HK1191342A - Method and constructs for the ph dependent passage of the blood-brain-barrier - Google Patents

Abstract

Herein is reported a fusion polypeptide comprising i) at least one binding site, e.g. which comprises an antibody heavy chain variable domain and an antibody light chain variable domain, and which binds to an internalizing cell surface receptor, and ii) at least one pharmaceutically active compound, whereby the EC50-value of the binding pair that binds to an internalizing cell surface receptor determined at pH 5.5 is higher than the EC50-value of the same binding pair determined at pH 7.4 and its use for delivering a pharmaceutically active compound across the blood brain-barrier.

Description

Methods and constructs for pH-dependent passage across the blood-brain barrier

Herein is reported a fusion polypeptide comprising at least one binding site and at least one pharmaceutically active compound, whereby the EC as determined by the binding site for binding to an internalized cell surface receptor at pH5.5₅₀EC at a value higher than that determined for this same binding site at pH7.4₅₀-values and reports the use of the fusion polypeptide for delivering a pharmaceutically active compound across the blood-brain barrier.

Background

The endothelial or epithelial cell layers, which are interconnected by tight junctions, represent a major barrier to the diffusion of large polar molecules (particularly proteins) into tissues behind these barriers. Although small molecules can be transported across these barriers by specialized channel proteins, the transport mechanism of the protein is still not well understood, but it is believed that the most physiologically important mechanism is receptor-mediated transcytosis (RMT).

During RMT, protein ligands bind to receptors expressed on the luminal side of barrier cells, which are then internalized by endocytosis. Sorting of endosomal content is accomplished in specialized vesicular compartments and depends on signals encoded by the receptor sequence that mediate transport of the receptor into recycling, degradation, or transcytosis pathways. One of the best known examples of RMTs is the transport of IgG across intestinal epithelial cells in rodents via neonatal Fc receptors.

For the Blood Brain Barrier (BBB), which consists of tightly sealed brain endothelial cells surrounded by pericytes and astrocytes, several RMT pathways have also been described, in particular for the receptors for transferrin, insulin or low density lipoprotein. Ligands for these receptors have been shown to include properties that promote transcytosis, one of which is pH-dependent binding to their receptor. For example, insulin is released from its receptor when the endosomal contents are acidified after internalization.

Researchers have attempted to study the transcytosis of receptors to deliver therapeutic molecules across the blood-brain barrier by coupling therapeutic agents to antibodies directed against these receptors. However, none of these antibodies has been used in commercially available drugs, probably due to their insufficient transport potential. Indeed, there is no clear demonstration that functional therapeutic proteins displayed independently in more than one pharmacodynamic model transcytosed across the BBB.

Kobayashi, et al (Kobayashi, N., et al, am.J.physiol.Renal.physiol.282(2002) F358-F365) reported that FcRn-mediated transcytosis of immunoglobulin G in human renal proximal tubular epithelial cells. The blood brain barrier specific properties of the human adult brain endothelial cell line (human adult brain endothielialcell line) are reported by Weksler, B.B., et al in FASEB J.19(2005) 1872-1874.

In US 6,030,613, receptor specific transepithelial transport of immunogens is reported. In WO02/060919, molecules, compositions and uses thereof with extended half-lives are reported. A method for preparing transferrin receptor specific antibody-neurological agent or diagnostic agent conjugates is reported in WO 93/010819. In WO 2008/119096, endocytosed immunoglobulins are reported. The human blood brain barrier model is reported in WO 2006/056879.

An endocytotic transport module antibody (transcytotic modular antibody) was reported in EP 1975178. Antibodies targeting the blood brain barrier are reported in US 2008/019984. Friden, PM., et al report the characterization, receptor localization and blood brain barrier transcytosis of antibodies to the human transferrin receptor (j. pharmacol. exp. therap.278(1996) 1491-1498). Pardridge et al (pharm. Res.12(1995)807-816) reported that human insulin receptor monoclonal antibodies undergo high affinity binding to human brain capillaries in vitro and rapid transcytosis across the blood brain barrier in vivo in primates.

Summary of The Invention

Herein it is reported that the pH-dependent binding pattern enables efficient crossing of a tight layer of barrier cells, in particular the blood-brain barrier, by antibodies directed against internalized cell surface receptors, in particular transcytosis receptors. For example, binding affinity (EC) at pH5.5 has been shown₅₀Higher) than its affinity (EC) at pH7.4₅₀Lower value), for example, an antibody that binds to human transferrin receptor (as an example of an internalized cell surface receptor), is transcytosed by endocytosis across blood brain barrier endothelial cells, while exhibiting approximately equal affinity (approximately equal binding efficiency, and thus, EC) at both pH values₅₀Approximately equal values) of different antibodies are degraded inside the cell. Thus, the binding sites for binding to internalized cell surface receptors are defined at pH5.5EC₅₀A value higher (greater) than the EC determined for this same binding site at pH7.4₅₀-a value. This criterion allows the generation and selection of antibodies against internalized cell surface receptors and/or transcytosis receptors that are not degraded intracellularly in endothelial or epithelial barrier cells due to altered sorting behavior resulting from pH-dependent, reversible binding to these receptors.

In one aspect, herein is reported a fusion polypeptide comprising:

-at least one binding site that binds to an internalized cell surface receptor, and

-at least one effector part,

EC as determined by the at least one binding site binding to an internalized cell surface receptor at pH5.5₅₀An EC value higher than that determined for the same binding site of the same receptor at pH7.4₅₀-a value.

In one embodiment of all aspects, the fusion polypeptide is characterized by i) the EC determined at pH5.5 for the binding site that binds to an internalized cell surface receptor₅₀The EC with value determined at pH7.4 for ii) the same binding site for the same receptor₅₀-the ratio of the values is at least 5. In one embodiment, the ratio is 10 or greater. In one embodiment, the ratio is 15 or greater. In one embodiment, the ratio is about 15.

In one embodiment of all aspects, the binding site for binding to an internalized cell surface receptor has an EC determined at pH5.5₅₀The value is the EC determined at pH7.4 for the same binding site of the same receptor₅₀-at least 5 times the value. In one embodiment, the EC is determined at pH5.5₅₀The value is the EC determined at pH7.4₅₀-at least 10 times the value. In one embodiment, the EC is determined at pH5.5₅₀EC at a value of about determined at pH7.4₅₀-15 times the value.

In one embodiment of all aspects, the effector moiety is a label, or a cytotoxin, or an enzyme, or a growth factor, or a transcription factor, or a drug, or a radionuclide, or a ligand, or an antibody fragment, or a liposome, or a nanoparticle, or a virion, or a cytokine.

In one embodiment of all aspects, the effector moiety is a pharmaceutically active compound. In one embodiment, the pharmaceutically active compound is an anti- Α β antibody, or an anti-tau antibody or an anti-alpha synuclein antibody.

In one embodiment of all aspects, the binding site for binding to an internalized cell surface receptor has an EC determined at pH5.5₅₀-a value of more than 100ng/ml, or more than 500ng/ml, or more than 1000 ng/ml.

In one embodiment of all aspects, the binding site for binding to an internalized cell surface receptor has an EC determined at pH7.4₅₀-a value of less than 100ng/ml, or less than 85ng/ml or less than 70 ng/ml.

In one embodiment of all aspects, the binding site is a binding pair comprising an antibody heavy chain variable domain and an antibody light chain variable domain. In one embodiment, the binding pair is selected from the group consisting of Fv, Fab '-SH, F (ab')₂A diabody, a linear antibody, a single chain antibody molecule, and a multispecific antibody, full-length heavy chain, full-length light chain, intact antibody, bispecific antibody, trispecific antibody, tetraspecific antibody, or hexaspecific antibody formed from an antibody fragment. In one embodiment, the binding pair is an intact monoclonal antibody. In one embodiment, the binding pair is at least a fragment of an intact antibody, a member of the immunoglobulin superfamily, or a polypeptide having an immunoglobulin-like structure that retains binding specificity for its antigen.

In one embodiment of all aspects, the binding site is selected from the group consisting of fibronectin, TCR, CTLA-4, single chain antigen receptors, e.g. those associated with T-cell receptors, antibody mimetics, transferrin, apolipoprotein, adnectin, anticalin-based molecules, phylomers, avimers, affibodies, ankyrin repeats, Kunitz domains, PDZ-domains, scorpion toxin immunity proteins (scorpio toxin immunity proteins), Knottin, Versabody, green fluorescent protein and other non-antibody protein scaffolds with binding properties.

In one aspect, herein is reported a nucleic acid encoding a fusion polypeptide as reported herein.

In one aspect, herein is reported a host cell comprising a nucleic acid as reported herein.

In one aspect, herein is reported a method for the preparation of a fusion polypeptide, said method comprising culturing a host cell as reported herein, thereby producing said fusion polypeptide.

In one aspect, herein is reported a pharmaceutical formulation comprising a fusion polypeptide as reported herein and optionally a pharmaceutically acceptable carrier.

In one aspect, the fusion polypeptides as reported herein are reported herein for use as a medicament.

In one aspect, herein is reported a fusion polypeptide as reported herein for use in the treatment of a CNS-related disease.

In one aspect, herein is reported a fusion polypeptide as reported herein for use in the delivery of a pharmaceutically active compound across the blood brain barrier.

In one aspect, the use of a fusion polypeptide as reported herein for the preparation of a medicament is reported herein.

In one embodiment, the medicament is for treating a CNS-related disease.

In one aspect, herein is reported a method of treating an individual having a CNS-related disease, said method comprising administering to said individual an effective amount of a fusion polypeptide reported herein.

In one aspect, herein is reported a method of delivering a pharmaceutically active compound across the blood-brain barrier in an individual, said method comprising administering to said individual an effective amount of a fusion polypeptide as reported herein, thereby delivering the pharmaceutically active compound across the blood-brain barrier.

In one aspect, herein is reported a method of delivering a pharmaceutically active compound across the blood-brain barrier in an individual or in the brain of a subject, the method comprising administering the pharmaceutically active compound fused to a binding pair comprising an antibody heavy chain variable domain and an antibody light chain variable domain and which binds to an internalizing cell surface receptor, whereby the binding pair that binds to the internalizing cell surface receptor has an EC determined at pH5.5₅₀EC at a value higher than that determined for this same binding pair at pH7.4₅₀-a value.

In one embodiment of all aspects, the fusion polypeptide is characterized by an EC determined at pH5.5 for the binding site that binds to an internalized cell surface receptor₅₀EC with value determined at pH7.4 for the same binding site of the same receptor₅₀-the ratio of values is at least 5. In one embodiment, the ratio is 10 or greater.

One aspect as reported herein is the use of a fusion polypeptide for delivering a pharmaceutically active compound across the blood-brain barrier, said fusion polypeptide comprising:

-at least one binding pair comprising an antibody heavy chain variable domain and an antibody light chain variable domain and which binds to an internalized cell surface receptor, and

-at least one pharmaceutically active compound,

EC determined at pH5.5 by the binding pair bound to the internalized cell surface receptor₅₀EC with value determined at pH7.4 for the same binding pair against the same receptor₅₀-a ratio of values above 10.

