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US20160347821A1 - P97 fusion proteins - Google Patents

P97 fusion proteins Download PDF

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US20160347821A1
US20160347821A1 US15/116,388 US201515116388A US2016347821A1 US 20160347821 A1 US20160347821 A1 US 20160347821A1 US 201515116388 A US201515116388 A US 201515116388A US 2016347821 A1 US2016347821 A1 US 2016347821A1
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trastuzumab
sequence
fusion protein
heavy chain
seq
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US15/116,388
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Timothy Z. Vitalis
Reinhard Gabathuler
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Bioasis Technologies Inc
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Bioasis Technologies Inc
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Assigned to BIOASIS TECHNOLOGIES, INC. reassignment BIOASIS TECHNOLOGIES, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GABATHULER, REINHARD, VITALIS, TIMOTHY Z.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor

Definitions

  • Embodiments of the present invention relate to p97 (melanotransferrin)-trastuzumab fusion proteins and antibody 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).
  • BBB blood-brain barrier
  • CNS central nervous system
  • BBB blood-brain barrier
  • Therapeutic molecules and genes that might otherwise be effective in diagnosis and therapy do not cross the BBB in adequate amounts and often have poor tissue penetration, even in peripheral tissues. It is reported that over 95% of all therapeutic molecules do not cross the blood-brain barrier.
  • compositions and methods that facilitate the delivery of therapeutic agents and other molecules across the blood-brain-barrier, for instance, to effectively treat certain diseases of the central nervous system (CNS) such as cancers, particularly those that have metastasized to the CNS.
  • CNS central nervous system
  • Embodiments of the present invention include p97 (melanotransferrin or MTf)-trastuzumab fusion proteins, comprising a trastuzumab heavy chain and/or light chain sequence fused to a p97 sequence and an optional linker in between.
  • the fusion protein comprises a trastuzumab heavy chain sequence fused to the N-terminus of the p97 sequence.
  • the fusion protein comprises a trastuzumab heavy chain sequence fused to the C-terminus of the p97 sequence.
  • the fusion protein comprises a truncated trastuzumab heavy chain sequence fused to the C-terminus of the p97 sequence.
  • 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. In certain embodiments, 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. In certain embodiments, the truncated trastuzumab heavy chain sequence consists essentially of the hinge region or a fragment thereof, the CH2 domain, and the CH3 domain.
  • the fusion protein comprises (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.
  • the fusion protein comprises a heavy chain amino acid sequence set forth in one or more of SEQ ID NOs:37-46 or 96-109.
  • the fusion protein comprises a trastuzumab light chain sequence fused to the N-terminus of the p97 sequence. In certain embodiments, the fusion protein comprises a trastuzumab light chain sequence fused to the C-terminus of the p97 sequence. In some embodiments, the fusion protein comprises (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. In some embodiments, the fusion protein comprises a light chain amino acid sequence set forth in SEQ ID NOs: 110-121.
  • the p97 sequence comprises, consists, or consists essentially of SEQ ID NO:2 (soluble MTf) or SEQ ID NO:14 (MTfp or MTfpep).
  • isolated polynucleotides which encode a p97 fusion protein described herein.
  • the isolated polynucleotides are codon-optimized for expression in a host cell.
  • the host cell is a mammalian cell, an insect cell, a yeast cell, or a bacterial cell.
  • recombinant host cells comprising an isolated polynucleotide described herein, optionally where the isolated polynucleotide is operably linked to one or more regulatory elements.
  • the recombinant host cell comprises an isolated polynucleotide that encodes a (non-fusion) trastuzumab light chain sequence, which is operably linked to one or more regulatory elements.
  • the recombinant host cell comprises an isolated polynucleotide that encodes a (non-fusion) trastuzumab heavy chain sequence, which is operably linked to one or more regulatory elements.
  • the recombinant host cell comprises 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.
  • vectors 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.
  • the vector comprises an isolated polynucleotide that encodes a (non-fusion) trastuzumab light chain sequence, which is operably linked to one or more regulatory elements.
  • the vector comprises an isolated polynucleotide that encodes a (non-fusion) trastuzumab heavy chain sequence, which is operably linked to one or more regulatory elements.
  • the vector comprises 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. Also included are recombinant host cells, comprising one or more vectors as described herein.
  • Some embodiments relate to p97-antibody fusion proteins that comprise two (non-fusion) trastuzumab light chain sequences, and one or two p97-trastuzumab heavy chain fusion proteins described herein, where the one or two p97-trastuzumab heavy chain fusion protein(s) comprise a trastuzumab heavy chain sequence fused to the N-terminus of a p97 sequence and an optional linker in between (see, e.g., FIGS. 1A & 1B ).
  • the p97-antibody fusion protein comprises two p97-trastuzumab heavy chain fusion proteins (see, e.g., FIGS. 1A and 1E ).
  • the p97-antibody fusion protein comprises one p97-trastuzumab heavy chain fusion protein and one (non-fusion) trastuzumab heavy chain sequence (see, e.g., FIG. 1B ).
  • p97-antibody fusion proteins that comprise one trastuzumab light chain sequence, one trastuzumab heavy chain sequence, and one p97-trastuzumab heavy chain fusion protein of any of the preceding claims, where p97-trastuzumab heavy chain fusion protein comprises a truncated trastuzumab heavy chain fused to the C-terminus of a p97 sequence and an optional linker in between (see, e.g., FIG. 1C ).
  • the p97-antibody fusion protein comprises two trastuzumab light chain sequences, and two p97-trastuzumab heavy chain fusion proteins described herein, where the p97-trastuzumab heavy chain fusion proteins comprise a trastuzumab heavy chain fused to the C-terminus of a p97 sequence and an optional linker in between (see, e.g., FIG. 1D ).
  • the p97-antibody fusion protein comprises two p97-trastuzumab light chain fusion proteins described herein, and two p97-trastuzumab heavy chain fusion proteins described herein, where the p97-trastuzumab light chain fusion proteins comprise a trastuzumab light chain fused to the N-terminus of a p97 sequence and an optional linker in between, and where the p97-trastuzumab heavy chain fusion proteins comprise a trastuzumab heavy chain fused to the N-terminus of a p97 sequence and an optional linker in between (see, e.g., FIG. 1F ).
  • p97-antibody fusion proteins include those that comprise two sets of heavy and light chains, where at least one set is selected from one or more of:
  • the p97-antibody fusion 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). In some embodiments, the p97-antibody fusion is a heterodimer that comprises any combination of a)-g) above.
  • the p97-antibody fusion is a heterodimer that comprises a first set of sets of heavy and light chains selected from a)-g) above, and a second set of trastuzumab (non-fusion) heavy and light chains, for example, SEQ ID NOs: 29-35 or 122 (heavy chains) and 36 or 123 (light chains).
  • the host cell is a mammalian cell, an insect cell, a yeast cell, or a bacterial cell.
  • the mammalian cell is a Chinese hamster ovary (CHO) cell or a HEK-293 cell.
  • compositions comprising a pharmaceutically-acceptable carrier and a p97-antibody fusion protein of any of the preceding claims.
  • Some embodiments include methods 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 described herein.
  • the HER2-overexpressing cancer is at risk for metastasizing to the CNS of the subject. In some embodiments, the HER2-overexpressing cancer has metastasized to the CNS of the subject. In certain embodiments, the HER2-overexpressing cancer is a breast cancer, ovarian cancer, gastric cancer, or uterine cancer.
  • the HER2-overexpressing cancer is a HER2-overexpressing metastatic breast cancer.
  • the HER2-overexpressing metastatic breast cancer is at risk for metastasizing to the CNS of the subject.
  • the HER2-overexpressing breast cancer has metastasized to the CNS of the subject.
  • the HER2-overexpressing cancer is a HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma.
  • the HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma is at risk for metastasizing to the CNS of the subject.
  • the HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma has metastasized to the CNS of the subject.
  • the HER2-overexpressing cancer is a HER2-overexpressing uterine serous carcinoma (USC).
  • the HER2-overexpressing USC is at risk for metastasizing to the CNS of the subject.
  • the HER2-overexpressing USC has metastasized to the CNS of the subject.
  • Certain methods include administering the p97-antibody fusion protein or pharmaceutical composition as part of an adjuvant treatment for a HER2-overexpressing breast cancer.
  • the adjuvant treatment comprises doxorubicin, cyclophosphamide, and either paclitaxel or docetaxel.
  • the adjuvant treatment comprises docetaxel and carboplatin.
  • Some methods include administering the p97-antibody fusion protein or pharmaceutical composition as a single agent following multi-modality anthracycline based therapy.
  • the subject is a female human.
  • Certain methods include administering the p97-antibody fusion protein or pharmaceutical composition by intravenous (IV) infusion.
  • IV intravenous
  • FIGS. 1A-1G illustrate the general structure of exemplary p97-antibody fusion proteins (black circles represent p97).
  • FIG. 1A illustrates a homodimeric antibody fusion protein composed of two trastuzumab light chains and two trastuzumab heavy chains each fused to the N-terminus of p97.
  • FIG. 1B illustrates a heterodimeric antibody fusion protein composed of two trastuzumab light chains, one non-fused trastuzumab heavy chain, and one trastuzumab heavy chain fused to the N-terminus of p97.
  • FIG. 1A illustrates a homodimeric antibody fusion protein composed of two trastuzumab light chains and two trastuzumab heavy chains each fused to the N-terminus of p97.
  • FIG. 1B illustrates a heterodimeric antibody fusion protein composed of two trastuzumab light chains, one non-fused trastuzumab heavy chain, and one trastuzum
  • FIG. 1C illustrates a heterodimeric antibody fusion protein composed of one trastuzumab light chain, one non-fused trastuzumab heavy chain, and one trastuzumab heavy chain fused to the C-terminus of p97.
  • FIGS. 1D-1E illustrate antibody fusions composed of two p97-trastuzumab heavy chain fusion proteins and two (non-fusion) trastuzumab light chains.
  • FIG. 1F illustrates an antibody fusion composed of two p97-trastuzumab light chain fusion proteins and two 97-trastuzumab heavy chain fusion proteins.
  • FIG. 1G illustrates an antibody fusion composed of two p97-trastuzumab light chain fusion proteins and two (non-fusion) trastuzumab heavy chain sequences.
  • FIGS. 2A-2D shows octet analysis demonstrating the affinity of antibody fusions for Her2 relative to IgG1 control.
  • FIG. 2A shows the results for human IgG1
  • FIG. 2B shows the results for TZM HC-MTf
  • FIG. 2C shows the results for MTfp NH-TZM
  • FIG. 2D shows the results for TZM HC-MTfp (see Example 1).
  • FIGS. 3A-3B show antibody-dependent cell-mediated cytotoxicity (ADCC) evaluation of antibody fusions in BT-474 breast cancer cells compared to Herceptin® and human IgG1 Fc controls (see Example 2). These data show that the p97-trastuzumab antibody fusions induced significant antibody-dependent cell-mediated cytotoxicity in breast cancer cells.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • an element means one element or more than one element.
  • amino acid is intended to mean both naturally occurring and non-naturally occurring amino acids as well as amino acid analogs and mimetics.
  • Naturally occurring amino acids include the 20 (L)-amino acids utilized during protein biosynthesis as well as others such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline and ornithine, for example.
  • Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine and the like, which are known to a person skilled in the art.
  • Amino acid analogs include modified forms of naturally and non-naturally occurring amino acids.
  • Such modifications can include, for example, substitution or replacement of chemical groups and moieties on the amino acid or by derivatization of the amino acid.
  • Amino acid mimetics include, for example, organic structures which exhibit functionally similar properties such as charge and charge spacing characteristic of the reference amino acid. For example, an organic structure which mimics Arginine (Arg or R) would have a positive charge moiety located in similar molecular space and having the same degree of mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid.
  • Mimetics also include constrained structures so as to maintain optimal spacing and charge interactions of the amino acid or of the amino acid functional groups. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.
  • conjugate is intended to refer to the entity formed as a result of covalent or non-covalent attachment or linkage of an agent or other molecule, e.g., a biologically active molecule, to a p97 polypeptide or p97 sequence.
  • an agent or other molecule e.g., a biologically active molecule
  • conjugate polypeptide is a “fusion protein” or “fusion polypeptide,” that is, a polypeptide that is created through the joining of two or more coding sequences, which originally coded for separate polypeptides; translation of the joined coding sequences results in a single, fusion polypeptide, typically with functional properties derived from each of the separate polypeptides.
  • fusion protein or “fusion polypeptide”
  • antibody fusion protein are used interchangeably herein to refer to an antibody or antibody-like molecule that comprises at least one fusion protein described herein.
  • the terms “function” and “functional” and the like refer to a biological, enzymatic, or therapeutic function.
  • Homology refers to the percentage number of amino acids that are identical or constitute conservative substitutions. Homology may be determined using sequence comparison programs such as GAP (Deveraux et al., Nucleic Acids Research. 12, 387-395, 1984), which is incorporated herein by reference. In this way sequences of a similar or substantially different length to those cited herein could be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.
  • isolated is meant material that is substantially or essentially free from components that normally accompany it in its native state.
  • an “isolated peptide” or an “isolated polypeptide” and the like, as used herein, includes the in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell; i.e., it is not significantly associated with in vivo substances.
  • linkage refers to a linker that can be used to separate a p97 polypeptide from an agent of interest, or to separate a first agent from another agent, for instance where two or more agents are linked to form a p97 conjugate.
  • the linker may be physiologically stable or may include a releasable linker such as an enzymatically degradable linker (e.g., proteolytically cleavable linkers).
  • the linker may be a peptide linker, for instance, as part of a p97 fusion protein.
  • the linker may be a non-peptide linker or non-proteinaceous linker.
  • the linker may be particle, such as a nanoparticle.
  • modulating and “altering” include “increasing,” “enhancing” or “stimulating,” as well as “decreasing” or “reducing,” typically in a statistically significant or a physiologically significant amount or degree relative to a control.
  • An “increased,” “stimulated” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7.
  • a “decreased” or “reduced” amount is typically a “statistically significant” amount, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease in the amount produced by no composition or a control composition, including all integers in between.
  • a control could compare the activity, such as the amount or rate of transport/delivery across the blood brain barrier, the rate and/or levels of distribution to central nervous system tissue, and/or the C max for plasma, central nervous system tissues, or any other systemic or peripheral non-central nervous system tissues, of a p97 fusion protein or antibody fusion relative to the agent/antibody alone.
  • Other examples of comparisons and “statistically significant” amounts are described herein.
  • the “purity” of any given agent (e.g., a p97 conjugate such as a fusion protein or antibody fusion) in a composition may be specifically defined.
  • certain compositions may comprise an agent that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure, including all decimals in between, as measured, for example and by no means limiting, by high pressure liquid chromatography (HPLC), a well-known form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds.
  • HPLC high pressure liquid chromatography
  • polypeptide and protein are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers.
  • the polypeptides described herein are not limited to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise.
  • polypeptides described herein may also comprise post-expression modifications, such as glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • a polypeptide may be an entire protein, or a subsequence, fragment, variant, or derivative thereof.
  • a “physiologically cleavable” or “hydrolyzable” or “degradable” bond is a bond that reacts with water (i.e., is hydrolyzed) under physiological conditions.
  • the tendency of a bond to hydrolyze in water will depend not only on the general type of linkage connecting two central atoms but also on the substituents attached to these central atoms.
  • Appropriate hydrolytically unstable or weak linkages include, but are not limited to: carboxylate ester, phosphate ester, anhydride, acetal, ketal, acyloxyalkyl ether, imine, orthoester, thio ester, thiol ester, carbonate, and hydrazone, peptides and oligonucleotides.
  • a “releasable linker” includes, but is not limited to, a physiologically cleavable linker and an enzymatically degradable linker.
  • a “releasable linker” is a linker that may undergo either spontaneous hydrolysis, or cleavage by some other mechanism (e.g., enzyme-catalyzed, acid-catalyzed, base-catalyzed, and so forth) under physiological conditions.
  • a “releasable linker” can involve an elimination reaction that has a base abstraction of a proton, (e.g., an ionizable hydrogen atom, H ⁇ ), as the driving force.
  • a “releasable linker” is synonymous with a “degradable linker.”
  • An “enzymatically degradable linkage” includes a linkage, e.g., amino acid sequence that is subject to degradation by one or more enzymes, e.g., peptidases or proteases.
  • a releasable linker has a half life at pH 7.4, 25° C., e.g., a physiological pH, human body temperature (e.g., in vivo), of about 30 minutes, about 1 hour, about 2 hour, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about 96 hours or less.
  • reference sequence refers generally to a nucleic acid coding sequence, or amino acid sequence, to which another sequence is being compared. All polypeptide and polynucleotide sequences described herein are included as references sequences, including those described by name and those described in the Sequence Listing.
  • sequence identity or, for example, comprising a “sequence 50% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, I
  • the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys
  • nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein (see, e.g., Sequence Listing), typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide.
  • references to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence,” “comparison window,” “sequence identity,” “percentage of sequence identity,” and “substantial identity.”
  • a “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length.
  • two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • the comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • GAP Garnier et al.
  • FASTA Altschul et al.
  • TFASTA Pearson-binding Alignment of sequences for aligning a comparison window
  • Statistical significance By “statistically significant,” it is meant that the result was unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or probability with which the observed event would occur, if the null hypothesis were true. If the obtained p-value is smaller than the significance level, then the null hypothesis is rejected. In simple cases, the significance level is defined at a p-value of 0.05 or less.
  • solubility refers to the property of a protein to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as a concentration, either by mass of solute per unit volume of solvent (g of solute per kg of solvent, g per dL (100 mL), mg/ml, etc.), molarity, molality, mole fraction or other similar descriptions of concentration.
  • the maximum equilibrium amount of solute that can dissolve per amount of solvent is the solubility of that solute in that solvent under the specified conditions, including temperature, pressure, pH, and the nature of the solvent. In certain embodiments, solubility is measured at physiological pH, or other pH, for example, at pH 5.0, pH 6.0, pH 7.0, or pH 7.4.
  • solubility is measured in water or a physiological buffer such as PBS or NaCl (with or without NaP). In specific embodiments, solubility is measured at relatively lower pH (e.g., pH 6.0) and relatively higher salt (e.g., 500 mM NaCl and 10 mM NaP). In certain embodiments, solubility is measured in a biological fluid (solvent) such as blood or serum. In certain embodiments, the temperature can be about room temperature (e.g., about 20, 21, 22, 23, 24, 25° C.) or about body temperature ( ⁇ 37° C.).
  • a p97 polypeptide or conjugate has a solubility of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 mg/ml at room temperature or at about 37° C.
  • a “subject,” as used herein, includes any animal that exhibits a symptom, or is at risk for exhibiting a symptom, which can be treated or diagnosed with a p97 fusion protein or related antibody fusion of the invention.
  • Suitable subjects include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog).
  • Non-human primates and, preferably, human patients, are included.
  • substantially or “essentially” means nearly totally or completely, for instance, 95%, 96%, 97%, 98%, 99% or greater of some given quantity.
  • compositions refers to the nearly complete or complete absence of a given quantity for instance, less than about 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or less of some given quantity.
  • certain compositions may be “substantially free” of cell proteins, membranes, nucleic acids, endotoxins, or other contaminants.
  • Treatment includes any desirable effect on the symptoms or pathology of a disease or condition, and may include even minimal changes or improvements in one or more measurable markers of the disease or condition being treated. “Treatment” or “treating” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. The subject receiving this treatment is any subject in need thereof. Exemplary markers of clinical improvement will be apparent to persons skilled in the art.
  • wild-type refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally-occurring source.
  • a wild type gene or gene product e.g., a polypeptide
  • a wild type gene or gene product is that which is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form of the gene.
  • Embodiments of the present invention relate generally to fusion proteins that comprise a human p97 (melanotransferrin; MTf) polypeptide sequence a trastuzumab sequence, or an antigen-binding fragment thereof, antibodies that comprise such fusion proteins (i.e., antibody fusions), polynucleotides encoding the fusion proteins, host cells and methods of producing fusion proteins/antibodies, and related compositions and methods of use thereof.
  • Exemplary p97 polypeptide sequences and trastuzumab sequences are described below. Also described are exemplary methods and components, such as linker peptides, for coupling a p97 polypeptide sequence to a trastuzumab sequence.
  • a p97 polypeptide sequence used in a composition and/or fusion protein of the invention comprises, consists essentially of, or consists of a human p97 reference sequence provided in Table 1 below. Also included are variants and fragments thereof.
  • a p97 polypeptide sequence comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity or homology, along its length, to a human p97 sequence in Table 1, or a fragment thereof.
  • a p97 polypeptide sequence comprises a fragment of a human p97 sequence in Table 1.
  • a p97 polypeptide fragment is about, at least about, or up to about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 100, 105, 110,
  • a p97 polypeptide fragment is about 5-700, 5-600, 5-500, 5-400, 5-300, 5-200, 5-100, 5-50, 5-40, 5-30, 5-25, 5-20, 5-15, 5-10, 10-700, 10-600, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 10-40, 10-30, 10-25, 10-20, 10-15, 20-700, 20-600, 20-500, 20-400, 20-300, 20-200, 20-100, 20-50, 20-40, 20-30, 20-25, 30-700, 30-600, 30-500, 30-400, 30-300, 30-200, 30-100, 30-50, 30-40, 40-700, 40-600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-50, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 60-700, 60-600, 60-500, 60-400, 60-300, 60-200, 60-100, 60-70, 70
  • p97 polypeptide sequences of interest include p97 amino acid sequences, subsequences, and/or variants of p97 that are effective for transporting an agent of interest across the blood brain barrier and into the central nervous system (CNS).
  • the variant or fragment comprises the N-lobe of human p97 (residues 20-361 of SEQ ID NO:1).
  • the variant or fragment comprises an intact and functional Fe 3+ -binding site.
  • a p97 polypeptide sequence is a soluble form of a p97 polypeptide (see Yang et al., Prot Exp Purif. 34:28-48, 2004), or a fragment or variant thereof.
  • the soluble p97 polypeptide has a deletion of the all or a portion of the hydrophobic domain (residues 710-738 of SEQ ID NO:1), alone or in combination with a deletion of all or a portion of the signal peptide (residues 1-19 of SEQ ID NO:1).
  • the soluble p97 polypeptide comprises or consists of SEQ ID NO:2 (residues 20-711 of SEQ ID NO:1), including variants and fragments thereof.
  • the p97 polypeptide comprises, consists, or consists essentially of the sequence DSSHAFTLDELR (SEQ ID NO:14 or MTfp), including variants and fragments thereof.
  • the DSSHAFTLDELR (SEQ ID NO:14) peptide comprises a C-terminal tyrosine (Y).
  • the p97 polypeptide sequence is a lipid soluble form of a p97 polypeptide.
  • certain of these and related embodiments include a p97 polypeptide that comprises all or a portion of the hydrophobic domain, optionally with or without the signal peptide.
  • the p97 fragment or variant is capable of specifically binding to a p97 receptor, an LRP1 receptor and/or an LRP1B receptor.
  • a trastuzumab antibody sequence used in a fusion protein of the invention comprises, consists essentially of, or consists of the trastuzumab light chain and/or heavy chain sequence(s) illustrated in Table 2 below.
  • trastuzumab heavy and light chain sequences described herein are also included.
  • the trastuzumab antibody, antigen-binding fragment thereof, or related fusion protein or antibody fusion specifically binds to Her2/neu or an epitope or fragment thereof.
  • the trastuzumab heavy chain fragment(s), e.g., the Fc regions of the heavy chain fragments, are modified to increase a preferred chain combination, for example, by using knobs-into-holes (KiH) technology (see, e.g., Klein et al., mAbs. 4:6, 653-663, 2012) or other technologies, such as those described in U.S. Application No. 2012/0149876.
  • a heterodimeric antibody fusion e.g., an antibody that comprises one p97-trastuzumab heavy chain fusion and one normal trastuzumab heavy chain; see, e.g., FIGS.
  • one of the heavy chains can have amino acid modifications to generate the “knob” and the other heavy chain can have amino acid modifications to form the “hole”.
  • KiH modifications to trastuzumab heavy chain sequences are illustrated in Table 2 above.
  • one heavy chain fragment could comprise a CH3 domain having amino acid modifications selected from one or more of T350V, L351Y, D399R, D399 (e.g., D399R, D399W, D399Y, D399K), 5400 (e.g., S400E, S400D, S400R, S400K), F405 (e.g., F4051, F405M, F405T, F405S, F405V, F405W), Y407A, Y4071, Y407V, including combinations thereof, and the other could comprise a CH3 domain having amino acid modifications selected from one or more of T350V, T366V, T3661, T366L, T366M, N390 (e.g., N390R, N390K, N390D), K392 (e.g., K392V, K392M, K392R, K392L, K392F, K392
  • an antigen-binding fragment refers to a polypeptide fragment that contains at least one CDR of an immunoglobulin heavy and/or light chains that bind to the antigen of interest.
  • an antigen-binding fragment of the herein described antibodies may comprise 1, 2, 3, 4, 5, or all 6 CDRs of a VH and VL sequence from antibodies that bind to a therapeutic or diagnostic target.
  • antigen refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and additionally capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen.
  • a selective binding agent such as an antibody
  • An antigen may have one or more epitopes.
  • epitope includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor.
  • An epitope is a region of an antigen that is bound by an antibody.
  • epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl, and may in certain embodiments have specific three-dimensional structural characteristics, and/or specific charge characteristics. Epitopes can be contiguous or non-contiguous in relation to the primary structure of the antigen.
  • An antibody, antigen-binding fragment thereof is said to exhibit “specific binding” or “preferential binding” if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances.
  • An antibody “specifically binds” or “preferentially binds” to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances.
  • an antibody that specifically or preferentially binds to a specific epitope is an antibody that binds that specific epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other epitopes.
  • an antibody or moiety or epitope that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target.
  • “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding.
  • reference to binding means preferential binding.
  • Immunological binding generally refers to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific, for example by way of illustration and not limitation, as a result of electrostatic, ionic, hydrophilic and/or hydrophobic attractions or repulsion, steric forces, hydrogen bonding, van der Waals forces, and other interactions.
  • the strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (K d ) of the interaction, wherein a smaller K d represents a greater affinity.
  • Immunological binding properties of selected polypeptides can be quantified using methods well known in the art.
  • One such method entails measuring the rates of antigen-binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and on geometric parameters that equally influence the rate in both directions.
  • both the “on rate constant” (K on ) and the “off rate constant” (K off ) can be determined by calculation of the concentrations and the actual rates of association and dissociation.
  • the ratio of K off /K on enables cancellation of all parameters not related to affinity, and is thus equal to the dissociation constant K d .
  • a protein is said to specifically bind an antigen or epitope thereof when the equilibrium dissociation constant is about ⁇ 10 ⁇ 7 or 10 ⁇ 8 M.
  • the equilibrium dissociation constant of a protein may be about ⁇ 10 ⁇ 9 M or 10 ⁇ 10 M.
  • a protein has an affinity (K d ) for an antigen or target described herein (to which it specifically binds) of at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM.
  • antibodies and antigen-binding fragments thereof as described herein include a heavy chain and a light chain CDR set, respectively interposed between a heavy chain and a light chain framework region (FR) set which provide support to the CDRs and define the spatial relationship of the CDRs relative to each other.
  • CDR set refers to the three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3” respectively.
  • An antigen-binding site therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region.
  • a polypeptide comprising a single CDR (e.g., a CDR1, CDR2 or CDR3) is referred to herein as a “molecular recognition unit.” Crystallographic analysis of a number of antigen-antibody complexes has demonstrated that the amino acid residues of CDRs form extensive contact with bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the molecular recognition units are primarily responsible for the specificity of an antigen-binding site.
  • FR set refers to the four flanking amino acid sequences which frame the CDRs of a CDR set of a heavy or light chain V region. Some FR residues may contact bound antigen; however, FRs are primarily responsible for folding the V region into the antigen-binding site, particularly the FR residues directly adjacent to the CDRs. Within FRs, certain amino residues and certain structural features are very highly conserved. In this regard, all V region sequences contain an internal disulfide loop of around 90 amino acid residues. When the V regions fold into a binding-site, the CDRs are displayed as projecting loop motifs which form an antigen-binding surface.
  • immunoglobulin variable domains may be determined by reference to Kabat, E. A. et al., Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and updates thereof.
  • a “monoclonal antibody” refers to a homogeneous antibody population wherein the monoclonal antibody is comprised of amino acids (naturally occurring and non-naturally occurring) that are involved in the selective binding of an epitope. Monoclonal antibodies are highly specific, being directed against a single epitope.
  • monoclonal antibody encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab′, F(ab′) 2 , Fv), single chain (ScFv), variants thereof, fusion proteins comprising an antigen-binding portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding fragment (epitope recognition site) of the required specificity and the ability to bind to an epitope.
  • fragments thereof such as Fab, Fab′, F(ab′) 2 , Fv), single chain (ScFv), variants thereof, fusion proteins comprising an antigen-binding portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding fragment (epitope recognition site) of the required specificity and the ability to bind to an epi
  • antibody it is not intended to be limited as regards the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals).
  • the term includes whole immunoglobulins as well as the fragments etc. described above under the definition of “antibody.”
  • the proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the F(ab) fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site.
  • the enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the F(ab′) 2 fragment which comprises both antigen-binding sites.
  • An Fv fragment for use according to certain embodiments of the present invention can be produced by preferential proteolytic cleavage of an IgM, and on rare occasions of an IgG or IgA immunoglobulin molecule. Fv fragments are, however, more commonly derived using recombinant techniques known in the art.
  • the Fv fragment includes a non-covalent V H ::V L heterodimer including an antigen-binding site which retains much of the antigen recognition and binding capabilities of the native antibody molecule. See Inbar et al., PNAS USA. 69:2659-2662, 1972; Hochman et al., Biochem. 15:2706-2710, 1976; and Ehrlich et al., Biochem. 19:4091-4096, 1980.
  • single chain Fv or scFV antibodies are contemplated.
  • Kappa bodies III et al., Prot. Eng. 10:949-57, 1997
  • minibodies Martin et al., EMBO J 13:5305-9, 1994
  • diabodies Holliger et al., PNAS 90: 6444-8, 1993
  • Janusins Traunecker et al., EMBO J 10: 3655-59, 1991; and Traunecker et al., Int. J. Cancer Suppl. 7:51-52, 1992
  • a single chain Fv (sFv) polypeptide is a covalently linked V H ::V L heterodimer which is expressed from a gene fusion including V H - and V L -encoding genes linked by a peptide-encoding linker.
  • Huston et al. PNAS USA. 85(16):5879-5883, 1988).
  • a number of methods have been described to discern chemical structures for converting the naturally aggregated—but chemically separated—light and heavy polypeptide chains from an antibody V region into an sFv molecule which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778, to Ladner et al.
  • the antibodies or antigen-binding fragments thereof are humanized. These embodiments refer to a chimeric molecule, generally prepared using recombinant techniques, having an antigen-binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based upon the structure and/or sequence of a human immunoglobulin.
  • the antigen-binding site may comprise either complete variable domains fused onto constant domains or only the CDRs grafted onto appropriate framework regions in the variable domains.
  • Epitope binding sites may be wild type or modified by one or more amino acid substitutions.
  • variable regions of both heavy and light chains contain three complementarity-determining regions (CDRs) which vary in response to the epitopes in question and determine binding capability, flanked by four framework regions (FRs) which are relatively conserved in a given species and which putatively provide a scaffolding for the CDRs.
  • CDRs complementarity-determining regions
  • FRs framework regions
  • the variable regions can be “reshaped” or “humanized” by grafting CDRs derived from nonhuman antibody on the FRs present in the human antibody to be modified.
