CD20-BINDING PROTEINS COMPRISING SHIGA TOXIN A SUBUNIT EFFECTOR REGIONS FOR INDUCING CELLULAR INTERNALIZATION AND METHODS USING SAME

• US 20150259428A1
• Filed: 03/10/2015
• Published: 09/17/2015
• Est. Priority Date: 03/11/2014
• Status: Active Grant

• Current Assignee: Molecular Templates, Inc.
• Original Assignee: Molecular Templates, Inc.
• Inventors: POMA, Eric, KIM, Jason, HIGGINS, Jack, WILLERT, Erin, RAJAGOPOLAN, Sangeetha
• Granted Patent: US 11,142,584 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 2039/6056 Antibodies
• CPC Class Codes: A61K 39/12 Viral antigens
• CPC Class Codes: A61K 47/6829 Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
• CPC Class Codes: A61K 47/6849 the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• CPC Class Codes: C07K 14/25 Shigella (G)
• CPC Class Codes: C07K 16/2887 against CD20
• 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 2319/22 containing a Strep-tag
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C07K 2319/55 containing a fusion with a toxin, e.g. diphteria toxin
• CPC Class Codes: C12N 2760/16134 Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: G01N 2333/70596 Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
• CPC Class Codes: G01N 33/57426 leukemia
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Abstract

The present invention provides CD20-binding proteins that bind to and rapidly internalize in a CD20-mediated fashion from a cell surface location to the interior of the 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, such as, e.g., antigens, and are capable of targeted delivery of these additional exogenous materials into the interior of CD20 expressing cells. These CD20-binding proteins have uses in methods such as, e.g., methods involving the efficient cellular internalization of CD20, targeted killing of CD20 expressing cells, delivering exogenous materials inside CD20 expressing cells, detecting CD20 expressing cells, and treating a variety of conditions involving CD20 expressing cells including cancers, tumors, growth abnormalities, and immune disorders.

Claims

1. A CD20-binding protein comprising

a) a CD20 binding region capable of specifically binding an extracellular part of CD20;

and

b) a Shiga toxin effector region comprising a polypeptide derived from an A Subunit of at least one member of the Shiga toxin family; and

whereby administration of CD20-binding protein to one or more CD20 positive cells, CD20-binding protein is internalized into one or more of said CD20 positive cells within five hours at 37 degrees Celsius.

2. The CD20-binding protein of claim 1, whereby administration of a plurality of the CD20-binding protein to a plurality of said CD20 positive cells at a concentration equivalent to 50% cell-surface CD20 occupancy, the majority of the CD20-binding protein is internalized into one or more of said CD20 positive cells within one hour at 37 degrees Celsius.

3. The CD20-binding protein of any one of claims 1-2, wherein the binding region comprises an immunoglobulin-type binding region comprising a polypeptide selected from the group consisting of:

single-domain antibody fragment, single-chain variable fragment, antibody variable fragment, complementary determining region 3 fragment, constrained FR3-CDR3-FR4 polypeptide, Fd fragment, antigen-binding fragment, fibronection-derived 10^th fibronectin type 111 domain, tenacsin 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, miniprotein, C-type lectin-like domain scaffold, engineered antibody mimic, and any genetically manipulated counterparts of any of the foregoing which retain binding functionality.

4. The CD20-binding protein of any one of claims 1-2 which lacks an Fc region or which comprises only Fc region effector domain(s) which lack Fc effector function.

5. The CD20-binding protein of any one of claims 1-2, wherein the CD20 positive cell is a descendant or member of a B-cell lineage.

6. The CD20-binding protein of any one of claims 1-2, wherein the CD20 positive cell is selected from the group consisting of:

