Human serum albumin linkers and conjugates thereof

Abstract

Disclosed is a human serum albumin (HSA) linker and HSA linker with binding, diagnostic, and therapeutic agents conjugated thereto. Also disclosed is a conjugate in which the HSA linker is covalently bonded to amino and carboxy terminal binding moieties that are first and second single-chain Fv molecules (scFvs). Exemplified conjugates are useful, e.g., in reducing tumor cell proliferation, e.g., for therapeutic therapeutic applications. Also disclosed are methods and kits for the diagnostic and therapeutic application of an HSA linker conjugate.

1 .- 36 . (canceled) 
     
     
         37 . An human serum albumin (HSA) linker conjugate comprising:
 i) an HSA linker having an amino terminus and a carboxy terminus, said HSA linker comprising an amino acid sequence set forth in any one of SEQ ID NOs:6- 15; and      ii) first and second binding moieties selected from the group consisting of whole antibodies or immunoglobulins, single-chain Fv molecules, bispecific single chain Fv molecules, domain antibodies, diabodies, triabodies, Fab fragments, F(ab′) 2  molecules, tandem scFv molecules, aptamers, receptors, ligands, hormones, and biologically-active fragments thereof, wherein said first binding moiety is bonded to the amino terminus of the HSA linker and said second binding moiety is bonded to the carboxy terminus of the HSA linker or said first binding moiety is bonded to the carboxy terminus of the HSA linker and said second binding moiety is bonded to the amino terminus of the HSA linker.   
     
     
         38 . The HSA linker conjugate of  claim 37 , wherein the HSA linker comprises the amino acid sequence set forth in SEQ ID NO:1. 
     
     
         39 . The HSA linker conjugate of  claim 37 , wherein said first or second binding moiety specifically binds a protein selected from the group consisting of insulin-like growth factor 1 receptor (IGF1R), IGF2R, insulin-like growth factor (IGF), mesenchymal epithelial transition factor receptor (c-met), hepatocyte growth factor (HGF), epidermal growth factor receptor (EGFR), ErbB2, ErbB3, epidermal growth factor (EGF), heregulin, fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR), platelet-derived growth factor (PDGF), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor (VEGF), tumor necrosis factor receptor (TNFR), tumor necrosis factor alpha (TNF-a), folate receptor (FOLR), folate, transferrin receptor (TfR), mesothelia, Fc receptor, c-kit receptor, c-kit, a4 integrin, P-selectin, sphingosine-1-phosphate receptor-1 (S1PR), hyaluronate receptor, leukocyte function antigen-1 (LFA-1), CD4, CD11, CD18, CD20, CD25, CD27, CD52, CD70, CD80, CD85, CD95 (Fas receptor), CD106 (vascular cell adhesion molecule 1 (VCAM1)), CD166 (activated leukocyte cell adhesion molecule (ALCAM)), CD 178 (Fas ligand), CD253 (TNF-related apoptosis-inducing ligand (TRAIL)), inducible costimulator (ICOS) ligand, CCR2, CXCR3, CCR5, CXCL12 (stromal cell-derived factor 1 (SDF-1)), interleukin 1 (IL-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), MART-1, gp100, MAGE-1, ephrin (Eph) receptor, mucosal addressin cell adhesion molecule 1 (MAdCAM-1), carcinoembryonic antigen (CEA), Lewis Y , MUC-1, epithelial cell adhesion molecule (EpCAM), cancer antigen 125 (CA125), prostate specific membrane antigen (PSMA), TAG-72 antigen, and fragments thereof. 
     
     
         40 . The HSA linker conjugate of  claim 37 , wherein said first or second binding moiety is selected from the group consisting of natalizumab, infliximab, adalimumab, rituximab, alemtuzumab, bevacizumab, daclizumab, efalizumab, golimumab, certolizumab, trastuzumab, abatacept, etanercept, pertuzumab, cetuximab, panitumumab, and anakinra. 
     
     
         41 . The HSA linker conjugate of  claim 37  further comprising a diagnostic or therapeutic agent. 
     
     
         42 . The HSA linker conjugate of  claim 41 , wherein said diagnostic agent is a detectable label that is radioactive, fluorescent, bioluminescent, a heavy metal, or an epitope tag. 
     
     
         43 . The HSA linker conjugate of  claim 39 , wherein said first binding moiety specifically binds ErbB3 and said second binding moiety specifically binds ErbB2 and said first and second binding moieties are single-chain Fv molecules. 
     
     
         44 . The HSA linker conjugate of  claim 43 , wherein said first binding moiety binds ErbB3 with a K d  of about 16 nM and said second binding moiety binds ErbB2 with a K d  of about 0.3 nM. 
     