One aspect as reported herein is a method for transcytosis of epithelial cells in a subject, said method comprising administering to said subject a fusion polypeptide comprising:

-at least one binding pair comprising an antibody heavy chain variable domain and an antibody light chain variable domain and which binds to an internalized cell surface receptor, and

-at least one pharmaceutically active compound,

EC determined at pH5.5 by the binding pair bound to the internalized cell surface receptor₅₀EC with value determined at pH7.4 for the same binding pair against the same receptor₅₀-a ratio of values above 10.

Herein is reported a method of delivering a pharmaceutically active compound across the blood-brain barrier in an individual, said method comprising administering to said individual an effective amount of a fusion polypeptide comprising:

-at least one binding pair comprising an antibody heavy chain variable domain and an antibody light chain variable domain and which binds to an internalized cell surface receptor, and

-at least one pharmaceutically active compound,

EC determined at pH5.5 by the binding pair bound to the internalized cell surface receptor₅₀EC with value determined at pH7.4 for the same binding pair against the same receptor₅₀-a ratio of values of 10 or more, such that the fusion polypeptide delivers the pharmaceutically active compound across the blood-brain barrier.

In one aspect the use of a fusion polypeptide comprising:

-at least one binding pair comprising an antibody heavy chain variable domain and an antibody light chain variable domain and which binds to an internalized cell surface receptor, and

-at least one pharmaceutically active compound,

thereby contacting the cell surface with internalizationBinding of receptor binding to EC determined at pH5.5₅₀EC with value determined at pH7.4 for the same binding pair against the same receptor₅₀-a ratio of values above 10.

In one aspect, herein is reported a method of increasing transport of at least one pharmaceutically active compound across the blood-brain barrier in an individual relative to transport of one or more pharmaceutically active compounds across the blood-brain barrier in unconjugated form, the method comprising administering to the individual an effective amount of a fusion polypeptide comprising:

-at least one binding pair comprising an antibody heavy chain variable domain and an antibody light chain variable domain and which binds to an internalized cell surface receptor, and

-at least one pharmaceutically active compound,

EC determined at pH5.5 by the binding pair bound to the internalized cell surface receptor₅₀EC with value determined at pH7.4 for the same binding pair against the same receptor₅₀-a ratio of values of 10 or more, such that the fusion polypeptide transports the pharmaceutically active compound across the blood-brain barrier.

In one aspect, herein is reported a method of selecting a binding pair for efficient blood brain barrier transport of one or more pharmaceutically active compounds, the method comprising measuring the EC of the binding of the one or more binding pairs to an internalized cell surface receptor at pH5.5 and pH7.4₅₀-a ratio of values, and selecting one or more binding pairs wherein the ratio is above 10.

In one embodiment of all aspects, the fusion polypeptide is characterized by an EC determined at pH5.5 for the binding site that binds to an internalized cell surface receptor₅₀EC with value determined at pH7.4 for the same binding site of the same receptor₅₀-the ratio of values is 15 or more. In yet another embodiment, the ratio is about 15.

In all aspects oneIn one embodiment, the binding pair that binds to an internalized cell surface receptor has an EC determined at pH5.5₅₀The value is the EC determined at pH7.4 for the same binding pair to the same receptor₅₀-at least 5 times the value. In one embodiment, the EC is determined at pH5.5₅₀The value is the EC determined at pH7.4₅₀-at least 10 times the value. In one embodiment, the EC is determined at pH5.5₅₀An EC value of about determined at pH7.4₅₀-15 times the value.

In one aspect, herein is reported the use of a fusion polypeptide as reported herein for the delivery of a pharmaceutically active compound across the blood-brain barrier.

In one aspect, herein is reported a method of transcytosis of epithelial cells of a subject, said method comprising administering to said subject a fusion polypeptide as reported herein.

In one aspect, herein is reported a method for selecting an antibody or fusion polypeptide comprising at least one binding site, wherein said antibody or fusion polypeptide has an EC determined at pH5.5 with respect to binding to an internalized cell surface receptor₅₀A value higher than the EC determined for the same antibody or the same fusion polypeptide against the same receptor at pH7.4₅₀-a value.

In one embodiment of all aspects, the antibody or fusion polypeptide is characterized by an EC determined at pH5.5 for the binding site that binds to an internalized cell surface receptor₅₀EC with value determined at pH7.4 for the same binding site of the same receptor₅₀-the ratio of the values is at least 5. In one embodiment, the ratio is 10 or greater. In one embodiment, the ratio is 15 or greater. In one embodiment, the ratio is about 15.

In one embodiment of all aspects, the binding site for binding to an internalized cell surface receptor has an EC determined at pH5.5₅₀The value is the EC determined at pH7.4 for the same binding site of the same receptor₅₀-at least of value5 times. In one embodiment, the EC is determined at pH5.5₅₀The value is the EC determined at pH7.4₅₀-at least 10 times the value. In one embodiment, the EC is determined at pH5.5₅₀An EC value of about determined at pH7.4₅₀-15 times the value.

In one embodiment of all aspects as reported herein, said CNS-related disease is selected from the group consisting of: (i) neurodegenerative diseases or disorders such as parkinson's disease, alzheimer's disease or huntington's disease, or (ii) psychiatric diseases such as depression, anxiety, schizophrenia, or (iii) neuroinflammation and other neurological disorders such as multiple Sclerosis (multiple Sclerosis), Amyotrophic Lateral Sclerosis (Amyotrophic late Sclerosis), autism (autism) or pain, or (iv) neoplasms of the CNS, or (v) viral and bacterial infections of the CNS.

Detailed Description

The present invention demonstrates that a pH-dependent binding pattern enables fusion polypeptide polypeptides comprising at least one binding site and antibodies directed against an endocytotic transport receptor to efficiently cross a tight layer of barrier cells. For example, it was shown that an antibody directed against the human transferrin receptor, which has a low binding affinity at pH5.5 compared to its affinity at pH7.4, is transcytosed across the blood brain barrier endothelial cells, while another antibody, which exhibits an equivalent effective binding to the transferrin receptor at both pH values, is degraded intracellularly. The present invention allows the selection and generation of antibodies against transcytosis receptors that avoid intracellular degradation in endothelial or epithelial barrier cells due to altered sorting behavior resulting from pH-dependent, reversible binding to these receptors.

I. Definition of

The term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a polypeptide or an antibody) and its binding partner (e.g., a target or an antigen). Unless otherwise indicated, "binding affinity" as used herein is meant to reflect the composition of the binding pairIntrinsic binding affinity for 1: 1 interactions between members (e.g., in a polypeptide-polynucleotide-complex, or between a polypeptide and its target, or between an antibody and its antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, such as surface plasmon resonance, and also includes those methods reported herein. The higher affinity of the molecule X for the alignment binding partner Y can be seen in the lower Kd and/or EC₅₀The value is obtained.

The term "antibody" includes various forms of antibody structures, including whole antibodies and antibody fragments. The antibodies reported and used herein may be human antibodies, humanized antibodies, chimeric antibodies or T cell antigen depleted antibodies (T cell antigen depleted antibodies). The term "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognized immunoglobulin genes include various constant region genes as well as numerous immunoglobulin variable region genes. Immunoglobulins can exist in a variety of forms, including, for example, Fv, Fab and F (ab)₂And single chain (scFv) or diabodies. Full-length antibodies typically comprise two so-called light chain polypeptides (light chains) and two so-called heavy chain polypeptides (heavy chains). Each of the heavy and light chain polypeptides comprises a variable domain (variable region), typically the amino-terminal portion of the polypeptide chain, which comprises a binding region capable of interacting with an antigen. Each of the heavy and light chain polypeptides comprises a constant region (typically the carboxy-terminal portion). The constant region of the heavy chain mediates i) binding of the antibody to cells (e.g., phagocytes) bearing Fc γ receptors (fcyr), or ii) binding of the antibody to cells bearing nascent Fc receptors (FcRn), also known as Brambell receptors. It also mediates binding to factors including those of the classical complement system, such as complement (Clq).

The variable domain of the light or heavy chain of an immunoglobulin in turn comprises different segments, i.e. four Framework Regions (FR) and three hypervariable regions (CDR).

The term "binding pair) "means a polypeptide comprising an antibody heavy chain variable domain and an antibody light chain variable domain. The variable domains may be linked to each other by suitable means such as peptide bonds, linkers, or linking non-peptide components. In one embodiment, the binding pair is selected from the group consisting of Fv, Fab, Fab ', Fab ' SH, F (ab ')₂A diabody, a linear antibody, a single chain antibody molecule and a multispecific antibody formed from antibody fragments, a full-length heavy chain, a full-length light chain, a whole antibody, a bispecific antibody, a trispecific antibody, a tetraspecific antibody, or a hexaspecific antibody. In one embodiment, the binding pair is a monoclonal antibody. In one embodiment, the binding pair is at least a fragment of an intact antibody, a member of the immunoglobulin superfamily, or a polypeptide having an immunoglobulin-like structure that retains binding specificity for its antigen.

The term "binding site" denotes a polypeptide capable of specifically binding another polypeptide. In one embodiment, the binding site is a binding pair. In one embodiment, the binding site is a polypeptide having an immunoglobulin-like modular structure, which may be selected from the group consisting of: fibronectin, TCR, CTLA-4, single chain antigen receptors, e.g., those associated with T cell receptors and antibodies, antibody mimetics, transferrin, apolipoproteins, adnectins, anticalins-based molecules, phylomes, avimers, affibodies, ankyrin repeats, Kunitz domains, PDZ-domains, scorpion toxin immunity proteins, Knottins, Versabodies, green fluorescent protein, and other non-antibody protein scaffolds with antigen binding properties.

The term "CNS-related disease" denotes a disease or disorder of the Central Nervous System (CNS). CNS-related diseases are, but are not limited to, in particular (i) neurodegenerative diseases or disorders such as parkinson's disease, alzheimer's disease or huntington's disease, (ii) psychiatric diseases such as depression, anxiety, schizophrenia, (iii) neuroinflammation and other neurological disorders such as multiple sclerosis, amyotrophic lateral sclerosis, autism or pain, (iv) tumors of the CNS, or (v) viral and bacterial infections of the CNS.

