, the library for anti-VP3 scFv was screened by four rounds of adsorption–elution–enrichment with the purified VP3 protein. The characters of binding ability, specificity and neutralization of soluble antibodies expressed were evaluated by ELISA. The results showed 7 VP3-specific scFvs have been screened and identified with high both sensitivity and specificity for binding DHAV-1. To our knowledge, this is the first report for VP3-specific scFv of DHAV-1 and potentially promising application used in prevention and treatment of duck viral hepatitis.

Production of a phage-displayed single chain variable fragment antibody against infectious bursal disease virus

Purpose : To develop specific single chain variable fragments (scFv) against infectious bursal disease virus (IBDV) via phage display technology. Methods : Purified viruses were initially applied for iterative panning rounds of scFv phage display libraries. The binding ability of the selected scFv antibody fragments against the IBDV particles was analyzed by indirect enzyme-linked immunosorbent assay (ELISA) followed by blotting assays. Threedimensional (3D) structure of the selected scFv antibody fragment and VP3 protein were predicted through in silico analysis. Structural characterization of the antibody-antigen complexes was carried out by computational docking analysis. Results : The serological results obtained from the ELISA and blotting analysis showed that the selected clones produced specific scFv antibody fragments that were capable of effectively detecting infectious bursal disease (IBD) in the infected animal tissue. Biodiversity analysis by BstNI finger printing and nucleotide sequencing revealed that there was no major difference in nucleotide sequences of the selected clones. Further analysis demonstrated that this recombinant fragment of the antibody was able to bind to VP3 structural protein of IBDV with a molecular weight of ~30 kDa. Molecular docking results revealed that the binding energy of scFv to IBDV-VP3 was 545 kj/mol. Conclusion : The developed scFv antibody fragments possess great potentials for the diagnosis of IBD. The findings of the present study confirm the feasibility of using phage display technology for rapid production of antibodies against IBD diseases by applying naive scFv libraries. Keywords : Antibody, Molecular docking, Phage display technology, Single chain variable fragments

Selection and Evaluation of Human Recombinant Antibodies against ErbB2 Antigen for Breast Cancer Immunotherapy

Background: Breast cancer is the most common cause of cancer-related death in women worldwide. ErBb2/HER2 breast cancer accounts for 25% - 30% of all cases of breast cancer. Approved anti-ErbB2 monoclonal antibodies, trastuzumab and pertuzumab, are currently used for the treatment of ErbB-positive breast cancer, although their clinical use is limited due to their immunogenicity. Human recombinant single-chain antibodies, which are produced by antibody engineering technologies, are new and effective antibodies in cancer immunotherapy. Objectives: To select specific single-chain variable fragments (scFvs) against 2 immunodominant ErbB2 epitopes, including trastuzumab and pertuzumab binding sites, and to evaluate their reactivity and specificity against ErbB2 epitopes. Methods: Escherichia coli bacteria, containing a phagemid with a scFv insert segment, were used to select specific high-affinity scFvs against 2 ErbB2 epitopes, using the panning process. Polymerase chain reaction (PCR) and DNA fingerprinting were performed on the obtained clones to select the positive ones and isolate the common patterns. The selected clones were evaluated via phage Elisa in terms of reactivity and specificity to epitopes. Results: Single-chain antibodies, scFvI and scFvII, which are ErbB2-specific with 40% and 45% frequencies, were selected against epitopes I and II, respectively. The results of phage ELISA demonstrated a significant difference in the optical density (OD) of scFvs in reaction with the related peptides and non-peptide wells. ODs of 0.65 and 0.71 were obtained for scFvI and scFvII reactions with the corresponding peptides, whereas the ODs of non-peptide wells were 0.1 and 0.13, respectively. Conclusions: Targeted cancer therapy, which acts on a specific molecule in cancer cells, minimizes the side effects of immunotherapy. Due to the unique properties of scFvs, these antibodies have been used in targeted therapy of several cancers. In this study, 2 specific scFvs were selected against 2 ErbB2 epitopes, which contained trastuzumab and pertuzumab binding sites. The results of the panning process demonstrated the selection of 2 specific scFvs (with frequencies of 40% and 45%, respectively), which significantly reacted with the corresponding epitopes in phage ELISA assay. These small, high-affinity, human antibodies, which were selected against regions containing the binding sites of 2 food and drug administration (FDA)-approved monoclonal antibodies for breast cancer immunotherapy, have the potential to be considered for breast cancer targeted therapy. However, in vitro and in vivo tests should be performed to evaluate the antitumor effects of these scFvs.

