Abstract
CD44 is a transmembrane glycoprotein that binds to hyaluronic acid, plays roles in a number of cellular processes and is expressed in a variety of cell types. It is up-regulated in stem cells and cancer. Anti-CD44 monoclonal antibodies affect cell motility and aggregation, and repress tumorigenesis and metastasis. Here we describe four new anti-CD44 monoclonal antibodies originating from B cells of a mouse injected with a plasmid expressing CD44 isoform 12. The four monoclonal antibodies bind to the terminal, extracellular, conserved domain of CD44 isoforms. Based on differences in western blot patterns of cancer cell lysates, the four anti-CD44 mAbs separated into three distinct categories that include P4G9, P3D2, and P3A7, and P3G4. Spot assay analysis with peptides generated in Escherichia coli support the conclusion that the monoclonal antibodies recognize unglycosylated sequences in the N-terminal conserved region between amino acid 21–220, and analyses with a peptide generated in human embryonic kidney 293 cells, demonstrate that these monoclonal antibodies bind to these peptides only after deglycosylation. Western blots with lysates from three cancer cell lines demonstrate that several CD44 isoforms are unglycosylated in the anti-CD44 target regions. The potential utility of the monoclonal antibodies in blocking tumorigenesis was tested by co-injection of cells of the breast cancer-derived tumorigenic cell line MDA-MB-231 with the anti-CD44 monoclonal antibody P3D2 into the mammary fat pads of mice. All five control mice injected with MDA-MB-231 cells plus anti-IgG formed palpable tumors, while only one of the six test mice injected with MDA-MB-231 cells plus P3D2 formed a tiny tumor, while the remaining five were tumor-free, indicating that the four anti-CD44 mAbs may be useful therapeutically.
Highlights
CD44, a transmembrane glycoprotein with a large extracellular domain [1,2,3,4,5], was originally identified as a receptor for the extracellular matrix molecule hyaluronic acid [6,7,8,9]
These results demonstrate that the anti-CD44 monoclonal antibodies (mAbs) P3D2, when co-injected with cells of the breast cancer-derived, tumorigenic cell line MB-231 blocked subsequent tumorigenesis
For all of the preceding reasons, we developed a strategy for generating new mAbs against CD44, in which we co-inoculated mice with both a 15 amino acid peptide from the proximal extracellular conserved domain (CED2) of CD44 and an expression plasmid that expressed a protein with the amino acid sequence of the smallest CD44 isoform, isf12, which lacked the extracellular variable region (V2-V10)
Summary
CD44, a transmembrane glycoprotein with a large extracellular domain [1,2,3,4,5], was originally identified as a receptor for the extracellular matrix molecule hyaluronic acid [6,7,8,9]. Anti-CD44 mAbs have been shown to affect cancer cell motility and aggregation of breast tumor and melanoma cell lines in a 3D Matrigel model, in the apparent absence of hyaluronic acid (HA) [14, 15]. For investigating the role of CD44 in tumorigenesis and metastasis, there are over 140 commercially available anti-CD44 mAbs (S1 Table), the majority generated against peptides representing conserved regions of the protein. At least 38 CD44 mRNAs have been identified, and by protein separation methods, at least 21 isoforms [42] Many of these isoforms probably play specialized roles in different cellular functions and are cell type-specific. The mAbs did not bind to a similar peptide generated in the human embryonic kidney cell line HEK293, which was glycosylated If this HEK293 recombinant protein, representing amino acids (aa) 1
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