HK1208235B - Anti-biotin antibodies and methods of use - Google Patents

Description

Anti-biotin antibodies and methods of use

Technical Field

The present invention relates to anti-biotin antibodies and anti-biotin derivative antibodies and methods of using the anti-biotin antibodies and anti-biotin derivative antibodies.

Background Art

Hapten binding antibodies can be used as capture modules for therapeutic and diagnostic applications. For example, hapten binding entities (such as fluorophores, chelating agents, peptides, nucleic acids, proteins, lipids, nanoparticles and many other active agents) can react with hapten binding antibodies and antibody derivatives. This enables effective detection of this type of "payload", as well as capture, accumulation at desired locations, cross-linking and other antibody-mediated effects. Since the characteristics and composition of the hapten binding entity can affect the composition and "behavior" (including size, solubility, activity, biophysical properties, PK, biological effects and more) of the hapten binding entity, it is highly desirable to develop a variety of different hapten binding entities. Thus, it is possible to produce optimized hapten conjugates with the selected hapten matching of a given payload. Subsequently, the most suitable hapten binding entity can be combined with the conjugate to produce the most suitable antibody-hapten-payload complex. It is also desirable to obtain hapten binding entities, such as humanized antibody derivatives. This enables application with significantly reduced interference risks (such as immunogenicity in therapeutic applications). The antibodies described herein bind biotin derivatives (rather than unmodified biotin). These antibodies are referred to in this document as "biotin-binding" or "anti-biotin" antibodies.

Biotinylated chemokine antibody complexes are reported in WO 00/50088.

Kohen, F. et al. reported the preparation and properties of anti-biotin antibodies (Methods Enzymol. 279 (1997) 451-463). Bagci, H. et al. (FEBS Lett. 322 (1993) 47-50) reported the mimetic affinity of monoclonal anti-biotin antibodies in recognizing biotin. Cao, Y. et al. reported the development of bispecific monoclonal antibodies as universal immunoprobes for detecting biotinylated macromolecules (J. Immunol. Meth. 220 (1998) 85-91).

In WO 01/34651 antibodies binding to a non-naturally occurring enantiomer (L-biotin) and their use as targeting agents are reported.

Dakshinamurti et al. reported the production and characterization of monoclonal antibodies against biotin (Biochem. J. 237 (1986) 477-482). Vincent, P. et al. (J. Immunol. Meth. 165 (1993) 177-182) reported a comparison of the binding of biotin and biotinylated macromolecular ligands to anti-biotin monoclonal antibodies and streptavidin. Berger, M. et al. (Biochem. 14 (1975) 2338-2342) reported the production of antibodies that bind to biotin and inhibit biotin-containing enzymes.

SUMMARY OF THE INVENTION

The present invention provides anti-biotin antibodies and anti-biotin derivative antibodies and methods of using the anti-biotin antibodies and anti-biotin derivative antibodies.

One aspect as reported herein is a humanized anti-biotin antibody, wherein the antibody comprises: (a) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (b) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15; and (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10. The antibody specifically binds biotin.

In one embodiment, the antibody further comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:09; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:10; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:11.

In one embodiment, the antibody further comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:13; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:14; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:15.

In one embodiment, the antibody comprises the amino acid residue serine at position 24 of the heavy chain variable domain according to Kabat numbering or/and the amino acid residue threonine at position 73 of the heavy chain variable domain according to Kabat numbering.

In one embodiment, the antibody comprises amino acid residue alanine at position 60 of the heavy chain variable domain according to Kabat numbering and amino acid residue glutamic acid at position 61 of the heavy chain variable domain according to Kabat numbering.

In one embodiment, the antibody: (1) comprises the amino acid residue serine at position 24 of the heavy chain variable domain according to the Kabat numbering and/or the amino acid residue threonine at position 73 of the heavy chain variable domain according to the Kabat numbering; (2) comprises the amino acid residue alanine at position 60 of the heavy chain variable domain according to the Kabat numbering; and (3) comprises the amino acid residue glutamic acid at position 61 of the heavy chain variable domain according to the Kabat numbering.

In one embodiment, the antibody comprises: (a) a VH sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 12; (b) a VL sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16; or (c) the VH sequence of (a) and the VL sequence of (b), wherein the amino acid residue at position 24 of the heavy chain variable domain according to the Kabat numbering is serine and/or the amino acid residue at position 73 of the heavy chain variable domain according to the Kabat numbering is threonine, the amino acid residue at position 60 of the heavy chain variable domain according to the Kabat numbering is alanine, and the amino acid residue at position 61 of the heavy chain variable domain according to the Kabat numbering is glutamic acid.

In one embodiment, the antibody comprises the VH sequence of SEQ ID NO:12.

In one embodiment, the antibody comprises the VL sequence of SEQ ID NO:16.

One aspect as reported herein is an antibody comprising the VH sequence of SEQ ID NO: 12 and the VL sequence of SEQ ID NO: 16.

In one embodiment, the antibody is a full-length IgG1 antibody or a full-length IgG4 antibody.

In one embodiment, the antibody is a monoclonal antibody.

In one embodiment, the antibody is an antibody fragment that binds biotin.

One aspect as reported herein is a pharmaceutical formulation comprising an antibody as reported herein and a pharmaceutically acceptable carrier.

One aspect as reported herein is the antibody as reported herein for use as a medicament.

One aspect as reported herein is the use of an antibody as reported herein in the preparation of a medicament.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Expression of humanized antibodies conjugated to biotin and biotin derivatives with and without cys mutations for covalent payload conjugation: Reducing and non-reducing SDS PAGE showing the composition and homogeneity of the humanized antibodies after purification with protein A and SEC. The antibody H chain (upper band at 50 k) and L chain (lower band at 25 k) can be detected as single bands in the SEC-purified fractions of both antibody derivatives, with no visible amounts of additional protein contamination.

Figure 2. Determination of the protein structure of a mouse anti-biotin antibody-Fab fragment in complex with biocytinamid: the complexed hapten is positioned in close proximity to a cluster of negatively charged amino acids; biotin derivatized as a hapten for efficient loading of conjugates at its carboxyl group binds with good efficiency since there is no charge repulsion at this position (due to the lack of a COOH group); in contrast, free (normal) biotin cannot bind efficiently to the antibody since its carboxyl group would be in close proximity to this negatively charged cluster and would therefore become repulsive.

Detailed Description of the Invention

I. Definition

"Acceptor human framework " for the purposes of this paper is a framework 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 a people's consensus framework as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a people's consensus framework can comprise the same amino acid sequence, or it can comprise amino acid sequence changes. In some embodiments, the number of amino acid changes 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 VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a people's consensus framework sequence.

"Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by methods well known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

An "affinity matured" antibody is one with one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody which does not possess such alterations, such alterations resulting in improvements in the affinity of the antibody for antigen.

The terms "anti-biotin antibody" and "antibody that binds to biotin" refer to antibodies that are capable of binding to biotin with sufficient affinity so that the antibody can be used as a diagnostic and/or therapeutic agent in targeting biotin. In one embodiment, the extent of binding of the anti-biotin antibody to unrelated non-biotin proteins is less than about 10% of the binding of the antibody to biotin, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to biotin has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 ^-8 M or less, e.g., from 10 ^-8 M to 10 ^-13 M, e.g., from 10 ^-9 M to 10 ^-13 M).

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

An "antibody fragment" refers to a molecule comprising a portion of an intact antibody, other than an 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 ; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

An "antibody that binds to the same epitope as a reference antibody" is one that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, whereas the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. Exemplary competition assays are provided herein.

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 "class" of an antibody refers to the type of constant domain, or constant region, possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as _IgG1 , _IgG2 , _IgG3 , _IgG4 , _IgA1 , and _IgA2 . The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioisotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various anti-tumor or anti-cancer agents disclosed below.

"Effector functions" refer to those biological activities attributable to the Fc region of an antibody and vary with the antibody class. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cellular cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

An "effective amount" of an active agent (eg, a pharmaceutical agent) refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

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

"Framework" or "FR" refers to the variable domain residues outside the hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the following order 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 to refer to an antibody that has a structure substantially similar to a native antibody structure or has heavy chains that comprise an Fc region as defined herein.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived therefrom, without regard to the number of passages. The nucleic acid content of the progeny may not be identical to that of the parent cell, but may contain mutations. Mutant progeny that have the same function and biological activity as screened or selected for in the originally transformed cell are included herein.

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

A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH framework sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is the subgroup described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda MD (1991), NIH Publication 91-3242, Volumes 1-3. In one embodiment, for VL, the subgroup is subgroup κI described in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III described in Kabat et al., supra.

"Humanized" antibodies refer to chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one and typically two variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may alternatively comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.

The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain that are highly variable in sequence and/or form structurally defined loops ("hypervariable loops"). Typically, a natural four-chain antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs generally comprise amino acid residues from hypervariable loops and/or from "complementarity determining regions" (CDRs), the latter of which have the highest sequence variability and/or are involved in antigen recognition. Exemplary hypervariable loops occur 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). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) are present at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of 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.). Except for CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. CDRs also comprise "specificity determining residues" or "SDRs," which are residues that contact the antigen. SDRs are contained in regions of the CDRs known as shortened CDRs or α-CDRs. Exemplary α-CDRs (α-CDR-L1, α-CDR-L2, α-CDR-L3, α-CDR-H1, α-CDR-H2, and α-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3 (see Almagro, J.C. and Fransson, J., Front. Biosci. 13 (2008) 1619-1633). Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domain are numbered herein according to Kabat et al., supra.

An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent.

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

An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99% as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). Methods for assessing antibody purity are reviewed in, for example, Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

An "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 ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

"Isolated nucleic acid encoding an anti-biotin antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including such (similar) nucleic acid molecules in a single vector or separate vectors, and such (similar) nucleic acid molecules present in one or more locations in a host cell.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, i.e., except for example comprising naturally occurring mutations or possible variant antibodies (such variants are generally present in smaller amounts) that occur during the production of monoclonal antibody preparations, the single antibody comprising the population is identical and/or binds to the same epitope. Unlike polyclonal antibody preparations that generally comprise different antibodies against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is against a single determinant on the antigen. Therefore, the modifier "monoclonal" refers to the characteristic that an antibody is obtained from a substantially homogeneous antibody population, and is not interpreted as requiring antibody production by any specific method. For example, the monoclonal antibody to be used according to the present invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of human immunoglobulin loci, and this paper describes such methods and other exemplary methods for preparing monoclonal antibodies.

A "naked antibody" is an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in pharmaceutical formulations.

"Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons consisting of two identical light chains and two identical heavy chains that form disulfide bonds. From N-terminus to C-terminus, each heavy chain has a variable region (VH), also referred to as a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also referred to as a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be assigned to one of two types, referred to as κ and λ.

The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning 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 the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the compared sequences. However, for purposes herein, the sequence comparison computer program ALIGN-2 is used to generate % amino acid sequence identity values. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been submitted with user documentation to the U.S. Copyright Office, Washington, D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

When ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (which can alternatively be stated as a given amino acid sequence A having or comprising a certain % amino acid sequence identity with a given amino acid sequence B) is calculated as follows:

Multiply 100 by the fraction X/Y

where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be understood that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless expressly stated otherwise, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

The term "pharmaceutical formulation" refers to a preparation which is in such form that the biological activity of the active ingredient contained therein is effective, and which contains no additional ingredients which are unacceptably toxic to a subject to which the formulation would be administered.

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

The term "biotin" as used herein refers to 5-[(3aS,4S,6aR)-2-oxohexahydroxy-1H-thieno[3,4-d]imidazol-4-yl]pentanoic acid. Biotin is also known as vitamin H or coenzyme R.