  • humanized antibodies preserve all CDR sequences (for example, a humanized mouse antibody which contains all six CDRs from the mouse antibodies).
  • humanized antibodies have one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody.
  • certain fusion proteins may employ one or more linker groups, including peptide linkers.
  • linkers can be stable linkers or releasable linkers.
  • peptide linkers can separate the components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures.
  • a peptide linker sequence may be incorporated into the fusion protein using standard techniques described herein and well-known in the art.
  • Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes.
  • Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180.
  • a peptide linker is between about 1 to 5 amino acids, between 5 to 10 amino acids, between 5 to 25 amino acids, between 5 to 50 amino acids, between 10 to 25 amino acids, between 10 to 50 amino acids, between 10 to 100 amino acids, or any intervening range of amino acids. In other illustrative embodiments, a peptide linker comprises about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids in length.
  • Particular linkers can have an overall amino acid length of about 1-200 amino acids, 1-150 amino acids, 1-100 amino acids, 1-90 amino acids, 1-80 amino acids, 1-70 amino acids, 1-60 amino acids, 1-50 amino acids, 1-40 amino acids, 1-30 amino acids, 1-20 amino acids, 1-10 amino acids, 1-5 amino acids, 1-4 amino acids, 1-3 amino acids, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100 or more amino acids.
  • a peptide linker may employ any one or more naturally-occurring amino acids, non-naturally occurring amino acid(s), amino acid analogs, and/or amino acid mimetics as described elsewhere herein and known in the art.
  • Certain amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., PNAS USA. 83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180.
  • Particular peptide linker sequences contain Gly, Ser, and/or Asn residues.
  • Other near neutral amino acids, such as Thr and Ala may also be employed in the peptide linker sequence, if desired.
  • Certain exemplary linkers include Gly, Ser and/or Asn-containing linkers, as follows: [G] x , [S] x , [N] x , [GS] x , [GGS] x , [GSS] x , [GSGS] x (SEQ ID NO:47), [GGSG] x (SEQ ID NO:48), [GGGS] x (SEQ ID NO:49), [GGGGS] x (SEQ ID NO:50), [GN] x , [GGN] x , [GNN] x , [GNGN] x (SEQ ID NO:51), [GGNG] x (SEQ ID NO:52), [GGGN] x (SEQ ID NO:53), [GGGGN] x (SEQ ID NO:54) linkers, where x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more.
  • the linker comprises or consists of a [GGGGS] 3 (SEQ ID NO:55) sequence, or GGGGSGGGGSGGGGS (SEQ ID NO:55).
  • the linker sequence comprises a Gly3 linker sequence, which includes three glycine residues.
  • flexible linkers can be rationally designed using a computer program capable of modeling both DNA-binding sites and the peptides themselves (Desjarlais & Berg, PNAS. 90:2256-2260, 1993; and PNAS. 91:11099-11103, 1994) or by phage display methods.
  • the peptide linkers may be physiologically stable or may include a releasable linker such as a physiologically degradable or enzymatically degradable linker (e.g., proteolytically cleavable linker).
  • a releasable linker such as a physiologically degradable or enzymatically degradable linker (e.g., proteolytically cleavable linker).
  • one or more releasable linkers can result in a shorter half-life and more rapid clearance of the conjugate.
  • Enzymatically degradable linkages suitable for use in particular embodiments of the present invention include, but are not limited to: an amino acid sequence cleaved by a serine protease such as thrombin, chymotrypsin, trypsin, elastase, kallikrein, or substilisin.
  • a serine protease such as thrombin, chymotrypsin, trypsin, elastase, kallikrein, or substilisin.
  • thrombin-cleavable amino acid sequences include, but are not limited to: -Gly-Arg-Gly-Asp-(SEQ ID NO:56), -Gly-Gly-Arg-, -Gly-Arg-Gly-Asp-Asn-Pro-(SEQ ID NO:57), -Gly-Arg-Gly-Asp-Ser-(SEQ ID NO:58), -Gly-Arg-Gly-Asp-Ser-Pro-Lys-(SEQ ID NO:59), -Gly-Pro-Arg-, -Val-Pro-Arg-, and -Phe-Val-Arg-.
  • elastase-cleavable amino acid sequences include, but are not limited to: -Ala-Ala-Ala-, -Ala-Ala-Pro-Val-(SEQ ID NO:60), -Ala-Ala-Pro-Leu-(SEQ ID NO:61), -Ala-Ala-Pro-Phe-(SEQ ID NO:62), -Ala-Ala-Pro-Ala-(SEQ ID NO:63), and -Ala-Tyr-Leu-Val-(SEQ ID NO:64).
  • Enzymatically degradable linkages suitable for use in particular embodiments of the present invention also include amino acid sequences that can be cleaved by a matrix metalloproteinase such as collagenase, stromelysin, and gelatinase.
  • a matrix metalloproteinase such as collagenase, stromelysin, and gelatinase.
  • matrix metalloproteinase-cleavable amino acid sequences include, but are not limited to: -Gly-Pro-Y-Gly-Pro-Z-(SEQ ID NO:65), -Gly-Pro-, Leu-Gly-Pro-Z-(SEQ ID NO:66), -Gly-Pro-Ile-Gly-Pro-Z-(SEQ ID NO:67), and -Ala-Pro-Gly-Leu-Z-(SEQ ID NO: 68), where Y and Z are amino acids.
  • collagenase-cleavable amino acid sequences include, but are not limited to: -Pro-Leu-Gly-Pro-D-Arg-Z-(SEQ ID NO:69), -Pro-Leu-Gly-Leu-Leu-Gly-Z-(SEQ ID NO:70), -Pro-Gln-Gly-Ile-Ala-Gly-Trp-(SEQ ID NO:71), -Pro-Leu-Gly-Cys(Me)-His-(SEQ ID NO:72), -Pro-Leu-Gly-Leu-Tyr-Ala-(SEQ ID NO:73), -Pro-Leu-Ala-Leu-Trp-Ala-Arg-(SEQ ID NO:74), and -Pro-Leu-Ala-Tyr-Trp-Ala-Arg-(SEQ ID NO:75), where Z is an amino acid.
  • stromelysin-cleavable amino acid sequence is -Pro-Tyr-Ala-Tyr-Tyr-Met-Arg-(SEQ ID NO:76); and an example of a gelatinase-cleavable amino acid sequence is -Pro-Leu-Gly-Met-Tyr-Ser-Arg-(SEQ ID NO:77).
  • Enzymatically degradable linkages suitable for use in particular embodiments of the present invention also include amino acid sequences that can be cleaved by an angiotensin converting enzyme, such as, for example, -Asp-Lys-Pro-, -Gly-Asp-Lys-Pro-(SEQ ID NO:78), and -Gly-Ser-Asp-Lys-Pro-(SEQ ID NO:79).
  • Enzymatically degradable linkages suitable for use in particular embodiments of the present invention also include amino acid sequences that can be degraded by cathepsin B, such as, for example, -Val-Cit-, -Ala-Leu-Ala-Leu- (SEQ ID NO:80), -Gly-Phe-Leu-Gly- (SEQ ID NO:81) and -Phe-Lys-.
  • the linker comprises, consists, or consists essentially of 125 (SEQ ID NO:124), including fragments and variants thereof.
  • any one or more of the non-peptide or peptide linkers are optional.
  • linker sequences may not be required in a fusion protein where the first and second polypeptides have non-essential N-terminal and/or C-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
  • fusion proteins comprising a p97 polypeptide sequence fused to a trastuzumab polypeptide sequence such as a trastuzumab heavy or light chain sequence, and antibody fusions comprising the same.
  • An “antibody fusion” refers to an antibody or antibody-like immunoglobulin molecule that comprises one or more p97-trastuzumab fusion proteins and optionally one or more non-fusion trastuzumab sequences, i.e., trastuzumab light chain or heavy chain sequences, or variants/fragments thereof, which are not fused to a p97 sequence.
  • an antibody fusion comprises two light chain sequences and two heavy chain sequences, which are individually selected from any of the light/heavy chain sequences and/or fusion protein sequences described herein. In some instances, an antibody fusion comprises one light chain sequence and two heavy chain sequences, which are individually selected from any of the light/heavy chain sequences and/or fusion protein sequences described herein.
  • p97-trastuzumab heavy chain fusion proteins are illustrated in Table 3 below, and in Table E1 (see Examples).
  • the p97-trastuzumab fusion protein comprises, consists, or consists essentially of a trastuzumab heavy chain sequence fused to the N-terminus of a p97 sequence.
  • the p97 sequence is human soluble p97, for example, comprising or consisting of SEQ ID NO:2, or a variant/fragment thereof.
  • the p97 sequence comprises or consists of SEQ ID NO:14, or a variant/fragment thereof.
  • the trastuzumab heavy chain sequence is selected from SEQ ID NO:29-35 or 122, or a variant/fragment thereof.
  • the fusion protein comprises a peptide linker in between the p97 and trastuzumab sequences.
  • the linker is a (GGGGS) 2 or (GGGGS) 3 linker or a EAAAKEAAAKEAAAK (SEQ ID NO:124) linker.
  • the p97-trastuzumab fusion protein comprises, consists, or consists essentially of SEQ ID NO:37 (trastuzumab heavy chain fused to the N-terminus of soluble p97) or a variant/fragment thereof.
  • the linker is a (GGGGS) 3 linker
  • the fusion protein optionally comprises, consists, or consists essentially of SEQ ID NO:38 (trastuzumab heavy chain fused to the N-terminus of soluble human p97, and separated by a (GGGGS) 3 linker), or a variant/fragment thereof.
  • SEQ ID NO:38 tacuzumab heavy chain fused to the N-terminus of soluble human p97, and separated by a (GGGGS) 3 linker
  • Other combinations will be apparent to persons skilled in the art.
  • the p97-trastuzumab fusion protein comprises, consists, or consists essentially of a trastuzumab heavy chain sequence fused to the C-terminus of a p97 sequence.
  • the p97 sequence is human soluble p97, for example, comprising or consisting of SEQ ID NO:2, or a variant/fragment thereof.
  • the p97 sequence comprises or consists of SEQ ID NO:14, or a variant/fragment thereof.
  • the trastuzumab heavy chain sequence is selected from SEQ ID NO:29-35 or 122, or a variant/fragment thereof.
  • the trastuzumab heavy chain sequence is a truncated sequence that comprises the polypeptide of SEQ ID NO:31-33 or 34, or a variant/fragment thereof.
  • the p97-trastuzumab fusion protein comprises, consists, or consists essentially of SEQ ID NO:39, 41, 43, or 45 (truncated trastuzumab heavy chain fused to the C-terminus of soluble human p97), or a variant/fragment thereof.
  • the fusion protein comprises a peptide linker in between the p97 and trastuzumab sequences.
  • the linker is a (GGGGS) 2 or (GGGGS) 3 linker or a EAAAKEAAAKEAAAK (SEQ ID NO:124) linker.
  • the linker is a (GGGGS) 3 linker, and the fusion protein optionally comprises, consists, or consists essentially of SEQ ID NO:40, 42, 44 or 46 (truncated trastuzumab heavy chain fused to the C-terminus of soluble human p97, and separated by a (GGGGS) 3 linker), or a variant/fragment thereof.
  • the p97-trastuzumab heavy chain fusion protein comprises, consists, or consists essentially of a polypeptide sequence selected from SEQ ID NOs:37-46 and 96-109, or a variant/fragment thereof.
  • p97-trastuzumab light chain fusion proteins are also included.
  • Specific, non-limiting examples of p97-trastuzumab light chain fusion proteins are illustrated in Table 4 below, and in Table E1 (see Examples).
  • the p97-trastuzumab fusion protein comprises, consists, or consists essentially of a trastuzumab light chain sequence fused to the N-terminus of a p97 sequence.
  • the p97 sequence is human soluble p97, for example, comprising or consisting of SEQ ID NO:2, or a variant/fragment thereof.
  • the p97 sequence comprises or consists of SEQ ID NO:14, or a variant/fragment thereof.
  • the trastuzumab light chain sequence comprises or consists of SEQ ID NO:36 or 123, or a variant/fragment thereof.
  • the p97-trastuzumab fusion protein comprises, consists, or consists essentially of a sequence selected from SEQ ID NOs:110-115 (trastuzumab light chain fused to the N-terminus of p97pep), or a variant/fragment thereof.
  • the fusion protein comprises a peptide linker in between the p97 and trastuzumab sequence, for example, as illustrated in SEQ ID NO:111-112 or 114-115.
  • the linker is a (GGGGS) 2 or (GGGGS) 3 linker or a EAAAKEAAAKEAAAK (SEQ ID NO:124) linker.
  • Other combinations will be apparent to persons skilled in the art.
  • the p97-trastuzumab fusion protein comprises, consists, or consists essentially of a trastuzumab light chain sequence fused to the C-terminus of a p97 sequence.
  • the p97 sequence is human soluble p97, for example, comprising or consisting of SEQ ID NO:2, or a variant/fragment thereof.
  • the p97 sequence comprises or consists of SEQ ID NO:14, or a variant/fragment thereof.
  • the trastuzumab light chain sequence comprises or consists of SEQ ID NO:36 or 123, or a variant/fragment thereof.
  • the p97-trastuzumab fusion protein comprises, consists, or consists essentially of SEQ ID NO:116-121 (trastuzumab light chain fused to the C-terminus of p97p), or a variant/fragment thereof.
  • the fusion protein comprises a peptide linker in between the p97 and trastuzumab sequences, as illustrated, for example, in SEQ ID NOs:117-118 and 120-121.
  • the linker is a (GGGGS) 2 or (GGGGS) 3 linker or a EAAAKEAAAKEAAAK (SEQ ID NO:124) linker.
  • Other combinations will be apparent to persons skilled in the art.
  • p97-antibody fusion proteins comprising one or more p97-trastuzumab heavy or light chain fusion proteins described herein.
  • the p97-antibody fusion protein comprises two (non-fusion) trastuzumab light chain sequences, and one or two p97-trastuzumab heavy chain fusion proteins described herein, where the one or two p97-trastuzumab heavy chain fusion protein(s) comprise a trastuzumab heavy chain sequence fused to the N-terminus of a p97 sequence and an optional linker in between.
  • the p97-antibody fusion comprises two p97-trastuzumab heavy chain fusion proteins (see, e.g., FIGS. 1A and 1E ), including homodimeric antibody fusions that comprise the same p97-trastuzumab heavy chain fusion proteins.
  • the p97-antibody fusion comprises one p97-trastuzumab heavy chain fusion protein (see, e.g., FIG. 1B ).
  • the p97-antibody fusion protein comprises one or two trastuzumab light chain sequences, one trastuzumab heavy chain sequence, and one p97-trastuzumab heavy chain fusion protein described herein, where 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.
  • the p97-antibody fusion protein comprises one trastuzumab light chain sequence, one trastuzumab heavy chain sequence, and one p97-trastuzumab heavy chain fusion protein that has a truncated trastuzumab heavy chain (see, e.g., FIG. 1C ).
  • the p97-antibody fusion protein comprises two trastuzumab light chain sequences, and two p97-trastuzumab heavy chain fusion proteins described herein, where the p97-trastuzumab heavy chain fusion proteins comprise a trastuzumab heavy chain fused to the C-terminus of a p97 sequence and an optional linker in between (see, e.g., FIG. 1D ).
  • the p97-antibody fusion protein comprises two p97-trastuzumab light chain fusion proteins described herein, and two p97-trastuzumab heavy chain fusion proteins described herein, where the p97-trastuzumab light chain fusion proteins comprise a trastuzumab light chain fused to the N-terminus of a p97 sequence and an optional linker in between, and where the p97-trastuzumab heavy chain fusion proteins comprise a trastuzumab heavy chain fused to the N-terminus of a p97 sequence and an optional linker in between (see, e.g., FIG. 1F ).
  • the p97-antibody fusion protein comprises one or two p97-trastuzumab light chain fusion proteins described herein, and two trastuzumab heavy chain sequences, where the one or two p97-trastuzumab light chain fusion proteins comprise a trastuzumab light chain fused to the N-terminus of a p97 sequence and an optional linker in between.
  • the p97-antibody fusion protein comprises two p97-trastuzumab light chain fusion proteins described herein (see, e.g., FIG. 1G ).
  • the p97-antibody fusion comprises one or two sets of heavy and light chains selected from one or more of the following:
  • the p97-antibody fusion 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).
  • the p97-antibody fusion is a heterodimer that comprises a first set of sets of heavy and light chains selected from a)-g) above, and a second set of heavy and light chains composed of any combination of the p97-trastuzumab heavy or light chains described herein (for example, a)-g) above; SEQ ID NOS:37-46, 96-109, and 110-121), and/or any of the trastuzumab (non-fusion) heavy and light chains (for example, SEQ ID NOs: 29-35 or 122 (heavy chains) and 36 or 123 (light chains)).
  • FIGS. 1A-1G Non-limiting examples of p97-antibody fusions are illustrated in FIGS. 1A-1G .
  • FIG. 1A illustrates an antibody fusion composed of two p97-trastuzumab heavy chain fusion proteins, and two (non-fusion) trastuzumab light chains.
  • FIG. 1B illustrates an antibody fusion composed of one p97-trastuzumab heavy chain fusion protein, one (non-fusion) trastuzumab heavy chain, and two (non-fusion) trastuzumab light chains.
  • FIG. 1A illustrates an antibody fusion composed of two p97-trastuzumab heavy chain fusion proteins, and two (non-fusion) trastuzumab light chains.
  • FIG. 1B illustrates an antibody fusion composed of one p97-trastuzumab heavy chain fusion protein, one (non-fusion) trastuzumab heavy chain, and two (non-fusion) trastuzumab
  • FIG. 1C illustrates an antibody fusion protein composed of one p97-trastuzumab heavy chain fusion protein (having a truncated trastuzumab heavy chain), one (non-fusion) trastuzumab light chain, and one (non-fusion) trastuzumab heavy chain.
  • FIGS. 1D and 1E illustrate antibody fusions composed of two p97-trastuzumab heavy chain fusion proteins and two (non-fusion) trastuzumab light chains.
  • FIG. 1F illustrates an antibody fusion composed of two p97-trastuzumab light chain fusion proteins and two 97-trastuzumab heavy chain fusion proteins.
  • 1G illustrates an antibody fusion composed of two p97-trastuzumab light chain fusion proteins and two (non-fusion) trastuzumab heavy chain sequences.
  • the first set of light/heavy chains can be the same as or different from the second set of light/heavy chains.
  • the p97-antibody fusion is a homodimer, for example, which is composed of two identical sets of heavy and/or light chains, at least one of which is a p97-trastuzumab fusion protein.
  • the p97-antibody fusion is a heterodimer, for example, which is composed of a first set of heavy and/or light chains and a second set of heavy/light chains, where the first set comprises at least one heavy and/or light chain p97-trastuzumab fusion protein and the second set comprises only trastuzumab heavy and/or light chain sequences.
  • any of the p97 sequences described herein can be combined with any of the trastuzumab sequences described herein, to generate a desired p97-trastuzumab light chain or heavy chain fusion protein, and any such fusion proteins can be combined with the same or different fusion protein(s) or with any of the trastuzumab heavy chain or light chain sequences to generate a desired antibody fusion.
  • the p97-antibody fusion protein specifically binds to the human HER2/neu receptor. In specific embodiments, the p97-antibody fusion protein specifically binds to domain IV of the extracellular segment of the human HER2/neu receptor (see Cho et al., Nature. 421:756-760, 2003). In particular embodiments, the p97-antibody fusion protein is an HER2/neu receptor antagonist.
  • the functional properties of the fusion proteins and antibody fusions described herein may be assessed using a variety of methods known to the skilled person, including, e.g., affinity/binding assays (for example, surface plasmon resonance, competitive inhibition assays); cytotoxicity assays, cell viability assays, cell proliferation or differentiation assays, cancer cell and/or tumor growth inhibition using in vitro or in vivo models.
  • affinity/binding assays for example, surface plasmon resonance, competitive inhibition assays
  • cytotoxicity assays for example, cell viability assays, cell proliferation or differentiation assays, cancer cell and/or tumor growth inhibition using in vitro or in vivo models.
  • the fusion proteins described herein may be tested for effects on receptor internalization, in vitro and in vivo efficacy, etc., including the rate of transport across the blood-brain barrier.
  • Such assays may be performed using well-established protocols known to the skilled person (see e.g., Current Protocols in Molecular Biology (Green
  • Certain embodiments include variants of the reference polypeptide and polynucleotide sequences described herein, whether described by name or by reference to a sequence identifier, including p97 sequences and trastuzumab sequences (see, e.g., the Sequence Listing).
  • the wild-type or most prevalent sequences of these polypeptides are known in the art, and can be used as a comparison for the variants and fragments described herein.
  • variant sequence refers to a polypeptide or polynucleotide sequence that differs from a reference sequence disclosed herein by one or more substitutions, deletions (e.g., truncations), additions, and/or insertions. Certain variants thus include fragments of a reference sequence described herein. Variant polypeptides are biologically active, that is, they continue to possess the enzymatic or binding activity of a reference polypeptide. Such variants may result from, for example, genetic polymorphism and/or from human manipulation.
  • a biologically active variant will contain one or more conservative substitutions.
  • a “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.
  • modifications may be made in the structure of the polynucleotides and polypeptides of the present invention and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics.
  • amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their utility.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte & Doolittle, 1982).
  • amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein.
  • substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine ( ⁇ 0.4); proline ( ⁇ 0.5 ⁇ 1); alanine ( ⁇ 0.5); histidine ( ⁇ 0.5); cysteine ( ⁇ 1.0); methionine ( ⁇ 1.3); valine ( ⁇ 1.5); leucine ( ⁇ 1.8); isoleucine ( ⁇ 1.8); tyrosine ( ⁇ 2.3); phenylalanine ( ⁇ 2.5); tryptophan ( ⁇ 3.4).
  • an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine.
  • amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
  • variant polypeptides may also, or alternatively, contain non-conservative changes.
  • variant polypeptides differ from a native or reference sequence by substitution, deletion or addition of fewer than about 10, 9, 8, 7, 6, 5, 4, 3, 2 amino acids, or even 1 amino acid.
  • Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure, enzymatic activity, and/or hydropathic nature of the polypeptide.
  • a polypeptide sequence is about, at least about, or up to about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640,
  • a polypeptide sequence consists of about or no more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650
  • a polypeptide sequence is about 10-1000, 10-900, 10-800, 10-700, 10-600, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 10-40, 10-30, 10-20, 20-1000, 20-900, 20-800, 20-700, 20-600, 20-500, 20-400, 20-300, 20-200, 20-100, 20-50, 20-40, 20-30, 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, or 200-300 contiguous amino acids, including all ranges in between, and comprises all or a portion of a reference sequence.
  • the C-terminal or N-terminal region of any reference polypeptide may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 or more amino acids, or by about 10-50, 20-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800 or more amino acids, including all integers and ranges in between (e.g., 101, 102, 103, 104, 105), so long as the truncated polypeptide retains the binding properties and/or activity of the reference polypeptide.
  • variants will display at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity or sequence identity or sequence homology to a reference polypeptide sequence.
  • sequences differing from the native or parent sequences by the addition e.g., C-terminal addition, N-terminal addition, both
  • deletion, truncation, insertion, or substitution e.g., conservative substitution
  • substitution e.g., conservative substitution
  • amino acids including all integers and ranges in between but which retain the properties or activities of a parent or reference polypeptide sequence
  • variant polypeptides differ from reference sequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s). In other embodiments, variant polypeptides differ from a reference sequence by at least 1% but less than 20%, 15%, 10% or 5% of the residues. (If this comparison requires alignment, the sequences should be aligned for maximum similarity. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.)
  • sequence similarity or sequence identity between sequences are performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence.
  • amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch, ( J. Mol. Biol. 48: 444-453, 1970) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • a particularly preferred set of parameters are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ( Cabios. 4:11-17, 1989) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences.
  • Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al., (1990 , J. Mol. Biol, 215: 403-10).
  • Gapped BLAST can be utilized as described in Altschul et al., ( Nucleic Acids Res. 25: 3389-3402, 1997).
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST can be used.
  • polynucleotides and/or polypeptides can be evaluated using a BLAST alignment tool.
  • a local alignment consists simply of a pair of sequence segments, one from each of the sequences being compared.
  • a modification of Smith-Waterman or Sellers algorithms will find all segment pairs whose scores cannot be improved by extension or trimming, called high-scoring segment pairs (HSPs).
  • HSPs high-scoring segment pairs
  • the results of the BLAST alignments include statistical measures to indicate the likelihood that the BLAST score can be expected from chance alone.
  • the raw score, S is calculated from the number of gaps and substitutions associated with each aligned sequence wherein higher similarity scores indicate a more significant alignment. Substitution scores are given by a look-up table (see PAM, BLOSUM).
  • Gap scores are typically calculated as the sum of G, the gap opening penalty and L, the gap extension penalty.
  • the gap cost would be G+Ln.
  • the choice of gap costs, G and L is empirical, but it is customary to choose a high value for G (10-15), e.g., 11, and a low value for L (1-2) e.g., 1.
  • bit score, S′ is derived from the raw alignment score S in which the statistical properties of the scoring system used have been taken into account. Bit scores are normalized with respect to the scoring system, therefore they can be used to compare alignment scores from different searches. The terms “bit score” and “similarity score” are used interchangeably. The bit score gives an indication of how good the alignment is; the higher the score, the better the alignment.
  • the E-Value describes the likelihood that a sequence with a similar score will occur in the database by chance. It is a prediction of the number of different alignments with scores equivalent to or better than S that are expected to occur in a database search by chance. The smaller the E-Value, the more significant the alignment. For example, an alignment having an E value of e ⁇ 117 means that a sequence with a similar score is very unlikely to occur simply by chance. Additionally, the expected score for aligning a random pair of amino acids is required to be negative, otherwise long alignments would tend to have high score independently of whether the segments aligned were related. Additionally, the BLAST algorithm uses an appropriate substitution matrix, nucleotide or amino acid and for gapped alignments uses gap creation and extension penalties. For example, BLAST alignment and comparison of polypeptide sequences are typically done using the BLOSUM62 matrix, a gap existence penalty of 11 and a gap extension penalty of 1.
  • sequence similarity scores are reported from BLAST analyses done using the BLOSUM62 matrix, a gap existence penalty of 11 and a gap extension penalty of 1.
  • sequence identity/similarity scores provided herein refer to the value obtained using GAP Version 10 (GCG, Accelrys, San Diego, Calif.) using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, PNAS USA. 89:10915-10919, 1992).
  • GAP uses the algorithm of Needleman and Wunsch ( J Mol Biol. 48:443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.
  • the variant polypeptide comprises an amino acid sequence that can be optimally aligned with a reference polypeptide sequence (see, e.g., Sequence Listing) to generate a BLAST bit scores or sequence similarity scores of at least about 50, 60, 70, 80, 90, 100, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750,
  • a reference polypeptide may be altered in various ways including amino acid substitutions, deletions, truncations, additions, and insertions. Methods for such manipulations are generally known in the art.
  • amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel ( PNAS USA. 82: 488-492, 1985); Kunkel et al., ( Methods in Enzymol. 154: 367-382, 1987), U.S. Pat. No. 4,873,192, Watson, J. D.
  • REM recursive ensemble mutagenesis
  • Certain embodiments relate to polynucleotides that encode the fusion proteins and antibody fusions described herein, and vectors that comprise such polynucleotides, for example, where the polynucleotides are operably linked to one or more regulatory elements. Also included are recombinant host cells that comprise such polynucleotides, vectors, fusion proteins, and antibody fusions, and methods of recombinant production of the foregoing.
  • Fusion proteins and antibody fusions may be prepared using standard techniques.
  • a fusion protein is expressed as a recombinant protein in an expression system, as described herein and known in the art.
  • Fusion proteins can contain one or multiple copies of a p97 sequence and one or multiple copies of a trastuzumab sequence, present in any desired arrangement.
  • Polynucleotides and fusion polynucleotides can contain one or multiple copies of a nucleic acid encoding a p97 polypeptide sequence, and/or may contain one or multiple copies of a nucleic acid encoding a trastuzumab sequence.
  • DNA sequences encoding the p97 polypeptide sequence, the trastuzumab sequence of interest, and optionally a peptide linker components may be assembled separately, and then ligated into an appropriate expression vector.
  • the 3′ end of the DNA sequence encoding one polypeptide component can be ligated, with or without a peptide linker, to the 5′ end of a DNA sequence encoding the other polypeptide component(s) so that the reading frames of the sequences are in frame.
  • the ligated DNA sequences are operably linked to suitable transcriptional and/or translational regulatory elements.
  • the regulatory elements responsible for expression of DNA are usually located only 5′ to the DNA sequence encoding the first polypeptides.
  • stop codons required to end translation and transcription termination signals are only present 3′ to the DNA sequence encoding the most C-terminal polypeptide. This permits translation into a single fusion polypeptide that retains the biological activity of both component polypeptides.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Vectors may be plasmids, viral e.g. phage, or phagemid, as appropriate.
  • plasmids viral e.g. phage, or phagemid, as appropriate.
  • Many known techniques and protocols for manipulation of nucleic acid for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992, or subsequent updates thereto.
  • codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
  • Such polynucleotides are commonly referred to as “codon-optimized.” Any of the polynucleotides described herein may be utilized in a codon-optimized form.
  • a polynucleotide can be codon optimized for use in specific bacteria such as E. coli or yeast such as S. cerevisiae (see, e.g., Burgess-Brown et al., Protein Expr Purif. 59:94-102, 2008).
  • Exemplary polynucleotide sequences are provided in Table 5 below.
  • a polynucleotide that encodes a fusion protein or antibody fusion described herein, or a portion thereof comprises one or more polynucleotide sequences from Table 5 (e.g., SEQ ID NOS:125-138), or a fragment/variant thereof.
  • one or more nucleic acids or vectors encoding a subject p97 polypeptide, a trastuzumab polypeptide (e.g., light chain polypeptide, heavy chain polypeptide), and/or a p97-trastuzumab fusion protein are introduced directly into a host cell, and the cell is incubated under conditions sufficient to induce expression of the encoded polypeptide(s).
  • a recombinant host cell which comprises a polynucleotide or a fusion polynucleotide that encodes one or more fusion proteins described herein, optionally in combination with other (non-fusion) components of an antibody, and which optionally comprises additional heterologous polynucleotide sequences.
  • fusion protein or antibody fusion in the host cell may be achieved by culturing the recombinant host cells (containing the polynucleotide(s)) under appropriate conditions. Following production by expression, the polypeptide(s), fusion proteins, and/or antibody fusions may be isolated and/or purified using any suitable technique, and then used as desired.
  • the term “host cell” is used to refer to a cell into which has been introduced, or which is capable of having introduced into it, a nucleic acid sequence encoding one or more of the polypeptides described herein, and which further expresses or is capable of expressing a selected gene of interest, such as a gene encoding any herein described polypeptide.
  • the term includes the progeny of the parent cell, whether or not the progeny are identical in morphology or in genetic make-up to the original parent, so long as the selected gene is present.
  • Host cells may be chosen for certain characteristics, for instance, the expression of aminoacyl tRNA synthetase(s) that can incorporate unnatural amino acids into the polypeptide.
  • Suitable host cells include mammalian cells, bacteria, yeast, and baculovirus systems.
  • Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, HEK-293 cells, NSO mouse melanoma cells and many others. Additional examples of useful mammalian host cell lines include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells sub-cloned for growth in suspension culture, Graham et al., J. Gen Virol.
