RU2799436C1 - Universal transmitter modules through the hematoencephalitic barrier - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: transporter module across the blood-brain barrier containing a molecule to be transported to the brain through the blood-brain barrier, and one monovalent antibody against the transferrin receptor or a fragment thereof that binds the transferrin receptor are proposed. The invention also relates to a pharmaceutical composition comprising the said carrier module and to the use of the carrier module for transporting a molecule to be transported to the brain.

EFFECT: invention can be used in medicine for efficient transport of an effector molecule across the blood-brain barrier.

12 cl, 1 dwg, 1 tbl, 12 ex

Description

This application is separated from the application No. 2016130707 for the grant of a patent of the Russian Federation for an invention, filed on December 29, 2014, with a priority claim by the filing date of the application PCT/CN2014/070183, filed on January 6, 2014.

Field of invention

The present invention relates to a blood brain barrier transporter module that has a single binding specificity that specifically binds to the blood brain barrier receptor (BBBR) and that is univalent with respect to this binding specificity, as well as methods of using this construct as carrier across the blood-brain barrier and in the treatment of neurological disorders.

State of the art

Penetration into the brain of drugs for the treatment of neurological disorders, such as, for example, large biotherapeutic drugs or small molecule drugs with low penetration into the brain, is strictly limited by the extensive and impenetrable blood brain barrier (BBB) along with another cellular component in the neurovascular block ( neurovascular unit, NVU). Many strategies have been tested to overcome this hurdle, and one of them is to use transcytosis pathways mediated by endogenous receptors expressed on the brain capillary endothelium (blood-brain barrier receptors). Recombinant proteins, such as monoclonal antibodies or peptides, have been developed against these receptors to provide receptor-mediated delivery of biotherapeutic drugs to the brain. However, strategies to maximize brain uptake while minimizing missorting in brain endothelial cells (BEC) and the degree of accumulation in certain organelles (especially organelles that lead to degradation of biotherapeutic drugs) in BEC remain unexplored.

Monoclonal antibodies and other biotherapeutic drugs have great therapeutic potential for the treatment of pathology in the central nervous system (CNS, central nervous system, CNS). However, their passage to the brain is prevented by the blood-brain barrier. Previous studies have shown that a very small percentage (about 0.1%) of IgG introduced into the circulation is able to enter the CNS compartment (FeIgenhauer, Klin. Wschr. 52 (1974) 1158-1164). This certainly limits any pharmacological effect due to the low concentration of the antibody in the CNS.

Thus, there is a need for BBB drug delivery systems for the treatment of neurological disorders that efficiently transport drugs to the brain.

WO 2014/033074 reports a transporter across the blood-brain barrier.

The murine antitransferrin antibody 8D3 and its light chain variable domain (VL) variant (L596V and L598I) are described in Boado, R.J., et al. (Biotechnol. Bioeng. 102 (2009) 1251-1258).

Short description

One aspect of the present invention is a blood-brain barrier transport module comprising a moiety active in the brain (effector moiety), a linker and one monovalent binding moiety that binds to the blood-brain barrier receptor, where the linker connects the effector moiety to the monovalent binding moiety that binds with a blood-brain barrier receptor, wherein the monovalent binding moiety does not contain the variable domains of an anti-transferrin 8D3 receptor antibody (SEQ ID NO 01 and SEQ ID NO 02) or a variant anti-transferrin 8D3v receptor antibody (SEQ ID NO 01 and SEQ ID NO 03).

The 8D3 transferrin receptor antibody has a heavy chain variable domain with the following amino acid sequence:

EVQLVESGGG LVQPGNSLTL SCVASGFTFS NYGMHWIRQA PKKGLEWIAM IYYDSSKMNY ADTVKGRFTI SRDNSKNTLY LEMNSLRSED TAMYYCAVPT SHYWDVWGQ GVSVTVSS

(SEQ ID NO 01).

The 8D3 transferrin receptor antibody has a light chain variable domain with the following amino acid sequence:

DIQMTQSPAS LSASLEEIVT ITCQASQDIG NWLAWYQQKP GKSPQLLIYG ATSLADGVPS RFSGSRSGTQ FSLKISRVQV EDIGIYYCLQ AYNTPWTFGG GTKLELK

(SEQ ID NO 02).

The variant anti-transferrin receptor antibody 8D3v has the same heavy chain variable domain as the 8D3 antibody and the light chain variable domain with the L104V and L106I mutants, which has the following amino acid sequence:

DIQMTQSPAS LSASLEEIVT ITCQASQDIG NWLAWYQQ KP GKSPQLLIYG ATSLADGVPS RFSGSRSGTQ FSLKISRVQV EDIGIYYCLQ AYNTPWTFGG GTKVEIK (SEQ ID NO 03).

In one embodiment of the blood brain barrier transport module, the monovalent binding moiety that binds to the blood brain barrier receptor is a polypeptide.

In one embodiment of the blood brain barrier transport module, the monovalent binding moiety that binds to the blood brain barrier receptor comprises a molecule selected from the group consisting of a blood brain barrier receptor ligand, full length antibody, scFv, Fv, scFab, and VHH.

In one embodiment of the blood brain barrier transport module, the blood brain barrier receptor is selected from the group consisting of: transferrin receptor, insulin receptor; an insulin-like growth factor receptor; protein 8 associated with the low density lipoprotein receptor; low-density lipoprotein receptor-associated protein 1; and a heparin-binding growth factor like epidermal growth factor. In one embodiment, the blood brain barrier receptor is a transferrin receptor.

In one embodiment, the monovalent binding moiety binds specifically to the human transferrin receptor and the cynomolgus transferrin receptor.

In one embodiment of the blood brain barrier transport module, the monovalent binding moiety that binds to the blood brain barrier receptor comprises a single scFab directed against the transferrin receptor, more specifically a scFab that specifically binds to an epitope on the transferrin receptor contained in the amino acid sequence of SEQ ID NO 04, 05 or 06.

In one embodiment of the blood-brain barrier transport module, the monovalent binding moiety that binds to the blood-brain barrier receptor comprises a single scFv directed against the transferrin receptor, more specifically, an scFv that recognizes an epitope on the transferrin receptor contained in the amino acid sequence of SEQ ID NO 04, 05 or 06.

In one embodiment of the blood-brain barrier transporter module, the brain-active moiety is selected from the group consisting of drugs for the treatment of neurological disorders, neurotrophic factors, growth factors, enzymes, cytotoxic agents, antibodies directed against targets in the brain, monoclonal antibodies directed against against targets in the brain, peptides directed against targets in the brain.

In one embodiment of the BBB transporter module, the target in the brain is selected from the group consisting of beta-secretase 1, Aβ (Abeta), epidermal growth factor, epidermal growth factor receptor 2, tau protein, phosphorylated tau protein, phosphorylated tau (p8422)-protein, apolipoprotein E4, alpha-synuclein, oligomeric fragments of alpha-synuclein, CD20, huntingtin, prion protein, leucine-rich repeat kinase 2, parkin, presenilin 2, gamma-secretase, death receptor 6, amyloid precursor protein , the p75 neurotrophin receptor, and caspase 6.

In a specific embodiment of the blood-brain barrier transport module, the moiety active in the brain is a polypeptide.

In one embodiment of the blood brain barrier transport module, the monovalent binding moiety that binds to the blood brain barrier receptor is a polypeptide, and the monovalent binding moiety is conjugated to the C-terminus of the brain active moiety, either directly or via a linker.

In one embodiment of the blood brain barrier transport module, the brain active moiety comprises a full length antibody directed against a brain target. In one embodiment, the full length antibody is an IgG.

In one preferred embodiment of the blood-brain barrier transporter module, the blood-brain barrier transporter comprises a full-length IgG antibody as a brain-acting moiety, a linker, and one scFab as a monovalent binding moiety that binds to the blood-brain barrier receptor, wherein the scFab is conjugated to the C-terminus of an Fc - region of one of the heavy chains of the IgG antibody through a linker.

In one embodiment of the blood-brain barrier transporter module, the first heavy chain of the antibody in the brain-targeted blood-brain barrier transporter comprises a first dimerization module, and the second heavy chain of the antibody in the brain-targeted blood-brain barrier transporter comprises a second module. dimerizations that allow heterodimerization of the two heavy chains.

In one embodiment of the blood-brain barrier transporter module, the first dimerization module of the first heavy chain of the antibody in the blood-brain barrier transporter directed against the target in the brain includes a "key", and the dimerization module of the second heavy chain of the antibody in the blood-brain barrier transporter directed against the target in the brain, turns on the "lock" in accordance with the "key in the lock" strategy.

In one embodiment of the blood brain barrier transport module, the linker is a peptide linker. In one embodiment, the peptide linker has an amino acid sequence of at least 25 amino acids. In one embodiment, the peptide linker has an amino acid sequence of 30 to 50 amino acids. In one embodiment, the peptide linker is (G4S)6G2 (SEQ ID NO 07) or (G4S)4 (SEQ ID NO 08).

The following three embodiments are directed to a blood-brain barrier transport module where the brain-active moiety is a polypeptide, provided that the brain-active moiety is not a full-length antibody, in particular not a full-length IgG.

In one embodiment of the blood-brain barrier transporter module, the monovalent binding moiety that binds to the blood-brain barrier receptor contains a CH2-CH3-Ig moiety and one scFab (containing the first linker) that binds to the blood-brain barrier receptor, where the scFab is linked to C -end of the CH2-CH3-Ig group with the second linker.

In one embodiment of the blood-brain barrier transporter module, the blood-brain barrier transporter comprises a brain-active moiety, a linker, a CH2-CH3-Ig domain, a second linker, and one scFab that binds to the blood-brain barrier receptor, where the brain-active moiety is conjugated to using a first linker to the N-terminus of the CH2-CH3-Ig domain, and the scFab is conjugated to the C-terminus of the CH2-CH3-Ig domain using a second linker.

In one embodiment of the blood-brain barrier transport module, the CH2-CH3-Ig moiety is an IgG CH2-CH3 moiety.

Other aspects of the invention are an (isolated) nucleic acid encoding the BBB transporter module described herein, a host cell comprising an (isolated) nucleic acid encoding the BBB transporter module, and a pharmaceutical composition comprising the module transporter across the blood-brain barrier.

The blood-brain barrier transport module described herein can be used as a drug, in particular it can be used to treat neurological disorders such as, for example, Alzheimer's disease.

The blood-brain barrier transport module described herein can be used to transport a brain-active moiety across the blood-brain barrier.

In a specific embodiment of the invention, the heavy chain of the IgG antibody of the blood-brain barrier transport module described herein, conjugated at its C-terminal Fc region with scFab as a monovalent binding moiety that binds to the blood-brain barrier receptor, has the following structure:

- heavy chain IgG,

- a linker conjugating the C-terminus of the Fc region of the IgG heavy chain with the N-terminus of the scFab VL domain,

- light chain variable domain (VL) and light chain C-kappa domain of scFab,

- a linker conjugating the C-terminus of the scFab light chain kappa C-terminus to the N-terminus of the scFab VH domain,

the heavy chain variable domain (VH) of the scFab antibody and the IgG heavy chain CH1 domain.

One aspect described herein is a fusion polypeptide for transporting a brain-active moiety across the blood-brain barrier, comprising a CH2-CH3-Ig moiety, a linker, and one scFab that specifically binds to the blood-brain barrier receptor, where the scFab is conjugated to C- the end of the CH2-CH3-Ig moiety through a linker, where the scFab does not contain the variable domains of the anti-transferrin receptor antibody 8D3 (SEQ ID NO 01 and SEQ ID NO 02) or the variant anti-transferrin receptor antibody 8D3v (SEQ ID NO 01 and SEQ ID NO 03 ).

One aspect described herein is a fusion polypeptide for transport across the blood-brain barrier of a brain-acting moiety, comprising a CH2-CH3-Ig moiety, a linker, and one scFv that specifically binds to the blood-brain barrier receptor, where the scFv is conjugated to C- the end of the CH2-CH3-Ig moiety through a linker, where the scFv does not contain the variable domains of the anti-transferrin receptor antibody 8D3 (SEQ ID NO 01 and SEQ ID NO 02) or the variant anti-transferrin receptor antibody 8D3v (SEQ ID NO 01 and SEQ ID NO03) .

In one embodiment, the fusion polypeptide also contains a linker at the N-terminus of the CH2-CH3-Ig moiety for conjugation of the brain-active moiety to the N-terminus of the CH2-CH3-Ig moiety.

In one embodiment of the fusion polypeptide, a brain-active moiety is selected from the group consisting of drugs for the treatment of neurological disorders, neurotrophic factors, growth factors, enzymes, cytotoxic agents, antibody fragments or peptides directed against a target in the brain, selected from the group consisting of from scFv, Fv, scFab, Fab, VHH, F(ab')2.

In one embodiment, the scFab or scFv fusion polypeptide that specifically binds to the blood-brain barrier receptor binds specifically to the transferrin receptor. In one embodiment, the scFab or scFv specifically binds to an epitope on the transferrin receptor contained in the amino acid sequence of SEQ ID NO 04, 05 or 06.

In an embodiment of the hybrid polypeptide, the linker is a peptide linker. In one embodiment, the peptide linker has an amino acid sequence of at least 15 amino acids. In one embodiment, the peptide linker has a length of 20 to 50 amino acids. (G4S) 4 (SEQ ID NO 08).

In one embodiment of the fusion polypeptide, the CH2-CH3-Ig moiety is an IgG CH2-CH3 moiety.

Other aspects of the present invention are an isolated nucleic acid encoding a fusion polypeptide described herein and a host cell containing a nucleic acid encoding a fusion polypeptide described herein.

One aspect described herein is a conjugate comprising the fusion polypeptide described herein and a brain-active moiety conjugated through a linker to the N-terminus of the CH2-CH3-Ig moiety of the fusion polypeptide described herein.

In one embodiment of the conjugate, the brain-active moiety is a neurotrophic factor and the linker conjugating the neurotrophic factor to the N-terminus of the CH2-CH3-Ig moiety is a peptide linker.

Other aspects described herein are a pharmaceutical composition containing a conjugate described herein and a pharmaceutical carrier, as well as the use of the conjugate described herein, in particular, the use of the conjugate for the treatment of a neurodegenerative disorder, in particular Alzheimer's disease.

A monovalent binding moiety that specifically binds to the blood-brain barrier receptor can be conjugated to either end of the light or heavy chain of an antibody, either directly or through a peptide linker. In one embodiment, the monovalent linking moiety is conjugated to the C-terminus of the heavy chain.

The C-terminus of an antibody heavy chain may be a complete C-terminus ending in PGK amino acid residues. The C-terminus of the heavy chain may be a truncated C-terminus in which one or two C-terminal amino acid residues have been removed. In one embodiment, the C-terminus of the heavy chain is a truncated C-terminus that terminates in PG amino acid residues.

The monovalent binding moiety can be conjugated to the corresponding antibody chain, either directly or through a peptide linker. In one embodiment, the peptide linker has the amino acid sequence GGSGGGGSGGGGSGGGGS (SEQ ID NO 09).

The monovalent binding moiety may be an scFv antibody fragment. In one embodiment, the monovalent binding moiety is an scFv containing in N-terminal to C-terminal direction: light chain variable domain - light chain constant domain - peptide linker - heavy chain variable domain - heavy chain constant domain 1.

In one embodiment, the monovalent binding moiety is an anti-transferrin receptor antibody scFv fragment with a (C4S) ₆ peptide linker (SEQ ID NO 10).

In one embodiment, the blood-brain barrier receptor is selected from the group consisting of a transferrin receptor; an insulin receptor; an insulin-like growth factor receptor; low-density lipoprotein receptor-associated protein 8; low-density lipoprotein receptor-associated protein 1; and a heparin-binding growth factor like epidermal growth factor. In one embodiment, the blood brain barrier receptor is a human blood brain barrier receptor. In one embodiment, the blood brain barrier receptor is a transferrin receptor and the antibody does not inhibit the binding of the transferrin receptor to transferrin. In one embodiment, the blood brain barrier receptor is a human transferrin receptor.

In one embodiment, the peptide linker conjugating the monovalent binding moiety to the brain-active moiety is an amino acid sequence of at least 15 amino acids in length. In one embodiment, the peptide linker is 18 to 25 amino acids in length.

In one embodiment, the brain-active moiety is a full length antibody. In one embodiment, the brain-active moiety is a full-length IgG1 or IgG4 subclass antibody.

In one embodiment, the monovalent binding moiety is an anti-blood-brain barrier receptor antibody, or a blood-brain barrier receptor-binding fragment thereof. In one embodiment, the blood-brain barrier receptor antibody, or fragment thereof, does not interfere with the binding of the blood-brain barrier receptor to one or more of its native ligands. In another embodiment, the anti-blood-brain barrier receptor antibody specifically binds to the human transferrin receptor such that it does not inhibit the binding of the human transferrin receptor to human transferrin.

In one embodiment, the blood brain barrier transport module is a silent effector.

In one embodiment, the brain-active moiety is a full-length antibody containing an Fc region, where, when the Fc region belongs to the human IgG1 subclass, the Fc region contains mutations L234A, L235A, and P329G (numbered according to the Kabat EC index). ), or in the case where the Fc region belongs to the human IgG4 subclass, the Fc region contains mutations S228P, L235E and P329G (numbering according to the Kabat EC index).

Detailed description of embodiments of the invention

I. Definitions

"Acceptor human framework" in the context of the present invention is a framework region comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or human consensus framework as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or consensus human framework may contain the same amino acid sequence, or it may contain amino acid substitutions. In some embodiments, the number of amino acid substitutions is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the human VL acceptor framework is identical in sequence to a human immunoglobulin VL framework or consensus human framework sequence.

The term "affinity" refers to the strength of the overall overall non-covalent interactions between one binding site of a molecule (eg, antibody) and its binding partner (eg, antigen). Unless otherwise indicated, the term "binding affinity" as used herein refers to intrinsic binding affinity, which reflects a 1:1 interaction between elements of a binding pair (eg, between an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments of measuring binding affinity will now be described.

The term “affinity matured antibody” refers to an antibody with one or more changes in one or more hypervariable regions (HVR) compared to a parent antibody that does not contain such changes, and such changes lead to an increase in the affinity of the antibody for the antigen. .

The term "antibody" is used herein in its broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

The term "antibody fragment" refers to a molecule, other than an intact antibody, that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; dimers; linear antibodies; single chain antibody molecules (eg scFv); and multispecific antibodies formed from antibody fragments.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The term "class" of an antibody refers to the type of constant domain or constant region that its heavy chain possesses. There are five main classes of antibodies: IgA, IgD, IgE, IgG and IgM, and some of them can be divided into subclasses (isotypes), for example, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . The heavy chain constant domains that correspond to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The term "effector functions" refers to the biological activities attributed to the Fc region of an antibody, which varies depending on the class of the antibody. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cellular cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (eg, B cell receptor); and B-cell activation.

The concept of an "effective amount" of an agent, such as a pharmaceutical composition, refers to an amount effective in doses and for a period of time necessary to achieve the desired therapeutic or prophylactic result.

The term "Fc region" is used herein to refer to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions or variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated herein, amino acid residues in the Fc region or constant region are numbered according to the EC numbering system, also referred to as the EC index, as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

The term "framework region", abbreviated as "FR", refers to variable domain residues excluding hypervariable region (HVR) residues. The variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically follow the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein and refer to an antibody having a structure that is substantially similar to that of a native antibody, or having heavy chains that contain an Fc region as defined herein. description.