As used herein, "treatment" (and grammatical variations thereof) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology. The desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and resolving or improving the prognosis. In some embodiments, the antibodies of the present invention are used to delay the development of the disease or slow the progression of the disease.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt, T.J. et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y. (2007), p. 91). A single VH or VL domain can be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated by screening libraries of complementary VL or VH domains, respectively, using VH or VL domains from antibodies that bind to the antigen (see, e.g., Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are autonomously replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

The term "hapten" refers to a small molecule that elicits an immune response only when attached to a large carrier, such as a protein. Exemplary haptens are aniline, o-, m-, and p-aminobenzoic acid, quinone, hydralazine, halothane, fluorescein, biotin, digitoxin, theophylline, and dinitrophenol. In one embodiment, the hapten is biotin or digitoxin or theophylline or a carborane.

The term "hapten conjugated to" or "haptenylated compound" refers to a hapten covalently linked to another moiety (e.g., a polypeptide or a label). Activated hapten derivatives are often used as starting materials to form such conjugates. In one embodiment, the hapten is digoxigenin, and it is conjugated to the moiety via a linker (in one embodiment, via its 3-hydroxyl group). In one embodiment, the linker comprises: a) one or more (in one embodiment, three to six) methylene-carboxy-methyl ( _-CH2 -C(O)-) groups; and/or b) 1 to 10 (in one embodiment, 1 to 5) amino acid residues (in one embodiment, selected from glycine, serine, glutamic acid, β-alanine, γ-aminobutyric acid, ε-aminocaproic acid, or lysine); and/or c) one or more (in one embodiment, one or two) compounds of the formula _NH2 -[( _CH2 ) _nO ] _xCH2 - _CH2 -COOH, wherein n is ₂ or 3 and x is 1 to 10, in one embodiment, 1 to 7. The last element (at least in part) produces a linker (moiety) _of the formula -NH-[( _CH2 ) _nO ] _xCH2 - _CH2 -C(O)-. An example of such a compound is, for example, 12-amino-4,7,10-trioxalaric acid (yielding a TEG (triethylene glycol) linker). In one embodiment, the linker further comprises a maleimide group. This linker has a stabilizing and solubilizing effect because it contains a charge and/or can form hydrogen bridges. In addition, it can sterically facilitate the binding of anti-hapten antibodies to the hapten-conjugated polypeptide. In one embodiment, the linker is positioned on the side chain of an amino acid of the polypeptide (e.g., conjugated to a lysine or cysteine side chain via an amino or sulfhydryl group). In one embodiment, the linker is positioned at the amino or carboxyl terminus of the polypeptide. The position of the linker on the polypeptide is generally selected in an area that does not affect the biological activity of the polypeptide. Therefore, the position of attachment of the linker depends on the nature of the polypeptide and the associated structural elements responsible for the biological activity. The biological activity of the polypeptide to which the hapten is attached can be tested in in vitro assays.

The term "covalent complex formation" refers to the formation of a covalent bond between the two partners in the complex after the formation of a non-covalent complex, such as between an anti-theophylline antibody and theophylline. The formation of the covalent bond occurs without the addition of other reactants.

II. Compositions and Methods

In one aspect, the present invention is based on antibodies that bind to biotin. These antibodies are provided herein. The antibodies of the present invention are useful, for example, as monospecific antibodies for binding to biotinylated compounds and as multispecific antibodies for diagnosing or treating a wide variety of diseases by using the binding specificity to the biotinylated compound as a universal payload characteristic of the antibody.

A. Exemplary Anti-Biotin Antibodies

On the one hand, the present invention provides isolated antibodies that bind to biotin. In certain embodiments, the anti-biotin antibody is a humanized anti-biotin antibody. In certain embodiments, the anti-biotin antibodies reported herein bind to biotinylated compounds without interfering with the biological activity of the compound that is conjugated to biotin and specifically bound by the antibody through the biotin residue. Therefore, if the antibody is a monospecific antibody, these antibodies can be used to improve the pharmacokinetics of the compound conjugated to biotin (biotinylated compound). In addition, if the antibody is a bispecific or multispecific antibody, these antibodies can be used for targeted delivery of biotinylated compounds, because one binding specificity is for biotin and can be used as a universal payload specificity, while the second binding specificity specifically binds to, for example, a cell surface molecule and provides the targeting feature/component of the bispecific or multispecific antibody.

In one aspect, the present invention provides an anti-biotin antibody comprising at least one, two, three, four, five or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:01; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:02; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:03; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:05; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:06; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:07.

In one aspect, the present invention provides an anti-biotin antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 01; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 02; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 03. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 03. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 03 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 07. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 03, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 07, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 02. In another embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:01; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:02; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:03.

In another aspect, the present invention provides an anti-biotin antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 05; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 06; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 07. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 05; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 06; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 07.

In another aspect, an anti-biotin antibody of the invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 01, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 02, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 03; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 05, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 06, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 07.

In another aspect, the present invention provides an anti-biotin antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:01; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:02; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:03; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:05; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:06; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:07.

In one embodiment, the anti-biotin antibody is humanized.

In one aspect, the present invention provides a humanized anti-biotin antibody comprising at least one, two, three, four, five or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 09; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15.

In one aspect, the present invention provides a humanized anti-biotin antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 09; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10. In another embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:09; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:10; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:11.

In another aspect, the present invention provides a humanized anti-biotin antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15.

In another aspect, an antibody of the invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 09, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15.

In another aspect, the present invention provides a humanized anti-biotin antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 09; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 15.

In another aspect, the present invention provides a humanized anti-biotin antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:01; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:02; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:03; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:05; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:06; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:07; wherein the amino acid residue at position 60 in HVR-H2 is A, and the amino acid residue at position 61 in HVR-H2 is Q.

The humanized anti-biotin antibody contains an A at Kabat position 60 and a Q at Kabat position 61. These changes (forward mutations) were introduced to increase the binding affinity of the humanized anti-biotin antibody.

In one embodiment, a humanized anti-biotin antibody comprises the HVRs of any one of the above embodiments, and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In one embodiment, a humanized anti-biotin antibody comprises a VH comprising the HVR-H of any one of the above embodiments, and further comprises one or more of the following:

- 24-bit S; and/or

-T at position 73.

Kabat position 24 corresponds to residue number 24 of SEQ ID NOs: 04, 12, and 20.

Kabat position 60 corresponds to residue number 61 of SEQ ID NOs: 04, 12, and 20.

Kabat position 61 corresponds to residue number 62 of SEQ ID NOs: 04, 12, and 20.

Kabat position 71 corresponds to residue number 72 of SEQ ID NOs: 04, 12, and 20.

These changes (forward mutations) were introduced to improve the binding affinity of the humanized anti-biotin antibody.

In another aspect, the anti-biotin antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 04. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-biotin antibody comprising the sequence retains the ability to bind to biotin. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO: 04. In certain embodiments, the substitutions, insertions or deletions occur in regions outside the HVR (i.e., in the FR). Alternatively, the anti-biotin antibody comprises the VH sequence in SEQ ID NO: 04, including post-translational modifications of the sequence. In a specific embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:01; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:02; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:03.

On the other hand, an anti-biotin antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 08. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises a substitution (e.g., a conservative substitution), insertion or deletion relative to the reference sequence, but the anti-biotin antibody comprising the sequence retains the ability to bind to biotin. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO: 08. In certain embodiments, the substitution, insertion or deletion occurs in a region outside the HVR (i.e., in the FR). Alternatively, the anti-biotin antibody comprises the VL sequence in SEQ ID NO: 08, including post-translational modifications of the sequence. In a specific embodiment, the VL comprises one, two or three HVRs selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:05; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:06; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:07.

In another aspect, an anti-biotin antibody is provided, wherein the antibody comprises the VH of any one of the embodiments provided above, and the VL of any one of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 04 and SEQ ID NO: 08, respectively, including post-translational modifications of those sequences.

On the other hand, the humanized anti-biotin antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises a substitution (e.g., a conservative substitution), insertion or deletion relative to the reference sequence, but the anti-biotin antibody comprising the sequence retains the ability to bind to biotin. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO: 12. In certain embodiments, the substitution, insertion or deletion occurs in a region outside the HVR (i.e., in the FR). Alternatively, the anti-biotin antibody comprises the VH sequence in SEQ ID NO: 12, including post-translational modifications of the sequence. In a specific embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:09; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:10; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:11.

On the other hand, a humanized anti-biotin antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-biotin antibody comprising the sequence retains the ability to bind to biotin. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO: 16. In certain embodiments, the substitution, insertion or deletion occurs in a region outside the HVR (i.e., in the FR). Alternatively, the anti-biotin antibody comprises the VL sequence in SEQ ID NO: 16, including post-translational modifications of the sequence. In a specific embodiment, the VL comprises one, two or three HVRs selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15.

In another aspect, a humanized anti-biotin antibody is provided, wherein the antibody comprises the VH of any one of the embodiments provided above, and the VL of any one of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 12 and SEQ ID NO: 16, respectively, including post-translational modifications of those sequences.

In another aspect of the invention, the anti-biotin antibody of any of the above embodiments is a monoclonal antibody, including a chimeric, humanized, or human antibody. In one embodiment, the anti-biotin antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab') ₂ fragment. In another embodiment, the antibody is a full-length antibody, such as an intact IgG1 or IgG4 antibody, or other antibody class or isotype as defined herein.

In another aspect, the anti-biotin antibody of any of the above embodiments may incorporate any of the features described in Sections 1-5 below, alone or in combination:

1. Antibody affinity

In certain embodiments, the antibodies provided herein have a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 ⁻⁸ M or less, e.g., 10 ⁻⁸ M to 10 ⁻¹³ M, e.g., 10 ⁻⁹ M to 10 ⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) using a Fab form of the antibody of interest and its antigen as described in the following assay. Solution binding affinity of the Fab for the antigen is measured by equilibrating the Fab with a minimal concentration of ( ¹²⁵ I)-labeled antigen in the presence of a titration series of unlabeled antigen, and then capturing the bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881). To determine the conditions for the assay, multiwell plates (Thermo Scientific) were coated overnight with 5 μg/ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w/v) bovine serum albumin in PBS for 2 to 5 hours at room temperature (approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [ ¹²⁵ I]-antigen is mixed with a serial dilution of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody, Fab-12 in Presta, LG et al., Cancer Res. 57 (1997) 4593-4599). The Fab of interest is then incubated overnight; however, the incubation can be continued for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., for 1 hour). The solution is then removed and the plate is washed 8 times with PBS containing 0.1% polysorbate 20. When the plate is dry, 150 μl/well of scintillant (MICROSCINT-20 ^™ ; Packard) is added and the plate is counted for 10 minutes on a TOPCOUNT ^™ gamma counter (Packard). A concentration of each Fab that gives less than or equal to 20% of maximum binding is selected for use in the competitive binding assay.

According to another embodiment, Kd is measured by surface plasmon resonance assay using a CM5 chip with immobilized antigen at 10 response units (RU) at 25°C using a BIAcore® or a BIAcore® (BIAcore, Inc., Piscataway, NJ). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Prior to injection at a flow rate of 5 μl/min, the antigen was diluted to 5 μg/ml (~0.2 μM) with 10 mM sodium citrate, pH 4.8, to achieve approximately 10 response units (RU) of coupled protein. Following antigen injection, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were injected into PBS containing 0.05% polysorbate 20 (TWEEN-20 ^™ ) surfactant (PBST) at a flow rate of approximately 25 μl/min at 25° C. The association rate (k _on ) and dissociation rate (k _off ) were calculated using a simple one-to-one Langmuir binding model (Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) was calculated as the ratio k _off /k _on . See, e.g., Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881. If the on-rate exceeds 10 ⁶ M ⁻¹ s ⁻¹ by the surface plasmon resonance assay above, the on-rate can be determined by using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 at 25° C. in the presence of increasing concentrations of antigen as measured by a spectrophotometer such as a spectrophotometer equipped with a dwell (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer with a stirred cuvette (ThermoSpectronic).

