Cancer Vaccines

Tim F. Greten1, Elizabeth M. Jaffee1

1 Johns Hopkins University School of Medicine, Baltimore, Maryland
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 13.8
DOI:  10.1002/0471142905.hg1308s14
Online Posting Date:  May, 2001
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Abstract

This unit describes the use of retroviral vectors that can be successfully employed for gene transfer into both primary tumor cultures and established cell lines. The unit includes procedures for assaying the stability of the vaccine following gene transfer. Techniques for maintaining the retroviral producer lines and titering the retroviral vectors are also described. A protocol for frozen storage of the vaccine (transduced tumor cells) is provided. In addition, methods are described for characterizing the vaccine cells following gene transfer. Directions for testing the expression of the transferred gene in the transfected tumor line are also given. The final protocol provides suggestions for designing in vivo animal experiments and discusses what has to be observed in a clinical setting.

     
 
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Table of Contents

  • Basic Protocol 1: Gene Transfer into Tumor Cells and Preparation of Vaccine for Administration In Vivo
  • Support Protocol 1: Phenotyping of Tumor Cells
  • Support Protocol 2: Maintenance and Storage of Retroviral Vector Producer Lines
  • Support Protocol 3: Titering Retroviral Vectors
  • Support Protocol 4: Storage of Transduced Tumor Cells
  • Support Protocol 5: In Vitro Expression Testing of Cytokine‐Secreting Tumor Vaccines
  • Support Protocol 6: In Vivo Evaluation of Vaccine by Standard Protection Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Gene Transfer into Tumor Cells and Preparation of Vaccine for Administration In Vivo

  Materials
  • Tumor cell line (established long‐term or primary tumor line, phenotyped as described in protocol 2)
  • Tumor cell growth medium: e.g., as a general guideline, RPMI/10% FBS ( appendix 3G) with 50 µg/ml gentamycin (medium must be optimized; see Critical Parameters)
  • Retroviral vector producer line containing gene of interest, growing in tissue culture (see protocol 3)
  • DEAE‐dextran stock solution (see recipe)
  • PBS, pH 7.2 (see recipe), 4°C and 25°C
  • 100‐mm tissue culture dishes
  • 25‐cm2 and 75‐cm2 tissue culture flasks
  • 0.45‐µm cellulose nitrate syringe filters (Costar)
  • Tabletop centrifuge
  • 15‐ml and 50‐ml conical centrifuge tubes
  • 60Co or 137Cs γ irradiator (e.g., Gammacell 1000, Nordion International)
  • Additional reagents and equipment for growing mammalian cells in tissue culture and determining number of viable cells by trypan blue exclusion ( appendix 3G), maintaining, trypsinizing, and subculturing producer cells (see protocol 3), testing vaccine cells for cytokine secretion ( protocol 6), phenotyping tumor cells (see protocol 2), and preparing frozen stocks of tumor cells (see protocol 5)
NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Phenotyping of Tumor Cells

  Materials
  • Tumor cell line (established long‐term or primary tumor line)
  • Tumor cell growth medium: e.g., as a general guideline, RPMI/10% FBS ( appendix 3G) with 50 µg/ml gentamycin (medium must be optimized; see Critical Parameters)
  • Trypsin/EDTA solution (see recipe)
  • 1% (w/v) BSA in PBS (pH 7.2), 4°C (stable 4 to 6 weeks at 4°C)
  • PBS, pH 7.2 (see recipe), 4°C
  • 2% paraformaldehyde in PBS, pH 7.2 (prepare fresh), 4°C
  • Detergent solution (see recipe), 4°C
  • Antibodies:
  •  Mouse whole IgG1 purified from B8‐24‐3 hybridoma supernatant (ATCC #TIB‐139), to block nonspecific binding
  •  Fluorescein isothiocyanate (FITC)‐labeled mouse anti–cytokeratin 7 (Sigma)
  •  FITC‐labeled monoclonal mouse IgG1 (irrelevant control antibody; Sigma)
  • Tabletop centrifuge
  • 12 × 75–mm polystyrene tubes (e.g., Falcon 2052)
  • Additional reagents and equipment for growing mammalian cells in tissue culture and trypsinizing cells ( appendix 3G)
NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 2: Maintenance and Storage of Retroviral Vector Producer Lines