The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived from them, regardless of the number of passages. The offspring may not be completely identical to the parent cell in terms of nucleic acid content, and may contain mutations. Mutant progeny that have the same function or biological activity when screened or selected as the original transformed cell are included herein.

A "human consensus framework" is a framework that represents the most frequently occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. Typically, a subgroup of sequences is the subgroup described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Bethesda MD (1991), NIH Publication 91-3242, Vols. 1-3. In one embodiment for VL, the subgroup is subgroup kappa I, as described in Kabat et al., see supra. In one embodiment for VH, the subgroup is subgroup III, as described in Kabat et al., see supra.

The term "humanized" antibody refers to a chimeric antibody containing amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, a humanized antibody will contain substantially all of at least one, and usually two, variable domains in which all or substantially all of the HVRs (e.g., CDRs) match those of the non-human antibody and all or substantially all of the FRs match those of the non-human antibody. in a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has been humanized.

The terms "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain that are hypervariable in sequence ("complementarity determining regions" or "CDRs") and/or form structurally defined loops ( "hypervariable loops") and/or contain antigen-contacting residues ("antigenic contacts"). Typically, antibodies contain six HVRs, three in VH (H1, H2, H3) htphbVL (L1, L2, L3).

HVR in this document include

(a) hypervariable loops arising at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2) and 96-101 (H3) ). (Chothia, C. and Lesk, A.M., J. Mol. Biol. 196 (1987) 901-917);

(b) CDRs arising at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2) and 95-102 (H3) (Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242);

(c) antigenic contacts occurring at amino acid residues 27 c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2) and 93-101 ( H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); And

(d) combinations of (a), (b) and/or (c) including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

"Individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (eg, cows, sheep, cats, dogs, and horses), primates (eg, humans and primates such as monkeys), rabbits, and rodents (eg, mice and rats). In some embodiments, the individual or subject is a human.

An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as determined, for example, by electrophoresis (eg, SDS-PAGE, isoelectrofocusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reverse phase HPLC). For a review of antibody purity assessment techniques, see, for example, Flatman, S. et al., J. Chromatogr. In 848 (2007) 79-87.

The term "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is located outside the chromosome or at a location on the chromosome that differs from its natural chromosomal location.

The term "monoclonal antibody" as used herein refers to an antibody derived from a population of substantially homogeneous antibodies, ie. an antibody population consisting of individual antibodies that are identical and/or bind the same epitope, excluding possible variant antibodies, e.g. in small quantities. Unlike polyclonal antibody preparations, which typically include 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 designator "monoclonal" indicates the nature of the antibody, which was obtained from a population of essentially homogeneous antibodies, and should not be construed as requiring the production of antibodies in any particular way. For example, monoclonal antibodies used in accordance with this invention can be obtained using various techniques, including, but not limited to, hybridoma technique, recombinant DNA techniques, phage display techniques, as well as methods using transgenic animals containing all or part of human immunoglobulin loci, and such techniques and other exemplary techniques for obtaining monoclonal antibodies are described in this document.

The term "native antibodies" refers to natural immunoglobulin molecules with different structures. For example, native IgG antibodies are heterotetrameric hl and co proteins of approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains that are linked by a disulfide bond. From the N-terminus to the C-terminus, each heavy chain contains a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, and then three constant domains (CH1, CH2 and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

The term "package insert" is used to refer to instructions commonly included in commercial packages of therapeutic products that contain information about the indications, use, dosage, administration, combination therapy, contraindications and/or warnings regarding the use of such therapeutic products.

"Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps as necessary to achieve the maximum percentage of sequence identity, and ignoring any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways that are available to those skilled in the art, for example using open source software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the compared sequences. However, for the purposes of this invention, % amino acid sequence identity values are obtained using the ALIGN-2 sequence comparison computer program. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc., and the source code was filed with the user documentation with the US Copyright Office, Washington, DC 20559, where it was registered under number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, CA, or may be compiled from source code. The ALIGN-2 program must be compiled for use on a UNIX operating system, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

In situations where ALIGN-2 is used to compare amino acid sequences, % amino acid sequence identity of a given amino acid sequence A with a given amino acid sequence B (alternatively, a given amino acid sequence A has or contains a certain % amino acid sequence identity with a given amino acid sequence B) is calculated as follows:

100 × share X/Y,

where X is the number of amino acid residues scored as identical matches by the ALIGN-2 sequence alignment program when A and B are aligned in that program, and where Y is the total number of amino acid residues in B. Note that if the length of the amino acid sequence A is not equal to the length of the amino acid sequence of B, then the % amino acid sequence identity of A with B will not be equal to the % amino acid sequence identity of B with A. Unless otherwise noted, all % amino acid sequence identity values used in this specification are derived as described in the previous paragraph, using the computer program ALIGN-2.

The term "pharmaceutical formulation" refers to a formulation that is in a form that permits the biological activity of the active ingredient contained therein and that does not contain additional components that are unacceptably toxic to the subject to whom the formulation will be administered.

The term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than the active ingredient, that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, "treatment" (and its grammatical variants such as "treat") refers to a clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, which may be performed either for prophylaxis or in the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of a disease, ameliorating symptoms, reducing any direct or indirect pathological consequence of a disease, preventing metastasis, reducing the rate of disease progression, improving or temporarily alleviating a disease state, achieving remission, or improving prognosis. In some embodiments, the antibodies of this invention are used to delay the development of a disease or to slow the progression of a disease.

The term "variable region" or "variable domain" refers to the heavy or light chain domain of an antibody that is involved in the binding of an antibody to an antigen. The heavy chain and light chain variable domains (VH and VL, respectively) of a native antibody typically have a similar structure, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt, T.J. et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y. (2007), page 91). A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, antibodies that bind a particular antigen can be isolated using the VH or VL domain from an antibody that binds to the antigen to screen for a library of complementary VL or VH domains, respectively. See, for example, Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., 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. This term includes a vector as a self-replicating nucleic acid structure, as well as a vector incorporated into the genome of the host cell into which it has been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

The term "blood-brain barrier" (BBB) refers to the physiological barrier between the peripheral circulation and the brain and spinal cord, which is formed by tight junctions of the plasma membranes of brain capillary endothelial cells, creating a tight barrier that limits the transport of molecules to the brain, even very small molecules such as urea (60 Da). The BBB in the brain, the blood-spinal barrier in the spinal cord, and the blood-retinal barrier in the retina are adjacent capillary barriers in the CNS and are collectively referred to herein as the blood-brain barrier, or BBB. BBB also encompasses the blood-CSF barrier (choroid plexus), where the barrier is composed of ependymal cells rather than capillary endothelial cells.

The term "central nervous system" (CNS, central nervous system, CNS) refers to the complex of nerve tissues that control physical function, and includes the brain and spinal cord.

The term "blood-brain barrier receptor" (BBBR) refers to an extracellular membrane-bound receptor protein expressed on brain endothelial cells that is capable of transporting molecules across the BBB or used to transport exogenously administered molecules. Examples of BBBR include, but are not limited to, transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF-R), low-density lipoprotein receptors including, but not limited to, low-density lipoprotein receptor-related protein 1 ( LRP1), and low-density lipoprotein receptor-associated protein 8 (LRP8), and heparin-binding growth factor like epidermal growth factor (HB-EGF). An exemplary BBBR is the transferrin receptor (TfR).

The term "active in the brain group" ("effector group") means a molecule that must be transported to the brain through the BBB. The effector moiety typically has a characteristic therapeutic activity to be delivered to the brain. Effector moieties include drugs for the treatment of neurological disorders and cytotoxic agents such as, for example, polypeptides and antibodies, in particular monoclonal antibodies or fragments thereof, directed to target in the brain.

The term "univalent binding moiety" means a molecule capable of binding to BBBR specifically and in a monovalent binding mode. The blood-brain barrier transport module and/or conjugate described herein are characterized by having one unit of a monovalent binding moiety, i. e. the blood-brain barrier transport module and/or the conjugate of the present invention contain exactly one unit of a monovalent binding moiety. The monovalent binding moiety includes, but is not limited to, polypeptides, full length antibodies, antibody fragments including Fab, Fab', Fv fragments, single chain antibody molecules such as, for example, single chain Fab, scFv. The monovalent binding moiety can be, for example, a scaffold protein designed using standard techniques in the art such as phage display or immunization. The monovalent linking moiety can also be a polypeptide. In some embodiments, the monovalent binding moiety comprises a CH2-CH3-Ig domain and a single chain Fab (scFab) directed against the blood-brain barrier receptor. scFab is linked to the C-terminus of the CH2-CH3-Ig domain via a linker. In some embodiments, the scFab is directed against the transferrin receptor.

The term "univalent binding mode" means specific binding to BBBR, where the interaction between the monovalent binding moiety and BBBR occurs through one single epitope. The univalent binding mode prevents changes in intracellular BBBR sorting.

The term "epitope" means any polypeptide determinant capable of specifically binding to an antibody. In some embodiments, antigenic determinants include reactive surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in some embodiments, they may have specific three-dimensional structural characteristics and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody.

The "transferrin receptor" (TfR) is a transmembrane glycoprotein (with a molecular weight of about 180,000 Da) that consists of two disulfide-linked subunits (each with an apparent molecular weight of about 90,000 Da) and is involved in iron uptake in vertebrates. In one of the embodiments of the TfR in this document is a human TfR containing the amino acid sequence specified in Schneider et al. (Nature 311 (1984) 675-678).

The term "neurological disorder" means a disease or disorder that affects the CNS and/or that has an etiology in the CNS. Exemplary CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, an eye disease or disorder, viral or bacterial infection, inflammation, ischemia, neurodegenerative disease, stroke, behavioral disorders, and lysosomal storage disease. In the context of this application, it should be understood that the CNS includes the eye, which is usually separated from the rest of the body by the blood-retinal barrier. Specific examples of neurological disorders include, but are not limited to, neurodegenerative diseases (including but not limited to Lewy body disease, post-polio syndrome, Shy-Drager syndrome, olivopontocerebral atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, tauopathies (including , but not limited to Alzheimer's disease and supranuclear palsy), prion diseases (including but not limited to bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease, and heterodegenerative disorders of the nervous system (including, but not limited to, Canavan's disease, Huntington's disease, neural waxy lipofuscinosis, Alexander's disease, Tourette's syndrome, Menkes' curly hair syndrome, Cockayne's syndrome, Hallervorden-Spatz, Lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nychen syndrome and Unferricht-Lundborg syndrome), dementia (including but not limited to Pick's disease and spinocerebellar ataxia), cancer (e.g., CNS and/or brain including brain metastases from cancer elsewhere in the body).

The term "drug for the treatment of a neurological disorder" means a drug or therapeutic agent that treats one or more neurological disorders. Drugs for the treatment of neurological disorders include, but are not limited to, small molecule compounds, antibodies, peptides, proteins, natural ligands of one or more CNS targets, modified variants of natural ligands of one or more CNS targets, aptamers that inhibit nucleic acids ( ie, small inhibitory RNAs (siRNAs) and short hairpin RNAs (shRNAs)), ribozymes, and small molecules or active fragments of any of the above compounds. Examples of drugs for the treatment of a neurological disorder are described herein and include, but are not limited to: antibodies, aptamers, proteins, peptides, inhibitory nucleic acids and small molecules, and active fragments of any of the above compounds that either act alone or specifically recognize and/or act on (i.e., inhibit, activate or detect) CNS antigen or target molecules such as, but not limited to, amyloid precursor protein or portions thereof, beta-amyloid, beta-secretase, gamma- secretase, tau protein, alpha-synuclein, parkin, huntingtin, DR6, presenilin, ApoE, markers of glioma or other CNS cancer, and neurotrophins. Non-limiting examples of drugs for the treatment of neurological disorders and related disorders for which they can be used: brain-derived neurotrophic factor (BDNF), chronic brain damage (neurogenesis), fibroblast growth factor 2 (FGF-2, from English - Fibroblast growth factor 2), brain cancer and antibody against epidermal growth factor receptor (EGFR), neuronal factor from the glial cell line (GDNF, from English - glial cell-line derived neural factor) with Parkinson's disease, brain-derived neurotrophic factor (BDNF) in amyotrophic lateral sclerosis, depression, lysosomal enzyme and lysosomal brain storage diseases, ciliary neurotrophic factor (CNTF) and amyotrophic lateral sclerosis, neuregulin-1 and schizophrenia , anti-HER2 antibody (eg, trastuzumab), and metastases of HER2-positive cancer in the brain.

The term "rendering agent" means a compound that has one or more properties that allow its presence and/or location to be detected, directly or indirectly. Examples of such imaging agents include proteins and small molecules containing a detectable labeled moiety.

The terms "CNS antigen" and "brain target" refer to an antigen and/or molecule expressed in the CNS, including the brain, to which an antibody or small molecule can be targeted. Examples of such antigens and/or molecules include, but are not limited to: beta-secretase 1 (BACE1), beta-amyloid (Abeta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2). human epidermal growth factor receptor 2), tau protein, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP, from English - prion protein), leucine-rich repeat kinase 2 (LRRK2, from English . - leucine rich repeat kinase 2), parkin, presenilin 1, presenilin 2, gamma-secretase, death receptor 6 (DR6, from English - death receptor 6), amyloid precursor protein (APP, from English - amyloid precursor protein ), the p75 neurotrophin receptor (p75NTR) and caspase 6. In one embodiment, the antigen is BACE1.

The term "which specifically binds" means an antibody that selectively or preferentially binds to an antigen. Binding affinity is typically determined using a standard assay, such as Scatchard or Surface Plasmon Resonance (eg, using Biacore®).

The term "CH2-CH3-Ig moiety" as used herein refers to a protein moiety derived from the CH2 or CH3 domain of an immunoglobulin. A "CH2-CH3-Ig moiety" includes two "CH2-CH3" polypeptides forming a dimer. The immunoglobulin may be IgG, IgA, IgD, IgE or IgM. In one embodiment, the CH2-CH3-Ig moiety is derived from an IgG immunoglobulin and is referred to herein as the "CH2-CH3-IgG moiety". The term includes native sequence CH2-CH3 domains and variant CH2-CH3 domains. In one embodiment, the "CH2-CH3-Ig moiety" is derived from a human IgG heavy chain CH2-CH3 domain that extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated, amino acid residues in the CH2-CH3 domain or constant region are numbered according to the EC numbering system, also referred to as the EC index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

A "conjugate" is a fusion protein of the invention conjugated to one or more heterologous molecules, including, but not limited to, a label, a drug for the treatment of a neurological disorder, or a cytotoxic agent.

The term "linker" means a chemical linker or single strand peptide linker that covalently links different moieties of a carrier module across the blood-brain barrier and/or a hybrid polypeptide and/or conjugate described herein. The linker links, for example, a moiety active in the brain to a monovalent linking moiety. For example, if the monovalent binding moiety contains a CH2-CH3-Ig moiety and a scFab directed against the blood-brain barrier receptor, then the linker conjugates the scFab to the C-terminus of the CH3-CH2-Ig moiety. The linker conjugating the brain active moiety to the monovalent binding moiety (first linker) and the linker connecting the scFab to the C-terminus of the CH2-CH3-Ig domain (second linker) may be the same or different.

Can be used single-stranded peptide linkers, comprising from one to twenty amino acid residues connected by peptide bonds. In some embodiments, the amino acids are selected from twenty naturally occurring amino acids. In some other embodiments, one or more of the amino acids is selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In other embodiments, the linker is a chemical linker. In some embodiments, the linker is a single-stranded peptide linker with an amino acid sequence of at least 25 amino acid residues, in one preferred embodiment, 32 to 50 amino acid residues in length. In one embodiment, the peptide linker is a (G×S) _n -linker, where G is glycine, S is series, (× is 3, n is 8, 9 or 10) or (x is 4 and n is 6, 7 or 8), in one embodiment, x is 4, n is 6 or 7, in one preferred embodiment, x is 4, n is 7. In one embodiment, the linker is (G4S)4 (SED ID No. 08). In one embodiment, the linker is (G4S)6G2 (SEQ ID NO 07).

Conjugation can be performed using various chemical linkers. For example, a monovalent binding moiety or a hybrid polypeptide and a brain-active moiety can be conjugated with a variety of bifunctional protein binding agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N- maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), difunctional imidoester derivatives (such as dimethyl adipimidate 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 bis-active fluorine compounds (such as 1,5- difluoro-2,4-dinitrobenzene). The linker may be a "cleavable linker" facilitating release of the effector moiety after delivery to the brain. 52 (1992) 127-131; US 5208020).

Covalent conjugation can be done directly or via a linker. In some embodiments, direct conjugation is accomplished by constructing a hybrid polypeptide (ie, by genetically fusing two genes encoding a monovalent anti-BBBR binding moiety and an effector moiety and expressing as a single polypeptide (chain)). In some embodiments, direct conjugation is accomplished by forming a covalent bond between a reactive group on one of the two portions of the anti-BBBR monovalent binding moiety and the corresponding moiety or acceptor on the brain active moiety. In some embodiments, direct conjugation is carried out by modification (i.e., genetic modification) of one of the two molecules to be conjugated, which includes a reactive group (as non-limiting examples, a sulfhydryl group or a carboxyl group) that forms a covalent bond with another molecule, which must be conjugated under appropriate conditions. As a non-limiting example, a molecule (i.e., an amino acid) with the desired reactive group (i.e., a cysteine residue) can be introduced, for example, into a monovalent binding moiety against a BBBR antibody, and a disulfide bond is formed with a drug to treat a neurological disorder. Methods for covalently conjugating nucleic acids to proteins are also known in the art (i.e., photochemical crosslinking, see, for example, Zatsepin et al. Russ. Chem. Rev. 74 (2005) 77-95). Conjugation can also be carried out using various linkers. For example, a monovalent binding moiety and an effector moiety can be conjugated with a variety of bifunctional protein binding agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1 -carboxylate (SMCC), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyl adipimidate 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 bis-active fluorine compounds (such as 1,5-difluoro-2,4 -dinitrobenzene). Peptide linkers containing from one to twenty amino acid residues connected by peptide bonds can also be used. In some such embodiments, the amino acid residues are selected from twenty naturally occurring amino acids. In some other such embodiments, one or more of the amino acids is selected from glycine, alanine, proline, asparagine, glutamine, and lysine. The linker may be a "cleavable linker" to facilitate release of the effector moiety after delivery to the brain. 52 (1992) 127-131; US 5208020).

II. Compositions and Methods

In one aspect, the invention is based in part on the discovery that the blood-brain barrier transport modules described herein can be used to deliver a brain-active moiety across the blood-brain barrier to the brain. In some embodiments, the blood-brain barrier transport module the barrier includes a monovalent binding moiety that specifically binds to a blood-brain barrier receptor, such as the transferrin receptor. The blood brain barrier transport modules described herein can be used, for example, in the diagnosis or treatment of neurological disorders such as Alzheimer's disease, Parkinson's disease, and Alzheimer's disease with concomitant Parkinson's disease.