Selection of single chain antibody fragments binding to the extracellular domain of 4-1BB receptor by phage display technology.

The 4-1BB is a surface glycoprotein that pertains to the tumor necrosis factor-receptor family. There is compelling evidence suggesting important roles for 4-1BB in the immune response, including cell activation and proliferation and also cytokine induction. Because of encouraging results of different agonistic monoclonal antibodies against 4-1BB in the treatment of cancer, infectious, and autoimmune diseases, 4-1BB has been suggested as an attractive target for immunotherapy. In this study, single chain variable fragment phage display libraries, Tomlinson I+J, were screened against specific synthetic oligopeptides (peptides I and II) designed from 4-1BB extracellular domain. Five rounds of panning led to selection of four 4-1BB specific single chain variable fragments (PI.12, PI.42, PII.16, and PII.29) which showed specific reaction to relevant peptides in phage enzyme-linked immunosorbent assay. The selected clones were successfully expressed in Escherichia coli Rosetta-gami 2, and their expression was confirmed by western blot analysis. Enzyme-linked immunosorbent assay experiments indicated that these antibodies were able to specifically recognize 4-1BB without any cross-reactivity with other antigens. Flow cytometry analysis demonstrated an acceptable specific binding of the single chain variable fragments to 4-1BB expressed on CCRF-CEM cells, while no binding was observed with an irrelevant antibody. Anti-4-1BB single chain variable fragments enhanced surface CD69 expression and interleukin-2 production in stimulated CCRF-CEM cells which confirmed the agonistic effect of the selected single chain variable fragments. The data from this study have provided a rationale for further experiments involving the biological functions of anti-4-1BB single chain variable fragments in future studies.

Anti-Metastatic and Anti-Invasion Effects of a Specific Anti-MUC18 scFv Antibody on Breast Cancer Cells.

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.

High-affinity FRβ-specific CAR T cells eradicate AML and normal myeloid lineage without HSC toxicity.

Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here, we isolated a high affinity (HA) folate receptor beta (FRβ)-specific scFv (2.48nM KD) for optimization of FRβ-redirected CAR T-cell therapy for AML. T-cells stably expressing the HA-FRβ CAR exhibited greatly enhanced antitumor activity against FRβ+ AML in vitro and in vivo compared to a low affinity (LA) FRβ CAR (54.3nM KD). Using the HA-FRβ IgG, FRβ expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34+ hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRβ CAR T-cells lysed mature CD14+ monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRβ CAR T-cells retained effective anti-tumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRβ CAR T-cells is highly effective against AML and reduces the risk for long-term myeloid toxicity.

Effects of an amyloid-beta 1-42 oligomers antibody screened from a phage display library in APP/PS1 transgenic mice

We screened anti-Aβ1-42 antibodies from a human Alzheimer’s disease (AD) specific single chain variable fragment (scFv) phage display library and assessed their effects in APP/PS1 transgenic mice. Reverse transcription-PCR was used to construct the scFv phage display library, and screening identified 11A5 as an anti-Aβ1-42 antibody. We mixed 11A5 and the monoclonal antibody 6E10 with Aβ1-42 and administered the mixture to Sprague-Dawley rats via intracerebroventricular injection. After 30 days, rats injected with the antibody/Aβ1-42 mixture and those injected with Aβ1-42 alone were tested on the Morris water maze. We also injected 11A5 and 6E10 into APP/PS1 transgenic mice and assessed the concentrations of Aβ in brain and peripheral blood by ELISA at 1-month intervals for 3 months. Finally we evaluated behavior changes in the Morris water maze. Rats injected with Aβ1-42 and mixed antibodies showed better performance in the Morris water maze than did rats injected with Aβ1-42 alone. In APP/PS1 transgenic mice, Aβ concentration was lower in the brains of the antibody-treated group than in the control group, but higher in the peripheral blood. The antibody-treated mice also exhibited improved behavioral performance in the Morris water maze. In conclusion, anti-Aβ1-42 antibodies (11A5) screened from the human scFv antibody phage display library promoted the efflux or clearance of Aβ1-42 and effectively decreased the cerebral Aβ burden in an AD mouse model.