  • MRC 5 cells MRC 5 cells
  • FS4 cells MRC 5 cells
  • FS4 cells a human hepatoma line
  • Hep G2 human hepatoma line
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., PNAS USA 77:4216 (1980)); and myeloma cell lines such as NSO and Sp2/0.
  • CHO Chinese hamster ovary
  • DHFR-CHO cells Urlaub et al., PNAS USA 77:4216 (1980)
  • myeloma cell lines such as NSO and Sp2/0.
  • mammalian cell expression systems include CHO and HEK293-cell based expression systems.
  • Mammalian expression systems can utilize attached cell lines, for example, in T-flasks, roller bottles, or cell factories, or suspension cultures, for example, in 1 L and 5 L spinners, 5 L, 14 L, 40 L, 100 L and 200 L stir tank bioreactors, or 20/50 L and 100/200 L WAVE bioreactors, among others known in the art.
  • a common, preferred bacterial host is E. coli .
  • the expression of proteins in prokaryotic cells such as E. coli is well established in the art. For a review, see for example Pluckthun, A. Bio/Technology. 9:545-551 (1991). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for recombinant production of polypeptides (see Ref, Curr. Opinion Biotech. 4:573-576, 1993; and Trill et al., Curr. Opinion Biotech. 6:553-560, 1995).
  • protein expression may be controlled by a T7 RNA polymerase (e.g., pET vector series).
  • These and related embodiments may utilize the expression host strain BL21(DE3), a ⁇ DE3 lysogen of BL21 that supports T7-mediated expression and is deficient in Ion and ompT proteases for improved target protein stability. Also included are expression host strains carrying plasmids encoding tRNAs rarely used in E. coli , such as RosettaTM (DE3) and Rosetta 2 (DE3) strains. Cell lysis and sample handling may also be improved using reagents such as Benzonase® nuclease and BugBuster® Protein Extraction Reagent. For cell culture, auto-inducing media can improve the efficiency of many expression systems, including high-throughput expression systems.
  • Media of this type gradually elicit protein expression through metabolic shift without the addition of artificial inducing agents such as IPTG.
  • Particular embodiments employ hexahistidine tags (such as His•Tag® fusions), followed by immobilized metal affinity chromatography (IMAC) purification, or related techniques.
  • IMAC immobilized metal affinity chromatography
  • clinical grade proteins can be isolated from E. coli inclusion bodies, without or without the use of affinity tags (see, e.g., Shimp et al., Protein Expr Purif. 50:58-67, 2006).
  • certain embodiments may employ a cold-shock induced E.
  • a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, post-translational modifications such as acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a “prepro” form of the protein may also be used to facilitate correct insertion, folding and/or function.
  • Different host cells such as yeast, CHO, HeLa, MDCK, HEK293, and W138, in addition to bacterial cells, which have or even lack specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the fusion protein or antibody fusion of interest.
  • cell lines that stably express a polynucleotide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which, successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type. Transient production, such as by transient transfection or infection, can also be employed. Exemplary mammalian expression systems that are suitable for transient production include HEK293 and CHO-based systems.
  • Host cells transformed with a polynucleotide sequence of interest may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • Certain specific embodiments utilize serum free cell expression systems. Examples include HEK293 cells and CHO cells that can grow on serum free medium (see, e.g., Rosser et al., Protein Expr. Purif. 40:237-43, 2005; and U.S. Pat. No. 6,210,922).
  • the protein(s) produced by a recombinant cell can be purified and characterized according to a variety of techniques known in the art.
  • Exemplary systems for performing protein purification and analyzing protein purity include fast protein liquid chromatography (FPLC) (e.g., AKTA and Bio-Rad FPLC systems), high-pressure liquid chromatography (HPLC) (e.g., Beckman and Waters HPLC).
  • FPLC fast protein liquid chromatography
  • HPLC high-pressure liquid chromatography
  • Exemplary chemistries for purification include ion exchange chromatography (e.g., Q, S), size exclusion chromatography, salt gradients, affinity purification (e.g., Ni, Co, FLAG, maltose, glutathione, protein A/G), gel filtration, reverse-phase, ceramic HyperD® ion exchange chromatography, and hydrophobic interaction columns (HIC), among others known in the art. Also included are analytical methods such as SDS-PAGE (e.g., coomassie, silver stain), immunoblot, Bradford, and ELISA, which may be utilized during any step of the production or purification process, typically to measure the purity of the protein composition.
  • lyophilization which is typically employed when the solution contains few soluble components other than the protein of interest. Lyophilization is often performed after HPLC run, and can remove most or all volatile components from the mixture.
  • ultrafiltration techniques typically employ one or more selective permeable membranes to concentrate a protein solution. The membrane allows water and small molecules to pass through and retains the protein; the solution can be forced against the membrane by mechanical pump, gas pressure, or centrifugation, among other techniques.
  • the fusion proteins or antibody fusion proteins have a purity of at least about 90%, as measured according to routine techniques in the art. In certain embodiments, such as diagnostic compositions or certain therapeutic compositions, the fusion proteins or antibody fusions have a purity of at least about 95%. In specific embodiments, such as therapeutic or pharmaceutical compositions, the fusion proteins or antibody fusions have a purity of at least about 97% or 98% or 99%. In other embodiments, such as when being used as reference or research reagents, fusion proteins or antibody fusions can be of lesser purity, and may have a purity of at least about 50%, 60%, 70%, or 80%. Purity can be measured overall or in relation to selected components, such as other proteins, e.g., purity on a protein basis.
  • compositions described here are about substantially endotoxin free, including, for example, about 95% endotoxin free, preferably about 99% endotoxin free, and more preferably about 99.99% endotoxin free.
  • endotoxins can be detected according to routine techniques in the art, as described herein.
  • the fusion proteins or antibody fusions are made from a eukaryotic cell such as a mammalian or human cell in substantially serum free media.
  • Certain embodiments relate to methods of using the p97-trastuzumab fusion proteins and/or related antibody fusion proteins described herein.
  • methods of treatment and methods of diagnosis include methods of treatment and methods of diagnosis, the latter including, for instance, the medical imaging of certain organs/tissues, such as those of the central nervous system.
  • Specific embodiments include methods of treating and/or diagnosing disorders or conditions of the central nervous system (CNS), or disorders or conditions having a CNS component.
  • pharmaceutical compositions comprising the p97-trastuzumab fusion proteins and/or related antibody fusion proteins described herein.
  • certain embodiments include methods of treating a subject in need thereof, comprising administering a p97-trastuzumab fusion protein or antibody fusion protein described herein.
  • the methods comprise administering a p97-antibody fusion protein, which comprises one or more p97-trastuzumab fusion proteins described herein (e.g., as at least one component of the antibody or antibody-like molecule), and optionally other non-fusion antibody components (e.g., non-fusion light chain(s), non-fusion heavy chain(s)).
  • methods of delivering such molecules to the nervous system (e.g., central nervous system tissues) of a subject comprising administering to the subject a p97-trastuzumab fusion protein or antibody fusion described herein.
  • the methods increase the rate and/or amount of delivery of the trastuzumab antibody (or antigen-binding fragment thereof) to the central nervous system tissues of the subject, relative, for example, to delivery by a composition that comprises the trastuzumab antibody (or antigen-binding fragment thereof) alone.
  • the methods reduce the distribution of trastuzumab antibody (or antigen-binding fragment thereof) to heart tissues of the subject, relative, for example, to distribution by a composition that comprises the trastuzumab antibody (or antigen-binding fragment thereof) alone, and thereby reduces the cardiotoxicity associated with trastuzumab.
  • a subject has a disease, disorder, or condition that is associated with the central nervous system (CNS) or that has a CNS component, where increased delivery of the trastuzumab antibody (or antigen-binding fragment thereof) across the blood brain barrier to CNS tissues relative to peripheral tissues can improve treatment, for instance, by increasing the tissue concentration of the antibody in the CNS, and/or by reducing side-effects associated with exposure of the antibody to peripheral tissues/organs.
  • CNS central nervous system
  • cancer relates generally to a class of diseases or conditions in which a group of cells display one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., intrusion on and destruction of adjacent tissues), and/or metastasis (i.e., spread to other locations in the body via lymph or blood).
  • uncontrolled growth i.e., division beyond normal limits
  • invasion i.e., intrusion on and destruction of adjacent tissues
  • metastasis i.e., spread to other locations in the body via lymph or blood.
  • CNS central nervous system
  • neurological cancers such as brain cancers.
  • the neurological cancer is a metastatic brain cancer.
  • cancers that can metastasize to the brain include, without limitation, breast cancers, lung cancers, genitourinary tract cancers, gastrointestinal tract cancers (e.g., colorectal cancers, pancreatic carcinomas), osteosarcomas, melanomas, head and neck cancers, prostate cancers (e.g., prostatic adenocarcinomas), and lymphomas.
  • Certain embodiments thus include methods for treating, inhibiting or preventing metastasis of a cancer by administering to a patient a therapeutically effective amount of a fusion protein described herein (e.g., in an amount that, following administration, inhibits, prevents or delays metastasis of a cancer in a statistically significant manner, i.e., relative to an appropriate control as will be known to those skilled in the art).
  • the subject has a cancer that is at risk for but has not yet metastasized to the central nervous system, including one or more of the above-described cancers, among others known in the art.
  • the subject has a cancer associated with expression of Her2/neu.
  • subject has a Her2/neu-expressing or Her2/neu-overexpressing cancer.
  • certain embodiments include methods for the treatment of a HER2-overexpressing cancer in a subject in need thereof, comprising administering to the subject a (e.g., therapeutically-effective amount of a) p97-antibody fusion protein (described herein) or a pharmaceutical composition comprising the same.
  • the HER2-overexpressing cancer is at risk for metastasizing to the CNS of the subject.
  • the HER2-overexpressing cancer has metastasized to the CNS of the subject.
  • the antibody fusion is administered in an amount that inhibits, prevents, or delays the progression and/or metastasis of the cancer in a statistically significant manner (i.e., relative to an appropriate control as will be known to those skilled in the art). Included are amounts that inhibit, prevent, or delay the progression and/or metastasis into tissues of the CNS, and those that inhibit, prevent, or delay progression and/or metastasis within tissues of the CNS.
  • the HER2-overexpressing cancer is a breast cancer, ovarian cancer, gastric cancer, or uterine cancer.
  • the HER2-overexpressing cancer is a metastatic breast cancer, metastatic ovarian cancer, metastatic gastric cancer, or metastatic or aggressive form of uterine cancer.
  • the HER2-overexpressing cancer is a HER2-overexpressing breast cancer, such as a HER2-overexpressing metastatic breast cancer.
  • the HER2-overexpressing metastatic breast cancer is at risk for metastasizing to the CNS of the subject.
  • the HER2-overexpressing metastatic breast cancer has already metastasized to the CNS of the subject.
  • the p97-antibody fusion is administered with paclitaxel for first-line treatment of HER2-overexpressing metastatic breast cancer.
  • the p97-antibody fusion is administered as a single agent for treatment of HER2-overexpressing metastatic breast cancer in patients who have received one or more chemotherapy regimens for metastatic disease.
  • Certain embodiments include administering the p97-antibody fusion or pharmaceutical composition as part of an adjuvant treatment for HER2-overexpressing breast cancer.
  • the adjuvant treatment comprises doxorubicin, cyclophosphamide, and either paclitaxel or docetaxel.
  • the adjuvant treatment comprises docetaxel and carboplatin.
  • Certain aspects include administering the p97-antibody fusion or pharmaceutical composition as a single agent following multi-modality anthracycline based therapy.
  • the HER2-overexpressing cancer is a HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma.
  • the HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma is at risk for metastasizing to the CNS of the subject.
  • the HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma has already metastasized to the CNS of the subject.
  • the p97-antibody fusion is administered in combination with cisplatin and capecitabine or 5-fluorouracil, optionally where the subject or patient has not received prior treatment for metastatic disease.
  • the HER2-overexpressing uterine cancer is a HER2-overexpressing uterine serous carcinoma (USC) (see, e.g., Santin et al., Inti Gynaecol Obstet. 102:128-31, 2008).
  • USC also known as uterine papillary serous carcinoma (UPSC) and uterine serous adenocarcinoma
  • UPSC uterine papillary serous carcinoma
  • uterine serous adenocarcinoma is a form of endometrial cancer that typically arises in postmenopausal women.
  • the HER2-overexpressing USC is at risk for metastasizing to the CNS of the subject.
  • the HER2-overexpressing USC has already metastasized to the CNS of the subject.
  • Also included are methods for imaging an organ or tissue component in a subject comprising (a) administering to the subject a composition comprising a fusion protein or antibody fusion described herein, which is conjugated to a detectable entity, and (b) visualizing the detectable entity in the subject, organ, or tissue.
  • the organ or tissue compartment comprises the central nervous system (e.g., brain, brainstem, spinal cord).
  • the organ or tissue compartment comprises the brain or a portion thereof, for instance, the parenchyma of the brain.
  • Non-invasive methods include radiography, such as fluoroscopy and projectional radiographs, CT-scanning or CAT-scanning (computed tomography (CT) or computed axial tomography (CAT)), whether employing X-ray CT-scanning, positron emission tomography (PET), or single photon emission computed tomography (SPECT), and certain types of magnetic resonance imaging (MRI), especially those that utilize contrast agents, including combinations thereof.
  • radiography such as fluoroscopy and projectional radiographs, CT-scanning or CAT-scanning (computed tomography (CT) or computed axial tomography (CAT)), whether employing X-ray CT-scanning, positron emission tomography (PET), or single photon emission computed tomography (SPECT), and certain types of magnetic resonance imaging (MRI), especially those that utilize contrast agents, including combinations thereof.
  • CT computed tomography
  • CAT computed axial tomography
  • PET positron emission tomography
  • PET can be performed with positron-emitting contrast agents or radioisotopes such as 18 F
  • SPECT can be performed with gamma-emitting contrast agents or radioisotopes such as 201 Tl, 99m TC, 123 I, and 67 Ga
  • MRI can be performed with contrast agents or radioisotopes such as 3 H, 13 C, 19 F, 17 O, 23 Na, 31 P, and 129 Xe, and Gd (gadolidinium; chelated organic Gd (III) complexes).
  • Any one or more of these exemplary contrast agents or radioisotopes can be conjugated to or otherwise incorporated into a p97 polypeptide and administered to a subject for imaging purposes.
  • p97 polypeptides can be directly labeled with one or more of these radioisotopes, or conjugated to molecules (e.g., small molecules) that comprise one or more of these radioisotopic contrast agents, or any others described herein.
  • fusion proteins or antibody fusions described herein are generally incorporated into a pharmaceutical composition prior to administration.
  • a pharmaceutical composition comprises one or more of the fusion proteins or antibody fusions described herein in combination with a physiologically-acceptable, pharmaceutically-acceptable, or pharmaceutical grade carrier or excipient.
  • a pharmaceutical composition an effective or desired amount of one or more fusion protein or antibody fusions is mixed with any pharmaceutical carrier(s) or excipient known to those skilled in the art to be suitable for the particular mode of administration.
  • a pharmaceutical carrier may be liquid, semi-liquid or solid.
  • Solutions or suspensions used for parenteral, intradermal, subcutaneous or topical application may include, for example, a sterile diluent (such as water), saline solution (e.g., phosphate buffered saline; PBS), fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvent; antimicrobial agents (such as benzyl alcohol and methyl parabens); antioxidants (such as ascorbic acid and sodium bisulfite) and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); buffers (such as acetates, citrates and phosphates).
  • a sterile diluent such as water
  • saline solution e.g., phosphate buffered saline; PBS
  • fixed oil polyethylene glycol, glycerin, propylene glycol or other synthetic solvent
  • antimicrobial agents such as benzyl alcohol and methyl parabens
  • suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, polypropylene glycol and mixtures thereof.
  • PBS physiological saline or phosphate buffered saline
  • fusion proteins or antibody fusions described herein in pure form or in an appropriate pharmaceutical composition, can be carried out via any of the accepted modes of administration of agents for serving similar utilities.
  • the pharmaceutical compositions can be prepared by combining a fusion protein or antibody fusion-containing composition with an appropriate physiologically acceptable carrier, diluent or excipient, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
  • other pharmaceutically active ingredients including other small molecules as described elsewhere herein
  • suitable excipients such as salts, buffers and stabilizers may, but need not, be present within the composition.
  • Administration may be achieved by a variety of different routes, including oral, parenteral, nasal, intravenous, intradermal, subcutaneous or topical. Preferred modes of administration depend upon the nature of the condition to be treated or prevented. Particular embodiments include administration by IV infusion.
  • Carriers can include, for example, pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed.
  • physiologically acceptable carrier is an aqueous pH buffered solution.
  • physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as polysorbate 20 (TWEENTM) polyethylene glycol (PEG), and poloxamers (PLURONICSTM), and the like.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • a fusion protein or antibody fusion is bound to or encapsulated within a particle, e.g., a nanoparticle, bead, lipid formulation, lipid particle, or liposome, e.g., immunoliposome.
  • the fusion proteins or antibody fusions may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate)microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions.
  • the particle(s) or liposomes may further comprise other therapeutic or diagnostic agents, such as cytotoxic agents.
  • the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by testing the compositions in model systems known in the art and extrapolating therefrom. Controlled clinical trials may also be performed. Dosages may also vary with the severity of the condition to be alleviated.
  • a pharmaceutical composition is generally formulated and administered to exert a therapeutically useful effect while minimizing undesirable side effects.
  • the composition may be administered one time, or may be divided into a number of smaller doses to be administered at intervals of time. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need.
  • compositions according to certain embodiments of the present invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient.
  • Compositions that will be administered to a subject or patient may take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a herein described conjugate in aerosol form may hold a plurality of dosage units.
  • composition to be administered will typically contain a therapeutically effective amount of a fusion protein or antibody fusion described herein, for treatment of a disease or condition of interest.
  • a pharmaceutical composition may be in the form of a solid or liquid.
  • the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form.
  • the carrier(s) may be liquid, with the compositions being, for example, an oral oil, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration.
  • the pharmaceutical composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
  • the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like.
  • a solid composition will typically contain one or more inert diluents or edible carriers.
  • binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.
  • a liquid carrier such as polyethylene glycol or oil.
  • the pharmaceutical composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension.
  • the liquid may be for oral administration or for delivery by injection, as two examples.
  • preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer.
  • a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
  • the liquid pharmaceutical compositions may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • Physiological saline is a preferred adjuvant.
  • a liquid pharmaceutical composition intended for either parenteral or oral administration should contain an amount of a fusion protein or antibody fusion such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of the agent of interest in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition. Certain oral pharmaceutical compositions contain between about 4% and about 75% of the agent of interest. In certain embodiments, pharmaceutical compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.01 to 10% by weight of the agent of interest prior to dilution.
  • the pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base.
  • the base for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers.
  • Thickening agents may be present in a pharmaceutical composition for topical administration.
  • the composition may include a transdermal patch or iontophoresis device.
  • the pharmaceutical composition may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug.
  • the composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient.
  • bases include, without limitation, lanolin, cocoa butter, and polyethylene glycol.
  • the pharmaceutical composition may include various materials, which modify the physical form of a solid or liquid dosage unit.
  • the composition may include materials that form a coating shell around the active ingredients.
  • the materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents.
  • the active ingredients may be encased in a gelatin capsule.
  • the pharmaceutical composition in solid or liquid form may include an agent that binds to the conjugate or agent and thereby assists in the delivery of the compound. Suitable agents that may act in this capacity include monoclonal or polyclonal antibodies, one or more proteins or a liposome.
  • the pharmaceutical composition may consist essentially of dosage units that can be administered as an aerosol.
  • aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. One of ordinary skill in the art, without undue experimentation may determine preferred aerosols.
  • compositions described herein may be prepared with carriers that protect the fusion proteins or antibody fusions against rapid elimination from the body, such as time release formulations or coatings.
  • carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others known to those of ordinary skill in the art.
  • compositions may be prepared by methodology well known in the pharmaceutical art.
  • a pharmaceutical composition intended to be administered by injection may comprise one or more of salts, buffers and/or stabilizers, with sterile, distilled water so as to form a solution.
  • a surfactant may be added to facilitate the formation of a homogeneous solution or suspension.
  • Surfactants are compounds that non-covalently interact with the conjugate so as to facilitate dissolution or homogeneous suspension of the conjugate in the aqueous delivery system.
  • compositions may be administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy.
  • a therapeutically effective daily dose is (for a 70 kg mammal) from about 0.001 mg/kg (i.e., ⁇ 0.07 mg) to about 100 mg/kg (i.e., ⁇ 7.0 g); preferably a therapeutically effective dose is (for a 70 kg mammal) from about 0.01 mg/kg (i.e., ⁇ 0.7 mg) to about 50 mg/kg (i.e., ⁇ 3.5 g); more preferably a therapeutically effective dose is (for a 70 kg mammal) from about 1 mg/kg (i.e., ⁇ 70 mg) to about 25 mg/kg (i.e., ⁇ 1.75 g).
  • compositions described herein may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents, as described herein.
  • the conjugate is administered with an anti-inflammatory agent.
  • Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate.
  • steroids and glucocorticoids including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocort
  • compositions disclosed herein may be administered in conjunction with any number of chemotherapeutic or cytotoxic agents.
  • chemotherapeutic or cytotoxic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, pheneste
  • paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid derivatives such as TargretinTM (bexarotene), PanretinTM (alitretinoin); ONTA
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • Such combination therapy may include administration of a single pharmaceutical dosage formulation, which contains a compound of the invention (i.e., fusion protein, antibody fusion protein) and one or more additional active agents, as well as administration of compositions comprising conjugates of the invention and each active agent in its own separate pharmaceutical dosage formulation.
  • a fusion protein or antibody fusion as described herein and the other active agent can be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent administered in separate oral dosage formulations.
  • a fusion protein or antibody fusion as described herein and the other active agent can be administered to the patient together in a single parenteral dosage composition such as in a saline solution or other physiologically acceptable solution, or each agent administered in separate parenteral dosage formulations.
  • compositions comprising fusion proteins or antibody fusions and one or more additional active agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially and in any order; combination therapy is understood to include all these regimens.
  • MTf-trastuzumab fusion constructs Human p97 (MTf)-trastuzumab fusion constructs were prepared and tested for activity.
  • the amino acid sequences of the MTf-trastuzumab fusion constructs are shown in Table E1 below.
  • TZM HC-MTf Heavy Chain 82 Homodimer Signal sequence (italics): TZM heavy chain: linker (C-terminal (underlined): MTf (bold) TZM heavy METDTLLLWVLLLWVPGSTG EVQLVESGGGLVQPGGSLRLSCAASGFNIKDT chain MTf; YIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMN See FIG.
  • Her2-His His-tagged human Her2/Neu protein
  • Her2-His was loaded onto penta-His biosensor at a concentration of 20 ⁇ g/mL and dipped into TZM HC-MTf, MTfp NH-TZM, and TZM HC-MTfp fusion proteins (see Table E1) and a human IgG1 control at varying concentrations.
  • MTf-trastuzumab fusion proteins demonstrated tight binding to human Her2, as shown by the equilibrium dissociation constant (K D ), association rate constant (K on ), and dissociation rate constant (K off ).
  • MTf-trastuzumab fusion constructs were tested for Antibody-dependent cell-mediated cytotoxicity (ADCC) in BT-474 breast cancer cells compared to Herceptin® (trastuzumab) and human IgG1 Fc as positive and negative controls, respectively.
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • BT-474 cells were purchased from ATCC and grown in Hybri-Care Medium supplemented with 1.5 g/L sodium bicarbonate and 10% fetal bovine serum.
  • Human peripheral blood mononuclear cells (PBMCs) were freshly isolated by Histopaque centrifugation and incubated with carboxyfluorescein succinimidyl ester (CFSE)-labeled target BT-474 cells at a ratio of about 30:1.
  • PBMCs peripheral blood mononuclear cells
  • CFSE carboxyfluorescein succinimidyl ester
  • Test samples were co-incubated with the effector:target cell mixture for four hours at ten different concentrations (up to 100 ⁇ g/mL). Negative controls included effector:target cells only as no antibody control, and 100 ⁇ g/mL IgG Fc. Each treatment was performed in duplicate. After the 4 hour co-incubation, cells were stained with propidium iodide (PI) and analyzed by flow cytometry (FACS). The percentage of PI/CFSE+ cells was quantitated as an indication of cytotoxicity. A dose-response curve was generated by plotting the mean ⁇ range of the data points for all ten different concentrations, and the EC 50 was calculated. The results are shown in FIGS. 3A-3B .
  • PI propidium iodide
  • FACS flow cytometry
  • FIG. 3A shows the ADCC evaluation of TZM/MTf fusion in BT-474 breast cancer cells compared to Herceptin® and human IgG1 Fc.
  • the TZM/MTf homodimer fusion induced ADCC with an EC 50 of 0.6 ⁇ g/mL compared to that of Herceptin® at 0.2 pg/mL.
  • FIG. 3B shows the ADCC evaluation of LC-MTfp/Fc-MTfp, LC-MTfp/Fc, and LC/Fc-MTfp fusions in BT-474 breast cancer cells compared to Herceptin® and human IgG1 Fc.
  • LC-MTfp/Fc-MTfp, LC-MTfp/Fc, and LC/Fc-MTfp induced ADCC with an EC 50 of 0.216 ⁇ g/mL, 0.171 ⁇ g/mL, and 1.025 ⁇ g/mL, respectively.
  • the p97-trastuzumab fusion proteins are tested for anti-tumor efficacy and effects on survival in mice that are intracranially injected with human BT-474 breast tumor cells.
  • the fusion constructs TZM HC-MTf and MTfp NH-TZM are tested relative to Herceptin® (Trastuzumab, AMM: 5621037, ROCHE).
  • Herceptin® is prepared as follows: one vial containing 150 mg of Herceptin® is diluted in NaCl 0.9% (Aguettant, Lyon, France) to a final concentration of 20 mg/mL. The stock solution is stored at 4° C. for the duration of the study. Each day of administration to mice, the stock solution is diluted in NaCl 0.9% to reach 3 mg/mL final concentration. Herceptin® is administrated at 30 mg/kg.
  • All compounds are administered by intravenous injection (IV, bolus) into the caudal vein of mice at a dose volume of 10 mL/kg/inj (i.e., for one mouse weighing 20 g, 200 ⁇ L of test substance is administered).
  • the BT-474 cell line is purchased from ATCC. It was originally isolated from a solid, invasive ductal carcinoma of the breast from a 60 year old Caucasian female patient (Lasfargues et al., J Natl Cancer Inst. 61:967-78, 1978). Tumor cells are grown as monolayer at 37° C. in a humidified atmosphere (5% CO2, 95% air).
  • the culture medium contains DMEM supplemented with 2 mM L glutamine (ref: BE12-604F, Lonza, Verviers, Belgium) and 10% fetal bovine serum (ref: 3302, Lonza). The cells are adherent to plastic flasks.
  • tumor cells are detached from the culture flask by a 5 minute treatment with trypsin-versene (ref: BE02-007E, Lonza), in Hanks' medium without calcium or magnesium (ref: BE10-543F, Lonza) and neutralized by addition of complete culture medium.
  • the cells are counted in a hemocytometer and their viability is assessed by 0.25% trypan blue exclusion assay.
  • mice Sixty-one healthy female Balb/C nu/nu (CByJ.Cg-Foxn1nu/J) mice, 5-6 weeks old, are obtained from CHARLES RIVER (L'Arbresles). Animals are maintained in housing rooms under controlled environmental conditions: Temperature: 22 ⁇ 2° C., Humidity 55 ⁇ 10%, Photoperiod (12 h light/12 h dark), HEPA filtered air, 15 air exchanges per hour with no recirculation.
  • Animal enclosures provide sterile and adequate space with bedding material, food and water, environmental and social enrichment (group housing) as described: Top filter polycarbonate Eurostandard Type III or IV cages, Corn cob bedding (ref: LAB COB 12, SERLAB, France), 25 kGy Irradiated diet (Ssniff® Soest, Germany), Complete food for immunodeficient rodents—NM Extrudate, Complete food for immunocompetent rodents—R/M-H Extrudate, Sterile, filtrated at 0.2 ⁇ m water (supplemented with 2.5 ⁇ g/mL estradiol), Environmental enrichment (SIZZLE-dri kraft-D20004 SERLAB, France).
  • mice are anesthetized by an intraperitoneal injection of Ketamine 70 mg/kg (Ketamine500®, Ref 043KET204, Centravet, France) and Xylazine 5 mg/kg (Rompun®, Ref 002ROM001, Centravet, France) in 0.9% NaCl solution at 10 mL/kg/inj.
  • Tumors are induced by stereotaxic injection of 1 ⁇ 10 5 of BT-474 cells in 2 ⁇ L of RPMI 1640 of 52 female animals.
  • BT-474 tumor cell implantation is performed 24 to 72 hours after a whole body irradiation with a ⁇ -source (2 Gy, 60 Co, BioMep, Bretenines, France).
  • the tumor cell suspension is injected into the caudate nucleus of the right cerebral hemisphere at a rate of 0.5 ⁇ L/min. Five minutes after the end of the injection, the needle is slowly withdrawn by 1 mm every minute. Carprofen (dose: 5 mg/kg) is injected subcutaneously at the end of the surgery and 24 h post-surgery. The day of tumor cell implantation is considered as day zero (D 0 ).
  • the treatment is started on day five (D 5 ). Forty animals (40) out of fifty-two (52) are randomized according to their individual body weight into 4 groups each of 10 animals using Vivo Manager® software (Biosystemes, Couternon, France). A statistical test (analysis of variance) is performed to test for homogeneity between groups.
  • the treatment schedule is as follows:
  • Treatment Group animals Treatment (mg/kg/adm) Route schedule 1 10 Vehicle (PBS) — IV TW ⁇ 6 2 10 TZM HC-MTf 60 IV TW ⁇ 6 3 10 MTfp NH-TZM 30 IV TW ⁇ 6 4 10 Herceptin ® 30 IV TW ⁇ 6
  • Necropsy (macroscopic examination) is performed on all terminated animal in the study, and, if possible, on all euthanized moribund or found dead animal.
  • Magnetic Resonance Imaging MRI
  • mice are positioned prone in a dedicated mouse body cradle which is slid in a volume coil (38 mm internal diameter) within the Pharmascan and images are acquired under ParaVision (PV5.1, Bruker Biospin).
  • mice are continuously anaesthetized using isoflurane (Minerve, Bondoufle, France) in a mixture of air via a nose piece.
  • Body temperature of the animals is maintained within physiological levels by a flow of warm air.
  • Breathing rate is continuously monitored using a pressure sensor taped on its abdomen.
  • Physiological signals are monitored via a laptop placed next to the MRI workstation and connected to the sensors by fiber optic cables (SA Instruments, USA).
  • the contrast agent Gadopentetate dimeglumine (Gd-DTPA, Magnevist, Bayer Healthcare Pharmaceuticals, Germany) is injected intravenously (IV) at 0.4 mmol/kg via the caudal vein of mice.
  • ROIs Regions of interest
  • Tumor volume is computed from the ROIs by multiplying the number of ROI voxels by the voxel volume (in mm 3 ). Tumor volumes in mm 3 are tabulated at each time point and for each animal.
  • mice are euthanized fourteen days after the last treatment.
  • Intracardiac blood collection is used in terminal procedures under deep gas anesthesia. Approximately 200 ⁇ L of blood from ten animals per group is collected into blood collection tubes with clot activator. Tubes are centrifuged 30 minutes after sampling at 1300 g for 10 minutes at room temperature to obtain serum. The serum samples are stored in propylene tubes at 80° C. until analysis. Immediately after termination, samples from the brain, heart, lung, liver and kidney of each animal are collected, weighed and stored for analysis.
