Humanization of an antibody recognizing a breast cancer specific epitope by CDR-grafting.
BACKGROUND
Muc1-H23 is a cell surface mucin that is expressed on normal breast luminal epithelial cells and over-expressed in most breast tumors. In addition, Muc-1 expressed by malignant cells is glycosylated differently than Muc-1 expressed by normal cells. This difference in glycosylation exposes a peptide epitope on malignant cells which is not exposed on normal cells. Murine monoclonal antibody H23 recognizes this epitope and stains 91% of breast cancers, but only 1/56 non-malignant breast tissue samples.


OBJECTIVE
To create a human antibody that was equivalent to H23 for potential uses in imaging and/or the therapy of breast cancer.


METHODS
We decided to humanize H23 by CDR-grafting using overlap PCR, and to this end, designed and constructed a bacterial expression vector that would allow V-regions, cloned via unique restriction sites, to be expressed as Fab fragments. In this way, we hoped to be able to rapidly evaluate Fab constructs for binding to Muc-1 and to cells and tissue sections that expressed the antigen.


RESULTS
A fully humanized Fab fragment was able to bind Muc-1 peptide, as well as breast cancer cells known to express the epitope and tissue sections, generally showing the same reactivity as the native antibody. In addition, an analysis of sFab expressed with a [His]6 tag preceded by a factor Xa proteolytic cleavage site suggested that E. coli periplasmic signal peptidase was able to cleave the factor Xa site, thereby removing the [His]6 tag.


CONCLUSIONS
We have generated a human antibody that is capable of recognizing a tumor specific epitope expressed by 91% of breast cancers.
Fiorentini
S
S
Instituto di Microbiologia, University degli Studi di Brescia, Italy.
Matczak
E
E
Gallo
R C
RC
Reitz
M S
MS
Keydar
I
I
Watkins
B A
BA
D000951:Antigens, Neoplasm / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000276:immunology*; D003001:Cloning, Molecular; D000939:Epitopes / Q000276:immunology*; D004926:Escherichia coli; D005260:Female; D005822:Genetic Vectors; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D018396:Mucin-1; D009077:Mucins; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification
D000951:Antigens, Neoplasm; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; C060192:MUC1 protein, human; D018396:Mucin-1; D009077:Mucins; D011993:Recombinant Fusion Proteins