Anti-Ly6E Antibodies, Immunoconjugates, and Their Use

Abstract

The present invention provides anti-Ly6E antibodies and immunoconjugates, and methods of using them.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 63 / 337,945, filed May 3, 2022, and U.S. Provisional Application No. 63 / 492,297, filed March 27, 2023, each of which is hereby incorporated by reference in its entirety for any purpose.

[0002] Sequence Listing This application includes a sequence listing that was electronically submitted in XML format. The XML copy created on April 14, 2023, is named “2023 - 04 - 14_01146 - 0114 - 00PCT_ST26.xml” and is 46,932 bytes in size. The information in the electronic form of the sequence listing is hereby incorporated by reference in its entirety.

[0003] Field The present invention relates to anti - Ly6E antibodies and immunoconjugates, and methods of using them.

Background Art

[0004] Background Lymphocyte antigen 6 complex, locus E (Ly6E), also known as retinoic acid - inducible gene E (RIG - E) and stem cell antigen 2 (SCA - 2). It has an unknown function with an unknown binding partner for a 131 - amino - acid - long, approximately 8.4 kDa protein that is GPI - linked. It was first identified as a transcript expressed in immature thymocytes and thymic medullary epithelial cells in mice. Mao et al., “RIG - E, a human homolog of the murine Ly - 6 family, is induced by retinoic acid during the differentiation of acute promyelocytic leukemia cell,” Proc. Natl. Acad. Sci. U.S.A. 93:5910 - 5914 (1996).

[0005] In the art, there is a need for agents that target Ly6E for the diagnosis and treatment of Ly6E-related conditions such as cancer. The present invention meets that need and provides other advantages. SUMMARY OF THE INVENTION

[0006] Summary The present invention provides anti-Ly6E antibodies, immunoconjugates, and methods of using them.

[0007] In some embodiments, the disclosure provides an isolated antibody that binds to Ly6E and comprises (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 8, (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 9, (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10, (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 12.

[0008] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized or chimeric antibody. In some embodiments, the antibody is an antibody fragment.

[0009] In some embodiments, the antibody comprises (a) a VH sequence having at least 95% sequence identity with SEQ ID NO: 32; (b) a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 31; or (c) a VH sequence as in (a) and a VL sequence as in (b). In some embodiments, the antibody comprises the VH sequence of SEQ ID NO: 32 and the VL sequence of SEQ ID NO: 31.

[0010] In some embodiments, the disclosure provides an isolated antibody comprising the VH sequence of SEQ ID NO: 32 and the VL sequence of SEQ ID NO: 31.

[0011] In some embodiments, the antibody is an IgG1, IgG2a, IgG2b, IgG3, or IgG4 antibody.

[0012] In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 5, and a light chain comprising an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO: 29. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a light chain comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the present disclosure provides an isolated antibody that binds to Ly6E, the antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a light chain comprising the amino acid sequence of SEQ ID NO: 3.

[0013] In some embodiments, the antibody is a multispecific antibody. In some embodiments, the multispecific antibody is a bispecific antibody. In some embodiments, the multispecific antibody binds to Ly6E and CD3.

[0014] In some embodiments, the present disclosure provides an isolated nucleic acid encoding the antibody. In some embodiments, the present disclosure provides an expression vector comprising a nucleic acid encoding the antibody. In some embodiments, the present disclosure provides a host cell comprising the nucleic acid or the expression vector. In some embodiments, the present disclosure provides a host cell expressing the antibody. In some embodiments, the present disclosure provides a method for producing an antibody, comprising culturing a host cell such that the antibody is produced.