     
         45 . The HSA linker conjugate of  claim 43 , wherein said first binding moiety is bonded to the amino terminus of the HSA linker and said second binding moiety is bonded to the carboxy terminus of the HSA linker. 
     
     
         46 . The HSA linker conjugate of  claim 45 , wherein said first binding moiety binds ErbB3 with a K d  of about 16 nM and said second binding moiety binds ErbB2 with a K d  of about 0.3 nM. 
     
     
         47 . Human serum albumin (HSA) linker conjugate B2B3-1 comprising the amino acid sequence set forth in SEQ ID NO:16. 
     
     
         48 . A composition comprising the HSA linker conjugate of  claim 40  admixed with a pharmaceutically acceptable carrier, excipient, or diluent. 
     
     
         49 . The composition of  claim 48  in a dosage form adapted to be administered to a patient to provide at least 10 mg/kg of the HSA linker conjugate. 
     
     
         50 . The composition of  claim 48  further comprising one or more additional therapeutic agents. 
     
     
         51 . The composition of  claim 50 , wherein the one or more additional therapeutic agents is trastuzumab, lapatinib, a taxane, or letrozole. 
     
     
         52 . The composition of  claim 49 , wherein said dosage form is further adapted to provide at least 1 mg/kg trastuzumab when administered to the patient. 
     
     
         53 . The composition of  claim 51 , wherein the taxane is paclitaxel or docetaxel. 
     
     
         54 . A method for treating cancer in a mammal in need thereof comprising administering to said mammal an effective amount of the composition of  claim 48 . 
     
     
         55 . The method of  claim 54 , wherein the cancer is a solid tumor. 
     
     
         56 . The method of  claim 54 , wherein the HSA linker conjugate is coadministered with one or more additional therapeutic agents. 
     
     
         57 . The method of  claim 56 , wherein the one or more additional therapeutic agents is selected from an antineoplastic agent or an anticancer agent, a topoisomerase inhibitor, an alkylating agent, a selective estrogen modulator, an antimetabolite, an antitumor antibiotic, an aromatase inhibitor, an anti-vascular endothelial growth factor (VEGF) agent, an anti-ErbB2 agent, an anti-ErbB3 agent, an antibody targeting insulin growth factor 1 receptor (IGF1R), a small molecule targeting IGF1R, and anti-endothelial growth factor receptor (EGFR) monoclonal antibody, a small molecule targeting EGFR, a mesenchymal epithelial transition factor receptor (cMET) antibody, a small molecule targeting cMET, a glucocorticoid, an immunotherapeutic, a microtubule targeting agent, an mTOR inhibitor, a protein synthesis inhibitor, a somatostatin analogue, a chemotherapeutic agent, a hormonal therapy, a protein kinase B inhibitor, a phosphatidylinositol 3-kinase inhibitor, a cyclin-dependent kinase inhibitor, an anti-tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) antibody, and derivatives thereof 
     
     
         58 . The method of  claim 56 , wherein said mammal is a human patient, the HSA linker conjugate is HSA linker conjugate B2B3-1 comprising the amino acid sequence set forth in SEQ ID NO:16, and the additional therapeutic agent is trastuzumab. 
     
     
         59 . The method of  claim 58 , wherein, when tested using human breast tumor cells that express ErbB3 and over-express ErbB2 in a nude mouse human xenograft model, the combination of the HSA linker conjugate with the additional therapeutic agent provides greater efficacy than is provided by the HSA linker conjugate alone or the additional therapeutic agent alone in the same model. 
     
     
         60 . The method of  claim 58 , wherein the HSA linker conjugate is administered to the patient so as to provide at least 3 mg/kg of B2B3-1 and the additional therapeutic agent is administered to the patient so as to provide at least 1 mg/kg trastuzumab. 
     
     
         61 . A method for treating cancer in a mammal in need thereof comprising administering to the mammal an effective amount of the HSA linker conjugate of  claim 43 . 
     
     
         62 . A method for treating cancer in a mammal in need thereof comprising administering to the mammal an effective amount of the composition of  claim 48 . 
     
     
         63 . A method for making an HSA linker conjugate comprising bonding a first binding moiety to the amino terminus and a second binding moiety to the carboxy terminus of the amino acid sequence set forth in any one of SEQ ID NOS:1, 3, or 6-15, wherein said first binding moiety or said second binding moiety is selected from the group consisting of whole antibodies or immunoglobulins, single-chain Fv molecules, bispecific single chain Fv molecules, domain antibodies, diabodies, triabodies, Fab fragments, F(ab′) 2  molecules, tandem scFv molecules, aptamers, receptors, ligands, hormones, and biologically-active fragments thereof