  • T/C % percent treated over control survival
  • T ⁇ / ⁇ C ⁇ % T ⁇ ( median ⁇ ⁇ survival ⁇ ⁇ time ⁇ ⁇ of ⁇ ⁇ treated ⁇ ⁇ animals ) C ⁇ ( median ⁇ ⁇ survival ⁇ ⁇ time ⁇ ⁇ of ⁇ ⁇ vehicle - treated ⁇ ⁇ animals ) ⁇ 100
  • Statistical analyses are performed using Vivo Manager® software (Biosystemes, Couternon, France). Statistical analyses of mean body weights, MBWC, mean tumor volumes V, are performed using ANOVA and pairwise tests are performed using the Bonferroni/Dunn correction in case of significant ANOVA results. The log-Rank (Kaplan-Meier) test is used to compare the survival curves. A p value ⁇ 0.05 is considered as significant.

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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).

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application claims priority under 35 U.S.C. §119(e) to U.S. Application No. 61/935,253, filed Feb. 3, 2014, which is incorporated by reference in its entirety.
    • SEQUENCE LISTING
  • The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is BIOA_008_01 WO_ST25.txt. The text file is about 340 KB, was created on Feb. 3, 2015, and is being submitted electronically via EFS-Web.
    • BACKGROUND
  • Technical Field
  • Embodiments of the present invention relate to p97 (melanotransferrin)-trastuzumab fusion proteins and antibody 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).
  • Description of the Related Art
  • Overcoming the difficulties of delivering therapeutic or diagnostic agents to specific regions of the brain represents a major challenge to treatment or diagnosis of many central nervous system (CNS) disorders, including those of the brain. In its neuroprotective role, the blood-brain barrier (BBB) functions to hinder the delivery of many potentially important diagnostic and therapeutic agents to the brain.
  • Therapeutic molecules and genes that might otherwise be effective in diagnosis and therapy do not cross the BBB in adequate amounts and often have poor tissue penetration, even in peripheral tissues. It is reported that over 95% of all therapeutic molecules do not cross the blood-brain barrier.
  • Accordingly, there is a need for compositions and methods that facilitate the delivery of therapeutic agents and other molecules across the blood-brain-barrier, for instance, to effectively treat certain diseases of the central nervous system (CNS) such as cancers, particularly those that have metastasized to the CNS. The present invention addresses these needs and offers other related advantages.
    • BRIEF SUMMARY OF THE INVENTION
  • Embodiments of the present invention include p97 (melanotransferrin or MTf)-trastuzumab fusion proteins, comprising a trastuzumab heavy chain and/or light chain sequence fused to a p97 sequence and an optional linker in between. In some embodiments, the fusion protein comprises a trastuzumab heavy chain sequence fused to the N-terminus of the p97 sequence. In certain embodiments, the fusion protein comprises a trastuzumab heavy chain sequence fused to the C-terminus of the p97 sequence. In some embodiments, the fusion protein comprises a truncated trastuzumab heavy chain sequence fused to the C-terminus of the p97 sequence.
  • In some embodiments, 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. In certain embodiments, 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. In certain embodiments, the truncated trastuzumab heavy chain sequence consists essentially of the hinge region or a fragment thereof, the CH2 domain, and the CH3 domain.
  • In some embodiments, the fusion protein comprises (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. In some embodiments, the fusion protein comprises a heavy chain amino acid sequence set forth in one or more of SEQ ID NOs:37-46 or 96-109.
  • In some embodiments, the fusion protein comprises a trastuzumab light chain sequence fused to the N-terminus of the p97 sequence. In certain embodiments, the fusion protein comprises a trastuzumab light chain sequence fused to the C-terminus of the p97 sequence. In some embodiments, the fusion protein comprises (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. In some embodiments, the fusion protein comprises a light chain amino acid sequence set forth in SEQ ID NOs: 110-121.
  • In specific embodiments, the p97 sequence comprises, consists, or consists essentially of SEQ ID NO:2 (soluble MTf) or SEQ ID NO:14 (MTfp or MTfpep).
  • Also included are isolated polynucleotides, which encode a p97 fusion protein described herein. In some aspects, the isolated polynucleotides are codon-optimized for expression in a host cell. In some embodiments, the host cell is a mammalian cell, an insect cell, a yeast cell, or a bacterial cell.
  • Also included are recombinant host cells, comprising an isolated polynucleotide described herein, optionally where the isolated polynucleotide is operably linked to one or more regulatory elements. In certain embodiments, the recombinant host cell comprises an isolated polynucleotide that encodes a (non-fusion) trastuzumab light chain sequence, which is operably linked to one or more regulatory elements. In certain embodiments, the recombinant host cell comprises an isolated polynucleotide that encodes a (non-fusion) trastuzumab heavy chain sequence, which is operably linked to one or more regulatory elements. In certain embodiments, the recombinant host cell comprises 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.
  • Certain embodiments relate to vectors, 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. In some embodiments, the vector comprises an isolated polynucleotide that encodes a (non-fusion) trastuzumab light chain sequence, which is operably linked to one or more regulatory elements. In some embodiments, the vector comprises an isolated polynucleotide that encodes a (non-fusion) trastuzumab heavy chain sequence, which is operably linked to one or more regulatory elements. In some embodiments, the vector comprises 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. Also included are recombinant host cells, comprising one or more vectors as described herein.
  • Some embodiments relate to p97-antibody fusion proteins that comprise two (non-fusion) trastuzumab light chain sequences, and one or two p97-trastuzumab heavy chain fusion proteins described herein, where the one or two p97-trastuzumab heavy chain fusion protein(s) comprise a trastuzumab heavy chain sequence fused to the N-terminus of a p97 sequence and an optional linker in between (see, e.g., FIGS. 1A & 1B). In some embodiments, the p97-antibody fusion protein comprises two p97-trastuzumab heavy chain fusion proteins (see, e.g., FIGS. 1A and 1E). In certain embodiments, the p97-antibody fusion protein comprises one p97-trastuzumab heavy chain fusion protein and one (non-fusion) trastuzumab heavy chain sequence (see, e.g., FIG. 1B).
  • Also included are p97-antibody fusion proteins that comprise one trastuzumab light chain sequence, one trastuzumab heavy chain sequence, and one p97-trastuzumab heavy chain fusion protein of any of the preceding claims, where p97-trastuzumab heavy chain fusion protein comprises a truncated trastuzumab heavy chain fused to the C-terminus of a p97 sequence and an optional linker in between (see, e.g., FIG. 1C).
  • In some embodiments, the p97-antibody fusion protein comprises two trastuzumab light chain sequences, and two p97-trastuzumab heavy chain fusion proteins described herein, where the p97-trastuzumab heavy chain fusion proteins comprise a trastuzumab heavy chain fused to the C-terminus of a p97 sequence and an optional linker in between (see, e.g., FIG. 1D).
  • In some embodiments, the p97-antibody fusion protein comprises two p97-trastuzumab light chain fusion proteins described herein, and two p97-trastuzumab heavy chain fusion proteins described herein, where the p97-trastuzumab light chain fusion proteins comprise a trastuzumab light chain fused to the N-terminus of a p97 sequence and an optional linker in between, and where the p97-trastuzumab heavy chain fusion proteins comprise a trastuzumab heavy chain fused to the N-terminus of a p97 sequence and an optional linker in between (see, e.g., FIG. 1F).
  • Specific examples of p97-antibody fusion proteins include those that comprise 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 of any of the foregoing. In some embodiments, the p97-antibody fusion 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). In some embodiments, the p97-antibody fusion is a heterodimer that comprises any combination of a)-g) above. In particular embodiments, the p97-antibody fusion is a heterodimer that comprises a first set of sets of heavy and light chains selected from a)-g) above, and a second set of trastuzumab (non-fusion) heavy and light chains, for example, SEQ ID NOs: 29-35 or 122 (heavy chains) and 36 or 123 (light chains).
  • Certain embodiments relate to recombinant host cells that comprises a p97-antibody fusion protein of any of the preceding claims. In certain embodiments, the host cell is a mammalian cell, an insect cell, a yeast cell, or a bacterial cell. In certain embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell or a HEK-293 cell.
  • Also included are pharmaceutical compositions, comprising a pharmaceutically-acceptable carrier and a p97-antibody fusion protein of any of the preceding claims.
  • Some embodiments include methods 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 described herein.
  • In certain embodiments, the HER2-overexpressing cancer is at risk for metastasizing to the CNS of the subject. In some embodiments, the HER2-overexpressing cancer has metastasized to the CNS of the subject. In certain embodiments, the HER2-overexpressing cancer is a breast cancer, ovarian cancer, gastric cancer, or uterine cancer.
  • In particular embodiments, the HER2-overexpressing cancer is a HER2-overexpressing metastatic breast cancer. In certain embodiments, the HER2-overexpressing metastatic breast cancer is at risk for metastasizing to the CNS of the subject. In some embodiments, the HER2-overexpressing breast cancer has metastasized to the CNS of the subject.
  • In certain embodiments, the HER2-overexpressing cancer is a HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma. In certain embodiments, the HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma is at risk for metastasizing to the CNS of the subject. In certain embodiments, the HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma has metastasized to the CNS of the subject.
  • In certain embodiments, the HER2-overexpressing cancer is a HER2-overexpressing uterine serous carcinoma (USC). In certain embodiments, the HER2-overexpressing USC is at risk for metastasizing to the CNS of the subject. In certain embodiments, the HER2-overexpressing USC has metastasized to the CNS of the subject.
  • Certain methods include administering the p97-antibody fusion protein or pharmaceutical composition as part of an adjuvant treatment for a HER2-overexpressing breast cancer. In certain embodiments, the adjuvant treatment comprises doxorubicin, cyclophosphamide, and either paclitaxel or docetaxel. In certain embodiments, the adjuvant treatment comprises docetaxel and carboplatin.
  • Some methods include administering the p97-antibody fusion protein or pharmaceutical composition as a single agent following multi-modality anthracycline based therapy.
  • In certain embodiments, the subject is a female human.
  • Certain methods include administering the p97-antibody fusion protein or pharmaceutical composition by intravenous (IV) infusion.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIGS. 1A-1G illustrate the general structure of exemplary p97-antibody fusion proteins (black circles represent p97). FIG. 1A illustrates a homodimeric antibody fusion protein composed of two trastuzumab light chains and two trastuzumab heavy chains each fused to the N-terminus of p97. FIG. 1B illustrates a heterodimeric antibody fusion protein composed of two trastuzumab light chains, one non-fused trastuzumab heavy chain, and one trastuzumab heavy chain fused to the N-terminus of p97. FIG. 1C illustrates a heterodimeric antibody fusion protein composed of one trastuzumab light chain, one non-fused trastuzumab heavy chain, and one trastuzumab heavy chain fused to the C-terminus of p97. FIGS. 1D-1E illustrate antibody fusions composed of two p97-trastuzumab heavy chain fusion proteins and two (non-fusion) trastuzumab light chains. FIG. 1F illustrates an antibody fusion composed of two p97-trastuzumab light chain fusion proteins and two 97-trastuzumab heavy chain fusion proteins. FIG. 1G illustrates an antibody fusion composed of two p97-trastuzumab light chain fusion proteins and two (non-fusion) trastuzumab heavy chain sequences.
  • FIGS. 2A-2D shows octet analysis demonstrating the affinity of antibody fusions for Her2 relative to IgG1 control. FIG. 2A shows the results for human IgG1, FIG. 2B shows the results for TZM HC-MTf, FIG. 2C shows the results for MTfp NH-TZM, and FIG. 2D shows the results for TZM HC-MTfp (see Example 1).
  • FIGS. 3A-3B show antibody-dependent cell-mediated cytotoxicity (ADCC) evaluation of antibody fusions in BT-474 breast cancer cells compared to Herceptin® and human IgG1 Fc controls (see Example 2). These data show that the p97-trastuzumab antibody fusions induced significant antibody-dependent cell-mediated cytotoxicity in breast cancer cells.
    •  DETAILED DESCRIPTION
  • The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2000); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Oligonucleotide Synthesis: Methods and Applications (P. Herdewijn, ed., 2004); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Nucleic Acid Hybridization: Modern Applications (Buzdin and Lukyanov, eds., 2009); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Freshney, R. I. (2005) Culture of Animal Cells, a Manual of Basic Technique, 5th Ed. Hoboken N.J., John Wiley & Sons; B. Perbal, A Practical Guide to Molecular Cloning (3rd Edition 2010); Farrell, R., RNA Methodologies: A Laboratory Guide for Isolation and Characterization (3rd Edition 2005).
  • All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.
    • DEFINITIONS
  • Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.
  • The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
  • By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • As used herein, the term “amino acid” is intended to mean both naturally occurring and non-naturally occurring amino acids as well as amino acid analogs and mimetics. Naturally occurring amino acids include the 20 (L)-amino acids utilized during protein biosynthesis as well as others such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline and ornithine, for example. Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine and the like, which are known to a person skilled in the art. Amino acid analogs include modified forms of naturally and non-naturally occurring amino acids. Such modifications can include, for example, substitution or replacement of chemical groups and moieties on the amino acid or by derivatization of the amino acid. Amino acid mimetics include, for example, organic structures which exhibit functionally similar properties such as charge and charge spacing characteristic of the reference amino acid. For example, an organic structure which mimics Arginine (Arg or R) would have a positive charge moiety located in similar molecular space and having the same degree of mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid. Mimetics also include constrained structures so as to maintain optimal spacing and charge interactions of the amino acid or of the amino acid functional groups. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.
  • Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.
  • The term “conjugate” is intended to refer to the entity formed as a result of covalent or non-covalent attachment or linkage of an agent or other molecule, e.g., a biologically active molecule, to a p97 polypeptide or p97 sequence. One example of a conjugate polypeptide is a “fusion protein” or “fusion polypeptide,” that is, a polypeptide that is created through the joining of two or more coding sequences, which originally coded for separate polypeptides; translation of the joined coding sequences results in a single, fusion polypeptide, typically with functional properties derived from each of the separate polypeptides. The terms “antibody fusion” and “antibody fusion protein” are used interchangeably herein to refer to an antibody or antibody-like molecule that comprises at least one fusion protein described herein.
  • As used herein, the terms “function” and “functional” and the like refer to a biological, enzymatic, or therapeutic function.
  • “Homology” refers to the percentage number of amino acids that are identical or constitute conservative substitutions. Homology may be determined using sequence comparison programs such as GAP (Deveraux et al., Nucleic Acids Research. 12, 387-395, 1984), which is incorporated herein by reference. In this way sequences of a similar or substantially different length to those cited herein could be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.
  • By “isolated” is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an “isolated peptide” or an “isolated polypeptide” and the like, as used herein, includes the in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell; i.e., it is not significantly associated with in vivo substances.
  • The term “linkage,” “linker,” “linker moiety,” or “1” is used herein to refer to a linker that can be used to separate a p97 polypeptide from an agent of interest, or to separate a first agent from another agent, for instance where two or more agents are linked to form a p97 conjugate. The linker may be physiologically stable or may include a releasable linker such as an enzymatically degradable linker (e.g., proteolytically cleavable linkers). In certain aspects, the linker may be a peptide linker, for instance, as part of a p97 fusion protein. In some aspects, the linker may be a non-peptide linker or non-proteinaceous linker. In some aspects, the linker may be particle, such as a nanoparticle.
  • The terms “modulating” and “altering” include “increasing,” “enhancing” or “stimulating,” as well as “decreasing” or “reducing,” typically in a statistically significant or a physiologically significant amount or degree relative to a control. An “increased,” “stimulated” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the amount produced by no composition (e.g., the absence of a fusion protein or antibody fusion of the invention) or a control composition, sample or test subject. A “decreased” or “reduced” amount is typically a “statistically significant” amount, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease in the amount produced by no composition or a control composition, including all integers in between. As one non-limiting example, a control could compare the activity, such as the amount or rate of transport/delivery across the blood brain barrier, the rate and/or levels of distribution to central nervous system tissue, and/or the Cmax for plasma, central nervous system tissues, or any other systemic or peripheral non-central nervous system tissues, of a p97 fusion protein or antibody fusion relative to the agent/antibody alone. Other examples of comparisons and “statistically significant” amounts are described herein.
  • In certain embodiments, the “purity” of any given agent (e.g., a p97 conjugate such as a fusion protein or antibody fusion) in a composition may be specifically defined. For instance, certain compositions may comprise an agent that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure, including all decimals in between, as measured, for example and by no means limiting, by high pressure liquid chromatography (HPLC), a well-known form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds.
  • The terms “polypeptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers. The polypeptides described herein are not limited to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise. The polypeptides described herein may also comprise post-expression modifications, such as glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A polypeptide may be an entire protein, or a subsequence, fragment, variant, or derivative thereof.
  • A “physiologically cleavable” or “hydrolyzable” or “degradable” bond is a bond that reacts with water (i.e., is hydrolyzed) under physiological conditions. The tendency of a bond to hydrolyze in water will depend not only on the general type of linkage connecting two central atoms but also on the substituents attached to these central atoms. Appropriate hydrolytically unstable or weak linkages include, but are not limited to: carboxylate ester, phosphate ester, anhydride, acetal, ketal, acyloxyalkyl ether, imine, orthoester, thio ester, thiol ester, carbonate, and hydrazone, peptides and oligonucleotides.
  • A “releasable linker” includes, but is not limited to, a physiologically cleavable linker and an enzymatically degradable linker. Thus, a “releasable linker” is a linker that may undergo either spontaneous hydrolysis, or cleavage by some other mechanism (e.g., enzyme-catalyzed, acid-catalyzed, base-catalyzed, and so forth) under physiological conditions. For example, a “releasable linker” can involve an elimination reaction that has a base abstraction of a proton, (e.g., an ionizable hydrogen atom, Hα), as the driving force. For purposes herein, a “releasable linker” is synonymous with a “degradable linker.” An “enzymatically degradable linkage” includes a linkage, e.g., amino acid sequence that is subject to degradation by one or more enzymes, e.g., peptidases or proteases. In particular embodiments, a releasable linker has a half life at pH 7.4, 25° C., e.g., a physiological pH, human body temperature (e.g., in vivo), of about 30 minutes, about 1 hour, about 2 hour, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about 96 hours or less.
  • The term “reference sequence” refers generally to a nucleic acid coding sequence, or amino acid sequence, to which another sequence is being compared. All polypeptide and polynucleotide sequences described herein are included as references sequences, including those described by name and those described in the Sequence Listing.
  • The terms “sequence identity” or, for example, comprising a “sequence 50% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein (see, e.g., Sequence Listing), typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide.
  • Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence,” “comparison window,” “sequence identity,” “percentage of sequence identity,” and “substantial identity.” A “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., Nucl. Acids Res. 25:3389, 1997. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al., “Current Protocols in Molecular Biology,” John Wiley & Sons Inc, 1994-1998, Chapter 15.
  • By “statistically significant,” it is meant that the result was unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or probability with which the observed event would occur, if the null hypothesis were true. If the obtained p-value is smaller than the significance level, then the null hypothesis is rejected. In simple cases, the significance level is defined at a p-value of 0.05 or less.
  • The term “solubility” refers to the property of a protein to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as a concentration, either by mass of solute per unit volume of solvent (g of solute per kg of solvent, g per dL (100 mL), mg/ml, etc.), molarity, molality, mole fraction or other similar descriptions of concentration. The maximum equilibrium amount of solute that can dissolve per amount of solvent is the solubility of that solute in that solvent under the specified conditions, including temperature, pressure, pH, and the nature of the solvent. In certain embodiments, solubility is measured at physiological pH, or other pH, for example, at pH 5.0, pH 6.0, pH 7.0, or pH 7.4. In certain embodiments, solubility is measured in water or a physiological buffer such as PBS or NaCl (with or without NaP). In specific embodiments, solubility is measured at relatively lower pH (e.g., pH 6.0) and relatively higher salt (e.g., 500 mM NaCl and 10 mM NaP). In certain embodiments, solubility is measured in a biological fluid (solvent) such as blood or serum. In certain embodiments, the temperature can be about room temperature (e.g., about 20, 21, 22, 23, 24, 25° C.) or about body temperature (˜37° C.). In certain embodiments, a p97 polypeptide or conjugate has a solubility of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 mg/ml at room temperature or at about 37° C.
  • A “subject,” as used herein, includes any animal that exhibits a symptom, or is at risk for exhibiting a symptom, which can be treated or diagnosed with a p97 fusion protein or related antibody fusion of the invention. Suitable subjects (patients) include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included.
  • “Substantially” or “essentially” means nearly totally or completely, for instance, 95%, 96%, 97%, 98%, 99% or greater of some given quantity.
  • “Substantially free” refers to the nearly complete or complete absence of a given quantity for instance, less than about 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or less of some given quantity. For example, certain compositions may be “substantially free” of cell proteins, membranes, nucleic acids, endotoxins, or other contaminants.
  • “Treatment” or “treating,” as used herein, includes any desirable effect on the symptoms or pathology of a disease or condition, and may include even minimal changes or improvements in one or more measurable markers of the disease or condition being treated. “Treatment” or “treating” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. The subject receiving this treatment is any subject in need thereof. Exemplary markers of clinical improvement will be apparent to persons skilled in the art.
  • The term “wild-type” refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally-occurring source. A wild type gene or gene product (e.g., a polypeptide) is that which is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form of the gene.
    • Fusion Proteins
  • Embodiments of the present invention relate generally to fusion proteins that comprise a human p97 (melanotransferrin; MTf) polypeptide sequence a trastuzumab sequence, or an antigen-binding fragment thereof, antibodies that comprise such fusion proteins (i.e., antibody fusions), polynucleotides encoding the fusion proteins, host cells and methods of producing fusion proteins/antibodies, and related compositions and methods of use thereof. Exemplary p97 polypeptide sequences and trastuzumab sequences are described below. Also described are exemplary methods and components, such as linker peptides, for coupling a p97 polypeptide sequence to a trastuzumab sequence.
  • p97 Sequences.
  • In certain embodiments, a p97 polypeptide sequence used in a composition and/or fusion protein of the invention comprises, consists essentially of, or consists of a human p97 reference sequence provided in Table 1 below. Also included are variants and fragments thereof.
  • TABLE 1
    Exemplary p97 Sequences
    SEQ ID
    Description Sequence NO:
    FL Human p97 MRGPSGALWLLLALRTVLGGMEVRWCATSDPEQHKCGNMSEAFREAGIQ  1
    PSLLCVRGTSADHCVQLIAAQEADAITLDGGAIYEAGKEHGLKPVVGEV
    YDQEVGTSYYAVAVVRRSSHVTIDTLKGVKSCHTGINRTVGWNVPVGYL
    VESGRLSVMGCDVLKAVSDYFGGSCVPGAGETSYSESLCRLCRGDSSGE
    GVCDKSPLERYYDYSGAFRCLAEGAGDVAFVKHSTVLENTDGKTLPSWG
    QALLSQDFELLCRDGSRADVTEWRQCHLARVPAHAVVVRADTDGGLIFR
    LLNEGQRLFSHEGSSFQMFSSEAYGQKDLLFKDSTSELVPIATQTYEAW
    LGHEYLHAMKGLLCDPNRLPPYLRWCVLSTPEIQKCGDMAVAFRRQRLK
    PEIQCVSAKSPQHCMERIQAEQVDAVTLSGEDIYTAGKTYGLVPAAGEH
    YAPEDSSNSYYVVAVVRRDSSHAFTLDELRGKRSCHAGFGSPAGWDVPV
    GALIQRGFIRPKDCDVLTAVSEFFNASCVPVNNPKNYPSSLCALCVGDE
    QGRNKCVGNSQERYYGYRGAFRCLVENAGDVAFVRHTTVFDNTNGHNSE
    PWAAELRSEDYELLCPNGARAEVSQFAACNLAQIPPHAVMVRPDTNIFT
    VYGLLDKAQDLFGDDHNKNGFKMFDSSNYHGQDLLFKDATVRAVPVGEK
    TTYRGWLGLDYVAALEGMSSQQCSGAAAPAPGAPLLPLLLPALAARLLP
    PAL
    Soluble GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA  2
    Human p97 AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS
    HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD
    YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR
    CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD
    VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF
    SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL
    PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ
    AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD
    SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA
    VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG
    AFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLCPNGA
    RAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHNKN
    GFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGLDYVAALEGMS
    SQQCSG
    P97 fragment WCATSDPEQHK  3
    P97 fragment RSSHVTIDTLK  4
    P97 fragment SSHVTIDTLKGVK  5
    P97 fragment LCRGDSSGEGVCDK  6
    P97 fragment GDSSGEGVCDKSPLER  7
    P97 fragment YYDYSGAFR  8
    P97 fragment ADVTEWR  9
    P97 fragment VPAHAVVVR 10
    P97 fragment ADTDGGLIFR 11
    P97 fragment CGDMAVAFR 12
    P97 fragment LKPEIQCVSAK 13
    P97 fragment DSSHAFTLDELR 14
    P97 fragment SEDYELLCPNGAR 15
    P97 fragment AQDLFGDDHNKNGFK 16
    P97 fragment FSSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAM 17
    P97 fragment ERIQAEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAV 18
    VRRDSSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCD
    VLTAVSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYY
    GYRGAFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLC
    PNGARAEVSQFAACNLAQIPPHAVM
    P97 fragment VRPDTNIFTVYGLLDKAQDLFGDDHNKNGFKM 19
    P97 fragment GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA 20
    AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS
    HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD
    YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR
    CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD
    VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF
    SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL
    PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ
    AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD
    SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA
    VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG
    AFRCLVENAGDVAFVRHTTVFDNTN
    P97 fragment GHNSEPWAAELRSEDYELLCPN 21
    P97 fragment GARAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHN 22
    KN
    P97 fragment GFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGLDYVAALEGMS 23
    SQQC
    P97 fragment GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA 24
    AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS
    HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD
    YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR
    CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD
    VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF
    SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL
    PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ
    AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD
    SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA
    VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG
    AFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLCPN
    P97 fragment GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA 25
    AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS
    HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD
    YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR
    CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD
    VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF
    SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL
    PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ
    AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD
    SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA
    VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG
    AFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLCPNGA
    RAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHNKN
    P97 fragment GHNSEPWAAELRSEDYELLCPNGARAEVSQFAACNLAQIPPHAVMVRPD 26
    TNIFTVYGLLDKAQDLFGDDHNKN
    P97 fragment GHNSEPWAAELRSEDYELLCPNGARAEVSQFAACNLAQIPPHAVMVRPD 27
    TNIFTVYGLLDKAQDLFGDDHNKNGFKMFDSSNYHGQDLLFKDATVRAV
    PVGEKTTYRGWLGLDYVAALEGMSSQQC
    P97 fragment GARAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHN 28
    KNGFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGLDYVAALEG
    MSSQQC
  • In some embodiments, a p97 polypeptide sequence comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity or homology, along its length, to a human p97 sequence in Table 1, or a fragment thereof.
  • In particular embodiments, a p97 polypeptide sequence comprises a fragment of a human p97 sequence in Table 1. In certain embodiments, a p97 polypeptide fragment is about, at least about, or up to about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700. 700, 710, 720, 730 or more amino acids in length, including all integers and ranges in between, and which may comprise all or a portion of the sequence of a p97 reference sequence.
  • In certain embodiments, a p97 polypeptide fragment is about 5-700, 5-600, 5-500, 5-400, 5-300, 5-200, 5-100, 5-50, 5-40, 5-30, 5-25, 5-20, 5-15, 5-10, 10-700, 10-600, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 10-40, 10-30, 10-25, 10-20, 10-15, 20-700, 20-600, 20-500, 20-400, 20-300, 20-200, 20-100, 20-50, 20-40, 20-30, 20-25, 30-700, 30-600, 30-500, 30-400, 30-300, 30-200, 30-100, 30-50, 30-40, 40-700, 40-600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-50, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 60-700, 60-600, 60-500, 60-400, 60-300, 60-200, 60-100, 60-70, 70-700, 70-600, 70-500, 70-400, 70-300, 70-200, 70-100, 70-80, 80-700, 80-600, 80-500, 80-400, 80-300, 80-200, 80-100, 80-90, 90-700, 90-600, 90-500, 90-400, 90-300, 90-200, 90-100, 100-700, 100-600, 100-500, 100-400, 100-300, 100-250, 100-200, 100-150, 200-700, 200-600, 200-500, 200-400, 200-300, or 200-250 amino acids in length, and comprises all or a portion of a p97 reference sequence.
  • In certain embodiments, p97 polypeptide sequences of interest include p97 amino acid sequences, subsequences, and/or variants of p97 that are effective for transporting an agent of interest across the blood brain barrier and into the central nervous system (CNS). In particular embodiments, the variant or fragment comprises the N-lobe of human p97 (residues 20-361 of SEQ ID NO:1). In specific aspects, the variant or fragment comprises an intact and functional Fe3+-binding site.
  • In some embodiments, a p97 polypeptide sequence is a soluble form of a p97 polypeptide (see Yang et al., Prot Exp Purif. 34:28-48, 2004), or a fragment or variant thereof. In some aspects, the soluble p97 polypeptide has a deletion of the all or a portion of the hydrophobic domain (residues 710-738 of SEQ ID NO:1), alone or in combination with a deletion of all or a portion of the signal peptide (residues 1-19 of SEQ ID NO:1). In specific aspects, the soluble p97 polypeptide comprises or consists of SEQ ID NO:2 (residues 20-711 of SEQ ID NO:1), including variants and fragments thereof.
  • In some embodiments, the p97 polypeptide comprises, consists, or consists essentially of the sequence DSSHAFTLDELR (SEQ ID NO:14 or MTfp), including variants and fragments thereof. In some embodiments, the DSSHAFTLDELR (SEQ ID NO:14) peptide comprises a C-terminal tyrosine (Y).
  • In certain embodiments, for instance, those that employ liposomes, the p97 polypeptide sequence is a lipid soluble form of a p97 polypeptide. For instance, certain of these and related embodiments include a p97 polypeptide that comprises all or a portion of the hydrophobic domain, optionally with or without the signal peptide.
  • In certain other embodiments, the p97 fragment or variant is capable of specifically binding to a p97 receptor, an LRP1 receptor and/or an LRP1B receptor.
  • Variants and fragments of reference p97 polypeptides and other reference polypeptides are described in greater detail below.
  • Trastuzumab Sequences.
  • In certain embodiments, a trastuzumab antibody sequence used in a fusion protein of the invention comprises, consists essentially of, or consists of the trastuzumab light chain and/or heavy chain sequence(s) illustrated in Table 2 below.
