Anti-αvβ6 antibodies and antibody-drug conjugates

Abstract

Provided are novel anti-αvβ6 antibodies and antibody-drug conjugates and methods of using such anti-αvβ6 antibodies and antibody-drug conjugates to treat cancer. Preferred anti-αvβ6 antibodies comprise the heavy chain CDR sequences of SEQ ID NOs: 31, 32, and 33 and the light chain CDR sequences of SEQ ID NOs: 37, 42, and 39, as determined by Kabat numbering.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefits of U.S. Provisional Application No. 62 / 943,959, filed December 5, 2019, and U.S. Provisional Application No. 63 / 012,584, filed April 20, 2020, the entire contents of which are incorporated herein by reference for all purposes.

[0003] References to sequence lists

[0004] This application includes an electronic sequence list of a file named AVB6-00212_ST25, created on November 16, 2020, and containing 52KB, which is incorporated herein by reference. Technical Field

[0005] This invention relates to novel anti-αvβ6 antibodies and antibody-drug conjugates, and methods of treating cancer using such anti-αvβ6 antibodies and antibody-drug conjugates. Background Technology

[0006] αvβ6, also known as α-vβ-6, is a cell adhesion receptor that binds to extracellular matrix proteins such as fibronectin. αvβ6 is composed of αv and β6 subunits and is upregulated in a variety of cancers, including non-small cell lung cancer (NSCLC).

[0007] Non-splenic lung cancer (NSCLC) is the most common type of lung cancer. In the past year, more than 200,000 people were diagnosed with lung cancer, making it a leading cause of cancer death. Therefore, there is a need to improve treatment options for NSCLC.

[0008] All references cited in this article, including patent applications, patent publications and scientific literature, are incorporated into this article in their entirety by reference as if each individual reference had been explicitly and individually indicated to be incorporated by reference. Summary of the Invention

[0009] This article provides anti-αvβ6 antibodies and αvβ6-directed antibody-drug conjugates (ADCs). It also provides methods for treating diseases expressing αvβ6, including cancer, using anti-αvβ6-directed antibodies and ADCs. Preferred anti-αvβ6 antibodies comprise the heavy chain CDR sequences of SEQ ID NO: 31, 32, and 33 and the light chain CDR sequences of SEQ ID NO: 37, 42, and 39, as identified by the Kabat number. Attached Figure Description

[0010] Figure 1 The results of LAP blocking ELISA assays using various anti-αvβ6 antibodies are shown.

[0011] Figure 2 The results of a saturation binding study of 293F cells expressing human and macaque (cyno) αvβ6 using mouse antibody clone 2A2 (referred to as m2A2) are shown.

[0012] Figure 3 The image shows the alignment of the variable region of the parental mouse mAb (referred to as m2A2) heavy chain with the amino acid sequences of the selected human germline receptor (referred to as hIGHV1-46 / HJ4) and the humanized heavy chain variant. An asterisk indicates a CDR determined by Kabat, and a cross indicates a CDR determined by IMGT.

[0013] Figure 4 The image shows the alignment of the variable region of the parental mouse mAb (referred to as m2A2) with the amino acid sequences of the selected human germline receptor (referred to as hIGKV1D-33 / KJ2) and the humanized light chain variant. An asterisk indicates a CDR determined by Kabat, and a cross indicates a CDR determined by IMGT.

[0014] Figure 5 The results show the competitive binding of humanized antibodies with LA and LB light chains and parental mouse and chimeric antibodies (named m2A2 and c2A2, respectively) on 293F cells expressing human αvβ6.

[0015] Figure 6 The results show the competitive binding of humanized antibodies with HA and HC heavy chains and parental mouse and chimeric antibodies (named m2A2 and c2A2, respectively) on 293F cells expressing human αvβ6.

[0016] Figure 7 This study demonstrates the replication results of a competitive binding study on 293F cells expressing human αvβ6 using a subset of humanized antibodies and a parental mouse antibody (referred to as m2A2).

[0017] Figure 8 The results show the saturation binding of 293F cells expressing human and macaque αvβ6 with the h2A2 HCLG humanized antibody and the parental mouse antibody (referred to as m2A2).

[0018] Figure 9 The results show the competitive binding of humanized HCLG antibody and ADC on 293F cells expressing human and macaque αvβ6.

[0019] Figure 10 The results of an ELISA study on the specific binding of human αvβ6 to αvβ1, αvβ3, αvβ5, αvβ6 and αvβ8 using the h2A2 HCLG humanized antibody are shown.

[0020] Figure 11The results showed that h2A2 HCLG anti-αvβ6 vcMMAE ADC exhibited in vitro cytotoxicity against cancer cell lines expressing αvβ6.

[0021] Figure 12 The results of a xenograft study of the Detroit 562 head and neck cancer cell line in nude mice are shown. The dosage and regimen are indicated on the figure.

[0022] Figure 13 The results of a xenograft study of the HPAFII pancreatic cancer cell line in nude mice are shown. The dosage and regimen are indicated on the figure.

[0023] Figure 14 The results of a xenograft study of the BxPC-3 pancreatic cancer cell line in nude mice are shown. The dosage and regimen are indicated on the figure.

[0024] Figure 15 The results of a xenograft study of the SW780 bladder cancer cell line in nude mice are shown. The dosage and regimen are indicated on the figure.

[0025] Figure 16 Results of a PDX study in nude mice using six NSCLC cell lines are shown. ADC was administered at a dose of 3 mg / kg q7dx3.

[0026] Figure 17 Results of a PDX study in nude mice were presented for six ovarian cancer cell lines. ADC was administered at a dose of 5 mg / kg q7dx3.

[0027] Figure 18 The results comparing h2A2 HCLG and h15H3 in two cell line xenograft models are shown. ADC was administered once at a dose of 3 mg / kg. Invention Details

[0029] I. Definition

[0030] To better understand this invention, certain terms are first defined. As used in this application, unless otherwise described herein, each of the following terms should have the following meanings. Additional definitions are described throughout this application.

[0031] The term “and / or” as used herein should be considered as specifically disclosing each of two features or components, with or without the other. Therefore, the term “and / or” as used in phrases such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0032] It should be understood that the aspects and embodiments of the invention described herein include aspects and embodiments described as “comprising,” “forming,” and “consistently consisting of.”

[0033] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art to which this disclosure pertains. For example, the *Concise Dictionary of Biomedicine and Molecular Biology*, Juo, Pei-Show, 2nd edition, 2002, CRC Press; the *Dictionary of Cell and Molecular Biology*, 3rd edition, 1999, Academic Press; and the *Oxford Dictionary of Biochemistry and Molecular Biology*, revised, 2000, Oxford University Press, provide those skilled in the art with common dictionaries of many of the terms used in this disclosure.

[0034] Units, prefixes, and symbols are represented in their accepted form in the International System of Units (SI). Numerical ranges include the numerical values ​​that define that range. The headings provided herein are not intended to limit various aspects of this disclosure, and all aspects or embodiments of this disclosure can be understood in their entirety with reference to the specification. Therefore, the terms defined immediately thereafter are defined entirely with reference to the full text of the specification.

[0035] The terms “αvβ6”, “avb6”, “α-vβ-6”, or “β6” are used interchangeably in this document unless otherwise stated, and include any variant, isotype, and species homologue of human αvβ6, which are generally expressed by cells or on cells transfected with the αvβ6 gene.

[0036] The term "immunoglobulin" refers to a class of structure-related glycoproteins composed of two pairs of polypeptide chains: a pair of light (L) low molecular weight chains and a pair of heavy (H) chains, all four chains being interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Chapter 7 of *Fundamental Immunology* (Paul, W., ed., 2nd ed., RavenPress, New York (1989)). In short, each heavy chain typically contains a heavy chain variable region (abbreviated as V in this text). H or VH) and heavy chain constant region (C H Or CH). Heavy chain constant regions typically contain three domains C. H 1. C H 2 and C H 3. Heavy chains are typically interconnected by disulfide bonds in so-called "hinge regions." Each light chain usually contains a light chain variable region (abbreviated as V in this article). L or VL) and light chain constant region (C L Or CL). The constant region of a light chain typically contains a domain C. L CL can be either κ (kappa) or λ (lambda) isotypes. The terms "constant domain" and "constant region" are used interchangeably herein. Immunoglobulins can be derived from any conventionally known isotype, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art, including but not limited to human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to an antibody class or subclass (such as IgM or IgG1) encoded by a heavy chain constant region gene.

[0037] The term "variable region" or "variable domain" refers to the heavy or light chain domain of an antibody involved in antibody-antigen binding. The variable regions of the heavy and light chains of natural antibodies (V1 and V2, respectively) H and V LThe variable region can be further subdivided into regions of hypervariability (or hypervariable regions, which can be highly variable in the sequence and / or in the loop form defined by the structure), also known as complementarity-determining regions (CDRs), interspersed with more conserved regions called frame regions (FRs). The terms “complementarity-determining region” and “CDR”, synonyms with “hypervariable region” or “HVR”, are known in the art to refer to the discontinuous sequence of amino acids within the antibody variable region that confers antigen specificity and / or binding affinity. Typically, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, CDR-H3), and each light chain variable region has three CDRs (CDR-L1, CDR-L2, CDR-L3). The terms “frame region” and “FR” are known in the art to refer to the non-CDR portions of the heavy and light chain variable regions. Typically, each full-length heavy chain variable region contains four FRs (FR-H1, FR-H2, FR-H3, and FR-H4), and each full-length light chain variable region contains four FRs (FR-L1, FR-L2, FR-L3, and FR-L4). Each V H and V L Inside, the three CDRs and four FRs are usually arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mot. Biol., 195, 901-917 (1987)).

[0038] In the context of this invention, the term "antibody" (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative thereof, which has the ability to specifically bind to an antigen under typical physiological conditions and has a long half-life, such as at least about 30 minutes, at least about 45 minutes, at least about 1 hour (h), at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours (h), about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, or any other related functionally defined time period (such as the time sufficient to induce, promote, enhance, and / or modulate the physiological response associated with antibody binding to an antigen and / or the time sufficient to recruit effector activity). The variable regions of the heavy and light chains of an immunoglobulin molecule contain binding domains that interact with the antigen. The constant regions of an antibody (Ab) can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system (such as C1q), which is the first component in the classical pathway of complement activation. Antibodies can be bispecific antibodies, biantibodies, multispecific antibodies, or similar molecules.

[0039] As used herein, the term "monoclonal antibody" refers to an antibody molecule produced by recombination with a single primary amino acid sequence, fused from mouse B cells. Monoclonal antibody compositions exhibit single binding specificity and affinity for a specific epitope. Therefore, the term "human monoclonal antibody" refers to an antibody exhibiting single binding specificity, having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced via hybridomas comprising B cells obtained from transgenic or transchromosomal nonhuman animals (such as transgenic mice) with genomes containing human heavy-chain and light-chain transgenes, fused with immortalized cells.

[0040] "Isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., isolated antibodies that specifically bind to αvβ6 are substantially free of antibodies that specifically bind to antigens other than αvβ6). However, isolated antibodies that specifically bind to αvβ6 may have cross-reactivity with other antigens (e.g., αvβ6 molecules from different species). Additionally, isolated antibodies may be substantially free of other cellular material and / or chemicals. In one embodiment, the isolated antibody comprises an antibody conjugate attached to another reagent (e.g., a small molecule drug). In some embodiments, the isolated anti-αvβ6 antibody comprises a conjugate of an anti-αvβ6 antibody and a small molecule drug (e.g., MMAE or MMAF).

[0041] "Human antibody" (HuMAb) refers to an antibody in which both the FR and CDR in the variable region are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, that constant region is also derived from a human germline immunoglobulin sequence. Human antibodies disclosed herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which a germline CDR sequence derived from another mammalian species (such as a mouse) has been grafted onto a human frame sequence. The terms "human antibody" and "fully human antibody" are used synonymously.

[0042] As used herein, the term "humanized antibody" refers to a genetically engineered nonhuman antibody containing a human antibody constant domain and a nonhuman variable domain modified to contain a high level of sequence homology with the human variable domain. This can be achieved by transplanting six nonhuman antibody complementarity-determining regions (CDRs) (which together form the antigen-binding site) onto a homologous human receptor frame region (FR) (see WO92 / 22653 and EP0629240). To fully reconstruct the binding affinity and specificity of the parent antibody, it may be necessary to replace the frame residues from the parent antibody (i.e., the nonhuman antibody) with the human frame region (reversion mutation). Structural homology modeling can help identify amino acid residues in the frame region that are important for the antibody's binding properties. Therefore, a humanized antibody may contain a nonhuman CDR sequence, primarily a human frame region, optionally containing one or more amino acid reversion mutations to a nonhuman amino acid sequence, and a fully human constant region. Optionally, other amino acid modifications (not necessarily reversion mutations) may be applied to obtain humanized antibodies with preferred properties (such as affinity and biochemical properties).

[0043] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region is derived from a non-human species (e.g., rodent-derived) and the constant region is derived from a different species, such as humans. Chimeric antibodies can be produced through antibody engineering. "Antibody engineering" is a general term for modifications of different types of antibodies and is a method well known to those skilled in the art. Specifically, chimeric antibodies can be generated using standard DNA techniques as described in Sambrook et al., 1989, *Molecular Cloning: A Laboratory Manual*, New York: Cold Spring Harbor Laboratory Press, Chapter 15. Thus, chimeric antibodies can be recombinant antibodies that have been genetically or enzymatically engineered. The generation of chimeric antibodies is within the knowledge of those skilled in the art, and therefore, chimeric antibodies according to the invention can be generated by methods other than those described herein. Chimeric monoclonal antibodies have been developed for therapeutic applications to reduce antibody immunogenicity. They typically contain a non-human (e.g., mouse) variable region specific to the antigen of interest, and human constant antibody heavy and light chain domains. In the context of chimeric antibodies, the term "variable region" or "variable domain" refers to the region containing both the CDR and framework regions of immunoglobulin heavy and light chains.

[0044] "Antigen antibody" refers to an antibody that binds to an antigen. For example, anti-αvβ6 antibody is an antibody that binds to the antigen αvβ6.

[0045] An antibody's "antigen-binding portion" or "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to antigens bound by the intact antibody. Examples of antibody fragments (such as antigen-binding fragments) include, but are not limited to, Fv, Fab, Fab′, Fab′-SH, F(ab′)2; biantibodies; linear antibodies; single-chain antibody molecules (such as scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, each with one antigen-binding site, and a residual "Fc" fragment, the name reflecting its tendency to crystallize. Pepsin treatment produces the F(ab′)2 fragment, which has two antigen-binding sites and is still able to cross-link antigens.

[0046] The "percentage of sequence identity (%)" relative to a reference peptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the reference peptide sequence after alignment and cleavage (if necessary) to achieve the maximum percentage of sequence identity, without considering any conservation substitutions as partial sequence identity. Various well-known methods exist for determining the percentage of amino acid sequence identity through alignment, such as publicly available computer software like BLAST, BLAST-2, SnapGene Align, or ClustalW BioEdit. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithms required to achieve full-length maximum alignment of the compared sequences. For example, the percentage of sequence identity of a given amino acid sequence A with, and or for a given amino acid sequence B (which can also be expressed alternatively as a given amino acid sequence A having or containing a specific percentage of sequence identity with, and or for a given amino acid sequence B) is calculated as follows:

[0047] 100 times the fraction X / Y

[0048] Where X is the number of amino acid residues that are sequence-identically matched in the alignment of A and B in this procedure, and Y is the total number of amino acid residues in B. It should be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the sequence identity percentage of A to B will not be equal to the sequence identity percentage of B to A.

[0049] In the context of antibody-antigen binding, the terms “binding,” “conjugation,” or “specific binding” as used herein generally refer to binding that is determined by, for example, biolayer interference measurement (BLI) techniques in an Octet HTX instrument using an antibody as a ligand and an antigen as an analyte, where the affinity of such binding corresponds to approximately 10. -6 M or smaller, for example, 10 -7 M or smaller, such as about 10 -8 M or smaller, such as about 10-9 M or smaller, approximately 10 -10 M or smaller, or about 10 -11 M or smaller K D Furthermore, the affinity of the antibody for binding to the predetermined antigen corresponds to such a K. D The K D The K-type antigen that binds to the antibody against non-specific antigens other than the predetermined antigen or closely related antigens (e.g., BSA, casein). D At least ten times lower, such as at least 100 times lower, such as at least 1,000 times lower, such as at least 10,000 times lower, such as at least 100,000 times lower. Combined with K D The lower amount depends on the antibody's K D Therefore, when the antibody's K D At very low levels, K binds to the antigen. D Below the K level that binds to non-specific antigens D The amount can be at least 10,000 times (meaning the antibody is highly specific).

[0050] The term "K" used in this article D "(M)" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. As used in this paper, affinity and K D Inversely proportional, that is, higher affinity is intended to represent lower K. D Lower affinity is intended to represent higher K. D .

[0051] The term "ADC" refers to antibody-drug conjugate, which in the context of this invention refers to an anti-αvβ6 antibody conjugated to a pharmaceutical moiety (e.g., MMAE or MMAF) as described in this application.

[0052] The abbreviations "vc" and "val-cit" refer to dipeptide valine-citrulline.

[0053] The abbreviation VKG refers to the tripeptide linker valine-lysine-glycine.

[0054] The abbreviation "PAB" refers to a self-immolative spacer.

