P97 fusion proteins

Abstract

Provided are p97 (melanotransferrin)-trastuzumab fusion proteins and related methods of use thereof, for instance, to facilitate delivery of trastuzumab across the blood-brain barrier (BBB) and/or improve tissue penetration of the antibody in CNS and peripheral tissues, and thereby treat and/or diagnose HER2-positive cancers, including those of the central nervous system (CNS).

1 . A p97 fusion protein, comprising a trastuzumab heavy chain or light chain sequence fused to a p97 sequence and an optional linker in between. 
     
     
         2 . The p97 fusion protein of  claim 1 , comprising SEQ ID NO:84 (MTfp NH-TZM) or 82 (TZM HC-MTf), or a variant/fragment thereof. 
     
     
         3 . The p97 fusion protein of  claim 1 , comprising a trastuzumab heavy chain sequence fused to the N-terminus of the p97 sequence. 
     
     
         4 . The p97 fusion protein of  claim 1 , comprising a trastuzumab heavy chain sequence fused to the C-terminus of the p97 sequence. 
     
     
         5 . The p97 fusion protein of  claim 4 , comprising a truncated trastuzumab heavy chain sequence fused to the C-terminus of the p97 sequence. 
     
     
         6 . The p97 fusion protein of  claim 5 , where the truncated trastuzumab heavy chain sequence consists essentially of the heavy chain constant region or a fragment thereof and substantially or entirely lacks the heavy chain variable region. 
     
     
         7 . The p97 fusion protein of  claim 6 , where the truncated trastuzumab heavy chain sequence consists essentially of the CH1 domain or a fragment thereof, the hinge region, the CH2 domain, and the CH3 domain. 
     
     
         8 . The p97 fusion protein of  claim 6 , where the truncated trastuzumab heavy chain sequence consists essentially of the hinge region or a fragment thereof, the CH2 domain, and the CH3 domain. 
     
     
         9 . The p97 fusion protein of any of the preceding claims, comprising (a) a heavy chain amino acid sequence set forth in SEQ ID NOs:37-46 or 96-109; (b) a heavy chain amino acid sequence at least 90% identical to a sequence set forth in SEQ ID NOs:37-46 or 96-109; (c) or a heavy chain amino acid sequence that differs from SEQ ID NOs:37-46 or 96-109 by addition, substitution, insertion, or deletion of about 1-50 amino acids. 
     
     
         10 . The p97 fusion protein of  claim 9 , comprising an amino acid sequence set forth in SEQ ID NOs:37-46 or 96-109. 
     
     
         11 . The p97 fusion protein of  claim 1 , comprising a trastuzumab light chain sequence fused to the N-terminus of the p97 sequence. 
     
     
         12 . The p97 fusion protein of  claim 1 , comprising a trastuzumab light chain sequence fused to the C-terminus of the p97 sequence. 
     
     
         13 . The p97 fusion protein of any of  claims 1  or  11 - 12 , comprising (a) a light amino acid sequence set forth in SEQ ID NOs:110-121; (b) a light chain amino acid sequence at least 90% identical to a sequence set forth in SEQ ID NOs: 110-121; (c) or a light chain amino acid sequence that differs from SEQ ID NOs: 110-121 by addition, substitution, insertion, or deletion of about 1-50 amino acids. 
     
     
         14 . The p97 fusion protein of  claim 13 , comprising an amino acid sequence set forth in SEQ ID NOs:110-121, or a variant/fragment thereof. 
     
     
         15 . The p97 fusion protein of any of the preceding claims, where the p97 sequence comprises SEQ ID NO:2 or 14, or a variant/fragment thereof. 
     
     
         16 . The p97 fusion protein of any of the preceding claims, comprising a peptide linker between the trastuzumab heavy chain or light chain and the p97 sequence. 
     
     
         17 . An isolated polynucleotide which encodes a p97 fusion protein of any of the preceding claims. 
     
     
         18 . The isolated polynucleotide of  claim 17 , which is codon-optimized for expression in a host cell. 
     
     
         19 . The isolated polynucleotide of  claim 18 , where the host cell is a mammalian cell, an insect cell, a yeast cell, or a bacterial cell. 
     
     
         20 . A recombinant host cell, comprising an isolated polynucleotide of any of the preceding claims, where the isolated polynucleotide is operably linked to one or more regulatory elements. 
     
     
         21 . The recombinant host cell of  claim 20 , further comprising an isolated polynucleotide that encodes a (non-fusion) trastuzumab light chain sequence, which is operably linked to one or more regulatory elements. 
     