A. Exemplary antibodies against the blood-brain barrier receptor

Monovalent binding moieties that specifically bind to the blood-brain barrier receptor can be characterized in terms of their binding and transcytosis properties:

- efficient binding of cells expressing BBBR as a monovalent binding group,

- efficient transcytosis in vitro as a monovalent binding group,

- human - cynomolgus monkey cross-reactivity (for example, in the BIAcore and FACS experiments).

Screening for transcytosis was performed in an assay based on hCMEC/D3. The analysis was carried out in the label displacement mode. Brain endothelial cells hCMEC/D3 were incubated with monovalent binding group for 1 hour, washed, and then the following parameters were determined at 0 hours and 4 hours after washing:

i) the amount of monovalent binding group taken up by the cells during the loading phase,

ii) the basolateral amount of the monovalent binding moiety 4 hours after loading and washing;

iii) the apical amount of the monovalent binding moiety 4 hours after loading and washing;

iv) the amount of monovalent binding group in the cells (by cell lysis) at 0 h and 4 h after loading and washing;

v) the total amount of monovalent linking group at 0 hours and 4 hours after loading and washing.

In order to act as a monovalent binding moiety in the blood-brain barrier transport module described herein, the monovalent binding moiety must be i) taken up by hCMEC/D3 cells (endocytosis), ii) transported outside of hCMEC/D3 cells (exocytosis), and iii) is stable within hCMEC/D3 cells (no or low transport to endosomes for degradation).

Thus, in one embodiment, the monovalent binding moiety is characterized in an hCMEC/D3 assay by i) (substantial) uptake by hCMEC/D3 cells during a loading period of one hour, ii) release in the apical and/or basolateral compartment after the loading step, and a washing step 4 hours after washing, and iii) a low (intracellular) degradation rate.

In one embodiment, loading is at a concentration of about 2.67 μg/mL monovalent binding moiety for one hour.

It was found that a monovalent binding moiety, in order to act as a monovalent binding moiety in the blood-brain barrier transport module described herein, should show the following thresholds in the hCMEC/D3-based assay described above:

i) the amount of monovalent binding group taken up by the cells during the loading phase, 400 pg or more,

ii) a basolateral amount of monovalent binding group 4 hours after loading and washing - 100 pg or more, and

iii) apical amount of monovalent binding group 4 hours after loading and washing - 150 pg or more.

A mouse anti-human transferrin receptor antibody 128.1 (see WO 93/10819 and SEQ ID NOs 11 and 12 for variable region sequences) can be taken as a reference. In this case, a monovalent binding moiety, in order to act as a monovalent binding moiety in the blood-brain barrier transport module described herein, must show the following thresholds in the hCMEC/D3-based assay described above:

i) the amount of monovalent binding group captured by the cells during the loading phase, 20% or more of the loading of antibody 128.1,

ii) the basolateral amount of the monovalent binding group 4 hours after loading and washing - 15% or more of the basolateral amount of antibody 128.1; And

iii) apical amount of monovalent binding group 4 hours after loading and washing - 15% or more of the apical amount of antibody 128.1.

The hCMEC/D3 based assay was performed as follows (this is one embodiment of all aspects described herein):

Medium and supplements for hCMEC/D3 (see WO 2006/056879 and Weksler, B.B., et al., FAS EB J. 19 (2005) 1872-1874) can be obtained from Lonza. hCMEC/D3 cells (passages 26-29) are/can be cultured to confluency on collagen-coated coverslips (for microscopy) or in flasks in EBM2 medium containing 2.5% FBS and a quarter of the supplied growth factors and fully supplemented with the supplied hydrocortisone, gentamicin and ascorbic acid.

For all transcytosis assays in 12-well cell culture plates, insert filters (pore size 0.4 μm, diameter 12 mm) with a high pore density PET membrane (1×10 ⁸ pores/cm ² ) are/can be used. Medium volumes are calculated as 400 µl and 1600 µl for the apical and basolateral chambers, respectively. The apical chambers of the filter inserts are/may be coated with rat tail collagen I (7.5 µg/cm ² ) followed by fibronectin (5 µg/ml) with each incubation for 1 hour at room temperature. The hCMEC/D3 cells are/can be grown to confluent monolayers (about 2×10 ⁵ cells/cm ² ) for 10-12 days in EBM2 medium. Empty filters are/can be blocked with PBS containing 1% BSA for 1 hour or overnight (o/n) before analysis and then calibrated in EBM2 for at least 1 hour before analysis.

The assay (assay scheme, see FIG. 1) was performed in serum-free EBM2 medium, which was otherwise reconstituted as described herein. On the day of analysis, cells were incubated under serum-free conditions for 60 minutes to deplete the native ligand of the blood-brain barrier receptor in question. Insert filters with or without cells (but blocked overnight in complete medium) were incubated apically with the monoclonal antibodies in question (monovalent binding moiety) for 1 h at 37°C. The monolayers were washed at room temperature (RT) in serum free medium apically (400 microns) and basolaterally (1600 microns) three times for 3-5 minutes each. The prewarmed medium was added to the apical chamber and the filters were transferred to a fresh 12-well plate (blocked overnight with PBS containing 1% BSA) containing 1600 μl of prewarmed medium. At this point, the filters with or without cells were lysed in 500 μl of RIPA buffer to determine the uptake of the specific antibody (monovalent binding moiety). The remaining filters were incubated at 37° C. or 4° C. and samples were collected at various time points to determine the apical and/or basolateral release of the antibody (monovalent binding moiety). The content of antibodies in the samples can be quantified using a highly sensitive ELISA for IgG (see example 10). For each time point, data should be obtained from two blank filters and three cell culture filters.

The results for 69 anti-transferrin receptor antibodies are shown in the table below. Antibody 128.1 was used as a reference.

Antibody 299 shows transcytosis loading of 705 pg, where after 4 hours 170 pg (corresponding to 24% loading) can be detected in the basolateral compartment and 294 pg (corresponding to 42% loading) can be detected in the apical compartment.

Antibody 494 shows a transcytotic loading of 3510 pg, where at 4 h 748 pg (corresponding to 21% loading) can be detected in the basolateral compartment and 1503 pg (corresponding to 43% loading) can be detected in the apical compartment.

Antibodies that meet the criteria above are embodiments of this invention.

Thus, one of the aspects described herein is an anti-transferrin receptor antibody or a fragment thereof that binds the transferrin receptor, containing

(1) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 13 and a light chain variable domain having the amino acid sequence of SEQ ID NO 14, or

(2) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 15 and a light chain variable domain having the amino acid sequence of SEQ ID NO 16, or

(3) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 17 and a light chain variable domain having the amino acid sequence of SEQ ID NO 18, or

(4) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 19 and a light chain variable domain having the amino acid sequence of SEQ ID NO 20, or

(5) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 21 and a light chain variable domain having the amino acid sequence of SEQ ID NO 22, or

(6) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 23 and a light chain variable domain having the amino acid sequence of SEQ ID NO 24, or

(7) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 25 and a light chain variable domain having the amino acid sequence of SEQ ID NO 26, or

(8) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 27 and a light chain variable domain having the amino acid sequence of SEQ ID NO 28, or

(9) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 29 and a light chain variable domain having the amino acid sequence of SEQ ID NO 30, or

(10) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 31 and a light chain variable domain having the amino acid sequence of SEQ ID NO 32, or

(11) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 33 and a light chain variable domain having the amino acid sequence of SEQ ID NO 34, or

(12) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 35 and a light chain variable domain having the amino acid sequence of SEQ ID NO 36, or

(13) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 37 and a light chain variable domain having the amino acid sequence of SEQ ID NO 38, or

(14) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 39 and a light chain variable domain having the amino acid sequence of SEQ ID NO 40, or

(15) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 41 and a light chain variable domain having the amino acid sequence of SEQ ID NO 42, or

(16) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 43 and a light chain variable domain having the amino acid sequence of SEQ ID NO 44, or

(17) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 45 and a light chain variable domain having the amino acid sequence of SEQ ID NO 46, or

(18) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 47 and a light chain variable domain having the amino acid sequence of SEQ ID NO 48, or

(19) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 49 and a light chain variable domain having the amino acid sequence of SEQ ID NO 50, or

(20) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 51 and a light chain variable domain having the amino acid sequence of SEQ ID NO 52, or

(21) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 53 and a light chain variable domain having the amino acid sequence of SEQ ID NO 54, or

(22) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 55 and a light chain variable domain having the amino acid sequence of SEQ ID NO 56, or

(23) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 57 and a light chain variable domain having the amino acid sequence of SEQ ID NO 58, or

(24) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 59 and a light chain variable domain having the amino acid sequence of SEQ ID NO 60, or

(25) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 61 and a light chain variable domain having the amino acid sequence of SEQ ID NO 62, or

(26) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 63 and a light chain variable domain having the amino acid sequence of SEQ ID NO 64, or

(27) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 65 and a light chain variable domain having the amino acid sequence of SEQ ID NO 66, or

(28) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 67 and a light chain variable domain having the amino acid sequence of SEQ ID NO 68, or

(29) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 69 and a light chain variable domain having the amino acid sequence of SEQ ID NO 70, or

(30) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 71 and a light chain variable domain having the amino acid sequence of SEQ ID NO 72, or

(31) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 73 and a light chain variable domain having the amino acid sequence of SEQ ID NO 74, or

(32) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 75 and a light chain variable domain having the amino acid sequence of SEQ ID NO 76, or

(33) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 77 and a light chain variable domain having the amino acid sequence of SEQ ID NO 78, or

(34) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 79 and a light chain variable domain having the amino acid sequence of SEQ ID NO 80, or

(35) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 81 and a light chain variable domain having the amino acid sequence of SEQ ID NO 82, or

(36) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 83 and a light chain variable domain having the amino acid sequence of SEQ ID NO 84, or

(37) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 85 and a light chain variable domain having the amino acid sequence of SEQ ID NO 86, or

(38) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 87 and a light chain variable domain having the amino acid sequence of SEQ ID NO 88, or

(39) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 89 and a light chain variable domain having the amino acid sequence of SEQ ID NO 90, or

(40) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 91 and a light chain variable domain having the amino acid sequence of SEQ ID NO 92.

(41) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 93 and a light chain variable domain having the amino acid sequence of SEQ ID NO 94, or

(42) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 95 and a light chain variable domain having the amino acid sequence of SEQ ID NO 96, or

(43) a heavy chain variable domain having the amino acid sequence of SEQ ID NO 97 and a light chain variable domain having the amino acid sequence of SEQ ID NO 98.

One of the preferred aspects described herein is an anti-transferrin receptor antibody or fragment thereof that binds the transferrin receptor, comprising a heavy chain variable domain having the amino acid sequence of SEQ ID NO 23 and a light chain variable domain having the amino acid sequence of SEQ ID NO. NO 24.

One of the preferred aspects described herein is an anti-transferrin receptor antibody or fragment thereof that binds the transferrin receptor, comprising a heavy chain variable domain having the amino acid sequence of SEQ ID NO 91 and a light chain variable domain having the amino acid sequence of SEQ ID NO. NO 92.

The corresponding amino acid sequences are shown in the following table.

One aspect described herein is a humanized anti-transferrin receptor antibody or fragment thereof that binds the transferrin receptor, comprising

(1) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 13 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 14, or

(2) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 15 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 16, or

(3) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 17 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 18, or

(4) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 19 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 20, or

(5) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 21 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 22, or

(6) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 23 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 24, or

(7) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 25 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 26, or

(8) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 27 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 28, or

(9) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 29 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 30, or

(10) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 31 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 32, or

(11) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 33 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 34, or

(12) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 35 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 36, or

(13) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 37 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 38, or

(14) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 39 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 40, or

(15) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 41 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 42, or

(16) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 43 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 44, or

(17) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 45 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 46, or

(18) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 47 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 48, or

(19) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 49 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 50, or

(20) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 51 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 52, or

(21) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 53 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 54, or

(22) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 55 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 56, or

(23) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 57 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 58, or

(24) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 59 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 60, or

(25) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 61 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 62, or

(26) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 63 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 64, or

(27) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 65 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 66, or

(28) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 67 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 68, or

(29) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 69 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 70, or

(30) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 71 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 72, or

(31) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 73 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 74, or

(32) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 75 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 76, or

(33) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 77 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 78, or

(34) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 79 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 80, or

(35) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 81 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 82, or

(36) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 83 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 84, or

(37) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 85 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 86, or

(38) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 87 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 88, or

(39) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 89 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 90, or

(40) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 91, and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 92.

(41) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 93 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 94, or

(42) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 95 and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 96, or

(43) a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 97, and a humanized light chain variable domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 98.

One of the preferred aspects described herein is a humanized anti-transferrin receptor antibody or fragment thereof that binds the transferrin receptor, comprising a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 23, and a humanized variable a light chain domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 24.

One aspect described herein is a humanized anti-human transferrin receptor antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 109; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO 110; and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO 112. In one embodiment, the antibody also contains (d) HVR-L1 containing the amino acid sequence of SEQ ID NO 113; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO 114; and (f) HVR-L3 containing the amino acid sequence of SEQ ID NO 115.

One aspect described herein is a humanized anti-human transferrin receptor antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 109; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO 111; and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO 112. In one embodiment, the antibody also contains (d) HVR-L1 containing the amino acid sequence of SEQ ID NO 113; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO 114; and (f) HVR-L3 containing the amino acid sequence of SEQ ID NO 115.

In one aspect, the invention provides an anti-transferrin receptor antibody comprising at least one, two, three, four, five, or six HVRs selected from the following: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO 109; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO 110; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO 112; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO 113; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO 114; and (f) HVR-L3 containing the amino acid sequence of SEQ ID NO 115.

In one aspect, the invention provides an antibody comprising at least one, two, or all three VH HVR sequences selected from the following: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 109; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO 110; and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO 112. In one embodiment, the antibody also comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO 112. In another embodiment, the antibody also comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO 112. 112, and HVR-L3 containing the amino acid sequence of SEQ ID NO 115. In another embodiment, the antibody also contains HVR-H3 containing the amino acid sequence of SEQ ID NO 112, HVR-L3 containing the amino acid sequence of SEQ ID NO 115, and HVR-H2, containing the amino acid sequence of SEQ ID NO 111. In another embodiment, the antibody also contains (a) HVR-H1 containing the amino acid sequence of SEQ ID NO 109; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO 111; and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO 112.

In another aspect, the invention provides an antibody comprising at least one, two, or all three HVR VL sequences selected from the following: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 113; (b) HVR-L2 containing the amino acid sequence of SEQ ID NO 114; and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO 115. In one embodiment, the antibody also contains (a) HVR-L1 containing the amino acid sequence of SEQ ID NO 113; (b) HVR-L2 containing the amino acid sequence of SEQ ID NO 114; and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO 115.

In another aspect, an antibody of the invention also comprises (a) a VH domain comprising at least one, two, or all three VH HVR sequences selected from the following: (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO 109, ( ii) HVR-H2 containing the amino acid sequence of SEQ ID NO 110, and (iii) HVR-H3 containing the amino acid sequence selected from SEQ ID NO 112; and (b) a VL domain containing at least one, two or all three VL HVR sequences selected from the following: (i) an HVR-L1 containing the amino acid sequence of SEQ ID NO 113, (ii) an HVR-L2 containing the amino acid sequence of SEQ ID NO 114, and (c) an HVR-L3 containing the amino acid sequence of SEQ ID NO 115.

In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 109; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO 110; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO 112; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO 113; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO 114; and (f) HVR-L3 containing an amino acid sequence selected from SEQ ID NO 115.

One of the preferred aspects described herein is a humanized anti-transferrin receptor antibody or fragment thereof that binds the transferrin receptor, comprising a humanized heavy chain variable domain derived from a heavy chain variable domain having the amino acid sequence of SEQ ID NO 91 and a humanized variable a light chain domain derived from a light chain variable domain having the amino acid sequence of SEQ ID NO 92.

One aspect described herein is a humanized anti-human transferrin receptor antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 116; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO 117; and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO 119. In one embodiment, the antibody also contains (d) HVR-L1 containing the amino acid sequence of SEQ ID NO 120; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO 121; and (f) HVR-L3 containing the amino acid sequence of SEQ ID NO 122.

One aspect described herein is a humanized anti-human transferrin receptor antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 116; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO 118; and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO 119. In one embodiment, the antibody also contains (d) HVR-L1 containing the amino acid sequence of SEQ ID NO 120; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO 121; and (f) HVR-L3 containing the amino acid sequence of SEQ ID NO 122.

In one aspect, the invention provides an anti-transferrin receptor antibody comprising at least one, two, three, four, five, or six HVRs selected from the following: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO 116; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO 117; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO 119; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO 120; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO 121; and (f) HVR-L3 containing the amino acid sequence of SEQ ID NO 122.

In one aspect, the invention provides an antibody comprising at least one, two, or all three VH HVR sequences selected from the following: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 116; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO 117; and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO 119. In one embodiment, the antibody also comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO 119. In another embodiment, the antibody also comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO 119. NO 119, and HVR-L3 containing the amino acid sequence of SEQ ID NO 122. In another embodiment, the antibody also contains HVR-H3 containing the amino acid sequence of SEQ ID NO 119, HVR-L3 containing the amino acid sequence of SEQ ID NO 122, and HVR-H2 containing the amino acid sequence of SEQ ID NO 118. In another embodiment, the antibody also contains (a) HVR-H1 containing the amino acid sequence of SEQ ID NO 116; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO 118; and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO 119.

In another aspect, the invention provides an antibody comprising at least one, two, or all three VL HVR sequences selected from the following: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 120; (b) HVR-L2 containing the amino acid sequence of SEQ ID NO 121; and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO 122. In one embodiment, the antibody also contains (a) HVR-L1 containing the amino acid sequence of SEQ ID NO 120; (b) HVR-L2 containing the amino acid sequence of SEQ ID NO 121; and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO 122.

In another aspect, an antibody of the invention also comprises (a) a VH domain comprising at least one, two, or all three VH HVR sequences selected from the following: (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO 116, ( ii) HVR-H2 containing the amino acid sequence of SEQ ID NO 117, and (iii) HVR-H3 containing the amino acid sequence selected from SEQ ID NO 119; and (b) a VL domain containing at least one, two or all three VL HVR sequences selected from the following: (i) HVR-L1 containing the amino acid sequence of SEQ ID NO 120, (ii) HVR-L2 containing the amino acid sequence of SEQ ID NO 121, and (c) an HVR-L3 containing the amino acid sequence of SEQ ID NO 122.

In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 116; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO 117; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO 119; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO 120; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO 121; and (f) HVR-L3 containing an amino acid sequence selected from SEQ ID NO 122.

One aspect described herein is a transferrin transporter module comprising a full length IgG antibody as a brain active moiety, a linker and one scFab as a monovalent binding moiety that binds to the transferrin receptor, wherein the scFab is conjugated to the C-terminus of an Fc - region of one of the heavy chains of the IgG antibody through a linker.

One aspect described herein is a transferrin transporter module comprising a full length IgG antibody as a brain active moiety, a linker and one scFv as a monovalent binding moiety that binds to the transferrin receptor, where the scFv is conjugated to the C-terminus of the Fc - region of one of the heavy chains of the IgG antibody through a linker.

In one preferred embodiment, the monovalent linking moiety comprises HVR of SEQ ID NOS 109, 110, 112, 113, 114 and 115 or SEQ ID NOS 116, 117, 119, 120, 121 and 122.

