Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Jan 7;27(2):250-9.
doi: 10.1016/j.vaccine.2008.10.055. Epub 2008 Nov 8.

Enhanced immune stimulation by a therapeutic lymphoma tumor antigen vaccine produced in insect cells involves mannose receptor targeting to antigen presenting cells

David J Betting  1 , Xi Y Mu, Kamran Kafi, Desmond McDonnel, Francisco Rosas, Daniel P Gold, John M Timmerman
Affiliations

Enhanced immune stimulation by a therapeutic lymphoma tumor antigen vaccine produced in insect cells involves mannose receptor targeting to antigen presenting cells

David J Betting et al. Vaccine. .

Abstract

Therapeutic vaccination of lymphoma patients with tumor-specific immunoglobulin (idiotype, Id) coupled to the carrier protein keyhole limpet hemocyanin (Id-KLH) is undergoing clinical investigation, and methods to improve the immunogenicity of these and other protein tumor antigen vaccines are being sought. Id proteins can be produced via tumor-myeloma hybridomas or recombinant methods in mammalian, bacteria, or insect cells. We now demonstrate that terminal mannose residues, characteristic of recombinant proteins produced in insect cells, yield Id proteins with significantly enhanced immunostimulatory properties compared to Id proteins derived from mammalian cells. Recombinant baculovirus-infected insect cell-derived Id showed higher binding to and activation of human dendritic cells mediated by mannose receptors. In vivo, insect cell-derived Id elicited higher levels of tumor-specific CD8+ cytotoxic T lymphocyte (CTL) and improved eradication of pre-established murine lymphoma. Insect cell and mammalian Id generated similar levels of tumor-specific antibodies, showing no impairment in antibody responses to native tumor antigen despite the glycoslylation differences in the immunogen. Combining insect cell production and maleimide-based KLH conjugation offered the highest levels of anti-tumor immunity. Our data comparing sources of recombinant Id protein tumor antigens used in therapeutic cancer vaccines demonstrate that insect cell-derived antigens can offer several immunologic advantages over proteins derived from mammalian sources.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Comparative carbohydrate analysis of hybridoma-derived (Hyb) and insect cell-derived (BV) A20 Id proteins. Oligosaccharides were cleaved from purified proteins using PNGase F, stained with APTS, and analyzed by capillary electrophoresis alongside oligosaccharide standards. The elution profile is depicted as relative fluorescence units (RFU) over time. Hyb-derived Id contains oligosaccharides with terminal galactose (G) residues typical of Igs. Only the insect cell-derived Id has a high content of terminal mannose (Man) residues.
Fig. 2
Fig. 2
Human DC mannose receptors allow for increased binding of insect cell-derived Igs. (A) Human DCs were incubated at 4 °C with FITC-labeled KLH, human IgG-KLH, or recombinant BV-derived human IgG-KLH. IgGs were conjugated to KLH via glutaraldehyde and evaluated for surface binding. (B) Murine A20 Id produced in both BV or Hyb systems were labeled with Alexa 647 and incubated for 2 h at 37 °C with human DCs to determine surface binding of the Igs. (C) BV A20 Id-Alexa 647, Hyb A20 Id-Alexa 647, or FITC-dextran were incubated with human DCs in the presence of various inhibitors including mannan, control IgG antibody, anti-MR blocking antibody, or cytochalasin D, and bound antigen quantitated by flow cytometry.
Fig. 3
Fig. 3
BV Id proteins stimulate expression of human DC activation markers over that seen with Hyb Id. Surface expression of (A) CD80 and (B) CCR7 in the presence of BV- or Hyb-derived glutaraldehyde Id-KLH conjugates. (C) CD80 and CCR7 surface expression after pre-treatment with an anti-MR blocking antibody. Error bars in A & B represent standard deviation of n = 5 replicates for 0 and 50 μg samples and n = 4 for 100 μg samples.
Fig. 4
Fig. 4
Both BV Id production and maleimide conjugation result in improvements over traditional Hyb Id production and glutaraldehyde conjugation. (A) Four days after s.c. inoculation with A20 tumor cells, groups of mice received either two or three weekly s.c. vaccinations with BV-derived (n = 96) or Hyb-derived (n = 96) Id conjugated to KLH via either maleimide (n = 72) or glutaraldehyde (n = 120), plus locally administered GM-CSF as an adjuvant. Data from three individual experiments having identical endpoint criteria were pooled. Tumor eradication was greatest using BV Id and maleimide conjugation. (B) BV and Hyb Id-KLH groups were pooled, showing a greater proportion of survivors with insect cell-derived Id proteins. (C) Id-KLH maleimide and glutaraldehyde groups were pooled, showing superiority of maleimide-conjugated Id proteins compared to glutaraldehyde conjugates.
Fig. 5
Fig. 5
CD8+ T cells in mice vaccinated with insect cell-derived Id are required for tumor eradication and demonstrate enhanced effector function. (A) BV glutaraldehyde Id-KLH elicits greater CTL activity than Hyb glutaraldehyde Id-KLH. Mice were given two bi-weekly vaccinations, after which CTLs from the spleen and lymph nodes were tested for their ability to lyse A20 lymphoma cells at an E:T ratio of 10:1. (B) Dependence of vaccine efficacy on CD8+ T cells. BALB/c mice were pre-depleted of CD4+ or CD8+ T cells, inoculated s.c. with A20 tumor and 4 days later given three weekly vaccinations of BV glutaraldehyde Id-KLH plus GM-CSF, and followed for survival.
Fig. 6
Fig. 6
Vaccination with BV Id-KLH maleimide conjugate results in higher tumor antigen-specific antibody titers than glutaraldehyde conjugates. Groups of four mice were vaccinated with BV or Hyb Id conjugated to KLH via either maleimide or glutaraldehyde weekly for 3 weeks together with GM-CSF. Mice were bled 10 days later and anti-Id antibody titers determined by ELISA. Error bars represent standard deviations among serum values from individual mice.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Timmerman J.M. Therapeutic idiotype vaccines for non-Hodgkin's lymphoma. Adv Pharmacol (San Diego, Calif) 2004;51:271–293. - PubMed
    1. Rosenberg S.A. Progress in human tumour immunology and immunotherapy. Nature. 2001;411(May(6835)):380–384. - PubMed
    1. Timmerman J.M., Levy R.L. The history of the development of vaccines for lymphoma. Clin Lymph. 2000;1:129–139. - PubMed
    1. Kwak L.W., Campbell M.J., Czerwinski D.K., Hart S., Miller R.A., Levy R. Induction of immune responses in patients with B-cell lymphoma against the surface-immunoglobulin idiotype expressed by their tumors. N Engl J Med. 1992;327(17):1209–1215. - PubMed
    1. Hsu F.J., Caspar C.B., Czerwinski D., Kwak L.W., Liles T.M., Syrengelas A. Tumor-specific idiotype vaccines in the treatment of patients with B-cell lymphoma—long-term results of a clinical trial. Blood. 1997;89(9):3129–3135. - PubMed

Publication types

MeSH terms