Production of Bispecific Antibodies

David M. Segal1, Bert J.E.G. Bast2

1 National Cancer Institute, Bethesda, Maryland, 2 University Hospital, Utrecht, The Netherlands
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 2.13
DOI:  10.1002/0471142735.im0213s14
Online Posting Date:  May, 2001
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Abstract

Bispecific antibodies (bsAbs) contain two different binding specificities within a single molecule and can specifically bind two different molecules together. BsAbs can be produced by chemically cross‐linking purified antibodies or Fab fragments with reducible disulfide bonds or nonreducible thioether bonds, both of which are described in this unit. Protocols are also presented for producing BsAbs by fusing two antibody‐producing hybridomas that can be selected for based on drug resistance, or by double labeling with fluorochromes and FACS. Support protocols describe screening and purification of bsAbs.

     
 
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Table of Contents

  • Basic Protocol 1: Chemically Cross‐Linking Antibodies with SPDP
  • Alternate Protocol 1: Chemically Cross‐Linking Antibodies with Nonreducible Thioether Bonds
  • Basic Protocol 2: Production of Hybrid Hybridoma–Derived Antibodies
  • Alternate Protocol 2: Selecting Hybrid Hybridomas Using FACS
  • Support Protocol 1: Screening Procedures for Bispecific Antibodies
  • Support Protocol 2: Isolation of Bispecific Antibodies
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Chemically Cross‐Linking Antibodies with SPDP

  Materials
  • N‐succinimidyl‐3‐(2‐pyridyldithiol)propionate (SPDP, Pierce or Sigma)
  • 100% ethanol
  • 1 to 5 mg each of two purified IgG MAbs, affinity‐purified conventional IgG antibodies (unit 2.7), or Fab fragments (unit 2.8) at 10 mg/ml in borate buffer
  • recipeBorate buffer (unit 2.7)
  • recipeReducing buffer (see recipe)
  • DTT (Sigma)
  • Cytochrome c (Sigma)
  • Iodoacetamide (Sigma)
  • PD‐10 prepoured disposable Sephadex G‐25 column (Pharmacia Biotech)
  • Centricon‐30 microconcentrator (Amicon)
  • Sorvall RC5C centrifuge with SA600 rotor or equivalent
  • FPLC system with a Superose 12 HR 10/30 column (Pharmacia Biotech) or equivalent
  • Additional reagents and equipment for testing for bsAb activity (see protocol 5)

Alternate Protocol 1: Chemically Cross‐Linking Antibodies with Nonreducible Thioether Bonds

  • Succinimidyl‐4‐(p‐maleimidophenyl)butyrate (SMPB, Pierce)
  • Dimethylsulfoxide (DMSO)

Basic Protocol 2: Production of Hybrid Hybridoma–Derived Antibodies

  Materials
  • Two Ig‐producing hybridoma cell lines (unit 2.5)
  • RPMI‐10/HECS/Aza: recipecomplete RPMI‐10 ( appendix 2A) supplemented with 10% (v/v) human endothelial cell supernatant (HECS; Costar) and 20 µg/ml 8‐azaguanine (Aza; Sigma)
  • RPMI‐10/HAT: recipecomplete RPMI‐10 with 1× hypoxanthine/amethopterin/thymidine (HAT) supplement (e.g., Sigma)
  • recipeComplete RPMI‐10 ( appendix 2A) and recipeRPMI‐20
  • G418 (GIBCO/BRL)
  • RPMI‐10/HT: recipecomplete RPMI‐10 with 1× hypoxanthine/thymidine (HT) supplement (e.g., Sigma)
  • 0.1 µg/µl linearized pRc/RSV vector (Invitrogen), or equivalent vector containing the neomycin resistance gene, in recipeTE buffer ( appendix 2A)
  • Selection medium: recipecomplete RPMI‐10 supplemented with 10% (v/v) HECS, 1× HAT, and G418 at the optimal concentration (as determined in step below)
  • 175‐cm2 flasks
  • 96‐well flat‐bottom tissue culture plates
  • 50‐ml conical polypropylene centrifuge tubes
  • Additional reagents and equipment for electroporation (unit 10.15), screening for known antibody and bsAb activities (see protocol 5), polyethylene glycol–mediated cell fusion, and growth and expansion of hybridomas (units 2.5 & 2.6)NOTE: All reagents and equipment coming into contact with live cells must be sterile.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 2: Selecting Hybrid Hybridomas Using FACS

  • Two Ig‐producing hybridoma cell lines (unit 2.5) in log‐phase growth
  • recipeComplete RPMI medium ( appendix 2A), pH 6.8 and pH 7.2 (adjust pH with 1 M HCl)
  • 0.1 mg/ml FITC in dimethylsulfoxide (DMSO)
  • 0.1 mg/ml TRITC in DMSO
  • recipeComplete RPMI medium (pH unadjusted)
  • Culture medium: recipecomplete RPMI‐10 ( appendix 2A) supplemented with 10% (v/v) human endothelial cell supernatant (HECS; Costar)
  • Fluorescence‐activated cell sorter (FACS), preferably but not necessarily equipped with a single‐cell deposition system
  • Additional reagents and equipment for Ficoll‐Hypaque centrifugation (unit 7.1), polyethylene glycol–mediated cell fusion and growth and expansion of hybridomas (units 2.5 & 2.6), and testing for bsAb activity (see 2.13)NOTE: All reagents and equipment coming into contact with live cells must be sterile.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Screening Procedures for Bispecific Antibodies