[0055]

[0056] The abbreviation "MC" refers to the stretcher maleimide hexanoyl group:

[0057]

[0058] The abbreviation "MP" refers to the stretcher maleimide propionyl group:

[0059]

[0060] As used herein, a “PEG unit” is an organic moiety consisting of repeating ethylene-oxygen subunits (PEG or PEG subunits), which can be polydisperse, monodisperse, or discrete (i.e., having a discrete number of ethylene-oxygen subunits). Polydisperse PEG is a heterogeneous mixture of sizes and molecular weights, while monodisperse PEG is typically purified from a heterogeneous mixture, thus providing a single chain length and molecular weight. Preferred PEG units include discrete PEG, i.e., compounds synthesized stepwise rather than through a polymerization process. Discrete PEG provides a single molecule with a defined and specified chain length.

[0061] The PEG units provided herein comprise one or more polyethylene glycol chains, each consisting of one or more ethylene oxide subunits covalently attached to each other. The polyethylene glycol chains may be linked together, for example, in a linear, branched, or star configuration. Typically, prior to incorporation of the camptothecin conjugate, at least one end of a polyethylene glycol chain is derivatized with an electrophilic alkyl portion to covalently attach to the urethane nitrogen of the methylene carbamate unit (i.e., an example representing R). Typically, the terminal ethylene oxide subunits in each polyethylene glycol chain that do not participate in covalent attachment to the remainder of the linker unit are modified with a PEG end-capping unit, typically an optionally substituted alkyl group, such as -CH3, CH2CH3, or CH2CH2CO2H. Preferred PEG units have a single polyethylene glycol chain in which 2-24 -CH2CH2O- subunits are covalently attached in tandem and terminated at one end with a PEG end-capping unit.

[0062] "Cancer" refers to a large group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. "Cancer" or "cancer tissue" can include tumors. Uncontrolled cell division and growth lead to the formation of malignant tumors, which invade adjacent tissues and can also metastasize to distant parts of the body via the lymphatic system or bloodstream. After metastasis, the distant tumor can be said to "originate" from the pre-metastatic tumor.

[0063] The term "antibody-dependent cytotoxicity," or ADCC, is a mechanism that induces cell death through the interaction of antibody-coated target cells with lytically active immune cells (also known as effector cells). These effector cells include natural killer cells, monocytes / macrophages, and neutrophils. Effector cells attach to one or more Fc effector domains of Ig bound to the target cell via their antigen-binding sites. Due to the activity of the effector cells, the antibody-coated target cells die.

[0064] The term "antibody-dependent phagocytosis," or ADCP, refers to the process by which antibody-coated cells are internalized, in whole or in part, by phagocytic immune cells (such as macrophages, neutrophils, and dendritic cells) that bind to one or more of the Fc effector domains of Ig.

[0065] The term "complement-dependent cytotoxicity," or CDC, refers to a mechanism that induces cell death in which the Fc effector domain of an antibody binding to a target activates a series of enzymatic reactions that ultimately create pores on the target cell membrane. Typically, antigen-antibody complexes, such as antibody-coated complexes on target cells, bind to and activate the complement component Clq, which in turn activates the complement cascade, leading to target cell death. Activation of complement can also result in the deposition of complement components on the target cell surface, promoting ADCC by binding to complement receptors (such as CR3) on leukocytes.

[0066] "Cellular inhibition effect" refers to the inhibition of cell proliferation. "Cellular inhibitors" are agents that inhibit cell growth, thereby suppressing the growth and / or expansion of specific cell subpopulations. Cellular inhibitors can be conjugated to antibodies or administered in combination with antibodies.

[0067] The term "treatment" or "therapy" for a subject refers to any type of intervention or process performed on the subject, or the administration of an active agent to the subject, with the aim of reversing, alleviating, improving, suppressing, slowing, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, signs, or biochemical indicators associated with a disease. In some embodiments, the disease is cancer.

[0068] "Object" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In some embodiments, the object is a human. The terms "object," "patient," and "individual" are used interchangeably herein.

[0069] An "effective amount," "therapeutic effective amount," or "therapeutic dose" of medicine or therapeutic agent is any amount of medicine that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of disease or promotes disease remission, as demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or prevention of impairment or disability caused by the suffering caused by the disease. The ability of a therapeutic agent to promote disease remission can be evaluated using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems predicting efficacy in humans, or by determining the activity of the agent in in vitro studies.

[0070] For example, in the treatment of tumors, a therapeutically effective amount of the anticancer agent inhibits at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 98%, or at least about 99% of cell or tumor growth in the treated subject (e.g., one or more treated subjects) relative to an untreated subject (e.g., one or more untreated subjects). In some embodiments, a therapeutically effective amount of the anticancer agent inhibits 100% of cell or tumor growth in the treated subject (e.g., one or more treated subjects) relative to an untreated subject (e.g., one or more untreated subjects).

[0071] In other embodiments of this disclosure, tumor regression can be observed and last for at least about 20 days, at least about 30 days, at least about 40 days, at least about 50 days, or at least about 60 days.

[0072] Therapeutic effective amounts of a drug (e.g., anti-αvβ6 antibody-drug conjugates) include "preventive effective amounts," which are any amounts of the drug, when administered alone or in combination with an anticancer agent to a subject at risk of developing cancer (such as a subject with a pre-malignant condition) or suffering from cancer recurrence, that inhibit the development or recurrence of cancer. In some embodiments, preventive effective amounts completely prevent the development or recurrence of cancer. "Inhibiting" the development or recurrence of cancer means reducing the likelihood of cancer development or recurrence, or completely preventing the development or recurrence of cancer.

[0073] As used herein, “subtherapeutic dose” refers to a dose of a therapeutic compound that is below the usual or typical dose of a therapeutic compound (e.g., an anti-αvβ6 antibody-drug conjugate) when administered alone to treat a hyperproliferative disease (such as cancer).

[0074] "Immune-associated remission" refers to a clinical remission pattern frequently observed in cancer patients treated with immunotherapy, which exerts its anti-tumor effect by inducing a cancer-specific immune response or by modifying innate immune processes. This remission pattern is characterized by a beneficial therapeutic effect following an initial increase in tumor burden or the appearance of new lesions. When evaluating conventional chemotherapy, this would be classified as disease progression and synonymous with drug failure. Therefore, proper evaluation of immunotherapy may require long-term monitoring of the effects of these agents on the target disease.

[0075] For example, an "anticancer agent" promotes cancer regression in a subject. In some implementations, a therapeutically effective amount of the drug promotes cancer regression to the point of cancer elimination. "Promoting cancer regression" means that administering an effective amount of the drug, alone or in combination with an anticancer agent, results in a reduction in tumor growth or size, tumor necrosis, a decrease in the severity of at least one disease symptom, an increase in the frequency and duration of symptom-free periods, or prevention of impairment or disability caused by the suffering caused by the disease. Furthermore, the terms "effective" and "efficacy" in relation to treatment include both pharmacological efficacy and physiological safety. Pharmacological efficacy refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to toxicity or other adverse physiological reactions (adverse reactions) at the cellular, organ, and / or biological level caused by administration.

[0076] "Sustained remission" refers to a sustained effect in reducing tumor growth after treatment has been discontinued. For example, the tumor size may remain the same or smaller compared to its size at the start of the administration phase. In some implementations, the duration of sustained remission is at least the same as the duration of treatment, or at least 1.5, 2.0, 2.5, or 3 times longer than the duration of treatment.

[0077] As used in this article, “complete remission” or “CR” means that all target lesions have disappeared; “partial remission” or “PR” means that the sum of the longest diameters (SLD) of the target lesions has decreased by at least 30% compared to the baseline SLD; “stable disease” or “SD” means that the target lesions have neither shrunk sufficiently to meet the PR criteria nor increased sufficiently to meet the PD criteria compared to the smallest SLD since the start of treatment.

[0078] As used in this article, “progression-free survival” or “PFS” refers to the length of time during and after treatment during which the treated disease (such as cancer) does not worsen. Progression-free survival can include the time a patient experiences complete or partial remission, as well as the time a patient experiences stable disease.

[0079] As used in this article, "overall response rate" or "ORR" refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

[0080] As used in this article, “overall survival” or “OS” refers to the percentage of individuals in a group who are likely to survive after a specific period of time.

[0081] The phrase “pharmaceutically acceptable” means that a substance or composition must be chemically and / or toxicologically compatible with other ingredients contained in the formulation and / or with the mammals to which it is treated.

[0082] The phrase "pharmaceutically acceptable salt" as used herein refers to a pharmaceutically acceptable organic or inorganic salt of the compounds of this invention. Exemplary salts include, but are not limited to: sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbic acid salts, succinates, maleates, gentianates, fumarates, gluconates, glucurons, sucrose salts, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, and pyrantel salts (i.e., 4,4'-methylene-bis(2- Pharmaceutically acceptable salts include hydroxy-3-naphthyl carboxylate salts, alkali metal (such as sodium and potassium) salts, alkaline earth metal (such as magnesium) salts, and ammonium salts. Pharmaceutically acceptable salts may involve another molecule, such as an acetate ion, a succinate ion, or other counterions. This counterion can be any organic or inorganic moiety capable of stabilizing the charge on the parent compound. Furthermore, pharmaceutically acceptable salts may have more than one charged atom in their structure. In the case of pharmaceutically acceptable salts containing multiple charged atoms, multiple counterions may be present. Therefore, pharmaceutically acceptable salts may have one or more charged atoms and / or one or more counterions.

[0083] "Administration" or "giving" means the physical introduction of a therapeutic agent into a subject using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for anti-αvβ6 antibody-drug conjugates include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes, such as by injection or infusion (e.g., intravenous infusion). The phrase "parenteral administration" as used herein refers to a form of administration other than enteric and local administration, typically by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraocular, intravascular, intralesional, intraocular, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as intracorporeal electroporation. Therapeutic agents can be administered via non-parenteral routes or orally. Other non-parenteral routes include local, epidermal, or mucosal routes of administration, such as intranasal, vaginal, rectal, sublingual, or topical administration. It can also be administered, for example, once, multiple times, and / or over one or more extended periods.

[0084] The terms “baseline” or “baseline value” used interchangeably in this document can refer to a measurement or characterization of symptoms prior to or at the start of treatment (such as the anti-αvβ6 antibody-drug conjugate described herein). Baseline values ​​can be compared to reference values ​​to determine the reduction or improvement of symptoms for αvβ6-related diseases (e.g., cancer) considered herein. The terms “reference” or “reference value” used interchangeably in this document can refer to an assessment or characterization of symptoms following treatment (such as the anti-αvβ6 antibody-drug conjugate described herein). Reference values ​​can be measured once or multiple times during a dosing regimen or treatment cycle or at the completion of a dosing regimen or treatment cycle. A “reference value” can be an absolute value; a relative value; a value with an upper and / or lower limit; a series of values; an average; a median; a mean; or a value compared to a baseline value.

[0085] Similarly, a “baseline value” can be an absolute value; a relative value; a value with an upper and / or lower limit; a series of values; an average; a median; a mean; or a value compared to a reference value. Reference values ​​and / or baseline values ​​can be obtained from one individual, two different individuals, or a group of individuals (such as a group of two, three, four, five, or more individuals).

[0086] As used in this article, "monotherapy" refers to the use of an anti-αvβ6 antibody-drug conjugate as the sole anticancer agent administered to the subject during a treatment cycle. However, other therapeutic agents may also be administered to the subject. For example, an anti-inflammatory agent or other medication may be given to a subject with cancer during monotherapy to treat cancer-related symptoms rather than the underlying cancer itself, including, for example, inflammation, pain, weight loss, and general malaise.

[0087] As used in this article, “adverse events” (AEs) are any unfavorable and often unexpected or undesirable indication (including abnormal laboratory findings), symptom, or illness associated with the use of a drug treatment. A drug treatment may have one or more associated AEs, and each AE may have the same or different levels of severity. The mention of methods capable of “modifying adverse events” refers to treatment regimens that reduce the incidence and / or severity of one or more AEs associated with the use of different treatment regimens.

[0088] The term "serious adverse event" or "SAE" as used in this article refers to an adverse event that meets one of the following criteria:

[0089] • Fatal or life-threatening (The term “life-threatening” as used in the definition of serious adverse events refers to an event in which the patient is at risk of death at the time of the event; it does not refer to an event that, if it were more serious, would presumably lead to death.)

[0090] • Causes persistent or significant disability / incapacity

[0091] • Constitutes congenital abnormalities / birth defects

[0092] • It is medically serious, defined as an event that endangers the patient or may require medical or surgical intervention to prevent one of the aforementioned outcomes. Medical and scientific judgment is required in determining whether an AE is "medically serious."

[0093] • Hospitalization or extension of existing hospitalization is required, except in the following circumstances: 1) routine treatment or monitoring of an underlying disease that is not associated with any worsening of the condition; 2) selective or pre-planned treatment of an existing condition that is not related to the indication being studied and has not worsened since the informed consent was signed; and 3) social reasons and suspension of care in the absence of any deterioration in the patient’s overall condition.

[0094] The use of alternatives (e.g., “or”) should be understood to mean one, both, or any combination of the alternatives. As used herein, the indefinite article “a” or “an” should be understood to mean “one or more” of any cited or enumerated components.

[0095] The terms “about” or “substantially comprise” refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending on the method of measurement or determination of that value or composition, i.e., the limits of the measurement system. For example, “about” or “substantially comprise” may, in practice in the art, mean within or greater than one standard deviation. Alternatively, “about” or “substantially comprise” may mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, these terms may mean up to an order of magnitude or up to five times a value. When a particular value or composition is provided in this application and claims, unless otherwise stated, it should be assumed that the meaning of “about” or “substantially comprise” is within an acceptable margin of error for that particular value or composition.

[0096] The word "about" when referring to a value or parameter includes (and discloses) the implementation relating to that value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0097] As stated herein, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range, and, where appropriate, fractional values ​​(such as one-tenth and one-hundredth of an integer), unless otherwise stated.

[0098] The various aspects of this disclosure are described in further detail in the following sections.

[0099] II. Overview

[0100] This invention provides antibodies that specifically bind to αvβ6. This invention is partly based on the discovery that antibody-drug conjugates targeting αvβ6, including the vcMMAE antibody-drug conjugate, are particularly effective in killing cells expressing αvβ6+. αvβ6 has been shown to be expressed in a variety of cancers, including non-small cell lung cancer (NSCLC) (squamous and adenocarcinoma), head and neck cancer (including head and neck squamous cell carcinoma), esophageal cancer, breast cancer (including invasive breast cancer), ovarian cancer, bladder cancer (including urothelial carcinoma), skin cancer (squamous cell carcinoma or SCC), kidney cancer (including clear cell, papillary, and chromophobe cells), cervical cancer, gastric cancer, prostate cancer (including prostate adenocarcinoma), endometrial cancer (including uterine sarcoma and endometrial cancer), rectal adenocarcinoma, thyroid cancer, colonic adenocarcinoma, gastric adenocarcinoma, and pancreatic cancer (including pancreatic adenocarcinoma).

[0101] III. Target Molecules

[0102] Unless otherwise stated, αvβ6 refers to human αvβ6. An exemplary β6 human sequence is the specified GenBank accession number AAA36122. An exemplary αv human sequence is the specified NCBI NP_002201.1.

[0103] IV. The antibody of the present invention

[0104] This invention provides mouse 2A2 antibodies, as well as chimeric, humanized, and human 2A2 antibodies.

[0105] The antibodies of the present invention (such as chimeric, humanized, and human forms of mouse 2A2 antibodies) preferably have an affinity for human αvβ6 comparable to, greater than, that of mouse 2A2 antibodies for human αvβ6, or within 10-fold, 5-fold, or 2-fold weaker than that of mouse 2A2 antibodies for human αvβ6. One method for measuring the affinity of an antibody for its target antigen is by determining the apparent dissociation constant of the antibody. The present invention includes antibodies (such as chimeric, humanized, and human forms of mouse 2A2 antibodies) having an apparent dissociation constant substantially the same as that of mouse 2A2 (i.e., within the experimental error range), and antibodies having a dissociation constant lower or higher than that of mouse 2A2 antibodies for human αvβ6. Like mouse 2A2 antibodies, chimeric, humanized, and human 2A2 antibodies specifically bind to human αvβ6 in its natural form and / or recombinantly expressed from CHO cells. Typically, chimeric, humanized, and human 2A2 anti-αvβ6 antibodies compete with mouse 2A2 for binding to human αvβ6.

[0106] The preferred antibodies of this invention inhibit cancer (such as cell growth, metastasis, and / or lethality to the organism), as demonstrated by cancer cells proliferating in cultures, animal models, or clinical trials. Animal models can be established by implanting human tumor cell lines expressing αvβ6 into appropriate immunodeficient rodent strains, such as athymic nude mice or SCID mice. These tumor cell lines can be established as solid tumors in immunodeficient rodent hosts, either subcutaneously or intravenously, as disseminated tumors. Once established in a host, these tumor models can be used to evaluate the therapeutic effects of the anti-αvβ6 antibodies or their conjugate forms described in the examples.