  • TABLE 2
    Exemplary Trastuzumab Sequences
    SEQ ID
    Description Sequence NO:
    FL heavy EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA  29
    chain RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR
    (hinge WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC
    underlined) LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
    GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL
    FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
    REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
    GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
    NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
    KSLSLSPGK
    FL heavy EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA  30
    chain RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR
    (hinge WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC
    underlined) LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
    with GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL
    substituted FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
    amino acids REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
    for ″hole″ GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
    (bold NYKT W PPVLDSDGSFFL T SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
    underlined) KSLSTSPGK
    Heavy chain NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT  31
    truncation PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV
    (hinge LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
    underlined) RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
    FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
    Heavy chain DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE  32
    truncation DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE
    (partial YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
    hinge LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
    underlined) WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
    Heavy chain NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT  33
    truncation PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV
    (hinge LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
    underlined) RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT W PPVLDSDGS
    with FFL T SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSTSPGK
    substituted
    amino acids
    for ″hole″
    (bold
    underlined)
    Heavy chain DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE  34
    truncation DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE
    (partial YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
    hinge LVKGFYPSDIAVEWESNGQPENNYKT W PPVLDSDGSFFL T SKLTVDKSR
    underlined) WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
    with
    substituted
    amino acids
    for ″hole″
    (bold
    underlined)
    FL heavy EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA  35
    chain (hinge RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR
    underlined) WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC
    with LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
    substituted GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL
    amino acids FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
    for ″knob″ REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
    (bold GQPREPQVYTLPPSRDELTKNQVSL Y CLVKGFYPSDIAVEWESNGQPEN
    underlined) NYKTTPPVLDSDGSF A LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
    KSLSLSPGK
    FL heavy METDTLLLWVLLLWVPGSTGEVQLVESGGGLVQPGGSLRLSCAASGFNI 122
    chain with KDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNT
    Signal AYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPS
    Sequence VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
    (underlined) LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCD
    KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
    PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
    KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL
    VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
    QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
    FL Light DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIY  36
    Chain SASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTF
    GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
    WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV
    THQGLSSPVTKSFNRGEC
    FL Light METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASQDV 123
    Chain with NTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSL
    Signal QPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKS
    Sequence GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
    (underlined) STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
  • Also included are antigen-binding variants and fragments of the trastuzumab heavy and light chain sequences described herein. In certain embodiments, the trastuzumab antibody, antigen-binding fragment thereof, or related fusion protein or antibody fusion specifically binds to Her2/neu or an epitope or fragment thereof.
  • In particular embodiments, the trastuzumab heavy chain fragment(s), e.g., the Fc regions of the heavy chain fragments, are modified to increase a preferred chain combination, for example, by using knobs-into-holes (KiH) technology (see, e.g., Klein et al., mAbs. 4:6, 653-663, 2012) or other technologies, such as those described in U.S. Application No. 2012/0149876. As one example, to increase the formation of a heterodimeric antibody fusion (e.g., an antibody that comprises one p97-trastuzumab heavy chain fusion and one normal trastuzumab heavy chain; see, e.g., FIGS. 1B and 1C), one of the heavy chains can have amino acid modifications to generate the “knob” and the other heavy chain can have amino acid modifications to form the “hole”. Specific, non-limiting examples of KiH modifications to trastuzumab heavy chain sequences are illustrated in Table 2 above. As another example, one heavy chain fragment could comprise a CH3 domain having amino acid modifications selected from one or more of T350V, L351Y, D399R, D399 (e.g., D399R, D399W, D399Y, D399K), 5400 (e.g., S400E, S400D, S400R, S400K), F405 (e.g., F4051, F405M, F405T, F405S, F405V, F405W), Y407A, Y4071, Y407V, including combinations thereof, and the other could comprise a CH3 domain having amino acid modifications selected from one or more of T350V, T366V, T3661, T366L, T366M, N390 (e.g., N390R, N390K, N390D), K392 (e.g., K392V, K392M, K392R, K392L, K392F, K392E), F405 (e.g., F4051, F405M, F405T, F405S, F405V, F405W), K409F, K409W, and T411 (e.g., T411N, T411R, T411Q, T411K, T411D, T411E, T411W) including combinations thereof (see U.S. Application No. 2012/0149876, hereby incorporated by reference in its entirety).
  • The term “antigen-binding fragment” as used herein refers to a polypeptide fragment that contains at least one CDR of an immunoglobulin heavy and/or light chains that bind to the antigen of interest. In this regard, an antigen-binding fragment of the herein described antibodies may comprise 1, 2, 3, 4, 5, or all 6 CDRs of a VH and VL sequence from antibodies that bind to a therapeutic or diagnostic target.
  • The term “antigen” refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and additionally capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen. An antigen may have one or more epitopes.
  • The term “epitope” includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl, and may in certain embodiments have specific three-dimensional structural characteristics, and/or specific charge characteristics. Epitopes can be contiguous or non-contiguous in relation to the primary structure of the antigen.
  • An antibody, antigen-binding fragment thereof, is said to exhibit “specific binding” or “preferential binding” if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances. An antibody “specifically binds” or “preferentially binds” to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to a specific epitope is an antibody that binds that specific epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other epitopes. It is also understood by reading this definition that, for example, an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.
  • Immunological binding generally refers to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific, for example by way of illustration and not limitation, as a result of electrostatic, ionic, hydrophilic and/or hydrophobic attractions or repulsion, steric forces, hydrogen bonding, van der Waals forces, and other interactions. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, wherein a smaller Kd represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and on geometric parameters that equally influence the rate in both directions. Thus, both the “on rate constant” (Kon) and the “off rate constant” (Koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. The ratio of Koff/Kon enables cancellation of all parameters not related to affinity, and is thus equal to the dissociation constant Kd.
  • Immunological binding properties of proteins such as trastuzumab, antigen-binding fragments thereof, and related fusion proteins and antibody fusions can be quantified using methods well known in the art (see Davies et al., Annual Rev. Biochem. 59:439-473, 1990). In some embodiments, a protein is said to specifically bind an antigen or epitope thereof when the equilibrium dissociation constant is about ≦10−7 or 10−8 M. In some embodiments, the equilibrium dissociation constant of a protein may be about ≦10−9 M or 10−10 M. In certain illustrative embodiments, a protein has an affinity (Kd) for an antigen or target described herein (to which it specifically binds) of at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM.
  • In certain embodiments, antibodies and antigen-binding fragments thereof as described herein include a heavy chain and a light chain CDR set, respectively interposed between a heavy chain and a light chain framework region (FR) set which provide support to the CDRs and define the spatial relationship of the CDRs relative to each other. As used herein, the term “CDR set” refers to the three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3” respectively. An antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3) is referred to herein as a “molecular recognition unit.” Crystallographic analysis of a number of antigen-antibody complexes has demonstrated that the amino acid residues of CDRs form extensive contact with bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the molecular recognition units are primarily responsible for the specificity of an antigen-binding site.
  • As used herein, the term “FR set” refers to the four flanking amino acid sequences which frame the CDRs of a CDR set of a heavy or light chain V region. Some FR residues may contact bound antigen; however, FRs are primarily responsible for folding the V region into the antigen-binding site, particularly the FR residues directly adjacent to the CDRs. Within FRs, certain amino residues and certain structural features are very highly conserved. In this regard, all V region sequences contain an internal disulfide loop of around 90 amino acid residues. When the V regions fold into a binding-site, the CDRs are displayed as projecting loop motifs which form an antigen-binding surface. It is generally recognized that there are conserved structural regions of FRs which influence the folded shape of the CDR loops into certain “canonical” structures—regardless of the precise CDR amino acid sequence. Further, certain FR residues are known to participate in non-covalent interdomain contacts which stabilize the interaction of the antibody heavy and light chains.
  • The structures and locations of immunoglobulin variable domains may be determined by reference to Kabat, E. A. et al., Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and updates thereof.
  • A “monoclonal antibody” refers to a homogeneous antibody population wherein the monoclonal antibody is comprised of amino acids (naturally occurring and non-naturally occurring) that are involved in the selective binding of an epitope. Monoclonal antibodies are highly specific, being directed against a single epitope. The term “monoclonal antibody” encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab′, F(ab′)2, Fv), single chain (ScFv), variants thereof, fusion proteins comprising an antigen-binding portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding fragment (epitope recognition site) of the required specificity and the ability to bind to an epitope. It is not intended to be limited as regards the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals). The term includes whole immunoglobulins as well as the fragments etc. described above under the definition of “antibody.”
  • The proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the F(ab) fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site. The enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the F(ab′)2 fragment which comprises both antigen-binding sites. An Fv fragment for use according to certain embodiments of the present invention can be produced by preferential proteolytic cleavage of an IgM, and on rare occasions of an IgG or IgA immunoglobulin molecule. Fv fragments are, however, more commonly derived using recombinant techniques known in the art. The Fv fragment includes a non-covalent VH::VL heterodimer including an antigen-binding site which retains much of the antigen recognition and binding capabilities of the native antibody molecule. See Inbar et al., PNAS USA. 69:2659-2662, 1972; Hochman et al., Biochem. 15:2706-2710, 1976; and Ehrlich et al., Biochem. 19:4091-4096, 1980.
  • In certain embodiments, single chain Fv or scFV antibodies are contemplated. For example, Kappa bodies (III et al., Prot. Eng. 10:949-57, 1997); minibodies (Martin et al., EMBO J 13:5305-9, 1994); diabodies (Holliger et al., PNAS 90: 6444-8, 1993); or Janusins (Traunecker et al., EMBO J 10: 3655-59, 1991; and Traunecker et al., Int. J. Cancer Suppl. 7:51-52, 1992), may be prepared using standard molecular biology techniques following the teachings of the present application with regard to selecting antibodies having the desired specificity.
  • A single chain Fv (sFv) polypeptide is a covalently linked VH::VL heterodimer which is expressed from a gene fusion including VH- and VL-encoding genes linked by a peptide-encoding linker. Huston et al. (PNAS USA. 85(16):5879-5883, 1988). A number of methods have been described to discern chemical structures for converting the naturally aggregated—but chemically separated—light and heavy polypeptide chains from an antibody V region into an sFv molecule which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778, to Ladner et al.
  • In certain embodiments, the antibodies or antigen-binding fragments thereof are humanized. These embodiments refer to a chimeric molecule, generally prepared using recombinant techniques, having an antigen-binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based upon the structure and/or sequence of a human immunoglobulin. The antigen-binding site may comprise either complete variable domains fused onto constant domains or only the CDRs grafted onto appropriate framework regions in the variable domains. Epitope binding sites may be wild type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but the possibility of an immune response to the foreign variable region remains (LoBuglio et al., PNAS USA 86:4220-4224, 1989; Queen et al., PNAS USA. 86:10029-10033, 1988; Riechmann et al., Nature. 332:323-327, 1988). Illustrative methods for humanization of antibodies include the methods described in U.S. Pat. No. 7,462,697.
  • Another approach focuses not only on providing human-derived constant regions, but modifying the variable regions as well so as to reshape them as closely as possible to human form. It is known that the variable regions of both heavy and light chains contain three complementarity-determining regions (CDRs) which vary in response to the epitopes in question and determine binding capability, flanked by four framework regions (FRs) which are relatively conserved in a given species and which putatively provide a scaffolding for the CDRs. When nonhuman antibodies are prepared with respect to a particular epitope, the variable regions can be “reshaped” or “humanized” by grafting CDRs derived from nonhuman antibody on the FRs present in the human antibody to be modified. Application of this approach to various antibodies has been reported by Sato et al., Cancer Res. 53:851-856, 1993; Riechmann et al., Nature 332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988; Kettleborough et al., Protein Engineering. 4:773-3783, 1991; Maeda et al., Human Antibodies Hybridoma 2:124-134, 1991; Gorman et al., PNAS USA. 88:4181-4185, 1991; Tempest et al., Bio/Technology 9:266-271, 1991; Co et al., PNAS USA. 88:2869-2873, 1991; Carter et al., PNAS USA. 89:4285-4289, 1992; and Co et al., J Immunol. 148:1149-1154, 1992. In some embodiments, humanized antibodies preserve all CDR sequences (for example, a humanized mouse antibody which contains all six CDRs from the mouse antibodies). In other embodiments, humanized antibodies have one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody.
  • Linkers.
  • As noted above, certain fusion proteins may employ one or more linker groups, including peptide linkers. Such linkers can be stable linkers or releasable linkers.
  • For instance, for polypeptide-polypeptide conjugates, peptide linkers can separate the components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence may be incorporated into the fusion protein using standard techniques described herein and well-known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180.
  • In certain illustrative embodiments, a peptide linker is between about 1 to 5 amino acids, between 5 to 10 amino acids, between 5 to 25 amino acids, between 5 to 50 amino acids, between 10 to 25 amino acids, between 10 to 50 amino acids, between 10 to 100 amino acids, or any intervening range of amino acids. In other illustrative embodiments, a peptide linker comprises about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids in length. Particular linkers can have an overall amino acid length of about 1-200 amino acids, 1-150 amino acids, 1-100 amino acids, 1-90 amino acids, 1-80 amino acids, 1-70 amino acids, 1-60 amino acids, 1-50 amino acids, 1-40 amino acids, 1-30 amino acids, 1-20 amino acids, 1-10 amino acids, 1-5 amino acids, 1-4 amino acids, 1-3 amino acids, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100 or more amino acids.
  • A peptide linker may employ any one or more naturally-occurring amino acids, non-naturally occurring amino acid(s), amino acid analogs, and/or amino acid mimetics as described elsewhere herein and known in the art. Certain amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., PNAS USA. 83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. Particular peptide linker sequences contain Gly, Ser, and/or Asn residues. Other near neutral amino acids, such as Thr and Ala may also be employed in the peptide linker sequence, if desired.
  • Certain exemplary linkers include Gly, Ser and/or Asn-containing linkers, as follows: [G]x, [S]x, [N]x, [GS]x, [GGS]x, [GSS]x, [GSGS]x (SEQ ID NO:47), [GGSG]x (SEQ ID NO:48), [GGGS]x (SEQ ID NO:49), [GGGGS]x(SEQ ID NO:50), [GN]x, [GGN]x, [GNN]x, [GNGN]x(SEQ ID NO:51), [GGNG]x(SEQ ID NO:52), [GGGN]x (SEQ ID NO:53), [GGGGN]x(SEQ ID NO:54) linkers, where x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more. Other combinations of these and related amino acids will be apparent to persons skilled in the art. In specific embodiments, the linker comprises or consists of a [GGGGS]3 (SEQ ID NO:55) sequence, or GGGGSGGGGSGGGGS (SEQ ID NO:55).
  • In specific embodiments, the linker sequence comprises a Gly3 linker sequence, which includes three glycine residues. In particular embodiments, flexible linkers can be rationally designed using a computer program capable of modeling both DNA-binding sites and the peptides themselves (Desjarlais & Berg, PNAS. 90:2256-2260, 1993; and PNAS. 91:11099-11103, 1994) or by phage display methods.
  • The peptide linkers may be physiologically stable or may include a releasable linker such as a physiologically degradable or enzymatically degradable linker (e.g., proteolytically cleavable linker). In certain embodiments, one or more releasable linkers can result in a shorter half-life and more rapid clearance of the conjugate. These and related embodiments can be used, for example, to enhance the solubility and blood circulation lifetime of p97 conjugates in the bloodstream, while also delivering an agent into the bloodstream (or across the BBB) that, subsequent to linker degradation, is substantially free of the p97 sequence. These aspects are especially useful in those cases where polypeptides or other agents, when permanently conjugated to a p97 sequence, demonstrate reduced activity. By using the linkers as provided herein, such antibodies can maintain their therapeutic activity when in conjugated form. In these and other ways, the properties of the p97 conjugates can be more effectively tailored to balance the bioactivity and circulating half-life of the antibodies over time.
  • Enzymatically degradable linkages suitable for use in particular embodiments of the present invention include, but are not limited to: an amino acid sequence cleaved by a serine protease such as thrombin, chymotrypsin, trypsin, elastase, kallikrein, or substilisin. Illustrative examples of thrombin-cleavable amino acid sequences include, but are not limited to: -Gly-Arg-Gly-Asp-(SEQ ID NO:56), -Gly-Gly-Arg-, -Gly-Arg-Gly-Asp-Asn-Pro-(SEQ ID NO:57), -Gly-Arg-Gly-Asp-Ser-(SEQ ID NO:58), -Gly-Arg-Gly-Asp-Ser-Pro-Lys-(SEQ ID NO:59), -Gly-Pro-Arg-, -Val-Pro-Arg-, and -Phe-Val-Arg-. Illustrative examples of elastase-cleavable amino acid sequences include, but are not limited to: -Ala-Ala-Ala-, -Ala-Ala-Pro-Val-(SEQ ID NO:60), -Ala-Ala-Pro-Leu-(SEQ ID NO:61), -Ala-Ala-Pro-Phe-(SEQ ID NO:62), -Ala-Ala-Pro-Ala-(SEQ ID NO:63), and -Ala-Tyr-Leu-Val-(SEQ ID NO:64).
  • Enzymatically degradable linkages suitable for use in particular embodiments of the present invention also include amino acid sequences that can be cleaved by a matrix metalloproteinase such as collagenase, stromelysin, and gelatinase. Illustrative examples of matrix metalloproteinase-cleavable amino acid sequences include, but are not limited to: -Gly-Pro-Y-Gly-Pro-Z-(SEQ ID NO:65), -Gly-Pro-, Leu-Gly-Pro-Z-(SEQ ID NO:66), -Gly-Pro-Ile-Gly-Pro-Z-(SEQ ID NO:67), and -Ala-Pro-Gly-Leu-Z-(SEQ ID NO: 68), where Y and Z are amino acids. Illustrative examples of collagenase-cleavable amino acid sequences include, but are not limited to: -Pro-Leu-Gly-Pro-D-Arg-Z-(SEQ ID NO:69), -Pro-Leu-Gly-Leu-Leu-Gly-Z-(SEQ ID NO:70), -Pro-Gln-Gly-Ile-Ala-Gly-Trp-(SEQ ID NO:71), -Pro-Leu-Gly-Cys(Me)-His-(SEQ ID NO:72), -Pro-Leu-Gly-Leu-Tyr-Ala-(SEQ ID NO:73), -Pro-Leu-Ala-Leu-Trp-Ala-Arg-(SEQ ID NO:74), and -Pro-Leu-Ala-Tyr-Trp-Ala-Arg-(SEQ ID NO:75), where Z is an amino acid. An illustrative example of a stromelysin-cleavable amino acid sequence is -Pro-Tyr-Ala-Tyr-Tyr-Met-Arg-(SEQ ID NO:76); and an example of a gelatinase-cleavable amino acid sequence is -Pro-Leu-Gly-Met-Tyr-Ser-Arg-(SEQ ID NO:77).
  • Enzymatically degradable linkages suitable for use in particular embodiments of the present invention also include amino acid sequences that can be cleaved by an angiotensin converting enzyme, such as, for example, -Asp-Lys-Pro-, -Gly-Asp-Lys-Pro-(SEQ ID NO:78), and -Gly-Ser-Asp-Lys-Pro-(SEQ ID NO:79).
  • Enzymatically degradable linkages suitable for use in particular embodiments of the present invention also include amino acid sequences that can be degraded by cathepsin B, such as, for example, -Val-Cit-, -Ala-Leu-Ala-Leu- (SEQ ID NO:80), -Gly-Phe-Leu-Gly- (SEQ ID NO:81) and -Phe-Lys-.
  • In some embodiments, the linker comprises, consists, or consists essentially of 125 (SEQ ID NO:124), including fragments and variants thereof.
  • In certain embodiments, however, any one or more of the non-peptide or peptide linkers are optional. For instance, linker sequences may not be required in a fusion protein where the first and second polypeptides have non-essential N-terminal and/or C-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
  • Fusion Proteins and Antibody Fusions.
  • Certain embodiments relate to fusion proteins, comprising a p97 polypeptide sequence fused to a trastuzumab polypeptide sequence such as a trastuzumab heavy or light chain sequence, and antibody fusions comprising the same. An “antibody fusion” refers to an antibody or antibody-like immunoglobulin molecule that comprises one or more p97-trastuzumab fusion proteins and optionally one or more non-fusion trastuzumab sequences, i.e., trastuzumab light chain or heavy chain sequences, or variants/fragments thereof, which are not fused to a p97 sequence. In some instances, an antibody fusion comprises two light chain sequences and two heavy chain sequences, which are individually selected from any of the light/heavy chain sequences and/or fusion protein sequences described herein. In some instances, an antibody fusion comprises one light chain sequence and two heavy chain sequences, which are individually selected from any of the light/heavy chain sequences and/or fusion protein sequences described herein.
  • Specific, non-limiting examples of p97-trastuzumab heavy chain fusion proteins are illustrated in Table 3 below, and in Table E1 (see Examples).
  • TABLE 3
    Exemplary p97-trastuzmab fusion protein sequences
    SEQ ID
    Description Sequence NO:
    Trastuzumab EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA  37
    FL heavy RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR
    chain fused WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC
    to N- LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
    terminus of GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL
    soluble FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
    human p97 (no REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
    linker) GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
    NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
    KSLSLSPGK/GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGT
    SADHCVQLIAAQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSY
    YAVAVVRRSSHVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVM
    GCDVLKAVSDYFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLE
    RYYDYSGAFRCLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFE
    LLCRDGSRADVTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLF
    SHEGSSFQMFSSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAM
    KGLLCDPNRLPPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAK
    SPQHCMERIQAEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNS
    YYVVAVVRRDSSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFI
    RPKDCDVLTAVSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGN
    SQERYYGYRGAFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSE
    DYELLCPNGARAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQ
    DLFGDDHNKNGFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGL
    DYVAALEGMSSQQCSG
    Trastuzumab EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA  38
    FL heavy RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR
    chain fused WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC
    to N- LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
    terminus of GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL
    soluble FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
    human p97 REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
    (linker GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
    underlined) NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
    KSLSLSPGKGGGGSGGGGSGGGGSGMEVRWCATSDPEQHKCGNMSEAFR
    EAGIQPSLLCVRGTSADHCVQLIAAQEADAITLDGGAIYEAGKEHGLKP
    VVGEVYDQEVGTSYYAVAVVRRSSHVTIDTLKGVKSCHTGINRTVGWNV
    PVGYLVESGRLSVMGCDVLKAVSDYFGGSCVPGAGETSYSESLCRLCRG
    DSSGEGVCDKSPLERYYDYSGAFRCLAEGAGDVAFVKHSTVLENTDGKT
    LPSWGQALLSQDFELLCRDGSRADVTEWRQCHLARVPAHAVVVRADTDG
    GLIFRLLNEGQRLFSHEGSSFQMFSSEAYGQKDLLFKDSTSELVPIATQ
    TYEAWLGHEYLHAMKGLLCDPNRLPPYLRWCVLSTPEIQKCGDMAVAFR
    RQRLKPEIQCVSAKSPQHCMERIQAEQVDAVTLSGEDIYTAGKTYGLVP
    AAGEHYAPEDSSNSYYVVAVVRRDSSHAFTLDELRGKRSCHAGFGSPAG
    WDVPVGALIQRGFIRPKDCDVLTAVSEFFNASCVPVNNPKNYPSSLCAL
    CVGDEQGRNKCVGNSQERYYGYRGAFRCLVENAGDVAFVRHTTVFDNTN
    GHNSEPWAAELRSEDYELLCPNGARAEVSQFAACNLAQIPPHAVMVRPD
    TNIFTVYGLLDKAQDLFGDDHNKNGFKMFDSSNYHGQDLLFKDATVRAV
    PVGEKTTYRGWLGLDYVAALEGMSSQQCSG
    Trastuzumab GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA  39
    truncated AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS
    heavy chain HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD
    fused to C- YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR
    terminus of CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD
    soluble VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF
    human p97 SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL
    (no linker) PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ
    AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD
    SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA
    VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG
    AFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLCPNGA
    RAEVSQFAACKLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHNKN
    GFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGLDYVAALEGMS
    SQQCG/NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT
    LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
    YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
    YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
    LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
    K
    Trastuzumab GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA  40
    truncated AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS
    heavy chain HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD
    fused to C- YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR
    terminus of CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD
    soluble VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF
    human p97 SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL
    (linker PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ
    underlined) AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD
    SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA
    VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG
    AFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLCPNGA
    RAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHNKN
    GFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGLDYVAALEGMS
    SQQCGGGGGSGGGGSGGGGSNTKVDKKVEPKSCDKTHTCPPCPAPELLG
    GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
    NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK
    TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
    NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL
    HNHYTQKSLSLSPGK
    Trastuzumab GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA  41
    partial AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS
    hinge HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD
    truncated YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR
    heavy chain CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD
    fused to C- VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF
    terminus of SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL
    soluble PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ
    human p97 AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD
    (no linker) SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA
    VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG
    AFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLCPNGA
    RAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHNKN
    GFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGLDYVAALEGMS
    SQQCG/DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV
    VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
    WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
    QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
    TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
    Trastuzumab GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA  42
    partial AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS
    hinge HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD
    truncated YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR
    heavy chain CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD
    fused to C- VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF
    terminus of SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL
    soluble PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ
    human p97 AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD
    (linker SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA
    underlined) VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG
    AFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLCPNGA
    RAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHNKN
    GFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGLDYVAALEGMS
    SQQCGGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKD
    TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
    TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
    VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
    VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
    GK
    Trastuzumab GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA  43
    truncated AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS
    heavy chain HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD
    with KiH YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR
    ″hole″ CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD
    variation VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF
    fused to C- SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL
    terminus of PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ
    soluble AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD
    human p97 SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA
    (no linker) VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG
    AFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLCPNGA
    RAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHNKN
    GFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGLDYVAALEGMS
    SQQCSG/NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD
    TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
    TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
    VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTWPP
    VLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
    GK
    Trastuzumab GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA  44
    truncated AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS
    heavy chain HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD
    with KiH YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR
    ″hole″ CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD
    variation VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF
    fused to C- SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL
    terminus of PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ
    soluble AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD
    human p97 SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA
    (linker VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG
    underlined) AFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLCPNGA
    RAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHNKN
    GFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGLDYVAALEGMS
    SQQCSGGGGGSGGGGSGGGGSNTKVDKKVEPKSCDKTHTCPPCPAPELL
    GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
    HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
    KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE
    SNGQPENNYKTWPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEA
    LHNHYTQKSLSLSPGK
    Trastuzumab GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA  45
    partial AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS
    hinge HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD
    truncated YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR
    heavy chain CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD
    with KiH VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF
    ″hole″ SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL
    variation PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ
    fused to C- AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD
    terminus of SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA
    soluble VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG
    human p97 AFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLCPNGA
    (no linker) RAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHNKN
    GFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGLDYVAALEGMS
    SQQCSG/DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV
    VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
    DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK
    NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTWPPVLDSDGSFFLTSK
    LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
    Trastuzumab GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA  46
    partial AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS
    hinge HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD
    truncated YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR
    heavy chain CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD
    with KiH VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF
    ″hole″ SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL
    variation PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ
    fused to C- AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD
    terminus of SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA
    soluble VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG
    human p97 AFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLCPNGA
    (linker RAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHNKN
    underlined) GFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGLDYVAALEGMS
    SQQCSGGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPK
    DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
    STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
    QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTWP
    PVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
    PGK
    TZM heavy EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA  96
    chain: RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR
    linker WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC
    (underlined): LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
    MTf (bold) GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL
    FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
    REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
    GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
    NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
    KSLSLSPGKGGGGSGGGGS GMEVRWCATSDPEQHKCGNMSEAFREAGIQ
    PSLLCVRGTSADHCVQLIAAQEADAITLDGGAIYEAGKEHGLKPVVGEV
    YDQEVGTSYYAVAVVRRSSHVTIDTLKGVKSCHTGINRTVGWNVPVGYL
    VESGRLSVMGCDVLKAVSDYFGGSCVPGAGETSYSESLCRLCRGDSSGE
    GVCDKSPLERYYDYSGAFRCLAEGAGDVAFVKHSTVLENTDGKTLPSWG
    QALLSQDFELLCRDGSRADVTEWRQCHLARVPAHAVVVRADTDGGLIFR
    LLNEGQRLFSHEGSSFQMFSSEAYGQKDLLFKDSTSELVPIATQTYEAW
    LGHEYLHAMKGLLCDPNRLPPYLRWCVLSTPEIQKCGDMAVAFRRQRLK
    PEIQCVSAKSPQHCMERIQAEQVDAVTLSGEDIYTAGKTYGLVPAAGEH
    YAPEDSSNSYYVVAVVRRDSSHAFTLDELRGKRSCHAGFGSPAGWDVPV
    GALIQRGFIRPKDCDVLTAVSEFFNASCVPVNNPKNYPSSLCALCVGDE
    QGRNKCVGNSQERYYGYRGAFRCLVENAGDVAFVRHTTVFDNTNGHNSE
    PWAAELRSEDYELLCPNGARAEVSQFAACNLAQIPPHAVMVRPDTNIFT
    VYGLLDKAQDLFGDDHNKNGFKMFDSSNYHGQDLLFKDATVRAVPVGEK
    TTYRGWLGLDYVAALEGMSSQQCS
    TZM heavy EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA  97
    chain: RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR
    linker WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC
    (underlined): LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
    MTf (bold) GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL
    FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
    REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
    GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
    NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
    KSLSLSPGKGGGGSGGGGSGGGGS GMEVRWCATSDPEQHKCGNMSEAFR
    EAGIQPSLLCVRGTSADHCVQLIAAQEADAITLDGGAIYEAGKEHGLKP
    VVGEVYDQEVGTSYYAVAVVRRSSHVTIDTLKGVKSCHTGINRTVGWNV
    PVGYLVESGRLSVMGCDVLKAVSDYFGGSCVPGAGETSYSESLCRLCRG
    DSSGEGVCDKSPLERYYDYSGAFRCLAEGAGDVAFVKHSTVLENTDGKT
    LPSWGQALLSQDFELLCRDGSRADVTEWRQCHLARVPAHAVVVRADTDG
    GLIFRLLNEGQRLFSHEGSSFQMFSSEAYGQKDLLFKDSTSELVPIATQ
    TYEAWLGHEYLHAMKGLLCDPNRLPPYLRWCVLSTPEIQKCGDMAVAFR
    RQRLKPEIQCVSAKSPQHCMERIQAEQVDAVTLSGEDIYTAGKTYGLVP
    AAGEHYAPEDSSNSYYVVAVVRRDSSHAFTLDELRGKRSCHAGFGSPAG
    WDVPVGALIQRGFIRPKDCDVLTAVSEFFNASCVPVNNPKNYPSSLCAL
    CVGDEQGRNKCVGNSQERYYGYRGAFRCLVENAGDVAFVRHTTVFDNTN
    GHNSEPWAAELRSEDYELLCPNGARAEVSQFAACNLAQIPPHAVMVRPD
    TNIFTVYGLLDKAQDLFGDDHNKNGFKMFDSSNYHGQDLLFKDATVRAV
    PVGEKTTYRGWLGLDYVAALEGMSSQQCSG
    MTfp DSSHAFTLDELRY GGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASG  98
    sequence FNIKDTYIHWVRQAPGKGLEWVARTYPTNGYTRYADSVKGRFTISADTS
    (bold): KNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTK
    linker GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
    (underlined): PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS
    TZM heavy CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
    chain EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
    EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT
    CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
    RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
    MTfp DSSHAFTLDELR GGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGF  99
    sequence NIKDTYIHWVRQAPGKGLEWVARTYPTNGYTRYADSVKGRFTISADTSK
    (bold): NTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKG
    linker PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
    (underlined): AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
    TZM heavy DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
    chain DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE
    YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
    LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
    WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
    MTfp DSSHAFTLDELRY EAAAKEAAAKEAAAKEVQLVESGGGLVQPGGSLRLS 100
    sequence CAASGFNIKDTYIHWVRQAPGKGLEWVARTYPTNGYTRYADSVKGRFTI
    (bold) SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS
    w/terminal Y SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
    (bold): GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK
    linker VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV
    (underlined): VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
    TZM heavy WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
    chain QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
    TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
    MTfp DSSHAFTLDELR EAAAKEAAAKEAAAKEVQLVESGGGLVQPGGSLRLSC 101
    sequence AASGFNIKDTYIHWVRQAPGKGLEWVARTYPTNGYTRYADSVKGRFTIS
    (bold) w/o ADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
    terminal Y: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
    linker VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
    (underlined) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
    : TZM heavy DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW
    chain LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ
    VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
    VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
    MTfp DSSHAFTLDELRYEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHW 102
    sequence VRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNS
    (bold): TZM LRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPS
    heavy chain SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY
    w/terminal Y SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC
    (bold) PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
    VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA
    LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD
    IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
    SVMHEALHNHYTQKSLSLSPGK
    MTfp DSSHAFTLDELREVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWV 103
    sequence RQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSL
    (bold) w/o RAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSS
    terminal Y: KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
    TZM heavy LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP
    chain APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
    DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
    PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI
    AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
    VMHEALHNHYTQKSLSLSPGK
    TZM heavy EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA 104
    chain : RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR
    linker WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC
    (underlined): LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
    MTfp GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL
    sequence FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
    without C- REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
    terminal Y GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
    (bold) NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
    KSLSLSPGKGGGGSGGGGS DSSHAFTLDELR
    TZM heavy EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA 105
    chain linker RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR
    (underlined): WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC
    MTfp LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
    sequence GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL
    w/terminal Y FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
    (bold) REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
    GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
    NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
    KSLSLSPGKGGGGSGGGGS DSSHAFTLDELRY
    TZM heavy EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA 106
    chain: RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR
    linker WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC
    (underlined): LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
    MTfp GTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLGGPSVF
    w/terminal Y LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
    (bold) PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
    KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
    NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
    QKSLSLSPGKEAAAKEAAAKEAAAK DSSHAFTLDELRY
    TZM heavy EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA 107
    chain: RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR
    linker WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC
    (underlined): LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
    MTfp w/o GTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLGGPSVF
    terminal LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
    Y(bold PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
    KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
    NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
    QKSLSLSPGKEAAAKEAAAKEAAAK DSSHAFTLDELR
    TZM heavy EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA 108
    chain: MTfp RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR
    sequence WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC
    without C- LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
    terminal Y GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL
    (bold) FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
    REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
    GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
    NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
    KSLSLSPGKDSSHAFTLDELR
    TZM heavy EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA 109
    chain: MTfp RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR
    sequence WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC
    w/terminal Y LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
    (bold) GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL
    FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
    REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
    GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
    NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
    KSLSLSPGKDSSHAFTLDELRY
  • In certain embodiments, the p97-trastuzumab fusion protein comprises, consists, or consists essentially of a trastuzumab heavy chain sequence fused to the N-terminus of a p97 sequence. In particular embodiments, the p97 sequence is human soluble p97, for example, comprising or consisting of SEQ ID NO:2, or a variant/fragment thereof. In some embodiments, the p97 sequence comprises or consists of SEQ ID NO:14, or a variant/fragment thereof. In some embodiments, the trastuzumab heavy chain sequence is selected from SEQ ID NO:29-35 or 122, or a variant/fragment thereof. Optionally, the fusion protein comprises a peptide linker in between the p97 and trastuzumab sequences. In specific embodiments, the linker is a (GGGGS)2 or (GGGGS)3 linker or a EAAAKEAAAKEAAAK (SEQ ID NO:124) linker. In specific embodiments, the p97-trastuzumab fusion protein comprises, consists, or consists essentially of SEQ ID NO:37 (trastuzumab heavy chain fused to the N-terminus of soluble p97) or a variant/fragment thereof. In specific embodiments, the linker is a (GGGGS)3 linker, and the fusion protein optionally comprises, consists, or consists essentially of SEQ ID NO:38 (trastuzumab heavy chain fused to the N-terminus of soluble human p97, and separated by a (GGGGS)3 linker), or a variant/fragment thereof. Other combinations will be apparent to persons skilled in the art.
