Human antibodies that have MN binding and cell adhesion-neutralizing activity
The invention is composed of monoclonal human MN antibodies or MN antibody fragments that target the GEEDLP (SEQ ID NO: 118) repeat within the proteoglycan domain. The proteoglycan domain of the MN cell surface protein contains four of these identical GEEDLP (SEQ ID NO: 118) repeats. Binding to the desired epitope is verified by competition ELISA, where ELISA signal can be attenuated by co-incubation with a peptide containing this repeat (PGEEDLPGEEDLP (SEQ ID NO: 119)). This inhibition of binding can also be verified using Biacore assays, where binding of desired antibodies to immobilized MN or proteoglycan peptides can be inhibited by the peptide repeat. In addition to binding to the peptide repeat, human anti-MN antibodies can inhibit the cell adhesion of CGL-1 cells to MN coated plastic plates. Human anti-MN antibodies have been used to diagnose and quantify MN expression in cancer cells and tumors using FACS and immunohistochemical methods. An example is also provided where a human anti-MN IgG1 mediates tumor cell lysis though antibody-dependent cell-mediated cytotoxicity. Therefore, these antibodies will be useful for the treatment of cancers in which MN is upregulated or can be useful for the diagnosis of cancers in which MN is upregulated.
1. A purified preparation of a human antibody comprising:
a) a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 100, wherein
the variable heavy chain CDR3 region of SEQ ID NO: 100 is replaced by an amino acid sequence selected from the group consisting of SEQ ID NOS: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, and 80, and
the variable heavy chain CDR1 region of SEQ ID NO: 100 is optionally replaced by an amino acid sequence selected from the group consisting of SEQ ID NOS: 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60; and
b) a variable light chain comprising an amino acid sequence of SEQ ID NO: 94 or 95, wherein
the variable light chain CDR3 region of SEO ID NO: 94 or 95 is replaced by an amino acid sequence selected from the group consisting of SEQ ID NOS: 81, 84, 85, 86, 87, 88, and 89, and
the variable light chain CDR1 region of SEQ ID NO: 94 or 95 is optionally replaced by an amino acid sequence of SEQ ID NO: 82 or 83, wherein the antibody binds to a GEEDLP (SEQ ID NO: 118) repeat region within a MN protein's proteoglycan domain.
2. The purified preparation of claim 1 wherein the variable heavy chain CDR3 region is the amino acid of SEQ ID NO: 64.
3. The purified preparation of claim 1 wherein the variable light chain CDR3 region is the amino acid sequence of SEQ ID NO: 81.
4. The purified preparation of claim 1 wherein the variable light chain CDR3 region is the amino acid sequence selected from the group consisting of SEQ ID NOS: 84,85,86,87,88, and 89.
5. The purified preparation of claim 1 wherein the variable heavy chain CDR3 region and the variable light chain CDR3 region, respectively, are the amino acid sequence pairs selected from the group consisting of SEQ ID NOS: 61 and 84, SEQ ID NOS: 62 and 87, SEQ ID NOS: 63 and 89, SEQ ID NOS: 64 and 84, SEQ ID NOS: 65 and 84, SEQ ID NOS: 66 and 85, and SEQ ID NOS: 67 and 88.
6. The purified preparation of claim 1 wherein the variable heavy chain CDR3 region and the variable light chain CDR3 region, respectively, are the amino acid sequence pairs selected from the group consisting of SEQ ID NOS: 61 and 86, SEQ ID NOS: 61 and 85, SEQ ID NOS: 61 and 87, SEQ ID NOS: 61 and 88, SEQ ID NOS: 61 and 89, SEQ ID NOS: 63 and 86, SEQ ID NOS: 63 and 85, SEQ ID NOS: 63 and 87, SEQ ID NOS: 63 and 88, and SEQ ID NOS: 63 and 84.
7. The purified preparation of claim 1 wherein the variable heavy chain CDR3 region and the variable light chain CDR3 region, respectively, are the amino acid sequence pairs selected from the group consisting of SEQ ID NOS: 71 and 87, SEQ ID NOS: 61 and 87, SEQ ID NOS: 72 and 87, SEQ ID NOS: 73 and 87, SEQ ID NOS: 74 and 87, SEQ ID NOS: 75 and 87, SEQ ID NOS: 76 and 87, SEQ ID NOS: 77 and 87, SEQ ID NOS: 78 and 87, SEQ ID NOS: 79 and 87, and SEQ ID NOS: 80 and 87.
8. The purified preparation of claim 1 wherein the variable heavy chain CDR3 region and the variable light chain CDR3 region, respectively, are the amino acid sequence pairs selected from the group consisting of SEQ ID NOS: 61 and 81, SEQ ID NOS: 69 and 81, and SEQ ID NOS: 70 and 81.
9. The purified preparation of claim 1 , wherein the variable heavy chain CDR3 region, the variable light chain CDR3 region, and the variable heavy chain CDR1 region, respectively, are the amino acid sequences selected from the group consisting of SEQ ID NOS: 61, 86 and 48; SEQ ID NOS: 61, 86, and 49; SEQ ID NOS: 61, 86, and 50; SEQ ID NOS: 61, 86, and 51; SEQ ID NOS: 61, 86, and 52; SEQ ID NOS: 61, 86, and 53; SEQ ID NOS: 61, 86, and 54; SEQ ID NOS: 61, 86, and 55; SEQ ID NOS: 61, 86, and 56; SEQ ID NOS: 61, 86, and 57; SEQ ID NOS: 61, 86, and 82; and SEQ ID 61, 86, and 83.
10. A composition comprising a human antibody of claim 1 and a parmaceutically acceptable carrier.