Antibodies binding to PSI domain of integrin β6, and therapeutic method using same

Abstract

The present invention relates to an antibodies that bind to the PSI domain of integrin β6, drugs comprising the antibodies, a method for screening for the antibodies, a method for producing the antibodies, a method for treating cancer comprising a step for administering the antibodies, and the like.

Description

Antibody that binds to the PSI domain of integrin β6 and therapeutic method using the same

[0001] The present invention relates to an antibody that binds to the PSI domain of integrin β6 and to a method of using the same.

[0002] Integrins are membrane-associated family proteins that play diverse roles in cell adhesion, motility, and cell division. Integrins exist as heterodimers consisting of α and β chains, with 18 α-chain isoforms and 8 β-chain isoforms known. Within this family, integrin αvβ6 is expressed only in epithelial cells. While its expression is limited in normal epithelial tissue, it increases significantly during the carcinogenesis of many epithelial cancers. Integrin αvβ6 expression correlates with reduced survival rates in many cancer types, including colorectal cancer, breast cancer, pancreatic ductal adenocarcinoma, non-small cell lung cancer, and cervical squamous cell carcinoma. One of the characteristic functions of integrin αvβ6 is the activation of transforming growth factor β (TGF-β). TGF-β signaling suppresses T cell proliferation and effector function, and inhibits T cell differentiation into the Th1 subtype, which mediates the T cell response to cancer. Due to its important role in tumor progression, integrin αvβ6 is known as a promising target for cancer treatment (Non-Patent Literature 1).

[0003] Meng Li et al., Acta Pharm Sin B 2021; 11: 2726-2737

[0004] The present invention provides an antibody that binds to the PSI domain of integrin β6 (hereinafter sometimes simply referred to as "anti-integrin β6 antibody") and a method for using the same.

[0005] The inventors have successfully produced an antibody that binds to the PSI domain of integrin β6, thereby completing the present invention. The present invention provides an antibody that binds to the PSI domain of integrin β6, as well as a method for using the antibody, a therapeutic method using the antibody, a screening method for the antibody, and a pharmaceutical preparation containing the antibody.

[0006] Specifically, the following embodiments are included as illustrative examples: [A1] An anti-integrin β6 antibody characterized by binding to the PSI domain of integrin β6. [A2] The antibody according to [A1], including the following: (1) HVR-H1 containing the amino acid sequence of (a) SEQ ID NO: 1; (b) HVR-H2 containing the amino acid sequence of (c) SEQ ID NO: 3; (d) HVR-L1 containing the amino acid sequence of (d) SEQ ID NO: 4; (e) HVR-L2 containing the amino acid sequence of (e) SEQ ID NO: 5; and (f) HVR-L3 containing the amino acid sequence of (f) SEQ ID NO: 6; or (2) a heavy chain variable region containing the amino acid sequence of (e) SEQ ID NO: 7, and a light chain variable region containing the amino acid sequence of (e) SEQ ID NO: 8. [A3] An anti-integrin β6 antibody that competes with the antibody according to [A2] (reference antibody) for binding to the PSI domain of integrin β6. [A4] An anti-integrin β6 antibody according to any one of [A1] to [A3] for cancer treatment. [A5] CAR cells containing an anti-integrin β6 antibody as described in any of [A1] to [A3]. [A6] A method for screening anti-integrin β6 antibodies, comprising the step of selecting an antibody that binds to the PSI domain of integrin β6. [A7] A nucleic acid encoding an anti-integrin β6 antibody as described in any of [A1] to [A3]. [A8] A host cell containing the nucleic acid described in [A7]. [A9] A method for producing an anti-integrin β6 antibody, comprising the step of culturing the host cell described in [A8]. [B1] An anti-integrin β6 antibody comprising a heavy chain variable region consisting of a sequence having 80%, 85%, 90%, or 95% or more identity with the amino acid sequence of SEQ ID NO: 7, and a light chain variable region consisting of a sequence having 80%, 85%, 90%, or 95% or more identity with the amino acid sequence of SEQ ID NO: 8, and having binding activity to the PSI domain of integrin β6. [B2] A monoclonal anti-integrin β6 antibody as described in any of [A1] to [A3] and [B1]. [B3] A human antibody, a humanized antibody, or a chimeric antibody as described in any of [A1] to [A3] and [B1] to [B2].[B4] An anti-integrin β6 antibody according to any of [A1] to [A3] and [B1] to [B3], which is an antibody fragment that binds to the PSI domain of integrin β6. [C1] A cell expressing a CAR containing the anti-integrin β6 antibody according to any of [A1] to [A3] and [B1] to [B4] (CAR cell). [C2] A T cell (CAR-T cell) according to [C1]. [D1] A composition, pharmaceutical composition, pharmaceutical, or drug containing the anti-integrin β6 antibody according to any of [A1] to [A3] and [B1] to [B4], or the CAR cell according to [A5], the CAR cell according to [C1], or the CAR-T cell according to [C2]. [D2] The composition, pharmaceutical composition, pharmaceutical, or drug according to [D1] for cancer treatment. [D3] A pharmaceutical preparation comprising an anti-integrin β6 antibody according to any one of [A1] to [A3] and [B1] to [B4], and a pharmaceutically acceptable carrier. [E1] A method for treating cancer, comprising the step of administering an effective amount of an anti-integrin β6 antibody according to any one of [A1] to [A3] and [B1] to [B4], or CAR cells according to [A5], CAR cells according to [C1], or CAR-T cells according to [C2] to a target. [E2] Use of an anti-integrin β6 antibody according to any one of [A1] to [A3] and [B1] to [B4] in the manufacture of a pharmaceutical for the treatment of cancer. [F1] The method for manufacturing according to [A9], further comprising the step of recovering the antibody from the host cells. [G1] An immunoconjugate comprising an anti-integrin β6 antibody according to any one of [A1] to [A3] and [B1] to [B4], and a cytotoxic agent.

[0007] This figure shows the interaction between MabITGB6 / / MabCD3 and the PSI domain fragment. This figure shows the results of T cell-dependent cytotoxic activity of an anti-integrin β6 PSI domain / CD3 bispecific antibody. This figure shows a schematic of the pMSGV1 MabITGB6 CAR construct. This figure shows confirmation of MabITGB6 CAR expression. This figure shows the results of cytotoxic activity of MabITGB6 CAR-T.

[0008] I. Definitions In the spirit of this specification, “acceptor human framework” 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 the human immunoglobulin framework or human consensus framework as defined below. An acceptor human framework “derived” from the human immunoglobulin framework or human consensus framework may include the same amino acid sequence or may include a modification of the amino acid sequence. In some embodiments, the number of amino acid modifications 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 sequence-identical to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0009] "Affinity" refers to the strength of the combined non-covalent interactions between one binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X to its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific examples and exemplary embodiments for measuring binding affinity are described below.

[0010] An "affinity-matured" antibody is an antibody that, compared to a parent antibody without modifications, has one or more modifications in one or more hypervariable regions (HVRs) that result in improved affinity of the antibody to the antigen.

[0011] The term "anti-integrin β6 antibody" or "antibody that binds to integrin β6" refers to an antibody that can bind to integrin β6 with sufficient affinity, and as a result, is useful as a diagnostic and / or therapeutic agent when it targets integrin β6. In one embodiment, the degree of binding of an anti-integrin β6 antibody to unrelated non-integrin β6 proteins is less than approximately 10% of the antibody's binding to integrin β6 when measured (e.g., by radioimmunoassay (RIA)). In a particular embodiment, an antibody that binds to integrin β6 has a concentration 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, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 It has a dissociation constant (Kd) of M. In certain embodiments, the anti-integrin β6 antibody binds to an integrin β6 epitope that is conserved among integrin β6 from different species. In certain embodiments, the anti-integrin β6 antibody binds to the PSI domain of integrin β6. In certain embodiments, the anti-integrin β6 antibody specifically binds to the PSI domain of integrin β6.

[0012] In this specification, the term "and / or" is used to indicate each subject or any combination thereof that is listed before or after "and / or". For example, "A, B and / or C" includes not only the subjects "A", "B", and "C", but also the combinations "A and B", "A and C", "B and C", and "A and B and C".

[0013] In this specification, the term “antibody” is used in its broadest sense and encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0014] An "antibody fragment" refers to a molecule other than the complete antibody that binds to the antigen to which the complete antibody binds, including a portion of that complete antibody. Examples of antibody fragments are not limited to these, but include 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. In one context, an example of an "antibody fragment" is VHH (Variable domain of heavy chain of heavy chain antibody).

[0015] The term "epitope" refers to a site on an antigen to which an antibody binds, whether proteinaceous or nonproteinaceous. For example, an epitope can be defined by its structure. Alternatively, an epitope can be defined by the binding activity of the antibody that recognizes it. If the antigen is a peptide or polypeptide, the epitope can also be identified by the amino acid residues that make up the epitope. Furthermore, if the epitope is a sugar chain, it can be identified by its specific sugar chain structure.

[0016] A linear epitope is an epitope that contains an epitope whose primary amino acid sequence has been recognized. A linear epitope typically contains at least three, and most commonly at least five, for example, about eight to about ten, or six to twenty amino acids in a specific sequence.

