scholarly journals Soluble Expression of a Neo2/15-Conjugated Single Chain Fv against PD-L1 in Escherichia coli

2022 ◽  
Vol 44 (1) ◽  
pp. 301-308
Author(s):  
Sun-Hee Kim ◽  
Hee-Jin Jeong
Keyword(s):  
Fusion Protein ◽  
Expression System ◽  
Antibody Fragment ◽  
Therapeutic Index ◽  
High Yield ◽  
Soluble Form ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Single Chain Fv ◽  
Programmed Death

Immunocytokines, antibody-cytokine fusion proteins, have the potential to improve the therapeutic index of cytokines by delivering the cytokine to the site of localized tumor cells using antibodies. In this study, we produced a recombinant anti-programmed death-ligand 1 (PD-L1) scFv, an antibody fragment against PD-L1 combined with a Neo2/15, which is an engineered interleukin with superior function using an E. coli expression system. We expressed the fusion protein in a soluble form and purified it, resulting in high yield and purity. The high PD-L1-binding efficiency of the fusion protein was confirmed via enzyme-linked immunosorbent assay, suggesting the application of this immunocytokine as a cancer-related therapeutic agent.

scholarly journals Cell-free production of scFv fusion proteins: an efficient approach for personalized lymphoma vaccines

Blood ◽  
2006 ◽  
Vol 109 (8) ◽  
pp. 3393-3399 ◽  
Author(s):  
Gregory Kanter ◽  
Junhao Yang ◽  
Alexei Voloshin ◽  
Shoshana Levy ◽  
James R. Swartz ◽  
...  
Keyword(s):  
B Cell ◽  
Expression System ◽  
Specific Antigen ◽  
Therapeutic Vaccine ◽  
Antibody Fragment ◽  
B Cell Lymphoma ◽  
Keyhole Limpet Hemocyanin ◽  
Single Chain ◽  
Single Chain Fv ◽  
Fv Antibody

Abstract The unique immunoglobulin (Ig) idiotype on the surface of each B-cell lymphoma represents an ideal tumor-specific antigen for use as a therapeutic vaccine. We have used an Escherichia coli—based, cell-free protein-expression system to produce a vaccine within hours of cloning the Ig genes from a B-cell tumor. We demonstrated that a fusion protein consisting of an idiotypic single chain Fv antibody fragment (scFv) linked to a cytokine (GM-CSF) or to an immunostimulatory peptide was an effective lymphoma vaccine. These vaccines elicited humoral immune responses against the native Ig protein displayed on the surface of a tumor and protected mice against tumor challenge with efficacy equal to that of the conventional Ig produced in a mammalian cell and chemically coupled to keyhole limpet hemocyanin. The cell-free E coli system offers a platform for rapidly generating individualized vaccines, thereby allowing much more efficient application in the clinic.

A Recombinant Soluble Form of the Integrin IIbβ3 (GPIIb-IIIa) Assumes an Active, Ligand-Binding Conformation and Is Recognized by GPIIb-IIIa–Specific Monoclonal, Allo-, Auto-, and Drug-Dependent Platelet Antibodies

Blood ◽  
1998 ◽  
Vol 92 (6) ◽  
pp. 2053-2063 ◽  
Author(s):  
Julie A. Peterson ◽  
Gian P. Visentin ◽  
Peter J. Newman ◽  
Richard H. Aster
Keyword(s):  
Ligand Binding ◽  
Expression System ◽  
Site Directed Mutagenesis ◽  
High Yield ◽  
Soluble Form ◽  
Specific Antibodies ◽  
Binding Conformation ◽  
Active Ligand ◽  
Extracellular Region ◽  
Drug Dependent

Abstract The IIb-IIIa glycoprotein complex is a favored target for allo-, auto-, and drug-dependent antibodies associated with immune thrombocytopenia. A soluble, recombinant form of the GPIIb-IIIa heterodimer that could be produced in large quantities and maintained in solution without detergent could provide a useful experimental tool for the study of platelet-reactive antibodies, but previous attempts to produce such a construct have yielded only small quantities of the end product. Using a baculovirus expression system and the dual-promoter transfer vector P2Bac, we were able to express soluble GPIIb-IIIa complex (srGPIIb-IIIa) lacking cytoplasmic and transmembrane domains in quantities of about 1,000 μg/L, about 40 times greater than reported previously. The high yield achieved may be related to inclusion of the entire extracellular region of the GPIIb light chain in the construct. srGPIIb-IIIa reacts spontaneously with fibrinogen, and this interaction is totally inhibited by the peptide RGDS. Reactions of 24 GPIIb-IIIa–specific antibodies evaluated (12 monoclonal, 3 allo-specific, 3 auto-specific, and 6 drug-dependent) with srGPIIb-IIIa were indistinguishable from reactions with platelet GPIIb-IIIa. Thus, srGPIIb-IIIa spontaneously assumes an active, ligand-binding conformation and contains epitopes for all monoclonal and human antibodies tested to date. srGPIIb-IIIa can be produced in large quantities, can readily be modified by site-directed mutagenesis, and should facilitate identification of epitopes recognized by GPIIb-IIIa–specific antibodies, study of the mechanism(s) by which certain drugs promote antibody binding to GPIIb-IIIa in drug-induced thrombocytopenia and structure-function relationships of GPIIb-IIIa. © 1998 by The American Society of Hematology.

