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.

Acquisition of potential N-glycosylation sites in the immunoglobulin variable region by somatic mutation is a distinctive feature of follicular lymphoma

Blood ◽  
2002 ◽  
Vol 99 (7) ◽  
pp. 2562-2568 ◽  
Author(s):  
Delin Zhu ◽  
Helen McCarthy ◽  
Christian H. Ottensmeier ◽  
Peter Johnson ◽  
Terry J. Hamblin ◽  
...  
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Somatic Mutation ◽  
Germinal Center ◽  
Variable Region ◽  
B Cell Lymphoma ◽  
Single Chain ◽  
Single Chain Fv ◽  
Glycosylation Sites ◽  
Vh Genes

Most patients with follicular lymphoma (FL) have somatically mutated V genes with intraclonal variation, consistent with location in the germinal center site. Using our own and published sequences, we have investigated the frequency of potential N-glycosylation sites introduced into functional VH genes as a consequence of somatic mutation. FL cells were compared with normal memory B cells or plasma cells matched for similar levels of mutation. Strikingly, novel sites were detected in 55 of 70 (79%) patients with FL, compared to 7 of 75 (9%) in the normal B-cell population (P < .001). Diffuse large B-cell lymphoma (DLCL) showed an intermediate frequency (13 of 32 [41%] patients). Myeloma and the mutated subset of chronic lymphocytic leukemia showed frequencies similar to those of normal cells in 5 of 64 (8%) patients and 5 of 40 (13%) patients, respectively. In 3 of 3 random patients with FL, immunoglobulin was expressed as recombinant single-chain Fv inPichia pastoris, and glycosylation was demonstrated. These findings indicate that N-glycosylation of the variable region may be common in FL and in a subset of DLCL. Most novel sites are located in the complementarity-determining regions. VH sequences of nonfunctional VH genes contained few sites, arguing for positive selection in FL. One possibility is that the added carbohydrate in the variable region contributes to interaction with elements in the germinal center environment. This common feature of FL may be critical for tumor behavior.

scholarly journals Idiotypic vaccination against human B-cell lymphoma. Rescue of variable region gene sequences from biopsy material for assembly as single-chain Fv personal vaccines

Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3279-3288 ◽  
Author(s):  
RE Hawkins ◽  
D Zhu ◽  
M Ovecka ◽  
G Winter ◽  
TJ Hamblin ◽  
...  
Keyword(s):  
B Cell ◽  
Tumor Antigens ◽  
Variable Region ◽  
B Cell Lymphoma ◽  
Repeated Sequences ◽  
Expression Vectors ◽  
Single Chain ◽  
Region Gene ◽  
Biopsy Material ◽  
Single Chain Fv

Abstract Idiotypic determinants on neoplastic B cells could provide tumor antigens for vaccination of patients with B-cell tumors. Because this approach requires an individual vaccine for each patient, simple methods for obtaining idiotypic antigen are desirable. Using polymerase chain reaction (PCR) with family-based V-gene and J-region primers, the variable region genes of heavy and light chains (VH and VL) of Ig have been obtained from biopsy material from 13 patients with B-cell tumors. In each case, analysis of random clones derived from the PCR product showed repeated, clonally-related sequences, whereas normal lymphoid tissue generated no repeated sequences. In 3/3 cases, the repeated sequences were found to be the same as those in a tumor-derived hybridoma. Mutational patterns in the V-genes differed among the tumors, with follicular lymphoma tending to be more highly mutated. The individual VH and VL sequences have been assembled with a flexible linker sequence to encode single-chain Fv (scFv). The scFv sequences can be cloned into bacterial expression vectors to produce protein, or into vectors suitable for direct vaccination using naked DNA. In a model system, expressed scFv protein retained all idiotypic determinants defined by a panel of five anti-idiotypic monoclonal antibodies (MoAbs). Similarly, expressed scFv proteins from two patients were shown to react with anti-idiotypic antibodies. This approach allows production of potential vaccines from surgical biopsies within 2 to 3 weeks.

An optimized fermentation process for high-level production of a single-chain Fv antibody fragment in Pichia pastoris

2004 ◽  
Vol 37 (1) ◽  
pp. 18-26 ◽  
Author(s):  
Leonardo M Damasceno ◽  
Itzcoatl Pla ◽  
Hyun-Joo Chang ◽  
Leonard Cohen ◽  
Gerd Ritter ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Fermentation Process ◽  
Antibody Fragment ◽  
Single Chain ◽  
Single Chain Fv ◽  
Fv Antibody ◽  
High Level ◽  
Level Production ◽  
Single Chain Fv Antibody

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.

Linker length effects in the conformation of M3 anti-CEA single chain Fv antibody fragment

2017 ◽  
Vol 176 (5) ◽  
Author(s):  
Lincidio Pérez Sánchez ◽  
Monica Bequet-Romero ◽  
Joem M. Serrano ◽  
Hanssel Bell Garcia ◽  
Marta Ayala Avila ◽  
...  
Keyword(s):  
Antibody Fragment ◽  
Single Chain ◽  
Linker Length ◽  
Single Chain Fv ◽  
Fv Antibody ◽  
Single Chain Fv Antibody ◽  
Length Effects
Sign in / Sign up

Export Citation Format

Share Document