[0017] In contrast to linear epitopes, structural epitopes typically consist of discontinuous amino acid residues rather than a continuous primary sequence (sometimes called discontinuous epitopes). Structural epitopes may contain an increased number of amino acids compared to linear epitopes. Regarding the recognition of structural epitopes, antibodies recognize the three-dimensional structure of peptides or proteins. For example, when a protein molecule folds to form a three-dimensional structure, certain amino acids and / or polypeptide backbone that form the structural epitope are parallel, allowing the antibody to recognize the epitope. Methods for determining the three-dimensional structure of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-specific spin labeling and electromagnetic paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

[0018] The structure of an antibody that binds to an epitope is called a paratope. The epitope and paratope bind stably due to hydrogen bonds, electrostatic forces, van der Waals forces, hydrophobic bonds, etc., acting between them. This binding force between the epitope and paratope is called affinity. The sum of the binding forces when multiple antibodies bind to multiple antigens is called avidity. For example, when an antibody containing multiple antigen-binding domains (i.e., a polyvalent antibody) binds to multiple epitopes, the affinity acts synergistically, resulting in avidity that is higher than the affinity.

[0019] A "competing antibody" with a reference antibody refers to an antibody that inhibits the binding of the reference antibody to its own antigen by 50% or more in a competition assay. Conversely, the reference antibody inhibits the binding of the aforementioned antibody to its own antigen by 50% or more in a competition assay. Competing antibodies include antibodies that bind to the same or overlapping epitopes as the reference antibody, and antibodies that bind to adjacent epitopes in close proximity to the epitope to which the reference antibody binds and cause steric hindrance. Exemplary competition assays are provided herein.

[0020] 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 remaining portion of the heavy and / or light chain is derived from a different source or species.

[0021] The "class" of an antibody refers to the type of constant domain or constant region present in the heavy chain of the antibody. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM. And some of these may be further divided into subclasses (isotypes). For example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively.

[0022] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or interferes with the function of cells and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212Radioisotopes of Pb and Lu); chemotherapeutic agents or chemotherapeutic drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes such as nuclease and fragments thereof; antibiotics; toxins such as, for example, low molecular weight toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including fragments and / or variants thereof); and various antitumor agents or anticancer agents disclosed below are included.

[0023] "Effector function" refers to the biological activities that vary depending on the antibody isotype, which are caused by the Fc region of the antibody. Examples of the effector functions of antibodies include the following: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0024] "Effective amount" of an agent (e.g., pharmaceutical formulation) refers to the amount at the required dosage and over the required period that is effective to achieve the desired therapeutic or prophylactic result.

[0025] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes both the native sequence Fc region and mutant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain, provided that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region is present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0026] The term "framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the sequences of HVR and FR usually appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. In one embodiment, the term "HVR" is used synonymously with "CDR." For example, the terms HVR-H1 to 3 (HVR-L1 to 3) are interchangeable to mean HCDR1 to 3 (LCDR1 to 3).

[0027] The terms "full-length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a native antibody, or having a heavy chain containing an Fc region as defined herein.

[0028] The terms “host cell,” “host cell line,” and “host cell culture” refer to cells (including their offspring) that are interchangeably used and into which foreign nucleic acids have been introduced. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring, regardless of passage number. Offspring do not have to be completely identical to the parent cells in terms of nucleic acid content and may contain mutations. Mutant offspring that have the same function or biological activity as those used when the original transformed cells were screened or selected are also included herein.

[0029] A "human antibody" is an antibody that possesses an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or an antibody derived from a non-human source that uses the human antibody repertoire or other human antibody coding sequences. This definition of a human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues.

[0030] The "Human Consensus Framework" is a framework that shows the most commonly occurring amino acid residues in selected human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from subgroups of variable domain sequences. Typically, the sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI by Kabat et al. As described above. In another embodiment, for VH, the subgroup is subgroup III by Kabat et al. As described above.

[0031] A “humanized” antibody is a chimeric antibody that contains amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, a humanized antibody contains substantially all of at least one, typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0032] As used herein, the term “hypervariable region” or “HVR” refers to each region of the variable domain of an antibody that is hypervariable in sequence (a “complementarity determining region” or “CDR”), and / or forms a structurally defined loop (a “hypervariable loop”), and / or contains an antigen contact residue (a “antigen contact”). Typically, an antibody contains six HVRs: three in the VH region (H1, H2, H3) and three in the VL region (L1, L2, L3). Examples of HVRs described herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) combinations of (a), (b), and / or (c) including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein in accordance with Kabat et al.

[0033] An "immunoconjugate" is an antibody that has been conjugated to one or more heterologous molecules (the heterologous molecules include, but are not limited to, cytotoxic agents).

[0034] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, 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.

[0035] "Isolated" antibodies are those separated from the components of their original environment. In some embodiments, antibodies are purified to a purity of over 95% or 99% by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion-exchange or reverse-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0036] "Isolated" nucleic acids are nucleic acid molecules that have been separated from the components of their original environment. Isolated nucleic acids include nucleic acid molecules that would normally be found in the cell containing them, but these nucleic acid molecules are located outside the chromosome or in a chromosomal location different from their original chromosomal location.

[0037] "Isolated nucleic acids encoding an anti-integrin β6 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, and includes nucleic acid molecules mounted on one or more vectors, and nucleic acid molecules present at one or more locations within a host cell.

[0038] • Polynucleotides (nucleic acids) As used interchangeably herein, “polynucleotide” or “nucleic acid” means a polymer of nucleotides of any length, including DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substance that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may include modified nucleotides such as methylated nucleotides and analogs thereof. Non-nucleotide components may be interposed in the sequence of nucleotides. Polynucleotides may include modifications made after synthesis, such as conjugation to labels. Other types of modifications include, for example, "caps," substitutions of one or more naturally occurring nucleotides with analogs, internucleotide modifications, such as those involving uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramic acids, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those involving pendant portions such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those involving intercalating agents (e.g., acridine, psoralen, etc.), those involving chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those involving alkylating agents, and those involving modified linkages (e.g., alpha-anomeric nucleic acids, etc.) or unmodified forms of polynucleotides. Furthermore, any hydroxyl group normally present in sugars may be substituted with, for example, a phosphonate group, a phosphate group, protected with a standard protecting group, activated to generate further linkages to further nucleotides, or conjugated to a solid or semi-solid support. The 5' and 3' terminal OH groups can be substituted with phosphorylation or with amines or organic cap groups of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups.Polynucleotides may also include analogous forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro-, or 2'-azid-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose or xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and basic nucleoside analogs such as methylriboside. One or more phosphodiester bonds may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by: P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H, or optionally an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl substituted or unsubstituted alkyl (1–20C). Not all linkages in a polynucleotide need to be identical. The above description applies to all polynucleotides referred to herein, including RNA and DNA.

[0039] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies constituting that population are identical and / or bind to the same epitope, except for any possible mutant antibodies (e.g., mutant antibodies containing naturally occurring mutations, or mutant antibodies that arise during the production of a monoclonal antibody preparation; such variants are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. Therefore, the modifier “monoclonal” indicates a characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by a variety of methods, including, but are not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0040] A "naked antibody" is an antibody that is not conjugated with a different part (e.g., a cytotoxic part) or a radioactive label. Naked antibodies may be present in pharmaceutical formulations.

[0041] "Natural antibodies" refer to immunoglobulin molecules with various structures that occur naturally. For example, a natural IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. Based on the amino acid sequence of its constant domain, the light chains of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ).

[0042] The term “package insert” is used to refer to instructions for use that are typically included in the commercial packaging of therapeutic products and contain information regarding indications, usage, dosage, method of administration, combination therapies, contraindications, and / or warnings relating to the use of such therapeutic products.

[0043] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage ratio of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after the sequences have been aligned to obtain the greatest possible percentage sequence identity and gaps have been introduced where necessary, and no conservative substitutions are considered part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved by using various methods within the scope of the art, such as publicly available computer software, including BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetics Co., Ltd.). A person skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve the greatest possible alignment over the entire length of the sequences being compared.

[0044] The ALIGN-2 sequence comparison computer program is copyrighted by Genentech, Inc., and its source code, along with user documentation, is filed with the U.S. Copyright Office (Washington DC, 20559) and registered under U.S. Copyright Registration Number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, and may also be compiled from the source code. The ALIGN-2 program is 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 change. In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, or with, or relative to, a given amino acid sequence B (or, a given amino acid sequence A having or containing a certain % amino acid sequence identity to, or with, or relative to, a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical in the alignment of A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B. It will be understood that if 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 is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0045] The term "pharmaceutical preparation" refers to a preparation in which the biological activity of the active ingredient contained therein can exert its effect, and which does not contain additional elements that are toxic to an extent unacceptable to the subject to which the preparation is administered.

[0046] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical preparation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0047] As used herein, the term “integrin β6” refers to any native integrin β6 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. This term encompasses both “full-length” integrin β6 that has not undergone processing and any form of integrin β6 resulting from processing within cells. This term also encompasses naturally occurring variants of integrin β6, such as splice variants and allele variants.

[0048] As used herein, “treatment” (and its grammatical derivatives, e.g., “to treat,” “to treat,” etc.) means a clinical intervention intended to modify the natural course of the individual being treated, and may be carried out for preventive purposes or during the course of a clinical condition. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, reduction of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction of the rate of disease progression, recovery or mitigation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of disease or to slow the progression of disease.

[0049] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) typically have a similar structure, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated by screening complementary libraries of VL or VH domains, respectively, using the VH or VL domains from antibodies that bind to that antigen. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0050] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures, and vectors that are incorporated into the genome of a host cell into which they are introduced. Some vectors can result in the expression of the nucleic acid to which they are operationally linked. Such vectors are also referred to herein as "expression vectors." Vectors can be introduced into host cells by methods such as viruses or electroporation, but vector introduction is not limited to in vitro; it is also possible to introduce vectors directly into living organisms.

[0051] II. Compositions and Methods In one aspect, the present invention relates to an antibody that binds to integrin β6. In a particular embodiment, an antibody that binds to the PSI domain of integrin β6 is provided. The antibody of the present invention is useful, for example, for the diagnosis or treatment of cancer.

