CD20-BINDING IMMUNOTOXINS FOR INDUCING CELLULAR INTERNALIZATION AND METHODS USING SAME

• US 20160017047A1
• Filed: 03/11/2014
• Published: 01/21/2016
• 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
• Granted Patent: US 10,450,354 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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

a) a CD20 binding region comprising an immunoglobulin-type binding region and capable of specifically binding an extracellular part of CD20 and

b) a Shiga toxin effector polypeptide;

whereby upon administration of the CD20-binding protein to a cell expressing CD20 on a cellular surface, the CD20-binding protein is capable of inducing rapid cellular internalization of a protein complex comprising the CD20-binding protein bound to CD20.

2. The CD20-binding protein of claim 1, wherein the CD20 binding region comprises a polypeptide selected from the group consisting of:

complementary determining region 3 fragment, constrained FR3-CDR3-FR4 polypeptide, single-domain antibody fragment, single-chain variable fragment, antibody variable fragment, antigen-binding fragment, Fd fragment, fibronectin-derived 10^th fibronectin type III domain, tenascin type III domain, ankyrin repeat motif domain, low-density-lipoprotein-receptor-derived A-domain, lipocalin, Kunitz domain, Protein-A-derived Z domain, gamma-B crystalline-derived domain, ubiquitin-derived domain, Sac7d-derived polypeptide, Fyn-derived SH2 domain, engineered antibody mimic, and any genetically manipulated counterparts of any of the foregoing that retain CD20 binding function.

3. The CD20-binding protein of claim 2, whereby upon administration of the CD20-binding protein to a cell, the CD20-binding protein is capable of inducing rapid cellular internalization of CD20 natively present on the surface of a cell;

optionally wherein the CD20-binding protein is capable of inducing, in less than about one hour, cellular internalization of a CD20 natively present on the surface of a cell.

5. The CD20-binding protein of claim 3, wherein the cell is a member of a B-cell lineage.

6. The CD20-binding protein of any one of claims 1-4, whereby upon administration of the CD20-binding protein to a cell which expresses CD20 on a cellular surface, the CD20-binding protein is capable of causing death of the cell.

7. The CD20-binding protein of claim 5, whereby upon administration of the CD20-binding protein to a first populations of cells whose members express CD20, and a second population of cells whose members do not express CD20, the cytotoxic effect of the CD20-binding protein to members of the first population of cells relative to members of the second population of cells is at least 3-fold greater.

8. The CD20-binding protein of claim 6, wherein the Shiga toxin effector polypeptide comprises or consists essentially of the polypeptide sequence selected from the group consisting of:

(i) amino acids 75 to 251 of SEQ ID NO;

1, SEQ ID NO;

25, or SEQ ID NO;

26;

(ii) amino acids 1 to 241 of SEQ ID NO;

1, SEQ ID NO;

25, or SEQ ID NO;

26;

(iii) amino acids 1 to 251 of SEQ ID NO;

1, SEQ ID NO;

25, or SEQ ID NO;

26; and

(iv) amino acids 1 to 261 of SEQ ID NO;

1, SEQ ID NO;

25, or SEQ ID NO 26.

12. The CD20-binding protein of claim 7, which comprises or consists essentially of the polypeptide shown in SEQ ID NO:

4, SEQ ID NO;

12, SEQ ID NO;

14, or SEQ ID NO;

16.

13. The CD20-binding protein of any one of claims 1-7, wherein the CD20 binding region comprises the polypeptides selected from the group consisting of:

a) a heavy chain variable (V_H) domain comprising HCDR1, HCDR2, and HCDR3 amino acid sequences as shown in SEQ ID NO;

6, SEQ ID NO;

7, and SEQ ID NO;

8, respectively, and a light chain variable (V_L) domain comprising LCDR1, LCDR2, and LCDR3 amino acid sequences as shown in SEQ ID NO;

9, SEQ ID NO;

10, and SEQ ID NO;

11, respectively;

b) a heavy chain variable (V_H) domain comprising HCDR1, HCDR2, and HCDR3 amino acid sequences as shown in SEQ ID NO;