[0107] Typically, the anti-αvβ6 antibodies and / or anti-αvβ6 antibody-drug conjugates of this disclosure bind to αvβ6 (e.g., human αvβ6) and exert cytotoxic and inhibitory effects on malignant cells such as cancer cells. The anti-αvβ6 antibodies of this disclosure are preferably monoclonal and can be multispecific, human, humanized, or chimeric antibodies, single-chain antibodies, Fab fragments, F(ab') fragments, fragments generated from Fab expression libraries, and any of the above-mentioned αvβ6-binding fragments. In some embodiments, the anti-αvβ6 antibodies of this disclosure specifically bind to αvβ6. The immunoglobulin molecules of this disclosure can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0108] In some embodiments of this disclosure, the anti-αvβ6 antibody is an antigen-binding fragment described herein (e.g., a human antigen-binding fragment), including but not limited to: Fab, Fab' and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibody, disulfide-linked Fv (sdFv), and V-containing antibodies. L Fragments of the VH domain or a single-chain antibody may be used. Antigen-binding fragments, including single-chain antibodies, may individually contain one or more variable regions, or may contain variable regions in combination with all or some of the following: hinge region, CH1, CH2, CH3, and CL domains. This disclosure also includes antigen-binding fragments comprising one or more variable regions in any combination of hinge region, CH1, CH2, CH3, and CL domains. In some embodiments, the anti-αvβ6 antibody or its antigen-binding fragment is human, mouse (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken.

[0109] The anti-αvβ6 antibody disclosed herein can be monospecific, bispecific, trispecific, or even more multispecific. Multispecific antibodies can be specific to different epitopes of αvβ6, or specific to both αvβ6 and heterologous proteins.

[0110] The anti-αvβ6 antibodies disclosed herein can be described or specified according to the specific CDRs they contain. The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of many known numbering schemes, including Kabat et al., (1991), Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, NIH, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol. 262, 732-745 (“Contact” numbering scheme); Lefranc MP et al., “Unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains”, Dev Comp Immunol, 2003 Jan; 27(1): 55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yetanother numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool”, J Mol Biol, 2001 Jun 8; 309(3): 657-70 (“Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm”, PNAS, 1989, 86(23): 9268-9272 (“AbM” numbering scheme). The boundaries of a given CDR can vary depending on the scheme used for identification.In some implementations, a “CDR” or “complementarity-determining region” or a separately designated CDR (e.g., CDR-H1, CDR-H2, CDR-H3) of a given antibody or its region (e.g., its variable region) should be understood to encompass (or specific) CDRs defined by any of the above schemes. For example, when describing a particular CDR (such as CDR-H3), it may include a given V. H or V L When determining the amino acid sequence of the corresponding CDR within the region's amino acid sequence, it should be understood that the CDR has the sequence of the corresponding CDR within the variable region (e.g., CDR-H3), as defined by any of the above-described schemes. Schemes for identifying one or more specific CDRs can be specified, such as those defined by the Kabat, Chothia, AbM, or IMGT methods.

[0111] In one embodiment, the CDR sequences of the anti-αvβ6 antibody and the anti-αvβ6 antibody-drug conjugate described herein are based on the Kabat numbering scheme. In another embodiment, the CDR sequences of the anti-αvβ6 antibody and the anti-αvβ6 antibody-drug conjugate described herein are based on the IMGT numbering scheme.

[0112] On one hand, this document provides an anti-αvβ6 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33; and / or wherein the light chain variable region comprises: (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 37, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 42, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 39; wherein the CDRs of the anti-αvβ6 antibody are defined by the Kabat numbering scheme. In other embodiments, CDR-L1 comprises the amino acid sequence of SEQ ID NO: 40. In other embodiments, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 38 or 41.

[0113] On the one hand, this document provides an anti-αvβ6 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; and / or wherein the light chain variable region comprises: (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 43, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 45; wherein the CDRs of the anti-αvβ6 antibody are defined by the IMGT numbering scheme.

[0114] The anti-αvβ6 antibodies described herein may contain any suitable frame variable domain sequence, as long as the antibody retains its ability to bind to αvβ6 (such as human αvβ6). In some embodiments of the anti-αvβ6 antibodies described herein, the heavy chain and light chain variable domains contain the amino acid sequences of SEQ ID NO: 6 and SEQ ID NO: 17, respectively. In some embodiments of the anti-αvβ6 antibodies described herein, the heavy and light chains contain the amino acid sequences of SEQ ID NO: 21 and SEQ ID NO: 29, respectively.

[0115] In some embodiments, this document provides anti-αvβ6 antibodies and / or anti-αvβ6 antibody-drug conjugates comprising a heavy chain variable domain containing an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the heavy chain variable domain containing an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 6 contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence and retains the ability to bind to αvβ6 (e.g., human αvβ6). In some embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 6 are substituted, inserted, and / or deleted. In other embodiments, a total of 3 to 10 amino acids in SEQ ID NO: 6 are substituted, inserted, and / or deleted. In some embodiments, the substitution, insertion, or deletion (e.g., 1, 2, 3, 4, or 5 amino acids) occurs within the CDR. In some embodiments, the substitution, insertion, or deletion (e.g., 1, 2, 3, 4, or 5 amino acids) occurs outside the CDR (i.e., in the FR). In some embodiments, the anti-αvβ6 antibody comprises a post-translational modified heavy chain variable domain sequence of SEQ ID NO: 6 containing the sequence.

[0116] In some embodiments, this document provides anti-αvβ6 antibodies and / or anti-αvβ6 antibody-drug conjugates comprising a light chain variable domain containing an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 17. In some embodiments, the light chain variable domain containing an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 17 contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence and retains the ability to bind to αvβ6 (e.g., human αvβ6). In some embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 17 are substituted, inserted, and / or deleted. In other embodiments, a total of 1 to 2 amino acids in SEQ ID NO: 17 are substituted, inserted, and / or deleted. In some embodiments, the substitution, insertion, or deletion (e.g., 1, 2, 3, 4, or 5 amino acids) occurs in a region outside the CDR (i.e., in the FR). In some embodiments, the anti-αvβ6 antibody comprises a light chain variable domain sequence of SEQ ID NO: 17 containing a post-translational modification of the sequence.

[0117] There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each with a heavy chain denoted as α, δ, ε, γ, and μ, respectively. The γ and α classes are further subdivided into subclasses, for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. IgG1 antibodies can exist in a variety of polymorphic variants called allotypes (reviewed in Jefferis and Lefranc 2009 mAbs Vol 1 Issue 41-7), any of which is suitable for use in some embodiments herein. Common allotype variants in the population are variants indicated by the letters a, f, n, z, or combinations thereof. In any embodiment herein, the antibody may include a heavy chain Fc region containing the human IgG Fc region. In other embodiments, the human IgG Fc region contains human IgG1.

[0118] The antibodies of this invention also include modified derivatives, i.e., modified by covalent attachment of any type of molecule to the antibody, such that the covalent attachment does not prevent the antibody from binding to αvβ6 or exerting cytotoxic or inhibitory effects on HD cells. For example, but not limited to, antibody derivatives include already modified antibodies, such as those derived by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or linkage with cellular ligands or other proteins. Any of a variety of chemical modifications can be performed using known techniques, including but not limited to: specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin. Furthermore, the derivatives may contain one or more non-classical amino acids.

[0119] Humanized antibodies

[0120] Humanized antibodies are genetically engineered antibodies in which a CDR derived from a non-human "donor" antibody is inserted into a human "recipient" antibody sequence (see, for example, Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539; Carter, US 6,407,213; Adair, US 5,859,205; and Foote, US 6,881,557). The recipient antibody sequence can be, for example, a mature human antibody sequence, a combination of such sequences, a common sequence of human antibody sequences, or a germline region sequence. Preferred recipient sequences for the heavy chain are: germline V... H Exon IGHV1-46, for exon J (J HThe preferred receptor sequence for the light chain is exon IGKV1D-33, and for exon J IGKJ2. Alternative preferred receptor sequences for the heavy chain include exon J (JH), IGHJ1, IGHJ2, IGHJ3, IGHJ5, or IGHJ6. Alternative preferred receptor sequences for the light chain include exon J IGKJ1, IGKJ3, IGKJ4, or IGKJ5. Therefore, a humanized antibody is an antibody in which some or all of the CDRs are entirely or substantially derived from the donor antibody, while the constant and variable region framework sequences, if present, are entirely or substantially derived from the human antibody sequence. Similarly, a humanized heavy chain has at least one, two, and usually all three CDRs, entirely or substantially derived from the donor antibody heavy chain, and the heavy chain variable region framework sequence and the heavy chain constant region, if present, substantially derived from the human heavy chain variable region framework and constant region sequences. Similarly, the humanized light chain contains at least one, two, and usually all three CDRs, entirely or substantially derived from the donor antibody light chain, as well as the light chain variable region framework sequence and the light chain constant region, if present, substantially derived from the human light chain variable region framework and constant region sequences. Except for nanobodies and dAbs, humanized antibodies comprise humanized heavy chains and humanized light chains. The CDRs in humanized antibodies are substantially derived from the corresponding CDRs in non-human antibodies, with at least 60%, 85%, 90%, 95%, or 100% of corresponding residues (as defined by Kabat) being identical among the CDRs. When there is at least 85%, 90%, 95%, or 100% identical corresponding residues as defined by Kabat, the variable region framework sequence of the antibody chain or the constant region of the antibody chain is substantially derived from the human variable region framework sequence or the human constant region, respectively.

[0121] Although humanized antibodies typically contain all six CDRs from mouse antibodies (preferably as defined by Kabat), they can also be prepared with fewer than all (e.g., at least 3, 4, or 5) CDRs from mouse antibodies (e.g., Pascalis, J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320:415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al., Journal of Immunology, 164:1432-1441, 2000).

[0122] Some amino acids of the human variable region framework residues can be substituted based on their potential impact on CDR conformation and / or antigen binding. The investigation of this potential impact is conducted through modeling, examining the characterization of amino acids at specific positions, or empirically observing the effects of substitution or site-directed mutagenesis of specific amino acids.

[0123] For example, when there are differences between amino acids in mouse variable region framework residues and selected human variable region framework residues, human framework amino acids can be substituted with equivalent framework amino acids from mouse antibodies, where it is reasonably expected that the amino acid will:

[0124] (1) Non-covalent direct binding to antigen,

[0125] (2) Adjacent to the CDR area,

[0126] (3) Interacting with the CDR region in other ways (e.g., within approximately 6 Å of the CDR region); or

[0127] (6) Mediates the interaction between heavy and light chains.

[0128] Although 2A2 antibodies have been identified as mouse antibodies, this application also includes human 2A2 antibodies. The term "human 2A2 antibody" refers to an antibody derived from a human immunoglobulin gene sequence, whose CDR is substantially identical to that of a mouse 2A2 antibody, and which exhibits similar properties, namely, specific binding to αvβ6. In some aspects, human 2A2 antibodies include a heavy chain variable region substantially identical to the heavy chain variable region described herein and / or a light chain variable region substantially identical to the light chain variable region described herein. In some embodiments, the 2A2 antibody of the present invention is not a human antibody; for example, the 2A2 antibody of the present invention is a mouse, chimeric, or humanized antibody.

[0129] One aspect of the present invention provides a humanized form of mouse antibody 2A2. One such humanized variant of mouse antibody 2A2 is named HCLG. HCLG comprises a mature heavy chain variable region containing the amino acid sequence of SEQ ID NO: 6 and a mature light chain variable region containing the amino acid sequence of SEQ ID NO: 17. The humanized antibodies of the present invention comprise variants of the HCLG humanized antibody, wherein the humanized heavy chain mature variable region exhibits at least 90%, 95%, or 99% identity with SEQ ID NO: 6, and the humanized light chain mature variable region exhibits at least 90%, 95%, or 99% sequence identity with SEQ ID NO: 17. Preferably, in such antibodies, the reversion mutation in HCLG is partially or completely retained. In other words, at least 1, 2, 3, 4, 5, 6, 7, 8, or preferably all nine positions of the heavy chain H2, H28, H48, H67, H69, H71, H73, H78, and H93 are occupied by F, S, I, A, L, V, K, A, and T, respectively. Similarly, position L69 is preferably occupied by R, and L71 is preferably occupied by Y. The CDR region can be defined by any conventional definition (e.g., Chothia), but is preferably defined by Kabat or IMGT. In one embodiment, the humanized antibody comprises a heavy chain containing three CDRs of SEQ ID NO: 6 and a variable region frame that is at least 95% identical to the variable region frame of SEQ ID NO: 6. In another embodiment, the humanized antibody comprises a light chain containing three CDRs of SEQ ID NO: 17 and a variable region frame that is at least 95% identical to the variable region frame of SEQ ID NO: 17. In other embodiments, the humanized antibody comprises a heavy chain containing three CDRs of SEQ ID NO: 6 and a variable region framework with at least 98% identity to the variable region framework of SEQ ID NO: 6, and a light chain containing three CDRs of SEQ ID NO: 17 and a variable region framework with at least 95% identity to the variable region framework of SEQ ID NO: 17. In one embodiment, the humanized antibody comprises a heavy chain containing three CDRs of SEQ ID NO: 6 and a variable region framework with at least 99% identity to the variable region framework of SEQ ID NO: 6. In another embodiment, the humanized antibody comprises a light chain containing three CDRs of SEQ ID NO: 17 and a variable region framework with at least 99% identity to the variable region framework of SEQ ID NO: 17.

[0130] One possible variation is to replace certain residues in the mouse antibody CDR with corresponding residues from the human CDR sequence, typically derived from the CDR of the human receptor sequence used in the design example humanized antibody. In some antibodies, only a subset of the CDRs, i.e., the CDR residues required for binding, called the SDR, needs to be retained in the humanized antibody. CDR residues that do not contact the antigen and are not in the SDR can be identified from the Kabat CDR region located outside the Chothia hypervariable loop (Chothia, J. Mol. Biol. 196: 901, 1987) by molecular modeling and / or experience, or as described in Gonzales et al., Mol. Immunol. 41: 863 (2004), based on previous studies (e.g., H60-H65 residues in CDR H2 are often not required). In such humanized antibodies, at positions where one or more donor CDR residues are absent or where the entire donor CDR is omitted, the amino acid occupying that position can be the amino acid occupying the corresponding position in the receptor antibody sequence (by Kabat numbering). The number of donor amino acids replaced by this receptor in the CDR reflects a balance of various competing factors. This substitution has a potential advantage in reducing the number of mouse amino acids in humanized antibodies, thereby reducing potential immunogenicity. However, substitution can also lead to altered affinity, and a significant reduction in affinity is preferably avoided. The substitution site within the CDR and the amino acid to be substituted can also be selected empirically.

[0131] While not preferred, other amino acid substitutions can be made, for example, in framework residues not in contact with the CDR, or even in some potentially CDR-contacting amino acids within the CDR. Typically, substitutions made in the variant humanized sequence are conserved relative to the substituted HCLG amino acid. Preferably, substitutions relative to HCLG (whether conserved or not) have no substantial effect on the binding affinity or potency of the humanized mAb (i.e., its ability to bind human αVβ6 and inhibit cancer cell growth).

[0132] Selection of constant region

[0133] The variable regions of the heavy and light chains of humanized antibodies can be linked to at least a portion of the human constant region. The choice of the constant region depends in part on whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent phagocytosis, and / or complement-dependent cytotoxicity are required. For example, human allotypes IgG1 and IgG3 have strong complement-dependent cytotoxicity, human allotype IgG2 has weak complement-dependent cytotoxicity, and human IgG4 lacks complement-dependent cytotoxicity. Human IgG1 and IgG3 also induce stronger cell-mediated effector function than human IgG2 and IgG4. The light chain constant region can be λ or κ. Antibodies can be expressed as tetramers containing two light chains and two heavy chains, or as separate heavy and light chains, such as Fab, Fab′, F(ab′)2, and Fv, or as single-chain antibodies in which the variable domains of the heavy and light chains are linked by spacers.

[0134] Human constant regions exhibit both allotypic variation and isoallotypic variation among individuals; that is, constant regions can differ at one or more polymorphic locations in different individuals. Isoallotypes differ from allotries in that serum identifying isoallotypes binds to one or more non-polymorphic regions of other isotypes.

[0135] One or more amino acids at the amino or carboxyl terminus of the light and / or heavy chains, such as the C-terminal lysine of the heavy chain, may be deleted or derivatized in part or all of the molecule. Substitutions may be made in the constant region to reduce or increase effector function, such as complement-mediated cytotoxicity or ADCC (see, for example, U.S. Patent No. 5,624,821 to Winter et al.; U.S. Patent No. 5,834,597 to Tso et al.; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to prolong the half-life in humans (see, for example, Hinton et al., J. Biol. Chem. 279:6213, 2004).

[0136] Exemplary substitutions include introducing a native amino acid at amino acid positions 234, 235, 237, 239, 267, 298, 299, 326, 330, or 332 to replace a cysteine ​​residue, preferably the S239C mutation of the human IgG1 isotype (US 20100158909). The presence of the additional cysteine ​​residue allows for the formation of interchain disulfide bonds. This formation of interchain disulfide bonds creates steric hindrance, thereby reducing the affinity of the Fc region-FcyR binding interaction. One or more cysteine ​​residues introduced within or adjacent to the Fc region of the IgG constant region can also serve as sites for conjugation with therapeutic agents (i.e., conjugation of cytotoxic drugs using thiol-specific agents, such as maleimide derivatives of the drug). The presence of the therapeutic agent creates steric hindrance, further reducing the affinity of the Fc region-FcyR binding interaction. Other substitutions at any position 234, 235, 236, and / or 237 reduce affinity for Fey receptors, particularly FcyRI receptors (see, for example, US 6,624,821, US 5,624,821).