[0052] The cancer terms “cancer” and “malignant” refer to or describe a physiological condition in mammals typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinomas, lymphomas (e.g., Hodgkin and non-Hodgkin lymphomas), blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, leukemia, and other lymphoproliferative disorders, as well as various types of head and neck cancers. In one embodiment, the anti-integrin β6 antibody of this disclosure is for cancer treatment. In one embodiment, cancers that can be treated with the anti-integrin β6 antibody of this disclosure include cancers of cells and tissues that express integrin αvβ6. Examples of cancers that can be treated include the cancers mentioned above. In one embodiment, cancers that can be treated include colorectal cancer, breast cancer, pancreatic ductal adenocarcinoma, esophageal cancer, head and neck cancer, non-small cell lung cancer, and cervical squamous cell carcinoma.

[0053] A. Exemplary Anti-Integrin β6 Antibodies In one aspect, the present invention provides an isolated antibody that binds to integrin β6. In a particular embodiment, the anti-integrin β6 antibody binds to the PSI domain of integrin β6. In one aspect, the anti-integrin β6 antibody of the present disclosure binds to a polypeptide comprising the amino acid sequence of SEQ ID NO: 15. In one aspect, the anti-integrin β6 antibody of the present disclosure binds to a variant of the PSI domain of integrin β6. In one aspect, the variant of the PSI domain of integrin β6 has one or more amino acid mutations (e.g., addition, deletion, substitution, etc.) relative to the wild-type PSI domain. In one aspect, the variant of the PSI domain of integrin β6 has 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more amino acid sequence identity (homology) relative to the wild-type PSI domain. In one aspect, variants of the integrin β6 PSI domain exhibit sequence identity of 80%, 85%, 90%, 95%, and 98% or more with respect to the amino acid sequence of SEQ ID NO: 15.

[0054] In one aspect, the present invention provides an anti-integrin β6 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: 1; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0055] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3. In one aspect, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3. In another aspect, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In a further aspect, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2. In a further aspect, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3.

[0056] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0057] In another aspect, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0058] In another aspect, the present invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 6.

[0059] In any of the embodiments described above, the anti-integrin β6 antibody is humanized. In one embodiment, the anti-integrin β6 antibody comprises HVR in any of the embodiments described above and further comprises an acceptor human framework (e.g., a human immunoglobulin framework or a human consensus framework).

[0060] In another aspect, the anti-integrin β6 antibody contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the reference sequence includes substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-integrin β6 antibody containing such sequence retains the ability to bind to integrin β6 (in one embodiment, the PSI domain of integrin β6). In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 7. In certain embodiments, substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the anti-integrin β6 antibody includes the VH sequence in SEQ ID NO: 7, including those with post-translational modifications of said sequence. In certain embodiments, VH includes one, two, or three HVRs selected from (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 1, (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 2, and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 3. Post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0061] In another aspect, an anti-integrin β6 antibody is provided, comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 8. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the reference sequence includes substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-integrin β6 antibody comprising such sequence retains the ability to bind to integrin β6 (in one embodiment, the PSI domain of integrin β6). In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 8. In certain embodiments, substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the anti-integrin β6 antibody includes the VL sequence in SEQ ID NO: 8, including those with post-translational modifications of said sequence. In certain embodiments, the VL includes one, two, or three HVRs selected from (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 4, (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 5, and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO: 6. Post-translational modifications include, but are not limited to, modifications to pyroglutamic acid by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0062] In another aspect, an anti-integrin β6 antibody is provided, comprising VH in any of the above embodiments and VL in any of the above embodiments. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 7 and SEQ ID NO: 8, respectively, including those with post-translational modifications of said sequences. Post-translational modifications include, but are not limited to, modifications to pyroglutamic acid by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0063] In a further aspect, the present invention provides an antibody that binds to the same epitope as the anti-integrin β6 antibody provided herein. In one embodiment, the “same epitope” is the PSI domain of integrin β6. For example, in a particular embodiment, an antibody is provided that binds to the same epitope as the anti-integrin β6 antibody comprising the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 8.

[0064] In a further aspect, the present invention provides antibodies that compete with the anti-integrin β6 antibodies provided herein for binding to integrin β6 (in one embodiment, the PSI domain of integrin β6). For example, in certain embodiments, antibodies are provided that compete with the anti-integrin β6 antibody comprising the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 8 for binding to integrin β6 (in one embodiment, the PSI domain of integrin β6).

[0065] In a further aspect of the present invention, the anti-integrin β6 antibody according to any of the above embodiments is a monoclonal antibody comprising a chimeric, humanized, or human antibody. In one embodiment, the anti-integrin β6 antibody is an antibody fragment, such as Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as a complete IgG1 antibody or another antibody class or isotype as defined herein.

[0066] In further developments, anti-integrin β6 antibodies, either alone or in combination, may incorporate any of the characteristics described in items 1 to 7 below.

[0067] 1. Antibody Affinity In a particular aspect, the antibodies provided herein have an affinity of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (for example, 10 -8 M or less, for example, 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 It has a dissociation constant (Kd) of M.

[0068] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using the Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of Fab to the antigen is measured in the presence of a gradual increase in the concentration of the unlabeled antigen. 125 I) Fab is equilibrated with a labeled antigen, and then the bound antigen is captured by a plate coated with anti-Fab antibody. (See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the measurement conditions, a MICROTITER® multiwell plate (Thermo Scientific) is 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 2–5 hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [ 125 Mix the [I]-antigen with serial dilutions of the Fab of interest (e.g., as in the evaluation of anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). Then incubate the Fab of interest overnight, although this incubation may be continued for a longer period (e.g., about 65 hours) to ensure equilibrium is achieved. Subsequently, transfer the mixture to a capture plate for incubation at room temperature (e.g., 1 hour). Then remove the solution and wash the plate eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate is dry, add 150 μl / well of scintillant (MICROSCINT-20®, Packard) and count the plate for 10 minutes on a TOPCOUNT® gamma counter (Packard). Select concentrations of each Fab that give less than 20% of maximum binding for use in competitive binding assays.

[0069] In another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, the assay using BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25°C using a CM5 chip immobilized with approximately 10 response units (RUs) of antigen. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8 before being injected at a flow rate of 5 μl / min to achieve binding of approximately 10 response units (RUs) of protein. After antigen injection, 1M ethanolamine is injected to block unreacted groups. For kinetics measurement, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20™) surfactant are injected at 25°C and a flow rate of approximately 25 μl / min. Binding rate (k on ) and dissociation rate (k off ) is calculated by simultaneously fitting the coupling and dissociation sensorgrams using a simple one-to-one Langmuir coupling model (BIACORE® evaluation software version 3.2). The equilibrium dissociation constant (Kd) is given by k off / k on It is calculated as a ratio. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). The on velocity is 10 by the surface plasmon resonance assay described above. 6 M -1 s -1If it exceeds this, the ON rate can be determined by measuring the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass 16 nm) at 25°C in the presence of gradually increasing concentrations of antigen using a spectrometer (e.g., a stop-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) using a stirred cuvette).

[0070] 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, as well as other fragments described below. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp.269-315 (1994); in addition, see WO93 / 16185; and U.S. Patents 5,571,894 and 5,587,458. For a discussion on the Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and exhibiting extended in vivo half-lives, see U.S. Patent No. 5,869,046.

[0071] A diabody is an antibody fragment containing two antigen-binding sites, which may be bivalent or bispecific. See, for example, EP404,097; WO1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0072] A single-domain antibody is an antibody fragment containing 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, a single-domain antibody is a human single-domain antibody (see, for example, Domantis, Inc., Waltham, MA; U.S. Patent No. 6,248,516B1).

[0073] Antibody fragments can be produced by various methods, including, but are not limited to, the proteolytic digestion of complete antibodies and production by recombinant host cells (e.g., Escherichia coli or phages) as described herein.

[0074] 3. Chimeric and Humanized Antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and in Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody includes a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In further examples, a chimeric antibody is a “class-switched” antibody in which the class or subclass has been changed from that of the parent antibody. A chimeric antibody also includes its antigen-binding fragment.

[0075] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity to humans while maintaining the specificity and affinity of the parent non-human antibody. A humanized antibody usually contains one or more variable domains, in which the HVR (e.g., CDR (or a portion thereof)) is derived from the non-human antibody and the FR (or a portion thereof) is derived from the human antibody sequence. The humanized antibody optionally contains at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues originated) to restore or improve the specificity or affinity of the antibody, for example.

[0076] Humanized antibodies and their production methods have been reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and also in, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patents No. 5,821,337, No. 7,527,791, No. 6,982,321, and No. 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describes specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991). (Resurfacing is described); Dall'Acqua et al., Methods 36:43-60 (2005) (FR shuffling is described); and further described in Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) ("Guided Selection" approach for FR shuffling is described).

[0077] The human framework regions that can be used for humanization are not limited to these, but include: framework regions selected using the "best fit" method (see Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light chain or heavy chain variable regions (see Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al. J. Immunol., 151:2623 (1993)); human maturation (somatic mutation) framework regions or human germline framework regions (see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (Baca et al., J. Biol. Chem. 272:10678-10684). (See also 1997 and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0078] 4. Human Antibodies In a particular embodiment, the antibodies provided herein are human antibodies. Human antibodies can be produced by various methods known in the art. Human antibodies are outlined in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

[0079] Human antibodies may be prepared by administering immunogens to transgenic animals modified to produce fully human antibodies or fully human antibodies with human variable regions in response to antigen challenge (loading). Such animals typically contain all or part of a human immunoglobulin locus, which either replaces an endogenous immunoglobulin locus or is randomly incorporated extrachromosomally or within the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE® technology; U.S. Patent No. 5,770,429 describing HUMAB® technology; U.S. Patent No. 7,041,870 describing KM MOUSE® technology; and U.S. Patent Application Publication 2007 / 0061900 describing VELOCIMOUSE® technology. Human variable regions from complete antibodies produced by such animals may be further modified, for example, by combining them with different human constant regions.