  Materials
  • Retroviral vector producer lines: MFG retroviral producer cell lines Psi CRIP and Psi CRE obtained from R.C. Mulligan at the above address (also see unit 12.5)
  • Retroviral producer line growth medium (see recipe)
  • Trypsin/EDTA solution (see recipe)
  • 162 cm2 tissue culture flasks
  • 50‐ml conical centrifuge tubes
  • Additional reagents and equipment for growing mammalian cells in tissue culture, trypsinizing cells, and determining number of viable cells by trypan blue exclusion ( appendix 3G)
NOTE: All incubations in this protocol should be carried out in a 37°C, 10% CO 2 incubator for optimal buffer capacity, as the retroviral producer cell growth medium is a DMEM‐containing medium.

Support Protocol 3: Titering Retroviral Vectors

  Materials
  • Transduced tumor cells (see protocol 1)
  • Trypsin/EDTA solution (see recipe)
  • 10% (v/v) DMSO/90% FBS (or other serum used for growth)
  • Liquid nitrogen
  • 1.8‐ml cryovials
  • Nalgene Cryo 1°C freezing container (e.g., Baxter) filled with isopropanol
  • Additional reagents and equipment for trypsinizing and determining number of viable cells by trypan blue exclusion ( appendix 3G)
NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 4: Storage of Transduced Tumor Cells

  Materials
  • Vaccine cells (see protocol 1)
  • Tumor cell growth medium: e.g., as a general guideline, RPMI/10% FBS ( appendix 3G) with 50 µg/ml gentamycin (medium must be optimized; see Critical Parameters)
  • 0.45‐µm cellulose nitrate syringe filter
  • 25‐cm2 tissue culture flasks
  • Commercially available cytokine‐specific ELISA kits (e.g., R&D Systems, Endogen, or Biosource)
  • Additional reagents and equipment for counting cells ( appendix 3G)
NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 5: In Vitro Expression Testing of Cytokine‐Secreting Tumor Vaccines

  • Mice (of same genetic background; strain depends on tumor model)
  • 1‐ml syringes with 26‐G needles
NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.
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Figures

