ABSTRACT
Human epidermal growth factor receptor (HER) family proteins are currently major targets of therapeutic monoclonal antibodies against various epithelial cancers. However, the resistance of cancer cells to HER family-targeted therapies, which may be caused by cancer heterogeneity and persistent HER phosphorylation, often reduces overall therapeutic effects. We herein showed that a newly discovered molecular complex between CD98 and HER2 affected HER function and cancer cell growth. The immunoprecipitation of the HER2 or HER3 protein from lysates of SKBR3 breast cancer (BrCa) cells revealed the HER2-CD98 or HER3-CD98 complex. The knockdown of CD98 by small interfering RNAs inhibited the phosphorylation of HER2 in SKBR3 cells. A bispecific antibody (BsAb) that recognized the HER2 and CD98 proteins was constructed from a humanized anti-HER2 (SER4) IgG and an anti-CD98 (HBJ127) single chain variable fragment, and this BsAb significantly inhibited the cell growth of SKBR3 cells. Prior to the inhibition of AKT phosphorylation, BsAb inhibited the phosphorylation of HER2, however, significant inhibition of HER2 phosphorylation was not observed in anti-HER2 pertuzumab, trastuzumab, SER4 or anti-CD98 HBJ127 in SKBR3 cells. The dual targeting of HER2 and CD98 has potential as a new therapeutic strategy for BrCa.
Subject(s)
Breast Neoplasms , Humans , Female , Breast Neoplasms/pathology , Receptor, ErbB-2/metabolism , Trastuzumab/pharmacology , Trastuzumab/metabolism , Trastuzumab/therapeutic use , Antibodies, Monoclonal/metabolism , Phosphorylation , Cell Line, TumorABSTRACT
BACKGROUND: Fibroblast growth factor 8-isoform b (FGF8b) has been detected in human clinical sex-organ related cancers including hormone-refractory prostate cancer. There are, however, few relevant experimental models. A murine monoclonal anti-FGF8 antibody, KM1334, has been shown to neutralize FGF8b and inhibit the growth of androgen-dependent mouse mammary SC-3 cells in vitro and in vivo. In the present study, we evaluated the anti-tumor activity of KM1334 against androgen-dependent and -independent progression of FGF8b-expressing human prostate cancer xenografts. METHODS: FGF8b cDNA was transfected into androgen-dependent human prostate cancer cell line LNCaP, and its xenograft tumors were established subcutaneously in SCID mice with or without castration. KM1334 at the dose of 400 microg/head was injected twice weekly. RESULTS: FGF8b-expressing LNCaP cells secreted FGF8b, showed enhanced level of Erk1/2 phosphorylation, and showed more potent growth properties than mock-expressing cells in vitro and in vivo. KM1334 reduced these properties in vitro, inhibited tumorigenecity in vivo (T/C=0.33), and showed anti-tumor activity against established tumors (T/C=0.47) of FGF8b-expressing cells. FGF8b-expressing LNCaP tumors were androgen-dependent. However, they recurred as androgen-independent FGF8b positive tumors after castration. KM1334 also inhibited the growth of established FGF8b-expressing tumors in the androgen-independent states (T/C=0.47). CONCLUSIONS: These results indicate that humanized monoclonal antibodies, conserving the paratope of KM1334, are a promising candidate for therapy of FGF8b-expressing clinical prostate cancers. Follow-up studies using xenograft models with clinical FGF8b-expressing tumors are required to validate these early findings.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Fibroblast Growth Factor 8/immunology , Prostatic Neoplasms/drug therapy , Animals , Antibodies, Monoclonal/immunology , Antineoplastic Agents/immunology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Immunologic , Female , Fibroblast Growth Factor 8/genetics , Fibroblast Growth Factor 8/metabolism , Humans , Immunohistochemistry , Male , Mice , Mice, Inbred BALB C , Mice, Inbred ICR , Mice, SCID , Mitogen-Activated Protein Kinase 3/metabolism , Mitogen-Activated Protein Kinase 6/metabolism , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Transfection , Xenograft Model Antitumor AssaysABSTRACT
Designing small molecules that mimic the receptor-binding local surface structure of large proteins such as cytokines or growth factors is fascinating and challenging. In this study, we designed cyclic peptides that reproduce the receptor-binding loop structures of G-CSF. We found it is important to select a suitable linker to join two or more discontinuous sequences and both termini of the peptide corresponding to the receptor-binding loop. Structural simulations based on the crystallographic structure of KW-2228, a stable and potent analog of human G-CSF, led us to choose 4-aminobenzoic acid (Abz) as a part of the linker. A combination of 4-Abz with beta-alanine or glycine, and disulfide bridges between cysteins or homocysteins, gave a structure suitable for receptor binding. In this structure, the side-chains of several amino acids important for the interactions with the receptor are protruding from one side of the peptide ring. This artificial peptide showed G-CSF antagonistic activity in a cell proliferation assay.
Subject(s)
Granulocyte Colony-Stimulating Factor/chemistry , Peptides, Cyclic/chemistry , Receptors, Granulocyte Colony-Stimulating Factor/chemistry , 4-Aminobenzoic Acid/chemistry , Amino Acids/chemistry , Binding Sites , Cell Proliferation , Crystallography, X-Ray , Cysteine/chemistry , Disulfides/chemistry , Glycine/chemistry , Homocysteine/chemistry , Humans , Ligands , Models, Chemical , Models, Molecular , Protein Binding , Protein Conformation , beta-Alanine/chemistryABSTRACT
PURPOSE: Fibroblast growth factor 8b (FGF8b) has been implicated in oncogenesis of sex hormone-related malignancies. A murine monoclonal anti-FGF8 antibody, KM1334, has been raised against a FGF8b-derived peptide and shown to neutralize FGF8b activity in an androgen-dependent mouse mammary cell line (SC-3) in vitro growth. The purpose of this study was to evaluate KM1334 as a therapeutic agent for FGF8-dependent cancer. EXPERIMENTAL DESIGN: Specificity and neutralizing activity of KM1334 were examined in vitro. In vivo therapeutic studies were done in nude mice bearing SC-3 tumors s.c. RESULTS: KM1334 recognized FGF8b and FGF8f specifically out of four human FGF8 isoforms and showed little binding to other members of FGF family. Neutralizing activity of KM1334 was confirmed by both blocking of FGF8b binding to its three receptors (FGFR2IIIc, FGFR3IIIc, and FGFR4) and FGF8b-induced phosphorylation of FGFR substrate 2alpha and extracellular signal-regulated kinase 1/2 in SC-3 cells. The in vitro inhibitory effect could be extended to in vivo tumor models, where KM1334 caused rapid regression of established SC-3 tumors in nude mice. This rapid regression of tumors after KM1334 treatment was explained by two independent mechanisms: (a) decreased DNA synthesis, as evidenced by a decrease in uptake of 5-bromo-2'-deoxyuridine, and (b) induction of apoptosis as shown by the terminal deoxynucleotidyl transferase-mediated nick end labeling assay. CONCLUSIONS: KM1334 possesses strong blocking activity in vitro and antitumor activity in vivo and therefore may be an effective therapeutic candidate for the treatment of cancers that are dependent on FGF8b signaling for growth and survival.