[0137] The in vivo half-life of an antibody can also affect its effector function. The half-life of an antibody can be increased or decreased to alter its therapeutic activity. FcRn is a receptor structurally similar to an MHC class I antigen and non-covalently associates with β2-microglobulin. FcRn regulates the catabolic metabolism of IgG and its transcellular transport (Ghetie and Ward, 2000, Annu. Rev. Immunol. 18: 739-766; Ghetie and Ward, 2002, Immunol. Res. 25: 97-113). IgG-FcRn interaction occurs at pH 6.0 (the pH of intracellular vesicles), rather than pH 7.4 (the pH of blood); this interaction allows IgG to be recycled into circulation (Ghetie and Ward, 2000, Ann. Rev. Immunol. 18: 739-766; Ghetie and Ward, 2002, Immunol. Res. 25: 97-113). Regions on human IgG1 involved in FcRn binding have been localized (Shields et al., 2001, J. Biol. Chem. 276: 6591-604). Alanine substitutions at the Pro238, Thr256, Thr307, Gln311, Asp312, Glu380, Glu382, or Asn434 positions of human IgG1 enhance FcRn binding (Shields et al., 2001, J. Biol. Chem. 276: 6591-604). IgG1 molecules containing these substitutions have a longer serum half-life. Therefore, these modified IgG1 molecules are able to perform their effector function for a longer period compared to unmodified IgG1, thereby exerting their therapeutic effect. Other exemplary substitutions that increase binding to FcRn include Gln at position 250 and / or Leu at position 428. EU numbers are used at all positions in the constant region.

[0138] Oligosaccharides covalently attached to conserved Asn297 are associated with the ability of Fc region of IgG to bind to FcyR (Lund et al., 1996, J. Immunol. 157: 4963-69; Wright and Morrison, 199', Trends Biotechnol. 15: 26-31). Engineering this glycoform on IgG can significantly improve IgG-mediated ADCC. Modification with bipartite N-acetylglucosamine (Umana et al., 1999, Nat. Biotechnol. 17: 176-180; Davies et al., 2001, Biotech. Bioeng. 74: 288-94) by adding to or removing fucose from the glycoform (Shields et al., 2002, J. Biol. Chem. 277: 26733-40; Shinkawa et al., 2003, J. Biol. Chem. 278: 6591-604; Niwa et al., 2004, Cancer Res. 64: 2127-33) are two examples of IgG Fc engineering that improves the binding between IgG Fc and FcyR, thereby enhancing Ig-mediated ADCC activity.

[0139] Systematic substitution of solvent-exposed amino acids in the Fc region of human IgG1 yielded IgG variants with altered FcyR binding affinity (Shields et al., 2001, J. Biol. Chem. 276: 6591-604). Among these variants, subsets involving Thr256 / Ser298, Ser298 / Glu333, Ser298 / Lys334, or Ser298 / Glu333, Lys334 substitutions for Ala showed increased binding affinity for FcγR and ADCC activity compared to parental IgG1 (Shields et al., 2001, J. Biol. Chem. 276: 6591-604; Okazaki et al., 2004, J. Mol. Biol. 336: 1239-49).

[0140] The complement fixation activity of antibodies (including Clq binding and CDC activity) can be improved by substituting Lys326 and Glu333 (Idusogie et al., 2001, J. Immunol. 166: 2571-2575). The same substitution on the human IgG2 backbone can convert antibody isotypes that bind poorly to Clq and are severely lacking in complement activation activity into isotypes that can both bind Clq and mediate CDC (Idusogie et al., 2001, J. Immunol. 166: 2571-75). Other methods have also been used to improve antibody complement fixation activity. For example, attaching the 18-amino acid tail piece of IgM to the C-terminus of IgG can significantly enhance its CDC activity. This can be observed even with IgG4, which typically has no detectable CDC activity (Smith et al., 1995, J. Immunol. 154: 2226-36). Furthermore, replacing Ser444, located near the C-terminus of the IgG1 heavy chain, with Cys induces tail-to-tail dimerization of IgG1, increasing CDC activity by 200-fold compared to monomeric IgG1 (Shopes et al., 1992, J. Immunol. 148: 2918-22). In addition, bispecific secondary antibody constructs with Clq specificity also exhibit CDC activity (Kontermann et al., 1997, Nat. Biotech. 15: 629-31).

[0141] Complement activity can be reduced by mutating at least one amino acid residue in the heavy chain 318, 320, and 322 to a residue with a different side chain, such as Ala. Other alkyl-substituted nonionic residues, such as Gly, He, Leu, or Val, or aromatic nonpolar residues such as Phe, Tyr, Trp, and Pro, replacing any of these three residues, will also reduce or eliminate Clq binding. Ser, Thr, Cys, and Met can be used at residues 320 and 322, but not at 318, to reduce or eliminate Clq binding activity.

[0142] Replacing the 318 (Glu) residue with a polar residue may alter, but not eliminate, Clq binding activity. Replacing the 297 (Asn) residue with Ala results in the removal of cleavage activity, but only slightly reduces (about three-fold) the affinity for Clq. This change disrupts the presence of the glycosylation site and the carbohydrate required for complement activation. Any other substitution at this site will also disrupt the glycosylation site. The following mutations and any combinations thereof also reduce Clq binding: D270A, K322A, P329A, and P31IS (see WO06 / 036291). The L234A / L235A mutation (or the LALA mutation) also reduces C1q binding, as well as FcyR binding.

[0143] The human constant region is defined as any arrangement of residues that occupy polymorphic positions within a natural allotype or any natural allotype. Additionally, there may be up to 1, 2, 5, or 10 mutations relative to the natural human constant region, such as those mentioned above, to reduce FcT receptor binding or increase binding to FcRN.

[0144] V. Expression of recombinant antibodies

[0145] Humanized antibodies are typically generated through recombinant expression. Recombinant polynucleotide constructs generally include an expression control sequence operatively linked to the coding sequence of the antibody chain, including a native or heterologous promoter region. Preferably, the expression control sequence is a eukaryotic promoter system within a vector capable of transforming or transfecting eukaryotic host cells. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high levels of nucleotide sequence expression, and cross-reactive antibodies are collected and purified.

[0146] Mammalian cells are the preferred hosts for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, *From Genes to Clones* (VCH Publishing, NY, 1987). Many suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, including CHO cell lines (e.g., DG44), various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-derived myeloma (including Sp2 / 0 and NSO). Preferably, the cells are non-human. Expression vectors for these cells may include expression control sequences, such as replication initiation sites, promoters, enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), and essential processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. Preferred expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, etc. See Co et al., J. Immunol. 148: 1149 (1992).

[0147] Once expressed, the antibody can be purified according to standard procedures in the field, including HPLC purification, column chromatography, gel electrophoresis, etc. (see Scopes, Protein Purification (Springer, NY, 1982)).

[0148] VI. Nucleic Acids

[0149] This invention further provides nucleic acids encoding any of the aforementioned humanized heavy and light chains. Typically, the nucleic acid also encodes a signal peptide fused to the mature heavy and light chains. The coding sequences on the nucleic acid can be operatively linked to regulatory sequences to ensure expression of the coding sequences, such as promoters, enhancers, ribosome binding sites, transcription termination signals, etc. The nucleic acids encoding the heavy and light chains can be present in a separate form or can be cloned into one or more vectors. These nucleic acids can be synthesized, for example, by solid-state synthesis or PCR of overlapping oligonucleotides. The nucleic acids encoding the heavy and light chains can be conjugated as a continuous nucleic acid, for example, within an expression vector, or they can be separate, for example, each cloned into its own expression vector.

[0150] In some respects, this document also provides nucleic acids encoding the anti-αvβ6 antibody described herein or an antigen-binding fragment thereof. This document also provides vectors comprising nucleic acids encoding the anti-αvβ6 antibody described herein or an antigen-binding fragment thereof. This document also provides host cells expressing nucleic acids encoding the anti-αvβ6 antibody described herein or an antigen-binding fragment thereof. This document also provides host cells comprising vectors containing nucleic acids encoding the anti-αvβ6 antibody described herein or an antigen-binding fragment thereof.

[0151] The anti-αvβ6 antibody described herein can be prepared using well-known expression vector systems and host cells via well-known recombinant techniques. In one embodiment, the antibody is prepared in CHO cells using a GS expression vector system, as described in De la Cruz Edmunds et al., 2006, Molecular Biotechnology 34; 179-190, EP216846, U.S. Patent No. 5,981,216, WO 87 / 04462, EP323997, U.S. Patent No. 5,591,639, U.S. Patent No. 5,658,759, EP338841, U.S. Patent No. 5,879,936, and U.S. Patent No. 5,891,693.

[0152] The monoclonal anti-αvβ6 antibody described herein can be produced, for example, by the hybridoma method described first by Kohler et al., Nature, 256, 495 (1975), or by a recombinant DNA method. Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, those described by Clackson et al., Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222(3): 581-597 (1991). Monoclonal antibodies can be obtained from any suitable source. Thus, for example, monoclonal antibodies can be obtained from hybridomas prepared from mouse spleen B cells obtained from mice immunized with an antigen of interest, for example, in the form of cells expressing the antigen on their surface or nucleic acids encoding the antigen of interest. Monoclonal antibodies can also be obtained from hybridomas derived from antibody-expressing cells of immunized human or non-human mammals (such as rats, dogs, primates, etc.).

[0153] VII. Antibody-drug conjugates

[0154] Anti-αvβ6 antibodies can be conjugated to cytotoxic or cytosuppressive moieties (including pharmaceutically compatible salts) to form antibody-drug conjugates (ADCs). Parts particularly suitable for antibody conjugation include cytotoxic agents (e.g., chemotherapeutic agents), prodrug-converting enzymes, radioisotopes, or compounds or toxins (these moieties are collectively referred to as therapeutic agents). For example, anti-αvβ6 antibodies can be conjugated to cytotoxic agents, such as chemotherapeutic agents, or toxins (e.g., cytosuppressive or cytotoxic agents such as abrin A, ricin A, Pseudomonas exotoxin, or diphtheria toxin).

[0155] Anti-αvβ6 antibodies can be conjugated to prodrug-converting enzymes. These enzymes can be recombinantly fused with the antibody or chemically conjugated using known methods. Exemplary prodrug-converting enzymes are carboxypeptidase G2, β-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, β-lactamase, β-glucosidase, nitroreductase, and carboxypeptidase A.

[0156] The technique of conjugating therapeutic agents to proteins, particularly antibodies, is well-known. (See, for example, Arnon et al., "Monoclonal Antibodies For Immunotargefing Of Drugs In Cancer Therapy," Reisfeld et al., eds., ARL (Alan R. Liss, Inc.), 1985; Hellstrom et al., "Antibodies For Drug Delivery," Robinson et al., Marcel Decker, 2nd ed., 1987; Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," Monoclonal Antibodies '84: Biological and Clinical Applications.) Review)," (Pinchera et al., eds., 1985); "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy" in Monoclonal Antibodies For Cancer Detection and Therapy," (Baldwin et al., eds., Academic Press, 1985); and Thorpe et al., 1982, Immunol. Rev. 62: 119-58. See also, for example, PCT Publication WO 89 / 12624).

[0157] Therapeutic agents can be conjugated in a manner that reduces their activity unless they are cleaved from the antibody (e.g., by hydrolysis, degradation by antibody hydrolysis, or by a cleaving agent). Such therapeutic agents are attached to the antibody by a cleavable linker that is sensitive to cleavage in the intracellular environment of αvβ6-expressing cancer cells but largely insensitive to the extracellular environment. Thus, when the antibody is internalized by αvβ6-expressing cancer cells (e.g., in endosomes, or, for example, in a lysosomal or caveolear environment with pH or protease sensitivity), the conjugate is cleaved from the antibody.

[0158] Typically, an ADC includes a linker region between the therapeutic agent and the anti-αvβ6 antibody. As mentioned above, the linker is usually cleavable under intracellular conditions, so that cleavage of the linker releases the therapeutic agent from the antibody in the intracellular environment (e.g., within lysosomes, endosomes, or pits). The linker can be, for example, a peptide linker that is cleaved by intracellular peptidases or proteases, including lysosomal or endosomal proteases. Typically, the peptide linker is at least two or at least three amino acids in length. Cleavage agents can include cathepsin B and D and plasmin (e.g., see Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123). Most typically, the peptide linker is cleavable by enzymes present in cells expressing αvβ6. For example, a peptide linker that is cleaved by the thiol-dependent protease cathepsin B, which is highly expressed in cancerous tissue (e.g., a linker including Phe-Leu or Gly-Phe-Leu-Gly peptides) can be used. Other such connectors are described, for example, in U.S. Patent No. 6,214,345. In specific embodiments, intracellular protease-cleavable peptide connectors include the Val-Cit connector or the Phe-Lys dipeptide (see, for example, U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin using the Val-Cit connector). One advantage of using intracellular protein hydrolysis release of the therapeutic agent is that the agent is typically attenuated when conjugated and the serum stability of the conjugate is generally high.

[0159] Degradable linkers can be pH-sensitive, meaning they are susceptible to hydrolysis at certain pH values. Typically, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, acid-instable linkers that are hydrolyzable in lysosomes (such as hydrazones, ureas, thioureas, cis-aconitamides, orthoesters, acetals, ketals, etc.) can be used. (See, for example, U.S. Patents 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661.). Such linkers are relatively stable under neutral pH conditions (such as those in blood) but unstable below pH 5.5 or 5.0 (the approximate pH of lysosomes). In some embodiments, the hydrolyzable connector is a thioether connector (such as a thioether that attaches a therapeutic agent via an acylhydrazone bond (see, for example, U.S. Patent No. 5,622,929)).

[0160] Other junctions are cleavable under reducing conditions (e.g., disulfide junctions). Disulfide junctions include those that can be formed using SATA (N-succinimide-S-thioacetate), SPDP (N-succinimide-3-(2-pyridyldithio)propionate), SPDB (N-succinimide-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimide-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio)toluene), SPDB, and SMPT. (See, for example, Thorpe et al., 1987, Cancer Res. 47: 5924-5931; Wawrzynczak et al., Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogel, ed., Oxford University Press, 1987. See also U.S. Patent No. 4,880,935.)

[0161] The connector can also be a malonic acid connector (Johnson et al., 1995, Anticancer Res. 15: 1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1299-1304) or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12). The connector can also be a malonic acid connector (Johnson et al., 1995, Anticancer Res. 15: 1387-93), a maleimide benzoyl connector (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1299-1304) or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12).

[0162] Linkages can also be non-cleavable, such as maleimide-alkylene or maleimide-aryl linkers that attach directly to therapeutic agents (e.g., drugs). Active drug-linkages are released through antibody degradation.

[0163] This linker promotes cell internalization. When conjugated with a therapeutic agent (i.e., in the context of the linker-therapeutic agent portion of the ADC or ADC derivative described herein), the linker promotes cell internalization. Alternatively, when conjugated with both a therapeutic agent and an anti-αvβ6 antibody (i.e., in the context of the ADC described herein), the linker promotes cell internalization.

[0164] Anti-αvβ6 antibodies can be coupled to linkers via heteroatoms on the antibody. These heteroatoms can be natively present on the antibody or introduced into it. In some cases, anti-αvβ6 antibodies are coupled to linkers via the nitrogen atom of a lysine residue. In others, anti-αvβ6 antibodies are coupled to linkers via the sulfur atom of a cysteine ​​residue. Cysteine ​​residues can be natively present or engineered into the antibody. Methods for coupling linkers and drug-linkers to antibodies via lysine and cysteine ​​residues are known in the art.

[0165] Exemplary antibody-drug conjugates include olistatin-based antibody-drug conjugates (i.e., the drug component is the olistatin drug). Olistatin binds to tubulin and has been shown to interfere with microtubule dynamics and nuclear and cell division, and possesses anticancer activity. Typically, olistatin-based antibody-drug conjugates include a linker between the olistatin drug and an anti-αvβ6 antibody. This linker can be, for example, a cleavable linker (e.g., a peptide linker, a carbohydrate linker) or a non-cleavable linker (e.g., a linker released through antibody degradation). Olistatin includes (but is not limited to) olistatin T, MMAF, and MMAE. Exemplary olistatin syntheses and structures are described in U.S. Publications 7,659,241, 7,498,298, 2009-0111756, 2009-0018086, and 7,968,687, the entire contents of which are incorporated herein by reference for all purposes.

[0166] Exemplary olritatin-based antibody-drug conjugates include vcMMAE (or 1006), vcMMAF, and mcMMAF antibody-drug conjugates, as shown below, where p represents the drug loading, Ab is the anti-αvβ6 antibody described herein, and val-cit or “vc” represents a valine-citrulline dipeptide:

[0167]

[0168]

[0169] Or a pharmaceutically acceptable salt thereof. Drug load is denoted by p, which is the number of drug-adaptor molecules per antibody. Referring to antibody-drug conjugates targeting αvβ6, the subscript p represents drug load, which, depending on the context, can represent the number of drug-adaptor molecules attached to a single antibody molecule and is therefore an integer value, or it can represent the average drug load and is therefore an integer or non-integer value, but is usually a non-integer value. Average drug load represents the average number of drug-adaptor molecules per antibody in the population. Usually, but not always, when we refer to antibodies, such as monoclonal antibodies, we are referring to the population of antibody molecules. In compositions consisting of a population of antibody-drug conjugate molecules, average drug load is an important quality characteristic because it determines the amount of drug that can be delivered to the target cells. The percentage of unconjugated antibody molecules in the composition is included in the average drug load value.

