CD20-binding immunotoxins for inducing cellular internalization and methods using same

• US 10,450,354 B2
• Filed: 03/11/2014
• Issued: 10/22/2019
• Est. Priority Date: 03/12/2013
• Status: Active Grant

• Current Assignee: Molecular Templates, Inc.
• Original Assignee: Molecular Templates, Inc.
• Inventors: Poma, Eric, Willert, Erin, Kim, Jason, Higgins, Jack, Rajagopalan, Sangeetha
• Primary Examiner(s): Huynh, Phuong
• Application Number:
  US14/774,609
• Publication Number:
  US 20160017047A1
• Time in Patent Office: 2,051 Days
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 38/00 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 Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 13/02 of urine or of the urinary tract, e.g. urine acidifiers
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/14 : of the thyroid hormones, e.g. T3, T4
• CPC Class Codes: A61P 7/00 : Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/245 : Escherichia (G)
• CPC Class Codes: C07K 14/25 : Shigella (G)
• CPC Class Codes: C07K 16/085 : Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
• CPC Class Codes: C07K 16/20 : from protozoa
• CPC Class Codes: C07K 16/2863 : against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/2866 : against receptors for cytokines, lymphokines, interferons
• CPC Class Codes: C07K 16/2887 : against CD20
• CPC Class Codes: C07K 16/2896 : against molecules with a "CD"-designation, not provided for elsewhere
• CPC Class Codes: C07K 16/30 : from tumour cells
• CPC Class Codes: C07K 16/32 : against translation products of oncogenes
• CPC Class Codes: C07K 16/40 : against enzymes
• CPC Class Codes: C07K 2317/56 : variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/569 : Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• CPC Class Codes: C07K 2317/622 : Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/73 : Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2317/77 : Internalization into the cell
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• CPC Class Codes: C07K 2319/33 : fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
• CPC Class Codes: C07K 2319/55 : containing a fusion with a toxin, e.g. diphteria toxin

Abstract

The present invention provides CD20-binding proteins that bind to and rapidly internalize CD20 antigens from a cell surface location to the interior of a cell. CD20-binding proteins of the invention comprise a CD20 binding region and a Shiga toxin effector region. Certain of the disclosed CD20-binding proteins kill cells that express CD20 on their surface. Further, the presently disclosed CD20-binding proteins can comprise additional exogenous materials and are capable of targeted delivery of these additional exogenous materials into the interior of CD20 expressing cells. Such additional materials may include peptides, antigens, enzymes, and polynucleotides. These CD20-binding proteins have uses in methods of internalizing themselves, targeted killing of CD20 expressing cells, delivering exogenous materials into CD20 expressing cells, and treating a variety of diseases involving CD20 expressing cells, such as cancers and immune disorders.

Claims

1. A CD20-binding protein comprising the amino acid sequence shown in SEQ ID NO:

4 or SEQ ID NO;

12.

2. The CD20-binding protein of claim 1, comprising the amino acid sequence shown in SEQ ID NO:

4.

3. A CD20-binding protein consisting essentially of the amino acid sequence shown in SEQ ID NO:

4 or SEQ ID NO;

12.

4. The CD20-binding protein of claim 3, consisting essentially of the polypeptide represented SEQ ID NO:

4.

5. A pharmaceutical composition comprising

(i) a CD20-binding protein comprising the amino acid sequence shown in SEQ ID NO:

4 or SEQ ID NO;

12; and

(ii) at least one pharmaceutically acceptable excipient or carrier.

6. The pharmaceutical composition of claim 5, wherein the excipient or carrier is a physiological acceptable:

solvent, dispersion medium, coating, antimicrobial agent, isotonic agent, absorption delaying agent, aqueous solution, or powder.

7. The pharmaceutical composition of claim 5, which further comprises a pharmaceutically acceptable solvent, vehicle, aqueous solution, buffer, powder, surfactant, antioxidant, chelating agent, antimicrobial agent, preservative, isotonic agent, tonicity adjusting agent, dispersion medium, coating, adjuvant, wetting agent, emulsifying agent, dispersing agent, absorption delaying agent, stabilizer, or additive.