[0015] In some embodiments, the present disclosure provides an immunoconjugate comprising the antibody and a cytotoxic agent. In some embodiments, the immunoconjugate has the formula Ab-(L-D)p, wherein (a) Ab is the antibody provided herein; (b) L is a linker; (c) D is a pyrrolobenzodiazepine; and (d) p ranges from 1 to 8. In some embodiments, D is a pyrrolobenzodiazepine of formula A: TIFF2025517612000002.tif28170 wherein R 2 is of formula II: TIFF2025517612000003.tif9170 wherein A is C5-7 is an aryl group, and X is OH, SH, CO 2 H, COH, N=C=O, NHR N , and (OC 2 H 4 ) m OCH 3 selected from the group consisting of, where R N is selected from the group consisting of H and C 1-4 alkyl, m is an integer from 1 to 3, (i) Q 1 is a single bond, and Q 2 is selected from a single bond and -Z-(CH 2 ) n -, where Z is a single bond, O, S, or NH, and n is an integer from 1 to 3; or (ii) Q 1 is -CH=CH-, and Q 2 is a single bond; R 2’ is aryl optionally substituted by one or more substituents selected from the group consisting of halo, nitro, cyano, ether, C 1-7 alkyl, C 3-7 heterocyclyl, and bis-oxy-C 1-3 alkylene; 5-10 is an aryl group; R 6 and R 9 are independently selected from H, R, OH, OR, SH, SR, NH 2 , NHR, NRR’, nitro, Me 3 Sn and halo; R 7 is selected from the group consisting of H, R, OH, OR, SH, SR, NH 2 , NHR, NHRR’, nitro, Me 3 Sn and halo; where R and R’ are independently selected from optionally substituted C 1-12 alkyl, C 3-20 heterocyclyl and C 5-20 aryl groups; any of the following: (a) R 10is H and R 11 is OH or OR A wherein R A is C 1-4 alkyl; or (b) R 10 and R 11 form a nitrogen-carbon double bond between the nitrogen atom and the carbon atom to which they are attached; or (c) R 10 is H and R 11 is SO Z M, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; R” is a C 3-12 alkylene group, and this chain may be interrupted by one or more heteroatoms independently selected from the group consisting of O, S, and NH, and / or one or more aromatic rings independently selected from the group consisting of benzene and pyridine; Y is O, S, or NH; R 6’ , R 7’ and R 9’ are each selected from the same groups as R 6 , R 7 and R 9 , and R 10’ and R 11’ are the same as R 10 and R 11 , and when R 11 and R 11’ are SO Z M, M may represent a divalent pharmaceutically acceptable cation; and the point of attachment to the linker L is through R 2 or R 2’ .

[0016] In some embodiments, D has the following structure: TIFF2025517612000004.tif29170In the formula, the wavy line indicates the point of attachment to the linker L.

[0017] In some embodiments, (L-D) has the following structure: In TIFF2025517612000005.tif, the wavy line indicates the binding point with the protein.

[0018] In some embodiments, p ranges from 1.5 to 5 or 1.5 to 6 or 1.5 to 4 or 2 to 3.

[0019] In some embodiments, the present disclosure provides a pharmaceutical formulation comprising an immunoconjugate and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation further comprises an additional therapeutic agent.

[0020] In some embodiments, the present disclosure provides a method of treating an individual having Ly6E-positive cancer, the method comprising administering to the individual an effective amount of an immunoconjugate or a pharmaceutical formulation. In some embodiments, the Ly6E-positive cancer is selected from breast cancer, pancreatic cancer, colon cancer, colorectal cancer, melanoma, ovarian cancer, non-small cell lung cancer, or gastric cancer. Breast cancer

[0021] In some embodiments, the method further comprises administering an additional therapeutic agent to the individual. In some embodiments, the additional therapeutic agent is a platinum complex.

[0022] In some embodiments, the present disclosure provides a method of inhibiting the proliferation of Ly6E-positive cells, the method comprising exposing the cells to an immunoconjugate under conditions that permit binding of the immunoconjugate to Ly6E on the surface of the cells, thereby inhibiting the proliferation of the cells. In some embodiments, the cells are breast cancer cells, pancreatic cancer cells, colon cancer cells, colorectal cancer cells, melanoma cells, ovarian cancer cells, non-small cell lung cancer cells, or gastric cancer cells.

[0023] In some embodiments, the present disclosure provides an antibody provided herein conjugated to a label. In some embodiments, the label is a positron emitter. In some embodiments, the positron emitter is 89Zr.

[0024] In some embodiments, the present disclosure provides a method for detecting human Ly6E in a biological sample, the method comprising contacting the biological sample with an anti-Ly6E antibody under conditions that permit binding of the anti-Ly6E antibody to naturally occurring human Ly6E, and detecting whether a complex is formed between the anti-Ly6E antibody and naturally occurring human Ly6E in the biological sample. In some embodiments, the biological sample is a breast cancer sample, a pancreatic cancer sample, a colon cancer sample, a colorectal cancer sample, a melanoma cancer sample, an ovarian cancer sample, a non-small cell lung cancer sample, or a gastric cancer sample.