[0080] Human antibodies can also be produced using hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have already been described. (See, for example, Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies produced via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include, for example, those described in U.S. Patent No. 7,189,826 (describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0081] Human antibodies can also be generated by isolating selected Fv clone variable domain sequences from a human-derived phage display library. Such variable domain sequences can then be combined with a desired human constant domain. A method for selecting human antibodies from an antibody library is described below.

[0082] 5. Library-Derived Antibodies The antibodies of the present invention may be isolated by screening a combinatorial library for antibodies with one or more desired activities. For example, various methods are known in the art for generating phage display libraries and for screening such libraries for antibodies with desired binding properties. Such methods have been reviewed in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further reviewed in, for example, McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. This is described in Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

[0083] In certain phage display methods, the VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and screened for antigen-binding phages as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). The phages typically present antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies against the immunosource without requiring the construction of hybridomas. Alternatively, as described in Griffiths et al., EMBO J, 12: 725-734 (1993), naive repertoires (e.g., from humans) can be cloned to provide a single source of antibodies against a wide range of non-self and self-antigens without immunization. Finally, naive libraries can also be synthesized synthetically, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992), by cloning the pre-reorganization V-gene segment from stem cells and using PCR primers containing random sequences that encode the hypervariable CDR3 region and achieve in vitro rearrangement. Patent documents describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, as well as U.S. Patent Application Publications 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0084] As a screening method for antibodies using binding activity as an indicator, the panning method using phage vectors is also suitably employed. When antibody genes are obtained from a polyclonal antibody-expressing cell population as a library of heavy chain and light chain subclasses, the screening method using phage vectors is advantageous. Genes encoding the variable regions of the heavy chain and light chain can be linked with a suitable linker sequence to form a single-chain Fv (scFv). By inserting the gene encoding scFv into a phage vector, a phage expressing scFv on its surface can be obtained. After contact between this phage and the desired antigen, the phage bound to the antigen can be recovered, thereby recovering the DNA encoding scFv with the desired binding activity. By repeating this operation as needed, scFv with the desired binding activity can be enriched.

[0085] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments in this specification.

[0086] 6. Multispecific Antibodies In a particular embodiment, the antibodies provided herein are multispecific antibodies (e.g., bispecific antibodies). A multispecific antibody is a monoclonal antibody having binding specificity to at least two different sites. In a particular embodiment, one of the binding specificities is to integrin β6 and the other is to any other antigen. In a particular embodiment, a bispecific antibody may bind to two different epitopes of integrin β6. A bispecific antibody may be used to localize a cytotoxic agent to cells expressing integrin β6. A bispecific antibody may be prepared as a full-length antibody or as an antibody fragment.

[0087] Methods for producing multispecific antibodies are not limited to these, but include, the recombination co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and knob-in-hole techniques (see, for example, U.S. Patent No. 5,731,168). Multispecific antibodies can be produced by manipulating electrostatic steering effects to create Fc heterodimer molecules (WO2009 / 089004A1); crosslinking two or more antibodies or fragments (see U.S. Patent No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); producing antibodies with two specificities using a leucine zipper (see Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); producing bispecific antibody fragments using "diabody" technology (see Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (Gruber et al., J. Immunol., (See 152:5368 (1994); and may also be prepared by preparing a trispecific antibody as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991).

[0088] Modified antibodies containing three or more functional antigen-binding sites, including "octopus antibodies," are also included herein (see, for example, U.S. Patent Application Publication 2006 / 0025576A1).

[0089] In this specification, an antibody or fragment also includes a “dual-acting Fab” or “DAF” comprising one antigen-binding site that binds to integrin β6 and another different antigen (see, for example, U.S. Patent Application Publication No. 2008 / 0069820).

[0090] 7. Antibody Variants In a particular aspect, amino acid sequence variants of antibodies provided herein are also considered. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies may be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from the amino acid sequence of the antibody, and / or insertions into the amino acid sequence of the antibody, and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be performed to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., antigen-binding ability).

[0091] a) Substitution, insertion, and deletion variants are provided in specific embodiments, including antibody variants having one or more amino acid substitutions. Target sites for substitutional mutagenesis include HVR and FR. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." More substantial modifications are provided in Table 1 under the heading "Exemplary Substitutions" and are described in detail below, with reference to the classes of amino acid side chains. Amino acid substitutions may be introduced into the antibody of interest, and the product may be screened for desired activity, such as retained / improved antigen-binding, reduced immunogenicity, or improved ADCC or CDC.

[0092]

[0093] Amino acids can be grouped according to their common side-chain properties: (1) Hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile); (2) Neutral hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln); (3) Acidic: aspartic acid (Asp), glutamic acid (Glu); (4) Basic: histidine (His), lysine (Lys), arginine (Arg); (5) Residues that affect chain orientation: glycine (Gly), proline (Pro); (6) Aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe). Non-conservative substitution refers to replacing a member of one class with one of another.

[0094] One type of substitution mutant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting mutants, and those selected for further study, will have modifications (e.g., improvements) in specific biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody, and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitution mutant is an affinity-matured antibody, which can be appropriately produced using, for example, a phage display-based affinity-mature technique (e.g., one described herein). Briefly, one or more HVR residues are mutated, and the mutant antibody is displayed on a phage and screened for specific biological activity (e.g., binding affinity).

[0095] Modifications (e.g., substitutions) may be made in HVRs, for example, to improve antibody affinity. Such modifications may be made in HVR "hot spots," i.e., residues encoded by codons that frequently mutate during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or residues that come into contact with the antigen, and the resulting mutant VH or VL may be tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into variable genes selected for maturation by any variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). Next, a secondary library is prepared. This library is then screened to identify any antibody variant with the desired affinity. Another method for introducing diversity involves an HVR-directed approach that randomizes several HVR residues (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.

[0096] In certain embodiments, substitutions, insertions, or deletions may be made within one or more HVRs, provided that such modifications do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative modifications that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made within an HVR. Such modifications may be, for example, outside the antigen-contact residue of the HVR. In certain embodiments of the mutant VH and VL sequences described above, each HVR is either unmodified or contains only one, two, or three amino acid substitutions.

[0097] A useful method for identifying antibody residues or regions that can be targeted for mutational introduction is called "alanine scanning mutagenesis," described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, one or a group of target residues (e.g., charged residues, e.g., arginine, aspartic acid, histidine, lysine, and glutamic acid) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to this initial substitution. Alternatively, the crystal structure of the antigen-antibody complex may be analyzed to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted as substitution candidates or excluded from the list of substitution candidates. Mutants may be screened to determine whether they possess the desired properties.

[0098] Amino acid sequence insertions include not only the insertion of single or multiple amino acid residues within a sequence, but also the fusion of polypeptides ranging in length from one to 100 or more residues at the amino-terminus and / or carboxyl-terminus. An example of terminal insertion is an antibody with a methionyl residue at the N-terminus. Other insertion variants of antibody molecules include those in which an enzyme (e.g., for ADEPT) or a polypeptide that increases the plasma half-life of the antibody is fused to the N- or C-terminus of the antibody.

[0099] b) Glycosylation variants In a particular embodiment, the antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. Adding or removing glycosylation sites to an antibody can be easily achieved by modifying the amino acid sequence to create or remove one or more glycosylation sites.

[0100] If the antibody contains an Fc region, the carbohydrate to which it is attached may be modified. Native antibodies produced by mammalian cells typically contain branched, bifurcated oligosaccharides, which are usually attached to Asn297 of the CH2 domain of the Fc region by N-linkage. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the bifurcated oligosaccharide structure. In some embodiments, the modification of the oligosaccharide in the antibody of the present invention may be carried out to produce antibody variants with specific improved properties.

[0101] In one embodiment, antibody variants are provided having a carbohydrate structure lacking fucose (directly or indirectly) attached to the Fc region. For example, the amount of fucose in such an antibody may be 1%–80%, 1%–65%, 5%–65%, or 20%–40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, measured by MALDI-TOF mass spectrometry, as described, for example, in WO2008 / 077546. Asn297 represents an asparagine residue located around position 297 of the Fc region (EU numbering of Fc region residues). However, due to slight sequence variability among multiple antibodies, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.) and No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications concerning "defucosylated" or "fucose-deficient" antibody variants include: US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication US2003 / 0157108A1, Presta, L; and WO2004 / 056312A1, Adams et al., particularly Example 11) and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0102] Further antibody variants are provided having a bifid oligosaccharide, for example, in which a bifid branched oligosaccharide attached to the Fc region of the antibody is bifid by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and U.S.2005 / 0123546 (Umana et al.). 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, for example, in WO1997 / 30087 (Patel et al.); WO1998 / 58964 (Raju, S.); and WO1999 / 22764 (Raju, S.).

[0103] c) Fc region variants In a particular embodiment, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein to generate an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG4) that includes amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0104] In certain embodiments, antibody variants possessing some, but not all, effector functions are also within consideration of the present invention, such effector functions making the antibody a desirable candidate for application when its in vivo half-life is important, but certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity measurements can be performed to confirm reduced / deficient CDC and / or ADCC activity. For example, Fc receptor (FcR) binding measurements may be performed to confirm that the antibody lacks FcγR binding (and therefore is likely to lack ADCC activity) while maintaining FcRn binding ability. NK cells, the primary cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of the target molecule are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); and U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive measurement methods may be used (see, for example, ACT1® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assays (Promega, Madison, WI)).Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, the ADCC activity of the molecule of interest may be evaluated in vivo in animal models, such as those described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. Furthermore, CDC measurements may be performed to evaluate complement activation (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). In addition, FcRn binding and in vivo clearance / half-life can be determined using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0105] Antibodies with reduced effector function include those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include the so-called "DANA" Fc variant (U.S. Patent No. 7,332,581) with alanine substitutions at residues 265 and 297, and Fc variants with two or more substitutions at amino acid positions 265, 269, 270, 297, and 327.

