Methods and compositions for bi-specific targeting of cd19/cd22

Abstract

Methods and composition involving genetically engineered targeting conjugates with reversed orientation of V L and V H chains are provided. For example, in certain aspects targeting conjugates comprising V L and V H chains of anti-CD22 and anti-CD19 are described. In a further aspect, the invention provides methods and targeting conjugates comprising therapeutic agents or diagnostic agents for delivery to B cells.

1 . A conjugate comprising a therapeutic agent conjugated to a targeting moiety comprising at least a first antigen-binding fragment that binds a first antigen and a second antigen-binding fragment that binds a second antigen, wherein the first antigen-binding fragment comprises a first V L  domain which is linked at its carboxy terminus to a first V H  domain (V L -V H  orientation), and/or the second antigen-binding fragment comprises a second V L  domain which is linked at its carboxy terminus to a second V H  domain (V L -V H  orientation). 
     
     
         2 . The conjugate of  claim 1 , wherein said conjugate is further defined as a fusion protein. 
     
     
         3 . The conjugate of  claim 2 , wherein said fusion protein is DT2219ARL having an amino acid sequence of SEQ ID NO:01. 
     
     
         4 . The conjugate of  claim 1 , wherein said agent and targeting moiety is chemically conjugated. 
     
     
         5 . The conjugate of  claim 1 , wherein the first and/or second antigen-binding fragment is an Fv fragment. 
     
     
         6 . The conjugate of  claim 1 , wherein the first and/or second antigen-binding fragment is an scFv fragment. 
     
     
         7 . The conjugate of  claim 1 , wherein the first V L  domain is linked to the first V H  domain via a first peptide linker. 
     
     
         8 . The conjugate of  claim 1 , wherein the second V L  domain is linked to the second V H  domain via a second peptide linker. 
     
     
         9 . The conjugate of  claim 7 , wherein the first peptide linker comprises at least three charged resides selected from the group consisting of lysine, arginine, glutamic acid, aspartic acid, and histidine. 
     
     
         10 . The conjugate of  claim 8 , wherein the second peptide linker comprises at least three charged resides selected from the group consisting of lysine, arginine, glutamic acid, aspartic acid, and histidine. 
     
     
         11 . The conjugate of  claim 9  or  10 , wherein the first or second peptide linker is a ARL linker (GSTSGSGKPGSGEGSTKG; SEQ ID NO:14). 
     
     
         12 . The conjugate of  claim 1 , wherein the first antigen-binding fragment is linked to the second antigen-binding fragment via a third peptide linker. 
     
     
         13 . The conjugate of  claim 12 , wherein the third peptide linker is G4S. 
     
     
         14 . The conjugate of  claim 1 , wherein the therapeutic agent comprises a therapeutic peptide, wherein the therapeutic peptide is linked at its carboxy terminus to the first or second antigen-binding fragment. 
     
     
         15 . The conjugate of  claim 1 , wherein the therapeutic agent comprises a therapeutic peptide, wherein the therapeutic peptide is linked at its amino terminus to the first or second antigen-binding fragment. 
     
     
         16 . The conjugate of  claim 1 , wherein the first antigen and second antigen are different. 
     
     
         17 . The conjugate of  claim 1 , wherein the first or second antigen is selected from the group consisting of CD19, CD22, CD45, CD10, CD5, CD79a, polymorphic HLA-DR. 
     
     
         18 . The conjugate of  claim 17 , wherein the first antigen is CD19, and the second antigen is CD22. 
     
     
         19 . The conjugate of  claim 1 , wherein the first antigen-binding fragment is linked at its carboxy terminus to the therapeutic agent or the second antigen-binding fragment. 
     
     
         20 . The conjugate of  claim 1 , wherein the second antigen-binding fragment is linked at its carboxy terminus to the therapeutic agent or the first antigen-binding fragment. 
     