[0170] In a preferred aspect of the invention, when referring to a composition comprising a group of antibody-drug conjugate compounds, the average drug loading is 1 to about 16, preferably about 2 to about 14, more preferably about 2 to about 10. In one embodiment, the DAR is about 2 to about 5. In another embodiment, the DAR is 4. In another embodiment, the DAR is about 6 to about 10. In yet another embodiment, the DAR is 8. The average drug loading of each antibody in the formulation can be characterized by conventional means, such as mass spectrometry, HIC, ELISA assays, and HPLC. In some aspects, the anti-αvβ6 antibody is attached to the drug linker via a cysteine ​​residue of the antibody. In some aspects, the cysteine ​​residue is engineered into the antibody. In other aspects, the cysteine ​​residue is an inter-chain dithiocysteine ​​residue.

[0171] In some embodiments, a polyethylene glycol polymer is incorporated as a side chain into a cleavable β-glucuronide MMAE drug-linker. Compared to a non-PEGylated control, this results in reduced plasma clearance and increased antitumor activity of the antibody-drug conjugate in a xenograft model. Therefore, a particularly advantageous drug-linker for attachment to the antibodies of the present invention is shown in Formula V:

[0172]

[0173] Or its pharmaceutically acceptable salt.

[0174] The preferred stereochemistry of this drug-connector is shown in the following formula Va:

[0175]

[0176] Or a pharmaceutically acceptable salt thereof, wherein for formulas V and Va, Z represents an organic moiety having a reaction site capable of reacting with functional groups on the antibody to form a covalently attached reaction site, n ranges from 8 to 36, with a most preferred range of 8 to 14 (most preferred 12), R 21 It is the end-capping unit of the polyethylene glycol moiety, preferably -CH3 or -CH2CH2CO2H.

[0177] The preferred Z portion is the maleimide-containing portion. Particularly preferred Z portions are shown in the drug connector below:

[0178]

[0179] Or its pharmaceutically acceptable salt.

[0180] The preferred stereochemistry of this drug-connector is shown below:

[0181]

[0182]

[0183] Or a pharmaceutically acceptable salt thereof, wherein for formulas VI, VIa, VII, and VIIa, n ranges from 8 to 36, with the most preferred range being 8 to 14 (most preferred being 12), R PR It is a hydrogen or a protecting group, for example, an acid-labile protecting group, such as BOC, R 21 It is the end-capping unit of the polyethylene glycol moiety, preferably -CH3 or -CH2CH2CO2H.

[0184] As mentioned above, R PR It can be hydrogen or a protecting group. The protecting group used in this article refers to a group that selectively (temporarily or permanently) blocks the reaction site in a polyfunctional compound. A suitable protecting group is one that can prevent or avoid unwanted side reactions or prematurely lose its protecting group under reaction conditions to achieve the chemical transformations required elsewhere in the molecule, and can be removed without adversely affecting the structural or stereochemical integrity of the newly formed molecule when purification is needed. Suitable amine protecting groups include acid-labile nitrogen protecting groups, including those described by Isidro-Llobel et al., “Aminoacid-protecting groups”, Chem. Rev. (2009) 109: 2455-2504. Typically, acid-labile nitrogen protecting groups convert primary or secondary amino groups to their corresponding carbamates, including tert-butyl, allyl, and benzyl carbamates.

[0185] As mentioned above, R 21 It is the end-capping unit of the polyethylene glycol moiety. As those skilled in the art will understand, polyethylene glycol units can be end-capped with a variety of organic moieties, typically those that are relatively non-reactive. Alkyl and substituted alkyl groups are preferred.

[0186] For MMAE-PEGylated ADCs, such as those exemplified herein, a particularly preferred average drug load is approximately 8. In exemplary embodiments, the drug-linker is coupled to a cysteine ​​residue of the reduced interchain disulfide. In some aspects, the actual drug load of a single antibody molecule in the antibody-drug conjugate compound population is 1 to 10 (or 6 to 10 or 6 to 8), with a predominantly drug load of 8. For example, in addition to the interchain disulfide, a higher drug load can be achieved if the drug-linker is coupled to an introduced cysteine ​​residue (e.g., a cysteine ​​residue introduced at position 239 according to EU indexing).

[0187] Exemplary ADCs include the following:

[0188]

[0189]

[0190]

[0191] Or a pharmaceutically acceptable salt thereof, wherein n is between 8 and 36, most preferably between 8 and 14 (12 being the most preferred), R PR It is a hydrogen or a protecting group, for example, an acid-labile protecting group, such as BOC, R 21 It is a capping unit of the polyethylene glycol moiety, preferably -CH3 or -CH2CH2CO2H, Ab represents anti-αVβ6 antibody, p represents an integer from 1 to 16, when referring to a single antibody molecule, preferably 1 to 14, 6 to 12, 6 to 10 or 8 to 10, or when referring to a population of antibody molecules, the average drug loading is about 4 or about 6 to about 14, preferably about 8.

[0192] As described above, the PEG (polyethylene glycol) portion of the drug connector can be 8 to 36; however, a PEG with 12 ethylene oxide units has been found to be particularly preferred. It has been found that longer PEG chains result in slower clearance, while shorter PEG chains result in reduced activity. Therefore, the subscript n in all the above embodiments is preferably 8 to 14, 8 to 12, 10 to 12, or 10 to 14, and most preferably 12.

[0193] The PEGylated antibody-drug conjugates of the present invention can be prepared using polydisperse PEG, monodisperse PEG, and discrete PEG. Polydisperse PEG is a heterogeneous mixture of sizes and molecular weights, while monodisperse PEG is typically purified from a heterogeneous mixture, thus providing a single chain length and molecular weight. The preferred PEG unit is discrete PEG, i.e., a compound synthesized in a stepwise manner rather than through a polymerization process. Discrete PEG provides a single molecule with a defined and specified chain length. Similar to the subscript "p", when referring to a group of antibody-drug conjugates, the value of the subscript "n" can be an average, an integer, or a non-integer.

[0194] In a preferred embodiment, the covalent attachment of the antibody to the drug-linker is accomplished through the interaction of the thiol functional group of the antibody with the maleimide functional group of the drug-linker to form a thiosuccinimide. The thiol functional group can exist natively on the ligand unit, for example, in naturally occurring residues (interchain disulfide residues), or can be introduced into the ligand through chemical modification or bioengineering, or a combination of both. It should be understood that the antibody-substituted succinimide can exist in one or more hydrolyzed forms. For example, in a preferred embodiment, the ADC comprises a succinimide moiety, which, when bonded to an antibody, is represented by the following structure:

[0195]

[0196] Or it may contain its corresponding acid-amide moiety, which, when bonded to an antibody, is represented by the following structure:

[0197]

[0198] The wavy line indicates the connection to the remaining part of the drug-connector.

[0199] In some embodiments, the anti-αVβ6 antibody of the present invention is coupled to monomethylolpropionate E via an MDpr-PEG(12)-gluc linker to form an antibody-drug conjugate having the following structure:

[0200]

[0201] Or a pharmaceutically acceptable salt thereof, wherein n is between 8 and 36, most preferably between 8 and 14 (most preferably 12), R PR It is a hydrogen or a protecting group, for example, an acid-labile protecting group, such as BOC, R 21 It is a capping unit of the polyethylene glycol moiety, preferably -CH3 or -CH2CH2CO2H, Ab represents anti-αVβ6 antibody, p represents an integer from 1 to 16, when referring to a single antibody molecule, preferably 1 to 14, 6 to 12, 6 to 10 or 8 to 10, or when referring to a population of antibody molecules, the average drug loading is from about 4 or about 6 to about 14, preferably about 8.

[0202] Exemplary antibody-drug conjugates also include camptothecin-based antibody-drug conjugates (i.e., the drug component is a camptothecin drug). Camptothecin is a topoisomerase inhibitor that has been shown to have anticancer activity. Typically, camptothecin-based antibody-drug conjugates comprise a linker between a camptothecin drug and an anti-αvβ6 antibody. This linker can be, for example, a cleavable linker (e.g., a peptide linker, a carbohydrate linker) or a non-cleavable linker (e.g., a linker released through antibody degradation). The synthesis and structure of an exemplary camptothecin drug linker are described in PCT / US19 / 025968 (filed April 5, 2019), which is incorporated herein by reference in its entirety for all purposes.

[0203] Exemplary anti-αvβ6 antibody-drug conjugates include camptothecin antibody-drug conjugates, where p represents drug loading and Ab represents anti-αvβ6 antibody:

[0204] In some embodiments, camptothecin ADC has the formula (IC):

[0205]

[0206] Or its pharmaceutically acceptable salt;

[0207] in

[0208] Ab is an anti-αvβ6 antibody;

[0209] Y is 1, 2, 3, or 4, or 1 or 4; and

[0210] Z is an integer from 2 to 12, or 2, 4, 8 or 12;

[0211] And p is 1-16.

[0212] In some aspects of these implementations, p is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In other aspects, p is 2, 4, or 8.

[0213] In some embodiments, camptothecin ADC has the formula:

[0214]

[0215] Or its pharmaceutically acceptable salt;

[0216] Where p is 2, 4 or 8, preferably p is 8.

[0217] In some embodiments, camptothecin ADC has the formula:

[0218]

[0219] Or its pharmaceutically acceptable salt:

[0220] Where p is 2, 4 or 8, preferably p is 8.

[0221] In some embodiments, the camptothecin drug-connector has the following formula:

[0222]

[0223] Or its pharmaceutically acceptable salt;

[0224] in

[0225] y is 1, 2, 3, or 4, or 1 or 4; and

[0226] z is an integer from 2 to 12, or 2, 4, 8 or 12.

[0227] In some embodiments, the camptothecin drug-connector has the following formula:

[0228]

[0229] MP-PEG8-VKG-camptothecin

[0230] In some embodiments, the camptothecin drug-connector has the following formula:

[0231]

[0232] MP-PEG4-VKG-camptothecin

[0233] In some embodiments, the camptothecin drug-connector has the following formula:

[0234]

[0235] MP-PEG12-VKG-camptothecin

[0236] Other exemplary antibody-drug conjugates include maytansinoid antibody-drug conjugates (i.e., the drug component is a maytansinoid drug) and benzodiazepines. (benzodiazepine) antibody-drug conjugate (i.e., the drug component is benzodiazepine) (For example, pyrrolo[1,4]benzodiazepine) Dimer (PBD dimer), indoline benzodiazepine (indolinobenzodiazepine) dimer and oxazolidinone benzodiazepine (oxazolidinobenzodiazepine) dimer).

[0237] In some embodiments, the PBD dimer used in this invention is represented by Formula I. Preferred stereochemistry of the PBD dimer is shown in Formula Ia:

[0238]

[0239] Or the medicinal salt, solvate, or solvate of the salt; wherein the subscript n is 1 or 3.

[0240] The solvates of formulas (I) and (Ia) are typically formed by adding water or an alcohol solvent to the imine functional groups of one or both PBD monomers to form one or more carbinolamines and / or carbinolamine ethers. For example, at the N10-C11 position, there can be an imine (N=C), a carbinolamine (NH-CH(OH)), or a carbinolamine ether (NH-CH(OMe)), as shown in formulas I′ and Ia′ below:

[0241]

[0242] Among them, or:

[0243] (a)R 10 It is H, R 11 Is it OH or OR? A , where R AIt is saturated C 1-4 Alkyl (preferably methyl); or

[0244] (b)R 10 and R 11 A nitrogen-carbon double bond is formed between the nitrogen and carbon atoms they are bonded to; or

[0245] (c)R 10 One of them is H,R 11 Is it OH or OR? A , where R A It is saturated C 1-4 Alkyl (preferably methyl); other R 10 and R 11 A nitrogen-carbon double bond is formed between the nitrogen and carbon atoms they are bonded to.

[0246] PBD dimers of Formula I or 1a (or their pharmaceutical salts, solvates, or solvates) are typically linked to antibodies via a linker unit LU. The linker unit functions to release the PBD dimer of Formula I or 1a (or its pharmaceutical salts, solvates, or solvates) at a target site (e.g., within cancer cells). The PBD drug-linker compounds used in this invention are represented by Formula II (preferably stereochemistry as shown in IIa), where LU is the linker unit. The linker unit can be, for example, a cleavable peptide linker unit (e.g., a linker comprising a valine-alanine peptide) or a cleavable disulfide linker unit.

[0247]

[0248] Or the medicinal salt, solvate, or solvate of the salt; wherein the subscript n is 1 or 3.

[0249] Preferred PBD drug-connector compounds used in this invention are represented by the following formula III:

[0250]

[0251] Or the medicinal salt, solvate, or solvate of the salt; wherein the subscript n is 1 or 3, and the subscript m is an integer from 2 to 5.

[0252] PBD drug-linkers are conjugated to anti-αVβ6 antibodies to produce antibody-drug conjugates targeting αVβ6. For example, the antibody can be conjugated to a drug-linker of formula II or formula III. Exemplary antibody-drug conjugates targeting αVβ6 are shown in formulas IV, IVa, and IVb:

[0253]

[0254]

[0255] Or the medicinal salt, solvate, or solvate of the salt; wherein the subscript n is 1 or 3; the subscript m is an integer from 2 to 5; and the subscript p is from 1 to 4.

[0256] Useful classes of cytotoxic agents conjugated to anti-αVβ6 antibodies include, for example, anti-microtubule agents, DNA minor groove binding agents, DNA replication inhibitors, and chemosensitizers. Other exemplary classes of cytotoxic agents include anthracyclines, oliquistatins, camptothecins, docamycins, etoposides, maytansinoids, and vinca alkaloids. Some exemplary cytotoxic agents include olistatins (such as olistatin T, olistatin E, AFP, monomethylolistatin F (MMAF), lipophilic monomethylolistatin F, monomethylolistatin E (MMAE)), DNA minor groove binding agents (such as enediynes and lexitropsins), docalamycins, taxanes (such as paclitaxel and docetaxel), vinca alkaloids, nicotinamide phosphoribosyltransferase inhibitors (NAMPTi), tubulin lysin M, doxorubicin, morpholino-doxorubicin, and cyanomorpholino-doxorubicin.

[0257] Cytotoxic agents can be chemotherapeutic agents, such as doxorubicin, paclitaxel, melphalan, vinca alkaloids, methotrexate, mitomycin C, or etoposide. The agent can also be a CC-1065 analog, calicheamicin, maytansine, an analog of salicylic acid 10, rhizoxin, or palytoxin.

[0258] Cytotoxic agents can also be olistatin. Olistatin can be an olistatin E derivative, such as an ester formed between olistatin E and a keto acid. For example, olistatin E can react with p-acetylbenzoic acid or benzoylvaleric acid to generate AEB and AEVB, respectively. Other typical olistatins include olistatin T, AFP, MMAF, and MMAE. The synthesis and structure of various olistatins are described in, for example, US 2005-0238649 and US 2006-0074008.

[0259] Cytotoxic agents can be DNA minor groove binders. (See, for example, U.S. Patent No. 6,130,237.) For example, minor groove binders can be CBI compounds or enediynes (e.g., kazimidox).

[0260] Cytotoxicity or cell inhibitors can be anti-tubulin agents. Examples of anti-tubulin agents include taxanes (e.g., taxanes). (paclitaxel) Docetaxel, T67 (Tularik), vinca alkaloids (e.g., vincristine, vinblastine, vinorelbine, and vinorelbine), and oliganthromycins (e.g., oliganthromycin E, AFP, MMAF, MMAE, AEB, and AEB). Exemplary oliganthromycins are shown in the following formulas III-XIII. Other suitable anti-microtubule agents include, for example, baccatin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colcimid, estramustine, cryptophysins, cemadotin, maytansine, compbretastatins, disccodermoide, and eleuthrobin.

[0261] Cytotoxic agents can be maytansines or another group of anti-microtubule agents (e.g., DM1, DM2, DM3, DM4). For example, maytansines can be maytansine or drug linkers containing maytansine, such as DM-1 or DM-4 (ImmunoGen, Inc.; see also Charr et al., 1992, Cancer Res.).

[0262] VIII. Therapeutic Use

[0263] The antibodies of the present invention, alone or as their anti-αvβ6 antibody-drug conjugates, can be used to treat cancer. Some such cancers exhibit detectable αvβ6 levels, measured at the protein level (e.g., by immunoassay using one of the exemplified antibodies) or mRNA level. Some such cancers show elevated αvβ6 levels relative to non-cancerous tissue of the same type (preferably from the same patient). An exemplary level of αvβ6 on cancer cells suitable for treatment is 5,000-500,000 αvβ6 molecules per cell, although higher or lower levels may also be treated. Optionally, the level of αvβ6 in the cancer is measured prior to treatment.

[0264] Examples of cancers associated with and suitable for treatment include non-small cell lung cancer (NSCLC) (squamous cell carcinoma and adenocarcinoma), head and neck cancer (including head and neck squamous cell carcinoma), esophageal cancer, breast cancer (including invasive breast cancer), ovarian cancer, bladder cancer (including urothelial carcinoma), skin cancer (squamous cell carcinoma or SCC), kidney cancer (including clear cell, papillary, and chromophobe renal cells), cervical cancer, gastric cancer, prostate cancer (including prostate adenocarcinoma), endometrial cancer (including uterine sarcoma and endometrial cancer), rectal adenocarcinoma, thyroid cancer, colonic adenocarcinoma, gastric adenocarcinoma, and pancreatic cancer (including pancreatic adenocarcinoma). In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used in methods of treating NSCLC. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used in methods of treating head and neck cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used in methods of treating skin cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used in methods of treating esophageal cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used in methods for treating breast cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used in methods for treating ovarian cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used in methods for treating bladder cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used in methods for treating cervical cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used in methods for treating gastric cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used in methods for treating renal cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used in methods for treating endometrial cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used in methods for treating gastric cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used in methods for treating pancreatic cancer. This treatment can be applied to patients with these types of primary or metastatic tumors. This treatment method can also be applied to patients refractory to conventional treatment or patients who have relapsed after remission with such treatment.