  Materials
  • Ascites fluid or supernatant containing bsAb (unit 2.6)
  • 25 mM 2‐(N‐morpholine)ethane sulfonic acid (MES; Sigma), pH 5.4
  • Bakerbond ABx column 7.75 × 100 mm (Baker Chemical) connected to an FPLC system (Pharmacia Biotech)
  • 1 M sodium acetate, pH 7.0 (or other buffer of choice)
  • Sodium azide
  • recipePBS ( appendix 2A)
  • Additional reagents and equipment for dialysis ( appendix 3H) and quantitating protein (unit 2.11)
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Figures

Videos

Literature Cited

Literature Cited
   Behrsing, O., Kaiser, G., Karawajew, L., and Micheel, B. 1992. Bispecific IgA/IgM antibodies and their use in enzyme immunoassay. J. Immunol. Methods 156:69‐77.
   Carlson, J., Drevin, H., and Aven, R. 1978. Protein thiolation and reversible protein‐protein conjugation. N ‐succinimidyl‐3‐(2‐pyridyldithiol)propionate, a new heterobifunctional reagent. Biochem. J. 173:723‐737.
   Carter, P., Kelley, R.F., Rodrigues, M.L., Snedecor, B., Covarrubias, M., Velligan, M.D., Wong, W.L.T., Rowland, A.M., Kotts, C.E., Carver, M.E., Yang, M., Bourell, J.H., Shepard, H.M., and Henner, D. 1992. High level Escherichia coli expression and production of a bivalent humanized antibody fragment. Bio/Technology 10:163‐167.
   Clark, M.R. and Waldmann, H. 1987. T‐cell killing of target cells induced by hybrid antibodies: Comparison of two bispecific monoclonal antibodies. J. Natl. Cancer Inst. 79:1393‐1401.
   De Lau, W.B., Van Loon, A.E., Heije, K., Valerio, D., and Bast, B.J. 1989. Production of hybrid hybridomas based on HAT(s)‐neomycin(r) double mutants. J. Immunol. Methods 117:1‐8.
   De Lau, W.B.M., Heije, K., Neefjes, J.J., Oosterwegel, M., Rozemuller, E., and Bast, B.J.E.G. 1991. Absence of preferential homologous H/L chain association in hybrid hybridomas. J. Immunol. 146:906‐914.
   Fanger, M.W. 1995. Bispecific Antibodies. R.G. Landes, Austin, Tex.
   Glennie, M.J., McBride, H.M., Worth, A.T., and Stevenson, G.T. 1987. Preparation and performance of bispecific F(ab)′2 antibody containing thioether‐linked Fab′ fragments. J. Immunol. 139:2367‐2375.
   Haagen, I.A., Van de Griend, R., Clark, M., Geerars, A., Bast, B., and De Gast, B. 1992. Killing of human leukaemia/lymphoma B cells by activated cytotoxic T lymphocytes in the presence of a bispecific monoclonal antibody (αCD3/αCD19). Clin. Exp. Immunol. 90:368‐375.
   Karawajew, L., Micheel, B., Behrsing, O., and Gaestel, M. 1987. Bispecificv antibody–producing hybrid hybridomas selected by a fluorescence activated cell sorter. J. Immunol. Methods 96:265‐270.
   Karpovsky, B., Titus, J.A., Stephany, D.A., and Segal, D.M. 1984. Production of target‐specific effector cells using hetero‐crosslinked aggregates containing anti‐target cell and anti‐Fc gamma receptor antibodies. J. Exp. Med. 160:1686‐1701.
   Koolwijk, P., Rozemuller, E., Stad, R.K., De Lau, W.B., and Bast, B.J. 1988. Enrichment and selection of hybrid hybridomas by Percoll density gradient centrifugation and fluorescent‐activated cell sorting. Hybridoma 7:217‐225.
   Koolwijk, P., Spierenburg, G.T., Frasa, H., Boot, J.H., van de Winkel, J.G., and Bast, B.J. 1989. Interaction between hybrid mouse monoclonal antibodies and the human high‐affinity IgG FcR, huFc gamma RI, on U937. Involvement of only one of the mIgG heavy chains in receptor binding. J. Immunol. 143:1656‐1662.
   Kostelny, S.A., Cole, M.S., and Tso, J.Y. 1992. Formation of a bispecific antibody by the use of leucine zippers. J. Immunol. 148:1547‐1553.
   Mallender, W.D. and Voss, E.W. Jr. 1994. Construction,expression, and activity of a bivalent bispecific single‐chain antibody. J. Biol. Chem. 269:199‐206.
   Ross, A.H., Herlyn, D., and Koprowski, H. 1987. Purification of monoclonal antibodies from ascites using ABx liquid chromatography column. J. Immunol. Methods 102:227‐231.
   Shalaby, M.R., Shepard, H.M., Presta, L., Rodrigues, M.L., Beverley, P.C.L., Feldmann, M., and Carter, P. 1992. Development of humanized bispecific antibodies reactive with cytotoxic lymphocytes and tumor cells overexpressing the HER2 protooncogene. J. Exp. Med. 175:217‐225.
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