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Immunology 2008 Lecture 27 Hybridomas 13 November

Immunology 2008 Lecture 27 Hybridomas 13 November. Properties of Conventional Antibodies (e.g. anti- k IgG3 myeloma protein). Polyclonal, heterogeneous, expensive. Ab to distinct targets : • anti- k isotype • anti-Km allotype • anti- g 3 isotype • anti-G3m allotype • anti-idiotype.

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Immunology 2008 Lecture 27 Hybridomas 13 November

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  1. Immunology 2008 Lecture 27 Hybridomas 13 November

  2. Properties of Conventional Antibodies (e.g. anti-kIgG3 myeloma protein) Polyclonal, heterogeneous, expensive Ab to distinct targets : • anti-k isotype • anti-Km allotype • anti-g3 isotype • anti-G3m allotype • anti-idiotype Each population of Abs may be directed against multiple epitopes and exhibits varying affinities.

  3. Properties of Conventional Antibodies (e.g. anti-kIgG3 myeloma protein) ctd. • Variation in amount and quality of Abs with time and between individuals. • Immunization, absorption, analysis are all labor-intensive. • Limited batches - always have to restart from scratch. Would like to be able to produce uniform Ab in unlimited amounts.

  4. How can we expand and immortalize the progeny of a single AFCP?

  5. “HAT” Selection “HAT” - Hypoxanthine+Aminopterin+Thymidine Begins with production of an aminopterin-sensitive “fusion partner”

  6. Only the products of cell fusion can grow in HAT Kohler and Milstein, 1975; Nobel Prize in Physiology or Medicine, 1984

  7. Production of Hybridoma Lines sample protocol • Immunize mice; final Ag boost @-24hr • Harvest spleens • Mix with standard fusion partner + PEG • Distribute into 96-well plates in HAT • Test supernatants for Ab • Identify desired clones • Subclone, expand, freeze ~108 cells ~107 cells 102-103 sups? 10-20 clones? Production of antibody: Culture supernatants: ~10mg/ml Ab Ascites fluids: ~10mg/ml Ab

  8. Properties of Hybridoma Antibodies ●Monoclonal and therefore homogenous (even if immunogen is complex or unknown) ● Immortal lines, produced in unlimited amounts ● Each has a unique specificity ● No batch-to-batch variation in specificity ● Can be shared, stored and propagated with no variation

  9. Examples of Screening Strategies For anti-Km: Immunize with kIgG myeloma Screen using ELISA against purified Km(1) and Km(2) k light chains For leukocyte markers: Immunize mice with rat WBCs Screen by IF against rat WBCs; look for Abs that bind only some target cells (aCD3,4; PMN or Mf markers, etc.) Other novel differentiation markers: Immunize with Hydra cells; Then screen for Ab which detects sub-populations

  10. Uses for Hybridoma Antibodies (1) Diagnostic & research reagents: ● Anti-drug and hormone Abs for assays (insulin, hCG, TSH, T3/T4, EGF, barbital, THC …) ● Reagents for blood and HLA typing Rare blood groups Replace fortuitous sera for HLA (?) ● Abs to tumor-associated antigens for diagnosis (e.g. PSA), for detection of metastases, etc. ● Define & detect new proteins & cell markers

  11. Uses for Hybridoma Antibodies (2) Therapeutic reagents: ● anti-CD3 – suppress renal & cardiac graft rejection ● anti-CD4 – immunosuppression; trials in RA ● anti-CD52 – purge BM grafts of T-cells (marker of lymphocytes & monocytes) ● anti-TSTA, conjugated (e.g. radio-label or toxin); tumor killing

  12. Immunotherapy for Follicular Lymphoma; anti-Idiotype as a Tumor-Specific Antibody (1) ● B-cell leukemia, bears membrane IgM ● Immunize mice with CML cells, screen hybridomas for anti-Ig idiotype ● Use for therapy Several early good responses, one 6-year remission; no longer-term cures.

  13. Immunotherapy for Follicular Lymphoma; anti-Idiotype as a Tumor-Specific Antibody (2) Limitations of such therapy: 1) Presence of secreted tumor IgM (“blocking factor”) 2) Immunoselection/immunomodulation (loss or modification of idiotype) (see “Tumor Immunology”…)

  14. Genetic Engineering of Monoclonal Antibodies (1) Human hybridomas are difficult to make, but rodent antibodies are immunogenic in humans and can cause serum sickness. Decrease immunogenicity by “humanizing” mAbs

  15. mouse anti-CD52 (immunogenic in humans) “transplant” CDRs Humanizing a Mouse Monoclonal Antibody generic monoclonal human IgG humanized anti-CD52 (“Alemtuzumab”)

  16. MONDAY Exam #2, 9:00 AM Covers all material since the first exam through this week (+ MLR & HLA genetics…) TUESDAY Tumor Immunology, Chapter 23 WEDNESDAY POPS #2, Transplantation Clinical Correlate #3, HIV/AIDS Dr. Robinson

  17. Fin

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