Videos

Literature Cited

   Boon, T., Cerottini, J.C., Van den Eynde, B., van der Bruggen, P., and Van, Pel, A. 1994. Tumor antigens recognized by T lymphocytes. Annu. Rev. Immunol. 12:337‐365.
   Coligan, J.E., Kruisbeek, A.M., Margulies, D.M., Shevach, E.M., and Strober, W. (eds.) 1997. Current Protocols in Immunology. John Wiley & Sons, New York.
   Cornetta, K. and Anderson, W.F. 1989. Protamine sulfate as an effective alternative to polybrene in retroviral‐mediated gene‐transfer: Implications for human gene therapy. J. Virol. Methods 23:187‐194.
   Degiovanni, G., Hainaut, P., Lahaye, T., Weynants, P., and Boon, T. 1990. Antigens recognized on a melanoma cell line by autologous cytolytic T lymphocytes are also expressed on freshly collected tumor cells. Eur. J. Immunol. 20:1865‐1868.
   Dranoff, G., Jaffee, E., Lazenby, A., Golumbek, P., Levitsky, H., Brose, K., Jackson, V., Hamada, H., Pardoll, D., and Mulligan, R.C. 1993. Vaccination with irradiated tumor cells engineered to secrete murine granulocyte–macrophage colony‐stimulating factor stimulates potent, specific, and long‐lasting anti‐tumor immunity. Proc. Natl. Acad. Sci. U.S.A. 90:3539‐3543.
   Farrar, J.D. and Street, N.E. 1995. A synthetic standard DNA construct for use in quantification of murine cytokine mRNA molecules. Mol. Immunol. 32:991‐1000.
   Fung‐Leung, W.P., Schilham, M.W., Rahemtulla, A., and Kundig, T.M. 1991. CD8 is needed for development of cytotoxic T cells but not helper T cells. Cell 65:443‐449.
   Gearing, A.J.H. and Bird, C.B. 1987. Lymphokines and Interferons: A Practical Approach (M.J. Clemens, A.G. Morris, and A.J.H. Gearing, eds.) p. 296. IRL Press, Oxford.
   Hodge, J.W., Abrams, S., Schlom, J., and Kantor, J.A. 1994. Induction of antitumor immunity by recombinant vaccinia viruses expressing B7‐1 or B7‐2 costimulatory molecules. Cancer Res. 54:5552‐5555.
   Itaya, T., Yamagiwa, S., Okada, F., Oikawa, T., Kuzumaki, N., Takeichi, N., Hosokawa, M., and Kobayashi, H. 1987. Xenogenization of a mouse lung carcinoma (3LL) by transfection with an allogeneic class I major histocompatibility complex gene (H‐2Ld). Cancer Res. 47:3136‐3140.
   Jaffee, E.M., Dranoff, G., Cohen, L.K., Hauda, K.M., Clift, S., Marshall, F.F., Mulligan, R.C., and Pardoll, D.M. 1993. High efficiency gene transfer into primary human tumor explants without cell selection. Cancer Res. 53:2221‐2226.
   Jaffee, E.M., Hurwitz, H., and Pardoll, D.M. 1995. Gene modification of tumors. In Biologic Therapy of Cancer, 2nd ed. (V.T. DeVita, Jr., S. Hellman, and S.A. Rosenberg, eds.) pp. 774‐783. Lippincott, Philadelphia.
   Jaffee, E.M., Thomas, M.C., Huang, A.Y.‐C., Hauda, K., Levitsky, H.I., and Pardoll, D.M. 1996. Enhanced immune priming with spatial distribution of paracrine cytokine vaccines. J. Immunother. 19:176‐183.
   Koo, G.C., Dumont, F.J., Tutt, M., Hackett, J. Jr., and Kumar, V. 1986. The NK‐1.1(‐) mouse: A model to study differentiation of murine NK cells. J. Immunol. 137:3742‐3747.
   Kotani, H., Newton, P.B. III, Zhang, S., Chiang, Y.L., Otto, E., Weaver, L., Blaese, R.M., Anderson, W.F., and McGarrity, G.J. 1994. Improved methods of retroviral vector transduction and production for gene therapy. Hum. Gene Therapy 5:19‐28.
   Pardoll, D.M. 1995. Paracrine cytokine adjuvants in cancer immunotherapy. Annu. Rev. Immunol. 13:399‐415.
   Rahemtulla, A., Fung‐Leung, W.P., Schilham, M.W., Kundig, T.M., Sambhara, S.R., Narendran, A., Arabian, A., Wakeham, A., Paige, C.J., Zinkernagel, R.M., Miller, R.G., and Mak, T.W. 1991. Normal development and function of CD8+ cells but markedly decreased helper cell activity in mice lacking CD4. Nature 353:180‐184.
   Robinson, J.P., Darzynkiewicz, Z., Dean, P.N., Dressler, L.G., Rabinovitch, P.S., Stewart, C.C., Tanke, H.J., and Wheeless, L.L. (eds.). 1997. Current Protocols in Cytometry. John Wiley & Sons, New York.
   Shevach, E.M. 1997. Labeling cells in microtiter plates for determination of [3]thymidine uptake. In Current Protocols in Immunology (Coligan, J.E., Kruisbeek, A.M., Margulies, D.M., Shevach, E.M., and Strober, W., eds.) p. A.3D.1. John Wiley & Sons, New York.
   Simons, J.W., Jaffee, E.M., Weber, C.E., Levitsky, H.I., Nelson, W.G., Carducci, M.A., Lazenby, A.J., Cohen, L.K., Finn, C.C., Clift, S.M., Hauda, K.M., Beck, L.A., Leiferman, K.M., Owens, A.H. Jr., Piantadosi, S., Dranoff, G., Mulligan, R.C., Pardoll, D.M., and Marshall, F.F. 1997. Bioactivity of autologous irradiated renal cell carcinoma vaccines generated by ex vivo granulocyte–macrophage colony‐stimulating factor gene transfer. Cancer Res. 57:1537‐1546.
   Wilson, J.M., Jefferson, D.M., Chowdhury, J.R., Novikoff, P.M., Johnston, D.E., and Mulligan, R.C. 1988. Retrovirus‐mediated transduction of adult hepatocytes. Proc. Natl. Acad Sci. U.S.A. 85:3014‐3018.
Key Reference
   Pardoll, D.M. 1993. Cancer vaccines. Immunol. Today 14:310‐316.
  This paper gives a broad overview on current basics in immunotherapy.
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