     
         21 . The conjugate of  claim 1 , wherein said agent is a cytotoxic agent, a cytokine, an anti-angiogenic agent, a chemotherapeutic agent, a pro-apoptosis agent, an enzyme, a hormone, a growth factor, a peptide, a protein, an antibiotic, an antibody, a Fab fragment of an antibody, an antigen, a survival factor, an anti-apoptotic agent, a hormone antagonist, a virus, a bacteriophage, a bacterium, a liposome, a cell, a nucleic acid or an expression vector. 
     
     
         22 . The conjugate of  claim 1 , wherein said agent is a cytotoxic agent. 
     
     
         23 . The conjugate of  claim 22 , wherein said cytotoxic agent comprises a peptide, a polypeptide, or a small molecule. 
     
     
         24 . The conjugate of  claim 23 , wherein said cytotoxic agent is selected from the group consisting of gelonin, ricin, abrin, diphtheria toxin,  Pseudomonas  exotoxin,  Clostridium perfringens  enterotoxin, dodecandrin, tricosanthin, tricokirin, bryodin, mirabilis antiviral protein, barley ribosome-inactivating protein (BRIP), pokeweed antiviral protein (PAPs), saporin, luffin, momordin, colicin, anthrax toxin, tetanus toxin, botulinum neurotoxin, and fragments thereof. 
     
     
         25 . The conjugate of  claim 24 , wherein said cytotoxic agent comprises diphtheria toxin. 
     
     
         26 . The conjugate of  claim 25 , wherein said cytotoxic agent comprises the translocation enhancer region of diphtheria toxin. 
     
     
         27 . The conjugate of  claim 25 , wherein said cytotoxic agent comprises the amino terminal 390 amino acids of diphtheria toxin. 
     
     
         28 . The conjugate of  claim 24 , wherein said cytotoxic agent comprises  Pseudomonas  exotoxin KDEL (SEQ ID NO:05). 
     
     
         29 . The conjugate of  claim 24 , wherein said cytotoxic agent comprises  Pseudomonas  exotoxin KDEL 7 mutant. 
     
     
         30 . The conjugate of  claim 21 , wherein said agent is an anti-angiogenic agent selected from the group consisting of thrombospondin, angiostatin, endostatin or pigment epithelium-derived factor, angiotensin, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin 12, platelet factor 4, IP-10, Gro-β, 2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron), interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP-470, paclitaxel, accutin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline. 
     
     
         31 . The conjugate of  claim 21 , wherein said agent is a cytokine selected from the group consisting of interleukin 1 (IL-1), IL-2, IL-5, IL-10, IL-11, IL-12, IL-18, interferon-γ (IF-γ), IF-α, IF-β, tumor necrosis factor-α (TNF-α), or GM-CSF (granulocyte macrophage colony stimulating factor). 
     
     
         32 . The conjugate of  claim 1 , further defined as being comprised in a pharmaceutically acceptable carrier. 
     
     
         33 . A pharmaceutical composition, comprising the conjugate of  claim 1 . 
     
     
         34 . A method of treating a human patient having a B-cell malignancy, comprising the step of administering to said patient with the conjugate of  claim 1 , wherein the conjugate targets B cells. 
     
     
         35 . The method of  claim 34 , wherein said B-cell malignancy is selected from the group consisting of B-cell subtype of non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), hairy cell leukemia, and prolymphocytic leukemia. 
     
     
         36 . The method of  claim 34 , wherein said conjugate exerts an anti-tumor activity. 
     
     
         37 . The method of  claim 36 , wherein said anti-tumor activity is selected from the group consisting of increasing tumor-free survival, killing a tumor cell or tissue, inducing apoptosis of a tumor cell or tissue, inhibiting tumor growth, inhibiting metastatic spread, reducing tumor burden and inducing tumor regression. 
     
     
         38 . The method of  claim 34 , further comprising the step of treating said subject with chemotherapy, radiotherapy, surgery, hormone therapy or gene therapy. 
     
     
         39 - 40 . (canceled) 
     
     
         41 . The conjugate of  claim 10 , wherein the second peptide linker is a ARL linker (GSTSGSGKPGSGEGSTKG; SEQ ID NO:14).