[0265] An effective regimen for administering the antibodies of the present invention (e.g., humanized antibodies, alone or as conjugates) refers to the dosage, route of administration, and frequency of administration for delaying the onset of cancer, reducing its severity, inhibiting further deterioration, and / or alleviating at least one sign or symptom. If the patient already has cancer, this regimen may be called a therapeutically effective regimen. If the patient has an increased risk of cancer relative to the general population but has not yet developed symptoms, this regimen may be called a preventatively effective regimen. In some cases, therapeutic or preventative effects can be observed in individual patients compared to historical controls or past experience with the same patient. In other cases, therapeutic or preventative effects can be demonstrated in preclinical or clinical trials compared to a control group of untreated patients.

[0266] Exemplary doses of monoclonal antibodies are 0.1 mg / kg to 50 mg / kg of patient body weight, more commonly 1 mg / kg to 30 mg / kg, 1 mg / kg to 20 mg / kg, 1 mg / kg to 15 mg / kg, 1 mg / kg to 12 mg / kg, or 1 mg / kg to 10 mg / kg, or 2 mg / kg to 30 mg / kg, 2 mg / kg to 20 mg / kg, 2 mg / kg to 15 mg / kg, 2 mg / kg to 12 mg / kg, or 2 mg / kg to 10 mg / kg, or 3 mg / kg to 30 mg / kg, 3 mg / kg to 20 mg / kg, 3 mg / kg to 15 mg / kg, 3 mg / kg to 12 mg / kg, or 3 mg / kg to 10 mg / kg. Exemplary doses of monoclonal antibodies or antibody-drug conjugates thereof are 1 mg / kg to 7.5 mg / kg, or 2 mg / kg to 7.5 mg / kg, or 3 mg / kg to 7.5 mg / kg of the subject's body weight, or 0.1-20, or 0.5-5 mg / kg of body weight (e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg), or 10-1500 or 200-1500 mg as a fixed dose. In some methods, patients are given a dose of at least 1.5 mg / kg, at least 2 mg / kg, or at least 3 mg / kg, every three weeks or longer. The dosage depends on the frequency of administration, the patient's condition and response to prior treatment (if any), whether the treatment is prophylactic or therapeutic, whether the disease is acute or chronic, and other factors.

[0267] The administration route can be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, local, intranasal, or intramuscular. Direct administration to the tumor site is also possible. Systemic circulation is preferably achieved via intravenous or subcutaneous administration. Intravenous administration can be performed, for example, by infusion over 30-90 minutes or a single bolus injection.

[0268] The frequency of administration depends on factors such as the half-life of the antibody or conjugate in circulation, the patient's condition, and the route of administration. Frequency can be daily, weekly, monthly, quarterly, or irregular intervals depending on changes in the patient's condition or the progression of the cancer being treated. Exemplary frequencies for intravenous administration range from twice a week to once a quarter during a continuous treatment period, although higher or lower frequencies are also possible. Other exemplary frequencies for intravenous administration are between once a week or three times every four weeks during a continuous treatment period, although higher or lower frequencies are also possible. For subcutaneous administration, exemplary frequencies range from daily to monthly, although higher or lower frequencies are also possible.

[0269] The number of doses administered depends on the nature of the cancer (e.g., whether acute or chronic symptoms occur) and the disease remission achieved with treatment. For acute symptoms or acute exacerbations of chronic symptoms, 1–10 doses are usually sufficient. Sometimes, for acute symptoms or acute exacerbations of chronic symptoms, a single bolus dose (or multiple doses) is sufficient. For relapses or acute exacerbations of acute disease, treatment may be repeated. For chronic disease, medication may be administered at regular intervals, such as weekly, bi-weekly, monthly, quarterly, or semi-annually, for at least 1 year, 5 years, or 10 years, or for the patient's lifetime.

[0270] Pharmaceutical compositions intended for parenteral administration are preferably sterile, substantially isotonic, and manufactured under GMP conditions. Pharmaceutical compositions may be provided in unit dosage forms (i.e., single-dose doses). Pharmaceutical compositions may be formulated with one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the chosen route of administration. For injection, antibodies may be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce discomfort at the injection site). The solution may contain formulation reagents such as suspending agents, stabilizers, and / or dispersants. Alternatively, antibodies may be in lyophilized form for use prior to preparation with a suitable carrier (e.g., sterile, pyrogen-free water). The antibody concentration in liquid formulations may be, for example, 1-100 mg / ml, such as 10 mg / ml.

[0271] The antibody therapy of the present invention can be combined with effective treatments for the treated disease, such as chemotherapy, radiotherapy, stem cell therapy, and surgery. Other useful classes of agents that can be administered together with the αvβ6 antibody and antibody-drug conjugates described herein include, for example, antibodies against other receptors expressed on cancer cells, anti-microtubule agents (such as oliquistatin), DNA minor groove binders, DNA replication inhibitors, alkylating agents (such as platinum complexes, such as cisplatin, mono(platinum), bis(platinum), and trinuclear platinum complexes, and carboplatin), anthracyclines, antibiotics, antifolate agents, antimetabolites, chemosensitizers, docamycins, etoposides, fluorinated pyrimidines, ionotropic agents, lexitropsins, nitrosoureas, platinols, pre-forming compounds, purine antimetabolites, puromycins, radiosensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, etc.

[0272] Treatment with anti-αvβ6 antibodies or antibody-drug conjugates, optionally alone or in combination with any other drugs or regimens mentioned above, can increase median progression-free survival or overall survival in cancer patients (e.g., non-small cell lung cancer (NSCLC) (squamous cell carcinoma and adenoma), head and neck cancer (including head and neck squamous cell carcinoma), esophageal cancer, breast cancer (including invasive breast cancer), ovarian cancer, bladder cancer (including urothelial carcinoma), skin cancer (squamous cell carcinoma or SCC), and kidney cancer (including hyaline cell carcinoma). This treatment, particularly in cases of relapsed or refractory cancer, increases the complete response rate, partial response rate, or objective response rate (complete + partial) of cancer patients by at least 30% or 40%, but preferably 50%, 60% to 70%, or even 100% or longer, compared to the same treatment (e.g., chemotherapy) without anti-αvβ6 antibodies alone or as conjugates. Furthermore, or alternatively, treatment including anti-αvβ6 antibodies alone or as conjugates (e.g., standard chemotherapy) can increase the complete response rate, partial response rate, or objective response rate (complete + partial) of cancer patients by at least 30% or 40%, but preferably 50%, 60% to 70%, or even 100%, compared to the same treatment (e.g., chemotherapy) without anti-αvβ6 antibodies alone or as conjugates.

[0273] Typically, in clinical trials (such as phase II, II / III, or III trials), the aforementioned increase in median progression-free survival and / or response rate in patients treated with standard therapy plus anti-αvβ6 antibodies, alone or as conjugates, is statistically significant, for example, at the p = 0.05, 0.01, or even 0.001 level, compared to a control group receiving standard therapy alone (or placebo). Complete and partial response rates are determined using objective criteria commonly used in cancer clinical trials, such as those listed or accepted by the National Cancer Institute and / or the Food and Drug Administration.

[0274] IX. Products and Reagent Kits

[0275] On the other hand, an article or kit is provided that includes the anti-αvβ6 antibody or anti-αvβ6 antibody-drug conjugate described herein. This article or kit may further include instructions for the use of the anti-αvβ6 antibody or anti-αvβ6 antibody-drug conjugate as described herein in the methods of the present invention. Therefore, in some embodiments, the article or kit includes the use of an anti-αvβ6 antibody or anti-αvβ6 antibody-drug conjugate as described herein in a method of treating a subject's cancer, such as non-small cell lung cancer (NSCLC) (squamous cell carcinoma and adenoma), head and neck cancer (including head and neck squamous cell carcinoma), esophageal cancer, breast cancer (including invasive breast cancer), ovarian cancer, bladder cancer (including urothelial carcinoma), skin cancer (squamous cell carcinoma or SCC), kidney cancer (including clear cell renal cancer, papillary renal cancer, and chromophobe renal cancer), cervical cancer, gastric cancer, prostate cancer (including prostate adenocarcinoma), endometrial cancer (including uterine sarcoma and endometrial cancer), rectal adenocarcinoma, thyroid cancer, colonic adenocarcinoma, gastric adenocarcinoma, and pancreatic cancer (including pancreatic adenocarcinoma), including administering an effective amount of the anti-αvβ6 antibody or anti-αvβ6 antibody-drug conjugate described herein to the subject. In some embodiments, the cancer is NSCLC. In a preferred embodiment, the cancer is head and neck cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used in a method of treating kidney cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used in a method of treating endometrial cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used in a method of treating gastric cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is skin cancer. In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the subject is a human being.

[0276] The product or reagent kit may further include a container. Suitable containers include, for example, bottles, vials (e.g., two-chamber vials), syringes (e.g., single-chamber or two-chamber syringes), and test tubes. In some embodiments, the container is a vial. The container can be formed from a variety of materials, such as glass or plastic. The container contains the formulation.

[0277] The formulation or kit may further include a label or packaging insert located on or attached to the container, which may indicate instructions for the preparation and / or use of the formulation. This label or packaging insert may further indicate that the formulation is intended for, or is intended for, subcutaneous, intravenous (e.g., intravenous infusion), or other routes of administration to treat cancers of a target group (e.g., non-small cell lung cancer (NSCLC) (squamous cell and adenocarcinoma), head and neck cancer (including head and neck squamous cell carcinoma), esophageal cancer, breast cancer (including invasive breast cancer), ovarian cancer, bladder cancer (including urothelial carcinoma), skin cancer (squamous cell carcinoma or SCC), renal cancer (including clear cell, papillary, and chromophobe renal cells), cervical cancer, gastric cancer, prostate cancer (including prostate adenocarcinoma), endometrial cancer (including uterine sarcoma and endometrial cancer), rectal adenocarcinoma, thyroid cancer, colonic adenocarcinoma, gastric adenocarcinoma, pancreatic cancer (including pancreatic adenocarcinoma)). The container containing the formulation may be a single-use vial or a reusable vial, allowing for repeated administration of the reconstituted formulation. The product or kit may also include a second container containing a suitable diluent. Products or kits may also include other materials desired from a commercial, therapeutic, and user perspective, including additional buffers, diluents, filters, needles, syringes, and packaging inserts with instructions for use.

[0278] Optionally, the article or kit further includes a container containing a second drug, wherein the anti-αvβ6 antibody or anti-αvβ6 antibody-drug conjugate is the first drug, and the article or kit further includes instructions for use of the second drug in an effective amount for treating the subject, located on a label or packaging insert. In some embodiments, the second drug is used to eliminate or reduce the severity of one or more adverse events.

[0279] In some embodiments, the anti-αvβ6 antibody or anti-αvβ6 antibody-drug conjugate is present in the container as a lyophilized powder. In some embodiments, the lyophilized powder is placed in an airtight container, such as a vial, ampoule, or pouch, indicating the amount of active agent. When administering the drug by injection, for example, a sac of sterile water for injection or saline may optionally be provided as part of the kit for mixing with the drug component prior to administration. If desired, such a kit may also include one or more of various conventional pharmaceutical kit components, such as containers having one or more pharmaceutically acceptable carriers, other containers, etc., as will be apparent to those skilled in the art. The kit may also include printed instructions, as inserts or labels, indicating the amount of component to be administered, instructions for administration, and / or instructions for mixing components.

[0280] X. Other Applications

[0281] The anti-αvβ6 antibodies described herein, such as humanized anti-αvβ6 antibodies, can be used to detect αvβ6 in clinical diagnostic, therapeutic, or research settings. αvβ6 expression in cancer provides an indication that the cancer can be treated with the antibodies of this invention. These antibodies can also be sold as research reagents for laboratory studies to detect αvβ6-loaded cells and their responses to various stimuli. In this use, the monoclonal antibody can be labeled with fluorescent molecules, white spiral-labeled molecules, enzymes, or radioisotopes, and can be provided in kit form with all the necessary reagents for αvβ6 assay. The antibodies described herein can be used to detect αvβ6 protein expression and determine whether cancer is suitable for treatment with αvβ6 ADCs.

[0282] All patent applications, websites, other publications, registration numbers, etc., cited above or below are incorporated herein by reference in their entirety for all purposes, to the same extent that each individual item is specifically and individually indicated as being incorporated by reference. If different versions of a sequence are associated with a registration number at different times, the version associated with the registration number on the effective filing date of this application refers to the earlier of the actual filing date or the priority filing date (if applicable) that refers to the registration number. Similarly, if different versions of publications, websites, etc., are published at different times, unless otherwise stated, the most recently published version on the effective filing date of this application refers to the version. Unless specifically indicated, any feature, step, element, implementation, or aspect of the invention may be used in any other combination with the others. While the invention has been described in detail by way of illustration and example for ease of understanding, it will be apparent that certain changes and modifications should fall within the scope of the appended claims. Example

[0283] Material

[0284] The cell lines described in the examples below were maintained in cultures under the conditions specifically indicated by the American Type Culture Collection (ATCC), the National Cancer Institute (NCI), or the German Collection of Microbial Cultures (DMSZ) in Braunschweig, Germany. The SW780, Detroit 562, HPAFII, and BxPC3 cell lines were obtained from the ATCC. TM 293-F (InVitrogen) human epithelial kidney cells and corresponding transfection reagents were maintained according to the manufacturer's instructions. Cell culture reagents were obtained from Invitrogen (Carlsbad, CA), Molecular Devices (Sun Valley, CA), and other suppliers. Secondary antibody reagents were purchased from Jackson ImmunoResearch Laboratories (Sigrove, PA). Recombinant αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8 were purchased from R&D Systems (Minneapolis, Minnesota). FreeStyle TM 293-F cells expressing endogenous integrin αv were stably transfected with full-length cDNA encoding human, cynomolgus, or mouse integrin β6, generating HEK293F:huβ6, HEK293F:cynoβ6, and HEK293F:muβ6 cell lines, respectively. HEK293F cells transfected with the empty vector (HEK293F:vector) served as a negative control. Mouse 3T3 and FDC-P1 cells expressing endogenous mouse integrin αv were transfected with full-length cDNA clones of human and mouse integrin β6, generating 3T3:huβ6 and FDC-P1:muβ6, respectively.

[0285] method:

[0286] Production of 2A2 antibodies

[0287] ICR (CD-1) mice were administered an intraperitoneal injection of ~5x10 6Three immunizations were performed using 3T3:huβ6 transfectants. Three days prior to fusion, mice received a final injection of purified recombinant human αvβ6, administered intravenously (6 μg) and intraperitoneally (30 μg). Lymphocytes harvested from the spleen and lymph nodes were fused into P3X63Ag8.653 myeloma cells using polyethylene glycol. The fused cells were convalescent overnight in hybridoma growth medium (IMDM containing 4 mM glutamine, 10% Fetal Clone I, 10% cloning factor, and penicillin / streptomycin). After convalescence, the cells were centrifuged and then seeded in a semi-solid medium consisting of CloneMatrix medium supplemented with hybridoma growth medium, plus HAT for hybridoma selection and CloneDetect for IgG production. The hybridomas were incubated at 37°C for 10 days. On day 10, IgG-producing hybridoma clones were selected using ClonePixFL (Molecular Instruments) and transferred to 96-well plates containing IgG-depleted hybridoma growth medium supplemented with HT. Hybridoma culture supernatants were screened on 293F:huβ6 transfectants and detected using Alexifluor-647-labeled secondary antibodies to identify positive clones. Plates were read in an FMAT 8200 (Applied Biosystems). Hybridomas bound to 293F:huβ6 and 293F:cynoβ6 but not to the 293F:vector were amplified for direct conjugation to the drug-linker. The directly conjugated antibody combinations were tested in binding and cytotoxicity assays.

[0288] LAP blocking ELISA

[0289] 96-well microtiter plates (Nunc) were coated overnight at 4°C with 0.3 μg / mL recombinant human latency-associated peptide (rhuLAP) (in-house manufactured; batch 09-19-09DS) in 1x PBS. After removing the coating, the plates were blocked with 3% BSA in Tris-buffered saline (TBS) for one hour at room temperature, and then washed five times with PBS + 0.05% Tween-20 (PBST) before use. In separate conical-bottom 96-well plates, 0.25 μg / mL recombinant human (rhu)αvβ6-biotin (in-house manufactured rhuαvβ6-biotin, batch 171030A) was pre-incubated with an increased concentration of purified antibody in TBS buffer containing 1 mM CaCl2, 1 mM MgCl2, and 1 mg / mL BSA for one hour at room temperature. The antibody / αvβ6-biotin mixture was then transferred to LAP-coated plates and incubated at room temperature for 1 hour. Plates were washed as described above and incubated at room temperature for 1 hour with 50 μL / well of peroxidase-conjugated streptavidin (Jackson Immunological Research Laboratory, No. 016-030-084) diluted 1:1000 in TBS + 1 mg / mL BSA. Binding proteins and signals were detected by incubation for 5.5 min using TMB (Invitrogen, No. 00-2023), followed by quenching with 1N H2SO4 (Fisher, No. SA212-1). Plates were immediately read using a 450 nm plate reader (Molecular Devices Vmax Dynamic Microplate Reader). Data were exported to Microsoft Excel and analyzed using GraphPad Prism v5.03.