[0106] Certain antibody variants exhibiting increased or decreased binding affinity to FcRs have been described. (See U.S. Patent No. 6,737,056; WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

[0107] In certain embodiments, the antibody variant includes an Fc region with one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and / or 334 (residue in EU numbering) of the Fc region).

[0108] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either increased or decreased) C1q binding and / or complement-dependent cell injury (CDC), as described, for example, in U.S. Patent No. 6,194,551, WO99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0109] Antibodies with increased half-life and increased binding affinity to the neonatal Fc receptor (FcRn: which plays a role in transferring maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994))) are described in U.S. Patent Application Publication No. 2005 / 0014934A1 (Hinton et al.). These antibodies contain an Fc region with one or more substitutions therein that increase the binding affinity of the Fc region to FcRn. Such Fc variants include those involving substitutions at one or more of the 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. Patent No. 7,371,826)).

[0110] For other examples of Fc region variants, see Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO94 / 29351.

[0111] d) Cysteine-Modified Antibody Variants In certain embodiments, it would be desirable to produce cysteine-modified antibodies (e.g., "thioMAbs") in which one or more residues of the antibody are substituted with cysteine ​​residues. In certain embodiments, the residues to be substituted occur in an accessible site of the antibody. By substituting these residues with cysteine, a reactive thiol group is located in an accessible site of the antibody, and this reactive thiol group may be used to conjugate the antibody to other parts (such as a drug part or a linker-drug part) to create an immunoconjugate as further detailed herein. In certain embodiments, any one or more of the following residues may be substituted with 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-modified antibodies may be produced, for example, as described in U.S. Patent No. 7,521,541.

[0112] e) Antibody Derivatives In certain embodiments, the antibodies provided herein may be further modified to include additional non-protein moieties known and readily available in the art. Suitable moieties for antibody derivatization 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), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde would be advantageous in production due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if one or more polymers are attached, they may be the same molecule or different molecules. Generally, the number and / or type of polymers used in derivatization can be determined based on considerations such as the specific properties or functions of the antibody to be improved, and whether the antibody derivative will be used for therapy under specified conditions, although these are not limited to these.

[0113] In another embodiment, a conjugate is provided of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be of any wavelength, but is not limited thereto, and includes wavelengths that heat the non-protein moiety to a temperature that does not harm normal cells but kills cells adjacent to the antibody-non-protein moiety.

[0114] The anti-integrin β6 antibody described herein can be combined with various existing technologies. One example of such a combination is the creation of cells expressing a chimeric antigen receptor (CAR) using the anti-integrin β6 antibody. Examples of such cells include T cells, γδT cells, NK cells, NKT cells, cytokine-induced killer (CIK) cells, and macrophages (Int J Mol Sci. (2019) 20(11), 2839, Nat Rev Drug Discov. (2020) 19(5), 308). One example of a non-limited method for creating T cells expressing the CAR (CAR-T) is to introduce a CAR containing an anti-integrin β6 antibody (e.g., scFv), the transmembrane domain of the TCR, and the intracellular signaling domain of a costimulatory molecule such as CD28 to enhance T cell activation into effector cells such as T cells using genetic modification technology.

[0115] One embodiment relates to T cells expressing a CAR containing an anti-integrin β6 antibody. These cells may be CAR-expressing T cells (hereinafter simply referred to as "CAR-T cells") produced by introducing a gene encoding a chimeric antigen receptor (CAR) into normal peripheral blood T cells (peripheral blood T lymphocytes). A CAR is a chimeric protein created by artificially fusing an antibody that recognizes cell surface antigens such as cancer cells with a signaling region that induces T cell activation. CAR-T cells are produced by introducing a gene encoding a CAR into peripheral blood T lymphocytes. CAR-expressing T cells produced by this method are used in the treatment of diseases such as cancer through adoptive immunotherapy. Such CAR-T cells are reactive to target cells that express antigens and can induce damage to target cells without relying on interaction with the major histocompatibility complex (MHC).

[0116] Clinical trials are underway worldwide for cancer immunotherapy using CAR-T cells, more specifically, a therapy in which T cells are collected from patients, genetically modified to encode CARs are introduced into these T cells, which are then cultured and reintroduced to the patients. Cancer immunotherapy using CAR-T cells has shown efficacy in hematopoietic malignancies such as leukemia and lymphoma. In 2017, Kymriah® (Novartis, tisagenlecleucel, CTL-019, CD3 zeta-CD137) and Yescarta® (KiTE, axicabtagene ciloleucel, CD3 zeta-CD28), both CAR-T cells that use CD19 as an antigen, were approved as pharmaceuticals in the United States.

[0117] CRS and cytokine release resulting from cancer immunotherapy with CAR-T cell administration have been reported (Lee DW, et al. Current concepts in the diagnosis and management of cytokine release syndrome. Blood. 2014 Jul 10; 124(2): 188-95.). Since the CRS mechanisms induced by the administration of pharmaceutical compositions containing anti-T cell antigen-binding molecules and those induced by CAR-T cell administration are suggested to be common, it is understood that cytokine release syndrome (CRS) associated with CAR-T cell administration can be prevented, mitigated, or treated by the administration of VEGF inhibitors.

[0118] In one embodiment, CAR is constructed by fusing the antigen recognition site of an anti-integrin β6 antibody with a molecule that activates T cells (CD28 or 4-1BB and CD3ζ). In one embodiment, the antigen recognition site included in CAR is, for example, the scFv of an anti-integrin β6 antibody. In one embodiment, CAR-T cells can be produced by expressing CAR in T cells.

[0119] In one embodiment, the CAR of the Disclosure includes an extracellular antigen-binding domain that binds to the PSI domain of integrin β6 (e.g., the heavy chain variable region and light chain variable region of the anti-integrin β6 antibody of the Disclosure), a co-stimulatory intracellular domain (e.g., 4-1BB), and an intracellular signaling domain (e.g., CD3 zeta).

[0120] B. Recombination Methods and Compositions For example, antibodies can be produced using recombinant methods and compositions as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding the anti-integrin β6 antibody described herein is provided. Such nucleic acid may encode an amino acid sequence containing VL and / or VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) containing such nucleic acid are provided. In a further embodiment, host cells containing such nucleic acid are provided. In one such embodiment, the host cell comprises (1) a vector containing nucleic acid encoding an amino acid sequence containing VL of the antibody and an amino acid sequence containing VH of the antibody, or (2) a first vector containing nucleic acid encoding an amino acid sequence containing VL of the antibody and a second vector containing nucleic acid encoding an amino acid sequence containing VH of the antibody (e.g., transformed). In one embodiment, the host cell is eukaryotic (e.g., Chinese hamster ovary (CHO) cells) or lymphoid cells (e.g., Y0, NS0, Sp2 / 0 cells). In one embodiment, a method is provided for producing an anti-integrin β6 antibody, comprising culturing host cells containing the nucleic acid encoding the antibody as described above under conditions suitable for the expression of the anti-integrin β6 antibody, and optionally recovering the antibody from the host cells (or host cell culture medium).

[0121] For the recombinant production of anti-integrin β6 antibodies, nucleic acids encoding the antibody (e.g., those described above) are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids will be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody).

[0122] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, especially when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, for the expression of antibody fragments in Escherichia coli.) After expression, antibodies may be isolated from bacterial cell paste in soluble fractions and further purified.

[0123] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts, including strains of fungi and yeasts whose glycosylation pathways have been "humanized" to produce antibodies with partial or complete human glycosylation patterns, are suitable cloning or expression hosts for antibody-coding vectors. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0124] Cells derived from multicellular organisms (invertebrates and vertebrates) are also suitable host cells for the expression of glycosylated antibodies. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use in conjugation with insect cells, particularly for the transformation of Spodoptera frugiperda cells.

[0125] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for antibody production in transgenic plants).

[0126] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in a suspension state would be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7); human embryonic kidney cell line (293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977), etc.); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980), etc.); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary cancer cells (MMT 060562); and TRI cells (e.g., Mather et al., Annals NY Acad. Sci. 383:44-68). Examples include MRC5 cells (described in 1982); and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0127] In a particular embodiment, the present invention provides an antibody that can be obtained by the method described above.

[0128] C. Assays The anti-integrin β6 antibodies provided herein may be identified, screened, or have their physical / chemical properties and / or biological activity elucidated by various assays known in the art.

[0129] 1. In terms of binding assays and other assay methods, the antibodies of the present invention are tested for their antigen-binding activity by known methods such as ELISA and Western blotting.

[0130] In another context, a competitive assay may be used to identify antibodies that compete with a reference antibody for binding to integrin β6 (in one embodiment, the PSI domain of integrin β6). In certain embodiments, such a competitive antibody binds to the same epitope (e.g., a linear or structural epitope) bound by the reference antibody. In certain embodiments, such a competitive antibody binds to an epitope (e.g., a linear or structural epitope) that overlaps with that bound by the reference antibody. Detailed exemplary methods for mapping the epitopes to which antibodies bind are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). In one embodiment, the reference antibody may include, for example, an anti-integrin β6 antibody, including: (1) HVR-H1 containing the amino acid sequence of (a) SEQ ID NO: 1; (b) HVR-H2 containing the amino acid sequence of (c) SEQ ID NO: 3; (d) HVR-L1 containing the amino acid sequence of (d) SEQ ID NO: 4; (e) HVR-L2 containing the amino acid sequence of (e) SEQ ID NO: 5; and (f) HVR-L3 containing the amino acid sequence of (f) SEQ ID NO: 6; or (2) a heavy chain variable region containing the amino acid sequence of (d) SEQ ID NO: 7, and a light chain variable region containing the amino acid sequence of (d) SEQ ID NO: 8.

