Cd20-binding immunotoxins for inducing cellular internalization and methods using same

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.

The invention is claimed as follows: 
     
         1 . A CD20-binding protein comprising:
 (a) a CD20 binding region comprising an immunoglobulin-type binding region that specifically binds an extracellular part of a CD20 polypeptide,   (b) a Shiga toxin A subunit effector polypeptide; and   (c) an additional exogenous material.   
     
     
         2 . The CD20-binding protein of  claim 1 , wherein the CD20-binding protein is capable of rapidly internalizing into a CD20-expressing cell within less than six hours when the CD20-binding protein is contacted with the CD20-expressing cell. 
     
     
         3 . The CD20-binding protein of  claim 1 , wherein the CD20-binding region comprises a complementary determining region 3 fragment, a constrained FR3-CDR3-FR4 polypeptide, a single-domain antibody fragment, a single-chain variable fragment, an antibody variable fragment, an antigen-binding fragment, an Fd fragment, a fibronectin-derived 10 th  fibronectin type III domain, a tenascin type III domain, ankyrin repeat motif domain, a low-density-lipoprotein-receptor-derived A-domain, a lipocalin, a Kunitz domain, a Protein-A-derived Z domain, a gamma-B crystallin-derived domain, a ubiquitin-derived domain, a Sac7d-derived polypeptide, a Fyn-derived SH2 domain, or any genetically manipulated counterparts of any of the foregoing that retain CD20 binding function. 
     
     
         4 . The CD20-binding protein of  claim 1 , wherein the Shiga toxin A subunit effector polypeptide comprises an amino acid sequence having at least 85% sequence identity to:
 (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; or   (iv) amino acids 1 to 261 of SEQ ID NO: 1, SEQ ID NO: 25 or SEQ ID NO:   26.   
     
     
         5 . The CD20-binding protein of  claim 1 , wherein the CD20-binding region comprises:
 (a) a heavy chain variable (VH) domain comprising an HCDR1 of SEQ ID NO: 6, an HCDR2 of SEQ ID NO: 7, and an HCDR3 of SEQ ID NO: 8, and a light chain variable (VL) domain comprising an LCDR1 of SEQ ID NO:9, an LCDR2 of SEQ ID NO: 10, and an LDCR3 of SEQ ID NO: 11,   (b) a VH domain comprising an HCDR1 of SEQ ID NO: 21, an HCDR2 of SEQ ID NO: 22, and an HCDR3 of SEQ ID NO: 27, and a VL domain comprising an LCDR1 of SEQ ID NO: 24, an LCDR2 of SEQ ID NO: 10, and an LDCR3 of SEQ ID NO: 11, or   (c) a VH domain comprising an HCDR1 of SEQ ID NO: 21, an HCDR2 of SEQ ID NO: 22, and an HCDR3 of SEQ ID NO: 23, and a VL domain comprising an LCDR1 of SEQ ID NO: 28, an LCDR2 of SEQ ID NO: 10, and an LDCR3 of SEQ ID NO: 29.   
     
     
         6 . A pharmaceutical composition comprising the CD20-binding protein of  claim 1  and at least one pharmaceutically acceptable excipient or carrier. 
     
     
         7 . The pharmaceutical composition of  claim 6 , which comprises a solvate, salt, ester or amide of the CD20-binding protein. 
     
     
         8 . The pharmaceutical composition of  claim 6 , wherein the excipient is: acetate, alcohol, alpha-tocopherol, aluminum monostearate, ascorbic acid, ascorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, chlorobutanol, citrate, cysteine hydrochloride, dextrose, ethanol, ethylenediaminetetraacetic acid, ethyloleate, gelatin, glycerine, glycerol, lactic acid, lecithin, mannitol, methyl parabens, monostearate salt, organic ester, paraben, phenol phosphate, phosphoric acid, polyalcohol, polyethylene glycol, polyol, propylene glycol, propylgallate, Ringer's solution, saline, sodium bisulfate, sodium bisulfite, sodium chloride, sodium metabisulfite, sodium sulfite, sorbic acid, sorbitol, sugar, tartaric acid, vegetable oil or water. 
     
     
         9 . The pharmaceutical composition of  claim 6 , which further comprises an acceptable solvent, vehicle, sterile aqueous solution, buffer, powder, sterile powder, surfactant, antioxidant, chelating agent, antimicrobial agent, preservative, isotonic agent, dispersion medium, coating, adjuvant, wetting agent, emulsifying agent, dispersing agent, adsorption delaying agent, stabilizer, or additive. 
     
     
         10 . A method for delivering an exogenous material into a CD20-expressing cell, the method comprising contacting a CD20-expressing cell having an interior with 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 a CD20 protein,   (b) a Shiga toxin A subunit effector polypeptide; and   (c) an additional exogenous material, and   wherein the contacting step results in the CD20-binding protein delivering the additional exogenous material into the interior of the CD20-expressing cell.   
     
     
         11 . The method of  claim 10 , wherein the CD20-binding region comprises: a complementary determining region 3 fragment, a constrained FR3-CDR3-FR4 polypeptide, a single-domain antibody fragment, a single-chain variable fragment, an antibody variable fragment, an antigen binding fragment, an Fd fragment, a fibronectin-derived 10 th  fibronectin type III domain, a tenascin type III domain, an ankyrin repeat motif domain, a low-density lipoprotein receptor-derived A domain, a lipocalin, a Kunitz domain, a Protein-A-derived Z domain, a gamma-B crystallin-derived domain, a ubiquitin-derived domain, a Sac7d-derived polypeptide, a Fyn-derived SH2 domain, or any genetically manipulated counterparts of any of the foregoing that retain CD20-binding function. 
     
     
         12 . The method of  claim 10 , wherein the contacting step results in the CD20-binding protein inducing cellular internalization of the CD20-binding protein in less than about an hour. 
     
     
         13 . The method of  claim 10 , wherein the Shiga toxin A subunit effector polypeptide comprises an amino acid sequence having at least 85% sequence identity to:
 (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, or   (iv) amino acids 1 to 261 of SEQ ID NO: 1, SEQ ID NO: 25 or SEQ ID NO: 26.   
     
     
         14 . The method of  claim 10 , wherein the CD20-binding region comprises:
 (a) a heavy chain variable (VH) domain comprising an HCDR1 of SEQ ID NO: 6, an HCDR2 of SEQ ID NO: 7, and an HCDR3 of SEQ ID NO: 8, and a light chain variable (VL) domain comprising an LCDR1 of SEQ ID NO:9, an LCDR2 of SEQ ID NO: 10, and an LDCR3 of SEQ ID NO: 11,   (b) a VH domain comprising an HCDR1 of SEQ ID NO: 21, an HCDR2 of SEQ ID NO: 22, and an HCDR3 of SEQ ID NO: 27, and a VL domain comprising an LCDR1 of SEQ ID NO: 24, an LCDR2 of SEQ ID NO: 10, and an LDCR3 of SEQ ID NO: 11, or   (c) a VH domain comprising an HCDR1 of SEQ ID NO: 21, an HCDR2 of SEQ ID NO: 22, and an HCDR3 of SEQ ID NO: 23, and a VL domain comprising an LCDR1 of SEQ ID NO: 28, an LCDR2 of SEQ ID NO: 10, and an LDCR3 of SEQ ID NO: 29.