[0290] Competitive binding assay - humanized 2A2 antibody variant

[0291] The competitive binding assay was performed using the 293F:huβ6 cell line. 0.1 x 10⁻⁶ cells were placed on ice. 6Cells expressing the antigen were aliquoted into each well of a 96-well V-bottom plate. Cells were incubated for 1 hour in FACS buffer (Ttis-buffered saline, 2% fetal bovine serum, 0.5 mM MnCl2, 0.02% NaN3) with 2 nM Alexa Fluor-647-labeled m2A2 and increasing concentrations (from 0.03 to 500 nM) of unlabeled humanized 2A2 variant antibody. Cells were pelleted and washed three times with TBS / FBS. The pelleted cells were then resuspended in 125 μL of TBS / FBS. The binding fluorescence signal was detected using a Becton Dickinson Biosciences LSR II (San Jose, CA). The percentage of saturated fluorescence signal was used to determine the percentage of labeled humanized 2A2 antibody binding, and the EC50 was subsequently derived by fitting the data to a sloped sigmoid dose-response curve using GraphPad software (La Jolla, CA).

[0292] Competitive binding experiment -- Humans and macaques αvβ6

[0293] Competition binding assays were performed using 293F:huβ6 and HEK293F:cynoβ6 cell lines. 0.1 x 10⁻⁶ cells were placed on ice. 6 Cells expressing the antigen were aliquoted into each well of a 96-well V-bottom plate. Cells were incubated for 1 hour in a buffer (Tris-buffered saline, 2% fetal bovine serum, 0.5 mM MnCl2, 0.02% NaN3) with 2 nM Alexa Fluor-647-labeled humanized 2A2 HCLG and increased concentrations (from 4 pM to 1 μM) of unlabeled humanized 2A2 HCLG antibody and h2A2-Mdpr-PEG(12)-gluc-MMAE(8). Cells were pelleted and washed three times with TBS / FBS. The pelleted cells were then resuspended in 125 μL TBS / FBS. Fluorescence binding was detected using a Becton Dickinson Biosciences LSR II (San Jose, CA) instrument. The percentage of saturated fluorescence signal was used to determine the percentage of labeled humanized 2A2 antibody bound to cells, and the EC50 was then derived by fitting the data into a variable-slope S-shaped dose-response curve using GraphPad software (La Jolla, California).

[0294] αvβ6 saturation binding experiment -- human and macaque αvβ6

[0295] Saturation binding studies were performed using the following cell lines expressing the antigen: 293F:huβ6 and 293F:cynoβ6. 0.1 x 10⁻⁶ cells were used. 6Cells expressing the antigen were aliquoted into each well of a 96-well V-bottom plate. m2A2 and h2A2 HCLG were directly labeled with Alexa Fluor-647 and added to the plate at concentrations ranging from 6 pM to 340 nM in a buffer solution (Tris-buffered saline, 2% fetal bovine serum, 0.5 μM MnCl2, 0.02% NaN3). Cells were incubated for 1 hour, then precipitated and washed three times with TBS. The precipitated cells were resuspended in 120 μL of TBS. The binding fluorescence signal was detected using a Becton Dickinson Biosciences LSRII (San Jose, CA). EC50 was calculated using GraphPad software (La Jolla, CA).

[0296] ELISA

[0297] 96-well Maxisorb plates (Nunc) were coated overnight at 4°C with 1 μg / mL recombinant human αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8 (R&D Systems, Minnesota) diluted in 50 mM carbonate buffer (Sigma, Missouri). Plates were washed with PBS + 0.05% Tween 20 (PBS-T). After removing the wash buffer, plates were blocked for 2 hours at room temperature with TBS blocking buffer (TBS, 0.05% Tween 20, 1% BSA). Plates were washed and then incubated for 2 hours with humanized 2A2 antibody diluted to 1 pM-67 nM (10 μg / mL) in TBS binding buffer (TBS, 0.05% Tween 20, 1% BSA, 1 mM MnCl2). Wash the plates, incubate with 1:5000 diluted anti-human Fc-HRP (Jackson Immunological Research Laboratory, Pennsylvania, PAN) for 1 hour, wash again, and then incubate with TMB substrate for 5 minutes. Stop the reaction with 1M HCl. Read the absorbance at 450 nm using a Fusion HT plate reader (Perkin Elmer, Walsham, MA).

[0298] Quantitative flow cytometry analysis

[0299] Mouse αvβ6 mAb was used as the primary antibody, and the αvβ6 copy number on the cell surface was indirectly determined using a DAKO QiFiKit flow cytometer according to the manufacturer's (DAKO A / S, Gloucestershire, Denmark) instructions, and evaluated using a Becton Dickinson FACScan flow cytometer.

[0300] In vitro cytotoxicity assay

[0301] Tumor cells were incubated with the anti-αvβ6 antibody-drug conjugate at 37°C for 96 hours. [Use / Other methods / etc.] Cell viability was determined by a luminescence assay (Promega Corporation, Madison, Wisconsin), and results were measured on an EnVision multi-label plate reader (Platinum Elmer, Walsham, Massachusetts). Results are expressed as IC50. s0 The report defines the concentration that results in half of the maximum growth inhibition during the titration process.

[0302] Production of antibody-drug conjugates

[0303] Antibody-drug conjugates against αvβ6 antibodies were prepared as described in US20050238649 and WO2015 / 057699. The drug linkers vcMMAE (also known as 1006) and mcMMAF are both described in US20050238649, which are incorporated herein by reference for all purposes. The drug linker MDpr-Lys(PEGx)-glucuronide-MMAE linker is described in WO2015 / 057699, which is incorporated herein by reference for all purposes.

[0304] In vivo activity study

[0305] In cell line-derived xenotransplantation therapeutic experiments, 5x10 6 ATCC cells were subcutaneously injected into 5-8 female nude (nu / nu) mice (Envigo) for BxPC3, Detroit 562, HPAF-II, and SW780 studies. Mice were randomly assigned to study groups, and tumors were introduced once they reached approximately 100 mm. 3 The test drug dose was administered via intraperitoneal injection. When the tumor volume reached approximately 500-1000 mm... 3 Animals were euthanized. Tumor volume was calculated using the formula (volume = 1 / 2 x length x width x width). Mice showing sustained regression were terminated approximately 40-65 days post-implantation. No weight loss or treatment-related toxicity was observed in mice treated with any experimental materials in all xenotransplantation studies. All animal procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee and in facilities accredited by the Laboratory Animal Care Assessment and Accreditation Association.

[0306] In addition to cell line-derived xenografts, the antitumor activity of h2A2 vcMMAE ADC against patient-derived xenograft (PDX) models of non-small cell lung cancer (NSCLC) was investigated using models maintained by Champions Oncology (Hackensack River, NJ). These PDX models included NSCLC samples of glandular and squamous histology. These models were established by implantation into immunocompromised mice and allowed to grow to 150–300 mm. 3 The tumor volume was measured, and mice were then randomly assigned to treatment and control groups, receiving either h2A2 vcMMAE or the unbound control h00 vcMMAE ADC dose. Mice were given 3 mg / kg of ADC weekly for a total of three doses. Tumor volume was measured twice weekly for 28 days after the first dose, or until the tumor reached 1500 mm². 3 The volume.

[0307] The antitumor activity of the h2A2 vcMMAE ADC was further evaluated in ovarian cancer PDX models maintained by Champions Oncology (Hackensack River, NJ). These models were established by implantation into immunocompromised mice and allowed to grow to 150–300 mm. 3 The tumor volume was measured, and mice were then randomly assigned to treatment and control groups and administered ADC. Mice were given 5 mg / kg of ADC weekly for a total of three doses. Tumor volume was measured twice weekly for 28 days after the first dose, or until the tumor reached 1500 mm. 3 The volume, at most 60 days.

[0308] result

[0309] The mouse clone m2A2 was selected from the hybridoma group because it exhibited cytotoxic activity as an ADC against various αvβ6-positive tumor cell lines and showed considerable affinity for antigens in human and macaque forms. The specificity of mouse 2A2 was confirmed in FMAT and flow cytometry binding studies, where the antibody was shown to bind to the 293F:huβ6 transfectant but not to the αvβ5-positive parental line (293F:vector). Integrin avb6 has been shown to be a receptor for the RGD site in fibronectin (Weinacker et al., 1994), tendinin (Prieto et al., 1993), fibronectin (Huang et al., 1998), and latent-related peptide (LAP) (Munger et al., 1999). Binding of integrin avb6 to LAP can induce sterically hindered activation of transforming growth factor β1 (TGFb) (Munger et al., 1999). Latent-related peptide (LAP) blocking assays were performed. Figure 1The results showed that m2A2 (SG-44.2A2) could not block LAP, unlike the anti-αvβ6 antibody m15H3 (SG-42.15H3) (see WO2013 / 123152) and the positive control 10D5. This indicates that 2A2 antibodies can be delivered independently of ligand binding. The negative control was an unbound IgG control, and SG-44.8B9, SG-33.20B8, SG-44.32A6, and SG-44.34D6 were other clones selected from the hybridoma group.

[0310] binding of mouse antibodies

[0311] Using genetically engineered cell lines (293F: huβ6, 293F: cynoβ6), saturation binding studies were conducted to determine the EC50 of mouse monoclonal antibody 2A2 binding to human and rhesus monkey αvβ6. Figure 2 Genetically engineered cell lines express endogenous αv, which pairs with recombinant β6 chains to produce a heterodimeric receptor composed of endogenous αv and recombinant β6.

[0312] Design and selection of humanized antibodies

[0313] In this embodiment, the starting or donor antibody for humanization is a mouse 2A2 antibody. Genomic sequences of the heavy chain provided by IGHV1-46 and IGHJ4 and genomic sequences of the light chain provided by IGKV1D-33 and IGKJ2 were used.

[0314] During the humanization process, 10 positions (H2, H28, H48, H67, H69, H71, H73, H74, H78, H93) were identified in the heavy chain framework. Figure 3 Two locations (L69 and L71) were identified within the light chain framework; Figure 4 At these locations, the human receptor sequence differs from the donor sequence, which may affect antibody binding because it directly contacts the antigen, affecting the conformation of the CDR or the packaging between the heavy and light chains. Four humanized heavy chain variants (vHA, vHB, vHC, vHD) exist. Figure 3 ) and eight humanized light chain variants (vLA, vLB, vLC, vLD, vLE, vLF, vLG, vLH); Figure 4 These reversion mutations are incorporated into different arrangements of these positions. Table 1-4 lists these reversion mutations. The remaining framework positions are occupied by residues of the human receptor sequence.

[0315] Table 1: Humanization mutations of h2A2 heavy chain variants

[0316]

[0317] Table 2: Specific mouse framework mutations in h2A2 heavy chain variants

[0318]

[0319]

[0320] Table 3: Humanization mutations of the h2A2κ light chain variant

[0321]

[0322] Table 4: Specific mouse framework mutations in h2A2κ light chain variants

[0323] variants 69 71 people% hvLA 84.2 hvLB R Y 82.1 hvLC R Y 83.2 hvLD Y 83.2 hvLE R Y 83.2 hvLF Y 85.3 hvLG R Y 83.2 hvLH R 83.2

[0324] Then humanized antibodies are expressed, representing the combined arrangement of these humanized heavy and light chain variants. Figure 5-7 The competitive binding curves of the resulting humanized antibody variants (along with mouse 2A2 antibody and human-mouse chimeric antibody) to human αvβ6 are shown. Four humanized variants were selected for further analysis in in vitro activity assays. The humanized variants HCLE, HCLG, HCLH, HALG, and the humanized anti-αvβ6 antibody 15H3-HTLC (each conjugated to a drug-linker. Via, n=12, R) PR =H,R 21 The in vitro anticancer activity of αvβ6 (=CH3) was determined by cytotoxicity assays in four cell lines expressing different levels of αvβ6 (pancreatic cancer HPAFII and BxPC-3 cells, head and neck cancer Detroit 562 cells, and bladder cancer SW780 cells) using quantitative flow cytometry analysis. The results are shown in Table 5.

[0325] Table 5: In vitro activity assays (x50, nM)

[0326] BxPC3 Detroit-562 HAPF-II SW780 HCLE 6 17.6 14.8 7.9 HCLG 5.1 13.2 10 5.2 HCLH 15.5 28.4 28.7 11.6 HALG 8.2 21.2 17.9 19.6 m2A2 11.1 19.4 15.6 11.7 h15H3-HTLC 8.4 305 8.9 13.8

[0327] Humanized variants containing the heavy chain variant hvHC and the light chain variant hvLG (h2A2 HCLG) were selected for further research.

[0328] The binding of h2A2 HCLG to human and macaque αvβ6 was confirmed through saturation binding. Figure 8 This included parental mouse 2A2 as a reference to demonstrate its comparable binding to humanized variants. The binding of h2A2 HCLG to human and rhesus monkey αβ6 was also confirmed by competitive binding with fluorescently labeled mouse 2A2. Figure 9The assay also included the binding of an ADC prepared with h2A2 HCLG and SGD-5088 drug-linker, by reducing interchain disulfide and conjugating to 8 copies of the drug-linker. The conjugation process had no effect on binding to αvβ6 in humans or macaques. The binding specificity of h2A2 HCLG was also confirmed by ELISA, where the antibody bound to recombinant human αvβ6 but not to αvβ1, αvβ3, αvβ5, or αvβ8. Figure 10 ).

[0329] In vitro anticancer activity of h2A2 ADC

[0330] The in vitro anticancer activity of the humanized variant HCLG conjugated to vcMMAE was determined by quantitative flow cytometry based on a cytotoxicity assay in four cell lines expressing different levels of αvβ6 (pancreatic cancer HPAFII and BxPC-3 cells, head and neck cancer Detroit 562 cells, and bladder cancer SW780 cells). Figure 11 The results showed that the humanized 2A2 anti-αvβ6 ADC exhibited cytotoxicity in these experiments.

[0331] In vivo anticancer activity of h2A2 ADC

[0332] Using the same four cell lines used in in vitro testing, the in vivo antitumor activity of humanized 2A2 HCLG antibodies conjugated with vcMMAE (average 4 doses per antibody) was demonstrated. Figure 12-15 Significant tumor growth delay or regression was observed compared with untreated and non-binding control ADCs. h2A2 HCLG-1006(4) refers to an antibody-drug conjugate of the humanized HCLG form of parental mouse antibody 2A2, with an average of 4 vcMMAE drug linker molecules per antibody. h00-1006(4) refers to an antibody-drug conjugate of the non-binding control antibody, with an average of 4 vcMMAE drug linker molecules per antibody.

[0333] In the PDX model of NSCLC, humanized 2A2 HCLG antibodies conjugated with vcMMAE (each antibody containing an average of 4 drugs) also showed antitumor activity. Figure 16 Significant tumor growth delay or regression was observed compared with untreated and non-binding control ADCs. h2A2 HCLG-1006(4) refers to an antibody-drug conjugate of the humanized HCLG form of parental mouse antibody 2A2, with an average of 4 vcMMAE drug linker molecules per antibody. h00-1006(4) refers to an antibody-drug conjugate of the non-binding control antibody, with an average of 4 vcMMAE drug linker molecules per antibody.

[0334] In a PDX model of ovarian cancer, humanized 2A2 HCLG antibodies conjugated with vcMMAE (each antibody containing an average of 4 drugs) also showed antitumor activity. Figure 17 Significant tumor growth delay or slight tumor reduction was observed compared to untreated tumors. h2A2 HCLG-1006(4) refers to an antibody-drug conjugate of the humanized form of parental mouse antibody 2A2 HCLG, with an average of 4 vcMMAE drug linker molecules per antibody.

[0335] When with drug-connector Via (n=12, R) PR =H,R 21 When H2A2HCLG was conjugated with DAR at 8, its antitumor activity was compared with that of H15H3. The study protocol was the same as the cell line-derived xenograft models described above, using HPAFII and BxPC3 cell lines. Animals were administered a single dose of 3 mg / kg of h2A2HCLG, h15H3, or a non-targeted control (h00) ADC. Figure 18 In both models, the h2A2 HCLG ADC showed sustained regression of implanted tumors, while the h15H3 ADC showed delayed growth.