In another aspect, an anti-transferrin receptor antibody according to any of the embodiments described above may include any of the features, alone or in combination, as described in sections 1-6 below:

1. Antibody affinity

In one embodiment, Kd is determined in a radiolabeled antigen binding assay (RIA) assay. In one embodiment, RIA is performed with a Fab version of the antibody of interest and its antigen. For example, the binding affinity of a Fab to an antigen in solution is measured by equilibrating the Fab with a minimum concentration of Labeled antigen in the presence of a series of titers of unlabeled antigen, then capturing the bound antigen on an aHTH-Fab antibody coated plate (see, e.g., Chen, Y. et al. , J Mol Biol 293 (1999) 865-881). To condition the assay, MICROTITER® multiwell plates (Thermo Scientific) were coated overnight with anti-Fab capture antibody (Cappel Labs) at 5 µg/mL in 50 mM sodium carbonate solution (pH 9.6) and then blocked. 2% (w/v) bovine serum albumin in PBS for 2-5 hours at room temperature (about 23°C). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [ ¹²⁵ I] antigen was mixed with serial dilutions of the Fab of interest (e.g., as assessed by the anti-VEGF antibody, Fab-12, in Presta, LG et al. ., Cancer Res. 57 (1997) 4593-4599). The Fab of interest was then incubated overnight; however, incubation may be continued for a longer period (eg, about 65 hours) to ensure equilibrium is reached. Thereafter, the mixtures were transferred to a capture plate and incubated at room temperature (eg, one hour). The solution was then removed and the plate was washed eight times with 0.1% polysorbate 20 (Tween-20®) in PBS. When the plates were dry, 150 μl/well of scintillant (MICROSCINT-20™; Packard) was added and analyzed on a TOPCOUNT™ gamma counter (Packard) for 10 minutes. Concentrations of each Fab that gave 20% of maximum binding or less were chosen for use in competitive binding assays.

According to another embodiment, Kd was measured in a surface plasmon resonance assay using ^BIACORE® -2000 or ^BIACORE® -3000 (BIAcore, Inc., Piscataway, NJ) at 25° C. on about 10 antigen immobilized CM5 chips. response units (RU). In one embodiment, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer's instructions. The antigen was diluted in 10 mM sodium acetate, pH 4.8, to a concentration of 5 μg/ml (about 0.2 μM) and then injected at a flow rate of 5 μl/min to achieve approximately 10 response units (RU) of bound protein. Following antigen challenge, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (from 0.78 nM to 500 nM) in PBS with 0.05% polysorbate 20 surfactant (TWEEN-20™) were added ( PBST) at 25° C. with a flow rate of about 25 µl/min. Association rates (k _on ) and dissociation rates (k _off ) were calculated using a simple 1:1 Langmuir binding model ( ^BIACORE® Evaluation Software version 3.2) by simultaneously fitting association and dissociation sensorgrams. The dissociation equilibrium constant (Kd) was calculated as the ratio k _off /k _on (see, for example, Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881). If the on-rate exceeded 10 ⁶ M ^-1 s ^-1 in the surface plasmon resonance assay described above, then the op-rate could be determined using a fluorescence quenching technique that measures the increase or decrease in fluorescence intensity (excitation equal to 295 nm; emission equal to 340 nm; 16 nm bandpass filter) at 25°C with 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen measured by a spectrometer, such as a spectrophotometer equipped with a rapid mixing system, stop-flow (Aviv Instruments) or SLM-AMINCO™ 8000 series spectrophotometer (ThermoSpectronic) with shaking cell.

2. Antibody fragments

In some embodiments, the antibody described herein is an antibody fragment. Antibody fragments include, but are not limited to, Fab-, Fab'-, Fab'-SH-, F(ab') ₂ -, Fv- and scFv-fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson, PJ et al., Nat. Med. 9 (2003) 129-134. For a review of scFv fragments, see, for example, Plueckthun, A., In; The Pharmacology of Monoclonal Antibodies, Vol.113, Rosenburg and Moore (eds.), Springer-Verlag, New York (1994), pp.269-315; see also WO 93/16185; US 5,571,894 and US 5,587,458. For a discussion of Fab- and P(ab') ₂ fragments containing receptor-binding reutilization antigens and having an increased in vivo half-life, see US 5,869,046.

Dimeric antibodies are antibody fragments with two antigen-binding sites, which may be divalent or bispecific. See, for example, EP 0404097; WO 1993/01161; Hudson, P.J. et al., Nat. Med. 9 (2003) 129-134; and Holliger, P. et al., Proc. Natl. Acad. sci. USA 90 (1993) 6444-6448. In Hudson, P.J. et al., Nat. Med. 9 (2003) 129-134) also describes trimeric and tetrameric antibodies.

Single domain antibodies are antibody fragments containing all or part of the heavy chain variable domains or all or part of the light chain variable domains of an antibody. In some embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, for example, US 6248516).

Antibody fragments can be obtained using various techniques, including, but not limited to, proteolytic cleavage of intact antibodies, as well as production by recombinant host cells (eg, E, coli or phage), as described in this document.

3. Chimeric and humanized antibodies

In some embodiments, the antibody provided herein is a chimeric antibody. Some chimeric antibodies are described, for example, in US 4816567; and in Morrison, S.L. et al., Proc. Natl. Acad. sci. USA 81 (1984) 6851-6855. In one example, a chimeric antibody contains a non-human variable region (eg, a variable region derived from a mouse, rat, hamster, rabbit, or primate, such as a monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from the parent antibody. Chimeric antibodies include their antigen-binding fragments.

In some embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody contains one or more variable domains in which the HVRs, such as CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody may also contain at least a portion of the human constant region. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (eg, the antibody from which the HVR residues are derived), eg, to restore or increase the specificity or affinity of the antibody.

Humanized antibodies and methods for their preparation are given, for example, in Almagro, J.C. and Fransson, J., Front. biosci. 13 (2008) 1619-1633 and also described in, for example, Riechmann, I. et al., Nature 332 (1988) 323-329; Queen, C. et al., Proc. Natl. Acad. sci. USA 86 (1989) 10029-10033; US 5821337, US 7527791, US 6982321 and US 7087409; Kashmiri, S.V. et al., Methods 36 (2005) 25-34 (describes grafting a specificity determining region (SDR)); Padlan, E.A., Mol. Immunol. 28 (1991) 489-498 (describes "transposition"); Dall'Acqua, W.F. et al., Methods 36 (2005) 43-60 (describes "FR shuffling"); and Osbourn, J. et al., Methods 36 (2005) 61-68 and Klimka, A. et al., Br. J. Cancer 83 (2000) 252-260 (describe a "guided selection" approach to FR shuffling).

Human frameworks that can be used for humanization include, but are not limited to: frameworks selected using a "best fit" technique (see, e.g., Sims, M.J. et al., J. Immunol. 151 (1993) 2296-2308); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter, P. et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Presta , L. G. et al., J. Immunol 151 (1993) 2623-2632); human mature (somatically mutant) frameworks or human germline frameworks (see, for example, Almagro, J.C. and Fransson, J., Front. Biosci. 13 (2008) 1619-1633); and framework regions obtained by screening the FR library (see, for example, Baca, M. et al., J. Biol. Chem. 272 (1997) 10678-10684, and Rosok, M. J. et al., J. Biol. Chem. 271 (19969 22611-22618).

4. Antibodies obtained from libraries

Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for creating phage display libraries and screening for such libraries of antibodies having the desired binding characteristics. Such techniques are described, for example, in Hoogenboom, H.R. et al., Methods in Molecular Biology 178 (2001) 1-37, and also, for example, the McCafferty, J. et al., Nature 348 (1990) 552-554; Clackson, T. et al., Nature 352 (1991) 624-628; Marks, J.D. et al., J. Mol. Biol. 222 (1992) 581-597; Marks, J.D. and Bradbury, A., Methods in Molecular Biology 248 (2003) 161-175; Sidhu, S.S. et al., J. Mol. Biol. 338 (2004) 299-310; Lee, C.V. et al., J. Mol. Biol. 340 (2004) 1073-1093; Fellouse, F.A., Proc. Natl. Acad. sci. USA 101 (2004) 12467-12472; and Lee, C.V. et al., J. Immunol. Methods 284 (2004) 119-132.

In some phage display techniques, the VH and VL gene repertoires have been separately cloned by polymerase chain reaction (PCR) and recombined randomly into phage libraries, which can then be screened for antigen-binding phage as described in Winter, G. et al., Ann. Rev. Immunol. 12 (1994) 433-455. Phage typically presents antibody fragments as either single chain Fv fragments (scFv) or Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need for hybridoma construction. Alternatively, a naive repertoire (e.g. from a human) can be cloned to provide a single source of antibodies to a wide range of non-self as well as self antigens without any immunization, as described in Griffiths, A.D. et al., EMBO J. 12 (1993) 725-734. Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing a random sequence to encode highly variable CDR3 regions and to perform in vitro rearrangement as described in Hoogenboom, H.R. and Winter, G., J. Mol. Biol. 227 (1992) 381-388. Patent publications describing human antibody phage libraries include, for example: US 5,750,373 and US 2005/0079574, US 2005/0119455, US 2005/0266000, US 2007/0117126, US 2007/0160598, US 2007/0237764, US 2007/0292936 and US 2009/0002360.

5. Multispecific antibodies

In some embodiments, the antibody provided herein is a multispecific antibody, such as a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In some embodiments, one of the binding specificities is for the transferrin receptor and the other for any other antigen. Bispecific antibodies can also be used to localize cytotoxic agents to cells that express the transferrin receptor. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant coexpression of two pairs of immunoglobulin heavy chains and light chains having different specificities (see Milstein, C. and Cuello, A.C., Nature 305 (1983) 537-540, WO 93/08829, and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659), and key-in-lock engineering (see, for example, US 5731168). Multispecific antibodies can also be made by using engineered electrostatic mixing effects to make Fc heterodimeric molecules (WO 2009/089004); crosslinking two or more antibodies or fragments (see, for example, US 4676980, and Brennan, M. et al., Science 229 (1985) 81-83); the use of leucine zippers to generate bispecific antibodies (see, for example, Kostelny, S. A. et al., J. Immunol. 148 (1992) 1547-1553); using the "dimeric antibody" technique to make bispecific antibody fragments (see, for example, Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and the use of single chain Fv(sFv)-flHMepoB (see, for example, Gruber, M et al., J. Immunol. 152 (1994) 5368-5374); and obtaining trispecific antibodies, as described, for example, in Tutt, A. et al., J. Immunol. 147 (1991) 60-69.

Designed antibodies with three or more functional antigen-binding sites, including "Octopus antibodies", are also included in this document (see, for example, US 2006/0025576).

An antibody or fragment herein also includes a "dual acting Fab", or "DAF" (dual acting Fab), containing an antigen-binding site that binds to the transferrin receptor as well as to another antigen other than it (see US 2008/ 0069820, for example).

An antibody or fragment herein also includes the multispecific antibodies described in WO 2009/080251, WO 2009/080252, WO 2009/080253, WO 2009/080254, WO 2010/112193, WO 2010/115589, WO 2010/1361 72, WO 2010 /145792 and WO 2010/145793.

In one embodiment of all aspects described herein, the anti-transferrin receptor antibody is a bispecific antibody.

One aspect described herein is a bivalent bispecific antibody containing

a) the first light chain and the first heavy chain of an antibody that specifically binds to the first antibody, and

b) a second light chain and a second heavy chain of an antibody that specifically binds to the second antibody, wherein the VL and VH variable domains of the second light chain and the second heavy chain replace each other,

where the first antigen or the second antigen is a human transferrin receptor.

The antibody according to a) does not contain the modification according to b), and the heavy chain and light chain according to a) are isolated chains.

In the antibody according to b)

in the light chain

the VL light chain variable domain is replaced by the VH heavy chain variable domain of said antibody, and

in the heavy chain

the VH heavy chain variable domain is replaced by the VL light chain variable domain of said antibody. In one incarnation

i) in the constant domain CL of the first light chain according to a) the amino acid at position 124 (numbering according to Kabat) is replaced by a positively charged amino acid, and in the constant domain CH1 of the first heavy chain according to a) the amino acid at position 147 or the amino acid at position 213 (numbering according to EC -Kabat index) is substituted with a negatively charged amino acid,

or

ii) in the constant domain CL of the second light chain according to b) the amino acid at position 124 (numbering according to Kabat) is replaced by a positively charged amino acid, and in the constant domain CH1 of the second heavy chain according to b) the amino acid at position 147 or the amino acid at position 213 (numbering according to EC -index Kabat) is substituted with a negatively charged amino acid.

In one preferred embodiment

i) in the CL constant domain of the first light chain according to a) the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (Kabat numbering) (in one preferred embodiment, independently with lysine (K) or arginine ( R)), and in the CH1 constant domain of the first heavy chain according to a) the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EC index),

or

ii) in the CL constant domain of the second light chain according to b) the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (Kabat numbering) (in one preferred embodiment, independently with lysine (K) or arginine ( R)), and in the CH1 constant domain of the second heavy chain according to b), the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EC index).

In one embodiment, in the CL constant domain of the second heavy chain, the amino acids at positions 124 and 123 are substituted with K (Kabat EC numbering).

In one embodiment, in the CH1 constant domain of the second light chain, the amino acids at positions 147 and 213 are substituted with E (numbering according to the Kabat EC index).

In one preferred embodiment, in the CL constant domain of the first light chain, the amino acids at positions 124 and 123 are substituted with K, and in the first heavy chain constant domain CH1, the amino acids at positions 147 and 213 are substituted with E (numbering according to the Kabat EC index).

In one embodiment, in the constant domain CL of the second heavy chain, the amino acids at positions 124 and 123 are substituted with K, in the constant domain CH1 of the second light chain, the amino acids at positions 147 and 213 are substituted with E, in the variable domain VL of the first light chain, the amino acid at position 38 is substituted with K, in In the VH variable domain of the first heavy chain, the amino acid at position 39 is substituted with E, in the VL variable domain of the second heavy chain, the amino acid at position 38 is substituted with K, and in the VH variable domain of the second light chain, the amino acid at position 39 is substituted with E (numbering according to the Kabat EC index).

One of the aspects described herein is a bivalent bispecific antibody containing

a) the first light chain and the first heavy chain of an antibody that specifically binds to the first antibody, and

b) a second light chain and a second heavy chain of an antibody that specifically binds to the second antibody, wherein the variable domains VL and VH of the second light chain and the second heavy chain are substituted for each other, and where the constant domains CL and CH1 of the second light chain and the second heavy chain are substituted for each other friend,

where the first antigen or the second antigen is a human transferrin receptor.

The antibody according to a) does not contain the modification according to b), and the heavy chain and light chain according to a) are isolated chains. In the antibody according to b) in the light chain

the light chain variable domain VL is replaced by the heavy chain variable domain VH of said antibody, and the light chain constant domain CL is replaced by the heavy chain constant domain CH1 of said antibody;

and in the heavy chain

the VH heavy chain variable domain is replaced by the VL light chain variable domain of said antibody, and the CH1 heavy chain constant domain is replaced by the CL light chain constant domain of said antibody.

One aspect described herein is a bivalent bispecific antibody containing

a) the first light chain and the first heavy chain of an antibody that specifically binds to the first antibody, and

b) a second light chain and a second heavy chain of an antibody that specifically binds to the second antibody, wherein the constant domains CL and CH1 of the second light chain and the second heavy chain replace each other,

where the first antigen or the second antigen is a human transferrin receptor.

The antibody according to a) does not contain the modification according to b), and the heavy chain and light chain according to a) are isolated chains. In the antibody according to b) in the light chain

the constant domain of the light chain CL is replaced by the constant domain of the heavy chain CH1 of the indicated antibody; and in the heavy chain

the CH1 heavy chain constant domain is replaced by the CL light chain constant domain of said antibody.

One aspect described herein is a mutispecific antibody containing

a) a full length antibody that specifically binds to the first antigen and consists of two antibody heavy chains and two antibody light chains, and

b) one, two, three or four single chain Fab fragments specifically binding to 1-4 other antigens (i.e. second and/or third and/or fourth and/or fifth antigen, preferably specifically binding to one additional antigen, i.e. with the second antigen),

wherein said single chain Fab fragments according to b) are fused to said full length antibody according to a) via a peptide linker at the C- or N-terminus of the heavy or light chain of said full length antibody,

where the first antigen or one of the additional antigens is the human transferrin receptor.

In one embodiment, one or two identical single chain Fab fragments binding to a second antigen are fused to said full length antibody via a peptidic linker at the C-terminus of the heavy or light chains of said full length antibody.

In one embodiment, one or two identical single chain Fab fragments that bind to the second antigen are fused to said full length antibody via a peptide linker at the C-terminus of said full length antibody heavy chains.

In one embodiment, one or two identical single chain Fab fragments that bind to the second antigen are fused to said full length antibody via a peptide linker at the C-terminus of said full length antibody light chains.

In one embodiment, two identical single chain Fab fragments that bind to a second antigen are fused to said full length antibody via a peptide linker at the C-terminus of each heavy or light chain of said full length antibody.

In one embodiment, two identical single chain Fab fragments that bind to a second antigen are fused to said full length antibody via a peptide linker at the C-terminus of each heavy chain of said full length antibody.

In one embodiment, two identical single chain Fab fragments binding to a second antigen are fused to said full length antibody via a peptide linker at the C-terminus of each light chain of said full length antibody.

One of the aspects described herein is a trivalent bispecific antibody containing

a) a full length antibody that specifically binds to the first antigen and consists of two antibody heavy chains and two antibody light chains,

b) the first polypeptide consisting of

ba) an antibody heavy chain variable domain (VH), or

bb) an antibody heavy chain variable domain (VH) and an antibody 1 constant domain (CH1),

wherein said first polypeptide is fused at the N-terminus of its VH domain via a peptide linker to the C-terminus of one of the two heavy chains of said full length antibody,

c) a second polypeptide consisting of

ca) an antibody light chain variable domain (VL),

or

cb) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL),

wherein said second polypeptide is fused at the N-terminus of its VL domain via a peptide linker to the C-terminus of the other of the two heavy chains of said full length antibody,

And

wherein the antibody heavy chain variable domain (VH) of the first polypeptide and the antibody light chain variable domain (VL) of the second polypeptide together form an antigen-binding site that specifically binds to the second antibody,

And

where the first antigen or the second antigen is a human transferrin receptor.

In one embodiment, the antibody heavy chain variable domain (VH) of the polypeptide according to b) and the antibody light chain variable domain (VL) of the polypeptide according to c) are linked and stabilized by an interchain disulfide bridge by introducing a disulfide bond between the following positions:

i) position 44 of the heavy chain variable domain and position 100 of the light chain variable domain, or

ii) position 105 of the heavy chain variable domain and position 43 of the light chain variable domain, or

iii) position 101 of the heavy chain variable domain and position 100 of the light chain variable domain (numbering is always according to the Kabat EC index).

Techniques for introducing non-natural disulfide bridges for stabilization are described, for example, in WO 94/029350, Rajagopal, V., et al., Prot. Eng. (1997) 1453-59; Kobayashi, H., et al., Nuclear Medicine & Biology, Vol. 25, (1998) 387-393; or Schmidt, M., et al., Oncogene (1999) 18 1711-1721. In one embodiment, the possible disulfide bond between the variable domains of the polypeptides of b) and c) is between position 44 of the heavy chain variable domain and position 100 of the light chain variable domain. In one embodiment, the possible disulfide bond between the variable domains of the polypeptides according to b) and c) is between position 105 of the heavy chain variable domain and position 43 of the light chain variable domain (numbering is always according to the Kabat EC index). In one embodiment, a trivalent bispecific antibody without said possible stabilization by disulfide bridges between the VH and VL variable domains of single chain Fab fragments is preferred.

