title: Immunoconjugates of geldanamycin and anti-HER2 monoclonal antibodies: antiproliferative activity on human breast carcinoma cell lines.
abstract: BACKGROUND
  HER2 is a membrane receptor whose overexpression is strongly associated with poor prognosis in breast carcinomas. Inhibition of HER2 activity can reduce tumor growth, which led to the development of Herceptin, an anti-HER2 monoclonal antibody (MAb) that is already in clinical use. However, the objective response rate to Herceptin monotherapy is quite low. HER2 activity can also be inhibited by the highly cytotoxic antibiotic geldanamycin (GA). However, GA is not used clinically because of its adverse toxicity. Our purpose was to enhance the inhibitory activity of anti-HER2 MAb by coupling it to GA.
  
  
  METHODS
  We synthesized 17-(3-aminopropylamino)GA (17-APA-GA) and conjugated it to the anti-HER2 MAb e21, to form e21 : GA. The noninternalizing anti-HER2 MAb AE1 was used as a control. Internalization assays and western blot analyses were used to determine whether the anti-HER2 MAbs and their immunoconjugates were internalized into HER2-expressing cells and reduced HER2 levels. All statistical tests were two-sided.
  
  
  RESULTS
  The immunoconjugate e21 : GA inhibited the proliferation of HER2-overexpressing cell lines better than unconjugated e21 (concentration required for 50% inhibition = 40 versus 1650 microg/mL, respectively). At 15 microg/mL, e21 : GA reduced HER2 levels by 86% within 16 hours, whereas unconjugated e21, 17-APA-GA, or AE1 : GA reduced HER2 levels by only 20%. These effects were not caused by release of 17-APA-GA from the immunoconjugate because immunoconjugates containing [(3)H]GA were stable in serum at 37 degrees C. Furthermore, e21 : GA did not significantly inhibit proliferation of the adult T-cell leukemia cell line HuT102, which is HER2 negative yet highly sensitive to GA.
  
  
  CONCLUSIONS
  Our findings suggest that conjugating GA to internalizing MAbs enhances the inhibitory effect of the MAbs. This approach might also be applied in cellular targeting via growth factors and may be of clinical interest.
authors.affiliation: Metabolism Branch, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. rayam@box-r.nih.gov
authors.forename: R
authors.initials: R
authors.lastname: Mandler
authors.forename: C
authors.initials: C
authors.lastname: Wu
authors.forename: E A
authors.initials: EA
authors.lastname: Sausville
authors.forename: A J
authors.initials: AJ
authors.lastname: Roettinger
authors.forename: D J
authors.initials: DJ
authors.lastname: Newman
authors.forename: D K
authors.initials: DK
authors.lastname: Ho
authors.forename: C R
authors.initials: CR
authors.lastname: King
authors.forename: D
authors.initials: D
authors.lastname: Yang
authors.forename: M E
authors.initials: ME
authors.lastname: Lippman
authors.forename: N F
authors.initials: NF
authors.lastname: Landolfi
authors.forename: E
authors.initials: E
authors.lastname: Dadachova
authors.forename: M W
authors.initials: MW
authors.lastname: Brechbiel
authors.forename: T A
authors.initials: TA
authors.lastname: Waldmann
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000276:immunology / Q000494:pharmacology*; D000911:Antibodies, Monoclonal / Q000494:pharmacology* / Q000627:therapeutic use; D016227:Benzoquinones; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000276:immunology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D018796:Immunoconjugates; D047029:Lactams, Macrocyclic; D051379:Mice; D008807:Mice, Inbred BALB C; D011809:Quinones / Q000276:immunology / Q000494:pharmacology*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
chemical_list: D000903:Antibiotics, Antineoplastic; D000911:Antibodies, Monoclonal; D016227:Benzoquinones; D018796:Immunoconjugates; D047029:Lactams, Macrocyclic; D011809:Quinones; D018719:Erb-b2 Receptor Tyrosine Kinases; C001277:geldanamycin