Anti-melanoma antibodies from melanoma patients immunized with genetically modified autologous tumor cells: selection of specific antibodies from single-chain Fv fusion phage libraries.

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.

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Increased non-major histocompatibility complex-restricted lytic activity in melanoma patients vaccinated with cytokine gene-transfected autologous tumor cells

Cancer Gene Therapy ◽

10.1038/sj.cgt.7700203 ◽

2000 ◽

Vol 7 (7) ◽

pp. 976-984 ◽

Cited By ~ 17

Author(s):

Peter Möller ◽

Hjördis Möller ◽

Yuansheng Sun ◽

Tomislav Dorbic ◽

Beate M Henz ◽

...

Keyword(s):

Major Histocompatibility Complex ◽

Tumor Cells ◽

Lytic Activity ◽

Autologous Tumor ◽

Cytokine Gene ◽

Major Histocompatibility ◽

Histocompatibility Complex ◽

Melanoma Patients

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Selection of human antitumor single-chain Fv antibodies from the B-cell repertoire of patients immunized against autologous neuroblastoma

Medical and Pediatric Oncology ◽

10.1002/1096-911x(20001201)35:6<692::aid-mpo45>3.0.co;2-7 ◽

2000 ◽

Vol 35 (6) ◽

pp. 692-695 ◽

Cited By ~ 4

Author(s):

Claudia Rossig ◽

Jed G. Nuchtern ◽

Malcolm K. Brenner

Keyword(s):

B Cell ◽

Single Chain ◽

Cell Repertoire ◽

B Cell Repertoire ◽

Single Chain Fv ◽

Selection Of

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Generation and Selection of a Panel of Pan-Filovirus Single-Chain Antibodies using Cell-Free Ribosome Display

10.1101/327296 ◽

2018 ◽

Author(s):

Adinarayana Kunamneni ◽

Elizabeth C. Clarke ◽

Chunyan Ye ◽

Steven B. Bradfute ◽

Ravi Durvasula

Keyword(s):

Hemorrhagic Fever ◽

Cross Reactivity ◽

Free Ribosome ◽

Rapid Diagnostics ◽

Single Chain ◽

Ribosome Display ◽

Single Chain Antibodies ◽

Single Chain Fv ◽

New Generation ◽

Selection Of

AbstractFiloviruses, which include ebolaviruses and marburgvirus, can cause outbreaks of highly lethal hemorrhagic fever. This disease causes significant morbidity and mortality in humans and non-human primates, with human fatality rates reaching 90% during some outbreaks. Currently, there are a lack of licensed vaccines or antivirals for these viruses. Since early symptoms of filovirus infection mimic more common diseases, there is a strong unmet public health and biodefense need for broad-spectrum filovirus rapid diagnostics. We have generated a panel of mouse single-chain Fv-antibodies (scFvs) to filovirus glycoproteins (GPs) using cell-free ribosome display and determined their cross-reactivity profiles to all known filovirus species. Two scFvs (4-2 and 22-1) were able to detect all known Ebolavirus and Marburgvirus species. This is the first report on ribosome display scFvs that can detect a broad set of filovirus GPs, which demonstrates their potential use in the development of a new generation of rapid diagnostic immunoassays.

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