malignant B-cell, B-cell leukemia cell, B-cell lymphoma cell, B-cell myeloma cell, acute myeloid leukemia cell, acute non-lymphocytic leukemia cell, B-cell chronic lymphocytic leukemia cell, B-cell lymphoma cell, B-cell non-Hodgkin'"'"'s lymphoma cell, B-cell precursor acute lymphoblastic leukemia cell, B-cell prolymphocytic leukemia cell, Burkitt'"'"'s lymphoma cell, chronic lymphocytic leukemia cell, chronic myeloid leukemia cell, diffuse large B-cell lymphoma cell, follicular lymphoma cell, hairy cell leukemia cell, Hodgkin'"'"'s lymphoma cell, immunoblastic large cell lymphoma cell, mantle cell lymphoma cell, melanoma cell, multiple myeloma cell, neoplastic plasma cell, nodular lymphocyte predominant Hodgkin'"'"'s lymphoma cell, non-Hodgkin'"'"'s lymphoma cell, plasmablastic lymphoma cell, plasma cell myeloma cell, precursor B-lymphoblastic lymphoma cell, small lymphocytic lymphoma cell, malignant T-cell, T-cell leukemia cell, T-cell lymphoma cell, T-cell large granular lymphocyte leukemia cell, T-cell prolymphocytic leukemia, healthy B-cell lineage cell, and healthy T-cell.

7. The CD20-binding protein of any one of claims 1-2, whereby administration of the CD20-binding protein to one or more CD20 positive cells at a physiological temperature for the cell results in one or more of the following behaviors in said one or more CD20 positive cells:

(i) internalizing CD20-binding protein inside the cell within one hour,

(ii) subcellular routing at least one Shiga toxin effector region to the cell'"'"'s cytosol,

(iii) disrupting the cell'"'"'s ribosome function, and

(iv) killing of the cell.

8. The CD20-binding protein of any one of claims 1-2 whereby administration of the CD20-binding protein to a first population of CD20 positive cells and a second population of cells whose members do not express a significant amount of a CD20 target of the CD20-binding protein at a cellular surface, 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.

9. The CD20-binding protein of any one of claims 1-2, wherein the Shiga toxin effector region comprises or consists essentially of a polypeptide comprising the amino acid sequence selected from the group consisting of:

a) amino acids 75 to 251 of any one of SEQ ID NO;

1, SEQ ID NO;

2, or SEQ ID NO;

3;

b) amino acids 1 to 241 of any one of SEQ ID NO;

1, SEQ ID NO;

2, or SEQ ID NO;

3;

c) amino acids 1 to 251 of any one of SEQ ID NO;

1, SEQ ID NO;

2, or SEQ ID NO;

3; and

d) amino acids 1 to 261 of any one of SEQ ID NO;

1, SEQ ID NO;

2, or SEQ ID NO;

3.

10. The CD20-binding protein of any one of claims 1-2, wherein the CD20 binding region comprises at least one heavy-chain variable domain polypeptide and at least one light-chain variable domain polypeptide selected from the group consisting of:

a) a heavy chain variable (V_H) domain comprising

i) HCDR1, HCDR2, and HCDR3 amino acid sequences shown in SEQ ID NO;

5, SEQ ID NO;

6, and SEQ ID NO;

7, respectively;

ii) HCDR1, HCDR2, and HCDR3 amino acid sequences as shown in SEQ ID NO;

11, SEQ ID NO;

12, or SEQ ID NO;

13, respectively;

iii) HCDR1, HCDR2, and HCDR3 amino acid sequences shown in SEQ ID NO;

17, SEQ ID NO;

18, and SEQ ID NO;

19, respectively;

iv) HCDR1, HCDR2, and HCDR3 amino acid sequences shown in SEQ ID NO;

23, SEQ ID NO;

24, and SEQ ID NO;

25, respectively;

v) HCDR1, HCDR2, and HCDR3 amino acid sequences shown in SEQ ID NO;

29, SEQ ID NO;

30, and SEQ ID NO;

31, respectively; and

vi) HCDR1, HCDR2, and HCDR3 amino acid sequences shown in SEQ ID NO;

35, SEQ ID NO;

36, and SEQ ID NO;

37, respectively; and

b) a light chain variable (V_L) domain comprising

i) LCDR1, LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO;

8, SEQ ID NO;

9, and SEQ ID NO;

10, respectively;

ii) LCDR1, LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO;

14, SEQ ID NO;

15, and SEQ ID NO;

16, respectively;

iii) LCDR1, LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO;

20, SEQ ID NO;

21, and SEQ ID NO;

22, respectively;

iv) LCDR1, LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO;

26, SEQ ID NO;

27, and SEQ ID NO;

28, respectively;

v) LCDR1, LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO;

32, SEQ ID NO;

33, and SEQ ID NO;

34, respectively; and

vi) LCDR1, LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO;

38, SEQ ID NO;

39, and SEQ ID NO;

40, respectively.

