Antibody-mediated transduction of heat shock proteins into living cells

Patent metadata

Field	Value
Publication number	US-9732146-B2
Application number	US-201313815829-A
Country	US
Kind code	B2
Filing date	Mar 15, 2013
Priority date	Mar 30, 2012
Publication date	Aug 15, 2017
Grant date	Aug 15, 2017

Abstract

The invention provides for a fusion protein comprising a 3E10 Fv joined to a Hsp-70, Hsp-27, Hsp-90 or GRP-78 or portion thereof, and optionally, the 3E10 Fv comprising an amino acid sequence AGIH at its amino terminus.

First claim

What is claimed is:

1. A fusion protein comprising a 3E10 Fv joined to a Hsp-70, a peptide linker comprising an immunoglobulin heavy chain constant domain CH1 or a portion thereof, and a swivel sequence, and optionally, the 3E10 Fv comprising an amino acid sequence AGIH, as shown in SEQ ID NO:37, at its amino terminus, wherein the swivel sequence of the linker is a peptide sequence that is located between the 3E10 Fv and the Hsp-70 and permits the 3E10 Fv and the Hsp-70 to swivel, wherein the swivel sequence is a peptide sequence consisting of LESSGS beginning at position 375 and ending at position 380 of SEQ ID NO: 2.

2. The fusion protein of claim 1 having an arrangement of functional peptide sequence from amino- to carboxyl terminus, AGIH-3E10 Fv-CH1-swivel-Hsp70.

3. The fusion protein of claim 1 , wherein the 3E10 Fv is a derivative of monoclonal antibody 3E10 from 3E10 hybridoma (ATCC Accession No. PTA 2439 hybridoma), wherein the derivative of monoclonal antibody 3E10 comprises a light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of 3E10 antibody, and wherein the derivative competes with monoclonal antibody 3E10.

4. The fusion protein of claim 1 , wherein the 3E10 Fv is a derivative of monoclonal antibody 3E10 from 3E10 hybridoma (ATCC Accession No. PTA 2439 hybridoma), wherein the derivative of monoclonal antibody 3E10 comprises a light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of 3E10 antibody, and wherein the derivative competes with monoclonal antibody 3E10.

5. The fusion protein of claim 4 , wherein the light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of 3E10 antibody is shown in SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID NO:24, respectively.

6. The fusion protein of claim 3 , wherein the antibody that competes with monoclonal antibody 3E10 is an antibody that competes with the ENT2-dependent cell penetrating property and epitope recognition of monoclonal antibody 3E10.

7. The fusion protein of claim 3 , wherein the derivative is obtained by using any of the sequences of a light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2, or heavy chain CDR3 of 3E10 antibody as shown in SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24, respectively, in an antibody phage display screen.

8. The fusion protein of claim 1 , wherein the portion of the immunoglobulin heavy chain constant domain CH1 comprises a sequence of amino acids starting from position 361 and ending with position 374 of SEQ ID NO:6 or a sequence of amino acid starting from position 361 and ending with position 373 of SEQ ID NO:30 and wherein the swivel sequence comprises a sequence of amino acids starting from position 375 and ending with position 380 of SEQ ID NO:6.

9. The fusion protein of claim 1 , which is joined to a therapeutic or diagnostic agent.

10. The fusion protein of claim 1 , wherein the peptide linker is selected from a group consisting of a sequence of amino acids starting from position 361 and ending with position 380 of SEQ ID NO:6 and a sequence of amino acids starting from position 361 and ending with position 379 of SEQ ID NO:30.

11. The fusion protein of claim 9 , wherein the therapeutic agent is a cytotoxic agent.

12. The fusion protein of claim 11 , wherein the cytotoxic agent is selected from a group consisting of ricin, ricin A-chain, doxorubicin, daunorubicin, taxol, ethiduim bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin D, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, restrictocin, phenomycin, enomycin, curacin, crotin, calicheamicin, sapaonaria officinalis inhibitor, maytansinoids, and glucocorticoidricin.

13. The fusion protein of claim 9 , wherein the diagnostic agent is a detectable marker.

14. The fusion protein of claim 13 , wherein the detectable marker is an enzyme, biotin, a fluorophore, a chromophore, a heavy metal, a paramagnetic isotope, or a radioisotope.

15. The fusion protein of claim 1 , wherein the light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2, or heavy chain CDR3 of 3E10 antibody is encoded by nucleic acid sequence shown in SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23, respectively.

16. The fusion protein of claim 1 , wherein the Fv sequence is a recombinant Fv, a chimeric Fv, a humanized Fv or a human Fv.

17. The fusion protein of claim 1 , wherein the swivel sequence is attached to a C-terminus of the CH1 sequence.

18. The fusion protein of claim 1 , wherein the swivel sequence is attached to a C-terminus of the CH1 sequence.

Assignees

• Nishimura Robert N
• Weisbart Richard H
• Hansen James
• Us Veterans Affairs

Inventors

• Nishimura Robert N
• Weisbart Richard H
• Hansen James

Classifications

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• C07K2317/56

  variable (Fv) region, i.e. VH and/or VL · CPC title

• C07K16/18 Primary

  against material from animals or humans · CPC title

• C07K2319/33

  fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies · CPC title

• C07K14/47

  from mammals · CPC title