"chemotherapeutic agents" are compounds that are useful for the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide^TM(ii) a Alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines (aziridines) such as benzotepa (benzodopa), carboquone (carboquone), meturedpa (meturedpa) and uredepa (uredpa); ethyleneimines (ethylenimines) and melamelamines, including altretamine, tritylamine, triethylenephosphoramide, thiotepa, and trimetylomelamine; nitrogen mustards such as chlorambucil (chlorambucil), chlorambucil (chlorenaphazine), cholorophosphamide (cholorophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), mechlorethamine oxydichloride (mechlorethamine), melphalan (melphalalan), neomustard (novembichin), benzene mustard cholesterol (phenylesterine), prednimustine, triamcinolone (trofosfamide), uramustine (uracil mustard); nitrosoureas (nitrosureas) such as carmustine (carmustine), chlorouretocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ramustine (ranimustine); antibiotics such as aclacinomycins (aclacinomycins), actinomycins (actinomycins), anthracyclines (aurramycins), azaserins (azaserines), bleomycin (bleomycin), actinomycin C (cactinomycin), calicheamicins (calicheamicins), carabicins, nordaunorubicins (carminomycin), carzinomycin (carzinophilins), chromomycins (chromomycins), actinomycin D (dactinomycins), daunorubicins (daunorubicin), ditorexin (detoubicin), 6-diazo-5-oxo-L-norleucine (6-diazo-5-oxo-L-norleucine), doxorubicin (daunorubicin), epirubicin (epirubicin), ruscin (olivomycin), pararubicin (idarubicin), actinomycin (mitomycin), mitomycin (mitomycin), mycomycin (mycomycin), mycomycin C (mycomycin), mycomycin (mycomycin), and a strain (mycomycin), or a strain (mycomycin), ain), potfiromycin, puromycin (puromycin), triiron doxorubicin (quelemycin), rodoricin (rodorubicin), streptomycin (streptonigrin), streptozotocin (streptozocin), tubercidin (tubicidin), ubenimex (ubenimex), zinostatin (zinostatin), zorubicin (zorubicin); antimetabolites such as methotrexate (methotrexate) and 5-fluorouracil (5-fluorouracil) (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine (fludarabine), 6-mercaptopurine (6-mercaptopurine), thiamiprine (thiamiprine), thioguanine (thioguanine); pyrimidine analogs such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine (6-azauridine), carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine), 5-FU; androgens such as carposterone (calusterone), drostandrosterone propionate (dromostanolone propionate), epithioandrostanol (epithiostanol), mepiquat (mepiquitane), testolactone (testolactone); anti-adrenal agents (anti-adrenals) such as aminoglutethimide, mitotane, trostane; folic acid compensators such as frillinic acid; acetoglucurolactone (acegultone); hydroxyaldehyde phosphoramidite glycoside (aldophosphamide glycoside); 5-aminolevulinic acid (aminolevulinic acid); amsacrine (amsacrine); bestrabuucil; bisantrene; edatrexate (edatraxate); defofamine; colchicine (demecolcine); diazaquinone (diaziqutone); elfornitine; ammonium etitanium acetate; etoglut (etoglucid); gallium nitrate (gallium nitrate); hydroxyurea (hydroxyurea); lentinan (lentinan); lonidamine (lonidainine); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidanol (mopidanmol); nitrerine (nitrarine); pentostatin (pentostatin); promethazine (phenamett); pirarubicin (pirarubicin); podophyllinic acid (podophyllic acid); 2-ethyl hydrazide (2-ethyl hydrazide); procarbazine (procarbazine); PSKRazoxane (rizoxane); sizofuran (sizofiran); germanium spiroamines (spirogyranium); blepharicic acid (tenuazonicacid); triimine quinone (triaziquone); 2,2 ', 2 "-trichlorotriethylamine (2, 2', 2" -trichlorotriethylamine); urethane (urethan); vindesine (vindesine); dacarbazine (dacarbazine); mannomustine (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); a polycytidysine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa (thiotepa); taxanes (taxanes), e.g. paclitaxel (TAXOL)Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (docetaxel) (TAXOTERE)Rh6ne-PoulencRorer, Antony, France); chlorambucil (chlorambucil); gemcitabine (gemcitabine); 6-thioguanine (6-thioguanine); mercaptopurine (mercaptoprine); methotrexate; platinum analogs such as cisplatin (cissplatin) and carboplatin (carboplatin); vinblastine (vinblastine); platinum; etoposide (VP-16); ifosfamide (ifosfamide); mitomycin c (mitomycins c); mitoxantrone (mitoxantrone); vincristine (vincristine); vinorelbine (vinorelbine); navelbine (navelbine); norfloxacin (novantrone); teniposide (teniposide); daunorubicin (daunomycin); aminopterin (aminopterin); (xiloda); ibandronate (ibandronate); CPT-II; 35 topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid (retinic acid); an enediyne anthracycline (esperamicins); capecitabine (capecitabine); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included in this definition are anti-hormonal agents whose action modulates or inhibits hormonal effects on tumors, such as anti-estrogenic agents, including, for example, tamoxifen (tamoxifen), raloxifene (raloxifene), aromatase inhibiting 4(5) -imidazoles (4(5) -imidazoles, 4-Hydroxyttamoxifen (4-hydroxytamoxifen), trioxifene (trioxifene), keoxifene, LY117018, onapristone (onapristone), and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

"anti-angiogenic agent" refers to a compound that blocks or interferes to some extent with the development of blood vessels. For example, the anti-angiogenic agent can be a small molecule or an antibody that binds to a growth factor or growth factor receptor involved in promoting angiogenesis. In one embodiment, the anti-angiogenic agent is an antibody that binds to Vascular Endothelial Growth Factor (VEGF).

The term "cytokine" is a generic term that refers to a protein released by one cell population that acts as an intercellular modulator for another cell. Examples of such cytokines are lymphokines (lymphokines), monokines (monokines) and traditional polypeptide hormones. Included among cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin (proinsulin); relaxin; (ii) prorelaxin; glycoprotein hormones such as Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH), and Luteinizing Hormone (LH); a liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-a and-P; mullerian-inhibitive substance (mullerian-inhibitive substance); mouse gonadotropin-related peptides; inhibin (inhibin); activin (activin); vascular endothelial growth factor; integrins (integrins); thrombopoietin (TPO); nerve growth factors such as NGF-p; platelet growth factor; transforming Growth Factors (TGFs) such as TGF-a and TGF-p; insulin-like growth factors-I and-II; erythropoietin (EPO); osteoinductive factors (osteoinductive factors); interferons such as interferon-a, -p and-y, Colony Stimulating Factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-csf (gcsf); interleukins (ILs) such as IL-I, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-II, IL-12; tumor necrosis factor such as TNF-alpha or TNF-P; and other polypeptide factors, including LIF and Kit Ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, as well as biologically active equivalents of the native sequence cytokines.

The term "fMLP" denotes a tripeptide consisting of N-formylmethionine, leucine and phenylalanine. In one embodiment, the effector moiety is fMLP or a derivative thereof.

The term "fusion polypeptide" denotes a polypeptide comprising or consisting of at least two discrete peptides or polypeptides (which do not occur together in the native polypeptide in such a way, i.e. the moieties do not occur naturally in the same polypeptide or in the same order) in nature. The various portions of the fusion polypeptide are linked by peptide bonds.

The term "peptide linker" denotes linkers of natural and/or synthetic origin, comprising amino acid residues linked to each other by peptide bonds. It consists of a linear chain of amino acids, of which 20 naturally occurring amino acids are monomeric building blocks. The length of the chain is from 1 to 50 amino acid residues, in one embodiment from 3 to 28 amino acid residues, in another embodiment from 4 to20 amino acid residues. The linker may comprise a repeating amino acid sequence or a sequence of a naturally occurring polypeptide. The function of the joint is to ensure that the two elements connected by the joint can be correctly folded and correctly presented due to the spatial and rotational degrees of freedom. In one embodiment, the linker is a "synthetic peptide linker" designed to be rich in glycine, glutamine and/or serine residues. For example, these residues are arranged as small repeats of up to five amino acids, such as (G) GGGS, (Q) QQQG, or (S) SSSG (SEQ ID NO: 1,2 and 3). Such small repeating units may be repeated two to five times to formForming polymer units. Other synthetic peptide linkers comprise a single amino acid repeated 10-20 times, such as, for example, serine in linker GSSSSSSSSSSSSSSSG (SEQ ID NO: 4). In one embodiment, the linker is selected from [ GQ₄]₃GNN(SEQ ID NO∶5),LSLSPGK(SEQ ID NO∶6),LSPNRGEC(SEQ ID NO∶7),LSLSGG(SEQ ID NO∶8),LSLSPGG(SEQID NO∶9),G₃[SG₄]₂SG (SEQ ID NO: 10) or G₃[SG₄]₂SG₂(SEQ ID NO∶11)。

The term "prodrug" as used herein refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells than the parent drug and is capable of being enzymatically activated or converted to the more active parent form. See, for example, Wilman, "Prodrugs in cancer chemotherapy" Biochemical society of therapeutics, 14, pp. 375. 382., 615th Meeting Belfast (1986) and Stella et al, "Prodrugs: A Chemical Approach to Targeted drug delivery" direct drug delivery, "Borchardt et al, (eds.), pp. 247. 267. HumanaPress (1985). Prodrugs that may be used as effector moieties include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, β -lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorocytosine prodrugs that can be converted to more active non-cytotoxic drugs. Examples of cytotoxic drugs that may be derivatized into prodrug forms for use in the present invention include, but are not limited to, those chemotherapeutic agents described herein.

The term "cytotoxic moiety" refers to a substance that inhibits or prevents cellular function and/or causes cell destruction or death. Cytotoxic agents include, but are not limited toRadioisotope (e.g., At)²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、p³²、pb²¹²And radioactive isotopes of Lu); chemotherapeutic agents or chemotherapeutic agents (e.g., methotrexate), doxorubicin (adriamycin), vinca alkaloids (vinca alkaloids) (vincristine), vinblastine (vinblastine), etoposide (etoposide)), doxorubicin (doxorubicin), melphalan (melphalan), mitomycin c (mitomycin c), chlorambucil (chlorembucil), daunorubicin (daunorubicin), or other intercalating agents (intercalarting agents)); a growth inhibitor; enzymes and fragments thereof, such as nuclear hydrolases; (ii) an antibiotic; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various antineoplastic or anticancer agents disclosed herein.

An "effective amount" of an agent, e.g., a pharmaceutical formulation, is an amount effective to achieve the desired therapeutic result or prophylactic structure at the desired dosage and for the desired period of time.

The term "EC₅₀The value "represents the half-maximal effective concentration of the polypeptide (e.g. antibody) in a determination system (e.g. ELISA) that induces a 50% response between the baseline value and the maximum value. This is a measure of the efficacy of a therapeutic drug. Thus, EC₅₀The value is the concentration calculated on the basis of experimental data corresponding to the concentration of the drug substance that causes 50% of the effect. Reduced EC₅₀Values represent higher drug affinity and efficacy.

A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source using the human antibody repertoire or other sequence encoding a human antibody. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues.

"humanized" antibodies refer to chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In particular embodiments, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody optionally will also comprise antibody constant regions derived at least in part from a human antibody. "humanized forms" of antibodies (e.g., non-human antibodies) refer to antibodies that have been humanized.

An "immunoconjugate" is an antibody or antibody fragment conjugated to one or more molecules of non-antibody origin, including, but not limited to, a binding pair member, a nucleic acid, or an effector moiety.

An "individual" or subject "is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

The term "internalized cell surface receptors" denotes a group of cell surface receptors comprising at least the following members: asialoglycoprotein receptor, α (2,3) sialoglycoprotein receptor, diphtheria toxin receptor (DTR, which is a membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor (HB-EGF)), folate receptor, glutamate receptor, glutathione receptor, insulin-like growth factor (IGF) receptor, leptin receptor, Low Density Lipoprotein (LDL) receptor, LDL-related protein 1 receptor (LRP1, type B), LRP2 receptor (also known as megalin or glycoprotein 330), LRP4 receptor, LRP5 receptor, LRP6 receptor, LRP8 receptor, mannose 6-phosphate receptor, clearance receptor (class a or B, I, II or type III, or CD36 or CD163), substance P receptor, thiamine transporter, transferrin-1 and-2 receptor, and vitamin B12 receptor.

The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies comprising the population are identical and bind the same epitope, except for possible variant antibodies (e.g., which comprise naturally occurring mutations or arise during the course of producing a monoclonal antibody preparation, such variants typically being present in minor amounts). In contrast to polyclonal antibody preparations, which typically comprise different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies or monoclonal antibody fragments to be used in the fusion polypeptides reported herein can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of a human immunoglobulin locus, as well as other exemplary methods for preparing monoclonal antibodies described herein.