19. The CD20-binding protein of any one of claims 1-18, wherein the Shiga toxin effector region 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 region, the mutation selected from at least one amino acid residue deletion or substitution.

20. The CD20-binding protein of claim 19, wherein said mutation reduces or eliminates cytotoxicity of the Shiga toxin effector region.

36. A pharmaceutical composition comprising the CD20-binding protein of any one of claims 1-35 and at least one pharmaceutically acceptable excipient or carrier.

37. A diagnostic composition comprising

the CD20-binding protein of any one of claims 45-112 and

a detection promoting agent.

38. A nucleic acid capable of encoding the CD20-binding protein of any one of claims 1-35, or a complement thereof, or a fragment of any of the foregoing.

39. An expression vector that comprises the nucleic acid of claim 38.

40. A host cell comprising one of the nucleic acids or expression vectors of claims 38-39.

41. A method of killing a CD20 positive cell, the method comprising the step of contacting the cell with the CD20-binding protein of any one of claims 1-35, the pharmaceutical composition of claim 36, or the diagnostic composition of claim 37.

42. A method of inducing cellular internalization of a CD20-binding protein into a CD20 positive cell within five hours at 37 degrees Celsius, the method comprising the step of contacting the cell with a CD20-binding protein of any one of claims 1-35, the pharmaceutical composition of claim 36, or the diagnostic composition of claim 37.

43. The method of claim 42, wherein the inducing cellular internalization occurs within one hour of the contacting step.

44. A method of delivering an exogenous material into a CD20 positive cell, the method comprising the step of contacting the cell with the CD20-binding protein of any one of claims 1-35, the pharmaceutical composition of claim 36, or the diagnostic composition of claim 37.

45. The method of claim 44, wherein the delivering an exogenous material occurs within one hour of the contacting step.

46. The method of any of one of claims 41-45, wherein the contacting occurs in vitro.

47. The method of any of one of claims 41-45, wherein the contacting occurs in vivo.

48. A method of treating a disease, disorder, or condition in a patient 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 1-35 or the pharmaceutical composition of claim 36.

49. The method of claim 48, wherein the disease, disorder, or condition involves a CD20 positive cancer cell, tumor cell, or immune cell.

50. The method of claim 49, wherein the disease, disorder, or condition is selected from the group consisting of:

hematologic cancer, leukemia, lymphoma, melanoma, myeloma, amyloidosis, ankylosing spondylitis, asthma, Crohn'"'"'s disease, diabetes, graft rejection, graft-versus-host disease, Graves'"'"' disease, Graves'"'"' ophthalmopathy, Hashimoto'"'"'s thyroiditis, hemolytic uremic syndrome, HIV-related disease, lupus erythematosus, multiple sclerosis, neuromyelitis optica spectrum disorders, N-methyl D-aspartate receptor encephalitis, opsoclonus myoclonus syndrome, paroxysmal nocturnal hemoglobinuria, polyarteritis nodosa, polyarthritis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleritis, scleroderma, septic shock, Sjorgren'"'"'s syndrome, ulcerative colitis, and vasculitis.

51. The method of claim 50, wherein the disease is the cancer selected from the group consisting of:

acute myeloid leukemia, acute non-lymphocytic leukemia, B-cell chronic lymphocytic leukemias, B-cell lymphoma, B-cell non-Hodgkin'"'"'s lymphoma, B-cell precursor acute lymphoblastic leukemia, B-cell prolymphocytic leukemia, Burkitt'"'"'s lymphoma, chronic lymphocytic leukemia, chronic myeloid leukemia, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, Hodgkin'"'"'s lymphoma, immunoblastic large cell lymphoma, mantle cell lymphoma, multiple myeloma, nodular lymphocyte predominant Hodgkin'"'"'s lymphoma, non-Hodgkin'"'"'s lymphoma, plasmablastic lymphoma, plasma cell neoplasma, plasma cell myeloma, precursor B-lymphoblastic lymphoma, small lymphocytic lymphoma, T-cell large granular lymphocyte leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, and Waldenströ

m'"'"'s macroglobulinemia.