[0025] In some embodiments, the present disclosure provides a method for detecting Ly6E-positive cancer, the method comprising: (i) administering to a subject having or suspected of having Ly6E-positive cancer a labeled anti-Ly6E antibody comprising the anti-Ly6E antibody provided herein; and (ii) detecting the labeled anti-Ly6E antibody in the subject, wherein detection of the labeled anti-Ly6E antibody indicates Ly6E-positive cancer in the subject. In some embodiments, the labeled anti-Ly6E antibody comprises an anti-Ly6E antibody conjugated to a positron emitter. In some embodiments, the positron emitter is 89 Zr. BRIEF DESCRIPTION OF THE DRAWINGS

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DETAILED DESCRIPTION OF THE INVENTION

[0041] I. Definitions For the purposes of this specification, "acceptor human framework" means a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may contain the same amino acid sequence or may contain alterations in the amino acid sequence. In some embodiments, the number of amino acid alterations is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

[0042] "Affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by methods common in the art, including those described herein. Specific illustrative descriptions and exemplary embodiments for measuring binding affinity are described below.

[0043] An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody that does not have such alterations, and such alterations improve the affinity of the antibody for an antigen.

[0044] The terms "anti-Ly6E antibody" and "antibody that binds Ly6E" refer to an antibody that can bind Ly6E with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting Ly6E. In one embodiment, the degree of binding of the anti-Ly6E antibody to an irrelevant non-Ly6E protein is less than about 10% of the binding of the antibody to Ly6E when measured, for example, by radioimmunoassay (RIA), or by Scatchard analysis, or by surface plasmon resonance (e.g., BIAcore™, etc.). In certain embodiments, the antibody that binds Ly6E has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M). In certain embodiments, the anti-Ly6E antibody binds to an epitope of Ly6E that is conserved among Ly6Es from different species.

[0045] The term "antibody" is used herein in the broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0046] The term "antibody-drug conjugate" (ADC) as used herein is equivalent to the term "immunoconjugate".

[0047] "Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0048] An antibody that binds to the same epitope as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay, and conversely, a reference antibody that blocks the binding of an antibody to its antigen by 50% or more in a competitive assay. Exemplary competitive assays are provided herein.

[0049] The terms “cancer” and “cancerous” refer to or describe a physiological condition in a mammal that is typically characterized by uncontrolled cell growth / proliferation. Examples of cancer include, but are not limited to, carcinomas, lymphomas (e.g., Hodgkin and non-Hodgkin lymphomas), blastomas, sarcomas, and leukemias. More specific examples of such cancers include eyes that overexpress Ly6E, for example, breast cancer and / or metastatic breast cancer (including Her2-negative breast cancer and / or triple-negative breast cancer), pancreatic cancer, colon cancer, colorectal cancer, melanoma, ovarian cancer, non-small cell lung cancer (either squamous or non-squamous), gastric cancer, squamous cell cancer, small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular cancer, gastrointestinal cancer, glioma, cervical cancer, liver cancer, bladder cancer, hepatoma, endometrial or uterine carcinoma, salivary adenocarcinoma, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocarcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[0050] The term “chimeric” antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species and the remaining portion of the heavy and / or light chain is derived from a different source or species.

[0051] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is of the IgG1 isotype. In certain embodiments, the antibody is of the IgG1 isotype having P329G, L234A, and L235A mutations to reduce Fc region effector function. In other embodiments, the antibody is of the IgG2 isotype. In certain embodiments, the antibody is of the IgG4 isotype having an S228P mutation in the hinge region to improve the stability of the IgG4 antibody. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The light chain of an antibody can be assigned to one of two types called kappa (κ) and lambda (λ) based on the amino acid sequence of its constant domain.

[0052] As used herein, the terms "human-derived constant region" or "human constant region" refer to the constant heavy chain regions of human antibodies of subclasses IgG1, IgG2, IgG3, or IgG4, and / or the constant light chain kappa or lambda regions. Such constant regions are known in the art and are described, for example, in Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also, e.g., Johnson, G., and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218; Kabat, E.A., et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788). Unless otherwise specified herein, the numbering of amino acid residues in the constant regions follows the EU numbering system (also referred to as the Kabat EU index) as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0053] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and / or causes cell death or destruction. Cytotoxic agents include radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212, and radioisotopes of Lu); chemotherapeutic agents or chemotherapeutic drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes such as nucleolytic enzymes and fragments thereof; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including fragments and / or variants thereof); and various antitumor agents or anticancer agents described below, but are not limited thereto.

[0054] "Effector function" refers to the biological activities resulting from the Fc region of an antibody that vary depending on the isotype of the antibody. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0055] The "effective amount" of a drug, e.g., a pharmaceutical formulation, refers to the amount effective in the dosage and for the period required to achieve the desired therapeutic or prophylactic result.