The term "pharmaceutical formulation" refers to a preparation which is present in a form which allows the biological activity of the active ingredient contained therein to be effective, and which does not contain additional ingredients which are unacceptably toxic to the subject to which the formulation is to be administered.

By "pharmaceutically acceptable carrier" is meant an ingredient of a pharmaceutical formulation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

The term "transcellular transport" refers to the multi-step process of transport of molecules, particularly macromolecules or biopolymers such as antibodies, through the cytoplasm of cells. In the first step of transcellular transport, the extracellular space material/molecule or cell surface-associated or-bound molecule is enclosed in a vesicle. This step is called endocytosis. The vesicles diffuse through the cytoplasm of the cell. The endocytosis step is then reversed, i.e. the vesicles fuse with the cell membrane and the interior of the vesicles is released into the extracellular space. For example, transcellular transport occurs in epithelial cells, cells of the blood-brain barrier, neurons, or intestinal cells.

As used herein, "treatment" (and grammatical variations thereof, such as "treating" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and may be performed for prophylactic purposes or during the course of clinical pathology. The desired therapeutic effect includes, but is not limited to, preventing occurrence or recurrence of disease, alleviating symptoms, eliminating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating a disease condition, and slowing or improving prognosis. In some embodiments, the fusion polypeptides as reported herein are used to slow down the onset of disease or slow down the progression of disease.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in the binding of an antibody to its antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, each domain comprising four conserved Framework Regions (FRs) and three hypervariable regions (HVRs) (see, e.g., t.j., et al, Kuby Immunology, 6)^thed., w.h.freeman and co., n.y. (2007), page 91). A single VH or VL domain may be sufficient to confer antigen binding specificity. Furthermore, antibodies that bind a particular antigen can be isolated from antigen-binding antibodies by screening libraries of complementary VH or VL domains with VH or VL domains, respectively (see, for example, Portolano, S., et al, J.Immunol. J.150 (1993) 880-628, Clackson, T., et al, Nature (Nature) 352(1991) 624-628).

The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors which are self-replicating nucleic acid structures as well as vectors which are incorporated into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

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. For example, "an antibody" means one antibody or more than one antibody.

A "polypeptide" is a polymer, whether naturally occurring or synthetically produced, consisting of amino acids joined by peptide bonds. Polypeptides of less than about 20 amino acid residues may be referred to as "peptides", while molecules consisting of two or more polypeptides or molecules comprising one polypeptide of more than 100 amino acid residues may be referred to as "proteins". The polypeptide may also comprise non-amino acid components, such as carbohydrate groups, metal ions or carboxylic acid esters. The non-amino acid components may be added by the cell expressing the polypeptide and may vary with the cell type. Polypeptides are defined herein in terms of their amino acid backbone structure or the nucleic acids that encode them. Such as the addition of carbohydrate groups, is not generally specified, but may also be present.

The term "specifically binds" means that the binding site or polypeptide or antibody fragment has a 10 relationship to its target^-5Dissociation constant (Kd) binding of M or less, in one embodiment at 10^-7M-10^-13Dissociation constant (Kd) binding of M, in another embodiment, at 10^-7M-10^-9The dissociation constant (Kd) of M binds. The term is also used to indicate that the polypeptide does not bind to other biomolecules present, i.e., it is present at 10^-4Dissociation constants (Kd) above M for binding to other biomolecules, in one embodiment at 10^-4The dissociation constant (Kd) of M to 1M binds to other biomolecules.

The term "pharmaceutically active compound" denotes any molecule or combination of molecules whose activity needs to be delivered to the site of action. Pharmaceutically active compounds include, but are not limited to, drugs (e.g., polypeptides, antibodies), labels, cytotoxins (e.g., Pseudomonas exotoxin, ricin, abrin, diphtheria toxin, etc.), enzymes, growth factors, transcription factors, radionuclides, ligands, liposomes, nanoparticles, viral particles, cytokines, and the like.

Compositions and methods

Herein reported are fusion polypeptides with which therapeutic agents (biologically active compounds) such as polypeptides, antibodies or toxins can be transported across cell membranes, in particular across the blood-brain barrier. Thus, the fusion polypeptides as reported herein utilize a universal transport mechanism, i.e. receptor-mediated endocytosis and endocytosis transport using internalized cell surface receptors.

In one embodiment, herein is reported a fusion polypeptide comprising:

-at least one binding site that binds to an internalized cell surface receptor, and

-at least one pharmaceutically active compound,

EC determined at pH5.5 by the binding site bound to the internalized cell surface receptor₅₀EC with value determined at pH7.4 for the same binding site of the same receptor₅₀-a ratio of values above 10.

In one embodiment, the ratio is 15 or greater.

In one embodiment, the ratio is 100 or greater.

In one embodiment, the ratio is from 10 to 100.

In one embodiment, the binding site has an EC determined at pH5.5₅₀-a value of 700ng/ml or more. In one embodiment, the binding site has an EC determined at pH5.5₅₀-a value of above 850 ng/ml. In one embodiment, the binding site has an EC determined at pH5.5₅₀-value is above 1000 ng/ml.

In one embodiment, the binding site is a binding pair comprising an antibody heavy chain variable domain and an antibody light chain variable domain. In one embodiment, the binding pair is selected from the group consisting of Fv, Fab '-SH, F (ab')₂Double antibody, lineSex antibodies, single chain antibody molecules and multispecific antibodies, full-length heavy chains, full-length light chains, intact antibodies, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, or hexaspecific antibodies formed from antibody fragments. In one embodiment, the binding pair is an intact monoclonal antibody. In one embodiment, the binding pair is at least a fragment of an intact antibody, a member of the immunoglobulin superfamily, or a polypeptide having an immunoglobulin-like structure that retains binding specificity for its antigen.

In one embodiment, the binding site is selected from the group consisting of fibronectin, TCR, CTLA-4, single chain antigen receptors, e.g., those associated with T cell receptors and antibodies, antibody mimetics, transferrin, apolipoproteins, adnectins, anticalins-based molecules, phylomes, avimers, affibodies, ankyrin repeats, Kunitz domains, PDZ-domains, scorpion toxin immunity proteins, Knottins, Versabodies, green fluorescence proteins, and other non-antibody protein scaffolds with binding properties.

In one embodiment, the binding site is a full length antibody or antibody fragment that specifically binds transferrin receptor.

Without wishing to be bound by theory, figure 1 shows a schematic diagram of the pH-dependent endocytic transport mechanism. Iron-loaded intact transferrin (holo-transferrin) (middle panel) is transcytosially transported with transferrin receptor from the apical-lateral membrane of brain endothelial cells. When endosomes are acidified, iron is released from the intact transferrin, which is initiated by a conformational change in the transferrin-binding domain of the receptor. Apo-transferrin remains bound to the receptor. After passing through the sorting endosomes, the receptor is either recycled to the apical membrane or transcytosed into the basolateral membrane. After vesicle membrane fusion, apo-transferrin, which has no affinity for the receptor at pH7.4, dissociates from the receptor and leaves the cell. In contrast (left panel), the transferrin-receptor antibody mAb128.1, which binds to the receptor with high affinity at pH7.4 and at pH5.5, i.e., has an EC of less than 5₅₀Value ratio (1.3) which forms a tight complex with the receptor in the region ofIt is also stable in endosomes at this pH. The presence of the pH-stable complex prevents recirculation and transcytosis, rather than inducing reentry of the receptor into the CD 63-positive late endosome. Anti-transferrin-receptor antibodies with pH dependent binding properties (exemplified by antibody MEM-189, which exhibits reduced receptor binding at reduced (acidic) pH values at the pH of the body; right panel, EC > 10₅₀Value ratio (15.6)) enables transcytosis and recycling, possibly by reversible, low affinity interaction with transferrin-receptor in the endosomal compartment, without being bound by theory.

In FIG. 3, validation of the transcytosis assay used herein, which is via transcytosis, is shown¹²⁵I-transferrin proceeds through hCMEC/D3 brain endothelial cells. Use of hCMEC/D3 cells on collagen-coated Filter inserts¹²⁵I-labeled transferrin was loaded for 1 hour. The inserts were then washed and transferred to new plates at 37 ℃ (fig. 3A) or 4 ℃ (fig. 3B). At the time points shown, the radioactivity in the cell lysates (black squares), apical (grey bars) and basolateral (white bars) media compartments was measured by gamma Counting (CPM) after TCA precipitation. The sum of the radioactivity at each time point is shown as a white triangle. Although at 37 ℃, transferrin leaves the cell layer in equal amounts into the apical or basolateral media compartment (a), at 4 ℃, it remains intracellular (B), demonstrating that the transport process is energy-dependent.

In FIG. 4, it is shown that mAb128.1 directed against the human transferrin receptor does not leave hCMEC/D3 cells.¹²⁵I-labelled mAb128.1 was allowed to be taken up by hCMEC/D3 cells and radioactivity was determined in the cell, apical and basolateral media compartments as described above (figure 3). No intact antibody leaves the cell into the apical or basolateral compartment. In contrast, the intracellular radioactivity decreased slowly, providing an indication of intracellular degradation of the antibody.

In figure 5, mAb128.1 directed against the human transferrin receptor, unlike transferrin, is shown to be co-localized after internalization at the late endosomal marker CD 63. hCMEC/D3 cells grown on collagen-coated coverslips were incubated with mAb128.1 or FITC-labeled transferrin for ten minutes and then treated for immunofluorescence. mAb128.1 was detected with Alexa-488-labeled secondary antibody (a), panel C showing transferrin-FITC fluorescence. Both preparations were counterstained with an antibody against the late endosomal marker CD63 and an Alexa-594-labeled secondary antibody (B, D). Although mAb128.1 was shown to be co-localized with CD63, no transferrin was found in the late endosomal/lysosomal compartment, suggesting that transferrin receptor exits recycling/endocytic transport through mAb128.1 and reenters the degradation trafficking pathway.

In FIG. 6, it is shown that the antibody directed against the human IGF-1 receptor (anti-IGF-1R antibody) is not transcytosed but is recycled into the extracellular medium. The transcytosis assay was performed as described above (see fig. 2 and 3), except that antibody quantification was not performed by radioactivity counting but by using a highly sensitive human IgG ELISA. It can be seen that the anti-IGF-1R antibody is not transcytosed, but is recycled into the apical compartment, demonstrating that IGF-1 receptor is exclusively recycled in blood brain barrier endothelial cells.

In FIG. 7, mAb MEM-189, directed against the human transferrin receptor, is shown to be recycled and transcytosed, unlike mAb 128.1. The experiment was performed as described above (see FIG. 6) and quantification was performed using mouse IgGELISA. In contrast to mAb128.1, mAb128.1 was not present in the apical or basolateral compartment (see above, fig. 4), mAb MEM-189 was recycled and transcytosed in equal amounts, with a transport rate slightly lower than that of transferrin (see also fig. 3A).