Abstract B74: Co-targeting the tumor and its associated vasculature in glioblastoma

Abstract Background: Glioblastoma (GBM) is the most common and most aggressive primary brain tumor in adults. With the current standard treatment, survival is poor with substantial treatment related toxicities underscoring the need for new targeted therapies. Preclinical and early clinical data demonstrate the efficacy of chimeric antigen receptor (CAR)-grafted T-cells as a novel approach against cancer. Previous work from our lab has demonstrated that HER2-specific T cells can induce tumor regression in an orthotopic xenogeneic model. However, tumors recurred in a subset of mice treated with HER2-specific T cells. It is now evident that tumors are heterogeneous cellular complexes whose growth is dependent upon reciprocal interactions between the tumor and its heterogeneous microenvironment. In particular, glioblastomas are highly vascularized tumors, known for their invasive phenotype. Tumor Endothelium Marker 8 (TEM8) is upregulated in tumor angiogenesis and conserved in mice, thus making it a target which can be studied pre-clinically for both safety and efficacy. Objective: The purpose of this study is to determine whether co-targeting the tumor antigen, HER2 and Tumor Endothelium Marker 8 (TEM8) using bispecific CAR T cells will result in improved tumor control in GBM. Methods: In order to generate TEM8 and HER2 bispecific CAR T cells we employed two methods. For the first method we designed a TEM8 CAR, to be coexpressed along with a separate HER2 CAR on the same T cells (TEM8-HER2 BiCAR T cells). For the second method we designed a bispecific tandem CAR (TEM8-HER2 TanCAR) in which TEM8 and HER2 specific single chain variable fragments have been placed in-tandem and are connected by a glycine-serine linker to make a single CAR to be expressed on T cells. CAR constructs were assembled on clone manager, synthesized, then sequence verified and force expressed on T cells using a retroviral system. CAR T cells were expanded in IL-2 in parallel with the control T cells from the same donor. Flow cytometry was then used to confirm CAR expression on T cells and binding specificity. ELISA and flow-based cytotoxicity assays were used to assess selectivity and killing potential of TEM8 and HER2 bispecific T cells. Results: We have successfully cloned in the TEM8 and TEM8-HER2 TanCAR constructs and generated TEM8-HER2 BiCAR T cells TEM8-HER2 specific TanCAR T cells, respectively. Up to 70% of T cells expressed the specific CAR on the cell surface. TEM8 and HER2 specific CAR T cells selectively recognize and kill TEM8 and HER2 positive targets and release the immuno-stimulatory cytokines interferon-gamma and interleukin-2 in vitro. Further, cytokine release was augmented when bispecific T cells were simultaneously exposed to both target targets. Conclusion: Heterogeneous nature of GBM favors co-targeting the tumor and its microenvironment. We have successfully generated the first TEM8 specific CAR T cell product to specifically target the tumor associated vasculature. Co-targeting tumor and its endothelium using bispecific CAR T cells could enhance T cell activation and can potentially be used to improve tumor control and have therapeutic application in GBM patients. Citation Format: Tiara T. Byrd, Kristen Fousek, Zakaria Grada, Kevin Aviles-Padilla, Kevin Bielamowicz, Stephen Gottschalk, Bradley St Croix, Bradley Fletcher, Meenakshi Hegde, Nabil Ahmed. Co-targeting the tumor and its associated vasculature in glioblastoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B74. doi:10.1158/1538-7445.CHTME14-B74

A chimeric antigen receptor for TRAIL-receptor 1 induces apoptosis in various types of tumor cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its associated receptors (TRAIL-R/TR) are attractive targets for cancer therapy because TRAIL induces apoptosis in tumor cells through TR while having little cytotoxicity on normal cells. Therefore, many agonistic monoclonal antibodies (mAbs) specific for TR have been produced, and these induce apoptosis in multiple tumor cell types. However, some TR-expressing tumor cells are resistant to TR-specific mAb-induced apoptosis. In this study, we constructed a chimeric antigen receptor (CAR) of a TRAIL-receptor 1 (TR1)-specific single chain variable fragment (scFv) antibody (TR1-scFv-CAR) and expressed it on a Jurkat T cell line, the KHYG-1 NK cell line, and human peripheral blood lymphocytes (PBLs). We found that the TR1-scFv-CAR-expressing Jurkat cells killed target cells via TR1-mediated apoptosis, whereas TR1-scFv-CAR-expressing KHYG-1 cells and PBLs killed target cells not only via TR1-mediated apoptosis but also via CAR signal-induced cytolysis, resulting in cytotoxicity on a broader range if target cells than with TR1-scFv-CAR-expressing Jurkat cells. The results suggest that TR1-scFv-CAR could be a new candidate for cancer gene therapy.