A vaccine approach for breast cancer immunotherapy by targeting HER2/neu antigen to B cells via CD19 molecule.

2011 ◽  
Vol 29 (27_suppl) ◽  
pp. 287-287
Author(s):  
D. Xiang ◽  
Y. Ma ◽  
C. Ding ◽  
G. H. Kloecker ◽  
J. Yan
Keyword(s):  
Breast Cancer ◽  
B Cells ◽  
Fusion Protein ◽  
Tumor Cells ◽  
Fusion Proteins ◽  
In Vivo Studies ◽  
Single Chain ◽  
Tumor Vaccines ◽  
Single Chain Fv ◽  
Her 2

287 Background: Trastuzumab has been proven to be effective in the immunotherapy for Her-2/neu-positive breast cancer. However, this adoptively transferred therapeutic Ab must be continuously given to the patient with huge financial cost. Thus, it would be desirable if tumor vaccines could elicit long-lasting trastuzumab-like Ab. Methods: Constructing fusion protein of anti-mouse CD19 single chain variable fragment (anti-CD19-scFv) with human Her-2/neu extracellular domain P3-4 (P3-4) by extracting total RNA from Rat anti-mouse CD19 hybridoma (1D3) and first strand cDNA was synthesized accordingly. VH and VL were amplified using designed primers. Single chain Fv (VL-VH) was then synthesized by overlapping PCR. Then constructs were sequenced and cloned into pET-20b(+) vectors. Fragments P3-4 of extracellular domains of human Her-2/neu was cloned from pcDNA3.1-Her2 and then cloned into pET-20b(+)-scFv. Series of constructs were expressed and purified according to standard protocol and verified by Western Blot. Results: Both in vitro and in vivo studies demonstrated that fusion proteins anti-CD19-scFv and anti-CD19-scFv-P3-4 but not P3-4 could specifically bind to B cells with high affinity. Mice immunized with anti-CD19-scFv-P3-4 secreted higher titers of IgG and IgM than those from controls (p<0.05). Studies also demonstrated that sera from anti-CD19-scFv-P3-4 but not anti-CD19-scFv or P3-4 immunized mice stained with Her-2/neu expressing SKOV-3 tumor cells. These Abs also competitively inhibited trastuzumab-mediated Ag binding, suggesting that trastuzumab-like Ab responses were elicited. WT mice immunized with anti-CD19-scFv-P3-4 fusion protein then challenged with D2/F2-Her-2 mammary tumor cells showed significantly reduced tumor burden compared to those immunized with control fusion proteins (p<0.05) and had enhanced median overall survival to 45 days versus 34 days in WT mice immunized with either anti-CD19-scFv or P3-4. Conclusions: Targeting of Her-2/neu antigens to B cells stimulates Th-dependent humoral immune responses with anti tumor effect in mouse model. These findings provide a novel avenue for successful development of breast cancer vaccination strategy and help to fight for cancer.

Prognostic value of serum soluble programmed death-ligand 1 (sPDL1) and dynamics during chemotherapy in advanced gastric cancer patients.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. 4034-4034 ◽  
Author(s):  
Woochan Park ◽  
Ju-Hee Bang ◽  
Ah-Rong Nam ◽  
Ji Eun Park ◽  
Mei Hua Kim ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Advanced Gastric Cancer ◽  
Prognostic Value ◽  
Soluble Form ◽  
Enzyme Linked Immunosorbent Assay ◽  
Programmed Death Ligand 1 ◽  
Neutrophil Lymphocyte Ratio ◽  
Programmed Death ◽  
Response To Chemotherapy ◽  
Line Chemotherapy