In FIG. 8, mAb MEM-189 is shown to bind to transferrin receptor in a pH-dependent manner, whereas mAb128.1 does not show pH-dependent binding. Binding of antibodies 128.1 and MEM-189 to the extracellular domain of human transferrin receptor was measured by ELISA at pH7.4 (extracellular pH) or pH5.5 (endosomal pH). Although mAb128.1 binds with similar affinity to the receptor at these two pH values (triangles at pH7.4, crosses at pH 5.5), mAb MEM-189 exhibits strongly reduced binding at pH5.5 (inverted triangles) compared to pH7.4 (circles). At pH7.4, mAb MEM-189 binds to the receptor less strongly than mAb128.1 (see EC in the Table below)₅₀-value). In the following table, the EC of various antibodies against cell surface receptors are shown₅₀Value and its respective EC₅₀Ratio of values.

Watch (A)

In FIG. 9, mAbs128.1 are shown to compete with MEM-189 for the same epitope on transferrin receptor. Transferrin receptor extracellular domain was coated onto microtiter plates and pre-incubated with mAbs128.1 or MEM-189 prior to detection of binding of various other mAbs. Pre-incubation with mAb128.1 completely blocked the binding of mAb MEM-189 to the receptor (inverted triangle) compared to binding in the absence of mAb128.1 (circle). In contrast, mAb128.1 binding to the receptor was not inhibited by pre-incubation with mAb MEM-189 (triangles and crosses, respectively). Taken together, mAb MEM-189 competes for the same epitope on human transferrin receptor as mAb 128.1. The fact that mAb MEM-189 was unable to prevent mAb128.1 from binding can be explained by the significantly higher affinity of mAb 128.1.

In figure 10, it is shown that antibodies M-a712 and 13E4 (both of which do not exhibit pH-dependent binding) directed against the human transferrin receptor are not transcytosed across hCMEC/D3 cells.

The above data clearly demonstrate that the essential feature for antibody transcytosis is not the receptor epitope, but rather the binding affinity to the receptor and the pH-dependent change in binding affinity.

1. Affinity of

In some embodiments, the binding site of a fusion polypeptide provided herein has a dissociation constant (Kd) of ≦ 10 μ M ≦ 1 μ M ≦ 100 μ M ≦ 10nM, or ≦ 1nM (e.g., in one embodiment, about 10nM^-5M toAbout 10^-9M, or in another embodiment, about 10^-7M or less, e.g. 10^-7M to 10^-13M, e.g. 10^-9M to 10^-13M)。

In one embodiment, Kd is measured by performing a radiolabeled antigen binding assay (RIA) as described in the assay described below, wherein the binding site is a Fab fragment of an antibody and its antigen. Solution binding affinity of FABs to antigen by using the lowest concentration of (in the presence of a titration series of unlabeled antigen)¹²⁵I) The labeled antigen equilibrates the Fab, which is then measured by capturing the bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen, Y, et al, J.Mol.biol. (J.Mol.Biol.) (J.Mol.) -293 (1999) 865-881). To determine the assay conditions, MICROTITER was usedMulti-well plates (Thermo Scientific) were coated overnight with 5. mu.g/ml capture anti-Fab antibodies (Cappel Labs) in 50mM sodium carbonate (pH9.6) and then blocked with 2% (w/v) bovine serum albumin in PBS for 2-5 hours at room temperature (about 23 ℃). In a non-absorbent plate (Nunc #269620), 100pM or 26pM [ alpha ] amino acid is prepared¹²⁵I]Mixing of antigen with serial dilutions of Fab of interest (e.g., consistent with evaluation of anti-VEGF antibody, Fab-12, in Presta, L.G., et al, Cancer Res. (Cancer research) 57(1997) 4593-. Then, the Fab of interest was incubated overnight; however, the incubation may be continued for a longer period of time (e.g., about 65 hours) to ensure that equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., one hour). Then, the solution was removed and the plate was plated with 0.1% polysorbate 20 in PBSAnd washing eight times. When the plates had dried, 150. mu.l/well of scintillator (MICROSCINT-20) was added^TM(ii) a Packard), plate on TOPCOUNT^TMCount on a gamma counter (Packard) for ten minutes. The concentration of each Fab giving less than or equal to 20% of the maximal binding was selected forCompetitive binding assays.

According to another embodiment, Kd is determined by surface plasmon resonance using BIACORE-2000 or BIACORE-3000(BIAcore, inc., Piscataway, NJ) measured at 25 ℃ using an immobilized antigen CM5 chip at-10 Response Units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen was diluted to 5. mu.g/ml (. about.0.2. mu.M) with 10mM sodium acetate pH4.8 and then injected at a flow rate of 5. mu.l/min to obtain about 10 Response Units (RU) of conjugated protein. After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, a solution containing 0.05% polysorbate 20 (TWEEN-20) was injected at 25 ℃ at a flow rate of about 25. mu.l/min^TM) Two-fold serial dilutions of Fab (0.78nM to 500nM) in surfactant pbs (pbst). Using a simple one-to-one Langmuir (Langmuir) binding model (BIACORE)Evaluation Software version 3.2) calculation of the Association Rate (k) by Simultaneous fitting of Association and dissociation sensorgrams_on) And dissociation rate (k)_off). Equilibrium dissociation constant (Kd) in the ratio k_off/k_onAnd (4) calculating. See, e.g., Chen, Y et al, J.mol.biol. (J.Mol.Biol.) (J.Mol.) -293 (1999) 865-881. If according to the above surface plasmon resonance determination, the binding rate exceeds 10⁶M^-1S^-1The rate of binding can then be determined using fluorescence quenching techniques, i.e.using a stirred cuvette (st) in a spectrometer such as a stop-flow-compensated spectrophotometer (Avivinstruments) or a 8000 series SLM-AMINO TM spectrophotometer (Thermospectric) equipped with a flow-breaking deviceirred cuvette), in the presence of increasing concentrations of antigen, PBS, 20nM anti-antigen antibody (Fab form) in pH 7.2, was measured for fluorescence emission intensity at 25 ℃ (excitation 295 nM; emission 340nm, 16nm bandpass) increase or decrease.

2. Antibody fragments

In certain embodiments, the fusion polypeptide as reported herein comprises an antibody fragment as binding site. Antibody fragments include, but are not limited to, Fab, Fab ', Fab ' -SH, F (ab ')₂Fv, and scFv fragments, as well as other fragments described below. For a review of specific antibody fragments, see Hudson, p.j., et al, nat. med. (natural medicine) 9(2003) 129-. For reviews of scFv fragments, see, e.g., Plueckthun, a, at: the Pharmacology of monoclonal antibodies (Pharmacology of monoclonal antibodies), Vol.113, Rosenburg and Moore (eds.), Springer-Verlag, New York (1994), p.269-315; see also WO 93/16185; and U.S. patent nos. 5,571,894 and 5,587,458. See U.S. Pat. No. 5,869,046 for a discussion of Fab and F (ab') 2 fragments that contain salvage receptor (salvaging receptor) binding epitope residues and have increased half-life in vivo.

Diabodies are antibody fragments with two antigen binding sites, which may be bivalent or bispecific. See, for example, EP 0404097; WO 1993/01161, Hudson, P.J., et al, Nat.Med. (Nature medicine) 9(2003) 129-. Trivalent and tetravalent antibodies have also been described in Hudson, P.J., et al, Nat.Med. (Nature medicine) 9(2003) 129-.

A single domain antibody is an antibody fragment comprising all or part of a heavy chain variable domain or all or part of a light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516B 1).

Antibody fragments can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and preparation by recombinant host cells (e.g., e.coli or phage), as described herein.

3. Multispecific antibodies

In certain embodiments, the fusion polypeptide as reported herein is a multi-specific fusion polypeptide, e.g. a bispecific fusion polypeptide. The multispecific fusion polypeptide has binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is directed to an internalized cell surface receptor and the other is directed to a therapeutic target. In certain embodiments, a bispecific fusion polypeptide can bind two different epitopes of the internalized cell surface receptor. Bispecific fusion polypeptides can be prepared as full-length fusion polypeptides or fusion polypeptide fragments.

In one embodiment, the binding site of the fusion polypeptide is an intact antibody.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein, C. and Cuello, A.C., Nature (Nature) 305(1983)537 540), WO 93/08829, and Traunecker, A., et al, EMBO J.10(1991)3655-3659), and "bump-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be made by: engineering the electrostatic steering effect for the production of antibody Fc-heterodimeric molecules (WO 2009/089004a 1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan, M., et al, Science, 229(1985) 81-83); the use of leucine zippers to generate bispecific antibodies (see, e.g., Kostelny, s.a., et al, j.immunol. (journal of immunology) 148(1992) 1547-; bispecific antibody fragments are prepared using the "diabody" technique (see, e.g., Holliger, P., et al, Proc. Natl. Acad. Sci. USA (Proc. Natl. Acad. Sci. USA) 90(1993) 6444-; and the use of single-chain fv (sFv) dimers (see, e.g., Gruber, M., et al, J.Immunol. (J.Immunol., 152 (1994)) 5368-5374); and trispecific antibodies prepared as described, for example, in Tutt, a., et al, j.

Also included herein are engineered antibodies with more than three functional antigen binding sites, including "Octopus antibodies" (see, e.g., US 2006/0025576a 1).

The antibodies or fragments also include "dual action FABs" or "DAFs" comprising an antigen binding site that binds to an internalized cell surface receptor and another, different antigen (see, e.g., US 2008/0069820).

The antibodies or fragments herein also include multispecific antibodies described in WO 2009/080251, WO 2009/080252, WO 2009/080253, WO 2009/080254, WO 2010/112193, WO 2010/115589, WO 2010/136172, PCT application No. PCT/EP2010/003559 or PCT application No. PCT/EP 2010/003560.

4. Derivatives of the same

In certain embodiments, the fusion polypeptides reported herein may be further modified to comprise additional non-protein like moieties known in the art and readily available. Suitable derivatized moieties for fusion polypeptides include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in preparation due to its stability in water. The polymer may appear to be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, it can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on the following considerations: such considerations include, but are not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative is to be used in a therapy under defined conditions, and the like.

In another embodiment, conjugates of fusion polypeptides and non-protein-like moieties that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-protein-like moiety is a carbon nanotube (Kam, n.w., et al, proc.natl.acad.sci.usa (proceedings of the national academy of sciences usa) 102(2005) 11600-. The radiation may be of any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells but which heat the non-protein like moiety to a temperature that kills cells adjacent to the antibody-non-protein like moiety.

5. Measurement of

The fusion polypeptides or components thereof as reported herein can be identified, screened or characterized for their physical/chemical properties and/or biological activity by various assays known in the art.

5.1 binding assays and other assays

In one aspect, the cell surface receptor binding activity of the binding site of the fusion polypeptides as reported herein is detected, e.g., by known methods such as ELISA, western blot, etc.

In one aspect, a competition assay may be used to identify other binding sites, particularly antibodies or antibody fragments that compete with mAb MEM-189 for binding to transferrin receptor. In certain embodiments, the competing antibody binds to the same epitope (e.g., a linear or conformational epitope) as mAb MEM-189. Morris, g.e., (ed.), "Epitope Mapping Protocols," in: detailed exemplary Methods for mapping epitopes are provided in Methods in Molecular Biology, Vol.66, Humana Press, Totowa, NJ (1996).

"antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of 50% or more of the reference antibody to its antigen in a competition assay, and conversely, blocks binding of 50% or more of the antibody to its antigen in a competition assay.