21, SEQ ID NO;

22, and SEQ ID NO;

23, respectively, and a light chain variable (V_L) domain comprising LCDR1, LCDR2, and LCDR3 amino acid sequences as shown in SEQ ID NO;

24, SEQ ID NO;

10, and SEQ ID NO;

11, respectively; and

c) a heavy chain variable (V_H) domain comprising HCDR1, HCDR2, and HCDR3 amino acid sequences as shown in SEQ ID NO;

21, SEQ ID NO;

22, and SEQ ID NO;

27, respectively, and a light chain variable (V_L) domain comprising LCDR1, LCDR2, and LCDR3 amino acid sequences as shown in SEQ ID NO;

28, SEQ ID NO;

10, and SEQ ID NO;

29, respectively.

17. The CD20-binding protein of any one of claims 1-12, wherein the Shiga toxin effector polypeptide comprises a mutation relative to a naturally occurring A Subunit of a member of the Shiga toxin family which changes the enzymatic activity of the Shiga toxin effector polypeptide, the mutation selected from at least one amino acid residue deletion or substitution.

32. A pharmaceutical composition comprising the CD20-binding protein of any one of claims 1-20 and at least one pharmaceutically acceptable excipient or carrier.

33. A nucleic acid capable of encoding the CD20-binding protein of any one of claims 1-20, or a complement thereof, or a fragment of any of the foregoing.

36. A method of rapidly inducing cellular internalization of a CD20-binding protein into a CD20 expressing cell, the method comprising the step of contacting the cell with the CD20-binding protein of any one of claims 1-20 or the pharmaceutical composition of claim 21.

37. A method of delivering an exogenous material into a CD20 expressing cell, the method comprising the step of contacting the cell with the CD20-binding protein of any one of claims 1-20 or the pharmaceutical composition of claim 21.

38. A method of killing a CD20 expressing cell, the method comprising the step of contacting the cell with the CD20-binding protein of any one of claims 5-20 or the pharmaceutical composition of claim 21.

42. A method of treating a disease, disorder, or condition in a patient, the method comprising the step of administering to a patient in need thereof a therapeutically effective amount of the CD20-binding protein of any one of claims 1-20 or the pharmaceutical composition of claim 21;

optionally wherein the disease, disorder, or condition involves a cancer cell, tumor cell, or immune cell which expresses CD20 on a cellular surface.

4. (canceled)

9-11. -11. (canceled)

18. A CD20-binding protein for the delivery of an additional exogenous material into a cell that expresses CD20 on a cellular surface, wherein the CD20-binding protein comprises

a) a CD20 binding region comprising an immunoglobulin-type polypeptide and capable of specifically binding an extracellular part of a CD20 molecule,

b) a Shiga toxin effector polypeptide, and

c) an additional exogenous material;

whereby upon administration of the CD20-binding protein to a CD20 expressing cell, the CD20-binding protein is capable of inducing rapid cellular internalization of a protein complex comprising the CD20-binding protein bound to CD20 and capable of delivering said additional exogenous material into the interior of said cell.

30. The CD20-binding protein of claim 18, wherein the antigen comprises SEQ ID NO:

3.

44. The method of claim 30, wherein the cancer or tumor cell is associated with the disease selected from the group consisting of:

bone cancer, leukemia, lymphoma, melanoma, and myeloma.

45. The method of claim 30, wherein the disorder is an immune disorder associated with a disease selected from the group consisting of:

amyloidosis, ankylosing spondylitis, asthma, Crohn'"'"'s disease, diabetes, graft rejection, graft-versus-host disease, Hashimoto'"'"'s thyroiditis, hemolytic uremic syndrome, HIV-related diseases, lupus erythematosus, multiple sclerosis, polyarteritis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma, septic shock, Sjorgren'"'"'s syndrome, ulcerative colitis, and vasculitis.

21-27. -27. (canceled)

39. (canceled)

43. (canceled)

46-47. -47. (canceled)