Antigen-binding molecules which bind epidermal growth factor receptor (egfr), vectors coding them and using them

FIELD: medicine.

SUBSTANCE: there are offered: recovered polynucleotides and polypeptides for binding human EGFR as a part of an antibody, a vector and a host cell for antibody expression, a method for producing an anti-EGFR antibody and an antibody fragment, the anti-EGFR antibody and the antibody fragment. There are discussed a composition containing the anti-EGFR antibody or its fragment, as well as applying the antibody and its fragment for treating EGFR-associated disorders. Besides, there are described versions of glycosylation of the anti-EGFR antibody or its fragment.

EFFECT: invention can find further application in therapy of various EGFR-mediated diseases.

30 cl, 6 ex, 32 dwg, 39 tbl

 

The text descriptions are given in facsimile form.

1. Selected polynucleotide encoding humanitarian variable region of the heavy chain, which is specific binds human EGFR as part gumanitarnogo anti-EGFR antibody or antibody fragment,
and specified olignucleotides encodes humanitarian variable region of the heavy chain, comprising a sequence that is selected from the group of SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138.

2. Selected polynucleotide encoding humanitarian variable region of the heavy chain and humanitarian variable region light chain-specific bind the human EGFR as part gumanitarnogo anti-EGFR antibody or antibody fragment,
moreover, the specified polynucleotide codes:
humanitarian variable region heavy chain comprising a sequence that is selected from the group comprising SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO: 138, humanitarian variable region light chain comprising a sequence that is selected from the group comprising SEQ ID NO:45, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, and SEQ ID NO:147.

3. The vector for expression of humanized variable region that is specific binds human EGFR as part gumanitarnogo anti-EGFR antibodies or fragments of antibodies, including polynucleotide according to claim 1 or 2.

4. A host cell for expression of humanized variable region that is specific binds human EGFR as part gumanitarnogo anti-EGFR antibodies or fragments of antibodies, including olignucleotides according to claim 1 or 2.

5. A host cell for expression of the antibody or antibody fragment that is specific binds to human EGFR, including the first polynucleotide and second polynucleotide,
where the first polynucleotide encodes humanitarian variable region heavy chain comprising a sequence selected from the group comprising SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, where the specified second polynucleotide encodes humanitarian region light chain comprising a sequence selected from group comprising SEQ ID NO:45, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, and SEQ ID NO:147.

6. A host cell according to claim 4, where specified a host cell is designed for the expression of antibodies and engineered to Express at least one nucleic acid that encodes a polypeptide having the activity of a β(1,4)-N-acetylglucosaminyltransferase III.

7. A host cell according to claim 6, where specified a host cell additionally designed for the expression of at least one nucleic acid that encodes a polypeptide having the activity of mannosidase II.

8. A method of obtaining antibodies or fragments of antibodies that can compete with monoclonal antibody rat ICR62 for binding to human EGFR, including:
(a) culturing cells and the host according to claim 5 in environment, allowing the expression of the specified first polynucleotide and the specified second polynucleotide, and
(b) the selection of the indicated antibody or antibody fragment.

9. The selected polypeptide to bind to human EGFR as part gumanitarnogo anti-EGFR antibody or antibody fragment encoded by polynucleotides according to claim 1 or 2.

10. The selected polypeptide to bind to human EGFR as part gumanitarnogo anti-EGFR antibody or antibody fragment containing a sequence selected from the group comprising SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138.

11. The antibody or antibody fragment that is specific binds to human EGFR, obtained using the method according to item 8.

12. The antibody or antibody fragment that is specific binds to human EGFR, where the aforementioned antibody or antibody fragment contains humanitarian variable region of the heavy chain and humanitarian variable region light chain, where the specified humanitariannet variable region of the heavy chain contains a sequence selected from the group comprising SEQ ID NO:128 and SEQ ID NO:129. SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138,
and the said humanitariannet variable region light chain contains a sequence, a selected is from the group comprising SEQ ID NO:45, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, and SEQ ID NO:147.

13. The fragment of the antibody according to item 12, where the antibody fragment is selected from the group comprising a scFv fragment, a Fv fragment, a fragment F(ab')2, Minitel, ditelo, triatlo and tetrathele.

14. Antibody para.12, with the indicated antibody comprises an Fc region of a human.

15. The antibody according to 14, where the specified antibody was subjected to glycoengineering
(a) to increase the percentage of bisecting oligosaccharides compared to corresponding not subjected to glycoengineering antibody, or
(b) to reduce the number of fucose residues compared to the corresponding not subjected to glycoengineering antibody, or
(C) to increase binding affinity to the Fc receptor in comparison with corresponding not subjected to glycoengineering antibody and/or
(g) to enhance the effector function compared to corresponding not subjected to glycoengineering antibody where any of paragraphs (a)to(g) is the result of the increase of β(1,4)-N-acetylglucosaminyltransferase III activity in the cell-host expressing a polypeptide having this activity.

16. The antibody that is specific binds to human EGFR, comprising an Fc region that has been subjected to glycoengineering to enhance effector function, or enhance binding the nd affinity to the Fc receptor, obtained by a method including:
(a) culturing the host cell according to claim 6 for the expression of anti-EGFR antibodies under conditions that allow production of antibodies and the change in oligosaccharide structure in the Fc region of the antibody, and
(b) isolation of antibody;
where the antibody contains
humanitarian variable region heavy chain comprising a sequence selected from the group comprising SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138,
and humanitarian variable region light chain comprising a sequence selected from the group comprising SEQ ID NO:45, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, and SEQ ID NO:147.

17. Composition for binding to human EGFR, comprising an effective amount of the antibody or antibody fragment according to item 12, and a pharmaceutically acceptable carrier.

18. The use of a composition according to 17 for the treatment of a disorder that is treatable by blocking EGFR-mediated cellular signal transmission.

19. Use p, where the specified disorder is a cellular proliferative disease.

20. The use of claim,19, where the specified cell proliferative disorder is cancer.

21. The application of claim 20, where the specified cancer selected from the group comprising breast cancer, bladder cancer, RA is the head and neck, skin cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, prostate cancer, kidney cancer and brain cancer.

22. The antibody or antibody fragment according to item 12, with the indicated antibody or antibody fragment does not cause clinically significant levels of toxicity when administered to a subject in a therapeutically effective amount.

23. The antibody or antibody fragment according to item 22, and the specified therapeutically effective amount leads to the concentration of the indicated antibody or fragment antibodies in serum from about 1 μg/ml to approximately 500 μg/ml, or from about 1 μg/ml to about 100 μg/ml for at least 4 weeks.

24. The antibody or antibody fragment according to item 22, and the specified therapeutically effective amount of the indicated antibody or antibody fragment is from about 1.0 mg/kg to about 15 mg/kg

25. The antibody or antibody fragment according to item 22, and the toxicity of the indicated antibodies or fragments of antibodies measured using a chemical blood parameters or histopathological indicators.

26. The antibody or antibody fragment according to item 12, for use in the treatment of a disorder that is treatable by blocking EGFR-mediated cellular signal transmission.

27. The use of the antibody or antibody fragment according to item 12, for the treatment of disorders which are amenable to treatment the Oia by blocking EGFR-mediated cellular signal transmission.

28. The application of item 27, where the specified disorder is a cellular proliferative disease.

29. Use p, where the specified cell proliferative disorder is cancer.

30. The application of clause 29, where the specified cancer selected from the group comprising breast cancer, bladder cancer, head and neck cancer, skin cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, prostate cancer, kidney cancer and brain cancer.



 

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