52. A composition of matter of any one of claims 1-40 for the treatment or prevention of a cancer, tumor, or immune disorder.

53. Use of a composition of matter of any one of claims 1-40 in the manufacture of a medicament for the treatment or prevention of a cancer, tumor, or immune disorder.

56. A method of detecting a CD20 expressing cell comprising the steps of:

contacting said cell with the diagnostic composition of claim 37 and

detecting the presence of said diagnostic composition.

57. The method of claim 56, wherein the contacting occurs in vitro.

58. The method of claim 56, wherein the detecting occurs in vivo.

59. Use of a composition of matter of any one of claims 1-40 in the diagnosis, prognosis, or characterization of a disease, disorder, or condition.

60. A kit comprising the composition of matter of any one of claims 1-40 and an additional reagent and/or pharmaceutical delivery device.

11. A cytotoxic CD20-binding protein

comprising

a) a CD20 binding region capable of specifically binding an extracellular part of CD20 and comprising an immunoglobulin-type binding region comprising a polypeptide selected from the group consisting of:

single-domain antibody fragment, single-chain variable fragment, antibody variable fragment, complementary determining region 3 fragment, constrained FR3-CDR3-FR4 polypeptide, Fd fragment, antigen-binding fragment, fibronection-derived 10^th fibronectin type III domain, tenacsin 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, miniprotein, C-type lectin-like domain scaffold, engineered antibody mimic, and any genetically manipulated counterparts of any of the foregoing which retain binding functionality; and

b) a Shiga toxin effector region comprising a polypeptide derived from an A Subunit of at least one member of the Shiga toxin family; and

wherein the cytotoxic CD20-binding protein does not comprise an Fc region or Fc region effector domain which retains Fc function; and

whereby administration of CD20-binding protein to one or more CD20 positive cells, CD20-binding protein is internalized into one or more of said CD20 positive cells within five hours at 37 degrees Celsius and kills one or more of said CD20 positive cells.

12. The cytotoxic CD20-binding protein of claim 11, whereby administration of a plurality of the CD20-binding protein to a plurality of CD20 positive cells, CD20-binding protein is internalized into and kills one or more of said CD20 positive cells.

13. The cytotoxic CD20-binding protein of claim 12, whereby administration of a plurality of the CD20-binding protein to a first populations of CD20 positive cells and a second population of cells whose members do not express a significant amount of a CD20 target of the CD20-binding protein at a cellular surface, 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.

14. The cytotoxic CD20-binding protein of any one of claims 11-13, wherein the Shiga toxin effector region comprises or consists essentially of the polypeptide selected from the group consisting of:

a) amino acids 75 to 251 of any one of SEQ ID NO;

1, SEQ ID NO;

2, or SEQ ID NO;

3;

b) amino acids 1 to 241 of any one of SEQ ID NO;

1, SEQ ID NO;

2, or SEQ ID NO;

3;

c) amino acids 1 to 251 of any one of SEQ ID NO;

1, SEQ ID NO;

2, or SEQ ID NO;

3; and

d) amino acids 1 to 261 of any one of SEQ ID NO;

1, SEQ ID NO;

2, or SEQ ID NO;

3.

15. The cytotoxic CD20-binding protein of any one of claims 11-13, wherein the CD20 binding region comprises at least one heavy-chain variable domain polypeptide and at least one light-chain variable domain polypeptide selected from the group consisting of:

a) a heavy chain variable (V_H) domain comprising

i) HCDR1, HCDR2, and HCDR3 amino acid sequences shown in SEQ ID NO;

5, SEQ ID NO;

6, and SEQ ID NO;

7, respectively;

ii) HCDR1, HCDR2, and HCDR3 amino acid sequences shown in SEQ ID NO;

11, SEQ ID NO;

12, or SEQ ID NO;

13, respectively;

iii) HCDR1, HCDR2, and HCDR3 amino acid sequences shown in SEQ ID NO;

17, SEQ ID NO;

18, and SEQ ID NO;

19, respectively;

iv) HCDR1, HCDR2, and HCDR3 amino acid sequences shown in SEQ ID NO;