2. Antibody fragments

In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, and 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., 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 F(ab') ₂ fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see US 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 0404,097; WO 1993/01161; Hudson, P.J. et al. Nat. Med. 9 (2003): 129-134; and Hollinger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993): 6444-6448. Triabodies and tetrabodies are also described in Hudson, P.J. et al., Nat. Med. 9 (2003): 129-134.

Single-domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody.In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl).

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

3. Chimeric and humanized antibodies

In certain embodiments, the antibodies provided herein are chimeric antibodies. For example, US 4,816,567; and Morrison, S.L. et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855) describe certain chimeric antibodies. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In another example, a chimeric antibody is a "class-swapped" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity to people while maintaining the specificity and affinity of the parent non-human antibody. Typically, humanized antibodies comprise one or more variable domains, wherein HVR, for example, CDR (or a portion thereof) is derived from a non-human antibody, and FR (or a portion thereof) is derived from a human antibody sequence. Humanized antibodies optionally also comprise at least a portion of human constant regions. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (for example, an antibody from which HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

Humanized antibodies and methods of making the same are reviewed, for example, in Almagro, J.C. and Fransson, J., Front.Biosci. 13 (2008) 1619-1633, and 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; U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321; U.S. 7,087,409; Kashmiri, S.V. et al., Methods 36 (2005) 25-34 (describing SDR (a-CDR) grafting); Padlan, E.A., Mol. Immunol. 28 (1991) 489-498 (describing "resurfacing"); Dall'Acqua, W.F. et al., Methods 36 (2005) 43-60 (describing "FR shuffling"); and Osbourn, J. et al., Methods 36 (2005) 61-68 and Klimka, A. et al., Br. J. Cancer 83 (2000) 252-260 (describing the "guided selection" method of FR shuffling).

Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (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 mutated) framework regions or human germline framework regions (see, e.g., Almagro, J.C. and Fransson, J., Front. Biosci. 13 (2008) 1619-1633); and framework regions derived from screening FR libraries (see, e.g., Baca, M. et al., J. Biol. Chem. 272 (1997): 10678-10684 and Rosok, M.J. et al., J. Biol. Chem. 271 (1996) 22611-22618).

4. Multispecific Antibodies

In certain embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies having binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for biotin and the other is for any other antigen. In certain embodiments, bispecific antibodies can bind to two different epitopes of biotin. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing biotin. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with 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 “knob-in-hole” engineering (see, e.g., US 5,731,168). Antibody Fc heterodimer molecules can also be prepared by engineering the electrostatic steering effect (WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., US 4,676,980 and Brennan, M. et al., Science 229 (1985) 81-83; using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny, S.A. et al., J. Immunol. 148 (1992) 1547-1553; using "diabody" technology for preparing bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and using single-chain Fv (sFv) dimers (see, e.g., Gruber, M et al., J. Immunol. 148 (1992) 1547-1553). et al., J. Immunol. 152 (1994) 5368-5374); and, for example, trispecific antibodies as described in Tutt, A. et al., J. Immunol. 147 (1991) 60-69, to prepare multispecific antibodies.

Also included herein are engineered antibodies having three or more functional antigen-binding sites, including "octopus antibodies" (see, e.g., US 2006/0025576).

The antibodies or fragments herein also include "dual-acting Fabs" or "DAFs," which contain an antigen binding site that binds biotin as well as another, different antigen (see, eg, US 2008/0069820).

The antibodies or fragments herein may also include the multispecific antibodies described in WO 2009/080251, WO 2009/080252, WO 2009/080253, WO 2009/080254, WO 2010/112193, WO 2010/115589, WO 2010/136172, WO 2010/145792, and WO 2010/145793.

5. Antibody variants

In certain embodiments, it is envisioned that amino acid sequence variants of the antibodies provided herein. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions, and/or insertions and/or replacement residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and replacements can be made to achieve the final construct, provided that the final construct possesses the desired characteristics, for example, antigen binding.

a) Substitution, insertion, and deletion variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitution mutagenesis include HVRs and FRs. Conservative substitutions are shown under the heading "Preferred Substitutions" in Table 1. More substantial changes are provided under the heading "Exemplary Substitutions" in Table 1, and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the target antibody and the product screened for desired activity, e.g., retained/improved antibody binding, reduced immunogenicity, or improved ADCC or CDC.

Table 1

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, Gln;

(3) Acidic: Asp, Glu;

(4) Basic: His, Lys, Arg;

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

(6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will involve exchanging a member of one of these classes for another class.

One class of substitution variants involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). In general, the variants obtained by (one or more) selection for further study will have modifications (e.g., improvements) and/or certain biological properties of the parent antibody that are substantially retained relative to the parent antibody in certain biological properties (e.g., increased affinity, reduced immunogenicity). Exemplary substitution variants are affinity-matured antibodies, which can be conveniently generated, for example, using affinity maturation techniques based on phage display such as those described herein. In short, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

Variants (e.g., replacements) can be made in HVRs, for example, to improve antibody affinity. Such variations can be made in HVR "hot spots," residues encoded by codons that undergo mutations at high frequencies during somatic maturation (see, e.g., Chowdhury, P.S., Methods Mol. Biol. 207 (2008) 179-196), and/or SDRs (a-CDRs), and the binding affinity of the obtained variants VH or VL is tested. Affinity maturation by constructing and reselecting from a second library has been described in, e.g., Hoogenboom, H.R. et al. in Methods in Molecular Biology 178 (2002) 1-37. In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A second library is then made. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introduce diversity involves an HVR-directed approach, in which several HVR residues are randomized (e.g., 4-6 residues at a time). HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, as long as such variations do not substantially reduce the ability of the antibody to bind to antigens. For example, conservative variations that do not substantially reduce binding affinity may be prepared in HVRs (e.g., conservative substitutions as provided herein). Such substitutions may be outside HVR "hot spots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR may be unchanged or contain no more than one, two, or three amino acid substitutions.

The available method of identifying residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis", as described in Cunningham, B.C. and Wells, J.A., Science 244 (1989) 1081-1085. In this method, a group of residues or target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Other replacements can be introduced at amino acid positions that appear to be functionally sensitive to the initial replacement. Alternatively, or additionally, the crystal structure of the antigen-antibody complex identifies the contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as replacement candidates. Variants can be screened to determine whether they contain desired properties.

Amino acid sequence insertions include amino or carboxyl terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of the antibody molecule include fusions of the N- or C-terminus of the antibody to an enzyme (e.g., ADEPT) or a polypeptide that increases the serum half-life of the antibody.

A preferred variant is a single cysteine variant, wherein the amino acid residue at position 53 according to Kabat in the heavy chain variable domain is cysteine.

b) Glycosylation variants

In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibodies are glycosylated. Adding or deleting glycosylation sites to an antibody can be conveniently achieved by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

When an antibody comprises an Fc region, the sugars attached thereto can be altered. Natural antibodies produced by mammalian cells typically comprise branched, biantennary oligosaccharides that are generally attached to Asn297 of the CH2 domain of the Fc region via an N-link. See, e.g., Wright, A. and Morrison, S.L., TIBTECH 15 (1997) 26-32. Oligosaccharides can include a variety of sugars, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the present invention can be made to create antibody variants with certain improved properties.

In one embodiment, the antibody variants provided have a sugar structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose at Asn297 within the sugar chain relative to the sum of all glycosyl structures attached to Asn 297 as measured by MALDI-TOF mass spectrometry (e.g., composite, hybrid and high mannose structures), as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylated variants may have improved ADCC function. See, e.g., US 2003/0157108; US 2004/0093621. Examples of disclosures concerning "defucosylated" or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; 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, particularly in Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., 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).

The antibody variants provided also have bisected oligosaccharides, for example, wherein the biantennary oligosaccharide attached to the antibody Fc region is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described in, for example, WO 2003/011878, US 6,602,684, and US 2005/0123546. Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described in, for example, WO 1997/30087; WO 1998/58964; and WO 1999/22764.

c) Fc region variants

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., replacement) at one or more amino acid positions.

In certain embodiments, the present invention contemplates antibody variants with some but not all effector functions, which makes it an ideal candidate for such applications, where the in vivo half-life of the antibody is important, but certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and/or in vivo cytotoxicity assays 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 lacks FcγR binding (and therefore may lack ADCC activity) but retains FcRn binding ability. The main cells mediating ADCC, NK cells, only express FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch, JV and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492. Non-limiting examples of in vitro assays for assessing ADCC activity of target molecules are described in US 5,500,362 (see, e.g., 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 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactive assays can be employed (see, e.g., the ACTI ^™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA; and the CytoTox non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the target molecule can be assessed in vivo, e.g., in an animal model such as that disclosed 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 to C1q and therefore lacks CDC activity. See, e.g., WO 2006/029879 and WO2005/100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro, H. et al., J. Immunol. Methods 202 (1996) 163-171; Cragg, MS et al., Blood 101 (2003) 1045-1052; and Cragg, MS and MJ Glennie, Blood 103 (2004) 2738-2743). FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int. Immunol. 18 (2006) 1759-1769).

Antibodies with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 ( U.S. Patent No. 6,737,056 ). Such Fc mutants include those with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant with residues 265 and 297 substituted with alanine ( U.S. Patent No. 7,332,581 ).

Certain antibody variants with improved or diminished binding to FcRs have been described (see, e.g., US 6,737,056; WO 2004/056312; and Shields, R.L. et al., J. Biol. Chem. 276 (2001) 6591-6604).

In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).

In some embodiments, alterations are made in the Fc region that result in altered (i.e., increased or decreased) C1q binding and/or complement dependent cytotoxicity (CDC), e.g., as described in US 6,194,551, WO 99/51642, and Idusogie, E.E. et al., J. Immunol. 164 (2000) 4178-4184.

Antibodies with increased half-life and improved 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 comprise an Fc region with one or more substitutions that improve its binding to FcRn. Such Fc variants include those having substitutions at one or more of 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, e.g., substitution of Fc region residue 434 ( U.S. Pat. No. 7,371,826 ).

For other examples of Fc region variants, see also Duncan, A.R. and Winter, G., Nature 322 (1988) 738-740; US 5,648,260; US 5,624,821; and WO 94/29351.

d) Cysteine engineered antibody variants

In certain embodiments, it is desirable to create cysteine engineered antibodies, e.g., "thioMAbs," wherein one or more residues of an antibody are replaced by cysteine residues. In particular embodiments, the replaced residues are present at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody and can be used to conjugate the antibody with other moieties, such as drug moieties or linker drug moieties, to create immunoconjugates, as described herein. In certain embodiments, any one or more of the following residues can be replaced by cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine engineered antibodies can be generated as described in US 7,521,541.

e) Antibody derivatives

In certain embodiments, the antibodies provided herein can also be further modified to contain non-protein moieties known in the art and readily available. Suitable derivatized portions of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly-(n-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde can have advantages in production due to its stability in water. Polymers can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, etc.