One of the aspects described herein is a trispecific or tetraspecific antibody containing

a) the first light chain and the first heavy chain of a full-length antibody that specifically binds to the first antigen, and

b) a second (modified) light chain and a second (modified) heavy chain of a full-length antibody that specifically binds to a second antigen, where the VL and VH variable domains replace each other, and/or where the CL and CH1 constant domains replace each other,

c) where 1-4 antigen-binding peptides that specifically bind to 1-4 antigens (i.e. the third and/or fourth antigen) are fused via a peptide linker to the C- or N-terminus of the light chains or heavy chains a) and/ or b)

where the first antigen or the second antigen or one of the additional antigens is the human transferrin receptor.

The antibody according to a) does not contain the modification according to b), and the heavy chain and light chain according to a) are isolated chains.

In one embodiment, the trispecific or tetraspecific antibody also comprises, according to c), one or two antigen-binding peptides that specifically bind to one or two additional antigens.

In one embodiment, the antigen binding peptides are selected from the group of scFv fragment and scFab fragment.

In one embodiment, the antigen binding peptides are scFv fragments.

In one embodiment, the antigen binding peptides are scFab fragments.

In one embodiment, the antigen-binding peptides are fused to the C-terminus of the heavy chains a) and/or b).

In one embodiment, the trispecific or tetraspecific antibody comprises, according to c), one or two antigen-binding peptides that specifically bind to 1-4 antigens.

In one embodiment, the trispecific or tetraspecific antibody comprises, according to c), two identical antigen-binding peptides that specifically bind to a third antigen. In one preferred embodiment, these two identical antigen-binding peptides are fused via the same peptide linker to the C-terminus of the heavy chains a) and b). In one preferred embodiment, the two identical antigen binding peptides are either scFv fragments or scFab fragments.

In one embodiment, the trispecific or tetraspecific antibody comprises, according to c), two antigen-binding peptides that specifically bind to a third and a fourth antigen. In one embodiment, the two antigen-binding peptides are fused via the same peptide linker to the C-terminus of the heavy chains a) and b). In one preferred embodiment, said two antigen-binding peptides are either scFv fragments or scFab fragments.

One of the aspects described herein is a bispecific tetravalent antibody containing

a) two light chains and two heavy chains of an antibody that specifically bind to the first antigen (and contain two Fab fragments),

b) two additional antibody Fab fragments that specifically bind to a second antigen, wherein said additional Fab fragments are both fused via a peptide linker to either the C- or N-terminus of the heavy chains a),

And

where the following modifications have been made in fab fragments

i) in both Fab fragments from a) or both Fab fragments from b) the variable domains VL and VH replace each other and/or the constant domains CL and CH1 replace each other,

or

ii) in both Fab fragments from a) the variable domains VL and VH replace each other, and the constant domains CL and CH1 replace each other,

And

in both Fab fragments from b) the variable domains VL and VH replace each other, or the constant domains CL and CH1 replace each other, or

iii) in both Fab fragments from a) the variable domains VL and VH replace each other or the constant domains CL and CH1 replace each other,

And

in both Fab fragments from b) the variable domains VL and VH replace each other, and the constant domains CL and CH1 replace each other, or

iv) in both Fab fragments from a) the variable domains VL and VH replace each other, and in both Fab fragments from b) the constant domains CL and CH1 replace each other,

or

v) in both Fab fragments from a) the constant domains CL and CH1 replace each other, and in both Fab fragments from b) the variable domains VL and VH replace each other,

where the first antigen or the second antigen is a human transferrin receptor.

In one embodiment, said additional Fab fragments are both fused via a peptide linker to either the C-terminus of the heavy chains of a) or the N-terminus of the heavy chains of a).

In one embodiment, said additional Fab fragments are both fused via a peptide linker to the C-terminus of the heavy chains of a).

In one embodiment, said additional Fab fragments are both fused via a peptide linker to the N-terminus of the heavy chains of a).

In one embodiment, the following modifications are made to the Fab fragments:

i) in both Fab fragments from a) or both Fab fragments from b) the VL and VH variable domains replace each other, and/or

constant domains CL and CH1 replace each other.

In one embodiment, the following modifications are made to the Fab fragments:

i) in both Fab fragments from a) the variable domains VL and VH replace each other,

and/or

constant domains CL and CH1 replace each other.

In one embodiment, the following modifications are made to the Fab fragments:

i) in both Fab fragments from a) the constant domains CL and CH1 replace each other.

In one embodiment, the following modifications are made to the Fab fragments:

i) in both Fab fragments from b) the variable domains VL and VH replace each other,

and/or

constant domains CL and CH1 replace each other.

In one embodiment, the following modifications are made to the Fab fragments:

i) in both Fab fragments from b) the constant domains CL and CH1 replace each other.

One aspect described herein is a bispecific tetravalent antibody comprising:

a) a (modified) heavy chain of a first antibody that specifically binds to a first antigen and contains a first pair of VH-CH1 domains, where the N-terminus of the second pair of VH-CH1 domains of said first antibody is fused to the C-terminus of said heavy chain via a peptide linker,

b) two light chains of said first antibody from a),

c) a (modified) heavy chain of a second antibody that specifically binds to a second antigen and contains a first pair of VH-CL domains, where the N-terminus of the second pair of VH-CL domains of said second antibody is fused to the C-terminus of said heavy chain via a peptide linker, And

d) two (modified) light chains of said second antibody from c), each containing a pair of CL-CH1 domains,

where the first antigen or the second antigen is a human transferrin receptor.

One of the aspects described herein is a bispecific antibody containing

a) the heavy chain and light chain of the first full-length antibody that specifically binds to the first antigen, and

b) a heavy chain and a light chain of a second full-length antibody that specifically binds to a second antigen, where the N-terminus of the heavy chain is connected to the C-terminus of the light chain via a peptide linker,

where the first antigen or the second antigen is a human transferrin receptor.

The antibody according to a) does not contain the modification according to b), and the heavy chain and light chain are isolated chains.

One of the aspects described herein is a bispecific antibody containing

a) a full length antibody that specifically binds to the first antigen and consists of two antibody heavy chains and two antibody light chains, and

b) an Fv fragment that specifically binds to the second antibody and contains a VH ² domain and a VL ² domain, where both domains are connected to each other through a disulfide bridge,

where only either the VH ² domain or the VL ² domain is fused via a peptide linker to the heavy or light chain of a full length antibody specifically binding to the first antigen,

where the first antigen or the second antigen is a human transferrin receptor.

In a bispecific antibody, the heavy chains and light chains according to a) are isolated chains.

In one embodiment, the other VH ² domain or VL ² domain is not fused via a peptide linker to the heavy or light chain of a full length antibody specifically binding to the first antigen.

In all aspects described herein, the first light chain contains a VL domain and a CL domain, and the first heavy chain contains a VH domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain.

In one embodiment of all aspects of the invention, the antibody described herein is a multispecific antibody that requires heterodimerization of at least two heavy polypeptide chains, wherein the antibody specifically binds to a human transferrin receptor and a second non-human transferrin receptor antigen.

Several approaches to CH3 modification have been described to support heterodimerization, e.g. 011 /90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO 2013/096291, which are incorporated herein by reference. Generally, in approaches known in the art, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain are designed in a complementary manner such that a heavy chain containing one engineered CH3 domain does not homodimerize with another heavy chain of the same structure. (e.g., a CH3-engineered first heavy chain can no longer homodimerize with another CH3-engineered first heavy chain; and a CH3-engineered second heavy chain can no longer homodimerize with another CH3-engineered second heavy chain). In this case, a heavy chain containing one designed CH3 domain is forced to heterodimerize with another heavy chain containing a CH3 domain that is designed in a complementary manner. For this embodiment of the invention, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain are designed in a complementary manner with amino acid substitutions such that the first heavy chain and the second heavy chain are forced to heterodimerize while the first heavy chain and the second heavy chain are larger. do not homodimerize (for example, for steric reasons).

The various approaches to maintaining heavy chain heterodimerization known in the art that have been cited and included above are contemplated as other alternatives used in a multispecific antibody of the invention that contains a "non-cross Fab region" derived from the first antibody. , which specifically binds to the first antigen, and a "cross-Fab region" derived from a second antibody that specifically binds to the second antigen, in combination with the specific amino acid substitutions described above in this invention.

The CH3 domains of the multispecific antibody described herein can be changed in a key-in-the-lock manner, which is detailed in several examples, e.g., WO 96/027011, Ridgway, J.B., et al., Protein Eng . 9 (1996) 617-621; and Merchant, A. M., et al., Nat Biotechnol 16 (1998) 677-681. In this method, the interacting surfaces of the two CH3 domains are altered in such a way as to increase the heterodimerization of both heavy chains containing the two CH3 domains. Each of the two CH3 domains (two heavy chains) can be a "key" and the other a "lock". The introduction of a disulfide bridge also stabilizes heterodimers (Merchant, A.M., et al., Nature Biotech 16 (1998) 677-681; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35) and increases exit.

In one preferred embodiment, the multispecific antibody described herein contains the T366W mutation in the key chain CH3 domain and the T366S, L368A, Y407V mutations in the lock chain CH3 domain (numbering according to the EC index according to Kabat ). An additional interstrand disulfide bridge can also be introduced between the CH3 domains (Merchant, A.M., et al., Nature Biotech 16 (1998) 677-681), for example, by introducing a Y349C mutation in the keychain CH3 domain and E356C mutations or S354C mutations in the lock chain CH3 domain. Thus, in another preferred embodiment, the multispecific antibody described herein contains the Y349C and T366W mutations in one of the two CH3 domains and the E356C, T366S, L368A and Y407V mutations in the other of the two CH3 domains, or the multispecific antibody described herein document contains mutations Y349C and T366W in one of the two CH3 domains and mutations S354C, T366S, L368A and Y407V in the other of the two CH3 domains (an additional Y349C mutation in one CH3 domain and an additional E356C or S354C mutation in the other CH3 domain to form an interchain disulfide bridge) (numbering according to the Kabat EC index).

Alternatively or additionally, other key-in-lock techniques, such as those described in EP 1870459A1, may also be used. In one embodiment, the multispecific antibody described herein contains R409D and K370E mutations in the key chain CH3 domain and D399K and E357K mutations in the lock chain CH3 domain (numbered according to the Kabat EC index).

In one embodiment, the multispecific antibody described herein comprises the T366W mutation in the key chain CH3 domain and the T366S, L368A, and Y407V mutations in the lock chain CH3 domain, and additionally the R409D and K370E mutations in the CH3 domain. "chain-key" and mutations D399K and E357K in the CH3-domain of the "chain-lock" (numbering in accordance with the Kabat EU index).

In one embodiment, the multispecific antibody described herein contains mutations Y349C and T366WB in one of two CH3 domains and mutations S354C, T366S, L368A, and Y407V in the other of the two CH3 domains, or the multispecific antibody described herein contains mutations Y349C and T366W in one of the two CH3 domains and mutations S354C, T366S, L368A, Y407V in the other of the two CH3 domains, and additional mutations R409D and K370E in the "key chain" CH3 domain and mutations D399K and E357K in CH3- "chain-lock" domain (numbering in accordance with the Kabat EU-index).

In addition to the "key in the lock" technique, other techniques are known in the art to modify the CH3 domains of the heavy chains of a multispecific antibody to allow for heterodimerization. These techniques, especially those described in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/ US Pat.

In one embodiment of the multispecific antibody described herein, the approach described in EP 1870459 is used to maintain heterodimerization of the first heavy chain and second heavy chain of the multispecific antibody. This approach is based on the introduction of charged amino acids with opposite charges at certain amino acid positions in the CH3/CH3 domain interaction surface between both the first and second heavy chains.

Accordingly, this embodiment relates to the multispecific antibody described herein, wherein in the tertiary structure of the antibody, the first heavy chain CH3 domain and the second heavy chain CH3 domain form an interaction surface that is located between the respective antibody CH3 domains, where each of the respective amino acids sequences of the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain contains a number of amino acids that are located inside the specified interaction surface in the tertiary structure of the antibody, where from the number of amino acids that are located in the interaction surface in the CH3 domain of one heavy chain, the first amino acid is substituted a positively charged amino acid, and from a number of amino acids that are located in the interaction surface in the CH3 domain of another heavy chain, the second amino acid is replaced by a negatively charged amino acid. The multispecific antibody of this embodiment is also referred to herein as "CH3(+/-)-engineered multispecific antibody" (wherein the abbreviation "+/-" denotes oppositely charged amino acids that have been introduced into the respective CH3 domains).

In one embodiment of said CH3(+/-) engineered multispecific antibody described herein, the positively charged amino acid is selected from K, R and H and the negatively charged amino acid is selected from E or D.

In one embodiment of said CH3(+/-) engineered multispecific antibody described herein, the positively charged amino acid is selected from K and R and the negatively charged amino acid is selected from E or D.

In one embodiment of said CH3(+/-)-engineered multispecific antibody described herein, the positively charged amino acid is K and the negatively charged amino acid is E.

In one embodiment of said CH3(+/-)-engineered multispecific antibody described herein, in the CH3 domain of one heavy chain, the R amino acid at position 409 is substituted by D, and the K amino acid at position is substituted by E, and in the CH3 domain of another heavy chain chain amino acid D at position 399 is substituted by K, and amino acid E at position 357 is substituted by K (numbering according to the Kabat EC index).

In one embodiment of the multispecific antibody described herein, the approach described in WO 2013/157953 is used to maintain heterodimerization of the first heavy chain and second heavy chain of the multispecific antibody. In one embodiment of said multispecific antibody described herein, in the CH3 domain of one heavy chain, the amino acid T at position 366 is substituted with K, and in the CH3 domain of the other heavy chain, the amino acid L at position 351 is substituted with D (numbering according to the Kabat EC index) . In another embodiment of said multispecific antibody described herein, in the CH3 domain of one heavy chain, the amino acid T at position 366 is substituted with K, and the amino acid L at position 351 is substituted with K, and in the CH3 domain of the other heavy chain, the amino acid L at position 351 is substituted D (numbering according to the Kabat EU index).

In another embodiment of said multispecific antibody described herein, in the CH3 domain of one heavy chain, the amino acid T at position 366 is substituted with K, and the amino acid L at position 351 is substituted with K, and in the CH3 domain of the other heavy chain, the amino acid L at position 351 is substituted D (numbering according to the Kabat EU index). In addition, at least one of the following substitutions is contained in the CH3 domain of another heavy chain: the Y amino acid at position 349 is substituted with E, the Y amino acid at position 349 is substituted with D, and the L amino acid at position 368 is substituted with E (numbering according to the Kabat EC index ). In one embodiment, the amino acid L at position 368 is replaced by E (numbering according to the Kabat EC index).

In one embodiment of the multispecific antibody described herein, the approach described in WO 2012/058768 is used to maintain heterodimerization of the first heavy chain and second heavy chain of the multispecific antibody. In one embodiment of said multispecific antibody described herein, in the CH3 domain of one heavy chain, the L amino acid at position 351 is substituted by Y, and the Y amino acid at position 407 is substituted by A, and in the CH3 domain of the other heavy chain, the T amino acid at position 366 is substituted A, and amino acid K at position 409 is replaced by F (numbering according to the Kabat EC index). In another embodiment, in addition to the substitutions described above, the CH3 domain of another heavy chain is substituted at least one of the amino acids at positions 411 (originally T), 399 (originally D), 400 (originally S), 405 (originally F), 390 (originally N) and 392 (originally K) (numbering according to Kabat EC index). The preferred substitutions are:

substitution of amino acid T at position 411 with an amino acid selected from N, R, Q, K, D, E and W (numbering according to the Kabat EC index),

substitution of the amino acid D at position 399 with an amino acid selected from R, W, Y and K (numbering according to the Kabat EC index),

substitution of the amino acid S at position 400 with an amino acid selected from among E, D, R and K (numbering according to the Kabat EC index),

substitution of the amino acid F at position 405 with an amino acid selected from I, M, T, S, V and W (numbering according to the Kabat EC index);

substitution of the amino acid N at position 390 with an amino acid selected from R, K and D (numbered according to the Kabat EC index);

- substitution of amino acid K at position 392 with an amino acid selected from among V, M, R, L, F and E (numbering according to the Kabat EC index).

In another embodiment of said multispecific antibody described herein (designed according to WO 2012/058768), in the CH3 domain of one heavy chain, the amino acid L at position 351 is replaced by Y, and the amino acid Y at position 407 is replaced by A, and in the CH3 domain by another heavy chain, the T amino acid at position 366 is substituted with V, and the K amino acid at position 409 is substituted with F (numbering according to the Kabat EC index). In another embodiment of said multispecific antibody described herein, in the CH3 domain of one heavy chain, the Y amino acid at position 407 is substituted by A, and in the CH3 domain of the other heavy chain, the T amino acid at position 366 is substituted by A, and the K amino acid at position 409 is substituted F (numbering according to the Kabat EU index). In the last of the above embodiments, in the CH3 domain of said other heavy chain, the K amino acid at position 392 is substituted with E, the T amino acid at position 411 is substituted with E, the D amino acid at position 399 is substituted with R, and the S amino acid at position 400 is substituted with R (EC numbering). -Kabat index).

In one embodiment of the multispecific antibody described herein, the approach described in WO 2011/143545 is used to maintain heterodimerization of the first heavy chain and second heavy chain of the multispecific antibody. In one embodiment of said mutispecific antibody described herein, amino acid modifications in the CH3 domains of both heavy chains are introduced at positions 368 and/or 409 (numbered according to the Kabat EC index).

In one embodiment of the multispecific antibody described herein, the approach described in WO 2011/090762 is used to maintain heterodimerization of the first heavy chain and second heavy chain of the multispecific antibody. WO 2011/090762 relates to amino acid modifications according to the "key in the lock" technique. In one embodiment of said CH3(K1H) engineered multispecific antibody described herein, in the CH3 domain of one heavy chain, the amino acid T at position 366 is substituted with W, and in the CH3 domain of the other heavy chain, the amino acid Y at position 407 is substituted with A (numbering according to the Kabat EU index). In another embodiment of said CH3(KiH)-engineered multispecific antibody described herein, in the CH3 domain of one heavy chain, the amino acid T at position 366 is substituted by Y, and in the CH3 domain of the other heavy chain, the amino acid Y at position 407 is substituted by T (numbering according to the Kabat EU index).

In one embodiment of the multispecific antibody described herein, which is of the IgG2 isotype, the approach described in WO 2011/090762 is used to maintain heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody.