  • In some embodiments, the p97-trastuzumab fusion protein comprises, consists, or consists essentially of a trastuzumab heavy chain sequence fused to the C-terminus of a p97 sequence. In particular embodiments, the p97 sequence is human soluble p97, for example, comprising or consisting of SEQ ID NO:2, or a variant/fragment thereof. In some embodiments, the p97 sequence comprises or consists of SEQ ID NO:14, or a variant/fragment thereof. In some embodiments, the trastuzumab heavy chain sequence is selected from SEQ ID NO:29-35 or 122, or a variant/fragment thereof. In some embodiments, the trastuzumab heavy chain sequence is a truncated sequence that comprises the polypeptide of SEQ ID NO:31-33 or 34, or a variant/fragment thereof. In specific embodiments, the p97-trastuzumab fusion protein comprises, consists, or consists essentially of SEQ ID NO:39, 41, 43, or 45 (truncated trastuzumab heavy chain fused to the C-terminus of soluble human p97), or a variant/fragment thereof. Optionally, the fusion protein comprises a peptide linker in between the p97 and trastuzumab sequences. In specific embodiments, the linker is a (GGGGS)2 or (GGGGS)3 linker or a EAAAKEAAAKEAAAK (SEQ ID NO:124) linker. In specific embodiments, the linker is a (GGGGS)3 linker, and the fusion protein optionally comprises, consists, or consists essentially of SEQ ID NO:40, 42, 44 or 46 (truncated trastuzumab heavy chain fused to the C-terminus of soluble human p97, and separated by a (GGGGS)3 linker), or a variant/fragment thereof.
  • In some embodiments, the p97-trastuzumab heavy chain fusion protein comprises, consists, or consists essentially of a polypeptide sequence selected from SEQ ID NOs:37-46 and 96-109, or a variant/fragment thereof.
  • Other combinations will be apparent to persons skilled in the art.
  • Also included are p97-trastuzumab light chain fusion proteins. Specific, non-limiting examples of p97-trastuzumab light chain fusion proteins are illustrated in Table 4 below, and in Table E1 (see Examples).
  • TABLE 4
    Exemplary p97-trastuzmab fusion protein sequences
    SEQ ID
    Sequence NO:
    Trastuzumab DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIY 110
    FL light SASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTF
    chain fused GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
    to N- WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV
    terminus of THQGLSSPVTKSFNRGECDSSHAFTLDELRY
    MTfp(no
    linker)
    Trastuzumab DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIY 111
    FL light SASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTF
    chain fused GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
    to N- WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV
    terminus of THQGLSSPVTKSFNRGECEAAAKEAAAKEAAAK DSSHAFTLDELRY
    MTfp(linker
    underlined)
    Trastuzumab DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIY 112
    FL light SASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTF
    chain fused GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
    to N- WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV
    terminus of THQGLSSPVTKSFNRGECGGGGSGGGGS DSSHAFTLDELRY
    MTfp(linker
    underlined)
    Trastuzumab DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIY 113
    FL light SASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTF
    chain fused GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
    to N- WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV
    terminus of THQGLSSPVTKSFNRGECDSSHAFTLDELR
    MTfp(no
    linker)
    Trastuzumab DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIY 114
    FL light SASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTF
    chain fused GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
    to N- WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV
    terminus of THQGLSSPVTKSFNRGECEAAAKEAAAKEAAAK DSSHAFTLDELR
    MTfp(linker
    underlined)
    Trastuzumab DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIY 115
    FL light SASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTF
    chain fused GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
    to N- WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV
    terminus of THQGLSSPVTKSFNRGECGGGGSGGGGS DSSHAFTLDELRY
    MTfp(linker
    underlined)
    Trastuzumab DSSHAFTLDELRYDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWY 116
    FL light QQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFAT
    chain fused YYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC
    to C- LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK
    terminus of ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
    MTfp(no
    linker)
    Trastuzumab DSSHAFTLDELRY EAAAKEAAAKEAAAKDIQMTQSPSSLSASVGDRVTI 117
    FL light TCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD
    chain fused FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFP
    to C- PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS
    terminus of KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
    MTfp(linker
    underlined)
    Trastuzumab DSSHAFTLDELRY GGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS 118
    FL light QDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTI
    chain fused SSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQ
    to C- LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY
    terminus of SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
    MTfp(linker
    underlined)
    Trastuzumab DSSHAFTLDELRDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQ 119
    FL light QKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATY
    chain fused YCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
    to C- LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA
    terminus of DYEKHKVYACEVTHQGLSSPVTKSFNRGEC
    MTfp(no
    linker)
    Trastuzumab DSSHAFTLDELR EAAAKEAAAKEAAAKDIQMTQSPSSLSASVGDRVTIT 120
    FL light CRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDF
    chain fused TLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPP
    to C- SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
    terminus of DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
    MTfp(linker
    underlined)
    Trastuzumab DSSHAFTLDELR GGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ 121
    FL light DVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTIS
    chain fused SLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQL
    to C- KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
    terminus of LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
    MTfp(linker
    underlined)
  • In some embodiments, the p97-trastuzumab fusion protein comprises, consists, or consists essentially of a trastuzumab light chain sequence fused to the N-terminus of a p97 sequence. In particular embodiments, the p97 sequence is human soluble p97, for example, comprising or consisting of SEQ ID NO:2, or a variant/fragment thereof. In some embodiments, the p97 sequence comprises or consists of SEQ ID NO:14, or a variant/fragment thereof. In some embodiments, the trastuzumab light chain sequence comprises or consists of SEQ ID NO:36 or 123, or a variant/fragment thereof. In specific embodiments, the p97-trastuzumab fusion protein comprises, consists, or consists essentially of a sequence selected from SEQ ID NOs:110-115 (trastuzumab light chain fused to the N-terminus of p97pep), or a variant/fragment thereof. Optionally, the fusion protein comprises a peptide linker in between the p97 and trastuzumab sequence, for example, as illustrated in SEQ ID NO:111-112 or 114-115. In specific embodiments, the linker is a (GGGGS)2 or (GGGGS)3 linker or a EAAAKEAAAKEAAAK (SEQ ID NO:124) linker. Other combinations will be apparent to persons skilled in the art.
  • In some embodiments, the p97-trastuzumab fusion protein comprises, consists, or consists essentially of a trastuzumab light chain sequence fused to the C-terminus of a p97 sequence. In particular embodiments, the p97 sequence is human soluble p97, for example, comprising or consisting of SEQ ID NO:2, or a variant/fragment thereof. In some embodiments, the p97 sequence comprises or consists of SEQ ID NO:14, or a variant/fragment thereof. In some embodiments, the trastuzumab light chain sequence comprises or consists of SEQ ID NO:36 or 123, or a variant/fragment thereof. In specific embodiments, the p97-trastuzumab fusion protein comprises, consists, or consists essentially of SEQ ID NO:116-121 (trastuzumab light chain fused to the C-terminus of p97p), or a variant/fragment thereof. Optionally, the fusion protein comprises a peptide linker in between the p97 and trastuzumab sequences, as illustrated, for example, in SEQ ID NOs:117-118 and 120-121. In specific embodiments, the linker is a (GGGGS)2 or (GGGGS)3 linker or a EAAAKEAAAKEAAAK (SEQ ID NO:124) linker. Other combinations will be apparent to persons skilled in the art.
  • Also included are p97-antibody fusion proteins, comprising one or more p97-trastuzumab heavy or light chain fusion proteins described herein. In particular embodiments, the p97-antibody fusion protein comprises two (non-fusion) trastuzumab light chain sequences, and one or two p97-trastuzumab heavy chain fusion proteins described herein, where the one or two p97-trastuzumab heavy chain fusion protein(s) comprise a trastuzumab heavy chain sequence fused to the N-terminus of a p97 sequence and an optional linker in between. In some embodiments, the p97-antibody fusion comprises two p97-trastuzumab heavy chain fusion proteins (see, e.g., FIGS. 1A and 1E), including homodimeric antibody fusions that comprise the same p97-trastuzumab heavy chain fusion proteins. In some embodiments, the p97-antibody fusion comprises one p97-trastuzumab heavy chain fusion protein (see, e.g., FIG. 1B).
  • In some embodiments, the p97-antibody fusion protein comprises one or two trastuzumab light chain sequences, one trastuzumab heavy chain sequence, and one p97-trastuzumab heavy chain fusion protein described herein, where 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. In some embodiments, the p97-antibody fusion protein comprises one trastuzumab light chain sequence, one trastuzumab heavy chain sequence, and one p97-trastuzumab heavy chain fusion protein that has a truncated trastuzumab heavy chain (see, e.g., FIG. 1C).
  • In some embodiments, the p97-antibody fusion protein comprises two trastuzumab light chain sequences, and two p97-trastuzumab heavy chain fusion proteins described herein, where the p97-trastuzumab heavy chain fusion proteins comprise a trastuzumab heavy chain fused to the C-terminus of a p97 sequence and an optional linker in between (see, e.g., FIG. 1D).
  • In some embodiments, the p97-antibody fusion protein comprises two p97-trastuzumab light chain fusion proteins described herein, and two p97-trastuzumab heavy chain fusion proteins described herein, where the p97-trastuzumab light chain fusion proteins comprise a trastuzumab light chain fused to the N-terminus of a p97 sequence and an optional linker in between, and where the p97-trastuzumab heavy chain fusion proteins comprise a trastuzumab heavy chain fused to the N-terminus of a p97 sequence and an optional linker in between (see, e.g., FIG. 1F).
  • In some embodiments, the p97-antibody fusion protein comprises one or two p97-trastuzumab light chain fusion proteins described herein, and two trastuzumab heavy chain sequences, where the one or two p97-trastuzumab light chain fusion proteins comprise a trastuzumab light chain fused to the N-terminus of a p97 sequence and an optional linker in between. In some embodiments, the p97-antibody fusion protein comprises two p97-trastuzumab light chain fusion proteins described herein (see, e.g., FIG. 1G).
  • In specific embodiments, the p97-antibody fusion comprises one or two sets of heavy and light chains selected from one or more of the following:
  • 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;
  • g) the heavy chain of SEQ ID NO:94 and the light chain of SEQ ID NO:95;
  • including fragments/variants thereof. In some embodiments, the p97-antibody fusion 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). In particular embodiments, the p97-antibody fusion is a heterodimer that comprises a first set of sets of heavy and light chains selected from a)-g) above, and a second set of heavy and light chains composed of any combination of the p97-trastuzumab heavy or light chains described herein (for example, a)-g) above; SEQ ID NOS:37-46, 96-109, and 110-121), and/or any of the trastuzumab (non-fusion) heavy and light chains (for example, SEQ ID NOs: 29-35 or 122 (heavy chains) and 36 or 123 (light chains)).
  • Non-limiting examples of p97-antibody fusions are illustrated in FIGS. 1A-1G. FIG. 1A illustrates an antibody fusion composed of two p97-trastuzumab heavy chain fusion proteins, and two (non-fusion) trastuzumab light chains. FIG. 1B illustrates an antibody fusion composed of one p97-trastuzumab heavy chain fusion protein, one (non-fusion) trastuzumab heavy chain, and two (non-fusion) trastuzumab light chains. FIG. 1C illustrates an antibody fusion protein composed of one p97-trastuzumab heavy chain fusion protein (having a truncated trastuzumab heavy chain), one (non-fusion) trastuzumab light chain, and one (non-fusion) trastuzumab heavy chain. FIGS. 1D and 1E illustrate antibody fusions composed of two p97-trastuzumab heavy chain fusion proteins and two (non-fusion) trastuzumab light chains. FIG. 1F illustrates an antibody fusion composed of two p97-trastuzumab light chain fusion proteins and two 97-trastuzumab heavy chain fusion proteins. FIG. 1G illustrates an antibody fusion composed of two p97-trastuzumab light chain fusion proteins and two (non-fusion) trastuzumab heavy chain sequences. In any of the antibody fusions described herein, the first set of light/heavy chains can be the same as or different from the second set of light/heavy chains.
  • In some embodiments, the p97-antibody fusion is a homodimer, for example, which is composed of two identical sets of heavy and/or light chains, at least one of which is a p97-trastuzumab fusion protein. In some embodiments, the p97-antibody fusion is a heterodimer, for example, which is composed of a first set of heavy and/or light chains and a second set of heavy/light chains, where the first set comprises at least one heavy and/or light chain p97-trastuzumab fusion protein and the second set comprises only trastuzumab heavy and/or light chain sequences.
  • Other combinations will be apparent to persons skilled in the art. Thus, any of the p97 sequences described herein can be combined with any of the trastuzumab sequences described herein, to generate a desired p97-trastuzumab light chain or heavy chain fusion protein, and any such fusion proteins can be combined with the same or different fusion protein(s) or with any of the trastuzumab heavy chain or light chain sequences to generate a desired antibody fusion.
  • In certain embodiments, the p97-antibody fusion protein specifically binds to the human HER2/neu receptor. In specific embodiments, the p97-antibody fusion protein specifically binds to domain IV of the extracellular segment of the human HER2/neu receptor (see Cho et al., Nature. 421:756-760, 2003). In particular embodiments, the p97-antibody fusion protein is an HER2/neu receptor antagonist.
  • The functional properties of the fusion proteins and antibody fusions described herein may be assessed using a variety of methods known to the skilled person, including, e.g., affinity/binding assays (for example, surface plasmon resonance, competitive inhibition assays); cytotoxicity assays, cell viability assays, cell proliferation or differentiation assays, cancer cell and/or tumor growth inhibition using in vitro or in vivo models. For instance, the fusion proteins described herein may be tested for effects on receptor internalization, in vitro and in vivo efficacy, etc., including the rate of transport across the blood-brain barrier. Such assays may be performed using well-established protocols known to the skilled person (see e.g., Current Protocols in Molecular Biology (Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., NY, N.Y.); Current Protocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober 2001 John Wiley & Sons, NY, N.Y.); or commercially available kits.
  • Variant Sequences.
  • Certain embodiments include variants of the reference polypeptide and polynucleotide sequences described herein, whether described by name or by reference to a sequence identifier, including p97 sequences and trastuzumab sequences (see, e.g., the Sequence Listing). The wild-type or most prevalent sequences of these polypeptides are known in the art, and can be used as a comparison for the variants and fragments described herein.
  • A “variant” sequence, as the term is used herein, refers to a polypeptide or polynucleotide sequence that differs from a reference sequence disclosed herein by one or more substitutions, deletions (e.g., truncations), additions, and/or insertions. Certain variants thus include fragments of a reference sequence described herein. Variant polypeptides are biologically active, that is, they continue to possess the enzymatic or binding activity of a reference polypeptide. Such variants may result from, for example, genetic polymorphism and/or from human manipulation.
  • In many instances, a biologically active variant will contain one or more conservative substitutions. A “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. As described above, modifications may be made in the structure of the polynucleotides and polypeptides of the present invention and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, variant or portion of a polypeptide of the invention, one skilled in the art will typically change one or more of the codons of the encoding DNA sequence according to Table A below.
  • TABLE A
    Amino Acids Codons
    Alanine Ala A GCA GCC GCG GCU 
    Cysteine Cys C UGC UGU 
    Aspartic acid Asp D GAC GAU 
    Glutamic acid Glu E GAA GAG 
    Phenylalanine Phe F UUC UUU 
    Glycine Gly G GGA GGC GGG GGU 
    Histidine His H CAC CAU 
    Isoleucine Ile I AUA AUC AUU 
    Lysine Lys K AAA AAG 
    Leucine Leu L UUA UUG CUA CUC CUG CUU
    Methionine Met M AUG 
    Asparagine Asn N AAC AAU 
    Proline Pro P CCA CCC CCG CCU 
    Glutamine Gln Q CAA CAG 
    Arginine Arg R AGA AGG CGA CGC CGG CGU
    Serine Ser S AGC AGU UCA UCC UCG UCU
    Threonine Thr T ACA ACC ACG ACU 
    Valine Val V GUA GUC GUG GUU 
    Tryptophan Trp W UGG 
    Tyrosine Tyr Y UAC UAU 
  • For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their utility.
  • In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte & Doolittle, 1982). These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5). It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
  • It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101 (specifically incorporated herein by reference in its entirety), states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
  • As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
  • A variant may also, or alternatively, contain non-conservative changes. In a preferred embodiment, variant polypeptides differ from a native or reference sequence by substitution, deletion or addition of fewer than about 10, 9, 8, 7, 6, 5, 4, 3, 2 amino acids, or even 1 amino acid. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure, enzymatic activity, and/or hydropathic nature of the polypeptide.
  • In certain embodiments, a polypeptide sequence is about, at least about, or up to about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700. 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800. 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 or more contiguous amino acids in length, including all integers in between, and which may comprise all or a portion of a reference sequence (see, e.g., Sequence Listing).
  • In other specific embodiments, a polypeptide sequence consists of about or no more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800. 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 or more contiguous amino acids, including all integers in between, and which may comprise all or a portion of a reference sequence (see, e.g., Sequence Listing).
  • In still other specific embodiments, a polypeptide sequence is about 10-1000, 10-900, 10-800, 10-700, 10-600, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 10-40, 10-30, 10-20, 20-1000, 20-900, 20-800, 20-700, 20-600, 20-500, 20-400, 20-300, 20-200, 20-100, 20-50, 20-40, 20-30, 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, or 200-300 contiguous amino acids, including all ranges in between, and comprises all or a portion of a reference sequence. In certain embodiments, the C-terminal or N-terminal region of any reference polypeptide may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 or more amino acids, or by about 10-50, 20-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800 or more amino acids, including all integers and ranges in between (e.g., 101, 102, 103, 104, 105), so long as the truncated polypeptide retains the binding properties and/or activity of the reference polypeptide. Typically, the biologically-active fragment has no less than about 1%, about 5%, about 10%, about 25%, or about 50% of an activity of the biologically-active reference polypeptide from which it is derived.
  • In general, variants will display at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity or sequence identity or sequence homology to a reference polypeptide sequence. Moreover, sequences differing from the native or parent sequences by the addition (e.g., C-terminal addition, N-terminal addition, both), deletion, truncation, insertion, or substitution (e.g., conservative substitution) of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids (including all integers and ranges in between) but which retain the properties or activities of a parent or reference polypeptide sequence are contemplated.
  • In some embodiments, variant polypeptides differ from reference sequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s). In other embodiments, variant polypeptides differ from a reference sequence by at least 1% but less than 20%, 15%, 10% or 5% of the residues. (If this comparison requires alignment, the sequences should be aligned for maximum similarity. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.)
  • Calculations of sequence similarity or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In certain embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch, (J. Mol. Biol. 48: 444-453, 1970) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (Cabios. 4:11-17, 1989) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al., (1990, J. Mol. Biol, 215: 403-10). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (Nucleic Acids Res. 25: 3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
  • In one embodiment, as noted above, polynucleotides and/or polypeptides can be evaluated using a BLAST alignment tool. A local alignment consists simply of a pair of sequence segments, one from each of the sequences being compared. A modification of Smith-Waterman or Sellers algorithms will find all segment pairs whose scores cannot be improved by extension or trimming, called high-scoring segment pairs (HSPs). The results of the BLAST alignments include statistical measures to indicate the likelihood that the BLAST score can be expected from chance alone.
  • The raw score, S, is calculated from the number of gaps and substitutions associated with each aligned sequence wherein higher similarity scores indicate a more significant alignment. Substitution scores are given by a look-up table (see PAM, BLOSUM).
  • Gap scores are typically calculated as the sum of G, the gap opening penalty and L, the gap extension penalty. For a gap of length n, the gap cost would be G+Ln. The choice of gap costs, G and L is empirical, but it is customary to choose a high value for G (10-15), e.g., 11, and a low value for L (1-2) e.g., 1.
  • The bit score, S′, is derived from the raw alignment score S in which the statistical properties of the scoring system used have been taken into account. Bit scores are normalized with respect to the scoring system, therefore they can be used to compare alignment scores from different searches. The terms “bit score” and “similarity score” are used interchangeably. The bit score gives an indication of how good the alignment is; the higher the score, the better the alignment.
  • The E-Value, or expected value, describes the likelihood that a sequence with a similar score will occur in the database by chance. It is a prediction of the number of different alignments with scores equivalent to or better than S that are expected to occur in a database search by chance. The smaller the E-Value, the more significant the alignment. For example, an alignment having an E value of e−117 means that a sequence with a similar score is very unlikely to occur simply by chance. Additionally, the expected score for aligning a random pair of amino acids is required to be negative, otherwise long alignments would tend to have high score independently of whether the segments aligned were related. Additionally, the BLAST algorithm uses an appropriate substitution matrix, nucleotide or amino acid and for gapped alignments uses gap creation and extension penalties. For example, BLAST alignment and comparison of polypeptide sequences are typically done using the BLOSUM62 matrix, a gap existence penalty of 11 and a gap extension penalty of 1.
  • In one embodiment, sequence similarity scores are reported from BLAST analyses done using the BLOSUM62 matrix, a gap existence penalty of 11 and a gap extension penalty of 1.
  • In a particular embodiment, sequence identity/similarity scores provided herein refer to the value obtained using GAP Version 10 (GCG, Accelrys, San Diego, Calif.) using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, PNAS USA. 89:10915-10919, 1992). GAP uses the algorithm of Needleman and Wunsch (J Mol Biol. 48:443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.
  • In one particular embodiment, the variant polypeptide comprises an amino acid sequence that can be optimally aligned with a reference polypeptide sequence (see, e.g., Sequence Listing) to generate a BLAST bit scores or sequence similarity scores of at least about 50, 60, 70, 80, 90, 100, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, or more, including all integers and ranges in between, wherein the BLAST alignment used the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1.
  • As noted above, a reference polypeptide may be altered in various ways including amino acid substitutions, deletions, truncations, additions, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (PNAS USA. 82: 488-492, 1985); Kunkel et al., (Methods in Enzymol. 154: 367-382, 1987), U.S. Pat. No. 4,873,192, Watson, J. D. et al., (“Molecular Biology of the Gene,” Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.).
  • Methods for screening gene products of combinatorial libraries made by such modifications, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of reference polypeptides. As one example, recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify polypeptide variants (Arkin and Yourvan, PNAS USA 89: 7811-7815, 1992; Delgrave et al., Protein Engineering. 6: 327-331, 1993).
  • Polynucleotides, Host Cells, and Methods of Production.
  • Certain embodiments relate to polynucleotides that encode the fusion proteins and antibody fusions described herein, and vectors that comprise such polynucleotides, for example, where the polynucleotides are operably linked to one or more regulatory elements. Also included are recombinant host cells that comprise such polynucleotides, vectors, fusion proteins, and antibody fusions, and methods of recombinant production of the foregoing.
  • Fusion proteins and antibody fusions may be prepared using standard techniques.
  • Preferably, however, a fusion protein is expressed as a recombinant protein in an expression system, as described herein and known in the art. Fusion proteins can contain one or multiple copies of a p97 sequence and one or multiple copies of a trastuzumab sequence, present in any desired arrangement.
  • Polynucleotides and fusion polynucleotides can contain one or multiple copies of a nucleic acid encoding a p97 polypeptide sequence, and/or may contain one or multiple copies of a nucleic acid encoding a trastuzumab sequence.
  • For fusion proteins, DNA sequences encoding the p97 polypeptide sequence, the trastuzumab sequence of interest, and optionally a peptide linker components may be assembled separately, and then ligated into an appropriate expression vector. The 3′ end of the DNA sequence encoding one polypeptide component can be ligated, with or without a peptide linker, to the 5′ end of a DNA sequence encoding the other polypeptide component(s) so that the reading frames of the sequences are in frame. The ligated DNA sequences are operably linked to suitable transcriptional and/or translational regulatory elements. The regulatory elements responsible for expression of DNA are usually located only 5′ to the DNA sequence encoding the first polypeptides. Similarly, stop codons required to end translation and transcription termination signals are only present 3′ to the DNA sequence encoding the most C-terminal polypeptide. This permits translation into a single fusion polypeptide that retains the biological activity of both component polypeptides.
  • Similar techniques, mainly the arrangement of regulatory elements such as promoters, stop codons, and transcription termination signals, can be applied to the recombinant production of non-fusion proteins, for instance, non-fusion trastuzumab sequences for the production of antibodies that comprise a fusion protein described herein.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992, or subsequent updates thereto.
  • As will be understood by those of skill in the art, it may be advantageous in some instances to produce polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence. Such polynucleotides are commonly referred to as “codon-optimized.” Any of the polynucleotides described herein may be utilized in a codon-optimized form. In certain embodiments, a polynucleotide can be codon optimized for use in specific bacteria such as E. coli or yeast such as S. cerevisiae (see, e.g., Burgess-Brown et al., Protein Expr Purif. 59:94-102, 2008).
  • Exemplary polynucleotide sequences are provided in Table 5 below.