For example, a fusion protein of the extracellular domain of the human transferrin receptor linked to human IgGl Fc can be coated onto a 96-well plate by incubating 50. mu.l of a 1. mu.g/ml solution in PBS for 1h at RT. After blocking with PBS/1% (w/v) BSA for 1h and washing four times with PBS/0.1% (w/v) Tween, different concentrations of the test antibody in PBS/0.1% (w/v) BSA adjusted to pH7.4 or pH5.5 can be added to the plate and incubated at RT for 1.5 h. After four washes with PBS/0.1% (w/v) tween, bound antibody can be detected with HRP-conjugated secondary antibody (30min., RT) and 50 μ l TMB substrate. The color development can be stopped by adding 50. mu.l of 1N hydrochloric acid (HCl) and the absorbance can be measured at 450nm in a plate reader.

5.2 Activity assay

Media and supplements for hCMEC/D3 (see WO 2006/056879 and Weksler, B.B., et al, FASEB J.19(2005)1872-1874) are available from Lonza. hCMEC/D3 cells (passage 26-29) can be cultured to confluence on collagen-coated coverslips (microscope) or flasks in EBM2 medium containing 2.5% FBS, one quarter and a supply of growth factor, gentamicin and ascorbic acid fully complementary to the supplemented hydrocortisone.

For all transcytosis assays, high density wells (1X 10) can be used in 12-well cell culture plates⁸Hole/cm²) PET membrane filter insert (0.4 μm pore size, 12mm diameter). The volume of medium used in the apical and basolateral chambers was calculated to be 400. mu.l and 1600. mu.l, respectively. The apical compartment of the filter insert may be treated with rat tail collagen I (7.5. mu.g/em)²) Coated and then coated with fibronectin (5. mu.g/ml) and each incubation was continued at RT for 1 h. hCMEC/D3 cells can grow to confluent cells in EBM2 medium for 10-12 daysLayer (2X 10)⁵Individual cell/cm²). The air filters can be blocked for 1h or overnight (o/n) in PBS containing 1% BSA before assay, and then corrected for at least 1h in EBM2 before assay.

The assay (see figure 2 for assay protocol) can be performed in serum-free EBM2 medium (which is otherwise reconstituted as described herein). On the day of the assay, cells were serum-starved for 60min to deplete the natural ligands of the internalized cell surface receptors to be detected. The filter insert with or without (but closed overnight in complete medium) cells is topcoated with the radiolabeled natural ligand of the internalized cell surface receptor to be tested, i.e.,¹²⁵i-labeled or unlabeled monoclonal antibodies were incubated at 37 ℃ for 1 h. Thereafter, the entire topside and bottom outside volumes are collected. The paracellular flow rate can be calculated from the determined value. Monolayers were washed three times at RT in serum free medium apical (400. mu.l) and basolateral (1600. mu.l), 3-5min each. All wash volumes were collected to monitor the efficiency of removal of unbound ligand or antibody. Pre-incubated medium was added to the apical chamber and the filter was transferred to a new 12-well plate (blocked overnight with PBS containing 1% BSA) containing 1600 μ l of pre-incubated medium. At this point, the filters with or without cells were lysed in 500 μ l RIPA buffer to determine specific ligand or antibody uptake. The remaining filters are incubated at 37 ℃ or at 4 ℃ and samples are collected at various time points to determine the release of the top side and/or bottom outside of the ligand or antibody. Evaluation of integrity and degradation Using trichloroacetic acid (TCA) precipitation¹²⁵I-natural ligands labeled or¹²⁵I-labeled antibodies. The amount of radioactive native ligand or antibody in the supernatant or lysate can be determined by gamma ray counting. The amount of unlabeled antibody in the sample can be quantified using a high selectivity IgG ELISA (see example 3). For each time point, data should be generated from two blank filters and three filter cell cultures.

6. Recombinant methods and compositions

Fusion polypeptides can be produced using recombinant methods and compositions. In one embodiment, an isolated nucleic acid encoding a fusion polypeptide as reported herein is provided. In another embodiment, one or more vectors (e.g., expression vectors) comprising the nucleic acids are provided. In another embodiment, a host cell comprising the nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with) one or more vectors comprising a nucleic acid encoding an amino acid sequence comprising the fusion polypeptide. In one embodiment, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, SP2/0 cell). In one embodiment, a method of producing a fusion polypeptide as reported herein is provided, wherein the method comprises culturing a host cell as provided above comprising a nucleic acid encoding the fusion polypeptide under conditions suitable for expression of the fusion polypeptide, and optionally recovering the fusion polypeptide from the host cell (or host cell culture medium).

For recombinant production of the fusion polypeptides as reported herein, the nucleic acid encoding the fusion polypeptide as reported herein is isolated, e.g. as described above, and inserted into one or more vectors for further cloning and/or expression in a host cell. The nucleic acids can be readily isolated and sequenced using conventional procedures.

Suitable host cells for cloning or expression of the polypeptide-encoding vector include prokaryotic or eukaryotic cells as described herein. For example, the polypeptide may be produced in bacteria, particularly when glycosylation is not required. For expression of antibody fragments and polypeptides, see, e.g., U.S. Pat. nos. 5,648,237,5,789,199, and 5,840,523. (see also Charlton, K.A., in: Methods in molecular biology, Vol.248, Lo, B.K.C., (ed.), Humana Press, Totowa, NJ (2003), page 245-. After expression, the polypeptide can be separated from the bacterial cell cytoplasm in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeast, are also suitable cloning or expression hosts for polypeptide-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized", resulting in the production of fusion polypeptides having a partially or fully human glycosylation pattern. See Gerngross, T.U., Nat.Biotech. (Nat Biotech.) 22(2004) 1409-shot 1414, and Li, H., et al, Nat.Biotech. (Nat Biotech.) 24(2006) 210-shot 215.

Host cells suitable for expression of glycosylated polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Various baculovirus strains have been identified which can be used in combination with these insect cells, in particular for transfecting Spodoptera frugiperda (Spodoptera frugiperda) cells.

Plant cell cultures may also be used as hosts. See, for example, U.S. Pat. Nos. 5,959,177,6,040,498,6,420,548,7,125,978 and 6,417,429 (which describe PLANTIBODIIES for antibody production in transgenic plants^TMA technique).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CVl cell line transformed with SV40 (COS-7); human embryonic kidney cell lines (293 or 293 cells as described, for example, in Graham, f.l., et al, j.gen.virol.36(1977) 59-74); baby hamster kidney cells (BHK); mouse support cells (TM4 cells, as described, for example, in Mather, J.P., biol. reprod.23(1980) 243-; monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); bovine rat (buffalo rat) hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described in Mather, J.P., et al, Annals N.Y.Acad.Sci.383(1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR^-CHO cells (Urlaub, g., et al., proc.natl.acad.sci.usa (national academy of sciences usa)Bulletin) 77(1980) 4216-; and myeloma cell lines, such as Y0, NS0 and Sp 2/0. For a review of specific mammalian host cell lines suitable for antibody production, see, e.g., Yazaki, p. and Wu, a.m., in Methods in molecular Biology, vol.248, Lo, b.k.c. (ed.), HumanaPress, Totowa, NJ (2004), page 255-268.

7. Immunoconjugates

Also provided herein are fusion polypeptides, wherein at least one of the components, such as the effector moiety, is, for example, a cytotoxic agent, such as a chemotherapeutic agent or chemotherapeutic drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioactive isotope.

In one embodiment, the effector moiety is a drug or pharmaceutically active compound, including, but not limited to, maytansinoids (see US5,208,020, US5,416,064, EP 0425235), auristatins, such as monomethyl auristatin drug moiety DE and DF (MMAE and MMAF, see US5,635,483, US5,780,588, US 7,498,298), dolastatin (dolastatin), calicheamicin (calicheamicin) or derivatives thereof (see US5,712,374, US5,714,586, US5,739,116, US5,767,285, US5,770,701, US5,770,710, US5,773,001, US5,877,296, Hinman, l.m., et al, Cancer Res 53 (331993) 53, lof 3342, lof 2942, n 2913, c 2927, c 2913, c 2975, c 2933, c 35, c 2933, c 2975, c 35, c 2933, c 35, mede.chem.letts (bio-organic chemical communication) 16(2006) 358-.

In another embodiment, the effector moiety is an enzymatically active toxin or fragment thereof, including, but are not limited to diphtheria a chain, non-binding active fragments of diphtheria toxin, exotoxin a chain (from Pseudomonas aeruginosa), ricin a chain, abrin a chain, modeccin a chain, α -fumagillin (sarcin), aleurites fordii (aleuties fordii) protein, dianthin protein, phytolacca americana (phytolacca americana) protein (PAPI, PAPII and PAP-S), momordica charantia (momordia) inhibitor, curcin (curcin), crotin (crotin), saponaria officinalis (sapaonaria officinalis) inhibitor, gelonin (gelonin), mitomycin (restrictocin), restrictocin (restrictocin), exotoxin (theomycin), and enomycin (enomycin).

In another embodiment, the effector moiety is a radioactive atom. A variety of radioactive isotopes are useful for preparing radioconjugates. Examples include At²¹¹，I¹³¹，I¹²⁵，Y⁹⁰，Re¹⁸⁶，Re¹⁸⁸，Sm¹⁵³，Bi²¹²，p³²，pb²¹²And radioactive isotopes of Lu. When detection is performed using a radioconjugate, a radioactive atom, e.g., Tc, for scintigraphic studies may be included⁹⁹m or I¹²³Or spin labels for Nuclear Magnetic Resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), as also I¹²³，I¹³¹，In¹¹¹，F¹⁹，C¹³，N¹⁵，O¹⁷Gadolinium, manganese or iron.

Effector moieties can be fused to the binding site of the fusion polypeptides reported herein using a variety of bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), Iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate hcl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis (p-diazoniumbenzoyl) ethylenediamine), diisocyanates (such as toluene 2, 6-diisocyanate), and a di-active fluoro compound component (such as 1, 5-difluoro-2, 4-dinitrobenzene). For example, ricin immunotoxins may be prepared as described in Vitetta, E.S., et al, Science 238(1987) 1098-. Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating a radioactive nucleotide to a fusion polypeptide (see WO 94/11026). The linker used to conjugate the toxic moiety to the fusion polypeptides reported herein may be a "cleavable linker" that facilitates the release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers (Chari, R.V., et al, Cancer Research 52(1992) 127-.

Effector moieties may be fused to the binding site of the fusion polypeptides reported herein by linkers, for example, but not limited to, the conjugates prepared with cross-linking linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, thio-EMCS, thio-GMBS, thio-KMUS, thio-MBS, thio-SIAB, thio-SMCC, and thio-SMPB, and SVSB (succinimidyl- (4-vinylsulfone) benzoate), these are (e.g., commercially available from Pierce Biotechnology, inc., Rockford, il., USA).

The effector moiety may be fused to the binding site by a peptide linker. In one embodiment, the peptide linker has 4-20 amino acid residues. In one embodiment, the linker is atThe repeat-motif-molecules are identical, and in another embodiment, the conjugate comprises a linker having two or more different amino acid sequences. In another embodiment, the linker is selected from (G)₃S)，(G₃S)₂，(G₃S)₃，(G₃S)₄，(G₃S)₅，(G₄S)，(G₄S)₂，(G₄S)₃，(G₄S)₄，(G₄S)₅(SEQ ID NO: 1 and SEQ ID NO: 12to20), in particular selected from (G)₄S)₃And (G)₄S)₄(SEQ ID NO: 18 vs SEQ ID NO: 19).