In one embodiment of the multispecific antibody described herein, the approach described in WO 2009/089004 is used to maintain heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody. In one embodiment of said multispecific antibody described herein, in the CH3 domain of one heavy chain, the amino acid K or N at position 392 is substituted with a negatively charged amino acid (in one preferred embodiment substituted with E or D, in one preferred embodiment with D), and in CH3 -domain of another heavy chain, amino acid D at position 399, amino acid E or D at position 356, or amino acid E at position 357 is substituted with a positively charged amino acid (in one preferred embodiment substituted for K or R, in one preferred embodiment K, in one preferred embodiment the amino acid at positions 399 or 356 are replaced by K) (numbering according to the Kabat EC index). In one additional embodiment, in addition to the above substitutions in the CH3 domain of one heavy chain, the amino acid K or R at position 409 is replaced with a negatively charged amino acid (in one preferred embodiment E or D, in one preferred embodiment D) (numbering according to the Kabat EC index). In yet another further embodiment, in addition to or alternative to the above substitutions in the CH3 domain of one heavy chain, amino acid K at position 439 and/or amino acid K at position 370 is independently substituted with a negatively charged amino acid (in one preferred embodiment E or D, in one preferred embodiment D ) (numbering according to the Kabat EC index).

In one embodiment of the multispecific antibody described herein, the approach described in WO 2007/147901 is used to maintain heterodimerization of the first heavy chain and second heavy chain of the multispecific antibody. In one embodiment of said multispecific antibody described herein, in the CH3 domain of one heavy chain, the K amino acid at position 253 is substituted with E, the D amino acid at position 282 is substituted with K, and the K amino acid at position 322 is substituted with D, and in the CH3 domain of the other heavy chain, the D amino acid at position 239 is substituted with K, the E amino acid at position 240 is substituted with K, and the K amino acid at position 292 is substituted with D (Kabat EC numbering).

In one embodiment of the multispecific antibody described herein, the approach described in WO 2007/110205 is used to maintain heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody.

In one embodiment of all aspects and embodiments described herein, the multispecific antibody is a bispecific antibody or a trispecific antibody. In one preferred embodiment of the present invention, the multispecific antibody is a bispecific antibody.

In one embodiment of all aspects described herein, the antibody is a divalent or trivalent antibody. In one embodiment, the antibody is a divalent antibody.

In one embodiment of all aspects described herein, the multispecific antibody has a constant domain structure from an IgG type antibody. In yet another embodiment of all aspects described herein, the multispecific antibody is characterized in that said multispecific antibody is a human IgG1 subclass or a human IgG1 subclass with mutations L234A and L235A. In yet another embodiment of all aspects described herein, the multispecific antibody is characterized in that said multispecific antibody is of the human IgG2 subclass. In yet another embodiment of all aspects described herein, the multispecific antibody is characterized in that said multispecific antibody is of the human IgG3 subclass. In yet another embodiment of all aspects described herein, the multispecific antibody is characterized in that said multispecific antibody is a human IgG4 subclass or a human IgG4 subclass with an additional S228P mutation. In yet another embodiment of all aspects described herein, the multispecific antibody is characterized in that said multispecific antibody is of the human IgG1 subclass or the human IgG4 subclass. In yet another embodiment of all aspects described herein, the multispecific antibody is characterized in that said multispecific antibody is of the human IgG1 subclass with mutations L234A and L235A (numbering according to the Kabat EC index). In yet another embodiment of all aspects described herein, the multispecific antibody is characterized in that said multispecific antibody is of the human IgG1 subclass with mutations L234A, L235A, and P329G (Kabat EC numbering). In yet another embodiment of all aspects described herein, the multispecific antibody is characterized in that said multispecific antibody is of the human IgG4 subclass with mutations S228P and L235E (numbering according to the Kabat EC index). In yet another embodiment of all aspects described herein, the multispecific antibody is characterized in that said multispecific antibody is of the human IgG4 subclass with mutations S228P, L235E and P329G (numbering according to the Kabat EC index).

In one embodiment of all aspects described herein, an antibody comprising a heavy chain comprising a CH3 domain as defined herein contains an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EC index). ). In one embodiment of all aspects described herein, an antibody comprising a heavy chain comprising a CH3 domain as defined herein contains an additional C-terminal glycine residue (G446, Kabat EC numbering).

6. Antibody variants

In some embodiments, variants of the amino acid sequences of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Variants of the amino acid sequence of an antibody can be obtained by introducing appropriate modifications to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequences of the antibody. Any combination of deletions, insertions, and substitutions can be used to produce the final construct, as long as the final construct has the desired characteristics, such as antigen binding.

a) Variants with substitution, insertion and deletion

In some embodiments, antibody variants are provided having one or more amino acid substitutions. Substitutional mutagenesis sites of interest include HVR and FR. Conservative substitutions are presented in Table 1 under the heading "conservative substitutions". More significant changes are presented in Table 1 under the heading "illustrative substitutions" and are further described below with reference to classes of amino acid side chains. Amino acid substitutions can be introduced into an antibody of interest and products can be tested for desired activity, improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

Amino acids can be grouped according to common side chain properties:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) main: His, Lys, Arg;

(5) residues that affect chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions entail replacing a member of one of these classes with a member of another class.

One type of substitution variant involves the replacement of one or more hypervariable region residues of the parent antibody (eg, a humanized or human antibody). Typically, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. antibodies. An example of a substitution variant is an affinity matured antibody, which can be easily generated, for example, using phage display based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and tested for a particular biological activity (eg, binding affinity).

Changes (eg substitutions) can be made to the HVR, eg to improve the affinity of the antibody. Such changes can be made in HVR hotspots, ie. in residues encoded by codons that undergo mutation at a high rate during the somatic maturation process (see, for example, Chowdhury, P.S., Methods Mol. Biol. 207 (2008) 179-196), and/or in residues that contact with antigen, thus obtaining variant VH or VL, which are examined for binding affinity. Affinity maturation by construction and reselection from secondary libraries has been described, for example, in Hoogenboom, H.R. et al. in Methods in Molecular Biology 178 (2002) 1-37. In some embodiments of affinity maturation, the diversity is introduced into the variable genes selected for maturation using any of a variety of techniques (eg, error-corrected PCR, strand shuffling, or oligonucleotide directed mutagenesis). Then a secondary library is created. This library is then screened for any antibody variants with the desired affinity. Another method of diversification involves HVR-directed approaches, in which several HVR residues are randomized (eg, 4-6 residues at a time). HVR residues involved in antigen binding can be specifically identified, for example, using alanine-scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

In some embodiments, substitutions, insertions, or deletions may occur in one or more HVRs, as long as such changes do not cause a significant reduction in the ability of the antibody to bind antigen. For example, conservative changes can be made to the HVR (eg conservative substitutions provided herein) that do not significantly reduce binding affinity. Such changes, for example, may be in the HVR outside the residues in contact with the antigen. In some embodiments of the variant VH and VL sequences above, each HVR is either unchanged or contains no more than one, two, or three amino acid substitutions.

A method that can be used to identify antibody residues or regions that can be targets of mutagenesis is called "alanine-scanning mutagenesis" and is described in Cunningham, B.C. and Wells, J.A., Science 244 (1989) 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine if they affect interaction of an antibody with an antigen. Further substitutions may be made at amino acid sites showing functional sensitivity to the original substitutions. Alternatively or additionally, the crystal structure of the antigen-antibody complex is determined to identify points of contact between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for replacement. Variants can be screened to determine if they exhibit desired properties.

Amino acid sequence insertions include amino- and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more hundred residues, as well as intrasequence insertions of one or more amino acid residues in length. Examples of end inserts include antibodies with an N-terminal methionine residue. Other insertion variants of the antibody molecule include fusion of the N- or C-terminus of the antibody with an enzyme (eg, ADEPT) or polypeptide that increases the serum half-life of the antibody.

b) Glycosylation variants

In some embodiments, an antibody provided herein is altered to increase or decrease the degree to which the antibody is glycosylated. The addition or removal of glycosylation sites to an antibody can conveniently be accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

If the antibody contains an Fc region, then the carbohydrate attached to it can be changed. Native antibodies produced by mammalian cells typically contain a branched, dual-antennary oligosaccharide, typically N-linked to the Asn297 CH2 domain of the Fc region (see, e.g., Wright, A. and Morrison, S.L., TIBTECH 15 (1997) 26-32). The oligosaccharide may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the two-antennary oligosaccharide structure. In some embodiments, modifications to the oligosaccharides in an antibody of the invention may be made in order to produce antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydrate structure lacking fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain per Asn297 relative to the sum of all gl and co structures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) measured by MALDI-TOF mass spectrometry as described, for example, in WO 2008/077546. Asn297 refers to an asparagine residue located approximately at position 297 in the Fc region (EC residue numbering of the Fc region); however, Asn297 can also be located at about plus/minus 3 amino acids before or after position 297, i. between positions 294 and 300, due to minor sequence changes in the antibodies. Such fucosylation variants may have improved ADCC function. See, for example, US 2003/0157108; US 2004/0093621. Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: US 2003/0157108; WO2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO2003/085119; WO2003/084570; WO2005/035586; W02005/035778; WO2005/053742; WO2002/031140; Okazaki, A. et al., J. Mol. Biol. 336 (2004) 1239-1249; Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 (2004) 614-622. Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka, J. et al., Arch. Biochem. Biophys. 249 (1986) 533-545; US 2003/0157108; and WO 2004 /056312, in particular, example 11), and knockout cell lines, such as CHO cells with a knockout of the alpha-1,6-fucosyltransferase gene, FUT8 (see, for example, Yamane-Ohnuki, N. et al., Biotech. Bioeng 87 (2004) 614-622; Kanda, Y. et al., Biotechnol Bioeng 94 (2006) 680-688; and WO 2003/085107).

Also provided are variants of antibodies with symmetrically branched oligosaccharides, for example, in which a two-antennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878; US 6602684; and US 2005/0123546. Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also contemplated. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087; WO 1998/58964; and WO 1999/22764.

c) Fc region variants

In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby producing a variant Fc region. A variant Fc region may comprise a human Fc region sequence (eg, an IgG1, IgG2, IgG3, or IgG4 human Fc region) containing an amino acid modification (eg, substitution) at one or more amino acid positions.

In some embodiments, the invention provides a variant of an antibody that has some but not all of the effector functions, making it a desirable candidate for applications where the in vivo half-life of the antibody is important and yet some effector functions (such as complement and ADCC) are not necessary or harmful. An in vitro and/or in vivo cytotoxicity assay can be performed to confirm the reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody does not bind FcyR (and therefore likely does not have ADCC activity) but retains FcRn binding capacity. The primary cells for mediating ADCC, NK cells, only express FcyRIII, while monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 in Ravetch, J.V. and Kinet, J.P., Annu. Rev. Immunol. 9 (1991) 457-492. Non-limiting examples of in vitro assays to assess the ADCC activity of a molecule of interest are described in US 5500362 (see, for example, Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 83 (1986) 7059-7063; and Hellstrom, I. et al., Proc Natl Acad Sci USA 82 (1985) 1499-1502); US 5821337 (see Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactive assays can be used (see, for example, non-radioactive ACTI™ cytotoxicity assay for flow cytometry (CellTechnology Inc., Mountain View, CA); and non-radioactive cytotoxicity assay CytoTox 96® (Promega, Madison, Wisconsin)). Useful effector cells for these assays include peripheral blood mononuclear cells (PBMCs) and natural killer cells (NK cells). Alternatively or additionally, the ADCC activity of a molecule of interest can be assessed in vivo, for example in an animal model such as that described in Clynes, R. et al., Proc. Natl. Acad. sci. USA 95 (1998) 652-656. C1q binding assays can also be performed to confirm that the antibody cannot bind C1q and therefore does not have CDC activity. See, for example, C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To evaluate complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro, H. et al., J. Immunol. Methods 202 (1996) 163-171; Cragg, M. S. et al., Blood 101 (2003) 1045 -1052 and Cragg, M.S. and M.J. Glennie, Blood 103 (2004) 2738-2743). Determination of FcRn binding and in vivo clearance/half-life can also be performed using methods known in the art (see, for example, Petkova, S. B. et al., Int. Immunol. 18 (2006) 1759-1769).

Antibodies with reduced effector function include those with the replacement of one or more of the Fc region residues among 238, 265, 269, 270, 297, 327 and 329 (US 6737056). Such Fc mutants include Fc mutants with substitutions at two or more amino acid positions selected from 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant with residues 265 and 297 replaced by alanine (US 7332581) .

Certain variants of antibodies with improved or reduced FcR binding have been described. (See, for example, US 6737056; WO 2004/056312, and Shields, R. L. et al., J. Biol. Chem. 276 (2001) 6591-6604).

In some embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improves ADCC, such as substitutions at positions 298, 333, and/or 334 in the Fc region (EC residue numbering).

In some embodiments, changes are made to the Fc region that result in altered (i.e., increased or decreased) Clq binding and/or complement dependent cytotoxicity (CDC), for example, as described in US 6194551, WO 99/51642 and Idusogie , HER. et al., J. Immunol. 164 (2000) 4178-4184.

Antibodies with increased half-life and increased binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer, R.L. et al., J. Immunol. 117 (1976) 587-593; and Kim, J.K. et al., J. Immunol. 24 (1994) 2429-2434) are described in US 2005/0014934. These antibodies contain an Fc region with one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376 , 378, 380, 382, 413, 424 or 434, for example, with the replacement of residue 434 in the Fc region (US 7371826).

See also Duncan, A.R. and Winter, G., Nature 322 (1988) 738-740; US 5648260; US 5624821 and WO 94/29351 for other examples of Fc region variants.

d) Cysteine-engineered antibody variants

In some embodiments, it may be desirable to create cysteine-engineered antibodies, such as "thioMAb" ("thioMAb"), in which one or more than one of the antibody residues is replaced by a cysteine residue. In specific embodiments, the substituted residues are located at accessible sites on the antibody. By replacing these residues with cysteine, the reactive thiol groups are thus located at accessible sites on the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker drug moieties, to form an immunoconjugate, as described below. In some embodiments, one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) light chain; A118 (EC number) of the heavy chain; and S400 (EC numbering) of the heavy chain Fc region. Cysteine-engineered antibodies can be prepared as described, for example, in US 7521541.

e) Antibody derivatives

In some embodiments, an antibody provided herein may be further modified to contain additional non-protein moieties that are known in the art and readily available. Groups suitable for obtaining derivatives of the antibody include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, 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 (eg glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde may offer manufacturing advantages due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. Generally, the number and/or type of polymers used to derivatize can be determined based on a number of considerations, including, but not limited to, the specific properties or functions of the antibody that will be improved, whether or not the antibody derivative is used in therapy under certain conditions. etc.

In another embodiment, conjugates of an antibody and a non-protein moiety are provided that can be selectively heated when exposed to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam, N. W. et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation can be of any wavelength, including, but not limited to, wavelengths that do not damage normal cells, but heat the non-protein moiety to a temperature at which cells closest to the non-protein moiety of the antibody die.

B. Recombinant Methods and Compositions

Antibodies can be produced using recombinant methods and compositions, such as those described in US 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-transferrin receptor antibody described herein is provided. Such a nucleic acid may encode an amino acid sequence containing the VL and/or an amino acid sequence containing the VH of an antibody (eg, an antibody light and/or heavy chain). In another embodiment, one or more vectors (eg, an expression vector) containing such a nucleic acid are provided. In another embodiment, a host cell is provided that contains such a nucleic acid. In one such embodiment, the host cell contains (for example, has been transformed by them): (1) a vector containing a nucleic acid that encodes an amino acid sequence containing the VL of the antibody and an amino acid sequence containing the VH of the antibody, or (2) a first vector containing a nucleic acid that encodes an amino acid sequence containing the VL of the antibody, and a second vector containing a nucleic acid that encodes an amino acid sequence containing the VH of the antibody. In one embodiment, the host cell is eukaryotic, eg, a Chinese hamster ovary (CHO) cell or a lymphoid cell (eg, Y0, NS0, Sp2/0). In one embodiment, a method for producing an anti-transferrin receptor antibody is provided, which comprises culturing a host cell containing a nucleic acid encoding the antibody as described above under conditions suitable for expression of the antibody, and optionally isolating the antibody from the host cell (or culture culture). host cell environment).

For recombinant production of an anti-transferrin receptor antibody, a nucleic acid encoding an antibody, such as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid can be easily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes that are capable of specifically binding to genes encoding antibody heavy and light chains).

Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be made in bacteria, particularly when glycosylation and Fc effector functions are not needed. For the expression of antibody fragments and polypeptides in bacteria, see, for example, US 5648237, US 5789199 and US 5840523. (See also Charlton, K.A., In: Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed. ), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the antibody can be isolated from the mass of bacterial cells in a soluble fraction and can be further purified.

In addition to prokaryotes, suitable cloning or expression hosts for antibody-encoding vectors are eukaryotic microorganisms such as filamentous fungi or yeasts, including strains of fungi and yeasts in which glycosylation pathways are "humanized" resulting in the production of an antibody with a partially or fully human character. glycosylation (see Gerngross, T. U., Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215).

Suitable host cells for expression of the glycosylated antibody are also obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used in combination with insect cells, in particular to transfect Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts. See, for example, US 5959177, US 6040498, US 6420548, US 7125978 and US 6417429 (describing the PLANTIBODIES™ technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to growth in suspension can be used. Other examples of useful mammalian host cell lines are the CV1 monkey kidney line transformed with SV40 (COS-7); human embryonic kidney line (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); murine Sertoli cells (TM4 cells described, for example, in Mather, J. P., Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma (HELA) cells; dog kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells, as described, for example, in Mather, J.P. et al., Annals N.Y. Acad. sci. 383 (1982) 44-68; MRC5 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 77 (1980) 4216-4220 ); and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of some mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2004), pp.255-268.

C. Analyzes

The anti-transferrin receptor antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activity using various assays known in the art.

1. Binding analysis

In one aspect, an antibody of the invention is assayed for antigen binding activity, for example, by known methods such as ELISA, alphaLISA, Western blot, direct (antibody) and reverse phase protein microarrays, etc.

In an exemplary ELISA or alphaLISA assay, a transferrin receptor in solution (cell supernatant, cell or tissue lysates, body fluids, etc.) is bound by a capture antibody that specifically binds to a first epitope on the transferrin receptor or to the transferrin receptor in a specific conformation, and to a detection antibody linked to a detection moiety that specifically binds to a second epitope or conformation of the transferrin receptor. The registration is based on grouping detection (chemiluminescence, fluorescence, energy transfer induced luminescence, etc.).

In the case of an antibody microchip, the antibodies are dotted onto glass or nitrocellulose chips. The slides are blocked and incubated with a solution containing the transferrin receptor, washed to remove unbound antibodies, and bound antibodies are detected with the appropriate fluorescently labeled secondary antibody. The fluorescent signal is measured using a fluorescent glass scanner. Similarly, for reverse phase microarrays, glass or nitrocellulose chips are dotted with recombinant transferrin receptor, cell supernatant, cell or tissue lysates, body fluids, etc. Slides are blocked and individual microarrays are incubated with antibodies against a specific epitope on the transferrin receptor. Unbound antibodies are washed away and bound antibodies are detected with the appropriate fluorescently labeled secondary antibody. The fluorescent signal is measured with a fluorescent glass scanner (Dernick, G., et al., J. Lipid Res. 52 (2011) 2323-2331).

D. Methods and compositions for diagnosis and detection

In some embodiments, any anti-transferrin receptor antibodies provided herein are used to detect the presence of a human transferrin receptor in a biological sample. The term "detection" as used herein includes quantitative or qualitative detection. In some embodiments, the biological sample contains a cell or tissue, such as brain tissue.