[0131] In an exemplary competition assay, immobilized integrin β6 is incubated in a solution containing a first labeled antibody that binds to integrin β6 (e.g., the anti-integrin β6 antibody described in the Examples) and a second unlabeled antibody to be tested for its ability to compete with the first antibody for binding to integrin β6. The second antibody may be present in the hybridoma supernatant. As a control, immobilized integrin β6 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 integrin β6, any excess unbound antibody is removed and the amount of label bound to the immobilized integrin β6 is measured. If the amount of label bound to the immobilized integrin β6 is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody is competing with the first antibody for binding to integrin β6. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0132] In some embodiments, if a competitive assay measures that a 1, 5, 10, 20, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, at least 75%, at least 90%, or even more than 99%, then the two antibodies are considered to bind to the same or overlapping epitopes. (See, for example, Junghans et al., Cancer Res. 50(1990) 1495-1502).

[0133] In some embodiments, if substantially all amino acid mutations in an antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, then the two antibodies are considered to bind to the same epitope. Two antibodies are considered to have an "overlapping epitope" if only a subset of amino acid mutations that reduce or eliminate the binding of one antibody also reduces or eliminates the binding of the other antibody.

[0134] D. Immunoconjugates The present invention also provides immunoconjugates comprising the anti-integrin β6 antibodies of this specification conjugated to one or more cytotoxic agents (e.g., chemotherapeutic agents or chemotherapeutic drugs, growth inhibitors, toxins (e.g., protein toxins of bacterial, fungal, plant or animal origin, enzymatically active toxins, or fragments thereof) or radioisotopes).

[0135] In one embodiment, an immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including, but not limited to, the following: These include: meitansinoids (see U.S. Patent Nos. 5,208,020, 5,416,064, and European Patent No. 0,425,235B1); auristatins such as monomethyl auristatin drug parts DE and DF (MMAE and MMAF) (see U.S. Patents Nos. 5,635,483, 5,780,588, and 7,498,298); drastatin; calicheamycin or its derivatives (see U.S. Patents Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342) (1993); and see Lode et al., Cancer Res. 58:2925-2928 (1998); anthracyclines such as daunomycin or doxorubicin (Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0136] In another embodiment, the immunoconjugate includes antibodies described herein, conjugated to enzymatically active toxins or fragments thereof, including, but not limited to, diphtheria A chain, unbound active fragments of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modesine A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, geronin, mitogellin, restrictosin, phenomycin, enomycin, and trichothecene.

[0137] In another embodiment, the immunoconjugate comprises an antibody described herein that has been conjugated to a radioactive atom to form a radioactive conjugate. Various radioisotopes are available for the production of radioactive conjugates. For example, 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 Contains radioactive isotopes of Pb and Lu. When using a radioactive conjugate for detection, the radioactive conjugate contains radioactive atoms (e.g., Tc-99m or) for scintigraphy examination. 123 I), or may include spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging or MRI) (e.g., iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron).

[0138] Antibody and cytotoxic agent conjugates can be prepared using a variety of difunctional protein conjugates. Examples include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), difunctional derivatives of imide esters (e.g., dimethyl HCl adipimidoate), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radionuclides to antibodies. See WO94 / 11026. Linkers may be “cleavable linkers” that facilitate the release of cytotoxic drugs within cells. For example, acid-unstable linkers, peptidase-sensitive linkers, photo-unstable linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) may be used.

[0139] The immunoconjugates or ADCs described herein expressly consider conjugates prepared using crosslinking reagents, 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, and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA).

[0140] E. Methods and Compositions for Diagnosis and Detection In certain embodiments, any of the anti-integrin β6 antibodies provided herein are useful for detecting the presence of integrin β6 in a biological sample. As used herein, “detection” includes quantitative or qualitative detection. In certain embodiments, the biological sample includes cells or tissues.

[0141] In one embodiment, an anti-integrin β6 antibody is provided for use in a diagnostic or detection method. In a further aspect, a method for detecting the presence of integrin β6 in a biological sample is provided. In a particular embodiment, the method comprises contacting a biological sample with the anti-integrin β6 antibody described herein under conditions in which binding of the anti-integrin β6 antibody to integrin β6 is permitted, and detecting whether a complex has been formed between the anti-integrin β6 antibody and integrin β6. Such a method may be an in vitro or in vivo method. In one embodiment, the anti-integrin β6 antibody is used to select subjects suitable for treatment using the anti-integrin β6 antibody, for example, when integrin β6 is a biomarker for patient selection.

[0142] In certain embodiments, labeled anti-integrin β6 antibodies are provided. Labeling includes, but is not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromogenic labels, high-electron-density labels, chemiluminescent labels, and radioactive labels) as well as moieties indirectly detectable through, for example, enzymatic reactions or intermolecular interactions (e.g., enzymes or ligands). Exemplary labels, but are not limited to, include: radioisotopes. 32 P, 14 C, 125 I, 3 H and 131 I, fluorescent phosphopoides such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, luciferases such as dansyl, umbelliferone, firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, monosaccharide oxidases (e.g., glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidases such as uricase and xanthine oxidase, enzymes linked to oxidizing pigment precursors with hydrogen peroxide (e.g., HRP, lactoperoxidase, or microperoxidase), biotin / avidin, spin-labeled, bacteriophage-labeled, stable free radicals, and similar substances.

[0143] F. Pharmaceutical Formulations The pharmaceutical formulations of the anti-integrin β6 antibody described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing the antibody having the desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the doses and concentrations used, and include, but are not limited to, the following: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and (e.g., m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, and 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 glycoproteins (sHASEGP) (e.g., human soluble PH-20 hyaluronidase glycoprotein such as rHuPH20 (HYLENEX®, Baxter International, Inc.)). Specific exemplary sHASEGPs and their uses (including rHuPH20) are described in U.S. Patent Publications 2005 / 0260186 and 2006 / 0104968.In one aspect, sHASEGP is combined with one or more additional glycosaminoglycans, such as chondroitinase.

[0144] An exemplary lyophilized antibody preparation is described in U.S. Patent No. 6,267,958. Aqueous aqueous antibody preparations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter of which contains a histidine-acetate buffer.

[0145] The formulations described herein may contain one or more active ingredients if necessary for the specific indication being treated. Preferably, these active ingredients have complementary activities that do not adversely affect each other. Such active ingredients are preferably present in combination in amounts effective for the intended purpose.

[0146] The active ingredient may be incorporated into microcapsules prepared, for example, by coacervation or interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules, and poly(methyl methacrylate) microcapsules, respectively), into colloidal drug delivery systems (e.g., liposomes, albumin spheres, microemulsions, nanoparticles, and nanocapsules), or into macroemulsions. Such methods are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0147] A sustained-release formulation may be prepared. A preferred example of a sustained-release formulation is one comprising a semipermeable matrix of a solid hydrophobic polymer containing an antibody, the matrix being in the form of a fabricated product such as a film or microcapsule.

[0148] Preparations used for in vivo administration are typically sterile. Sterility can be easily achieved, for example, by filtering through a sterile filtration membrane.

[0149] G. Therapeutic Methods and Therapeutic Compositions Any of the anti-integrin β6 antibodies provided herein may be used in therapeutic methods. In one aspect, an anti-integrin β6 antibody is provided for use as a pharmaceutical. In a further aspect, an anti-integrin β6 antibody is provided for use in the treatment of cancer. In a particular embodiment, an anti-integrin β6 antibody is provided for use in a therapeutic method. In a particular embodiment, the present invention provides an anti-integrin β6 antibody for use in a method of treating an individual having cancer, the method comprising the step of administering to the individual an effective amount of the anti-integrin β6 antibody. In one such embodiment, the method further comprises the step of administering to the individual an effective amount of at least one additional therapeutic agent (for example, as described below). The “individual” in any of the above embodiments is preferably a human.

[0150] In a further aspect, the present invention provides the use of an anti-integrin β6 antibody in the manufacture or preparation of a pharmaceutical product. In one embodiment, the pharmaceutical product is for the treatment of cancer. In a further embodiment, the pharmaceutical product is for use in a method of treating cancer, the method comprising the step of administering an effective amount of the pharmaceutical product to an individual having cancer. In one such embodiment, the method further comprises the step of administering an effective amount of at least one additional therapeutic agent (for example, as described below) to the individual. The “individual” in any of the above embodiments may be a human.

[0151] In a further aspect, the present invention provides a method for treating cancer. In one embodiment, the method comprises administering an effective amount of an anti-integrin β6 antibody to an individual having such cancer. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent (as described below) to the individual. The “individual” in any of the above embodiments may be a human.

[0152] In a further aspect, the present invention provides a pharmaceutical formulation comprising any of the anti-integrin β6 antibodies provided herein (for use, for example, in any of the therapeutic methods described above). In one embodiment, the pharmaceutical formulation comprises any of the anti-integrin β6 antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-integrin β6 antibodies provided herein and at least one additional therapeutic agent (for example, as described below).

[0153] The antibodies of the present invention can be used in therapy either alone or in combination with other agents. For example, the antibodies of the present invention may be administered concurrently with at least one additional therapeutic agent.