8. Methods and compositions for diagnosis and detection

In certain embodiments, any of the fusion polypeptides provided herein can be used to detect the presence of a target in a biological sample that is specifically bound by a binding site in the fusion polypeptide. The term "detecting" as used herein encompasses quantitative or qualitative detection.

In one embodiment, the fusion polypeptide is provided for use in a diagnostic or detection method. In another aspect, there is provided a method of detecting the presence of a target of a binding site or effector portion of a fusion polypeptide as reported herein in a biological sample. In certain embodiments, the method comprises contacting a biological sample with a fusion polypeptide as reported herein under conditions allowing binding of the binding site or effector moiety to the intended target, and detecting whether a complex is formed between the fusion polypeptide and the target. The method may be an in vitro or in vivo method. In one embodiment, the fusion polypeptides as reported herein are used for selecting a subject suitable for treatment with an isolated polypeptide comprised in the fusion polypeptide, e.g. wherein the target is a biomarker for selecting a patient.

In certain embodiments, a labeled fusion polypeptide is provided, i.e., a fusion polypeptide in which the effector moiety is a label. Labels include, but are not limited to, directly detectable labels (e.g., fluorescent labels, luminescent labels, fluorescent labels,electron density labels, chemiluminescent labels, and radioactive labels) as well as indirectly detectable labels (e.g., through an enzymatic reaction or molecular interaction), such as an enzyme or a ligand. Exemplary labels include, but are not limited to, the radioisotope p³²，C¹⁴，I¹²⁵，H³And I¹³¹Fluorophores such as rare earth chelators or luciferin and derivatives thereof, rhodamine and derivatives thereof, dansyl, umbelliferone, luciferases, e.g., firefly luciferase and bacterial luciferase (US 4,737,456), luciferin, 2, 3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β -galactosidase, glucoamylase, lysozyme, carbohydrate oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, which are coupled with an enzyme using hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, phage labels, stable free radicals, and the like.

Pharmaceutical preparation

Pharmaceutical formulations of the fusion polypeptides as reported herein (Osol, a., (ed.) Remington's Pharmaceutical Sciences 16th edition (1980)) are prepared by mixing said fusion polypeptides with the required purity with one or more optional Pharmaceutical carriers, in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers, which are generally non-toxic to recipients at the dosages and concentrations employed, include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexane diamine chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl parabens, such as methyl or propyl parabens, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin or immunoglobulins, hydrophilic polymers, such as polyvinyl pyrroleAlkanones, amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine, mono-, di-and other carbohydrates including glucose, mannose or dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutical carriers herein also include interstitial drug dispersing agents, such as soluble neutral-active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, e.g., rhuPH20 (h: (r))Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rhuPH20, are described in US 2005/0260186 and US 2006/0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases (chondroitinases), such as chondroitinase.

Exemplary lyophilized antibody formulations are described in US 6,267,958. Aqueous antibody formulations include those described in US 6, 171,586 and WO 2006/044908, the latter formulation comprising a histidine-acetate buffer.

The formulations herein may also contain more than one active ingredient necessary for the particular indication being treated, particularly those having complementary activities that do not adversely affect each other. The active ingredients are suitably present in combination in an amount effective for the intended purpose.

The active ingredient may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Osol, A., (ed.) Remington's Pharmaceutical Sciences 16th edition (1980).

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

Formulations to be used for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

Methods and compositions of treatment

Any of the fusion polypeptides reported herein, wherein the effector moiety is a therapeutically active compound or a detectable label, may be used in a method of treatment.

In one aspect, there is provided a fusion polypeptide as reported herein for use as a medicament. In another aspect, fusion polypeptides for use in treating CNS-related diseases are provided. In certain embodiments, fusion polypeptides for use in a method of treatment are provided. In certain embodiments, the present invention provides a fusion polypeptide for use in a method of treating an individual having a CNS-related disease, the method comprising administering to the individual an effective amount of the fusion polypeptide. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In other embodiments, the invention provides a fusion polypeptide as reported herein for use in reversing or stabilizing a CNS-related disease. In certain embodiments, the present invention provides a fusion polypeptide for reversing or stabilizing a CNS-related disorder in an individual, comprising administering to the individual an effective amount of the fusion polypeptide, thereby reversing or stabilizing the CNS-related disorder. An "individual" according to any of the above embodiments is in particular a human.

CNS-related diseases include, for example, viral or bacterial diseases (such as encephalitis, meningitis), cancers (such as brain cancer), neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, huntington's disease, amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), multiple sclerosis), acute diseases (such as stroke, physical trauma, spinal cord injury), psychoses (such as anxiety, depression, epilepsy, seizure disorders (seibustidorder), schizophrenia, sleep disorders), cognitive diseases (such as memory diseases, cognitive diseases), cerebrovascular disorders (ischemic attacks), intracerebral hemorrhage, subarachnoid hemorrhage (subarachnoid hemorrhage)), pain-related diseases, prion diseases (such as Creutzfeldt-Jakob disease, encephalopathy (spongiform encephalopathy) or spongiform encephalopathy (alcoholic intoxication).

The fusion polypeptides reported herein are therapeutically effective if they lead to the reversal or stabilization of CNS-related diseases.

In one embodiment, the effector moiety is a therapeutically active compound that binds to or modifies the activity of brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), insulin-like growth factor (IGF), or Nerve Growth Factor (NGF).

In one embodiment, the effector moiety is a therapeutically active compound selected from the group consisting of: cholecystokinin (CCK), dopamine, endorphin, enkephalin, gamma-amino-butyric acid (GABA), neuropeptide Y, substance P, thyroid stimulating hormone releasing hormone (TRH) or Vasoactive Intestinal Peptide (VIP).

In one embodiment, the effector moiety is a therapeutically active compound selected from the group consisting of: anticonvulsants, anxiolytics, cytokines, or polynucleotides, such as siRNA.

In other aspects as reported herein, there is provided the use of a fusion polypeptide as reported herein for the manufacture or preparation of a medicament. In one embodiment, the medicament is for treating a CNS-related disease. In another embodiment, the medicament is for use in a method of treating a CNS-related disease, the method comprising administering to an individual having a CNS-related disease an effective amount of the medicament. In another embodiment, the medicament is for reversing or stabilizing a CNS-related disorder. In another embodiment, the medicament is for use in a method of reversing or stabilizing a CNS-related disorder in an individual, the method comprising administering to the individual an effective amount of the medicament to reverse or stabilize the CNS-related disorder. An "individual" according to any of the above embodiments may be a human.

In other aspects as reported herein, methods for treating CNS-related diseases are provided. In one embodiment, the method comprises administering to an individual having the disease an effective amount of a fusion polypeptide as reported herein. An "individual" according to any of the above embodiments may be a human.

In other aspects as reported herein, a method for reversing or stabilizing a CNS-related disease in an individual is provided. In one embodiment, the method comprises administering to the individual an effective amount of a fusion polypeptide as reported herein to reverse or stabilize a CNS-related disease. In one embodiment, the "individual" is a human.

In other aspects as reported herein, a pharmaceutical formulation comprising any one of the fusion polypeptides as reported herein is provided, e.g., for use in any one of the above-mentioned methods of treatment. In one embodiment, the pharmaceutical formulation comprises any of the fusion polypeptides provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any one of the fusion polypeptides reported herein and at least one additional therapeutic agent.

The fusion polypeptides reported herein may be used in therapy, alone or in combination with other agents. For example, the fusion polypeptide as reported herein may be co-administered with at least one further therapeutic agent.

Such combination therapies described above include combined administration (two or more therapeutic agents contained in the same formulation or in separate formulations), and separate administration, in which case administration of the antibody of the invention may occur prior to, concurrently with, and/or after administration of the other therapeutic agent and/or adjuvant. The fusion polypeptides as reported herein may also be used in combination with radiotherapy.

The fusion polypeptides reported herein may be administered by appropriate methods, including parenteral, intrapulmonary and intranasal, and if required for topical treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is transient or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulse infusion.

The fusion polypeptides reported herein are formulated, administered and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disease being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the time schedule of administration and other factors known to medical practitioners. The fusion polypeptide need not be, but is optionally formulated with one or more agents for the simultaneous prevention or treatment of the disorder in question. The effective amount of the additional agent will depend on the amount of fusion polypeptide present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used at the same dosage, or by the route of administration described herein, or about 1% to 99% of the dosage described herein, or by any route at any dosage and as determined empirically/clinically appropriate.

For the prevention or treatment of disease, the appropriate dosage of the fusion polypeptide reported herein (when used alone or in combination with one or more additional therapeutic agents) will depend on the type of disease to be treated, the type of fusion polypeptide, the severity and course of the disease, whether the fusion polypeptide is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the fusion polypeptide, and the judgment of the attending physician. The fusion polypeptide is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1. mu.g/kg to 15mg/kg (e.g., 0.1mg/kg-10mg/kg) of the fusion polypeptide can be an initial candidate dose for administration to a patient, e.g., whether administered by one or more divided administrations or by continuous infusion. A typical daily dose may be in the range of about 1. mu.g/kg to 100mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment will generally continue until suppression of the need for disease symptoms occurs. An exemplary dose of the fusion polypeptide will be in the range of about 0.05mg/kg to about 10 mg/kg. Thus, a dose of about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg or 10mg/kg (or any combination thereof) may be administered to a patient one or more times. The dose can be administered intermittently, e.g., weekly or every three weeks (e.g., such that the patient receives from about 2to about 20, or, e.g., about 6, doses of the fusion polypeptide). A higher initial loading dose may be administered followed by one or more lower doses. The progress of such treatment can be readily monitored by conventional techniques and assays.

Article of manufacture

In another aspect as reported herein, an article of manufacture comprising a substance for use in the treatment, prevention and/or diagnosis of a condition as reported above is provided. The article comprises a container and a label or package insert on or with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be made of various materials such as glass or plastic. The container contains a composition effective for treating, preventing and/or diagnosing a condition by itself or in combination with another composition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a fusion polypeptide as reported herein. The label or package insert indicates that the composition is for use in treating the selected condition. Furthermore, the article of manufacture may comprise (a) a first container having a composition therein, wherein the composition comprises a fusion polypeptide as reported herein; and (b) a second container having a composition therein, wherein the composition comprises another cytotoxic agent or an additional therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise package inserts indicating that the composition may be used to treat a particular condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. It may further include other materials from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It will be appreciated that modifications may be made in the method described without departing from the spirit of the invention.

Description of the drawings

FIG. 1 is a schematic diagram of the pH-dependent endocytic transport mechanism.

FIG. 2 setup of hCMEC/D3 endocytosis transport assay.

FIG. 3¹²⁵Validation of I-transferrin transcytosis across hCMEC/D3 brain endothelial cells.

Figure 4 mAb128.1 against human transferrin receptor did not leave hCMEC/D3 cells.

Figure 5 mAb128.1 against human transferrin receptor, unlike transferrin, colocalizes with the late endosomal marker CD63 after internalization.

FIG. 6 anti-IGF-1R antibodies to human IGF-1 receptor are not transcytosed, but are instead recycled.

FIG. 7 mAb MEM-189 directed to the human transferrin receptor, unlike mAb128.1, is recycled and transcytosed.

Figure 8 mAb MEM-189 binds transferrin receptor in a pH-dependent manner, whereas mAb128.1 does not bind transferrin receptor in a pH-dependent manner.