In another embodiment, a conjugate of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation is provided. 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 and includes, but is not limited to, a wavelength that does not harm normal cells but heats the non-protein moiety to a temperature that kills cells in the vicinity of the antibody-non-protein moiety.

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-biotin antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light chain and/or heavy chain of the antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic organism, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell). In one embodiment, a method of preparing an anti-biotin antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of anti-biotin antibodies, for example, nucleic acids encoding the antibodies as described above are isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibodies).

Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expressing antibody fragments and polypeptides in bacteria, see, for example, US 5,648,237, US 5,789,199, and US 5,840,523 (see also Charlton, K.A., in: Methods in Molecular Biology, 248 volumes, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, describing expression of antibodies in E. coli). After expression, antibodies can be separated from the bacterial cell paste of the soluble fraction and can be further purified.

In addition to prokaryotic microorganisms, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partially or fully human glycosylation patterns. 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 expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A variety of baculovirus strains have been identified that can be used with insect cells, particularly for transfecting Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts. See, for example, US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (describing 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 monkey kidney CV1 cell line (COS-7) transformed by SV40; human embryonic kidney cell line (described in, for example, Graham, FL et al., 293 or 293 cells in J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse supporting cells (described in, for example, TM4 cells in Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); described in, for example, Mather, JP et al., Annals of TRI cells (NY Acad. Sci. 383 (1982) 44-68); MRC 5 cells; and FS4 cells. Other useful mammalian 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 certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

C. Determination

The physical/chemical properties and/or biological activities of the anti-biotin antibodies provided herein can be identified, screened, or characterized by a variety of assays known in the art.

Binding assays and other assays

In one aspect, antibodies of the invention are tested for their antigen binding activity, for example, by known methods (eg, ELISA, Western blot, etc.).

In another aspect, competition assays can be used to identify antibodies that compete with the antibodies reported herein for binding to biotin.

In an exemplary competition assay, immobilized biotin is incubated in a solution containing a first labeled antibody that binds to biotin and a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding to biotin. The second antibody can be present in the hybridoma supernatant. As a control, immobilized biotin is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to biotin, excess unbound antibody is removed and the amount of label bound to the immobilized biotin is measured. If the amount of label bound to the immobilized biotin in the test sample is substantially reduced relative to the control sample, it indicates that the second antibody is competing with the first antibody for binding to biotin. See Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988).

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-biotin antibody herein conjugated to one or more cytotoxic agents (e.g., chemotherapeutic agents or drugs), growth inhibitory agents, toxins (e.g., protein toxins of bacterial, fungal, plant, or animal origin, enzymatically active toxins, or fragments thereof), or radioactive isotopes.

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see US 5,208,020, US 5,416,064 and EP 0 425 235 B1); auristatins, such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see US 5,635,483, US 5,780,588 and US 7,498,298); dolastatin; calicheamicin or its derivatives (see US 5,712,374, US 5,714,586, US 5,739,116, US 5,767,285, US 5,770,701, US 5,770,710, US 5,773,001 and US 5,877,296; Hinman, L.M. et al., Cancer Res. 53 (1993) 3336-3342; and Lode, H.N. et al., Cancer Res. 58 (1998) 2925-2928); anthracyclines such as daunorubicin or doxorubicin (see Kratz, F. et al., Curr. Med. Chem. 13 (2006) 477-523; Jeffrey, S.C. et al., Bioorg. Med. Chem. Lett. 16 (2006) 358-362; Torgov, M.Y. et al., Bioconjug. Chem. 16 (2005) 717-721; Nagy, A. et al., Proc. Natl. Acad. Sci. USA 97 (2000) 829-834; Dubowchik, G.M. et al., Bioorg. & Med. Chem. Letters 12 (2002) 1529-1532; King, H.D. et al., J. Med. Chem. 45 (20029 4336-4343; and US 6,630,579); methotrexate; vindesine; taxanes, such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, a nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, euphorin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. Radioconjugates can be produced using a variety of radioisotopes. Examples include At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioisotopes of Lu. When the radioconjugate is used for detection, it can contain a radioactive atom for scintigraphic studies, such as TC ^99m or I ¹²³ , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Conjugates of antibodies and cytotoxic agents can be produced using a variety of bifunctional protein coupling agents, such as N-succinimidyl 3-(2-pyridyldithiol) propionate (SPDP), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), imidosulfolane (IT), bifunctional derivatives of imidoesters (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 2,6-diisocyanatotoluene), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta, E.S. et al., Science 238 (1987) 1098-1104. Carbon-14 labeled 3-methyldiethylenetriaminepentaacetic acid 1-isothiocyanatobenzyl ester (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94/11026. The linker can be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, an acid-sensitive linker, a peptidase-sensitive linker, a photosensitive linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari, R.V. et al., Cancer Res. 52 (1992) 127-131; US Pat. No. 5,208,020).

The immunoconjugates or ADCs herein specifically contemplate, but are not limited to, such conjugates prepared with cross-linkers including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and commercially available cross-linkers of SVSB (succinimidyl-(4-vinylsulfone)benzoate) (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A.).

E. Methods and compositions for diagnosis and detection

As used herein, the term "detecting" encompasses quantitative or qualitative detection.

In one embodiment, an anti-biotin antibody is provided for use in a method of diagnosis or detection. The method can be an in vitro or in vivo method.

In certain embodiments, a labeled anti-biotin antibody is provided. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromogenic labels, electron density labels, chemiluminescent labels, and radioactive labels), as well as moieties that are indirectly detected, for example, by enzymatic reactions or molecular interactions (e.g., enzymes or ligands). Exemplary 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 (e.g., firefly luciferase and bacterial luciferase ( US 4,737,456 )), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., urate oxidase and xanthine oxidase), conjugated to enzymes that utilize hydrogen peroxide to oxidize dye precursors (e.g., HRP, lactoperoxidase, or microperoxidase), biotin/avidin, spin labels, phage labels, stable free radicals, and the like.

F. Pharmaceutical Preparations

Pharmaceutical formulations of the anti-biotin antibodies described herein are prepared by mixing such antibodies of the desired purity with one or more optional pharmaceutically acceptable carriers (Osol, A. (ed.) Remington's Pharmaceutical Sciences 16th edition, (1980)) in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to: buffers such as phosphate, citric acid, 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) polyols; Peptides; 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 sugars including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (Baxter International, Inc.). Certain exemplary sHASEGPs, including rhuPH20, and methods of use 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.

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

The formulations herein may also contain more than one active ingredient, as needed for the specific indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it would be desirable to also provide a list of drugs that can be combined with anti-biotin antibodies. Such active ingredients are suitably combined in amounts effective for their intended purpose.

The active ingredient can be encapsulated in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by coacervation techniques or by interfacial polymerization, 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).

Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg films, or microcapsules.

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

G. Methods of Treatment and Compositions

Any of the anti-biotin antibodies provided herein can be used in therapeutic methods.

In one aspect, an anti-biotin antibody for use as a medicament is provided. In certain embodiments, an anti-biotin antibody for use in a method of treatment is provided.

In another aspect, the present invention provides use of an anti-biotin antibody in the manufacture or preparation of a medicament.

In another aspect, the present invention provides a pharmaceutical formulation comprising any of the anti-biotin antibodies provided herein. In one embodiment, the pharmaceutical formulation comprises any of the anti-biotin antibodies provided herein and a pharmaceutically acceptable carrier.

The antibodies of the present invention can be used alone or in combination with other active agents in therapy. For example, the antibodies of the present invention can be co-administered with at least one additional therapeutic agent.

Such combination therapies as noted above encompass both combined administration (two or more therapeutic agents contained in the same or separate formulations) and separate administration, in which case administration of the antibodies of the invention may occur before, simultaneously with, and/or after administration of the additional therapeutic agent and/or adjuvant. The antibodies of the invention may also be used in combination with radiation therapy.

The antibody of the present invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and if desired for local treatment, then intralesional administration can be performed. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be performed by any suitable approach, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is short-lived or long-term. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations, bolus administration and pulse infusions at multiple time points.

The antibodies of the present invention will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered in this context include the specific condition to be treated, the specific mammal to be treated, the clinical condition of the individual patient, the cause of the disease, the site of delivery of the active agent, the method of administration, the schedule of administration, and other factors known to medical practitioners. The antibodies need not be formulated with one or more active agents currently used to prevent or treat the condition in question, but are optionally formulated with the active agent. The effective amount of such other active agents depends on the amount of antibody present in the formulation, the type of condition or treatment, and the other factors discussed above. These are generally administered at the same dose and using the routes of administration described herein, or about 1% to 99% of the doses described herein, or at any dose determined empirically/clinically appropriate and administered by any route.

For the prevention or treatment of disease, the appropriate dosage of the antibody of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether to administer the antibody for preventive or therapeutic purposes, previous treatment, the patient's clinical history and the reaction to the antibody and the judgment of the attending physician. The antibody is suitably administered to the patient in one or a series of treatments. Depending on the type and severity of the disease, an antibody of about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg-10 mg/kg) can be the initial candidate dose administered to the patient, for example, by one or more separate administrations or by continuous infusion. Depending on the above factors, a typical daily dose can be from about 1 μg/kg to 100 mg/kg or more. For repeated administration over several days or longer, depending on the disease, treatment will generally continue until the desired disease symptoms are suppressed. An exemplary dosage of the antibody will be in the range of from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) can be administered to the patient. Such doses can be administered intermittently, for example weekly or every three weeks (e.g., so that the patient receives about 2 to about 20, or for example, about 6 doses of the antibody). An initial higher loading dose can be administered, followed by one or more lower doses. The progress of this treatment can be easily monitored by conventional techniques and assays.

It will be understood that any of the above formulations or treatment methods can be performed using an immunoconjugate of the invention in place of or in addition to an anti-biotin antibody.

III. Finished Products

In another aspect of the present invention, an article of manufacture comprising materials for treating, preventing and/or diagnosing the above-mentioned conditions is provided. The article of manufacture comprises a container and a label or package insert on or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition that is effective for treating, preventing and/or diagnosing the condition when it is alone or in combination with another composition, and can have a sterile access (for example, the container can be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is an antibody of the present invention. The label or package insert indicates that the composition is used to treat a selected condition. In addition, the article of manufacture may comprise (a) a first container containing a composition comprising an antibody of the present invention; and (b) a second container containing a composition comprising another cytotoxic agent or therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the composition can be used to treat a specific condition. Alternatively or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may also contain other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

It will be appreciated that any of the above articles of manufacture may comprise an immunoconjugate of the invention in place of or in addition to an anti-biotin antibody.

IV. Examples

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

Example 1

Isolation and characterization of cDNA encoding the VH and VL domains of an IgG1 class murine anti-biotin antibody with a kappa light chain from a mouse hybridoma

The protein and (DNA) sequence information for the VH and VL domains of the murine hapten-biotin antibody was obtained directly from hybridoma clones. The following experimental steps were performed: (i) RNA was isolated from the antibody-producing hybridoma cells; (ii) this RNA was converted into cDNA and PCR fragments containing the VH and VL domains were inserted; and (iii) these PCR fragments were incorporated into plasmid vectors for propagation in E. coli and their DNA (and deduced protein) sequences were determined.