In one embodiment, an anti-transferrin receptor antibody is provided for use in a diagnostic or detection method. In another aspect, a method is provided for detecting the presence of a transferrin receptor in a biological sample. In some embodiments, the method includes contacting a biological sample with an anti-transferrin receptor antibody described herein under conditions that permit the binding of the anti-transferrin receptor antibody to the transferrin receptor and detecting whether a complex is formed between the anti-transferrin receptor antibody and the transferrin receptor. Such a method may be an in vitro or in vivo method. In one embodiment, an anti-transferrin receptor antibody is used to select subjects eligible for anti-transferrin receptor antibody therapy, eg, where transferrin receptor is a biomarker for patient selection.

Examples of disorders that can be diagnosed using an antibody of the invention include neurodegeneration with brain iron storage type 1 (NBIA1), idiopathic autonomic failure, Down's syndrome, Guam's complex, as well as several disorders with Leo bodies and such as diffuse body disease. Lewy disease (DLBD), Lewy body variant of Alzheimer's disease (LBVAD), some forms of Gaucher's disease, and Parkinson's disease with dementia (PDD).

In some embodiments, labeled antibodies against the transferrin receptor are provided. Labels include, but are not limited to, labels or moieties that are directly detectable (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels) as well as moieties such as enzymes or ligands that are detectable indirectly, such as through enzymatic reaction or molecular interaction. Examples of labels include, but are not limited to, radioisotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases, for example, firefly luciferase and bacterial luciferase (US 4737456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, (3-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g. glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogen azu , heterocyclic oxidases, e.g., uricase and xanthine oxidase, in combination with an enzyme that uses hydrogen peroxide to oxidize a label precursor, such as HRP, lactoperoxidase or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, etc.

E. Pharmaceutical formulations

Pharmaceutical formulations of an anti-transferrin receptor antibody described herein are prepared by mixing such an antibody, having the desired purity, with one or more possible pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) ( 1980)), as lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; 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 polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides 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 (eg Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersing agents such as soluble neutrally active hyaluronidase glycoproteins (sHASEGP), e.g. human soluble PH-20 hyaluronidase glycoproteins such as rhuPH20 (HYLENEX®, Baxter International, Inc. ). Some examples of sHASEGP and methods of using them, including rhuPH20, are described in US 2005/0260186 and US 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

Illustrative 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 formulations including histidine acetate buffer.

The composition herein may also contain more than one active ingredient needed in the treatment of specific indications, especially with complementary activities that do not interfere with each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose.

The active ingredients can be microcapsuled, for example, by coacervation or interfacial polymerization techniques, for example, hydroxymethyl cellulose or gelatin microcapsules and polymethyl methacrylate microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. (ed.), (1980).

Sustained release preparations can be made. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing the antibody or conjugate, the matrices being in the form of molded articles such as films or microcapsules.

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

III. Production products

In another aspect of the invention, there is provided a product of manufacture containing materials used in the treatment, prevention and/or diagnosis of the disorders described above. The product of manufacture contains a container and a label or package insert attached to or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV bags, and the like. Containers can be made from various materials such as glass or plastic. The container contains the composition alone or in combination with another composition effective for the treatment, prevention, and/or diagnosis of the condition and may have a sterile entry port (for example, the container may be an intravenous solution bag or a vial with a needle-piercing stopper for subcutaneous injection). At least one active agent in the composition is an antibody according to this invention. The label or package insert indicates that the composition is used to treat the selected condition. In addition, the product of manufacture may include: (a) the first container with the composition contained therein, where the composition contains an antibody according to this invention; and (b) a second container with the composition contained therein, where the composition also contains a cytotoxic or other therapeutic agent. The product of manufacture in this embodiment of the invention may also contain a package insert indicating that the compositions can be used to treat a particular condition. Alternatively or additionally, the product may also include 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 also include other materials commercially and consumerly desirable, including other buffers, diluents, filters, needles, and syringes.

It is understood that any of the above products of manufacture may include the immunoconjugate of the invention in place of, or in addition to, an anti-transferrin receptor antibody.

VI. Examples

The following are examples of methods and compositions according to the invention. It is understood that various other embodiments may be practiced given the general description given above.

Materials and methods

Recombinant DNA techniques

For DNA manipulation, standard methods were used as described in Sambrook, J., et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions.

Gene and oligonucleotide synthesis

The desired gene segments were obtained by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into a plasmid for propagation/amplification in E. coli. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or by PCR. The corresponding oligonucleotides were obtained using Metabion GmbH (Planegg-Martinsried, Germany)

Reagents

All commercial chemicals, antibodies, and kits were used according to the manufacturers' protocols unless otherwise noted.

Example 1

Immunization of rabbits and mice

Immunization of mice

NMRI mice were genetically immunized using an expression plasmid vector encoding a full-length human or cynomolgus TfR by intradermal injection of vector DNA at a dose of 100 μg followed by electroporation (2 square pulses, 1000 V/cm, duration 0.1 ms, interval 0.125 s ; then 4 rectangular pulses, 287.5 V/cm, duration 10 ms, interval 0.125 sec). Mice were subjected to 6 or 7 consecutive immunizations on days 0, 14, 28, 42, 56, 70 and 84. The fourth and sixth immunizations were performed with a vector encoding cynomolgus TfR; a vector encoding human TfR was used for all other immunizations. Blood was taken on days 36, 78 and 92, and sera were obtained from it, which were used to determine the titer by ELISA (see below). Animals with the highest titers were selected for boost on day 96 by intravenous administration of either 10 ⁶ human TF-1 cells or 50 µg of recombinant human soluble TfR lacking the helical domain (human TfR extracellular domain starting at Leu122, ending at Asn608, expressed in HEK293F cells as an N-terminal fusion with the human Fc region and purified by protein A affinity chromatography and size exclusion chromatography); monoclonal antibodies were isolated by hybridoma technology based on their ability to bind the human and cynomolgus monkey transferrin receptor expressed on the surface of stably transfected CHO-K1 cells (see example 4).

Immunization of rabbits

New Zealand white rabbits or transgenic rabbits expressing a repertoire of humanized antibodies were genetically immunized using an expression plasmid vector encoding a full-length human or cynomolgus TfR by intradermal injection of vector DNA at a dose of 400 μg followed by electroporation (5 rectangular pulses, 750 V/cm , duration 10 ms, interval 1 s). Rabbits were subjected to 6 consecutive immunizations on days 0, 14, 28, 56, 84 and 112. The fourth and sixth immunizations were performed with a vector encoding cynomolgus TfR; for all other immunizations, a vector encoding human TfR was used. Blood (10% of estimated total blood volume) was drawn on days 35, 63, 91, and 119. Serums were obtained, which were used for titer determination by ELISA (see below), and peripheral mononuclear cells were isolated, which were used as a source of antigen -specific b-cells in the process of cloning b-cells (see example 2).

Determination of serum titers (ELISA)

Human recombinant soluble TfR (R&D Systems, cat. no. 2474-TP) was immobilized on a NUNC Maxisorb 96-well plate at 3 μg/ml, 100 μl/well, in PBS, and then the plate was blocked with 2% CroteinC in PBS , 200 µl/well; made serial dilutions of antisera in duplicate, in 0.5% CroteinC in PBS, 100 μl/well; detection was performed with (1) HRP-conjugated goat anti-mouse antibody (Jackson Immunoresearch/Dianova 115-036-071; 1/16000) for all mouse sera, (2) HRP-conjugated donkey anti-rabbit IgG antibody (Jackson Immunoresearch/Dianova 711 -036-152; 1/16000) for all rabbit sera, (3) rabbit anti-human IgG antibody (Pierce/Thermo Scientific 31423; 1/5000) for transgenic rabbit sera only, (4) biotinylated goat anti-human kappa- region (Southern Biotech/Biozol 2063-08, 1/5000) and streptavidin-HRP for sera from transgenic rabbits only; diluted in 0.5% CroteinC in PBS, 100 µl/well. At all stages, the plates were incubated for 1 h at 37°C. Between all steps, the plates were washed 3 times with 0.05% Tween 20 in PBS. The signal was obtained by adding soluble BM Blue POD Substrate (Roche), 100 μl/well; and stopped by adding 1 M HCl, 100 μl/well. Absorbance was read at 450 nm against 690 nm as a reference. The titer was defined as the dilution of the antiserum giving half the maximum signal.

Example 2

Cloning of B cells from rabbits

Isolation of Rabbit Peripheral Blood Mononuclear Cells (PBMCs)

Blood samples were taken from a total of 6 animals (2 wild-type (wt) and 4 transgenic (tg) rabbits). These rabbits were obtained from two different immunization programs: the first program with two wt rabbits and two tg rabbits and the second program with two tg rabbits (see also the example "Immunization of rabbits"). Whole blood containing EDTA was diluted 2-fold in 1x PBS (PAA, Pasching, Austria) and density gradient centrifuged using mammalian lymphocytes (Cedarlane Laboratories, Burlington, Ontario, Canada) according to the manufacturer's instructions. The PBMCs were washed twice with 1x PBS.

Medium EL-4 B5

RPMI 1640 medium (Pan Biotech, Eidenbach, Germany) supplemented with 10% FCS (Hyclone, Logan, Utah, USA), 2 mM glutamine, 1% penicillin/streptomycin solution (PAA, Pasching, Austria), 2 mM sodium pyruvate, 10 mM HEPES (PAN Biotech, Eidenbach, Germany) and 0.05 mM (3-mercaptoethanol (Gibco, Paisley, Scotland)

Cell depletion

First Immunization Program: Sterile 6-well plates (for cell culture) coated with a confluent monolayer of CHO cells were used to deplete macrophages/monocytes by non-specific adhesion as well as non-specific binding of lymphocytes.

Second immunization program: Skipped the depletion step with CHO cell coated wells as we could not rule out that B cells producing antibodies that cross-react with anti-hamster transferrin receptor antibodies would not be eliminated. Thus, empty sterile 6-well plates (for cell culture) were used to deplete macrophages and monocytes by non-specific adhesion, allowing potential B-lymphocytes producing antibodies with hamster cross-reactivity (and possibly mouse cross-reactivity), reach the next step in the workflow.

For each immunization program: a maximum of 4 ml of media and up to 6×10 ⁶ PBMC from an immunized rabbit was added to each well and allowed to bind for 1 hour at 37° C. in an incubator. The cells in the supernatant (peripheral blood lymphocytes (PBL)) were used in the antigen panning step.

Enrichment of B cells at the human transferrin receptor

6-well tissue culture plates coated with a monolayer of human transferrin receptor positive CHO cells were seeded to a density of 6×10 ⁶ PBL per 4 ml of medium and allowed to bind for 1 hour at 37° C. in an incubator. Unattached cells were removed by thoroughly washing the wells 1-2 times with 1x PBS. The remaining attached cells were separated by treatment with trypsin for 10 minutes at 37°C in an incubator. Trypsinization was stopped with EL-4 B5 medium. Cells were kept on ice until immunofluorescent staining. Immunofluorescent staining and flow cytometry Anti-IgG-FITC (ABD Serotec, Düsseldorf, Germany) was used to sort single cells. For surface staining, cells from the depletion and enrichment step were incubated with anti-IgG-FITC antibody in PBS and incubated for 45 minutes in the dark at 4°C. After staining, the PBMCs were washed twice with ice-cold PBS. Finally, PBMCs were resuspended in ice-cold PBS and immediately subjected to FACS analysis. Prior to FACS analysis, propidium iodide was added at a concentration of 5 μg/ml (BD Pharmingen, San Diego, CA, USA) to distinguish between dead and living cells.

Single cells were sorted using a Becton Dickinson FACSAria equipped with a computer and FACSDiva software (BD Biosciences, USA).

Cultivation of B cells

Cultivation of rabbit B cells was carried out in the same way as described in Zubler et al. (1985). Briefly, single sorted rabbit B cells were incubated in 96-well plates with 200 µl/well of EL-4 B5 medium containing Pansorbin cells (1:100,000) (Calbiochem (Merck), Darmstadt, Germany), 5% rabbit thymocyte supernatant ( TSN-M13 (10242), MicroCoat, Bernried, Germany) and gamma-irradiated murine thymoma cells EL-4-B5 (2.5 x 10 ⁴ /well) for 7 days at 37°C in an atmosphere of 5% CO ₂ in incubator. Cultured B cell supernatants were removed for screening and the remaining cells were immediately harvested and frozen at -80° C. in 100 μl of RLT buffer (Qiagen, Hilden, Germany).

Example 3

Phage display for selection and production of monovalent anti-TfR-IgG

Selection by phage display

Antibodies binding to human and cynomolgus TfRs were generated by phage display using standard protocols (Silacci et al, Proteomics, 5 (2005) 2340-2350). The synthesized gene for the hTfR-Fc(KiH)-Avi antigen (KiH - "key in the lock", Avi - Avi-tag) was cloned by connecting the hTfR ECD to the N-terminal hinge of the human Fc-region of the "lock" type, which carries C- an Avi end tag (SEQ ID NO 99) and ligated into a vector for expression in mammalian cells. All of our mammalian expression vectors carry the MPSV promoter to initiate transcription and translation, where transcription ends with a synthetic polyA signal sequence located after the ORF. In addition, the vector contains the EBV op'R sequence for autonomous replication in cell lines expressing EBV-EBNA. The correct ORF was confirmed by sequencing. This vector was used to express EBNA HEK293 suspension cells by co-expression with an empty key-type Fc region and BirA protein and adding 1 mM biotin to the culture medium. The resulting biotinylated protein was purified by protein A affinity chromatography followed by size exclusion chromatography. The cyTfR-Fc(KiH)-Avi antigen was prepared respectively (SEQ ID NO 100).

The library used was a fully synthetic library using the human framework regions of the VH1, 3, 4, and 5 families, as well as V-kappa-1, -2 and -3 and V-lambda-3. The randomized amino acids were in CDR-H3 and CDR-L3. Library pools were created using each VH heavy chain along with all the various light chain libraries. Selection was carried out in solution according to the following procedure: 1. Binding approximately 1012 phagemid particles from each library to 100 nM biotinylated TfR-avi-his for 0.5 h in a total volume of 1 ml; 2. capturing biotinylated TfR-avi-his and specifically bound phage particles by adding 5.4 x 10 ⁷ streptavidin-coated magnetic beads over 10 minutes; 3. washing the beads 5-10 times with 1 ml PBS/Tween 20 and 5-10 times with 1 ml PBS; 4. elution of phage particles by adding 1 ml of 100 mm TEA (triethylamine) for 10 minutes and neutralization by adding 500 μl of 1 M Tris/HCl, pH 7.4; and 5. re-infection of exponentially growing bacteria E, coli TG1, infection with helper phage VCSM13 and subsequent precipitation of phagemid particles with PEG/NaCl for use in subsequent rounds of selection. The selection was carried out for 3-5 rounds using a constant or decreasing (from 10 ^-7 M to 2×10 ^-9 M) antigen concentration. In the second round, capture of antigen/phage complexes was performed using neutravidin plates instead of streptavidin beads. Since binders made against the recombinant soluble antigen often showed only weak binding on cells, we introduced two additional selection steps using cells displaying the native antigen after a second or third round of enrichment on the recombinant antigen.

Two cell lines were used, TF-1 and NCl-H460 (both available from ATCC). Briefly, 1×10 ⁶ cells were incubated with about 1012 phage particles for 1 hour on ice to avoid receptor-mediated internalization. Washing was performed by 5-10 centrifugation steps using PBST buffer (PBS containing 1% Tween-20). Whole cells were used to infect bacteria with TG1 to release the phage.

Specific binding agents were identified by ELISA as follows: 100 μl of 10 nM biotinylated TfR-avi-his per well was applied to neutravidin plates. Fab-containing bacterial supernatants were added and Fab binding was recorded by their FLAG tags with an anti-FLAG/HRP-BTopn4Horo antibody. ELISA-positive clones were expressed in bacteria as soluble Fab fragments in a 96-well format, and supernatants were subjected to a kinetic screening experiment using SPR analysis using ProteOn XPR36 (BioRad). Clones expressing the Fab with the highest affinity constants were identified and the corresponding phagemids were sequenced.

Purification of Fab antibodies

Top10 cells were individually transfected with phagemid plasmids from all clones positive in cell ELISA. The cells were grown in the medium and the production of Fab antibodies was induced. Fab antibodies were isolated by periplasm preparation and purified by IMAC.

FACS analysis

Purified Fab antibodies were added to TF-1 cells at various concentrations. Cell-bound Fab antibodies were detected with a fluorescent labeled anti-Fab antibody and measured by FACS. EC ₅₀ values were calculated.

Production and characterization of selected clones

Convert to IgG format

Selected clones with a complex half-life (t1/2) of 6 to 20 minutes or an EC ₅₀ on cells from 10 nM to 500 nM were selected for conversion to IgG format, with a clear cell binding signal either on FACS or in cellular ELISA, and with cross-reactive linking against both antigens, hTfR-Fc(KiH)-Avi and cyTfR-Fc(KiH)-Avi.

To do this, the VL domain was amplified by PCR and cloned into a vector for expression in mammalian cells, as described above, immediately before the CL domain. In addition, the VH domain was amplified by PCR and cloned immediately before the CH-Fc domain. The sequences of both expression vectors were determined.

Production of IgG antibodies

EBNA HEK293 suspension cells were transfected with both LC and HC coding plasmids and cultured for 7 days. The supernatant was clarified by sterile filtration. IgG concentration was determined by protein A chromatography.

Determination of the EU ₅₀ using the TagLite method

The hTfR or cyTfR ECD gene, respectively, including the transmembrane domain, was ligated into a vector for expression in mammalian cells after a SNAP tag. This vector was used to transfect HEK293 suspension cells with EBNA, producing a hybrid TfR display with a C-terminal SNAP tag (SEQ ID NO 101, SEQ ID NO 102). The SNAP tag was specifically labeled with SNAP-Lumi4Tb (Cisbio). Labeling efficiency was determined by measuring the terbium emission at 615 nm. Labeled cells were stored at -80°C.

In the presence of anti-human Fc-d2 IgG antibody, the labeled cells were incubated with various dilutions of IgG supernatant and FRET signals were measured after 4 hours (Lumi4Tb donor label emission: 615 nm and d2 acceptor label: 665 nm). EC ₅₀ values were calculated to give 25 IgGs that bind to both hTfR and cyTfR.

Determination of cell binding by FACS

The full-length hTfR or cyTfR gene, respectively, was cloned into a vector for expression in mammalian cells. This vector was used to transfect CHO EBNA suspension cells. The IgG supernatant was directly applied to TfR-bearing cells. After washing with PBST, the antigen-antibody complex was detected using an anti-huFc-IgG-HRP-antibody conjugate, followed by staining with 3,3'-5,5'-tetramethylbenzidine. Functional display was tested using a commercially available anti-TfR antibody. All 25 Tag Lite-positive clones showed a strong binding signal.

Production and Purification of Univalent IgG-Like Molecules The VH domain was cloned into a mammalian expression vector encoding a key human IgG1 HC that carries mutations L234A, L235A and P329G. This vector was used to express in suspension HEK293 EBNA cells co-expressing LC (the vector described above) and an empty lock Fc domain carrying mutations L234A, L235A, P329G, H435R and Y436F. The resulting protein was purified by protein A affinity chromatography. In cases where the monomeric protein was more than 95%, as determined by analytical size exclusion chromatography, the protein was further purified by size exclusion chromatography.