  • TABLE 5
    Exemplary polynucleotide sequences
    SEQ ID
    Name Sequence NO:
    TZM HC- Heavy Chain 125
    MTf ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC
    GGAGAGGTGCAGCTGGTGGAGAGCGGCGGAGGCCTCGTGCAGCCCGGCGGATCTCTG
    CGGCTGAGCTGCGCCGCTAGCGGCTTCAACATCAAGGACACCTACATCCACTGGGTG
    CGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCCGGATCTACCCCACCAACGGC
    TACACCCGCTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCGCCGACACCTCC
    AAGAACACCGCCTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTAC
    TACTGCAGCCGGTGGGGCGGCGACGGATTCTACGCCATGGACTACTGGGGACAGGGC
    ACCCTGGTGACCGTGAGCAGCGCCTCTACCAAGGGCCCCAGCGTGTTCCCTCTGGCC
    CCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGAC
    TACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTG
    CACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTG
    ACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAG
    CCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCAC
    ACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTC
    CCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTG
    GTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGC
    GTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTAC
    CGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTAC
    AAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAG
    GCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAG
    CTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGAC
    ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCT
    CCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAG
    TCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCAC
    AACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGAAAGGGTGGCGGAGGATCT
    GGCGGAGGCGGATCCGGCATGGAAGTGCGTTGGTGCGCCACCTCTGACCCCGAGCAG
    CACAAGTGCGGCAACATGTCCGAGGCCTTCAGAGAGGCCGGCATCCAGCCTTCTCTG
    CTGTGTGTGCGGGGCACCTCTGCCGACCATTGCGTGCAGCTGATCGCCGCCCAGGAA
    GCCGACGCTATCACACTGGATGGCGGCGCTATCTACGAGGCTGGCAAAGAGCACGGC
    CTGAAGCCCGTCGTGGGCGAGGTGTACGATCAGGAAGTGGGCACCTCCTACTACGCC
    GTGGCTGTCGTGCGGAGATCCTCCCACGTGACCATCGACACCCTGAAGGGCGTGAAG
    TCCTGCCACACCGGCATCAACAGAACCGTGGGCTGGAACGTGCCCGTGGGCTACCTG
    GTGGAATCCGGCAGACTGTCCGTGATGGGCTGCGACGTGCTGAAGGCCGTGTCCGAT
    TACTTCGGCGGCTCTTGTGTGCCTGGCGCTGGCGAGACATCCTACTCCGAGTCCCTG
    TGCAGACTGTGCAGGGGCGACTCTTCTGGCGAGGGCGTGTGCGACAAGTCCCCTCTG
    GAACGGTACTACGACTACTCCGGCGCCTTCAGATGCCTGGCTGAAGGTGCTGGCGAC
    GTGGCCTTCGTGAAGCACTCCACCGTGCTGGAAAACACCGACGGCAAGACCCTGCCT
    TCTTGGGGCCAGGCACTGCTGTCCCAGGACTTCGAGCTGCTGTGCCGGGATGGCTCC
    AGAGCCGATGTGACAGAGTGGCGGCAGTGCCACCTGGCCAGAGTGCCTGCCCATGCT
    GTGGTCGTGCGCGCCGATACAGATGGCGGCCTGATCTTCCGGCTGCTGAACGAGGGC
    CAGCGGCTGTTCTCTCACGAGGGCTCCAGCTTCCAGATGTTCTCCAGCGAGGCCTAC
    GGCCAGAAGGACCTGCTGTTCAAGGACTCCACCTCCGAGCTGGTGCCTATCGCCACC
    CAGACCTATGAGGCTTGGCTGGGCCACGAGTACCTGCACGCTATGAAGGGACTGCTG
    TGCGACCCCAACCGGCTGCCTCCTTATCTGAGGTGGTGCGTGCTGTCCACCCCCGAG
    ATCCAGAAATGCGGCGATATGGCCGTGGCCTTTCGGCGGCAGAGACTGAAGCCTGAG
    ATCCAGTGCGTGTCTGCCAAGAGCCCTCAGCACTGCATGGAACGGATCCAGGCCGAA
    CAGGTGGACGCCGTGACACTGTCCGGCGAGGATATCTACACCGCCGGAAAGACCTAC
    GGCCTGGTGCCAGCTGCTGGCGAGCATTACGCCCCTGAGGACTCCTCCAACAGCTAC
    TACGTGGTGGCAGTCGTGCGCCGGGACTCCTCTCACGCCTTTACCCTGGATGAGCTG
    CGGGGCAAGAGAAGCTGTCACGCCGGCTTTGGAAGCCCTGCCGGATGGGATGTGCCT
    GTGGGCGCTCTGATCCAGCGGGGCTTCATCAGACCCAAGGACTGTGATGTGCTGACC
    GCCGTGTCTGAGTTCTTCAACGCCTCCTGTGTGCCCGTGAACAACCCCAAGAACTAC
    CCCTCCAGCCTGTGCGCCCTGTGTGTGGGAGATGAGCAGGGCCGGAACAAATGCGTG
    GGCAACTCCCAGGAAAGATATTACGGCTACAGAGGCGCCTTCCGGTGTCTGGTGGAA
    AACGCCGGGGATGTGGCTTTTGTGCGGCACACCACCGTGTTCGACAACACCAATGGC
    CACAACTCCGAGCCTTGGGCCGCTGAGCTGAGATCCGAGGATTACGAACTGCTGTGT
    CCCAACGGCGCCAGGGCTGAGGTGTCCCAGTTTGCCGCCTGTAACCTGGCCCAGATC
    CCTCCCCACGCTGTGATGGTGCGACCCGACACCAACATCTTCACCGTGTACGGCCTG
    CTGGACAAGGCCCAGGATCTGTTCGGCGACGACCACAACAAGAACGGGTTCAAGATG
    TTCGACTCCAGCAACTACCACGGACAGGATCTGCTGTTTAAAGATGCCACCGTGCGG
    GCCGTGCCAGTGGGCGAAAAGACCACCTACAGAGGATGGCTGGGACTGGACTACGTG
    GCCGCCCTGGAAGGCATGTCCTCCCAGCAGTGTTCCGGCTAG
    Light Chain 126
    ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC
    GGAGACATCCAGATGACCCAGAGCCCTTCCAGCCTGAGCGCCAGCGTGGGCGACCGG
    GTGACCATCACCTGCCGCGCTAGCCAGGACGTGAACACCGCCGTGGCCTGGTACCAG
    CAGAAGCCCGGAAAGGCCCCCAAGCTGCTGATCTACTCTGCTAGCTTCCTGTACAGC
    GGCGTGCCCAGCCGGTTCAGCGGATCTCGCAGCGGCACCGACTTCACCCTGACCATC
    AGCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACG
    CCTCCCACCTTCGGACAGGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCGCCCCC
    AGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTG
    GTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGAC
    AACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGAC
    AGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCAC
    AAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGC
    TTCAACCGGGGCGAGTGCTAA
    MTfp Heavy Chain 127
    NH-TZM ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC
    GGAGACTCCTCTCACGCCTTCACCCTGGACGAGCTGCGGTACGGTGGCGGAGGATCT
    GGCGGAGGCGGATCCGAGGTGCAGCTGGTGGAGAGCGGCGGAGGCCTCGTGCAGCCC
    GGCGGATCTCTGCGGCTGAGCTGCGCCGCTAGCGGCTTCAACATCAAGGACACCTAC
    ATCCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCCGGATCTAC
    CCCACCAACGGCTACACCCGCTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGC
    GCCGACACCTCCAAGAACACCGCCTACCTGCAGATGAACAGCCTGCGCGCCGAGGAC
    ACCGCCGTGTACTACTGCAGCCGGTGGGGCGGCGACGGATTCTACGCCATGGACTAC
    TGGGGACAGGGCACCCTGGTGACCGTGAGCAGCGCCTCTACCAAGGGCCCCAGCGTG
    TTCCCTCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGC
    CTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTG
    ACCAGCGGAGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTG
    AGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAAC
    GTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGC
    GACAAGACCCACACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGC
    GTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAG
    GTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGG
    TACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTAC
    AACTCCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAAC
    GGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAG
    ACCATCAGCAAGGCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCC
    AGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTC
    TACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTAC
    AAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTG
    ACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCAC
    GAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGAAAGTAA
    Light Chain 128
    ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC
    GGAGACATCCAGATGACCCAGAGCCCTTCCAGCCTGAGCGCCAGCGTGGGCGACCGG
    GTGACCATCACCTGCCGCGCTAGCCAGGACGTGAACACCGCCGTGGCCTGGTACCAG
    CAGAAGCCCGGAAAGGCCCCCAAGCTGCTGATCTACTCTGCTAGCTTCCTGTACAGC
    GGCGTGCCCAGCCGGTTCAGCGGATCTCGCAGCGGCACCGACTTCACCCTGACCATC
    AGCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACG
    CCTCCCACCTTCGGACAGGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCGCCCCC
    AGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTG
    GTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGAC
    AACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGAC
    AGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCAC
    AAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGC
    TTCAACCGGGGCGAGTGCTAA
    TZM HC- Heavy Chain 129
    MTfp ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC
    GGAGAGGTGCAGCTGGTGGAGAGCGGCGGAGGCCTCGTGCAGCCCGGCGGATCTCTG
    CGGCTGAGCTGCGCCGCTAGCGGCTTCAACATCAAGGACACCTACATCCACTGGGTG
    CGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCCGGATCTACCCCACCAACGGC
    TACACCCGCTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCGCCGACACCTCC
    AAGAACACCGCCTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTAC
    TACTGCAGCCGGTGGGGCGGCGACGGATTCTACGCCATGGACTACTGGGGACAGGGC
    ACCCTGGTGACCGTGAGCAGCGCCTCTACCAAGGGCCCCAGCGTGTTCCCTCTGGCC
    CCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGAC
    TACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTG
    CACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTG
    ACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAG
    CCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCAC
    ACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTC
    CCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTG
    GTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGC
    GTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTAC
    CGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTAC
    AAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAG
    GCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAG
    CTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGAC
    ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCT
    CCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAG
    TCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCAC
    AACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGAAAGGGTGGCGGAGGATCT
    GGCGGAGGCGGATCCGACTCCTCTCACGCCTTCACCCTGGACGAGCTGCGGTAG
    Light Chain 130
    ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC
    GGAGACATCCAGATGACCCAGAGCCCTTCCAGCCTGAGCGCCAGCGTGGGCGACCGG
    GTGACCATCACCTGCCGCGCTAGCCAGGACGTGAACACCGCCGTGGCCTGGTACCAG
    CAGAAGCCCGGAAAGGCCCCCAAGCTGCTGATCTACTCTGCTAGCTTCCTGTACAGC
    GGCGTGCCCAGCCGGTTCAGCGGATCTCGCAGCGGCACCGACTTCACCCTGACCATC
    AGCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACG
    CCTCCCACCTTCGGACAGGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCGCCCCC
    AGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTG
    GTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGAC
    AACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGAC
    AGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCAC
    AAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGC
    TTCAACCGGGGCGAGTGCTAA
    TZM/MTf Heavy Chain 131
    ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC
    GGAGAGGTGCAGCTGGTGGAGAGCGGCGGAGGCCTCGTGCAGCCCGGCGGATCTCTG
    CGGCTGAGCTGCGCCGCTAGCGGCTTCAACATCAAGGACACCTACATCCACTGGGTG
    CGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCCGGATCTACCCCACCAACGGC
    TACACCCGCTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCGCCGACACCTCC
    AAGAACACCGCCTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTAC
    TACTGCAGCCGGTGGGGCGGCGACGGATTCTACGCCATGGACTACTGGGGACAGGGC
    ACCCTGGTGACCGTGAGCAGCGCCTCTACCAAGGGCCCCAGCGTGTTCCCTCTGGCC
    CCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGAC
    TACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTG
    CACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTG
    ACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAG
    CCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCAC
    ACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTC
    CCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTG
    GTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGC
    GTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTAC
    CGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTAC
    AAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAG
    GCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAG
    CTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGAC
    ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCT
    CCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAG
    TCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCAC
    AACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGAAAGGGAGGTGGCGGTTCT
    GGTGGCGGAGGATCTGGCGGAGGCGGATCCGGCATGGAAGTGCGTTGGTGCGCCACC
    TCTGACCCCGAGCAGCACAAGTGCGGCAACATGTCCGAGGCCTTCAGAGAGGCCGGC
    ATCCAGCCTTCTCTGCTGTGTGTGCGGGGCACCTCTGCCGACCATTGCGTGCAGCTG
    ATCGCCGCCCAGGAAGCCGACGCTATCACACTGGATGGCGGCGCTATCTACGAGGCT
    GGCAAAGAGCACGGCCTGAAGCCCGTCGTGGGCGAGGTGTACGATCAGGAAGTGGGC
    ACCTCCTACTACGCCGTGGCTGTCGTGCGGAGATCCTCCCACGTGACCATCGACACC
    CTGAAGGGCGTGAAGTCCTGCCACACCGGCATCAACAGAACCGTGGGCTGGAACGTG
    CCCGTGGGCTACCTGGTGGAATCCGGCAGACTGTCCGTGATGGGCTGCGACGTGCTG
    AAGGCCGTGTCCGATTACTTCGGCGGCTCTTGTGTGCCTGGCGCTGGCGAGACATCC
    TACTCCGAGTCCCTGTGCAGACTGTGCAGGGGCGACTCTTCTGGCGAGGGCGTGTGC
    GACAAGTCCCCTCTGGAACGGTACTACGACTACTCCGGCGCCTTCAGATGCCTGGCT
    GAAGGTGCTGGCGACGTGGCCTTCGTGAAGCACTCCACCGTGCTGGAAAACACCGAC
    GGCAAGACCCTGCCTTCTTGGGGCCAGGCACTGCTGTCCCAGGACTTCGAGCTGCTG
    TGCCGGGATGGCTCCAGAGCCGATGTGACAGAGTGGCGGCAGTGCCACCTGGCCAGA
    GTGCCTGCTCATGCTGTGGTCGTGCGCGCCGATACAGATGGCGGCCTGATCTTCCGG
    CTGCTGAACGAGGGCCAGCGGCTGTTCTCTCACGAGGGCTCCAGCTTCCAGATGTTC
    TCCAGCGAGGCCTACGGCCAGAAGGACCTGCTGTTCAAGGACTCCACCTCCGAGCTG
    GTGCCTATCGCCACCCAGACCTATGAGGCTTGGCTGGGCCACGAGTACCTGCACGCT
    ATGAAGGGACTGCTGTGCGACCCCAACCGGCTGCCTCCTTATCTGAGGTGGTGCGTG
    CTGTCCACCCCCGAGATCCAGAAATGCGGCGATATGGCCGTGGCCTTTCGGCGGCAG
    AGACTGAAGCCTGAGATCCAGTGCGTGTCCGCCAAGAGCCCTCAGCACTGCATGGAA
    CGGATCCAGGCCGAACAGGTGGACGCCGTGACACTGTCCGGCGAGGATATCTACACC
    GCCGGAAAGACCTACGGCCTGGTGCCAGCTGCTGGCGAGCATTACGCCCCTGAGGAC
    TCCTCCAACAGCTACTACGTGGTGGCAGTCGTGCGCCGGGACTCCTCTCACGCCTTT
    ACCCTGGATGAGCTGCGGGGCAAGAGAAGCTGTCACGCCGGCTTTGGAAGCCCTGCC
    GGATGGGATGTGCCTGTGGGCGCTCTGATCCAGCGGGGCTTCATCAGACCCAAGGAC
    TGTGATGTGCTGACCGCCGTGTCTGAGTTCTTCAACGCCTCCTGTGTGCCCGTGAAC
    AACCCCAAGAACTACCCCTCCAGCCTGTGCGCCCTGTGTGTGGGAGATGAGCAGGGC
    CGGAACAAATGCGTGGGCAACTCCCAGGAAAGATATTACGGCTACAGAGGCGCCTTC
    CGGTGTCTGGTGGAAAACGCCGGGGATGTGGCTTTTGTGCGGCACACCACCGTGTTC
    GACAACACCAATGGCCACAACTCCGAGCCTTGGGCCGCTGAGCTGAGATCCGAGGAT
    TACGAACTGCTGTGTCCCAACGGCGCCAGGGCTGAGGTGTCCCAGTTTGCCGCCTGT
    AACCTGGCCCAGATCCCTCCCCACGCTGTGATGGTGCGACCCGACACCAACATCTTC
    ACCGTGTACGGCCTGCTGGACAAGGCCCAGGATCTGTTCGGCGACGACCACAACAAG
    AACGGGTTCAAGATGTTCGACTCCAGCAACTACCACGGACAGGATCTGCTGTTTAAA
    GATGCCACCGTGCGGGCCGTGCCAGTGGGCGAAAAGACCACCTACAGAGGATGGCTG
    GGACTGGACTACGTGGCCGCCCTGGAAGGCATGTCCTCCCAGCAGTGTTCCGGCTAG
    Light Chain 132
    ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC
    GGAGACATCCAGATGACCCAGAGCCCTTCCAGCCTGAGCGCCAGCGTGGGCGACCGG
    GTGACCATCACCTGCCGCGCTAGCCAGGACGTGAACACCGCCGTGGCCTGGTACCAG
    CAGAAGCCCGGAAAGGCCCCCAAGCTGCTGATCTACTCTGCTAGCTTCCTGTACAGC
    GGCGTGCCCAGCCGGTTCAGCGGATCTCGCAGCGGCACCGACTTCACCCTGACCATC
    AGCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACG
    CCTCCCACCTTCGGACAGGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCGCCCCC
    AGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTG
    GTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGAC
    AACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGAC
    AGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCAC
    AAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGC
    TTCAACCGGGGCGAGTGCTAA
    LC- Heavy Chain 133
    MTfp/Fc- ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC
    MTfp GGAGAGGTGCAGCTGGTGGAGAGCGGCGGAGGCCTCGTGCAGCCCGGCGGATCTCTG
    CGGCTGAGCTGCGCCGCTAGCGGCTTCAACATCAAGGACACCTACATCCACTGGGTG
    CGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCCGGATCTACCCCACCAACGGC
    TACACCCGCTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCGCCGACACCTCC
    AAGAACACCGCCTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTAC
    TACTGCAGCCGGTGGGGCGGCGACGGATTCTACGCCATGGACTACTGGGGACAGGGC
    ACCCTGGTGACCGTGAGCAGCGCCTCTACCAAGGGCCCCAGCGTGTTCCCTCTGGCC
    CCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGAC
    TACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTG
    CACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTG
    ACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAG
    CCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCAC
    ACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTC
    CCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTG
    GTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGC
    GTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTAC
    CGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTAC
    AAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAG
    GCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAG
    CTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGAC
    ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCT
    CCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAG
    TCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCAC
    AACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGAAAGGAGGCCGCTGCTAAA
    GAGGCTGCCGCCAAAGAAGCCGCCGCTAAGGACTCCTCTCACGCCTTCACCCTGGAC
    GAGCTGCGGTACTAA
    Light Chain 134
    ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC
    GGAGACATCCAGATGACCCAGAGCCCTTCCAGCCTGAGCGCCAGCGTGGGCGACCGG
    GTGACCATCACCTGCCGCGCTAGCCAGGACGTGAACACCGCCGTGGCCTGGTACCAG
    CAGAAGCCCGGAAAGGCCCCCAAGCTGCTGATCTACTCTGCTAGCTTCCTGTACAGC
    GGCGTGCCCAGCCGGTTCAGCGGATCTCGCAGCGGCACCGACTTCACCCTGACCATC
    AGCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACG
    CCTCCCACCTTCGGACAGGGCACCAAGGTAGAGATCAAGCGGACCGTGGCCGCCCCC
    AGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTG
    GTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGAC
    AACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGAC
    AGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCAC
    AAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGC
    TTCAACCGGGGCGAGTGCGAGGCCGCTGCTAAAGAGGCTGCCGCCAAAGAAGCCGCC
    GCTAAGGACTCCTCTCACGCCTTCACCCTGGACGAGCTGCGGTACTAA
    LC/Fc- Heavy Chain 135
    MTfp ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC
    GGAGAGGTGCAGCTGGTGGAGAGCGGCGGAGGCCTCGTGCAGCCCGGCGGATCTCTG
    CGGCTGAGCTGCGCCGCTAGCGGCTTCAACATCAAGGACACCTACATCCACTGGGTG
    CGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCCGGATCTACCCCACCAACGGC
    TACACCCGCTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCGCCGACACCTCC
    AAGAACACCGCCTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTAC
    TACTGCAGCCGGTGGGGCGGCGACGGATTCTACGCCATGGACTACTGGGGACAGGGC
    ACCCTGGTGACCGTGAGCAGCGCCTCTACCAAGGGCCCCAGCGTGTTCCCTCTGGCC
    CCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGAC
    TACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTG
    CACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTG
    ACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAG
    CCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCAC
    ACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTC
    CCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTG
    GTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGC
    GTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTAC
    CGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTAC
    AAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAG
    GCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAG
    CTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGAC
    ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCT
    CCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAG
    TCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCAC
    AACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGAAAGGAGGCCGCTGCTAAA
    GAGGCTGCCGCCAAAGAAGCCGCCGCTAAGGACTCCTCTCACGCCTTCACCCTGGAC
    GAGCTGCGGTACTAA
    Light Chain 136
    ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC
    GGAGACATCCAGATGACCCAGAGCCCTTCCAGCCTGAGCGCCAGCGTGGGCGACCGG
    GTGACCATCACCTGCCGCGCTAGCCAGGACGTGAACACCGCCGTGGCCTGGTACCAG
    CAGAAGCCCGGAAAGGCCCCCAAGCTGCTGATCTACTCTGCTAGCTTCCTGTACAGC
    GGCGTGCCCAGCCGGTTCAGCGGATCTCGCAGCGGCACCGACTTCACCCTGACCATC
    AGCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACG
    CCTCCCACCTTCGGACAGGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCGCCCCC
    AGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTG
    GTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGAC
    AACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGAC
    AGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCAC
    AAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGC
    TTCAACCGGGGCGAGTGCTAA
    LC- Heavy Chain 137
    MTfp/Fc ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC
    GGAGAGGTGCAGCTGGTGGAGAGCGGCGGAGGCCTCGTGCAGCCCGGCGGATCTCTG
    CGGCTGAGCTGCGCCGCTAGCGGCTTCAACATCAAGGACACCTACATCCACTGGGTG
    CGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCCGGATCTACCCCACCAACGGC
    TACACCCGCTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCGCCGACACCTCC
    AAGAACACCGCCTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTAC
    TACTGCAGCCGGTGGGGCGGCGACGGATTCTACGCCATGGACTACTGGGGACAGGGC
    ACCCTGGTGACCGTGAGCAGCGCCTCTACCAAGGGCCCCAGCGTGTTCCCTCTGGCC
    CCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGAC
    TACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTG
    CACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTG
    ACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAG
    CCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCAC
    ACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTC
    CCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTG
    GTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGC
    GTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTAC
    CGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTAC
    AAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAG
    GCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAG
    CTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGAC
    ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCT
    CCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAG
    TCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCAC
    AACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGAAAGTAA
    Light Chain 138
    ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC
    GGAGACATCCAGATGACCCAGAGCCCTTCCAGCCTGAGCGCCAGCGTGGGCGACCGG
    GTGACCATCACCTGCCGCGCTAGCCAGGACGTGAACACCGCCGTGGCCTGGTACCAG
    CAGAAGCCCGGAAAGGCCCCCAAGCTGCTGATCTACTCTGCTAGCTTCCTGTACAGC
    GGCGTGCCCAGCCGGTTCAGCGGATCTCGCAGCGGCACCGACTTCACCCTGACCATC
    AGCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACG
    CCTCCCACCTTCGGACAGGGCACCAAGGTAGAGATCAAGCGGACCGTGGCCGCCCCC
    AGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTG
    GTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGAC
    AACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGAC
    AGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCAC
    AAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGC
    TTCAACCGGGGCGAGTGCGAGGCCGCTGCTAAAGAGGCTGCCGCCAAAGAAGCCGCC
    GCTAAGGACTCCTCTCACGCCTTCACCCTGGACGAGCTGCGGTACTAA
  • Thus, in certain embodiments, a polynucleotide that encodes a fusion protein or antibody fusion described herein, or a portion thereof, comprises one or more polynucleotide sequences from Table 5 (e.g., SEQ ID NOS:125-138), or a fragment/variant thereof.
  • In some embodiments, one or more nucleic acids or vectors encoding a subject p97 polypeptide, a trastuzumab polypeptide (e.g., light chain polypeptide, heavy chain polypeptide), and/or a p97-trastuzumab fusion protein are introduced directly into a host cell, and the cell is incubated under conditions sufficient to induce expression of the encoded polypeptide(s). Therefore, according to certain related embodiments, there is provided a recombinant host cell which comprises a polynucleotide or a fusion polynucleotide that encodes one or more fusion proteins described herein, optionally in combination with other (non-fusion) components of an antibody, and which optionally comprises additional heterologous polynucleotide sequences.
  • Expression of a fusion protein or antibody fusion in the host cell may be achieved by culturing the recombinant host cells (containing the polynucleotide(s)) under appropriate conditions. Following production by expression, the polypeptide(s), fusion proteins, and/or antibody fusions may be isolated and/or purified using any suitable technique, and then used as desired. The term “host cell” is used to refer to a cell into which has been introduced, or which is capable of having introduced into it, a nucleic acid sequence encoding one or more of the polypeptides described herein, and which further expresses or is capable of expressing a selected gene of interest, such as a gene encoding any herein described polypeptide. The term includes the progeny of the parent cell, whether or not the progeny are identical in morphology or in genetic make-up to the original parent, so long as the selected gene is present. Host cells may be chosen for certain characteristics, for instance, the expression of aminoacyl tRNA synthetase(s) that can incorporate unnatural amino acids into the polypeptide.
  • Systems for cloning and expression of a protein in a variety of different host cells are well known. Suitable host cells include mammalian cells, bacteria, yeast, and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, HEK-293 cells, NSO mouse melanoma cells and many others. Additional examples of useful mammalian host cell lines include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells sub-cloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., PNAS USA 77:4216 (1980)); and myeloma cell lines such as NSO and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 255-268. Certain preferred mammalian cell expression systems include CHO and HEK293-cell based expression systems. Mammalian expression systems can utilize attached cell lines, for example, in T-flasks, roller bottles, or cell factories, or suspension cultures, for example, in 1 L and 5 L spinners, 5 L, 14 L, 40 L, 100 L and 200 L stir tank bioreactors, or 20/50 L and 100/200 L WAVE bioreactors, among others known in the art.
  • A common, preferred bacterial host is E. coli. The expression of proteins in prokaryotic cells such as E. coli is well established in the art. For a review, see for example Pluckthun, A. Bio/Technology. 9:545-551 (1991). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for recombinant production of polypeptides (see Ref, Curr. Opinion Biotech. 4:573-576, 1993; and Trill et al., Curr. Opinion Biotech. 6:553-560, 1995). In specific embodiments, protein expression may be controlled by a T7 RNA polymerase (e.g., pET vector series). These and related embodiments may utilize the expression host strain BL21(DE3), a λDE3 lysogen of BL21 that supports T7-mediated expression and is deficient in Ion and ompT proteases for improved target protein stability. Also included are expression host strains carrying plasmids encoding tRNAs rarely used in E. coli, such as Rosetta™ (DE3) and Rosetta 2 (DE3) strains. Cell lysis and sample handling may also be improved using reagents such as Benzonase® nuclease and BugBuster® Protein Extraction Reagent. For cell culture, auto-inducing media can improve the efficiency of many expression systems, including high-throughput expression systems. Media of this type (e.g., Overnight Express™ Autoinduction System) gradually elicit protein expression through metabolic shift without the addition of artificial inducing agents such as IPTG. Particular embodiments employ hexahistidine tags (such as His•Tag® fusions), followed by immobilized metal affinity chromatography (IMAC) purification, or related techniques. In certain aspects, however, clinical grade proteins can be isolated from E. coli inclusion bodies, without or without the use of affinity tags (see, e.g., Shimp et al., Protein Expr Purif. 50:58-67, 2006). As a further example, certain embodiments may employ a cold-shock induced E. coli high-yield production system, because over-expression of proteins in Escherichia coli at low temperature improves their solubility and stability (see, e.g., Qing et al., Nature Biotechnology. 22:877-882, 2004).
  • In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, post-translational modifications such as acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing, which cleaves a “prepro” form of the protein may also be used to facilitate correct insertion, folding and/or function. Different host cells such as yeast, CHO, HeLa, MDCK, HEK293, and W138, in addition to bacterial cells, which have or even lack specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the fusion protein or antibody fusion of interest.
  • For long-term, high-yield production of recombinant proteins, stable expression is generally preferred. For example, cell lines that stably express a polynucleotide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which, successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type. Transient production, such as by transient transfection or infection, can also be employed. Exemplary mammalian expression systems that are suitable for transient production include HEK293 and CHO-based systems.
  • Host cells transformed with a polynucleotide sequence of interest may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. Certain specific embodiments utilize serum free cell expression systems. Examples include HEK293 cells and CHO cells that can grow on serum free medium (see, e.g., Rosser et al., Protein Expr. Purif. 40:237-43, 2005; and U.S. Pat. No. 6,210,922).
  • The protein(s) produced by a recombinant cell can be purified and characterized according to a variety of techniques known in the art. Exemplary systems for performing protein purification and analyzing protein purity include fast protein liquid chromatography (FPLC) (e.g., AKTA and Bio-Rad FPLC systems), high-pressure liquid chromatography (HPLC) (e.g., Beckman and Waters HPLC). Exemplary chemistries for purification include ion exchange chromatography (e.g., Q, S), size exclusion chromatography, salt gradients, affinity purification (e.g., Ni, Co, FLAG, maltose, glutathione, protein A/G), gel filtration, reverse-phase, ceramic HyperD® ion exchange chromatography, and hydrophobic interaction columns (HIC), among others known in the art. Also included are analytical methods such as SDS-PAGE (e.g., coomassie, silver stain), immunoblot, Bradford, and ELISA, which may be utilized during any step of the production or purification process, typically to measure the purity of the protein composition.
  • Also included are methods of concentrating recombinantly produced proteins, e.g., antibodies. Examples include lyophilization, which is typically employed when the solution contains few soluble components other than the protein of interest. Lyophilization is often performed after HPLC run, and can remove most or all volatile components from the mixture. Also included are ultrafiltration techniques, which typically employ one or more selective permeable membranes to concentrate a protein solution. The membrane allows water and small molecules to pass through and retains the protein; the solution can be forced against the membrane by mechanical pump, gas pressure, or centrifugation, among other techniques.
  • In certain embodiments, the fusion proteins or antibody fusion proteins have a purity of at least about 90%, as measured according to routine techniques in the art. In certain embodiments, such as diagnostic compositions or certain therapeutic compositions, the fusion proteins or antibody fusions have a purity of at least about 95%. In specific embodiments, such as therapeutic or pharmaceutical compositions, the fusion proteins or antibody fusions have a purity of at least about 97% or 98% or 99%. In other embodiments, such as when being used as reference or research reagents, fusion proteins or antibody fusions can be of lesser purity, and may have a purity of at least about 50%, 60%, 70%, or 80%. Purity can be measured overall or in relation to selected components, such as other proteins, e.g., purity on a protein basis.
  • In certain embodiments, as noted above, the compositions described here are about substantially endotoxin free, including, for example, about 95% endotoxin free, preferably about 99% endotoxin free, and more preferably about 99.99% endotoxin free. The presence of endotoxins can be detected according to routine techniques in the art, as described herein. In specific embodiments, the fusion proteins or antibody fusions are made from a eukaryotic cell such as a mammalian or human cell in substantially serum free media.
  • Methods of Use Pharmaceutical Compositions
  • Certain embodiments relate to methods of using the p97-trastuzumab fusion proteins and/or related antibody fusion proteins described herein. Examples of such methods include methods of treatment and methods of diagnosis, the latter including, for instance, the medical imaging of certain organs/tissues, such as those of the central nervous system. Specific embodiments include methods of treating and/or diagnosing disorders or conditions of the central nervous system (CNS), or disorders or conditions having a CNS component. Also included are pharmaceutical compositions comprising the p97-trastuzumab fusion proteins and/or related antibody fusion proteins described herein.
  • Accordingly, certain embodiments include methods of treating a subject in need thereof, comprising administering a p97-trastuzumab fusion protein or antibody fusion protein described herein. In particular embodiments, the methods comprise administering a p97-antibody fusion protein, which comprises one or more p97-trastuzumab fusion proteins described herein (e.g., as at least one component of the antibody or antibody-like molecule), and optionally other non-fusion antibody components (e.g., non-fusion light chain(s), non-fusion heavy chain(s)). Also included are methods of delivering such molecules to the nervous system (e.g., central nervous system tissues) of a subject, comprising administering to the subject a p97-trastuzumab fusion protein or antibody fusion described herein.
  • In some embodiments, the methods increase the rate and/or amount of delivery of the trastuzumab antibody (or antigen-binding fragment thereof) to the central nervous system tissues of the subject, relative, for example, to delivery by a composition that comprises the trastuzumab antibody (or antigen-binding fragment thereof) alone. In certain embodiments, the methods reduce the distribution of trastuzumab antibody (or antigen-binding fragment thereof) to heart tissues of the subject, relative, for example, to distribution by a composition that comprises the trastuzumab antibody (or antigen-binding fragment thereof) alone, and thereby reduces the cardiotoxicity associated with trastuzumab.
  • In some instances, a subject has a disease, disorder, or condition that is associated with the central nervous system (CNS) or that has a CNS component, where increased delivery of the trastuzumab antibody (or antigen-binding fragment thereof) across the blood brain barrier to CNS tissues relative to peripheral tissues can improve treatment, for instance, by increasing the tissue concentration of the antibody in the CNS, and/or by reducing side-effects associated with exposure of the antibody to peripheral tissues/organs.
  • Certain embodiments include the treatment of various cancers. “Cancer” relates generally to a class of diseases or conditions in which a group of cells display one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., intrusion on and destruction of adjacent tissues), and/or metastasis (i.e., spread to other locations in the body via lymph or blood). These malignant properties of cancers differentiate them from benign cancers, which are self-limited, and typically do not invade or metastasize. Included are cancers of the central nervous system (CNS), or neurological cancers, such as brain cancers.
  • In some instances, the neurological cancer is a metastatic brain cancer. Examples of cancers that can metastasize to the brain include, without limitation, breast cancers, lung cancers, genitourinary tract cancers, gastrointestinal tract cancers (e.g., colorectal cancers, pancreatic carcinomas), osteosarcomas, melanomas, head and neck cancers, prostate cancers (e.g., prostatic adenocarcinomas), and lymphomas. Certain embodiments thus include methods for treating, inhibiting or preventing metastasis of a cancer by administering to a patient a therapeutically effective amount of a fusion protein described herein (e.g., in an amount that, following administration, inhibits, prevents or delays metastasis of a cancer in a statistically significant manner, i.e., relative to an appropriate control as will be known to those skilled in the art). In particular embodiments, the subject has a cancer that is at risk for but has not yet metastasized to the central nervous system, including one or more of the above-described cancers, among others known in the art.