     
         22 . The recombinant host cell of  claim 20 , further comprising an isolated polynucleotide that encodes a (non-fusion) trastuzumab heavy chain sequence, which is operably linked to one or more regulatory elements. 
     
     
         23 . The recombinant host cell of  claim 20 , further comprising an isolated polynucleotide that encodes a (non-fusion) trastuzumab light chain sequence, and an isolated polynucleotide that encodes a (non-fusion) trastuzumab heavy chain sequence, which are operably linked to one or more regulatory elements. 
     
     
         24 . A vector, comprising an isolated polynucleotide, which encodes a p97 fusion protein of any of the preceding claims, which is operably linked to one or more regulatory elements. 
     
     
         25 . The vector of  claim 24 , further comprising an isolated polynucleotide that encodes a (non-fusion) trastuzumab light chain sequence, which is operably linked to one or more regulatory elements. 
     
     
         26 . The vector of  claim 24 , further comprising an isolated polynucleotide that encodes a (non-fusion) trastuzumab heavy chain sequence, which is operably linked to one or more regulatory elements. 
     
     
         27 . The vector of  claim 24 , further comprising an isolated polynucleotide that encodes a (non-fusion) trastuzumab light chain sequence, and an isolated polynucleotide that encodes a (non-fusion) trastuzumab heavy chain sequence, which are operably linked to one or more regulatory elements. 
     
     
         28 . A recombinant host cell, comprising a vector of any of the preceding claims. 
     
     
         29 . A p97-antibody fusion protein that comprises two (non-fusion) trastuzumab light chain sequences, and one or two p97-trastuzumab heavy chain fusion proteins of any of the preceding claims, where the one or two p97-trastuzumab heavy chain fusion protein(s) comprise a trastuzumab heavy chain sequence fused to the N-terminus or C-terminus of a p97 sequence and an optional linker in between. 
     
     
         30 . The p97-antibody fusion protein of  claim 29 , comprising two p97-trastuzumab heavy chain fusion proteins. 
     
     
         31 . The p97-antibody fusion protein of  claim 29 , comprising one p97-trastuzumab heavy chain fusion protein and one (non-fusion) trastuzumab heavy chain sequence. 
     
     
         32 . A p97-antibody fusion protein that comprises one or two trastuzumab light chain sequences, one trastuzumab heavy chain sequence, and one p97-trastuzumab heavy chain fusion protein of any of the preceding claims, where the p97-trastuzumab heavy chain fusion protein comprises a trastuzumab heavy chain fused to the C-terminus of a p97 sequence and an optional linker in between. 
     
     
         33 . The p97-antibody fusion protein of  claim 32 , comprising only one light chain sequence, where the trastuzumab heavy chain is a truncated trastuzumab heavy chain. 
     
     
         34 . A p97-antibody fusion protein that comprises one or two p97-trastuzumab light chain fusion proteins of any of the preceding claims, and two trastuzumab heavy chain sequences, where the one or two p97-trastuzumab light chain fusion protein(s) comprise a trastuzumab light chain sequence fused to the N-terminus a p97 sequence and an optional linker in between. 
     
     
         35 . The p97-antibody fusion protein of  claim 34 , comprising two p97-trastuzumab light chain fusion proteins. 
     
     
         36 . The p97-antibody fusion protein of  claim 34 , comprising one p97-trastuzumab light chain fusion protein and one (non-fusion) trastuzumab light chain sequence. 
     
     
         37 . A p97-antibody fusion protein that comprises one or two p97-trastuzumab light chain fusion proteins of any of the preceding claims, and one or two p97-trastuzumab heavy chain fusion proteins of any of the preceding claims, where the one or two p97-trastuzumab light chain fusion protein(s) comprise a trastuzumab light chain sequence fused to the N-terminus a p97 sequence and an optional linker in between, and where the one or two p97-trastuzumab heavy chain fusion protein(s) comprise a trastuzumab heavy chain sequence fused to the N-terminus or C-terminus of a p97 sequence and an optional linker in between. 
     
     
         38 . The p97-antibody fusion protein of  claim 37 , comprising two p97-trastuzumab light chain fusion proteins and two p97-trastuzumab heavy chain fusion proteins, where the two p97-trastuzumab heavy chain fusion protein comprise a trastuzumab heavy chain sequence fused to the N-terminus of a p97 sequence and an optional linker in between. 
     