[0154] The combination therapies described above include combined administration (two or more therapeutic agents contained in the same or separate formulations) and individual administration, in which case the antibody of the present invention may be administered prior to, simultaneously with, and / or subsequently to the administration of the additional therapeutic agent. In one embodiment, the administration of the anti-integrin β6 antibody and the administration of the additional therapeutic agent are performed within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days. The antibody of the present invention may also be used in combination with radiotherapy.

[0155] The antibodies (and any additional therapeutic agents) of the present invention may be administered by any preferred means, including parenteral administration, intrapulmonary administration, and nasal administration, and, if desired for local treatment, intrafocal administration. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Dosage may be made by any preferred route, such as injection, including intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules, including single doses, repeated doses over various time points, bolus administration, and pulse infusion, are within consideration herein, but are not limited to these.

[0156] The antibodies of the present invention are formulated, administered, and given in a manner consistent with good medical practice. Factors to be considered from this perspective include the specific disorder being treated, the specific mammal being treated, the clinical symptoms of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to healthcare professionals. The antibodies are formulated, optionally but not necessarily, with one or more agents already in use to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and the other factors discussed above. These are typically used in the same doses and routes of administration as described herein, or at about 1 to 99% of the doses described herein, or in any dose and route deemed empirically / clinically appropriate.

[0157] For the prevention or treatment of a disease, the appropriate dose of the antibody of the present invention (when used alone or with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, the patient's medical history, clinical history and response to the antibody, and the discretion of the attending physician. The antibody is preferably administered to the patient in a single dose or over a series of treatments. Depending on the type and severity of the disease, for example, whether by single or multiple separate doses or by continuous infusion, an antibody dose of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) may be the initial candidate dose for administration to the patient. A typical daily dose may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors described above. In the case of repeated administrations over several days or longer, treatment is usually maintained, depending on the situation, until the desired suppression of disease symptoms occurs. One exemplary dose of the antibody is in the range of approximately 0.05 mg / kg to approximately 10 mg / kg. Therefore, one or more doses (or any combination thereof) of approximately 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg may be administered to the patient. Such doses may be administered intermittently, for example, every week or every three weeks (for example, so that the patient receives approximately 2 to approximately 20, or for example, approximately 6, doses of antibody). One or more low doses may be administered after a high initial loading dose. The course of this therapy is readily monitored by conventional methods and measurements.

[0158] It will be understood that either of the above-described formulations or therapeutic methods may be carried out using the immunoconjugate of the present invention instead of or in addition to the anti-integrin β6 antibody.

[0159] The anti-integrin β6 antibody described herein can be administered by administering or incorporating nucleic acids encoding the anti-integrin β6 antibody into a living body using a vector, etc., and directly expressing the anti-integrin β6 antibody in the living body; however, it may also be administered without using a vector. Examples of vectors include viral vectors, plasmid vectors, and adenovirus vectors. The nucleic acids encoding the anti-integrin β6 antibody may be administered directly to the living body, or cells into which nucleic acids encoding the anti-integrin β6 antibody have been introduced may be administered to the living body. For example, the anti-integrin β6 antibody can be administered by chemically modifying mRNA encoding the anti-integrin β6 antibody to enhance its stability in vivo, and then directly administering the mRNA to a human to express the anti-integrin β6 antibody in vivo (see EP2101823B, WO2013 / 120629). Alternatively, B cells into which nucleic acids encoding the anti-integrin β6 antibody have been introduced may be administered (Sci Immunol. (2019) 4(35), eaax0644). Alternatively, bacteria into which nucleic acids encoding anti-integrin β6 antibodies have been introduced may be administered (Nature Reviews Cancer (2018) 18, 727-743).

[0160] As an example of a non-limiting technique that can be combined with the anti-integrin β6 antibody described herein, the creation of T cells that secrete T cell redirecting antibodies using the anti-integrin β6 antibody is exemplified (Trends Immunol. (2019) 40(3) 243-257). One non-limiting method of creation is to introduce a nucleic acid encoding a bispecific antibody containing a binding domain to one of the constituent subunits of the T cell receptor (TCR) complex on T cells, particularly a binding domain to the CD3 epsilon chain within CD3, and a binding domain to an antigen on target cancer cells, into effector cells such as T cells using genetic modification technology.

[0161] H. Product In another aspect of the present invention, a product is provided comprising equipment useful for the treatment, prevention, and / or diagnosis of the above-mentioned disorders. The product comprises a container and a label on the container or a document accompanying the container. Preferred containers include, for example, bottles, vials, syringes, and IV solution bags. Containers may be formed from a variety of materials, such as glass or plastic. A container may hold the composition alone or in combination with another composition effective for the treatment, prevention, and / or diagnosis of the symptoms, and may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper that can be punctured by a subcutaneous needle). At least one active ingredient in the composition is the antibody of the present invention. The label or document indicates that the composition is used to treat a selected symptom. The product further comprises (a) a first container comprising a composition containing the antibody of the present invention; and (b) a second container comprising a composition containing a further cytotoxic agent or other therapeutic agent. The product in this embodiment of the present invention may further include a package insert indicating that the composition may be used to treat a particular condition. Alternatively, the product may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other buffers, diluents, filters, needles, and syringes, or other equipment desirable from a commercial or user perspective.

[0162] It will be understood that any of the above-mentioned products may contain the immunoconjugate of the present invention in place of or in addition to the anti-integrin β6 antibody.

[0163] The following are examples of the methods and compositions of the present invention. In light of the general description above, it will be understood that various other embodiments may be implemented.

[0164] [Example 1] Preparation of anti-integrin β6 antibody An expression vector encoding MabITGB6 (heavy chain: AM1stFL677H1356-F760mnN17.S1 (SEQ ID NO: 9), light chain: AM1stFL677L0090-lamL.pLibpAFabHybrid (SEQ ID NO: 10)), a human integrin β6 (hITGB6) PSI domain-binding antibody, was prepared by a method known to the art. An expression vector encoding MabKLH (heavy chain: IC17HdK-F760mnN17 (SEQ ID NO: 11), light chain: IC17L-k0 (SEQ ID NO: 12)), an anti-keyhole limpet hemocyanin (KLH) antibody used as an isotype control antibody, was also prepared by a method known to the art. Expression vectors encoding MabCD3 (heavy chain: TR01H113-F760mnP17 (SEQ ID NO: 13), light chain: L0011-k0a (SEQ ID NO: 14)), an anti-human CD3 (hCD3) antibody for use as a T cell engager, were prepared by methods known to the art. The Fc domains of these antibodies were modified to include mutations for preparing bispecific antibodies via Fab-arm exchange (FAE) and mutations to suppress FcγR binding. MabITGB6, MabKLH, and MabCD3 were expressed transiently using the HEK293 cell line by methods known to the art and purified using protein A by methods known to the art. FAE was performed using the purified antibodies by methods known to the art to prepare bispecific T cell engagers. MabITGB6 / / MabCD3 is an anti-hITGB6 and anti-hCD3 bispecific antibody prepared using MabITGB6 and MabCD3. MabKLH / / MabCD3 is a bispecific anti-KLH and anti-hCD3 antibody prepared using MabKLH and MabCD3.

[0165] [Example 2] Preparation of Integrin β6 PSI Domain Fragment To reproduce the native disulfide bond, amino acids 22-80 and 453-455 of human integrin β6 (hITGB6, Uniprot ID: P18564 isoform 1) were linked by a GGG linker consisting of three glycine molecules (SEQ ID NO: 15). Furthermore, two glycine molecules were added to the N-terminus to facilitate tag cleavage. To facilitate the purification of the integrin β6 PSI domain fragment, the amino acid sequence of the above 67-amino acid integrin β6 PSI domain fragment was modified in the following order at the N-terminus: Avi tag (GLNDIFEAQKIEWHE (SEQ ID NO: 16)), 6His tag (HHHHHH (SEQ ID NO: 17)), HRV3C protease cleavage site (LEVLFQGP (SEQ ID NO: 18)), modified MBP tag (M. Bokhove, H. Sadat Al Hosseini, T. Saito, E. Dioguardi, K. Gegenschatz-Schmid, K. Nishimura, I. Raj, D. de Sanctis, L. Han, L. Jovine; Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins; J. Struct. Biol., 194 (2016), pp. 1-7), and WELQut protease cleavage site (WELQ (SEQ ID NO: 19)). The integrin β6 PSI domain fragment to which a tag sequence and protease cleavage site have been added was modified to secrete expression by further adding a Crypa secretion signal sequence (M. Bokhove et al., same literature) to its N-terminus (Sequence ID: 20). The PSI domain fragment to which the tag sequence and protease cleavage site have been added was expressed transiently using the HEK293 cell line by a method known to those skilled in the art, and purified by a method known to those skilled in the art using nickel affinity resin and StrepTactin affinity resin.The tag sequence and protease cleavage site were cleaved from the integrin β6 PSI domain fragment by WELQut protease and removed by gel filtration chromatography (Cytiva), resulting in the preparation of the integrin β6 PSI domain fragment consisting of the 67-amino acid peptide chain described above as the final product.