23, SEQ ID NO;

24, and SEQ ID NO;

25, respectively;

v) HCDR1, HCDR2, and HCDR3 amino acid sequences shown in SEQ ID NO;

29, SEQ ID NO;

30, and SEQ ID NO;

31, respectively; and

vi) HCDR1, HCDR2, and HCDR3 amino acid sequences shown in SEQ ID NO;

35, SEQ ID NO;

36, and SEQ ID NO;

37, respectively; and

b) a light chain variable (V_L) domain comprising

i) LCDR1, LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO;

8, SEQ ID NO;

9, and SEQ ID NO;

10, respectively;

ii) LCDR1, LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO;

14, SEQ ID NO;

15, and SEQ ID NO;

16, respectively;

iii) LCDR1, LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO;

20, SEQ ID NO;

21, and SEQ ID NO;

22, respectively;

iv) LCDR1, LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO;

26, SEQ ID NO;

27, and SEQ ID NO;

28, respectively;

v) LCDR1, LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO;

32, SEQ ID NO;

33, and SEQ ID NO;

34, respectively; and

vi) LCDR1, LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO;

38, SEQ ID NO;

39, and SEQ ID NO;

40, respectively.

16. The cytotoxic CD20-binding protein of any one of claims 11-13, wherein the CD20 binding region comprises or consists essentially of amino acids 2 to 245 of any one of SEQ ID NOs:

46-112.

17. The cytotoxic CD20-binding protein of any one of claims 11-13, wherein

a) the CD20 binding region comprises or consists essentially of amino acids 2 to 245 of any one of SEQ ID NOs:

46-112, and

b) the Shiga toxin effector region comprises or consists essentially of amino acids 75 to 251 of SEQ ID NO;

1.

18. The cytotoxic CD20-binding protein of any one of claims 11-13, which comprises or consists essentially of the polypeptide shown in any one of SEQ ID NOs:

46-112.

21. A CD20-binding protein for the delivery of an additional exogenous material into a CD20 positive cell, wherein the CD20-binding protein comprises

a) a CD20 binding region capable of specifically binding an extracellular part of CD20 and comprising an immunoglobulin-type binding region comprising a polypeptide selected from the group consisting of:

single-domain antibody fragment, single-chain variable fragment, antibody variable fragment, complementary determining region 3 fragment, constrained FR3-CDR3-FR4 polypeptide, Fd fragment, antigen-binding fragment, fibronection-derived 10^th fibronectin type III domain, tenacsin 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, miniprotein, C-type lectin-like domain scaffold, engineered antibody mimic, and any genetically manipulated counterparts of any of the foregoing which retain binding functionality;

b) a Shiga toxin effector region comprising a polypeptide derived from an A Subunit of at least one member of the Shiga toxin family; and

c) an additional exogenous material; and

whereby administration of CD20-binding protein to one or more CD20 positive cells, CD20-binding protein is

internalized into said one or more cells and

capable of delivering the additional exogenous material into the interior of

the cell within five hours at 37 degrees Celsius.

22. The CD20-binding protein of claim 21, whereby administration of a plurality of the CD20-binding protein to a plurality of said CD20 positive cells at a concentration equivalent to 50% cell-surface CD20 occupancy, the majority of the CD20-binding protein is internalized into one or more of said CD20 positive cells within one hour at 37 degrees Celsius.

23. The CD20-binding protein of claim 21 or 22, wherein the exogenous material is selected from the group consisting of a peptide, a polypeptide, a protein, and a nucleic acid.

24. The CD20-binding protein of claim 23, wherein the exogenous material is a protein or polypeptide comprising an enzyme.

25. CD20-binding protein of claim 23, wherein the exogenous material is a nucleic acid comprising a ribonucleic acid.

26. The CD20-binding protein of claim 23, wherein the exogenous material is a peptide comprising an antigen.

27. The CD20-binding protein of claim 26, wherein the antigen is derived from a molecule selected from the group consisting of:

a) bacterial protein,

b) protein mutated in cancer,

c) protein aberrantly expressed in cancer,

d) T-cell complementary determining region polypeptide, and

e) viral protein.

28. The CD20-binding protein of claim 26, wherein the antigen is located within the protein between the CD20-binding region and the Shiga toxin effector region.