RNA preparation from hybridoma cells

RNA was prepared from 5x10 ⁶ hybridoma cells expressing the antibody using RNeasy-Kit (Qiagen). Briefly, the settled cells were washed once and precipitated in PBS and then resuspended in 500 μl RLT-Puffer (+β-ME) for lysis. The cells were thoroughly lysed by passing through Qiashredder (Qiagen) as described in the manufacturer's manual, followed by a matrix-mediated purification method (ETOH, RNeasy column). After the final washing step, RNA was recovered from the column in 50 μl of RNase-free water. The concentration of the recovered RNA was determined by quantitatively measuring the A260 and A280 of the sample diluted 1:20. The integrity (quality, degree of degradation) of the isolated RNA sample was analyzed by denaturing RNA gel electrophoresis on formamide-agarose gel (see Maniatis manual). Discrete bands representing complete 18s and 28s ribosomal RNA were obtained, and the integrity of these bands (intensity ratio of approximately 2:1) indicated that the RNA preparation was of good quality. RNA isolated from hybridomas was frozen in aliquots at -80°C.

DNA fragments encoding VH and VH were generated by RACE PCR, cloned into plasmids, and their DNA and amino acid sequences were determined.

cDNA for subsequent (RACE-)PCR reactions was prepared from RNA preparations using the techniques described in International Patent Application PCT/EP2011/074273. Subsequently, PCR fragments encoding VH and VL were isolated by agarose gel extraction and subsequently purified using standard molecular biology techniques. The purified PCR fragments generated in PWO were inserted into the vector pCR bluntII topo using the pCR bluntII topoKit (Invitrogen) in full accordance with the manufacturer's instructions. The Topo ligation reaction was transformed into E. coli Topo10-one-shot competent cells. E. coli clones containing vectors containing the VL or VH inserts were then identified as colonies on LB-kanamycin agar plates. Plasmids were prepared from these clones, and the presence of the desired insert in the vector was confirmed by EcoRI restriction enzyme digestion. Since the vector backbone contains EcoRI restriction enzyme recognition sites on each side of the insert, plasmids containing the insert were identified by the presence of an EcoRI-releasable insert of approximately 800 bp (for VL) or 600 bp (for VH). The DNA sequences and deduced protein sequences of VL and VH were determined by automated DNA sequencing of multiple clones of VH and VL.

The murine VL sequence of the anti-biotin antibody is shown in SEQ ID NO: 08. The murine VH sequence of the anti-biotin antibody is shown in SEQ ID NO: 04.

Example 2

Humanization of the VH and VL domains of a murine anti-biotin antibody

The humanized murine biotin-binding antibody muM33 was prepared as follows: WO 2011/003557 and WO 2011/003780 describe the generation and characterization of the coding and amino acid sequences of the VH and VL domains of an IgG1-class murine anti-biotin antibody with a kappa light chain from a mouse hybridoma. Based on this information, the corresponding humanized anti-biotin antibody was generated based on the human germline frameworks IGHV1-69-02 and IGKV1-27-01. For the VL, no backmutations were incorporated into the framework of human IGKV1-27-01 and the human J elements of the IGKJ2-01 germline. The humanized VH was based on human J elements from the IGHV1-69-02 germline and the IGHJ4-01-3 germline. Two backmutations were introduced at position 24 (A24S) in framework region 1 and position 73 (K73T) in framework region 3. The amino acid sequence of humanized VH is shown in SEQ ID NO: 12, and the amino acid sequence of humanized VL is shown in SEQ ID NO: 16.

Example 3

Crystallization and X-ray Structure Determination of the Binding Region of Mouse Anti-Biotin Fv in the Presence of Biotin

The structure of the mouse anti-biotin antibody was determined. Therefore, Fab fragments were generated by protease digestion of the purified IgG using a well-known prior art method (papain digestion) and subsequently purified.

To crystallize the apo Fab fragment (purified Fab), the apo Fab fragment was concentrated to 13 mg/ml in 20 mM His-HCl, 140 mM NaCl, pH 6.0. Crystallization drops were established at 21° C. by mixing 0.2 μl of protein solution with 0.2 μl of storage solution in a vapor diffusion sitting drop experiment. Crystals appeared in 0.1 M Tris pH 8.5, 0.01 M cobalt chloride, 20% polyvinylpyrrolidone K15 within 5 days and grew to a final size of 0.3 mm x 0.06 mm x 0.03 mm within 8 days.

Crystals were harvested using 15% glycerol as a cryoprotectant and then flash-frozen in liquid nitrogen. Diffraction patterns were collected at 100 K using a Pilatus 6M detector under X10SA light from a Swiss Light Source. Data were processed using the program XDS [Kabsch, W., J. Appl. Cryst. 26 (1993) 795-800] and scaled using SCALA [obtained from BRUKER AXS] to generate high-resolution data. Crystals of this Fab fragment belong to the monoclinic space group P21 with unit cell dimensions of α and β = 117.53°, and each crystallographic asymmetric unit contains four Fab molecules (see Table 2).

The structure was solved by molecular replacement using PDB entry 3PQP as a search model, using standard crystallographic programs from the CCP4 software suite to calculate electron density and refine the x-ray structure [CCP4 (Collaborative Computational Project, N. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D (1994) 760-763]. The structural model was rebuilt into the electron density using COOT (Emsley, P. et al., Acta Crystallogr. D Biol. Crystallogr. 60 (2010) 486-501). Coordinates were refined using REFMAC5 (Murshudov, G.N. et al., Acta Crystallogr. D Biol. Crystallogr. 53 (1997) 240-255) and autoBUSTER (Global Phasing Ltd.).

Table 2: Data collection and structure refinement statistics for monoclinic muM33Fab fragment apo crystals

^1The values in parentheses refer to the highest resolution bin.

² R _fusion = Σ|I-<I>|/ΣI, where I is the intensity.

³ R _crystal = Σ|F _o - <F _c >|/ΣF _o , where F _o is the observed structure factor amplitude and F _c is the calculated structure factor amplitude.

⁴ R _free was calculated based on 5% of the total data omitted during the refinement process.

⁵ Calculated using PROCHECK [Laskowski, RA, MacArthur, MW, Moss, DS & Thornton, JM PROCHECK: a program to check the stereochemical quality of protein structure. J. Appl. Crystallogr. 26, 283-291 (1993)].

For crystallization of Fab fragments complexed with biotin derivatives, apo crystals of the Fab fragments used for soaking experiments were derived from 0.8 M succinate within 3 days of screening and grown to a final size of 0.25 mm x 0.04 mm x 0.04 mm within 5 days. Biocytinamid was dissolved in water at 100 mM. Subsequently, the compound was diluted in crystallization solution to a 10 mM working concentration and applied to the crystals in the crystallization droplet. The crystals were washed three times with 2 μl of 10 mM compound solution and finally incubated with biocytinamid at 21°C for 16 hours.

Crystals were harvested using 15% glycerol as a cryoprotectant and then flash-frozen in liquid nitrogen. Diffraction patterns were collected at 100 K using a Pilatus 6M detector under X10SA light from a Swiss Light Source. Data were processed using the program XDS [Kabsch, W., J. Appl. Cryst. 26 (1993) 795-800] and scaled using SCALA [obtained from BRUKER AXS] to generate high-resolution data. Crystals of this Fab fragment belong to the monoclinic space group P21 with unit cell dimensions of α and β = 117.15°, and each crystallographic asymmetric unit contains four Fab molecules (see Table 3).

The coordinates of the apo Fab fragment were used as a search model and the structure was solved by molecular replacement using standard crystallographic programs from the CCP4 software suite to calculate electron density and refine the x-ray structure to a resolution of 400 nm [CCP4 (Collaborative Computational Project)]. The structural model was rebuilt into the electron density using COOT (Emsley, P. et al., Acta Crystallogr. D Biol. Crystallogr. 60 (2010) 486-501). The coordinates were refined using REFMAC5 (Murshudov, G.N. et al., Acta Crystallogr. D Biol. Crystallogr. 53 (1997) 240-255) and autoBUSTER (Global Phasing Ltd.).

Table 3: Data collection and structure refinement statistics for the monoclinic muM33Fab fragment biocytinamid complex crystal

^1The values in parentheses refer to the highest resolution bin.

² R _fusion = Σ|I-<I>|/ΣI, where I is the intensity.

³ R _crystal = Σ|F _o - <F _c >|/ΣF _o , where F _o is the observed structure factor amplitude and F _c is the calculated structure factor amplitude.

⁴ R _free was calculated based on 5% of the total data omitted during the refinement process.

⁵ Calculated using PROCHECK [Laskowski, RA et al., J. Appl. Crystallogr. 26 (1993) 283-291].

The crystal form of the complex contains four independent biocytinamid:antibiotin Fab complexes within the asymmetric unit, with all Fab molecules similarly binding biocytinamid. Biocytidinamide binds in a pocket formed by CDRs 1 and 3 of the heavy chain and all three light chain CDRs. The ligand-binding pocket is defined by residues ASN29, ASP31, THR32, PHE33, GLN35, TRP99, and TRP106 from the heavy chain, and ASN31, TYR32, LEU33, SER34, TYR49, SER50, PHE91, and TYR96 from the light chain. The biotin headgroup forms hydrogen bonds with residues in CDR2 and CDR1 at one end of the pocket: N3 of the biocytinamid interacts with the hydroxyl oxygen of Ser50, while O22 contacts the backbone amide nitrogen of the same residue. Additionally, O22 of the biocytinamid forms a hydrogen bond with the hydroxyl oxygen of Ser34. In addition, hydrophobic interactions were observed between the biocytinamid and the aromatic side chains inside the binding pocket. The amide bond at the end of the biotin ( _CH2 ) ₄ aliphatic tail stacks onto PHE33 of the heavy chain CDR1 and is stabilized by additional hydrogen bonds to the backbone amide nitrogen of PHE33 and to Asp31. This positions the amide nitrogen (which is the site of attachment to the active entity) in such a way that the atom following the nitrogen is directed away from the binding pocket toward the solvent.

The experimental determination of the binding region at high resolution allowed the characterization of the binding mode of the ligand to its antibody, which is a prerequisite for detailed modeling and further improvement of the recombinant biotin-binding module through protein engineering.

Example 4

Composition, expression and purification of recombinant anti-biotin antibodies

The variable regions of murine and humanized anti-biotin antibodies are combined with constant regions of human origin to form monospecific or bispecific chimeric or humanized antibodies.

The production of monospecific and bispecific humanized anti-biotin antibodies that specifically bind to biotin and to different non-biotin targets (e.g., receptor tyrosine kinases or IGF-1R) requires: (i) designing and defining the amino acid and nucleotide sequences of such molecules; (ii) expressing these molecules in transfected cultured mammalian cells; and (iii) purifying these molecules from the supernatant of the transfected cells. These steps were performed as previously described in PCT/EP2011/074273.

Typically, to generate humanized antibodies of the IgG class with the binding specificity of the (original) mouse anti-biotin antibody, the humanized VH sequence is fused in frame to the N-terminus of the CH1-hinge-CH2-CH3 of the human Fc region of the IgG1 subclass. Similarly, the humanized VL sequence is fused in frame to the N-terminus of the human CLκ constant region.

To generate bispecific antibody derivatives containing biotin binding specificity as well as specificity for other targets, anti-biotin antibodies, scFvs or Fab fragments were fused in frame to the C-terminus of the heavy chain of the previously described antibodies. In many cases, the anti-hapten scFv used was further stabilized by introducing a VH44-VL100 disulfide bond as previously described (e.g., Reiter, Y., et al., Nature biotechnology 14 (1996) 1239-1245).

Expression plasmid

Expression plasmids containing expression cassettes for the heavy and light chains are individually assembled into mammalian cell expression vectors.

Gene segments encoding individual elements were thereby linked as described above.

General information on the nucleotide sequences of human light and heavy chains from which codon usage can be inferred is given in: Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication No 91-3242.