Example 4

Identification of Antibodies Binding Human and Cynomolgus TfRs by Cellular ELISA

Cellular ELISA with stably transfected CHO-K1 cells was used to screen rabbit B cells or mouse hybridoma supernatants for antibodies recognizing human and cynomolgus TfRs. Stable transfectants were obtained by transfection of CHO-K1 cells with expression plasmids containing expression cassettes for human and cynomolgus TfRs and for neomycin phosphotransferase. After transfection, cells were diluted in growth medium containing 500 μg/ml G418 (Life Technologies). After the emergence of growing clones, cells were separated, stained with MEM-75 (Abeam) or 13E4 (Life Technologies) and PE-labeled anti-human and cynomolgus TfR secondary antibodies, and highly fluorescent cells were sorted as single cells into wells of a 96-well plate (FACS Aria). After 7 days of growth, the clones were again checked for TfR expression, and clones with the best expression were selected for cell ELISA experiments.

Briefly, 15,000 cells/well were plated in a 384-well plate and incubated for 18 hours at 37° C., 5% CO ₂ . The supernatant was removed using an automated washer (BIOTEK) and 30 μl of the supernatant containing the antibody was added to each well followed by 24 μl of growth medium. After 2 hours of incubation, the wells were emptied and 30 μl of 0.05% glutaraldehyde in PBS was added for 45 minutes at room temperature. After 3 washes with PBS/0.025% Tween 20 (PBST), 30 μl of anti-rabbit HRP or anti-mouse HRP (Southern Biotech) diluted 1:5000 in blocking buffer was added and the plates were incubated for 1 hour at room temperature. The wells were washed 6 times with PBST, a signal was obtained by adding 30 μl of TMB per well, and the absorbance at 450 nm was measured.

Example 5

Cloning and expression of aHTH-TfR antibodies

Recombinant DNA techniques

For DNA manipulation, standard methods were used as described in Sambrook, J., et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions.

Gene and oligonucleotide synthesis

The desired gene segments were obtained by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into a plasmid for propagation/amplification in E. coli. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or by PCR. The corresponding oligonucleotides were obtained using Metabion GmbH (Planegg-Martinsried, Germany)

PCR amplification of V domains

Total RNA was prepared from B cell lysates (resuspended in RLT buffer, Qiagen, Cat #79216) using the NucleoSpin 8/96 RNA kit (Macherey &Nagel; 740709.4, 740698) according to the manufacturer's protocol. RNA was eluted in 60 µl without RNase water. 6 µl of RNA was used to prepare cDNA by reverse transcription reaction using Superscript III First-Strand Synthesis SuperMix reagents (Invitrogen 18080-400) and oligo-dT primer according to the manufacturer's instructions. All steps were performed on a Hamilton ML Star System instrument. 4 µl cDNA was used to amplify the immunoglobulin heavy and light chain variable regions (VH and VL) with AccuPrime SuperMix reagents (Invitrogen 12344-040) in a final volume of 50 µl using primers rbHC.up and rbHC.do for the heavy chain, rbLC. up and rbLC.do for light chain wild-type rabbit B cells, and BcPCR_FHLC_leader.fw and BcPCR_huCkappa.rev for light chain transgenic rabbit B cells (see table below). All forward primers were specific for the signal peptide (VH and VL, respectively), while the reverse primers were specific for constant regions (VH and VL, respectively). PCR conditions for RbVH+RbVL were as follows: hot start at 94°C for 5 minutes; 35 cycles: 20 seconds at 94°C, 20 seconds at 70°C, 45 seconds at 68°C; final elongation at 68°C for 7 minutes. The PCR conditions for HuVL were as follows: hot start at 94° C. for 5 minutes; 40 cycles: 20 seconds at 94°C, 20 seconds at 52°C, 45 seconds at 68°C; final elongation at 68°C for 7 minutes.

8 µl of the 50 µl PCR mixture was loaded onto 48 E-Gel 2% (Invitrogen G8008-02). Positive PCR reactions were purified with the NucleoSpin Extract II kit (Macherey&Nagel; 740609250) according to the manufacturer's protocol and eluted in 50 µl elution buffer. All purification steps were performed on a Hamilton ML Starlet System.

Recombinant expression of rabbit monoclonal divalent antibodies

For recombinant expression of rabbit monoclonal divalent antibodies, PCR products encoding VH or VL were cloned as cDNA into expression vectors using a sticky-end cloning technique (RS Haun et al., BioTechniques (1992) 13, 515- 518; MZ Li et al., Nature Methods (2007) 4, 251-256). The expression vectors contained an expression cassette consisting of a 5'-CM\/-promoter, including intron A, and 3' polyadenylation sequences from BGH. In addition to the expression cassette, the plasmids contained an origin of replication derived from pUC18 and a beta-lactamase gene conferring ampicillin resistance to amplify the plasmid in E. coli. Three variants of the main plasmid were used: one plasmid containing a rabbit IgG constant region to introduce the VH regions, and two additional plasmids containing a rabbit or human LC kappa constant region to introduce the VL regions.

Linearized expression plasmids encoding the kappa or gamma constant region and VL/VH inserts were amplified by PCR using overlapping primers.

Purified PCR products were incubated with T4 DNA polymerase, which created single-stranded overhangs. The reaction was stopped by adding dCTP.

At the next stage, the plasmid and insert were combined and incubated with hesA, which induced site-specific recombination. The recombinant plasmids were transformed into E. coli. The next day grown colonies were collected and analyzed for the correctness of the recombined plasmid by plasmid isolation, restriction analysis and DNA sequencing.

HEK293 cells were transiently co-transfected with isolated HC and LC plasmids for antibody expression, and supernatants were harvested 1 week later.

Creation of vectors for expression of rabbit monocponal monovalent antibodies

For recombinant expression of selected candidates as monocponal monovalent antibodies, the rabbit constant regions of all VH chains were converted to human constant regions surrounding the key mutation in the CH3 segment. For VL chains derived from wild-type rabbit B cells, rabbit C-kappa constant regions were converted to human. 4 µl of selected candidate cDNA was used to amplify immunoglobulin heavy and light chain variable regions with AccuPrime SuperMix reagents (Invitrogen 12344-040) in a final volume of 50 µl with forward primers specific for the signal peptide and reverse primers specific for CDR3-J- o6nacrn with a 20 bp (3'-terminal) overlapping sequence homologous to human constant regions (VH and VL, respectively). The PCR conditions for the amplification of the VH and VL chains were as follows: hot start at 94° C. for 5 minutes; 35 cycles: 20 seconds at 94°C, 20 seconds at 68°C, 45 seconds at 68°C; final elongation at 68°C for 7 minutes.

PCR products encoding VH or VL were cloned as cDNA into expression vectors using overhang ("sticky-end") cloning techniques (RS Haun et al., BioTechniques (1992) 13, 515-518; MZ Li et al. , Nature Methods (2007) 4, 251-256). The expression vectors contained an expression cassette consisting of a 5'CMV promoter including intron A and a 3'polyadenylation sequence from BGH. In addition to the expression cassette, the plasmids contained an origin of replication derived from pUC18 and a beta-lactamase gene conferring ampicillin resistance to amplify the plasmid in E. coli. Two versions of the main plasmid were used: one plasmid containing the human IgG constant region designed to introduce the new amplified VH chain, and a second plasmid containing the human kappa LC constant region to introduce the VL chain.

Linearized expression plasmids encoding the kappa or gamma constant region and VL/VH inserts were amplified by PCR using overlapping primers.

Purified PCR products were incubated with T4 DNA polymerase, which created single-stranded overhangs. The reaction was stopped by adding dCTP.

In the next step, the plasmid and insert were combined and incubated with recA, which induced site-specific recombination. The recombinant plasmids were transformed into E. coli. The next day grown colonies were collected and analyzed for the correctness of the recombined plasmid by plasmid isolation, restriction analysis and DNA sequencing.

Example 6

Transient expression of monovalent anti-TfR antibodies

Antibodies were generated in vivo in transiently transfected HEK293 cells (derived from human embryonic kidney cell line 293) cultured in F17 medium (Invitrogen Corp.). The "293-Free" Transfection Reagent (Novagen) was used for transfection. The antibodies and modified antibody molecules described above were expressed from separate expression plasmids. Transfection was performed as directed by the manufacturer's instructions. Cell culture supenatants containing the recombinant protein were harvested 3-7 days after transfection. The supernatants were stored at reduced temperature (eg -80°C) until purification.

General information regarding the recombinant expression of human immunoglobulins, for example in HEK293 cells, is given in Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203.

Example 7

High throughput purification of incomplete antibodies (single arm) against transferrin receptor

50 ml of clarified single-arm antibody supernatants were loaded into 96-well deep plates on a 200 μl MabSelectSuRe column. After a washing step with PBS at pH 7.4, the proteins were eluted with 2.5 mM HCl using a Tecan/Atoll system to give 0.5 ml of eluate. The eluate was neutralized with 2M Tris pH 8. Purified proteins were quantified using a Nanodrop spectrophotometer and analyzed by denaturing and reducing CE-SDS and by analytical SEC. To obtain a protein with a high degree of purity (greater than 95%), a significant proportion of the antibodies had to be further purified by size exclusion chromatography to separate from semi-antibodies, "key-key" antibodies and high molecular weight aggregates. Then 500 µl of samples were injected onto a Superdex200 10/300GL column in 20 mM histidine containing 140 mM NaCl, pH 6.0, using a Dionex UltiMate 3000. screened hits in single branch format. Fractions were collected and analyzed again as described above.

Example 8

hCMEC/D3 cell culture for transcytosis assays

Medium and supplements for hCMEC/D3 (Weksler, B. B. et al., FAS EB J. 19 (2005), 1872-1874) were obtained from Lonza. hCMEC/D3 cells (passages 26-29) were cultured to confluency on collagen-coated coverslips (for microscopy) or in flasks in EBM2 medium containing 2.5% FBS, one-quarter supplemented with supplied growth factors and fully supplemented with supplied hydrocortisone, gentamicin, and ascorbic acid.

For all transcytosis assays in 12-well cell culture plates, insert filters (pore size 0.4 μm, diameter 12 mm) with a high pore density PET membrane (1×10 ⁸ pores/cm ² ) were used. Optimal medium volumes were calculated as 400 µl and 1600 µl for the apical and basolateral chambers, respectively. The apical chambers of the filter inserts were coated with rat tail collagen I (7.5 μg/cm ² ) followed by fibronectin (5 μg/ml) with each incubation for 1 hour at room temperature. hCMEC/D3 cells were grown to confluent monolayers (approximately 2×10 ⁵ cells/cm2) for 10-12 days in EBM2 medium.

Example 9

Monovalent antibody transcytosis assay

All assays were performed in serum-free EBM2 medium, which was otherwise reconstituted as described in Example 1. Cell inserts were incubated apically with monovalent antibodies (concentration: 2.67 μg/ml) for 1 h at 37°C, after which all apical and basolateral media were collected. From these values, the paracellular current was calculated. The monolayers were washed at room temperature in serum-free medium apically (400 μl) and basolaterally (1600 μl) 3 times for 3-5 minutes each. All washes were collected to monitor the removal efficiency of unbound antibody. Prewarmed medium was added to the apical chamber and the filters were transferred to a fresh 12-well plate (blocked overnight with PBS containing 1% BSA) containing 1600 μl of prewarmed medium. At this point, the cells on the filters were lysed in 500 μl of RIPA buffer in order to determine the specific uptake of the antibody. The remaining filters were incubated at 37°C and samples were collected at various time points to determine the apical and/or basolateral release of the antibody. The content of antibodies in the samples was quantified using a highly sensitive ELISA for IgG (see example 3). For each time point received data from cell cultures with three filters.

Example 10

Sensitive IgG ELISA after transcytosis assays

The entire procedure was carried out at room temperature using an automated washer in the washing steps. A 384-well plate was coated with 30 μl/well of anti-human/anti-mouse IgG, Fc-specific, at a concentration of 1 μg/ml in PBS for 2 h, and then incubated for 1 h in PBS blocking buffer containing 1% BSA or 1% CroteinC in human and mouse IgG assays, respectively). Serially diluted samples from the transcytosis assay and standard antibody concentrations used in the transcytosis assay were plated and incubated for ₂ h. in blocking buffer and incubated for another 2 hours. After 6 washes, 30 μl/well 50 ng/ml (huIgG assay) or 100 ng/ml (mIgG assay) poly-HPP40-streptavidin (Fitzgerald, in PBS containing 1% BSA and 0.05% Tween-20) and incubated for 30 minutes. After 4 washes, immune complexes were detected by adding 30 μl/well of BM Chemiluminescence Substrate (Roche). The luminescence signal was measured with a fluorescent plate reader and the concentrations were calculated using the fitted standard curve. The sensitivity of the assay varied from 10 pg/mL to 10 ng/mL.

Example 11

Epitope Mapping by Cellular ELISA on CHO Cells Transfected with Mutant hTfR

To be able to define epitope regions on the human transferrin receptor (hTfR), mutations were introduced into the hTfR sequence at positions where the cluster of surface-exposed amino acids had different amino acids compared to the aligned mouse TfR sequence (see table below), following the rationale, that, despite the significant homology between human and mouse TfR (77% identity), no such antibodies are known directed against the extracellular part, which would demonstrate a high degree of cross-reactivity between both orthologues. Cloning of plasmids with the appropriate mutations is described above. To map the binding of human TfR binding molecules to these epitopes, CHO-K1 cells were transiently transfected with the described plasmids and antibody binding was measured in cell-based ELISA. Briefly, the day before the experiment, 10 ⁴ cells per well were seeded in a 96-well plate in normal growth medium (RPMI/10% FCS). The next day, the medium was changed to reduced serum OPTI-MEM medium (Gibco) and, after pre-incubation for 30 minutes, 10 µl of a mixture consisting of 1200 µl of OPTI-MEM, 12 µg of plasmid DNA and 12 µl of reagent for XtremeGENE transfections (Roche). Cells were incubated for 2 days at 37°C/7.5% CO ₂ and then the medium was removed and TfR antibodies were added at concentrations from 1 nM to 100 nM in growth medium, after which they were incubated for 2 h at 4°C . The antibody solutions were then replaced with 0.05% glutaraldehyde in PBS and the cells were fixed for 15 minutes at room temperature and then washed twice with PBS and incubated with HRP-conjugated anti-human Fc secondary antibody (BioRad; 1:2000 in ELISA blocking reagent (Roche)) for 1.5 hours at room temperature. After three washes in PBS, a signal was obtained by adding 50 μl of TMB per well and the absorbance at 450 nm was measured.

Example 12

Surface Plasmon Resonance Binding Assay for Anti-Human TfR Antibody Interaction

The binding experiment was performed on a BIAcore B 4000 instrument (GE Healthcare) equipped with a C1 sensor chip (GE Healthcare, cat. no. BR1005-35) pretreated with an anti-human Fab antibody (GE Healthcare, cat. no. 28-9583-25), using a standard amine coupling procedure according to the supplier's manual.

For kinetic measurements, the antibody was immobilized by coating with a contact time of 60 seconds and a flow rate of 10 μl/min in phosphate-buffered saline, pH 7.4, 0.05% Tween 20, at 25°C. Recombinant H1e6 labeled human transferrin receptor (R&D Systems cat. no. 2474-TR-050) was applied in increasing concentrations and the signal was monitored over time. An average association time of 150 seconds and a dissociation time of 600 seconds were recorded at a flow rate of 30 μl/min. Data were fitted using a 1:1 binding model (Langmuir isotherm).

Claims (13)

1. A blood-brain barrier transporter module containing a molecule to be transported to the brain across the blood-brain barrier and exactly one (univalent) anti-transferrin receptor antibody or fragment thereof that binds the transferrin receptor, wherein the anti-transferrin receptor antibody or fragment thereof that binds the transferrin receptor , contains (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 109; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 110; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 112; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 113; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 114; and (f) HVR-L3 containing the amino acid sequence of SEQ ID NO: 115, where the specified molecule to be transported to the brain through the blood-brain barrier is selected from the group consisting of drugs for the treatment of neurological disorders, neurotrophic factors, growth factors, enzymes, cytotoxic agents, antibodies directed against targets in the brain, monoclonal antibodies directed against targets in the brain, peptides directed against targets in the brain. 2. The blood-brain barrier transport module of claim 1, wherein the blood-brain barrier transport module further comprises a linker, and wherein the linker connects the molecule to be transported to the brain across the blood-brain barrier to a monovalent anti-transferrin receptor antibody or a transferrin receptor-binding fragment thereof . 3. The blood brain barrier transport module of claim 1, wherein the monovalent anti-transferrin receptor antibody or transferrin receptor binding fragment thereof comprises a molecule selected from the group consisting of full length antibody, scFv, Fv, scFab, and VHH. 4. The blood brain barrier transport module of claim 1, wherein the monovalent anti-transferrin receptor antibody or transferrin receptor binding fragment thereof specifically binds to the human transferrin receptor and to the cynomolgus monkey transferrin receptor. 5. The blood-brain barrier transport module of claim 1, wherein the monovalent anti-transferrin receptor antibody or transferrin receptor-binding fragment thereof contains a single scFab directed against the transferrin receptor. 6. The blood-brain barrier transport module of claim 1, wherein the monovalent anti-transferrin receptor antibody or transferrin receptor-binding fragment thereof contains a single scFv directed against the transferrin receptor. 7. The blood-brain barrier transport module according to claim 1, wherein the target in the brain is selected from the group consisting of: beta-secretase 1, Aβ (Abeta), epidermal growth factor, epidermal growth factor receptor 2, tau protein, phosphorylated tau -protein, phosphorylated Tay(pS422) protein, apolipoprotein E4, alpha-synuclein, oligomeric fragments of alpha-synuclein, CD20, huntingtin, prion protein, leucine-rich repeat kinase 2, parkin, presenilin 2, gamma-secretase, death receptor 6 , amyloid precursor protein, p75 neurotrophin receptor and caspase 6. 8. The blood-brain barrier transport module of claim 1, wherein the monovalent anti-transferrin receptor antibody or transferrin receptor-binding fragment thereof is conjugated to the C-terminus of a brain-active moiety, either directly or via a linker. 9. The blood-brain barrier transport module of claim 1, wherein the molecule to be transported to the brain across the blood-brain barrier contains a full length antibody directed against a target in the brain. 10. Pharmaceutical composition for transporting a molecule to be transported to the brain across the blood-brain barrier, selected from the group consisting of drugs for the treatment of neurological disorders, neurotrophic factors, growth factors, enzymes, cytotoxic agents, antibodies directed against targets in the brain, monoclonal antibodies directed against targets in the brain, peptides directed against targets in the brain, where the composition contains an effective amount of the carrier module through the blood-brain barrier according to any one of paragraphs. 1-9. 11. The use of a carrier module across the blood-brain barrier according to any one of paragraphs. 1-9 as a drug for transporting a molecule to be transported to the brain across the blood-brain barrier, selected from the group consisting of drugs for the treatment of neurological disorders, neurotrophic factors, growth factors, enzymes, cytotoxic agents, antibodies directed against targets in the brain , monoclonal antibodies directed against targets in the brain, peptides directed against targets in the brain. 12. Use according to claim 11, where the drug is for the treatment of a neurological disorder.

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