[0166] [Example 3] Binding Analysis of Anti-Integrin β6 Antibody to Integrin β6 PSI Domain Fragments The binding characteristics of MabITGB6 / / MabCD3 and MabKLH / / MabCD3 were determined at 25°C using a BIACORE® T200 instrument (Cytiva). First, Anti-Human IgG (Fc) antibody was immobilized on all flow cells of the CM5 sensor chip using the Amine Coupling Kit, type 2 (Cytiva) and Human antibody capture kit (Cytiva) according to the method recommended by Cytiva. The antibody and PSI domain fragment were diluted with the running buffer, HBS-P+ (HBS-P+, 1 mM CaCl2, 1 mM MgCl2, 10 mg / mL CMD, 0.5 mg / mL BSA, pH 7.4). Each antibody was captured on the sensor chip surface by the Anti-Human IgG (Fc) antibody. Generally, the antibody capture amount was 1250 Resonance units (RU). Next, integrin β6 PSI domain fragments at concentrations of 75, 150, 300, 600, and 1200 nM were injected and subsequently dissociated. The sensor chip surface was regenerated using 3 M MgCl2. The results are shown in Figure 1. Sensorgrams were acquired using BIACORE® T200 Evaluation Software, version 3.2.1 (Cytiva). Reaction rate parameters were determined by equilibrium value analysis using BIACORE® T200 Evaluation Software, version 3.2.1 (Cytiva). MabKLH / / MabCD3 did not show binding to the integrin β6 PSI domain fragment, but MabITGB6 / / MabCD3 did bind to the integrin β6 PSI domain fragment. D = 4.221 × 10 -7 The bond was shown to be of this strength.

[0167] [Example 4] In vitro evaluation of T cell-dependent cytotoxic activity of anti-integrin β6 PSI domain / CD3 bispecific antibodies by lactate dehydrogenase (LDH) release assay. The cytotoxic activity of MabITGB6 / / MabCD3 and MabKLH / / MabCD3 was evaluated by lactate dehydrogenase (LDH) release assay. The human lung cancer cell line NCI-H2009 (ATCC) expressing hITGB6 was used as the target cell. Frozen PBMCs (STEMCELL) were washed with RPMI-1640 medium (SIGMA) and adjusted to 4 x 10^6 cells / mL. These PBMCs were used as effector cells. The target cells were detached from the culture flask and seeded in 100 μL portions in each well of a flat-bottom clear 96-well plate (Corning) containing 1 x 10^4 cells. 50 μL of human PBMC solution (2 x 10^5 cells) and 50 μL of prepared antibody at concentrations selected from 0.27, 0.82, 2.47, 7.41, 22.22, 66.67, and 200 nM were added to each well. After incubation at 37°C for 48 hours, the plate was centrifuged, and 4 μL of the supernatant from each well was transferred to 196 μL of LDH storage buffer to prepare 50-fold diluted samples. 50 μL of the diluted samples were transferred to a new 96-well opaque-walled, opaque-bottom assay plate (Corning), and 50 μL of LDH detection reagent (LDH Detection Enzyme Mix containing Reductase Substrate; Promega) was added to each well. The plate was incubated at room temperature for 60 minutes. Luminescence was measured using EnVision (PerkinElmer Japan). The cytotoxicity rate (%) was calculated using the following formula: Cytotoxicity (%) = (ABC) x 100 / (DC) where "A" is the average luminescence value of wells treated with antibody and PBMCs, "B" is the average luminescence value of wells with only effector cells (PBMCs), "C" is the average luminescence value of wells with only untreated target cells, and "D" is the average luminescence value of wells with target cells lysed with Triton-X. The average luminescence value of the culture medium was subtracted from all luminescence values.MabKLH / / MabCD3 did not exhibit T cell-dependent cytotoxic activity against NCI-H2009, but MabITGB6 / / MabCD3 did. The results are shown in Figure 2. This indicates that MabITGB6 has cytotoxic activity in the form of a T cell engager against cancer cells expressing integrin β6.

[0168] [Example 5] Production of anti-integrin β6 CAR-T cells 1. Production of MabITGB6 CAR vector A retroviral vector of a chimeric receptor that recognizes the integrin β6 PSI domain was produced. The pMSGV1 retroviral expression vector (Tamada k et al., Clin Cancer Res 18:6436-6445 (2012)) was used as the retroviral vector skeleton. Figure 3 is a schematic diagram of the vector construct and CAR construct. A CAR construct was constructed comprising, in order from the N-terminus, a signal sequence, the MabITGB6 heavy chain variable region AM1stFL677H1356, the heavy chain CH1 region, a linker sequence, the hinge and transmembrane regions of the human CD8 alpha chain (nucleotide sequences 1301-1507, Genbank NM001768.6), the human 4-1BB costimulatory intracellular domain (nucleotide sequences 901-1026, Genbank NM001561.5), the human CD3 zeta intracellular signaling domain (nucleotide sequences 299-634, Genbank NM000734.3), 2A peptide (T2A) (derived from thosea signa virus), a signal sequence, the MabITGB6 light chain variable region AM1stFL677L0090, and the light chain CLλ region. The gene encoding the MabITGB6 CAR (SEQ ID NO: 21) was synthesized by methods known to the art. This sequence was ligated to pMSGV1, and the MabITGB6 CAR retrovirus vector was constructed.

[0169] 2. Transduction into Human T Cells Using a retroviral transduction method for human T cells, the retroviral vector constructed in Example 5-1 was used to create MabITGB6 CAR-T cells that recognize integrin β6. Specifically, the above-mentioned MabITGB6 CAR vector and p-Ampho plasmid (Takara Bio Inc.) were first transfected into GP2-293 packaging cell lines (Takara Bio Inc.) using lipofectamine 2000 or 3000 (Life Technologies Inc.) to create retroviruses into which the MabITGB6 CAR vector had been introduced. 48 hours after transfection, the supernatant containing the retrovirus was collected and transduction plates were prepared by adsorbing it onto two 24-well plates. Next, for the transduction of human T cells, 2 × 10^6 peripheral blood mononuclear cells per well were cultured for 72 hours in the presence of IL-2 on a 6-well plate immobilized with anti-CD3 monoclonal antibody and retronectin (registered trademark: Takara Bio). After culture, the cells were harvested and cultured overnight in the presence of IL-2 on a transduction plate to which the MabITGB6 CAR retrovirus prepared as described above had been adsorbed. The following day, the cells were transferred to another transduction plate and cultured for another overnight to obtain human T cells (MabITGB6 CAR-expressing T cells) to which anti-integrin β6 CAR had been introduced. Simultaneously, as a CAR-negative cell control, CAR-non-expressing T cells (ActT) were prepared by activating peripheral blood mononuclear cells using the same method but without retrovirus infection.

[0170] 3. Confirmation of CAR Expression Rate Surface expression of MabITGB6 CAR in transduced human T cells produced in Example 5-2 was determined by flow cytometry after the cells were stained with Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Biotin (ThermoFisher). The results are shown in Figure 4. BD FACS Lyric (BD Biosciences) was used as the flow cytometer, and FlowJo software (Tree Star) was used for data analysis. On average, 57% of all T cells expressed MabITGB6 CAR.

[0171] [Example 6] In vitro evaluation of T cell-dependent cytotoxic activity of anti-integrin β6 CAR-T by lactate dehydrogenase (LDH) release assay The cytotoxic activity of MabITGB6 CAR-T and ActT prepared in Example 5 was evaluated by lactate dehydrogenase (LDH) release assay. Human lung cancer cell line NCI-H2009 (ATCC) expressing integrin β6 was used as the target cell. MabITGB6 CAR-T and ActT were used as effector cells. The target cells were detached from the culture flask and seeded in 100 μL portions in each well of a flat-bottom clear 96-well plate (Corning) so that each well contained 1 x 10^4 cells. 100 μL of CAR-T solution at concentrations selected from 3 x 10^2 cells / mL, 1 x 10^3 cells / mL, 3 x 10^3 cells / mL, 1 x 10^4 cells / mL, and 3 x 10^4 cells / mL was added to each well. After incubation at 37°C for 48 hours, the plate was centrifuged, and 50 μL of the supernatant from each well was transferred to a new 96-well opaque-walled, opaque-bottom assay plate (Corning). 50 μL of LDH detection reagent (Cytotoxicity Detection kit PLUS (LDH); Roche) was added to each well, and the plate was incubated at room temperature for 15 minutes. Luminescence was measured using EnVision (PerkinElmer Japan). The cytotoxicity rate (%) was calculated using the following formula: Cytotoxicity (%) = (ABC) x 100 / (DC) where "A" is the average luminescence value of wells treated with CAR-T, "B" is the average luminescence value of wells with CAR-T only, "C" is the average luminescence value of wells with untreated target cells only, and "D" is the average luminescence value of wells with target cells lysed with Triton-X. The average luminescence value of the culture medium was subtracted from all luminescence values. ActT did not show T cell-dependent cytotoxicity against NCI-H2009, but anti-integrin β6 CAR-T showed T cell-dependent cytotoxicity against NCI-H2009. The results are shown in Figure 5.This indicates that MabITGB6 CAR-T has cytotoxic activity in the CAR-T format against cancer cells expressing integrin β6.

[0172] The invention described herein has been described in detail with examples and illustrations for the purpose of aiding clear understanding, but the descriptions and illustrations herein should not be construed as limiting the scope of the invention. All disclosures of patent and scientific documents cited herein are expressly incorporated herein by reference throughout.

[0173] In one embodiment, the antibody that binds to the PSI domain of integrin β6 of the present invention can be used in pharmaceuticals for the treatment of cancer, etc.

Claims

1. An anti-integrin β6 antibody characterized by binding to the PSI domain of integrin β6.

2. The antibody according to claim 1, comprising: (1) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or (2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

8.

3. An anti-integrin β6 antibody that competes with the antibody described in claim 2 for binding to the PSI domain of integrin β6.

4. An anti-integrin β6 antibody for cancer treatment according to any one of claims 1 to 3.

5. CAR cells comprising the anti-integrin β6 antibody according to any one of claims 1 to 3.

6. A method for screening anti-integrin β6 antibodies, comprising the step of selecting an antibody that binds to the PSI domain of integrin β6.

7. A nucleic acid encoding an anti-integrin β6 antibody according to any one of claims 1 to 3.

8. A host cell comprising the nucleic acid described in claim 7.

9. A method for producing an anti-integrin β6 antibody, comprising the step of culturing the host cells described in claim 8.

Images