The kappa light chain transcription unit consists of the following elements:

- the immediate early enhancer and promoter from human cytomegalovirus (hCMV);

- Synthetic 5'-UT including Kozak sequence;

- a murine immunoglobulin heavy chain signal sequence including a signal sequence intron;

- A cloned variable light chain cDNA aligned with a unique BsmI restriction site and splice donor site at the 5' end and a unique NotI restriction site at the 3' end;

- the genomic human kappa gene constant region, including intron 2 mouse Ig-kappa enhancer (Picard, D. and Schaffner, W. Nature 307 (1984) 80-82); and

- human immunoglobulin kappa polyadenylation ("poly A") signal sequence.

The transcription unit of the gamma-l heavy chain consists of the following elements:

- the immediate early enhancer and promoter from human cytomegalovirus (hCMV);

- Synthetic 5'-UT including Kozak sequence;

- a modified murine immunoglobulin heavy chain signal sequence including a signal sequence intron;

- a cloned monospecific variable heavy chain cDNA or a cloned bispecific fusion scFv-variable heavy chain cDNA aligned with a unique BsmI restriction site and a splice donor site at the 5' end and a unique NotI restriction site at the 3' end;

- genomic human gamma 1 heavy chain gene constant region, including the mouse Ig-μ enhancer (Neuberger, M.S., EMBO J. 2 (1983) 1373-1378); and

- Human gamma 1 immunoglobulin polyadenylation ("poly A") signal sequence.

In addition to the kappa light chain or gamma 1 heavy chain expression cassette, these plasmids contain:

- Hygromycin resistance gene;

-oriP, the origin of replication of Epstein-Barr virus (EBV);

- an origin of replication from the vector pUC18 allowing replication of this plasmid in E. coli; and

-β-lactamase gene that confers ampicillin resistance in Escherichia coli.

Recombinant DNA technology

Cloning was performed using standard cloning techniques as described in Sambrook, J. et al., (Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press (1989). All molecular biology reagents were commercially available (if not otherwise indicated) and used according to the manufacturer's instructions.

DNA containing coding sequences, mutations or other genetic elements was synthesized by Geneart AG, Regensburg.

DNA sequences were determined by double-strand sequencing performed at SequiServe (SequiServe GmbH, Germany).

DNA and protein sequence analysis and sequence data management

Sequence creation, mapping, analysis, annotation, and interpretation were performed using Vector NTI Advance suite version 9.0.

Expression of anti-biotin antibodies and derivatives

Anti-biotin antibodies were expressed by transient transfection of suspended human embryonic kidney 293 (HEK293) cells. To this end, the light and heavy chains of the corresponding monospecific or bispecific antibodies were constructed in the above-mentioned expression vectors carrying prokaryotic and eukaryotic selection markers. These plasmids were amplified in Escherichia coli, purified, and then used for transient transfection. Cells were processed using standard cell culture techniques as described in Current Protocols in Cell Biology (2000), Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott-Schwartz, J. and Yamada, K.M. (eds.), John Wiley & Sons, Inc.

Cells were cultured at 37°C/8% _CO2 in an appropriate expression medium. On the day of transfection, cells were seeded in fresh culture medium at a density of ^1-2x106 viable cells/ml. A complex of DNA and transfection reagent was prepared in Opti-MEM I medium (Invitrogen, USA) containing 250 μg of heavy and light chain plasmid DNA at a molar ratio of 1:1 for a final transfection volume of 250 ml. Seven days after transfection, the cell culture supernatant containing the monospecific or bispecific antibody was clarified by centrifugation at 14,000g for 30 minutes and filtering through a sterile filter membrane (0.22 μm). The supernatant was stored at -20°C until purification.

To determine the concentration of antibodies and derivatives in cell culture supernatants, affinity HPLC chromatography was used. To this end, cell culture supernatants containing monospecific or bispecific antibodies or derivatives thereof bound to Protein A were added to an Applied Biosystems Poros A/20 _column in a solution containing 200 mM _KH2PO4 , 100 mM sodium citrate, pH 7.4. Elution from the chromatography material was performed using a solution containing 200 mM NaCl, 100 mM citric acid, pH 2.5. An UltiMate 3000 HPLC system (Dionex) was used. Eluted proteins were quantified by UV absorbance and peak area integration. Purified IgG1 antibodies were used as standards.

Purification of anti-biotin antibodies

Seven days after transfection, HEK 293 cell supernatants were harvested. The recombinant antibodies contained therein were purified from the supernatant in two steps by affinity chromatography using Protein A-Sepharose ^™ affinity chromatography (GE Healthcare, Sweden) and Superdex200 size exclusion chromatography. Briefly, the clarified culture supernatant containing the antibody was added to a MabSelect SuRe Protein A (5-50 ml) column equilibrated with PBS buffer (10 mM Na ₂ HPO ₄ , 1 mM KH ₂ PO ₄ , 137 mM NaCl, and 2.7 mM KCl, pH 7.4). Unbound proteins were washed away with the equilibration buffer. The antibody (or derivative) was eluted with 50 mM citric acid buffer, pH 3.2. The protein-containing fraction was neutralized with 0.1 ml of 2 M Tris buffer, pH 9.0. The eluted protein fractions were then mixed, concentrated using an Amicon Ultra centrifugal filter device (MWCO: 30K, Millipore), and loaded onto a Superdex200 HiLoad 26/60 gel filtration column (GE Healthcare, Sweden) equilibrated with 20 mM histidine, 140 mM NaCl, pH 6.0. The protein concentration of the purified antibodies and derivatives was determined by measuring the optical density (OD) at 280 nm using the molar extinction coefficient calculated based on the amino acid sequence according to Pace et al., Protein Science, 4 (1995) 2411-2423, with 320 nm OD as background correction. The monomeric antibody fractions were mixed, flash-frozen, and stored at -80 ° C. A portion of the sample was provided for subsequent protein analysis and characterization.

Antibody homogeneity was confirmed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1,4-dithiothreitol) and Coomassie blue staining using the Pre-Cast gel system (Invitrogen, USA) (4-20% Tris-glycine gel) according to the manufacturer's instructions.

Under reducing conditions, polypeptide chains associated with IgG were displayed on SDS-PAGE as apparent molecular sizes similar to the calculated molecular weights. Expression levels of all constructs were analyzed by protein A. In these non-optimized transient expression experiments, average protein yields ranged from 6 mg to 35 mg of purified protein per liter of cell culture supernatant.

Figure 1 shows the results of expression and purification of humanized antibodies that bind to biotin and biotin derivatives. Reducing and non-reducing SDS PAGE demonstrate the composition and homogeneity of humanized antibodies with and without cysteine at Kabat position 53 after purification using Protein A (MabSelect) and SEC. Molecular weight markers are in the non-marker lane. Under reducing conditions, the antibody H chain (upper band at 50 kDa) and L chain (lower band at 25 kDa) are detectable as single bands, with no visible amounts of additional protein contaminants.

Example 5

Binding of recombinant humanized anti-biotin antibodies to biotin-labeled compounds (biotinylated compounds)

The binding properties of recombinant chimeric and humanized anti-biotin antibodies and their variants (which have a cysteine at position 53 in HVR-H2 according to Kabat numbering) were analyzed using an Octet QK instrument (Fortebio Inc.) by biolayer interferometry (BLI) technology. This system has been well established for studying molecular interactions. The BLI technology is based on the measurement of the interference pattern of white light reflected from the surface of the biosensor end and an internal reference. The binding of the molecule to the biosensor end results in a measurable migration of the interference pattern. In order to analyze whether the humanization method described above reduces the ability of the anti-biotin antibody to bind biotin, the chimeric and humanized forms of the antibody were directly compared in their ability to bind to biotinylated proteins. Binding studies were performed by capturing the anti-biotin antibody on an anti-huIgG Fc antibody capture (AHC) biosensor (Fortebio Inc.). First, the biosensor was incubated in an antibody solution at a concentration of 0.5 mg/ml in 20 mM histidine, 140 mM NaCl, pH 6.0 for 1 minute. The biosensor was then incubated in 1x PBS, pH 7.4, for 1 minute to achieve a stable baseline. Binding was measured by incubating the antibody-coated biosensor in a solution containing 0.06 mg/ml of biotinylated protein in 20 mM histidine, 140 mM NaCl, pH 6.0, for 5 minutes. Dissociation was monitored in 1x PBS, pH 7.4, for 5 minutes. The resulting binding curves for the chimeric and humanized anti-biotin antibodies were directly compared.

The humanized form of the antibody showed binding to the biotinylated antigen that was equivalent to or even better than the chimeric antibody. This was also true for the humanized antibody with a Cys mutation at Kabat position VH53. The biotinylated protein showed residual nonspecific binding to the biosensor, which was reduced when the biosensor was coated with Herceptin, which does not bind biotin. Thus, the functionality of the anti-biotin antibody was retained in its humanized variants (which are defined by the sequences shown in SEQ ID NOs: 12 and 16, SEQ ID NOs: 20 and 24).

surface plasmon resonance

Surface plasmon resonance measurements were carried out on T200 instruments (GE Healthcare Biosciences AB, Sweden) at 25 ° C. By using the standard amine coupling test kit (BR-1000-50) provided by GE Healthcare, at pH 5.0, the capture system of about 4300 resonance units (RU) (10 μg/ml anti-human capture from human antibody capture test kit BR-1008-39, GE Healthcare Biosciences AB, Sweden) is coupled to a CM3 chip (GE Healthcare, BR-1005-36). The running buffer for amine coupling is HBS-N (10mM HEPES, pH 7.4, 150mM NaCl, GE Healthcare, BR-1006-70). The operation and dilution buffer for subsequent binding studies are PBS-T (10mM phosphate buffered saline comprising 0.05% Tween 20) pH 7.4. Humanized anti-biotin antibodies were captured by injecting a 2 nM solution at a flow rate of 5 μl/min for 60 seconds. Biotinylated siRNA was diluted with PBS-T at a concentration of 0.14-100 nM (1:3 serial dilution). Binding was measured by injecting each concentration at a flow rate of 30 μl/min for 180 seconds with a dissociation time of 600 seconds. The surface was regenerated by washing with _a 3M MgCl solution at a flow rate of 5 μl/min for 30 seconds. Data were evaluated using BIAevaluation software (GE Healthcare Biosciences AB, Sweden). Bulk refractive index differences were corrected by deducting the response obtained from the anti-human IgG Fc surface. Blank injections (= double reference) were also deducted. For the calculation of KD and kinetic parameters, the Langmuir 1:1 model was used.

Surface plasmon resonance (SPR) kinetic binding analysis was performed on humanized anti-biotin antibodies SEQ ID NOs: 12 and 16 and humanized anti-biotin antibody VH53C SEQ ID NOs: 20 and 24. The anti-biotin antibodies were captured at a concentration of 2 nM by an anti-human IgG Fc antibody bound to a CM3 sensor chip. Binding of biotinylated siRNA (Mw: 13868 Da) was recorded at concentrations of 0.41, 1.23, 3.7, 11.1, 33.3, 100, and 300 nM. Measurements were performed in duplicate. The calculated K _values for the humanized anti-biotin antibodies and humanized anti-biotin antibody VH53C were 0.633 nM and 0.654 nM, respectively.

Example 6

Generation of non-covalent complexes of biotinylated compounds with anti-biotin antibodies

General approach

The production of complexes of anti-biotin antibodies with biotinylated compounds (= biotin conjugated to payloads) should produce defined complexes and ensure that the compounds (= payloads) in these complexes retain their activity. To produce complexes of biotinylated compounds with anti-biotin antibodies, dissolve the biotinylated compound in H ₂ O to a final concentration of 1 mg/ml. Concentrate the antibody to a final concentration of 1 mg/ml (4.85 μM) in 20 mM histidine buffer, 140 mM NaCl, pH = 6.0. Mix the biotinylated payload and antibody to a 2:1 molar ratio (compound to antibody) by pipetting up and down and incubate at RT for 15 minutes.