  • In some aspects, the subject has a cancer associated with expression of Her2/neu. In particular aspects, subject has a Her2/neu-expressing or Her2/neu-overexpressing cancer. Hence, certain embodiments include methods for the treatment of a HER2-overexpressing cancer in a subject in need thereof, comprising administering to the subject a (e.g., therapeutically-effective amount of a) p97-antibody fusion protein (described herein) or a pharmaceutical composition comprising the same. In some embodiments, the HER2-overexpressing cancer is at risk for metastasizing to the CNS of the subject. In particular embodiments, the HER2-overexpressing cancer has metastasized to the CNS of the subject. In some aspects, the antibody fusion is administered in an amount that inhibits, prevents, or delays the progression and/or metastasis of the cancer in a statistically significant manner (i.e., relative to an appropriate control as will be known to those skilled in the art). Included are amounts that inhibit, prevent, or delay the progression and/or metastasis into tissues of the CNS, and those that inhibit, prevent, or delay progression and/or metastasis within tissues of the CNS.
  • In certain aspects, the HER2-overexpressing cancer is a breast cancer, ovarian cancer, gastric cancer, or uterine cancer. In particular aspects, the HER2-overexpressing cancer is a metastatic breast cancer, metastatic ovarian cancer, metastatic gastric cancer, or metastatic or aggressive form of uterine cancer.
  • In some aspects, the HER2-overexpressing cancer is a HER2-overexpressing breast cancer, such as a HER2-overexpressing metastatic breast cancer. In certain instances, the HER2-overexpressing metastatic breast cancer is at risk for metastasizing to the CNS of the subject. In certain instances, the HER2-overexpressing metastatic breast cancer has already metastasized to the CNS of the subject. In some instances, the p97-antibody fusion is administered with paclitaxel for first-line treatment of HER2-overexpressing metastatic breast cancer. In particular instances, the p97-antibody fusion is administered as a single agent for treatment of HER2-overexpressing metastatic breast cancer in patients who have received one or more chemotherapy regimens for metastatic disease.
  • Certain embodiments include administering the p97-antibody fusion or pharmaceutical composition as part of an adjuvant treatment for HER2-overexpressing breast cancer. In some aspects, the adjuvant treatment comprises doxorubicin, cyclophosphamide, and either paclitaxel or docetaxel. In some aspects, the adjuvant treatment comprises docetaxel and carboplatin. Certain aspects include administering the p97-antibody fusion or pharmaceutical composition as a single agent following multi-modality anthracycline based therapy.
  • In some aspects, the HER2-overexpressing cancer is a HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma. In some instances, the HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma is at risk for metastasizing to the CNS of the subject. In certain instances, the HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma has already metastasized to the CNS of the subject. In some instances, the p97-antibody fusion is administered in combination with cisplatin and capecitabine or 5-fluorouracil, optionally where the subject or patient has not received prior treatment for metastatic disease.
  • In certain aspects, the HER2-overexpressing uterine cancer is a HER2-overexpressing uterine serous carcinoma (USC) (see, e.g., Santin et al., Inti Gynaecol Obstet. 102:128-31, 2008). USC, also known as uterine papillary serous carcinoma (UPSC) and uterine serous adenocarcinoma, is a form of endometrial cancer that typically arises in postmenopausal women. In some instances, the HER2-overexpressing USC is at risk for metastasizing to the CNS of the subject. In certain instances, the HER2-overexpressing USC has already metastasized to the CNS of the subject.
  • Methods for identifying subjects with one or more of the diseases or conditions described herein are known in the art.
  • Also included are methods for imaging an organ or tissue component in a subject, comprising (a) administering to the subject a composition comprising a fusion protein or antibody fusion described herein, which is conjugated to a detectable entity, and (b) visualizing the detectable entity in the subject, organ, or tissue.
  • In particular embodiments, the organ or tissue compartment comprises the central nervous system (e.g., brain, brainstem, spinal cord). In specific embodiments, the organ or tissue compartment comprises the brain or a portion thereof, for instance, the parenchyma of the brain.
  • A variety of methods can be employed to visualize the detectable entity in the subject, organ, or tissue. Exemplary non-invasive methods include radiography, such as fluoroscopy and projectional radiographs, CT-scanning or CAT-scanning (computed tomography (CT) or computed axial tomography (CAT)), whether employing X-ray CT-scanning, positron emission tomography (PET), or single photon emission computed tomography (SPECT), and certain types of magnetic resonance imaging (MRI), especially those that utilize contrast agents, including combinations thereof.
  • Merely by way of example, PET can be performed with positron-emitting contrast agents or radioisotopes such as 18F, SPECT can be performed with gamma-emitting contrast agents or radioisotopes such as 201Tl, 99mTC, 123I, and 67Ga, and MRI can be performed with contrast agents or radioisotopes such as 3H, 13C, 19F, 17O, 23Na, 31P, and 129Xe, and Gd (gadolidinium; chelated organic Gd (III) complexes). Any one or more of these exemplary contrast agents or radioisotopes can be conjugated to or otherwise incorporated into a p97 polypeptide and administered to a subject for imaging purposes. For instance, p97 polypeptides can be directly labeled with one or more of these radioisotopes, or conjugated to molecules (e.g., small molecules) that comprise one or more of these radioisotopic contrast agents, or any others described herein.
  • For in vivo use, for instance, for the treatment of human disease, medical imaging, or testing, the fusion proteins or antibody fusions described herein are generally incorporated into a pharmaceutical composition prior to administration. A pharmaceutical composition comprises one or more of the fusion proteins or antibody fusions described herein in combination with a physiologically-acceptable, pharmaceutically-acceptable, or pharmaceutical grade carrier or excipient.
  • To prepare a pharmaceutical composition, an effective or desired amount of one or more fusion protein or antibody fusions is mixed with any pharmaceutical carrier(s) or excipient known to those skilled in the art to be suitable for the particular mode of administration. A pharmaceutical carrier may be liquid, semi-liquid or solid. Solutions or suspensions used for parenteral, intradermal, subcutaneous or topical application may include, for example, a sterile diluent (such as water), saline solution (e.g., phosphate buffered saline; PBS), fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvent; antimicrobial agents (such as benzyl alcohol and methyl parabens); antioxidants (such as ascorbic acid and sodium bisulfite) and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); buffers (such as acetates, citrates and phosphates). If administered intravenously (e.g., by IV infusion), suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, polypropylene glycol and mixtures thereof.
  • Administration of fusion proteins or antibody fusions described herein, in pure form or in an appropriate pharmaceutical composition, can be carried out via any of the accepted modes of administration of agents for serving similar utilities. The pharmaceutical compositions can be prepared by combining a fusion protein or antibody fusion-containing composition with an appropriate physiologically acceptable carrier, diluent or excipient, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. In addition, other pharmaceutically active ingredients (including other small molecules as described elsewhere herein) and/or suitable excipients such as salts, buffers and stabilizers may, but need not, be present within the composition.
  • Administration may be achieved by a variety of different routes, including oral, parenteral, nasal, intravenous, intradermal, subcutaneous or topical. Preferred modes of administration depend upon the nature of the condition to be treated or prevented. Particular embodiments include administration by IV infusion.
  • Carriers can include, for example, pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as polysorbate 20 (TWEEN™) polyethylene glycol (PEG), and poloxamers (PLURONICS™), and the like.
  • In certain aspects, a fusion protein or antibody fusion is bound to or encapsulated within a particle, e.g., a nanoparticle, bead, lipid formulation, lipid particle, or liposome, e.g., immunoliposome. The fusion proteins or antibody fusions may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate)microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980). The particle(s) or liposomes may further comprise other therapeutic or diagnostic agents, such as cytotoxic agents.
  • The precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by testing the compositions in model systems known in the art and extrapolating therefrom. Controlled clinical trials may also be performed. Dosages may also vary with the severity of the condition to be alleviated. A pharmaceutical composition is generally formulated and administered to exert a therapeutically useful effect while minimizing undesirable side effects. The composition may be administered one time, or may be divided into a number of smaller doses to be administered at intervals of time. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need.
  • Typical routes of administering these and related pharmaceutical compositions thus include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. Pharmaceutical compositions according to certain embodiments of the present invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a subject or patient may take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a herein described conjugate in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will typically contain a therapeutically effective amount of a fusion protein or antibody fusion described herein, for treatment of a disease or condition of interest.
  • A pharmaceutical composition may be in the form of a solid or liquid. In one embodiment, the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, an oral oil, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration. When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
  • As a solid composition for oral administration, the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.
  • The pharmaceutical composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
  • The liquid pharmaceutical compositions, whether they be solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.
  • A liquid pharmaceutical composition intended for either parenteral or oral administration should contain an amount of a fusion protein or antibody fusion such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of the agent of interest in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition. Certain oral pharmaceutical compositions contain between about 4% and about 75% of the agent of interest. In certain embodiments, pharmaceutical compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.01 to 10% by weight of the agent of interest prior to dilution.
  • The pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device.
  • The pharmaceutical composition may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter, and polyethylene glycol.
  • The pharmaceutical composition may include various materials, which modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients may be encased in a gelatin capsule. The pharmaceutical composition in solid or liquid form may include an agent that binds to the conjugate or agent and thereby assists in the delivery of the compound. Suitable agents that may act in this capacity include monoclonal or polyclonal antibodies, one or more proteins or a liposome.
  • The pharmaceutical composition may consist essentially of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. One of ordinary skill in the art, without undue experimentation may determine preferred aerosols.
  • The compositions described herein may be prepared with carriers that protect the fusion proteins or antibody fusions against rapid elimination from the body, such as time release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others known to those of ordinary skill in the art.
  • The pharmaceutical compositions may be prepared by methodology well known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administered by injection may comprise one or more of salts, buffers and/or stabilizers, with sterile, distilled water so as to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the conjugate so as to facilitate dissolution or homogeneous suspension of the conjugate in the aqueous delivery system.
  • The compositions may be administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy. Generally, a therapeutically effective daily dose is (for a 70 kg mammal) from about 0.001 mg/kg (i.e., ˜0.07 mg) to about 100 mg/kg (i.e., ˜7.0 g); preferably a therapeutically effective dose is (for a 70 kg mammal) from about 0.01 mg/kg (i.e., ˜0.7 mg) to about 50 mg/kg (i.e., ˜3.5 g); more preferably a therapeutically effective dose is (for a 70 kg mammal) from about 1 mg/kg (i.e., ˜70 mg) to about 25 mg/kg (i.e., ˜1.75 g).
  • Compositions described herein may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents, as described herein. For instance, in one embodiment, the conjugate is administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate.
  • In certain embodiments, the compositions disclosed herein may be administered in conjunction with any number of chemotherapeutic or cytotoxic agents. Examples of chemotherapeutic or cytotoxic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™ (alitretinoin); ONTAK™ (denileukin diftitox); esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • Such combination therapy may include administration of a single pharmaceutical dosage formulation, which contains a compound of the invention (i.e., fusion protein, antibody fusion protein) and one or more additional active agents, as well as administration of compositions comprising conjugates of the invention and each active agent in its own separate pharmaceutical dosage formulation. For example, a fusion protein or antibody fusion as described herein and the other active agent can be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent administered in separate oral dosage formulations. Similarly, a fusion protein or antibody fusion as described herein and the other active agent can be administered to the patient together in a single parenteral dosage composition such as in a saline solution or other physiologically acceptable solution, or each agent administered in separate parenteral dosage formulations. Where separate dosage formulations are used, the compositions comprising fusion proteins or antibody fusions and one or more additional active agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially and in any order; combination therapy is understood to include all these regimens.
  • The various embodiments described herein can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent application, foreign patents, foreign patent application and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, application and publications to provide yet further embodiments.
  • These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.
    • EXAMPLES Example 1 Binding of p97-Trastuzumab Fusion Proteins to Human Her2/Neu
  • Human p97 (MTf)-trastuzumab fusion constructs were prepared and tested for activity. The amino acid sequences of the MTf-trastuzumab fusion constructs are shown in Table E1 below.
  • TABLE E1
    SEQ ID
    Name Heavy and Light Chain Sequences NO
    TZM HC-MTf Heavy Chain 82
    Homodimer Signal sequence (italics): TZM heavy chain: linker
    (C-terminal (underlined): MTf (bold)
    TZM heavy METDTLLLWVLLLWVPGSTGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDT
    chain MTf; YIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMN
    See FIG. SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSK
    1A for STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
    general VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
    structure) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
    KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
    QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
    TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
    GKGGGGSGGGGS GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTS
    ADHCVQLIAAQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVA
    VVRRSSHVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKA
    VSDYFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR
    CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRADVTE
    WRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMFSSEAYG
    QKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRLPPYLRWCVL
    STPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQAEQVDAVTLSGE
    DIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRDSSHAFTLDELRGKRS
    CHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTAVSEFFNASCVPVNNPKNY
    PSSLCALCVGDEQGRNKCVGNSQERYYGYRGAFRCLVENAGDVAFVRHTTVF
    DNTNGHNSEPWAAELRSEDYELLCPNGARAEVSQFAACNLAQIPPHAVMVRP
    DTNIFTVYGLLDKAQDLFGDDHNKNGFKMFDSSNYHGQDLLFKDATVRAVPV
    GEKTTYRGWLGLDYVAALEGMSSQQCS
    Light Chain 83
    Signal sequence (italics): TZM light chain
    METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTA
    VAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFA
    TYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL
    NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK
    HKVYACEVTHQGLSSPVTKSFNRGEC
    MTfp NH-TZM Heavy Chain 84
    (N-terminal Signal sequence(italics): MTfp sequence (bold):
    MTfp TZM linker (underlined): TZM heavy chain
    heavy
    chain; see METDTLLLWVLLLWVPGSTGDSSHAFTLDELRY GGGGSGGGGSEVQLVESGG
    FIG. 1D GLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYA
    for general DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQG
    structure) TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
    LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK
    VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
    SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE
    YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
    GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
    FSCSVMHEALHNHYTQKSLSLSPGK
    Light Chain 85
    Signal sequence(italics): TZM light chain
    METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTA
    VAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFA
    TYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL
    NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK
    HKVYACEVTHQGLSSPVTKSFNRGEC
    TZM HC-MTfp Heavy Chain 86
    (C-terminal Signal sequence (italics): TZM heavy chain : linker
    TZM heavy (underlined): MTfp sequence without C-terminal Y
    chain MTfp; (bold)
    see FIG. METDTLLLWVLLLWVPGSTGEVQLVESGGGLVQPGGSLRLSCAASGENIKDT
    1E for YIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMN
    general SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSK
    structure) STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
    VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
    PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
    KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
    QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
    TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
    GKGGGGSGGGGS DSSHAFTLDELR
    Light Chain 87
    Signal sequence (italics): TZM light chain
    METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTA
    VAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFA
    TYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL
    NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK
    HKVYACEVTHQGLSSPVTKSFNRGEC
    TZM/MTf Heavy Chain 88
    Homodimer Signal sequence (italics): TZM heavy chain: linker
    (C-terminal (underlined): MTf (bold)
    TZM heavy METDTLLLWVLLLWVPGSTGEVQLVESGGGLVQPGGSLRLSCAASGENIKDT
    chain MTf; YIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMN
    see FIG. SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSK
    1A for STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
    general VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
    structure) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
    KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
    QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
    TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
    GKGGGGSGGGGSGGGGS GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLC
    VRGTSADHCVQLIAAQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTS
    YYAVAVVRRSSHVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGC
    DVLKAVSDYFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDY
    SGAFRCLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSR
    ADVTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMFS
    SEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRLPPYL
    RWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQAEQVDAV
    TLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRDSSHAFTLDEL
    RGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTAVSEFFNASCVPVN
    NPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRGAFRCLVENAGDVAFVR
    HTTVFDNTNGHNSEPWAAELRSEDYELLCPNGARAEVSQFAACNLAQIPPHA
    VMVRPDTNIFTVYGLLDKAQDLFGDDHNKNGFKMFDSSNYHGQDLLFKDATV
    RAVPVGEKTTYRGWLGLDYVAALEGMSSQQCSG
    Light Chain 89
    Signal sequence: TZM light chain
    METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTA
    VAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFA
    TYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL
    NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK
    HKVYACEVTHQGLSSPVTKSFNRGEC
    LC-MTfp/Fc- Heavy Chain 90
    MTfp Signal sequence (italics): TZM heavy chain: linker
    (C-terminal (underlined): Mtfp (bold)
    TZM light METDTLLLWVLLLWVPGSTGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDT
    chain YIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMN
    MTfp/C- SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSK
    terminal STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
    TZM heavy VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLG
    chain MTfp; GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
    see FIG. TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
    1F for GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
    general TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
    structure) PGKEAAAKEAAAKEAAAK DSSHAFTLDELRY
    Light Chain 91
    Signal sequence (italics): TZM light chain: linker
    (underlined): Mtfp (bold)
    METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTA
    VAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFA
    TYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL
    NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK
    HKVYACEVTHQGLSSPVTKSFNRGECEAAAKEAAAKEAAAK DSSHAFTLDEL
    RY
    LC/Fc-MTfp Heavy Chain 92
    (TZM light Signal sequence (italics): TZM heavy chain: linker
    chain/C- (underlined): Mtfp (bold)
    terminal METDTLLLWVLLLWVPGSTGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDT
    TZM heavy YIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMN
    chain MTfp; SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSK
    see FIG. STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
    1E for VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLG
    general GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
    structure) TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
    GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
    TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
    PGKEAAAKEAAAKEAAAK DSSHAFTLDELRY
    Light Chain 93
    Signal sequence (italics): TZM heavy chain
    METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTA
    VAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFA
    TYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL
    NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK
    HKVYACEVTHQGLSSPVTKSFNRGEC
    LC-MTfp/Fc Heavy Chain 94
    (C-terminal Signal sequence (italics): TZM heavy chain: linker: 
    TZM light MTfp
    chain MTfp/ METDTLLLWVLLLWVPGSTGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDT
    TZM heavy YIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMN
    chain; see SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSK
    FIG. 1G STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
    for general VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLG
    structure) GPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
    PGK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
    GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
    TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
    Light Chain 95
    Signal sequence (italics): TZM light chain
    METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTA
    VAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFA
    TYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL
    NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK
    HKVYACEVTHQGLSSPVTKSFNRGECEAAAKEAAAKEAAAK DSSHAFTLDEL
    RY
    TZM-Trastuzumab
    MTf-p97
    MTfp-p97 fragment
    HC-Heavy Chain
    LC-Light Chain
  • In vitro binding assays were performed to measure the binding of MTf-trastuzumab fusion constructs to His-tagged human Her2/Neu protein (Her2-His). Her2-His was loaded onto penta-His biosensor at a concentration of 20 μg/mL and dipped into TZM HC-MTf, MTfp NH-TZM, and TZM HC-MTfp fusion proteins (see Table E1) and a human IgG1 control at varying concentrations.
  • The results of octet analysis in FIGS. 2A-2D and Table E2 (below) demonstrate the affinity of the MTf-trastuzumab fusions for Her2.
  • TABLE E2
    Loading Full
    Sample ID Sample ID KD (M) Kon (1/Ms) Koff (1/s) Full R{circumflex over ( )}2 X{circumflex over ( )}2
    Her2-His TZM HC-MTf <1.0E−12 4.94E+04 <1.0E−07 0.999074 0.002797
    Her2-His MTfp NH-TZM <1.0E−12 9.06E+04 <1.0E−07 0.997464 0.014593
    Her2-His TZM HC-MTfp <1.0E−12 1.21E+05 <1.0E−07 0.997177 0.019048
    Her2-His Human IgG1 <1.0E−12 1.04E+06 <1.0E−07 0.997389 0.010269
  • MTf-trastuzumab fusion proteins demonstrated tight binding to human Her2, as shown by the equilibrium dissociation constant (KD), association rate constant (Kon), and dissociation rate constant (Koff).
    • Example 2 ADCC Cell-Killing Activity of p97-Trastuzumab Fusion Proteins
  • MTf-trastuzumab fusion constructs were tested for Antibody-dependent cell-mediated cytotoxicity (ADCC) in BT-474 breast cancer cells compared to Herceptin® (trastuzumab) and human IgG1 Fc as positive and negative controls, respectively.
  • BT-474 cells were purchased from ATCC and grown in Hybri-Care Medium supplemented with 1.5 g/L sodium bicarbonate and 10% fetal bovine serum. Human peripheral blood mononuclear cells (PBMCs) were freshly isolated by Histopaque centrifugation and incubated with carboxyfluorescein succinimidyl ester (CFSE)-labeled target BT-474 cells at a ratio of about 30:1.
  • Test samples were co-incubated with the effector:target cell mixture for four hours at ten different concentrations (up to 100 μg/mL). Negative controls included effector:target cells only as no antibody control, and 100 μg/mL IgG Fc. Each treatment was performed in duplicate. After the 4 hour co-incubation, cells were stained with propidium iodide (PI) and analyzed by flow cytometry (FACS). The percentage of PI/CFSE+ cells was quantitated as an indication of cytotoxicity. A dose-response curve was generated by plotting the mean±range of the data points for all ten different concentrations, and the EC50 was calculated. The results are shown in FIGS. 3A-3B.
  • FIG. 3A shows the ADCC evaluation of TZM/MTf fusion in BT-474 breast cancer cells compared to Herceptin® and human IgG1 Fc. Here, the TZM/MTf homodimer fusion induced ADCC with an EC50 of 0.6 μg/mL compared to that of Herceptin® at 0.2 pg/mL.
  • FIG. 3B shows the ADCC evaluation of LC-MTfp/Fc-MTfp, LC-MTfp/Fc, and LC/Fc-MTfp fusions in BT-474 breast cancer cells compared to Herceptin® and human IgG1 Fc. LC-MTfp/Fc-MTfp, LC-MTfp/Fc, and LC/Fc-MTfp induced ADCC with an EC50 of 0.216 μg/mL, 0.171 μg/mL, and 1.025 μg/mL, respectively. These data show that MTf-trastuzumab fusion constructs induce significant antibody-dependent cell-mediated cytotoxicity in breast cancer cells.
    • Example 3 In Vivo Anti-Tumor Efficacy of p97-Trastuzumab Fusion Proteins
  • The p97-trastuzumab fusion proteins are tested for anti-tumor efficacy and effects on survival in mice that are intracranially injected with human BT-474 breast tumor cells. Specifically, the fusion constructs TZM HC-MTf and MTfp NH-TZM are tested relative to Herceptin® (Trastuzumab, AMM: 5621037, ROCHE).
  • Test Compounds.
  • The stock solutions of TZM HC-MTf and MTfp NH-TZM constructs are diluted in PBS and administered respectively at 60 mg/kg and 30 mg/kg (equivalent dose of 30 mg/kg Herceptin®). Herceptin® is prepared as follows: one vial containing 150 mg of Herceptin® is diluted in NaCl 0.9% (Aguettant, Lyon, France) to a final concentration of 20 mg/mL. The stock solution is stored at 4° C. for the duration of the study. Each day of administration to mice, the stock solution is diluted in NaCl 0.9% to reach 3 mg/mL final concentration. Herceptin® is administrated at 30 mg/kg.
  • All compounds are administered by intravenous injection (IV, bolus) into the caudal vein of mice at a dose volume of 10 mL/kg/inj (i.e., for one mouse weighing 20 g, 200 μL of test substance is administered).
  • Cancer Cell Line.
  • The BT-474 cell line is purchased from ATCC. It was originally isolated from a solid, invasive ductal carcinoma of the breast from a 60 year old Caucasian female patient (Lasfargues et al., J Natl Cancer Inst. 61:967-78, 1978). Tumor cells are grown as monolayer at 37° C. in a humidified atmosphere (5% CO2, 95% air). The culture medium contains DMEM supplemented with 2 mM L glutamine (ref: BE12-604F, Lonza, Verviers, Belgium) and 10% fetal bovine serum (ref: 3302, Lonza). The cells are adherent to plastic flasks. For experimental use, tumor cells are detached from the culture flask by a 5 minute treatment with trypsin-versene (ref: BE02-007E, Lonza), in Hanks' medium without calcium or magnesium (ref: BE10-543F, Lonza) and neutralized by addition of complete culture medium. The cells are counted in a hemocytometer and their viability is assessed by 0.25% trypan blue exclusion assay.
  • Animals.
  • Sixty-one healthy female Balb/C nu/nu (CByJ.Cg-Foxn1nu/J) mice, 5-6 weeks old, are obtained from CHARLES RIVER (L'Arbresles). Animals are maintained in housing rooms under controlled environmental conditions: Temperature: 22±2° C., Humidity 55±10%, Photoperiod (12 h light/12 h dark), HEPA filtered air, 15 air exchanges per hour with no recirculation.
  • Animal enclosures provide sterile and adequate space with bedding material, food and water, environmental and social enrichment (group housing) as described: Top filter polycarbonate Eurostandard Type III or IV cages, Corn cob bedding (ref: LAB COB 12, SERLAB, France), 25 kGy Irradiated diet (Ssniff® Soest, Germany), Complete food for immunodeficient rodents—NM Extrudate, Complete food for immunocompetent rodents—R/M-H Extrudate, Sterile, filtrated at 0.2 μm water (supplemented with 2.5 μg/mL estradiol), Environmental enrichment (SIZZLE-dri kraft-D20004 SERLAB, France).
  • Induction of BT-474 Tumors.
  • For stereotaxic injection of tumor cells, mice are anesthetized by an intraperitoneal injection of Ketamine 70 mg/kg (Ketamine500®, Ref 043KET204, Centravet, France) and Xylazine 5 mg/kg (Rompun®, Ref 002ROM001, Centravet, France) in 0.9% NaCl solution at 10 mL/kg/inj. Tumors are induced by stereotaxic injection of 1×105 of BT-474 cells in 2 μL of RPMI 1640 of 52 female animals. BT-474 tumor cell implantation is performed 24 to 72 hours after a whole body irradiation with a γ-source (2 Gy, 60Co, BioMep, Bretenières, France).
  • The tumor cell suspension is injected into the caudate nucleus of the right cerebral hemisphere at a rate of 0.5 μL/min. Five minutes after the end of the injection, the needle is slowly withdrawn by 1 mm every minute. Carprofen (dose: 5 mg/kg) is injected subcutaneously at the end of the surgery and 24 h post-surgery. The day of tumor cell implantation is considered as day zero (D0).
  • Treatment Schedule.
  • The treatment is started on day five (D5). Forty animals (40) out of fifty-two (52) are randomized according to their individual body weight into 4 groups each of 10 animals using Vivo Manager® software (Biosystemes, Couternon, France). A statistical test (analysis of variance) is performed to test for homogeneity between groups. The treatment schedule is as follows:
      • Group 1 mice receive two weekly IV administration of vehicle for 6 consecutive weeks.
      • Group 2 mice receive two weekly IV administrations of TZM HC-MTf at equivalent dose of 30 mg/kg Herceptin® for 6 consecutive weeks.
      • Group 3 mice receive two weekly IV administrations of MTfp NH-TZM at equivalent dose of 30 mg/kg Herceptin® for 6 consecutive weeks.
      • Group 4 mice receive two weekly IV administrations of Herceptin® at 30 mg/kg for 6 consecutive weeks.
  • The treatment schedule is summarized in Table E3 below:
  • TABLE E3
    No. Dose Adm. Treatment
    Group animals Treatment (mg/kg/adm) Route schedule
    1 10 Vehicle (PBS) — IV TW × 6
    2 10 TZM HC-MTf 60 IV TW × 6
    3 10 MTfp NH-TZM 30 IV TW × 6
    4 10 Herceptin ® 30 IV TW × 6
  • Monitoring.
  • All study data, including animal body weight measurements, clinical and mortality records, and treatment are scheduled and recorded on Vivo Manager® database (Biosystemes, Dijon, France). The viability and behavior is recorded every day. Body weights are measured twice a week.
  • The following Human endpoints are measured (Workman et al., Br J Cancer. 102:1555-77, 2010).
      • Signs of pain, suffering or distress: pain posture, pain face mask, behavior
      • 20% body weight loss remaining for 3 consecutive days
      • Poor body condition, emaciation, cachexia, dehydration
      • Prolonged absence of voluntary responses to external stimuli
      • Rapid labored breathing, anemia, significant bleeding
      • Neurologic signs: circling, convulsion, paralysis
      • Sustained decrease in body temperature
      • Abdominal distension
  • Necropsy (macroscopic examination) is performed on all terminated animal in the study, and, if possible, on all euthanized moribund or found dead animal.
  • The following evaluation criteria of health are determined using Vivo Manager® software (Biosystemes, Couternon, France).
      • Individual and mean body weights of animals
      • Mean body weight change (MBWC): Average weight change of treated animals in percent (weight at day B minus weight at day A divided by weight at day A) is calculated. The intervals over which MBWC is calculated are chosen as a function of body weight curves and the days of body weight measurement
  • Magnetic Resonance Imaging (MRI).
  • Imaging experiments are performed on a 4.7T horizontal magnet (PharmaScan, Bruker Biospin GmbH, Germany) equipped with an actively shielded gradient system. For image analysis, mice are positioned prone in a dedicated mouse body cradle which is slid in a volume coil (38 mm internal diameter) within the Pharmascan and images are acquired under ParaVision (PV5.1, Bruker Biospin).
  • During all the image acquisitions, mice are continuously anaesthetized using isoflurane (Minerve, Bondoufle, France) in a mixture of air via a nose piece. Body temperature of the animals is maintained within physiological levels by a flow of warm air. Breathing rate is continuously monitored using a pressure sensor taped on its abdomen. Physiological signals are monitored via a laptop placed next to the MRI workstation and connected to the sensors by fiber optic cables (SA Instruments, USA).
  • The contrast agent Gadopentetate dimeglumine (Gd-DTPA, Magnevist, Bayer Healthcare Pharmaceuticals, Germany) is injected intravenously (IV) at 0.4 mmol/kg via the caudal vein of mice.
  • Images are transferred to a workstation to be analyzed under ImageJ (4). Regions of interest (ROIs) are drawn manually on anatomical images. Tumor volume is computed from the ROIs by multiplying the number of ROI voxels by the voxel volume (in mm3). Tumor volumes in mm3 are tabulated at each time point and for each animal.
  • Sample Collection.
  • All mice are euthanized fourteen days after the last treatment. Intracardiac blood collection is used in terminal procedures under deep gas anesthesia. Approximately 200 μL of blood from ten animals per group is collected into blood collection tubes with clot activator. Tubes are centrifuged 30 minutes after sampling at 1300 g for 10 minutes at room temperature to obtain serum. The serum samples are stored in propylene tubes at 80° C. until analysis. Immediately after termination, samples from the brain, heart, lung, liver and kidney of each animal are collected, weighed and stored for analysis.
  • Efficacy Parameters.
  • The treatment efficacy is assessed in terms of the effects of the test substance on the tumor volumes of treated animals relative to control animals, as measured by MRI (see below). The efficacy parameters are expressed as a percent treated over control survival (T/C %). T is the median of the survival times of animals treated with test substances and C is the median survival times of control animals treated with vehicle. Survival systems indicate a degree of success when T/C percent exceed 125% (7). T/C % is expressed as follows:
  • T / C % = T ( median survival time of treated animals ) C ( median survival time of vehicle - treated animals ) × 100
  • Survival curves are drawn, and mean and median survival times are calculated.
  • Statistical Tests.
  • Statistical analyses are performed using Vivo Manager® software (Biosystemes, Couternon, France). Statistical analyses of mean body weights, MBWC, mean tumor volumes V, are performed using ANOVA and pairwise tests are performed using the Bonferroni/Dunn correction in case of significant ANOVA results. The log-Rank (Kaplan-Meier) test is used to compare the survival curves. A p value<0.05 is considered as significant.

Claims (64)

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.
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