[0336] sequence

[0337] SEQ ID NO: 1-m2A2 vH

[0338]

[0339] SEQ ID NO: 2-mIGHV1-39 (closest mouse strain V-gene)

[0340]

[0341] SEQ ID NO: 3-hIGHV1-46 / HJ4

[0342]

[0343] SEQ ID NO: 4-h2A2 vHA

[0344]

[0345] SEQ ID NO: 5-h2A2 vHB

[0346]

[0347] SEQ ID NO: 6-h2A2 vHC

[0348]

[0349] SEQ ID NO: 7-h2A2 vHD

[0350]

[0351] SEQ ID NO: 8-m2A2 vL

[0352]

[0353] SEQ ID NO: 9-mIGKV12-89 (closest mouse strain V-gene)

[0354]

[0355] SEQ ID NO: 10-hIGKV1D-33 / KJ2

[0356]

[0357] SEQ ID NO: 11-h2A2 vLA

[0358]

[0359] SEQ ID NO: 12-h2A2 vLB

[0360]

[0361] SEQ ID NO: 13-h2A2 vLC

[0362]

[0363] SEQ ID NO: 14-h2A2 vLD

[0364]

[0365] SEQ ID No: 15-h2A2 vLE

[0366]

[0367] SEQ ID NO: 16-h2A2 vLF

[0368]

[0369] SEQ ID NO: 17-h2A2 vLG

[0370]

[0371] SEQ ID NO: 18-h2A2 vLH

[0372]

[0373] SEQ ID NO: 19-h2A2 HA heavy chain

[0374]

[0375] SEQ ID NO: 20-h2A2 HB heavy chain

[0376]

[0377]

[0378] SEQ ID NO: 21-h2A2 HC heavy chain

[0379]

[0380] SEQ ID NO: 22-h2A2 HD heavy chain

[0381]

[0382] SEQ ID NO: 23-h2A2 LA light chain

[0383]

[0384] SEQ ID NO: 24-h2A2 LB light chain

[0385]

[0386] SEQ ID NO: 25-h2A2 LC light chain

[0387]

[0388] SEQ ID NO: 26-h2A2 LD light chain

[0389]

[0390] SEQ ID NO: 27-h2A2 LE light chain

[0391]

[0392] SEQ ID NO: 28-h2A2 LF light chain

[0393]

[0394]

[0395] SEQ ID NO: 29-h2A2 LG Light Chain

[0396]

[0397] SEQ ID NO: 30-h2A2 LH light chain

[0398]

[0399]

[0400]

[0401] sequence list <110> Seagen Inc. <120> Anti-AVB6 antibodies and antibody-drug conjugates <130> AVB6-00212PC <150> US 62 / 943,959 <151> 2019-12-05 <150> 63 / 012,584 <151> 2020-04-20 <160> 45 <170> PatentIn version 3.5 <210> 1 <211> 116 <212> PRT <213> Artificial sequence <220> <223> m2A2 vH <400> 1 Glu Phe Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Val Asn Trp Val Lys Gln Ser Asn Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Val Ile Asn Pro Lys Tyr Gly Thr Thr Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Pro Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Leu Asn Ala Trp Asp Tyr Trp Gly Gln Gly Ala Ser Val 100 105 110 Thr Val Ser Ser 115 <210> 2 <211> 98 <212> PRT <213> Synthetic Sequence <220> <223> mIGHV1-39 (Closest murine germline V-gene) <400> 2 Glu Phe Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Met Asn Trp Val Lys Gln Ser Asn Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Val Ile Asn Pro Asn Tyr Gly Thr Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Gln Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> 3 <211> 113 <212> PRT <213> Artificial sequence <220> <223> hIGHV1-46 / HJ4 <400> 3 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Looking <210> 4 <211> 116 <212> PRT <213> artificial sequence <220> <223> h2A2 vHA <400> 4 Gln Phe Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Val Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Val Ile Asn Pro Lys Tyr Gly Thr Thr Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Leu Asn Ala Trp Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 5 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> h2A2 vHB <400> 5 Gln Phe Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Val Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Val Ile Asn Pro Lys Tyr Gly Thr Thr Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Pro Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Leu Asn Ala Trp Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 6 <211> 116 <212> PRT <213> artificial sequence <220> <223> h2A2 vHC <400> 6 Gln Phe Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Val Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Asn Pro Lys Tyr Gly Thr Thr Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Leu Asn Ala Trp Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 7 <211> 116 <212> PRT <213> artificial sequence <220> <223> h2A2 vHD <400> 7 Gln Phe Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr i 20 25 30 Asn Val Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Asn Pro Lys Tyr Gly Thr Thr Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Pro Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Leu Asn Ala Trp Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 8 <211> 107 <212> PRT <213> Artificial sequence <220> <223> m2A2 vL <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Arg Lys Gln Gly Lys Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Met Ser Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Gln Tyr Ser Phe Lys Ile Ser Ser Leu His Pro 65 70 75 80 Asp Asp Val Ala Thr Tyr Tyr Cys Gln Asn Val Leu Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 9 <211> 95 <212> PRT <213> Artificial Sequence <220> <223> mIGKV12-89 (Closest murine germline V-gene) <400> 9 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Arg Lys Gln Gly Lys Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Met Ser Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Gln Tyr Ser Leu Lys Ile Ser Ser Leu His Pro 65 70 75 80 Asp Asp Val Ala Thr Tyr Tyr Cys Gln Asn Val Leu Ser Thr Pro 85 90 95 <210> 10 <211> 107 <212> PRT <213> artificial sequence <220> <223> hIGKV1D‑33 / KJ2 <400> 10 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 11 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> h2A2 vLA <400> 11 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Asn Val Leu Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 12 <211> 107 <212> PRT <213> Synthetic Sequence <220> <223> h2A2 vLB <400> 12 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Asn Val Leu Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 13 <211> 107 <212> PRT <213> Synthetic Sequence <220> <223> h2A2 vLC <400> 13 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Asn Val Leu Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 14 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> h2A2 vLD <400> 14 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Asn Val Leu Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 15 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> h2A2 vLE <400> 15 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Asn Val Leu Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 16 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> h2A2 vLF <400> 16 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Asn Val Leu Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 17 <211> 107 <212> PRT <213> Artificial sequence <220> <223> h2A2 vLG <400> 17 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Asn Val Leu Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 18 <211> 107 <212> PRT <213> Artificial sequence <220> <223> h2A2 vLH <400> 18 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Asn Val Leu Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 19 <211> 446 <212> PRT <213> Artificial sequence <220> <223> h2A2 HA heavy chain <400> 19 Gln Phe Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Val Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Val Ile Asn Pro Lys Tyr Gly Thr Thr Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Leu Asn Ala Trp Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 20 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> h2A2 HB heavy chain <400> 20 Gln Phe Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Val Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Val Ile Asn Pro Lys Tyr Gly Thr Thr Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Pro Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Leu Asn Ala Trp Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 21 <211> 446 <212> PRT <213> Synthetic Sequence <220> <223> h2A2 HC Heavy Chain <400> 21 Gln Phe Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Val Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Asn Pro Lys Tyr Gly Thr Thr Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Leu Asn Ala Trp Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 22 <211> 446 <212> PRT <213> Artificial Sequence<​​​​​​Gln Phe Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Val Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Asn Pro Lys Tyr Gly Thr Thr Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Pro Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Leu Asn Ala Trp Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 23 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> h2A2 LA Light Chain <400> 23 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Asn Val Leu Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 24 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> h2A2 LB Light Chain <4​​​​​​​​​​​​​​​​​​​​​Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Asn Val Leu Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 25 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> h2A2 LC light chain <400> 25 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Asn Val Leu Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 26 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> h2A2 LD Light Chain <400> 26<l Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 6θ Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Asn Val Leu Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 27 <211> 214 <212> PRT <213> Synthetic Sequence <220> <223> h2A2 LE Light Chain <400> 27 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Asn Val Leu Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 28 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> h2A2 LF light chain <400> 28 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Asn Val Leu Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 !60 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 29 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> h2A2 LG light chain <400> 29 It should be noted that there seems to be an "!" in the original text at position 160 which might be an error. The translation is done as accurately as possible based on the provided text. Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Asn Val Leu Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 30 [[ID=Z18]]<211> 214 <212> PRT <213> Artificial sequence <220> <223> h2A2 LH light chain <400> 30 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Asn Val Leu Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 31 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> HA, HB, HC, HD CDR1, KABAT <400> 31 Asp Tyr Asn Val Asn 1 5 <210> 32 <211> 17 <212> PRT <213> Artificial sequence <220> <223> HA, HB, HC, HD CDR2, KABAT <400> 32 Val Ile Asn Pro Lys Tyr Gly Thr Thr Arg Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 33 <211> 7 <212> PRT <213> Artificial sequence <220> <223> HA, HB, HC, HD CDR3, KABAT <400> 33 Gly Leu Asn Ala Trp Asp Tyr 1 5 <210> 34 <211> 8 <212> PRT <213> Artificial sequence <220> <223> HA, HB, HC, HD CDR1, IMGT <400> 34 Gly Tyr Ser Phe Thr Asp Tyr Asn 1 5 <210> 35 <211> 8 <212> PRT <213> Artificial sequence <220> <223> HA, HB, HC, HD CDR2, IMGT <400> 35 Ile Asn Pro Lys Tyr Gly Thr Thr 1 5 <210> 36 <211> 9 <212> PRT <213> Artificial sequence <220> <223> HA, HB, HC, HD CDR3, IMGT <400> 36 Thr Arg Gly Leu Asn Ala Trp Asp Tyr 1 5 <210> 37 <211> 11 <212> PRT <213> Artificial sequence <220> <223> LA, LB, LC, LD, LG, LH CDR1, KABAT <400> 37 Gly Ala Ser Glu Asn Ile Tyr Gly Ala Leu Asn 1 5 10 <210> 38 <211> 7 <212> PRT <213> Artificial sequence <220> <223> LA, LB, LD, LE, LH CDR2, KABAT <400> 38 Gly Ala Thr Asn Leu Ala Asp 1 5 <210> 39 <211> 9 <212> PRT <213> Artificial sequence <220> <223> LA, LB, LC, LD, LE, LF, LG, LH CDR3, KABAT <400> 39 Gln Asn Val Leu Thr Thr Pro Tyr Thr 1 5 <210> 40 <211> 11 <212> PRT <213> Artificial sequence <220> <223> LE, LF CDR1, KABAT <400> 40 Gln Ala Ser Glu Asn Ile Tyr Gly Ala Leu Asn 1 5 10 <210> 41 <211> 7 <212> PRT <213> Artificial sequence <220> <223> LC, LF CDR2, KABAT <400> 41 Gly Ala Thr Asn Leu Ala Thr 1 5 <210> 42 <211> 7 <212> PRT <213> Artificial sequence <220> <223> LG CDR2, KABAT <400> 42 Gly Ala Thr Asn Leu Glu Asp 1 5 <210> 43 <211> 6 <212> PRT <213> Artificial sequence <220> <223> LA, LB, LC, LD, LE, LF, LG, LH CDR1, IMGT <400> 43 Glu Asn Ile Tyr Gly Ala 1 5 <210> 44 <211> 3 <212> PRT <213> Artificial sequence <220> <223> LA, LB, LC, LD, LE, LF, LG, LH CDR2, IMGT <400> 44 Gly Ala Thr 1 <210> 45 <211> 9 <212> PRT <213> Artificial sequence <220> <223> LA, LB, LC, LD, LE, LF, LG, LH CDR3, IMGT <400> 45 Gln Asn Val Leu Thr Thr Pro Tyr Thr 1 5

Claims

1. An isolated anti-αvβ6 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: (i) The amino acid sequence is CDR-H1 of SEQ ID NO:31; (ii) The amino acid sequence is CDR-H2 of SEQ ID NO:32; as well as (iii) The amino acid sequence is CDR-H3 of SEQ ID NO:33; and The light chain variable region includes: (i) The amino acid sequence is CDR-L1 of SEQ ID NO:37; (ii) The amino acid sequence is CDR-L2 of SEQ ID NO:42; and (iii) The amino acid sequence is CDR-L3 of SEQ ID NO:39 The CDR mentioned above is determined by Kabat.

2. An isolated anti-αvβ6 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: (i) The amino acid sequence is CDR-H1 of SEQ ID NO:34; (ii) The amino acid sequence is CDR-H2 of SEQ ID NO:35; as well as (iii) The amino acid sequence is CDR-H3 of SEQ ID NO:36; and The light chain variable region includes: (i) The amino acid sequence is CDR-L1 of SEQ ID NO:43; (ii) CDR-L2 with the amino acid sequence SEQ ID NO:44; and (iii) CDR-L3 with the amino acid sequence SEQ ID NO:45 The CDR mentioned above is determined by IMGT.

3. The antibody or antigen-binding fragment as described in claim 1 or 2, wherein the antibody is humanized.

4. The antibody or antigen-binding fragment of claim 1 or 2, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:6, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

17.

5. The antibody or antigen-binding fragment of claim 1 or 2, wherein the heavy chain has an amino acid sequence comprising SEQ ID NO:21 and the light chain has an amino acid sequence comprising SEQ ID NO:

29.

6. The antibody or antigen-binding fragment of claim 1 or 2, wherein the antibody or antigen-binding fragment is an antigen-binding fragment selected from the group consisting of: Fab, Fab', F(ab')2, Fab'-SH, Fv, biantibody, linear antibody, and single-chain antibody fragment.

7. The antibody or antigen-binding fragment of claim 1 or 2, wherein the heavy chain variable region of the antibody is fused to the heavy chain constant region, and the light chain variable region is fused to the light chain constant region.

8. The antibody or antigen-binding fragment of claim 7, wherein the heavy chain constant region is the heavy chain constant region of the IgG1 isotype.

9. An antibody-drug conjugate comprising an antibody or antigen-binding fragment conjugated to a cytotoxic or cell-inhibiting agent as described in any one of claims 1-8.

10. The antibody-drug conjugate of claim 9, wherein the antibody or antigen-binding fragment is conjugated to a cytotoxic or cell-inhibiting agent via a linker.

11. The antibody-drug conjugate according to any one of claims 9-10, wherein the cytotoxicity or cell inhibitor is monomethylolpropionate.

12. The antibody-drug conjugate of claim 11, wherein the monomethylolpropamine is monomethylolpropamine E (MMAE).

13. The antibody-drug conjugate of claim 12, wherein the antibody or its antigen-binding fragment is conjugated to MMAE via an enzyme-cleavable linker unit.

14. The antibody-drug conjugate of claim 13, wherein the enzyme-cleavable linker unit comprises a Val-Cit linker.

15. The antibody-drug conjugate of claim 14, wherein the antibody or its antigen-binding fragment is coupled to the MMAE via a connector unit having the formula: –A a –W w –Y y –; where –A– is the elongation unit, a is 0 or 1; –W– is the amino acid unit, w is an integer from 0 to 12; –Y– is the spacer unit, y is 0, 1 or 2.

16. The antibody-drug conjugate of claim 15, wherein the extended unit has the structure of Formula I; wherein the amino acid unit is Val-Cit; and wherein the spacer subunit is a p-aminobenzyl alcohol (PAB) group having the structure of Formula II. Equation I; Formula II.

17. The antibody-drug conjugate of claim 10, wherein the linker is attached to monomethylolpropionate E to form the antibody-drug conjugate, having the following structure: Where Ab represents antibody h2A2 and p represents a number from 1 to 16.

18. The antibody-drug conjugate of claim 17, wherein the average value of p in the population of the antibody-drug conjugate is approximately 4.

19. A nucleic acid encoding the heavy chain variable region and / or the light chain variable region as defined in any one of claims 1-8.

20. A vector comprising the nucleic acid as described in claim 19.

21. The carrier of claim 20, wherein the carrier is an expression carrier.

22. A host cell comprising the nucleic acid of claim 21.

23. The host cell of claim 22, wherein the host cell is a Chinese hamster ovary (CHO) cell.

24. A method for producing an anti-αvβ6 antibody or an antigen-binding fragment thereof, comprising culturing a host cell as described in claim 22 or 23 under conditions suitable for producing the anti-αvβ6 antibody or an antigen-binding fragment thereof.

25. The method of claim 24, further comprising isolating the anti-αvβ6 antibody or its antigen-binding fragment produced by the host cells.

26. A method for producing an anti-αvβ6 antibody-drug conjugate, comprising culturing a host cell as described in claim 22 or 23 under conditions suitable for producing the anti-αvβ6 antibody; isolating the anti-αvβ6 antibody produced from the host cell; and conjugating the anti-αvβ6 antibody to a cytotoxic or cell inhibitor.

27. The method of claim 26, wherein the anti-αvβ6 antibody is coupled to a cytotoxic or cytotoxic inhibitor via a linker.

28. The method of claim 27, wherein the connector is a Val-Cit connector.

29. The method of any one of claims 26-28, wherein the cytotoxicity or cell inhibitor is monomethylolpropionate E (MMAE).

30. Use of the antibody-drug conjugate of any one of claims 9-18 in the preparation of a medicament for treating a target cancer, wherein the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), head and neck cancer, ovarian cancer, bladder cancer, and pancreatic cancer.

31. The use as described in claim 30, wherein the lung cancer is non-small cell lung cancer.

32. The use as described in claim 30, wherein the cancer is head and neck cancer.

33. The use according to any one of claims 30-32, wherein the antibody or antigen-binding fragment or antibody-drug conjugate is in the pharmaceutical composition, the pharmaceutical composition comprising the antibody or antigen-binding fragment or antibody-drug conjugate and a pharmaceutically acceptable carrier.

34. The use as described in any one of claims 30-32, wherein the object is a person.

35. A pharmaceutical composition comprising an antibody or antigen-binding fragment as described in any one of claims 1-8, or an antibody-drug conjugate as described in any one of claims 9-18, and one or more agents selected from the group consisting of physiologically acceptable carriers, diluents, and adjuvants.

36. A pharmaceutical composition comprising an antibody or antigen-binding fragment as described in any one of claims 1-8, or an antibody-drug conjugate as described in any one of claims 9-18, and one or more agents, wherein the agents are excipients.

37. The pharmaceutical composition of claim 35 or 36, wherein the composition is administered in combination with a radiation or chemotherapy agent.

38. An antibody-drug conjugate comprising an isolated anti-αvβ6 antibody conjugated to vcMMAE, wherein the antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17, wherein the antibody-drug conjugate has the following structure: Where Ab is the antibody and p represents a number from 1 to 16.

Images