Alternatively, dissolve the biotinylated compound in 100% DMF to a final concentration of 10 mg/ml. Concentrate the antibody in 50 mM Tris-HCl, 1 mM EDTA, pH = 8.2 to a final concentration of 10 mg/ml. Mix the biotinylated compound and antibody to a 2.5:1 molar ratio (compound to antibody) by pipetting up and down and incubate at RT and 350 rpm for 60 minutes.

Exemplary Method for Forming a Complex of a Biotinylated Fluorescent Dye and an Anti-Biotin Antibody (Biotin-Cy5/Chimeric Anti-Biotin Antibody (Human IgG Subclass) Complex)

To generate a complex of biotin-derivatized Cy5 containing a cysteine linker (biotin-Cys-Cy5), 0.16 mg of biotin-Cys-Cy5 was dissolved in 100% DMF to a concentration of 10 mg/ml. 1 mg of antibody was used at a concentration of 10.1 mg/ml (approximately 69 μM) in a buffer consisting of 50 mM Tris-HCl, 1 mM EDTA, pH 8.2. Biotin-Cys-Cy5 and antibody were mixed at a molar ratio of 2.5:1 (biotin-Cys-Cy5 to antibody) and incubated at 350 rpm for 60 minutes at room temperature. The resulting conjugate was analyzed by SDS-PAGE as described in Example 7. Fluorescence detection was performed as described in Example 7.

Exemplary Method for Forming a Complex of Biotinylated Fluorescent Dye and Anti-Biotin Antibody (Biotin-Cys-Cy5/Humanized Anti-Biotin Antibody)

To generate a complex of biotin-derivatized Cy5 containing a cysteine linker (biotin-Cys-Cy5), 0.16 mg of biotin-Cys-Cy5 was dissolved in 100% DMF to a concentration of 10 mg/ml. 1 mg of antibody was used at a concentration of 5.5 mg/ml (approximately 38 μM) in a buffer consisting of 50 mM Tris-HCl, 1 mM EDTA, pH 8.2. Biotin-Cys-Cy5 and antibody were mixed at a molar ratio of 2.5:1 (biotin-Cys-Cy5 to antibody) and incubated at 350 rpm for 60 minutes at room temperature. The resulting conjugate was analyzed by SDS-PAGE as described in Example 7. Fluorescence detection was performed as described in Example 7.

Exemplary Method for Forming a Complex of Biotinylated Polypeptide and Anti-Biotin Antibody (Ac-PYY-PEG3-Cys-β-Ala-Biotin/Chimeric Anti-Biotin Antibody Complex)

To generate a non-covalent complex of a biotinylated PYY polypeptide containing a cysteine linker, 0.19 mg of Ac-PYY-PEG3-Cys-β-Ala-biotin was dissolved in 100% DMF to a concentration of 10 mg/ml. The antibody was used at a concentration of 10.7 mg/ml (approximately 73 μM) in a buffer consisting of 50 mM Tris-HCl, 1 mM EDTA, pH 8.2. Ac-PYY-PEG3-Cys-β-Ala-biotin and antibody were mixed at a molar ratio of 2.5:1 (Ac-PYY-PEG3-Cys-β-Ala-biotin to antibody) and incubated at RT and 350 rpm for 60 minutes. The resulting complex was defined as a monomeric IgG-like molecule (95% monomeric) by the presence of a single peak in size exclusion chromatography. The resulting complex was further analyzed by SDS-PAGE followed by Western blot analysis. 10 μg of the complex was mixed with 4x LDS sample buffer (Invitrogen) and incubated at 95°C for 5 minutes. The sample was applied to a 4-12% Bis-Tris polyacrylamide gel (NuPAGE, Invitrogen) and electrophoresed at 200 V and 120 mA for 35 minutes. The molecules separated in the polyacrylamide gel were transferred to a PVDF membrane (0.2 μm pore size, Invitrogen) at 25 V and 160 mA for 40 minutes. The membrane was blocked in 1x PBST (1x PBS + 0.1% Tween 20) containing 1% (w/v) skim milk for 1 hour at room temperature. The membrane was washed 3x in 1x PBST for 5 minutes each and then incubated with streptavidin-POD-conjugate (2900 U/ml, Roche) at a 1:2000 dilution. Detection of streptavidin-POD bound to biotin on the membrane was performed using Lumi-Light Western blotting substrate (Roche).

Exemplary Method for Forming a Complex of Biotinylated Polypeptide and Anti-Biotin Antibody (Ac-PYY-PEG3-Cys-PEG2-Biotin/Chimeric Anti-Biotin Antibody Complex)

To generate a non-covalent complex of a biotinylated PYY polypeptide containing a cysteine linker, 0.16 mg of Ac-PYY-PEG3-Cys-PEG2-biotin was dissolved in 100% DMF to a concentration of 10 mg/ml. The antibody was used at a concentration of 10.7 mg/ml (approximately 73 μM) in a buffer consisting of 50 mM Tris-HCl, 1 mM EDTA, pH 8.2. Ac-PYY-PEG3-Cys-PEG2-biotin and antibody were mixed at a molar ratio of 2.5:1 (Ac-PYY-PEG3-Cys-PEG2-biotin to antibody) and incubated at RT and 350 rpm for 60 minutes. The resulting complex was defined by size exclusion chromatography as 63% monomeric IgG-like molecules and 37% dimeric soluble aggregates. The resulting complex was further analyzed by SDS-PAGE followed by Western blot analysis. 10 μg of the complex was mixed with 4x LDS sample buffer (Invitrogen) and incubated at 95°C for 5 minutes. The sample was applied to a 4-12% Bis-Tris polyacrylamide gel (NuPAGE, Invitrogen) and electrophoresed at 200 V and 120 mA for 35 minutes. The molecules separated in the polyacrylamide gel were transferred to a PVDF membrane (0.2 μm pore size, Invitrogen) at 25 V and 160 mA for 40 minutes. The membrane was blocked in 1x PBST (1x PBS + 0.1% Tween 20) containing 1% (w/v) skim milk for 1 hour at room temperature. The membrane was washed 3x in 1x PBST for 5 minutes each and then incubated with streptavidin-POD-conjugate (2900 U/ml, Roche) at a 1:2000 dilution. Detection of streptavidin-POD bound to biotin on the membrane was performed using Lumi-Light Western blotting substrate (Roche).

Generation of defined covalent conjugates of haptenated dyes and peptides with the anti-hapten antibody VH53C in the absence of redox agents

In order to produce covalent anti-biotin antibody/biotinylated polypeptide or biotinylated dye disulfide-linked conjugates, it is necessary to: (i) couple biotin to the polypeptide or dye via a linker containing a suitable reactive group (such as, for example, cysteine, maleimide) that allows the polypeptide to be exposed above the antibody surface, thereby retaining its activity; (ii) produce a covalent site-specific conjugate of the biotinylated polypeptide with an anti-biotin antibody with cysteine mutations (= antibody VH52bC/VH53C), wherein the biological activity of the polypeptide is retained; and (iii) perform the reaction in the absence of a reducing agent to avoid reduction of the antibody interchain disulfide bonds.

General approach

The production of conjugates of anti-biotin antibodies with biotinylated compounds should produce conjugates with defined stoichiometry and should ensure that the compounds in these conjugates retain their activity. To produce conjugates of biotinylated compounds with anti-biotin antibodies, dissolve the biotinylated compound in 100% DMF to a final concentration of 10 mg/ml. Bring the anti-biotin antibody VH52bC/VH53C to a concentration of 10 mg/ml in 50 mM Tris-HCl, 1 mM EDTA, pH = 8.2. Mix the biotinylated compound and anti-biotin antibody VH52bC/VH53C at a molar ratio of 2.5:1 (compound to antibody) by pipetting up and down and incubate at RT and 350 rpm for 60 minutes.

A polypeptide conjugated to biotin via a cysteine-containing linker is hereinafter referred to as biotin-Cys-polypeptide or polypeptide-Cys-biotin. The polypeptide may have a free N-terminus or an N-terminus capped, for example, with an acetyl group (Ac-polypeptide-Cys-biotin) or a PEG residue (PEG-polypeptide-Cys-biotin).

Fluorescent dyes conjugated to biotin via a cysteine-containing linker are referred to hereinafter as dye-Cys-biotin or biotin-Cys-dye.

Exemplary Method for Forming a Conjugate of a Biotinylated Fluorescent Dye and an Anti-Biotin Antibody (Biotin-Ser-Cy5/Humanized Anti-Biotin Antibody)

To generate a complex of biotin-derivatized Cy5 containing a serine residue in the linker (biotin-Ser-Cy5), 0.61 mg of biotin-Ser-Cy5 was dissolved in 20 mM histidine, 140 mM NaCl, pH 6.0 to a concentration of 10 mg/ml. 18.5 mg of humanized anti-biotin antibody was used at a concentration of 10 mg/ml (approximately 69 μM) in a buffer consisting of 50 mM Tris-HCl, 1 mM EDTA, pH 8.2. Biotin-Ser-Cy5 and antibody were mixed at a molar ratio of 2.5:1 (biotin-Ser-Cy5 to antibody) and incubated at RT with shaking at 350 rpm for 60 minutes. The sample was then size-exclusion chromatographed on a Superdex200 16/60 high-capacity preparative column (GE Healthcare) at a flow rate of 1.5 ml/min using 20 mM histidine, 140 mM NaCl, pH 6.0 as the mobile phase. Peak fractions were collected and analyzed for purity by SDS-PAGE. The dye-to-antibody ratio was calculated by (1) measuring the absorbance of the sample at 280 nm (protein) and 650 nm (Cy5) wavelengths; (2) using the formula: A ₆₅₀ of labeled protein / ε(Cy5) * protein concentration (M) = moles of dye per mole of protein, where ε(Cy5) = 250,000 ^{M -1} cm ^-1 , A ₆₅₀ of the complex = 47.0, and the protein concentration was 86.67 μM. The resulting dye-to-protein ratio was 2.17, indicating that all antibody paratopes were saturated with biotin-Cy5 molecules.

Exemplary Method for Forming a Conjugate of a Biotinylated Fluorescent Dye and an Anti-Biotin Antibody (Biotin-Cys-Cy5/Chimeric Anti-Biotin Antibody VH53C)

To generate a biotin-derivatized Cy5 conjugate containing a cysteine linker, 0.16 mg of biotin-Cys-Cy5 was dissolved in 100% DMF to a concentration of 10 mg/ml. 1 mg of anti-biotin antibody VH53C was used at a concentration of 9.7 mg/ml (approximately 68 μM) in a buffer consisting of 50 mM Tris-HCl, 1 mM EDTA, pH 8.2. Biotin-Cys-Cy5 and antibody were mixed at a molar ratio of 2.5:1 (Ac-biotin-Cys-Cy5 to antibody) and incubated at 350 rpm for 60 minutes at room temperature. The resulting conjugate was analyzed by SDS-PAGE as described in Example 7. Fluorescence detection was performed as described in Example 7.