4034 Background: The soluble form Programmed Death-Ligand 1(sPDL1) is suggested to have immunosuppressive activity and under investigation as candidate biomarker for immuno-oncology drug development. In this study, we measured the serum sPDL1 at pre-and post-chemotherapy and evaluated its prognostic implication and dynamics during chemotherapy in advanced gastric cancer (GC). Methods: We prospectively enrolled 68 GC patients who were candidates for palliative standard 1st-line chemotherapy, and blood was serially collected at pre-and post-one cycle of chemotherapy, at best response and disease progression. sPDL1 was measured using an enzyme-linked immunosorbent assay. Response to chemotherapy, overall survival (OS), progression-free survival (PFS) and other prognostic factors including neutrophil-lymphocyte ratio (NLR) were obtained. The cut-off values of sPDL1 levels and changes for survivals were found using C-statistics. Results: The median baseline sPDL1 was 0.8ng/mL(range, 0.06 - 6.06ng/mL). The median OS and PFS were 14.9 months (95% CI: 7.33-22.47) and 8.0 months (95% CI: 5.96-10.0), respectively. sPDL1 and NLR showed a positive correlation. Patients with low levels of sPD-L1 at diagnosis ( < 1.92 ng/mL) showed a better OS and PFS than the patients with a high sPDL1 (OS: 18.3 vs. 95 months, P = 0.057, PFS: 8.9 vs. 6.0 months, P = 0.04). The baseline sPDL1 before treatment were higher in the PD group than in the SD and PR groups (mean:2.91, 1.17, 1.19, P = 0.019). Patients whose sPDL1 increased after 1st cycle of chemotherapy showed the tendency of worse PFS and OS. When disease progressed, sPDL1 increased compared with baseline (mean:1.31, 1.45, P = 0.029). Conclusions: sPDL1 at pre-chemotherapy confers the prognostic value for PFS and OS in GC patients under palliative 1st-line chemotherapy. The dynamics of sPDL1 during chemotherapy correlates with disease courses.

scholarly journals Immunogenicity and Protective Efficacy of a Recombinant Subunit West Nile Virus Vaccine in Rhesus Monkeys

2009 ◽  
Vol 16 (9) ◽  
pp. 1332-1337 ◽  
Author(s):  
Michael M. Lieberman ◽  
Vivek R. Nerurkar ◽  
Haiyan Luo ◽  
Bruce Cropp ◽  
Ricardo Carrion ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Cellular Immunity ◽  
Cytokine Production ◽  
Protective Efficacy ◽  
Expression System ◽  
High Yield ◽  
Separate Experiment ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
West Nile

ABSTRACT The immunogenicity and protective efficacy of a recombinant subunit West Nile virus (WNV) vaccine was evaluated in rhesus macaques (Macaca mulatta). The vaccine consisted of a recombinant envelope (E) protein truncated at the C-terminal end, resulting in a polypeptide containing 80% of the N-terminal amino acids of the native WNV protein (WN-80E), mixed with an adjuvant (GPI-0100). WN-80E was produced in a Drosophila melanogaster expression system with high yield and purified by immunoaffinity chromatography using a monoclonal antibody specific for flavivirus E proteins. Groups of monkeys were vaccinated with formulations containing 1 or 25 μg of WN-80E antigen, and both humoral and cellular immunity were assessed after vaccination. The results demonstrated potent antibody responses to vaccination, as determined by both enzyme-linked immunosorbent assay and virus-neutralizing antibody assays. All vaccinated animals responded favorably, and there was little difference in response between animals immunized with 1 or 25 μg of WN-80E. Cellular immunity was determined by lymphocyte proliferation and cytokine production assays using peripheral blood mononuclear cells from vaccinated animals stimulated in vitro with WN-80E. Cell-mediated immune responses varied from animal to animal within each group. About half of the animals responded with lymphoproliferation, cytokine production, or both. Again, there was little difference in response between animals immunized with a 1- or 25-μg dose of WN-80E in the vaccine formulations. In a separate experiment, groups of monkeys were immunized with the WN-80E/GPI-0100 vaccine or an adjuvant-only control formulation. Animals were then challenged by inoculation of wild-type WNV, and the level of viremia in each animal was monitored daily for 10 days. The results showed that whereas all animals in the control group had detectable viremia for at least 3 days after challenge, all of the vaccinated animals were negative on all days after challenge. Thus, the WN-80E vaccine was 100% efficacious in protecting monkeys against infection with WNV.

In Vivo Tumor Delivery of a Recombinant Single-Chain Fv::Tumor Necrosis Factor:  A Fusion Protein

2002 ◽  
Vol 13 (1) ◽  
pp. 7-15 ◽  
Author(s):  
Stephen P. Cooke ◽  
R. Barbara Pedley ◽  
R. Boden ◽  
Richard H. J. Begent ◽  
Kerry A. Chester
Keyword(s):  
Tumor Necrosis Factor ◽  
Fusion Protein ◽  
Tumor Necrosis ◽  
Single Chain ◽  
Single Chain Fv ◽  
Tumor Delivery ◽  
Necrosis Factor
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