     
         39 . A p97-antibody fusion protein that comprises two sets of heavy and light chains, where at least one set is selected from one or more of:
 a) the heavy chain of SEQ ID NO:82 and the light chain of SEQ ID NO:83;   b) the heavy chain of SEQ ID NO:84 and the light chain of SEQ ID NO:85;   c) the heavy chain of SEQ ID NO:86 and the light chain of SEQ ID NO:87;   d) the heavy chain of SEQ ID NO:88 and the light chain of SEQ ID NO:89;   e) the heavy chain of SEQ ID NO:90 and the light chain of SEQ ID NO:91;   f) the heavy chain of SEQ ID NO:92 and the light chain of SEQ ID NO:93; and   g) the heavy chain of SEQ ID NO:94 and the light chain of SEQ ID NO:95; including fragments/variants thereof.   
     
     
         40 . The p97-antibody fusion protein of  claim 39 , where the fusion protein is a homodimer that comprises two sets of a), two sets of b), two sets of c), two sets of d), two sets of e), two sets of f), or two sets of g). 
     
     
         41 . The p97-antibody fusion protein of  claim 39 , where the fusion protein is a heterodimer that comprises a first set of heavy and light chains selected from a)-g), and a second set of heavy and light chains selected from any combination of (i) the p97-trastuzumab heavy and light chains of SEQ ID NOS:37-46, 82-109, or 110-121, and (ii) the trastuzumab (non-fusion) heavy and light chains of SEQ ID NOs: 29-35 or 122 (heavy chains) and 36 or 123 (light chains). 
     
     
         42 . A recombinant host cell, comprising a p97-antibody fusion protein of any of the preceding claims. 
     
     
         43 . The recombinant host cell of any of the preceding claims, where the host cell is a mammalian cell, an insect cell, a yeast cell, or a bacterial cell. 
     
     
         44 . The recombinant host cell of  claim 43 , where the mammalian cell is a Chinese hamster ovary (CHO) cell or a HEK-293 cell. 
     
     
         45 . A pharmaceutical composition, comprising a pharmaceutically-acceptable carrier and a p97-antibody fusion protein of any of the preceding claims. 
     
     
         46 . A method for the treatment of a HER2-overexpressing cancer in a subject in need thereof, comprising administering to the subject a p97-antibody fusion protein or pharmaceutical composition of any of the preceding claims. 
     
     
         47 . The method of  claim 46 , where the HER2-overexpressing cancer is at risk for metastasizing to the CNS of the subject. 
     
     
         48 . The method of  claim 46 , where the HER2-overexpressing cancer has metastasized to the CNS of the subject. 
     
     
         49 . The method of any of the preceding claims, where the HER2-overexpressing cancer is a breast cancer, ovarian cancer, gastric cancer, or uterine cancer. 
     
     
         50 . The method of any of  claims 46 - 48 , where the HER2-overexpressing cancer is a HER2-overexpressing metastatic breast cancer. 
     
     
         51 . The method of  claim 50 , where the HER2-overexpressing metastatic breast cancer is at risk for metastasizing to the CNS of the subject. 
     
     
         52 . The method of  claim 50 , where the HER2-overexpressing breast cancer has metastasized to the CNS of the subject. 
     
     
         53 . The method of any of  claims 46 - 48 , where the HER2-overexpressing cancer is a HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma. 
     
     
         54 . The method of  claim 53 , where the HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma is at risk for metastasizing to the CNS of the subject. 
     
     
         55 . The method of  claim 53 , where the HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma has metastasized to the CNS of the subject. 
     
     
         56 . The method of any of  claims 46 - 48 , where the HER2-overexpressing cancer is a HER2-overexpressing uterine serous carcinoma (USC). 
     
     
         57 . The method of  claim 56 , where the HER2-overexpressing USC is at risk for metastasizing to the CNS of the subject. 
     
     
         58 . The method of  claim 56 , where the HER2-overexpressing USC has metastasized to the CNS of the subject. 
     
     
         59 . The method of any of  claims 46 - 48 , comprising administering the p97-antibody fusion protein or pharmaceutical composition as part of an adjuvant treatment for a HER2-overexpressing breast cancer. 
     
     
         60 . The method of  claim 59 , where the adjuvant treatment comprises doxorubicin, cyclophosphamide, and either paclitaxel or docetaxel. 
     
     
         61 . The method of  claim 59 , where the adjuvant treatment comprises docetaxel and carboplatin. 
     
     
         62 . The method of  claim 59 , comprising administering the p97-antibody fusion protein or pharmaceutical composition as a single agent following multi-modality anthracycline based therapy. 
     
     
         63 . The method of any of the preceding claims, where the subject is a female human. 
     
     
         64 . The method of any of the preceding claims, comprising administering the p97-antibody fusion protein or pharmaceutical composition by intravenous (IV) infusion.