Exemplary Method for Forming a Conjugate of a Biotinylated Fluorescent Dye and an Anti-Biotin Antibody (Biotin-Cys-Cy5/Humanized Anti-Biotin Antibody VH53C)

To generate a biotin-derivatized Cy5 conjugate containing a cysteine linker, dissolve 0.16 mg of biotin-Cys-Cy5 in 100% DMF to a concentration of 10 mg/ml. Use 1 mg of the humanized anti-biotin antibody VH53C at a concentration of 7.4 mg/ml (approximately 51 μM) in a buffer consisting of 50 mM Tris-HCl, 1 mM EDTA, pH 8.2. Mix the biotin-Cys-Cy5 and antibody at a molar ratio of 2.5:1 (Ac-biotin-Cys-Cy5 to antibody) and incubate at 350 rpm for 60 minutes at room temperature. Analyze the resulting conjugate by SDS-PAGE as described in Example 7. Fluorescence detection is performed as described in Example 7.

Exemplary Method for Forming a Conjugate of a Biotinylated Polypeptide and an Anti-Biotin Antibody (Ac-PYY(PEG3-Cys-βAla-Biotin)/Chimeric Anti-Biotin Antibody VH53C)

To generate a conjugate of a biotin-derivatized PYY polypeptide containing a cysteine linker, 0.19 mg of Ac-PYY (PEG3-Cys-βAla-biotin) was dissolved in 100% DMF to a concentration of 10 mg/ml. 1 mg of the chimeric anti-biotin antibody VH53C was used at a concentration of 9.7 mg/ml (approximately 67 μM) in a buffer consisting of 50 mM Tris-HCl, 1 mM EDTA, pH 8.2. Ac-PYY (PEG3-Cys-βAla-biotin) and antibody were mixed at a molar ratio of 2.5:1 (Ac-PYY (PEG3-Cys-βAla-biotin) to antibody) and incubated at room temperature with shaking at 350 rpm for 60 minutes. The resulting conjugate was analyzed by mass spectrometry. 87.7% of the detected species were identified as antibody conjugated to two peptide molecules, and 12.3% were identified as antibody conjugated to one peptide molecule.

Exemplary Method for Forming a Conjugate of a Biotinylated Polypeptide and an Anti-Biotin Antibody (Ac-PYY(PEG3-Cys-PEG2-Biotin)/Chimeric Anti-Biotin Antibody VH53C)

To generate a conjugate of a biotin-derivatized PYY polypeptide containing a cysteine linker, 0.16 mg of Ac-PYY(PEG3-Cys-PEG2-biotin) was dissolved in 100% DMF to a concentration of 10 mg/ml. 1 mg of the chimeric anti-biotin antibody VH53C was used at a concentration of 9.9 mg/ml (approximately 68 μM) in a buffer consisting of 50 mM Tris-HCl, 1 mM EDTA, pH 8.2. Ac-PYY(PEG3-Cys-PEG2-biotin) and antibody were mixed at a molar ratio of 2.5:1 (Ac-PYY(PEG3-Cys-PEG2-biotin) to antibody) and incubated at room temperature with shaking at 350 rpm for 60 minutes. The resulting conjugate was analyzed by mass spectrometry. 100% of the detected species were identified as antibody conjugated to two peptide molecules.

Exemplary Method for Forming a Conjugate of a Biotinylated Polypeptide and an Anti-Biotin Antibody (Ac-PYY(PEG3-Cys-βAla-Biotin)/Humanized Anti-Biotin Antibody VH53C)

To generate a conjugate of a biotin-derivatized PYY polypeptide containing a cysteine linker, 0.06 mg of Ac-PYY (PEG3-Cys-βAla-biotin) was dissolved in 100% DMF to a concentration of 10 mg/ml. 0.8 mg of the humanized anti-biotin antibody VH53C was used at a concentration of 9 mg/ml (approximately 62 μM) in a buffer consisting of 50 mM Tris-HCl, 1 mM EDTA, pH 8.2. Ac-PYY (PEG3-Cys-βAla-biotin) and antibody were mixed at a molar ratio of 2.5:1 (Ac-PYY (PEG3-Cys-βAla-biotin) to antibody) and incubated at room temperature with shaking at 350 rpm for 60 minutes. The resulting conjugate was analyzed by mass spectrometry. 62.2% of the detected species were identified as antibodies conjugated to two peptide molecules, 33.9% were identified as antibodies conjugated to one peptide molecule, and 3.9% were identified as unconjugated antibodies.

Exemplary Method for Forming a Conjugate of a Biotinylated Polypeptide and an Anti-Biotin Antibody (Ac-PYY(PEG3-Cys-PEG2-Biotin)/Humanized Anti-Biotin Antibody VH53C)

To generate a conjugate of a biotin-derivatized PYY polypeptide containing a cysteine linker, 0.08 mg of Ac-PYY(PEG3-Cys-PEG2-biotin) was dissolved in 100% DMF to a concentration of 10 mg/ml. 0.8 mg of the humanized anti-biotin antibody VH53C was used at a concentration of 9 mg/ml (approximately 62 μM) in a buffer consisting of 50 mM Tris-HCl, 1 mM EDTA, pH 8.2. Ac-PYY(PEG3-Cys-PEG2-biotin) and antibody were mixed at a molar ratio of 2.5:1 (Ac-PYY(PEG3-Cys-PEG2-biotin) to antibody) and incubated at room temperature with shaking at 350 rpm for 60 minutes. The resulting conjugate was analyzed by mass spectrometry. 71.4% of the detected species were identified as antibodies conjugated to two peptide molecules, 26% were identified as antibodies conjugated to one peptide molecule, and 2.5% were identified as unconjugated antibodies.

Example 7

Detection method

SDS gel electrophoresis

For SDS gel electrophoresis, 4x LDS sample buffer (Invitrogen) was added to the samples. For each sample, a reduced form was also prepared by adding 10x NuPAGE sample reducing agent (Invitrogen). All samples were incubated at 70°C for 5 minutes and then electrophoresed on 4-12% Bis-Tris polyacrylamide gels (NuPAGE, Invitrogen) using 1x MOPS buffer (Invitrogen).

Fluorescence detection

The Cy5-related fluorescence in the gel was detected using a LumiImager F1 device (Roche) at an excitation wavelength of 645 nm. After fluorescence detection, the gel was stained with SimplyBlue SafeStain (Invitrogen).

Example 8

Serum stability

Comparison of serum stability of the complex of biotinylated Cy5 and humanized anti-biotin antibody with the covalent conjugate of biotinylated Cy5 and humanized anti-biotin antibody VH53C

The purpose of the peptide modification technology is to improve the therapeutic applicability of peptides. The main bottleneck of the therapeutic application of peptides is the currently limited in vivo stability and/or short serum half-life and rapid clearance. The PK parameters of the fluorophore antibody conjugate were determined in vivo and compared with the PK of the non-covalent antibody-fluorophore complex. Therefore, (i) the anti-biotin antibody VH53C was covalently conjugated to the biotinylated fluorophore Biot-Cys-Cy5; (ii) a non-covalent complex of the anti-biotin antibody and the biotinylated fluorophore Biot-Cy5 was generated; (iii) the covalently conjugated and non-covalently complexed compound was applied to animals; and (iv) the changes in the serum concentration of the compound in these animals over time were analyzed.

Experimental methods

To analyze the effect of antibody complexation with a small fluorescent substrate on PK parameters, 13 nmol of Cy5-biotin/humanized anti-biotin antibody disulfide-linked conjugate or the corresponding antibody non-covalently complexed compound in 20 mM histidine/140 mM NaCl, pH 6.0 was applied to six female mice (NRMI strain) for each substance. Approximately 0.1 ml blood samples were collected after the following time points: 0.08 h, 8 h, and 48 h for mice 1 and 2, 0.08 h, 24 h, and 48 h for mice 3 and 4, and 0.08 h, 36 h, and 48 h for mice 5 and 6. After 1 h at RT, serum samples of at least 40 μl were obtained by centrifugation (9,300 x g, 3 min, 4°C). Serum samples were stored at -80°C.

To determine the amount of compound in serum at a given time point, the fluorescence properties of Cy5 were utilized: Cy5-related fluorescence in serum samples was measured in a 120 μl quartz cuvette at room temperature using a Cary Eclipse fluorescence spectrophotometer (Varian). Excitation was at 649 nm, and emission was measured at 670 nm. Serum samples were diluted in 1x PBS to achieve the appropriate emission intensity range. Untreated mouse serum diluted in 1x PBS at the same dilution as each sample was used as a blank probe.

Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Example 9

X-ray Structure Determination of the Fab Fragment of Mouse Anti-Biotin Antibody Complexed with Biocytinamid

The protein structure of a murine anti-biotin antibody Fab fragment complexed with biocytinamid was determined. Crystals of the Fab fragment were incubated in 0.8 M succinic acid, and then charged with biocytidinamide (diluted to a working concentration of 10 mM in crystallization solution and added to the crystals in the crystallization droplet). The crystals were washed three times with 2 μl of a 10 mM biocytidinamide solution and finally incubated with biocytidinamide at 21°C for 16 hours. The crystals were harvested using 15% glycerol as a cryoprotectant and flash-frozen in liquid nitrogen. The processed diffraction images yielded the protein structure at high resolution. Figure 2 shows the structure and charge composition of the biotin-bound variable region: biotin binds to a surface pocket flanked by charged regions composed of amino acids from the CDR regions. The complexed hapten is positioned in close proximity to a cluster of negatively charged amino acids. Derivatization of the hapten for payload conjugation at its carboxyl group binds efficiently because there is no charge repulsion at this position (due to the lack of a COOH group). In contrast, free (normal) biotin cannot bind to the antibody efficiently because its carboxyl group will be in close proximity to this negatively charged cluster and thus become repelled.

Claims (10)

1. Humanized anti-biotin antibody, wherein the antibody comprises: (a) HVR-H1 of the amino acid sequence of SEQ ID NO:09; (b) HVR-H2 of the amino acid sequence of SEQ ID NO:10; and (c) HVR-H3 of the amino acid sequence of SEQ ID NO:11; (d) HVR-L1 of the amino acid sequence of SEQ ID NO:13; (e) HVR-L2 of the amino acid sequence of SEQ ID NO:14; and (f) HVR-L3 of the amino acid sequence of SEQ ID NO:15. The antibody contains the amino acid residue serine at position 24 of the heavy chain variable domain (numbered according to Kabat) and the amino acid residue threonine at position 73 of the heavy chain variable domain (numbered according to Kabat). The antibody contains the amino acid residue alanine at position 60 of the heavy chain variable domain numbered according to Kabat and the amino acid residue glutamine at position 61 of the heavy chain variable domain numbered according to Kabat. 2. The antibody of claim 1, comprising: The VH sequence has at least 95% sequence identity with the amino acid sequence of SEQ ID NO:12; and A VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:16; The 24th amino acid residue of the Kabat-numbered heavy chain variable domain is serine, and the 73rd amino acid residue of the Kabat-numbered heavy chain variable domain is threonine. The heavy chain variable domain, numbered according to Kabat, contains the amino acid residue alanine at position 60 and the amino acid residue glutamine at position 61. 3. The antibody of claim 1, comprising the VH sequence of SEQ ID NO:12. 4. The antibody of any one of claims 1 to 3, comprising the VL sequence of SEQ ID NO:16. 5. An antibody comprising the VH sequence of SEQ ID NO:12 and the VL sequence of SEQ ID NO:16. 6. The antibody of claim 1, wherein it is a full-length IgG1 antibody or a full-length IgG4 antibody. 7. The antibody of any one of claims 1 to 3 and 5 to 6 is a monoclonal antibody. 8. The antibody of any one of claims 1 to 3 and 5 to 6, wherein it is an antibody fragment that binds to biotin. 9. A pharmaceutical preparation comprising an antibody and a pharmaceutically acceptable carrier of any one of claims 1 to 8. 10. The antibody of any one of claims 1 to 3 and 5 to 6, which is used as a medicine.