8. The pharmaceutical composition of claim 5, which comprises

acetate, acetic acid, alcohol, alpha-tocopherol, aluminum monostearate, antibacterial agent, antifungal agent, ascorbic acid, ascorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, chlorobutanol, citrate, citric acid, cysteine hydrochloride, dextrose, ethanol, ethylenediaminetetraacetic acid, ethyloleate, fixed oil, gelatin, glycerine, glycerol, lactic acid, lecithin, liquid polyethylene glycol, mannitol, methyl parabens, metal chelating agent, monostearate salt, organic ester, paraben, phosphate, phosphoric acid, polyalcohol, polyethylene glycol, polyol, propylene glycol, propylgallate, Ringer'"'"'s solution, saline, small-molecular organic species, sodium bisulfate, sodium bisulfite, sodium chloride, sodium metabisulfite, sodium sulfite, sorbitol, sugar, synthetic solvent, tartaric acid, vegetable oil, or water.

9. The pharmaceutical composition of claim 5, which comprises citrate, propylene glycol, and sugar.

10. The pharmaceutical composition of claim 5, which comprises citrate, propylene glycol, sorbitol, and water.

11. The pharmaceutical composition of claim 5, is formulated for parenteral administration, oral administration, rectal administration, nasal administration, subcutaneous administration, intramuscular administration, intravenous administration, intradermal administration, or transdermal administration.

12. The pharmaceutical composition of claim 5, which comprises a solvate, salt, ester, or amide of the CD20-binding protein.

13. The pharmaceutical composition of any one of claims 5-10, 11-12, which comprises the CD20-binding protein comprising the amino acid sequence shown in SEQ ID NO:

4.

14. A pharmaceutical composition comprising

(i) a CD20-binding protein consisting essentially of the amino acid sequence shown in SEQ ID NO:

4 or SEQ ID NO;

12; and

(ii) at least one pharmaceutically acceptable excipient or carrier.

15. The pharmaceutical composition of claim 14, wherein the excipient or carrier is a physiological acceptable:

solvent, dispersion medium, coating, antimicrobial agent, isotonic agent, absorption delaying agent, aqueous solution, or powder.

16. The pharmaceutical composition of claim 14, which further comprises a pharmaceutically acceptable solvent, vehicle, aqueous solution, buffer, powder, surfactant, antioxidant, chelating agent, antimicrobial agent, preservative, isotonic agent, tonicity adjusting agent, dispersion medium, coating, adjuvant, wetting agent, emulsifying agent, dispersing agent, absorption delaying agent, stabilizer, or additive.

17. The pharmaceutical composition of claim 14, which comprises

acetate, acetic acid, alcohol, alpha-tocopherol, aluminum monostearate, antibacterial agent, antifungal agent, ascorbic acid, ascorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, chlorobutanol, citrate, citric acid, cysteine hydrochloride, dextrose, ethanol, ethylenediaminetetraacetic acid, ethyloleate, fixed oil, gelatin, glycerine, glycerol, lactic acid, lecithin, liquid polyethylene glycol, mannitol, methyl parabens, metal chelating agent, monostearate salt, organic ester, paraben, phosphate, phosphoric acid, polyalcohol, polyethylene glycol, polyol, propylene glycol, propylgallate, Ringer'"'"'s solution, saline, small-molecular organic species, sodium bisulfate, sodium bisulfite, sodium chloride, sodium metabisulfite, sodium sulfite, sorbitol, sugar, synthetic solvent, tartaric acid, vegetable oil, or water.

18. The pharmaceutical composition of claim 14, which comprises citrate, propylene glycol, and sugar.

19. The pharmaceutical composition of claim 14, which comprises citrate, propylene glycol, sorbitol, and water.

20. The pharmaceutical composition of claim 14, which is formulated for oral, rectal, nasal parenteral subcutaneous, intramuscular, intravenous, intradermal, or transdermal administration.

21. The pharmaceutical composition of claim 14, which comprises a solvate, salt, ester, or amide of the CD20-binding protein.

22. The pharmaceutical composition of any one of claims 14-19, which comprises the CD20-binding protein consisting essentially of the amino acid sequence shown in SEQ ID NO:

4.

23. A composition comprising

(i) a CD20-binding protein comprising the amino acid sequence shown in SEQ ID NO:

4 or SEQ ID NO;

12; and

(ii) a solvent or salt.

