Fusion proteins comprising antibody and HPV E6 and E7 antigens
The present invention includes compositions and methods for the expression, secretion and use of novel compositions for use as, e.g., vaccines and antigen delivery vectors, to delivery antigens to antigen presenting cells. In one embodiment, the vector is an anti-CD40 antibody, or fragments thereof, and one or more antigenic peptides linked to the anti-CD40 antibody or fragments thereof, including humanized antibodies.
1. A fusion protein comprising the formula:
Ab-(Ag-PL-Ag)x
wherein Ab is an antibody or antigen binding fragment thereof;
PL is at least one peptide linker comprising at least one glycosylation site;
Ag is at least one cancer antigen; and
x is an integer from 1 to 20,
wherein the Ag is selected from tumor associated antigens selected from human papilloma virus (HPV) E6 and E7 and the PL is selected from SSVSPTTSVHPTPTSVPPTPTKSSP (SEQ ID NO.: 11), PTSTPADSSTITPTATPTATPTIKG (SEQ ID NO.: 12), TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO.: 13) and TNGSITVAATAPTVTPTVNATPSAA (SEQ ID NO.: 14).
2. The fusion protein of claim 1 , wherein the fusion protein has more stability in solution than the same fusion protein without the glycosylation site.
3. The fusion protein of claim 1 , wherein the Ag is 19 to 32 amino acids long.
4. The fusion protein of claim 1 , wherein x is 2, 3, 4, 5, 6, 7, 8, 9, or 10.
5. The fusion protein of claim 1 , wherein the fusion protein comprises an E6 and E7 antigen.
6. The fusion protein of claim 1 , wherein the Ab comprises at least the variable region of the antibody anti-CD40 — 12E12.3F3 (ATCC Accession No. PTA-9854), anti-CD40 — 12B4.2C10 (ATCC Submission No. HS446, Accession No. PTA-10653), or anti-CD40 — 11B6.1C3 (ATCC Submission No. HS440, Accession No. PTA-10652).
7. The fusion protein of claim 1 , wherein the Ab comprises at least one variable domain having 100% sequence identity with a heavy chain variable domain of SEQ II) NOS.: 148, 150 or 153 or a light chain variable domains of SEQ ID NOS.: 149, 151, 152 or 154, or both.
8. The fusion protein of claim 7 , wherein the Ab comprises at least one light chain variable domain comprising the CDR1 L having the amino acid sequence SASQGISNYLN (SEQ ID NO.:41), the CDR2 L having the amino acid sequence YTSILHS (SEQ ID NO.:42) and the CDR3 L having the amino acid sequence QQFNKLPPT (SEQ ID NO.:43); and one heavy chain variable domain comprising the CDR1 H having the amino acid sequence GFTFSDYYMY (SEQ ID NO.:45), the CDR2 H having the amino acid sequence YINSGGGSTYYPDTVKG (SEQ ID NO.:46), and the CDR3 H having the amino acid sequence RGLPFHAMDY (SEQ ID NO.:47).
9. An anti-CD40 fusion protein comprising an anti-CD40 antibody or antigen binding fragment thereof and two or more cancer peptides at the carboxy-terminus of the anti-CD40 antibody, wherein the cancer peptides are separated by the one or more linker peptides that comprise at least one glycosylation site, wherein the cancer peptide is selected from tumor associated antigens selected from human papilloma virus (HPV) E6 and E7 and the linker is selected from SSVSPTTSVHPTPTSVPPTPTKSSP (SEQ ID NO.: 11), PTSTPADSSTITPTATPTATPTIKG (SEQ ID NO.: 12), TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO.: 13) and TNGSITVAATAPTVTPTVNATPSAA (SEQ ID NO.: 14).
10. The fusion protein of claim 9 , wherein the antibody comprises at least the variable region of the antibody anti-CD40 — 12E12.3F3 (ATCC Accession No. PTA-9854), anti-CD40 — 12B4.2C10 (ATCC Submission No. HS446, Accession No. PTA-10653), or anti-CD40 — 11B6.1C3 (ATCC Submission No. HS440, Accession No. PTA-10652).
11. The fusion protein of claim 9 , wherein the Ab comprises at least one variable domain having 100% sequence identity with a heavy chain variable domain of SEQ ID NOS.: 148, 150 or 153 or a light chain variable domains of SEQ ID NOS.: 149, 151, 152 or 154, or both.
12. The fusion protein of claim 11 , wherein the Ab comprises at least one light chain variable domain comprising the CDR1 L having the amino acid sequence SASQGISNYLN (SEQ ID NO.:41), the CDR2 L having the amino acid sequence YTSILHS (SEQ ID NO.:42) and the CDR3 L having the amino acid sequence QQFNKLPPT (SEQ ID NO.:43); and one heavy chain variable domain comprising the CDR1 H having the amino acid sequence GFTFSDYYMY (SEQ ID NO.:45), the CDR2 H having the amino acid sequence YINSGGGSTYYPDTVKG (SEQ ID NO.:46), and the CDR3 H having the amino acid sequence RGLPFHAMDY (SEQ ID NO.:47).
13. The fusion protein of claim 9 , wherein the fusion protein comprises an E6 and E7 antigen.