FIG. 9 mAbs128.1 competes with MEM-189 for the same epitope.

FIG. 10 mAbs 13E4 and M-A712 directed against the human transferrin receptor were not transcytosed.

Description of sequences

Examples

Example 1

hCMEC/D3 cell culture for transcytosis assay or fluorescence microscopy

Media and supplements for hCMEC/D3 (see WO 2006/056879 and Weksler, B.B., et al, FASEB J.19(2005)1872-1874) were obtained from Lonza. hCMEC/D3 cells (passage 26-29) can be cultured to confluence on collagen-coated coverslips (microscope) or flasks in EBM2 medium containing 2.5% FBS, one quarter and a supply of growth factor, gentamicin and ascorbic acid fully complementary to the supplemented hydrocortisone.

For all transcytosis assays, high density wells (1X 10) can be used in 12-well cell culture plates⁸Hole/cm²) PET membrane filter insert (0.4 μm pore size, 12mm diameter). The volume of medium used in the apical and basolateral chambers was calculated to be 400. mu.l and 1600. mu.l, respectively. The apical compartment of the filter insert may be treated with rat tail collagen I (7.5. mu.g/cm)²) Coated and then coated with fibronectin (5. mu.g/ml) and each incubation was continued at RT for 1 h. In EBM2 culture medium for 10-12 days,hCMEC/D3 cells were grown to confluent monolayers (. about.2X 10)⁵Individual cell/cm²). The air filters can be blocked for 1h or overnight (o/n) in PBS containing 1% BSA before assay, and then corrected for at least 1h in EBM2 before assay.

Example 2

¹²⁵I-transferrin and monoclonal antibody transcytosis assay

¹²⁵I-transferrin (Tfn) was obtained from Perkin Elmer (Perkin Elmer, Rodgau, Germany, # NEX212050 UC). mAb128.1 against human transferrin receptor and mAb 8D3 against mouse transferrin receptor were transiently expressed in vectors transfected with contiguous open reading frames comprising the coding sequences of human IgG1 heavy and light chain constant regions and HEK cells comprising vectors for mouse anti-human transferrin receptor antibody 128.1 (see WO 93/10819 and SEQ ID NOs: 21 and 22 for variable region sequences) or rat anti-mouse transferrin receptor antibody 8D3, respectively (Boado et al (2009), biotechnol.bioeng.102, 1251-1258), and purified as previously reported. mAb128.1 also used¹²⁵And I, marking. Monoclonal antibodies directed against the human IGF-1 receptor were expressed and purified as described in US 7,572,897. Mouse monoclonal mAbs MEM-189 and 13E4 directed against the human transferrin receptor were obtained from Abcam (Cambridge, England, # ab1086 and # ab38171, respectively) and mAb M-A712 from BD Biosciences (Heidelberg, Germany, # 555534). The entire assay (see FIG. 2 for assay protocol) was performed in serum-free EBM2 medium, which was otherwise reconstituted as described in example 1. On the day of assay, cells were serum-starved for 60min to deplete transferrin (only for transferrin transcytosis). Filter insert with or without (but closed overnight in complete medium) cells with radiolabelled transferrin on the apical side, i.e.¹²⁵I-labeled or unlabeled monoclonal antibodies were incubated at 37 ℃ for 1 h. Thereafter, the entire topside and bottom outside volumes are collected. The paracellular flux and stability of the radioiodinated ligand can be calculated from the determined values. Monolayers were washed three times apical (400. mu.l) and basolateral (1600. mu.l) in serum-free medium at RT, each time3-5 min. All wash volumes were collected to monitor the efficiency of removal of unbound ligand or antibody. Pre-incubated medium was added to the apical chamber and the filter was transferred to a new 12-well plate (blocked overnight with PBS containing 1% BSA) containing 1600 μ l of pre-incubated medium. At this point, the filters with or without cells were lysed in 500 μ l RIPA buffer (Sigma, Munich, germany, # R0278) to determine specific ligand or antibody uptake. The remaining filters are incubated at 37 ℃ or at 4 ℃ and samples are collected at various time points to determine the release of the top side and/or bottom outside of the ligand or antibody. Evaluation of integrity and degradation Using trichloroacetic acid (TCA) precipitation¹²⁵I-transferrin or¹²⁵I-mAb 128.1. The amount of radioactive transferrin or mAb128.1 in the supernatant or lysate can be determined by gamma ray counting. The amount of unlabeled antibody in the sample was quantified using a high selectivity IgG ELISA (see example 3). For each time point, data should be generated from two blank filters and three filter cell cultures.

Example 3

Sensitive IgG ELISA after transcytosis assay

The entire procedure was performed at RT, with the washing step using an automated washing machine. The 384 well plates were coated with 30. mu.l/well of 1. mu.g/ml anti-human/mouse-IgG, Fc γ -specific (Dianova, Hamburg, Germany, # 109-. Serial diluted samples from the transcytosis assay and standard concentrations of antibody for the transcytosis assay were added to the plates and incubated for 2 h. After four washes, 30. mu.l/well of 50ng/ml anti-human/mouse-F (ab) in blocking buffer (see above) was added₂Biotin-conjugate (Dianova, Hamburg, Germany, # 109-. After six washes, 30. mu.l/well of 50ng/ml (huIgG assay) or 100ng/ml (mIgG assay) poly-HRP 40-streptavidin were added(Fitzgerald, Acton (MA), USA, #65R-S104 PHRPx; in PBS containing 1% (w/v) BSA and 0.05% (w/v) Tween-20) and incubated for 30min. After four washes, the immune complexes were detected by adding 30. mu.l/well of BM chemiluminescent substrate (Roche Diagnostics GmbH, Mannheim, Germany). Luminescence signals were measured using a luminescence plate reader and concentrations were calculated using a fitted standard curve. The range of assay was from 10pg/ml to 10 ng/ml.

Example 4

Confocal fluorescence microscopy

To investigate the localization of the 128.1 antibody and intact transferrin, monolayers of hCMEC/D3 cells grown to confluence on collagen-coated coverslips were incubated with 5 μ g/ml FITC-labeled intact transferrin (Invitrogen, Darmstadt, Germany, # T-2871) or 1 μ g/ml mAb128.1 for 10min. Then, the medium was removed and replaced with fresh medium. After 1h at 37 ℃ the monolayers were fixed in 4% Paraformaldehyde (PFA) for 15min at RT, permeabilized for 10min (PBS/0.1% (w/v) Triton X-100(Sigma, Munich, Germany, #93443) and washed with antibodies against late endosome/lysosome marker CD63 (R & D Systems, Wiesbaden, Germany, # MAB5417) at RT 45min the cells were washed in PBS/0.1% (w/v) Triton X-100 for 15min, and if necessary further incubated with secondary antibodies (goat anti-human IgG-Alexa Fluor 488 and/or chicken anti-mouse IgG-Alexa Fluor Invitrogen, Darmstadt, Germany, # A11013 or # A21201 respectively) at RT 45min the cells in PBS/0.1% (w/v) Triton X-100 and then washed in blocking medium for 30min A series of single, representative slices.

Example 5

pH-dependent binding and competition ELISA

Fusion proteins of the extracellular domain of the human transferrin receptor linked to human IgGl Fc (R & D Systems, Wiesbaden, Germany, #2474-TR-050) or to the extracellular domain of the mouse transferrin receptor (SinoBiological, Beijing, China, #50741-M07H) or human insulin receptor (R & D Systems, Wiesbaden, Germany, # 4-IR/CF) were coated onto 96-well plates by incubation at RT for 1h with 50 μ l of a1 μ g/ml solution in PBS. After blocking for 1h with PBS/1% (w/v) BSA and washing four times with PBS/0.1% (w/v) Tween 20, the following antibodies at different concentrations in PBS/0.1% (w/v) BSA adjusted to pH7.4 or pH5.5 were added to the plates: MEM-189, 128.1, 13E4, M-A712, LT-71, MEM-75 (both available from Abcam, # ab9179 and # ab38446, respectively), OKT-9(eBioscience, Frankfurt, Germany, # 16-0719; all for human TfR), 8D3, R17217(Santa Cruz, Heidelberg, Germany, # sc-52504; both for mouse TfR), 83-13(Invitrogen, Darmstadt, Germany, # AHR0221) and 243524(R & D Systems, # MABl 544; both for human insulin receptor) and incubated at RT for 1.5 h. Alternatively, each well was incubated with mAb MEM-189 or mAb128.1 as blocking antibody at a fixed concentration of 5. mu.g/ml, washed four times, and then incubated with different concentrations of other antibodies not used for blocking for 30min at RT. After washing four more times with PBS/0.1% (w/v) Tween 20, bound antibody was detected with HRP-conjugated secondary antibody (Dianova, Hamburg, Germany, # 109-. The color development was stopped by adding 50. mu.l of 1N hydrochloric acid (HCl) and the absorbance was measured at 450nm in a plate reader.

Claims (8)

1. A method of delivering a pharmaceutically active compound across the blood-brain barrier in an individual, the method comprising administering to the individual an effective amount of a fusion polypeptide comprising:

-at least one binding pair comprising an antibody heavy chain variable domain and an antibody light chain variable domain, and which binds to an internalized cell surface receptor, and

-at least one pharmaceutically active compound,

whereby the binding pair that binds to the internalized cell surface receptor is at pH5.5Determined EC₅₀EC with value determined at pH7.4 for the same binding pair against the same receptor₅₀-a ratio of values above 10, thereby delivering the pharmaceutically active compound across the blood-brain barrier.

2. Use of a fusion polypeptide for delivering a pharmaceutically active compound across the blood-brain barrier, wherein the fusion polypeptide comprises:

-at least one binding pair comprising an antibody heavy chain variable domain and an antibody light chain variable domain, and which binds to an internalized cell surface receptor, and

-at least one pharmaceutically active compound,

EC determined at pH5.5 by the binding pair bound to the internalized cell surface receptor₅₀EC with value determined at pH7.4 for the same binding pair against the same receptor₅₀-a ratio of values above 10.

3. A method of transcytosis of epithelial cells in a subject, the method comprising administering to the subject a fusion polypeptide comprising:

-at least one binding pair comprising an antibody heavy chain variable domain and an antibody light chain variable domain, and which binds to an internalized cell surface receptor, and

-at least one pharmaceutically active compound,

EC as determined at pH5.5 for the binding pair bound by this internalized cell surface receptor₅₀EC with value determined at pH7.4 for the same binding pair against the same receptor₅₀-a ratio of values above 10.

4. Use of a fusion polypeptide for the manufacture of a medicament, said polypeptide comprising:

-at least one binding pair comprising an antibody heavy chain variable domain and an antibody light chain variable domain, and which binds to an internalized cell surface receptor, and

-at least one pharmaceutically active compound,

EC determined at pH5.5 by the binding pair bound to the internalized cell surface receptor₅₀EC with value determined at pH7.4 for the same binding pair against the same receptor₅₀-a ratio of values above 10.

5. Use according to claim 4, characterized in that the medicament is for the treatment of CNS-related diseases.

6. The method or use according to any one of the preceding claims, characterized in that said ratio is above 15.

7. The method or use according to any one of the preceding claims, characterised in that said ratio is about 15.

8. The method or use according to any of the preceding claims, characterized in that the binding to an internalized cell surface receptor is responsible for the EC determined at pH5.5₅₀-value is above 1000 ng/ml.