29. The CD20-binding protein of any one of claims 21-28, wherein the Shiga toxin effector region comprises or consists essentially of the polypeptide selected from the group consisting of:

a) amino acids 75 to 251 of SEQ ID NO;

1, SEQ ID NO;

2, or SEQ ID NO;

3;

b) amino acids 1 to 241 of SEQ ID NO;

1, SEQ ID NO;

2, or SEQ ID NO;

3;

c) amino acids 1 to 251 of SEQ ID NO;

1, SEQ ID NO;

2, or SEQ ID NO;

3; and

d) amino acids 1 to 261 of SEQ ID NO;

1, SEQ ID NO;

2, or SEQ ID NO;

3.

30. The CD20-binding protein of any one of claims 21-29, wherein the CD20 binding region comprises at least one heavy-chain variable domain polypeptide and at least one light-chain variable domain polypeptide selected from the group consisting of:

a) a heavy chain variable (V_H) domain comprising

i) HCDR1, HCDR2, and HCDR3 amino acid sequences shown in SEQ ID NO;

5, SEQ ID NO;

6, and SEQ ID NO;

7, respectively;

ii) HCDR1, HCDR2, and HCDR3 amino acid sequences shown in SEQ ID NO;

11, SEQ ID NO;

12, or SEQ ID NO;

13, respectively;

iii) HCDR1, HCDR2, and HCDR3 amino acid sequences shown in SEQ ID NO;

17, SEQ ID NO;

18, and SEQ ID NO;

19, respectively;

iv) HCDR1, HCDR2, and HCDR3 amino acid sequences shown in SEQ ID NO;

23, SEQ ID NO;

24, and SEQ ID NO;

25, respectively;

v) HCDR1, HCDR2, and HCDR3 amino acid sequences shown in SEQ ID NO;

29, SEQ ID NO;

30, and SEQ ID NO;

31, respectively; and

vi) HCDR1, HCDR2, and HCDR3 amino acid sequences shown in SEQ ID NO;

35, SEQ ID NO;

36, and SEQ ID NO;

37, respectively; and

b) a light chain variable (V_L) domain comprising

i) LCDR1, LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO;

8, SEQ ID NO;

9, and SEQ ID NO;

10, respectively;

ii) LCDR1, LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO;

14, SEQ ID NO;

15, and SEQ ID NO;

16, respectively;

iii) LCDR1, LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO;

20, SEQ ID NO;

21, and SEQ ID NO;

22, respectively;

iv) LCDR1, LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO;

26, SEQ ID NO;

27, and SEQ ID NO;

28, respectively;

v) LCDR1, LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO;

32, SEQ ID NO;

33, and SEQ ID NO;

34, respectively; and

vi) LCDR1, LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO;

38, SEQ ID NO;

39, and SEQ ID NO;

40, respectively.

31. The CD20-binding protein of any one of claims 21-30, wherein the CD20 binding region comprises or consists essentially of amino acids 2 to 245 of any one of SEQ ID NOs:

46-112.

32. The CD20-binding protein of claim any one of claims 26-28, wherein the antigen comprises or consists essentially of the amino acid sequence shown in SEQ ID NO:

44.

33. The CD20-binding protein of claim 32 comprising or consisting essentially of the amino acid sequence shown in any one of SEQ ID NO:

50, SEQ ID NO;

54, SEQ ID NO;

55, SEQ ID NO;

59, SEQ ID NO;

62, SEQ ID NO;

67, SEQ ID NO;

71, SEQ ID NO;

74, SEQ ID NO;

78, SEQ ID NO;

89, and SEQ ID NO;

110.

34. The CD20-binding protein of any one of claims 21-33, wherein the Shiga toxin effector region 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 region, the mutation selected from at least one amino acid residue deletion or substitution.

35. The CD20-binding protein of claim 34, wherein said mutation reduces or eliminates cytotoxicity.

54. A method of producing a CD20-binding protein comprising the step purifying the CD20-binding protein using a chitin binding interaction.

55. The method of claim 54, wherein the purifying step involves the protein comprising or consisting essentially of any one of the polypeptides shown in SEQ ID NOs:

90-102.