24. The composition of claim 23, wherein the solvent is water, alcohol, saline, small-molecular organic species, fixed oil, polyethylene glycol, glycerine, propylene glycol, ethanol, polyol, glycerol, acetic acid, lactic acid, vegetable oil, organic ester, or ethyloleate.

25. The composition of claim 23, which comprises:

a vehicle, aqueous solution, buffer, surfactant, antioxidant, chelating agent, antimicrobial agent, preservative, isotonic agent, dispersion medium, coating, adjuvant, tonicity adjusting agent, wetting agent, emulsifying agent, dispersing agent, absorption delaying agent, stabilizer, or additive.

26. The composition of claim 23, which comprises:

acetate, acetic acid, alcohol, alpha-tocopherol, aluminum monostearate, antibacterial agent, antifungal agent, ascorbic acid, ascorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, chlorobutanol, citrate, citric acid, cysteine hydrochloride, dextrose, ethanol, ethylenediaminetetraacetic acid, ethyloleate, fixed oil, gelatin, glycerine, glycerol, lactic acid, lecithin, liquid polyethylene glycol, mannitol, metal chelating agent, methyl parabens, monostearate salt, organic ester, paraben, phosphate, phosphoric acid, polyalcohol, polyethylene glycol, polyol, propylene glycol, propylgallate, Ringer'"'"'s solution, saline, small-molecular organic species, sodium bisulfate, sodium bisulfite, sodium chloride, sodium metabisulfite, sodium sulfite, sorbitol, sugar, synthetic solvent, tartaric acid, vegetable oil, or water.

27. The composition of claim 23, which comprises citrate, propylene glycol, and sugar.

28. The composition of claim 23, which comprises citrate, propylene glycol, sugar, and water.

29. The composition of claim 23, which comprises citrate, propylene glycol, and sorbitol.

30. The composition of claim 23, which comprises citrate, propylene glycol, sorbitol, and water.

31. The composition of any one of claims 23-30, which comprises the CD20-binding protein comprising the amino acid sequence shown in SEQ ID NO:

4.

32. A composition comprising

(i) a CD20-binding protein consisting essentially of the amino acid sequence shown in SEQ ID NO:

4 or SEQ ID NO;

12; and

(ii) a solvent or salt.

33. The composition of claim 32, wherein the solvent is water, alcohol, saline, small-molecular organic species, fixed oil, polyethylene glycol, glycerine, propylene glycol, ethanol, polyol, glycerol, acetic acid, lactic acid, vegetable oil, organic ester, or ethyloleate.

34. The composition of claim 33, which comprises:

a vehicle, aqueous solution, buffer, surfactant, antioxidant, chelating agent, antimicrobial agent, preservative, isotonic agent, dispersion medium, coating, adjuvant, tonicity adjusting agent, wetting agent, emulsifying agent, dispersing agent, absorption delaying agent, stabilizer, or additive.

35. The composition of claim 33, which comprises:

acetate, acetic acid, alcohol, alpha-tocopherol, aluminum monostearate, antibacterial agent, antifungal agent, ascorbic acid, ascorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, chlorobutanol, citrate, citric acid, cysteine hydrochloride, dextrose, ethanol, ethylenediaminetetraacetic acid, ethyloleate, fixed oil, gelatin, glycerine, glycerol, lactic acid, lecithin, liquid polyethylene glycol, mannitol, metal chelating agent, methyl parabens, monostearate salt, organic ester, paraben, phosphate, phosphoric acid, polyalcohol, polyethylene glycol, polyol, propylene glycol, propylgallate, Ringer'"'"'s solution, saline, small-molecular organic species, sodium bisulfate, sodium bisulfite, sodium chloride, sodium metabisulfite, sodium sulfite, sorbitol, sugar, synthetic solvent, tartaric acid, vegetable oil, or water.

36. The composition of claim 33, which comprises citrate, propylene glycol, and sugar.

37. The composition of claim 33, which comprises citrate, propylene glycol, sugar, and water.

38. The composition of claim 33, which comprises citrate, propylene glycol, and sorbitol.

39. The composition of claim 33, which comprises citrate, propylene glycol, sorbitol, and water.

40. The composition of any one of claims 32-39, which comprises the CD20-binding protein consisting essentially of the amino acid sequence shown in SEQ ID NO:

4.