[0056] The term "epitope" refers to a specific site on an antigen molecule to which an antibody binds.

[0057] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0058] The term "framework" or "FR" refers to the variable domain residues other than hypervariable region (HVR) residues. The FRs of the variable domain generally consist of the following four FR domains: FR1, FR2, FR3, and FR4. Thus, HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0059] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially the same as a native antibody structure or having a heavy chain that contains an Fc region as defined herein.

[0060] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, and the progeny of such a cell. Host cells include "transformants" and "transformed cells", and these cells include primary transformed cells and their progeny, regardless of the number of passages. The progeny may not be identical to the parental cell in terms of nucleic acid content and may include mutations. As used herein, progeny of mutants having the same function or biological activity as those screened or selected in the originally transformed cell are included.

[0061] "Human antibody" refers to an antibody produced by a human or human cell, or an antibody having an amino acid sequence corresponding to a non-human-derived antibody that utilizes a sequence encoding a human antibody such as a human antibody repertoire. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0062] "Human consensus framework" is a framework that represents the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup such as that in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I in Kabat et al. (supra). In one embodiment, for VH, the subgroup is subgroup III as described in Kabat et al. (supra).

[0063] A "humanized" antibody refers to a chimeric antibody that includes amino acid residues derived from non-human HVRs and amino acid residues derived from human FRs. In certain embodiments, a humanized antibody includes substantially all of at least one, typically two, variable domains, with all or substantially all of the HVRs (e.g., CDRs) corresponding to a non-human antibody and all or substantially all of the FRs corresponding to a human antibody. A humanized antibody may optionally include at least a portion of an antibody constant region derived from a human antibody. An "humanized form" of an antibody, e.g., as opposed to a non-human antibody, refers to an antibody that has been humanized.

[0064] The term "hypervariable region" or "HVR" as used herein refers to each of the regions of the variable domains of an antibody where the sequences are hypervariable and / or form structurally defined loops ("hypervariable loops"). Generally, native four-chain antibodies contain six HVRs, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs generally contain amino acid residues derived from hypervariable loops and / or "complementary determining regions" (CDRs), the latter having the highest sequence variability and / or being involved in antigen recognition. Exemplary hypervariable loops are found at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3) are found at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2 and 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).) Except for CDR1 in VH, CDRs generally contain the amino acid residues that form hypervariable loops. CDRs also include "specificity determining regions", i.e., "SDRs", which are the residues that contact the antigen. SDRs are contained within a region of the CDR called the abbreviated-CDR, i.e., a-CDR. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2 and a-CDR-H3) are found at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2 and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008).)Unless otherwise indicated, HVR residues and other residues within the variable domain (e.g., FR residues) are numbered herein according to Kabat et al. as described above.

[0065] An “immunoconjugate” is an antibody conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent. Immunoconjugates correspond to the term “antibody-drug conjugate” (ADC).

[0066] An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is human.

[0067] An “isolated antibody” is one that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or greater than 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing electrophoresis (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0068] An “isolated nucleic acid” is a nucleic acid molecule that has been separated from the components of its natural environment. Isolated nucleic acids include nucleic acid molecules that are normally contained within cells that contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally, or at a chromosomal location that is different from its natural chromosomal location.

[0069] "Isolated nucleic acid encoding an anti-Ly6E antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains of the antibody (or fragments thereof), including such nucleic acid molecule(s) within a single vector or separate vectors, and such nucleic acid molecule(s) are present at one or more locations within a host cell.

[0070] The term "Ly6E" as used herein refers to any native mature Ly6E resulting from the processing of the intracellular Ly6E precursor protein. This term includes Ly6E from any vertebrate source, including, but not limited to, mammals such as primates (e.g., humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats). This term also encompasses naturally occurring variants of Ly6E, such as splice variants or allelic variants. The amino acid sequence of an exemplary human Ly6E precursor protein having a signal sequence (amino acids 1-20 = signal sequence) is shown in SEQ ID NO: 1. The amino acid sequence of an exemplary mature human Ly6E is shown in SEQ ID NO: 24. The sequence of amino acids 1-131 of an exemplary cynomolgus monkey Ly6E is shown in SEQ ID NO: 2. The amino acid sequence of an exemplary mature cynomolgus monkey Ly6E is shown in SEQ ID NO: 25. The amino acid sequences of an exemplary rat Ly6E precursor (having a signal sequence, amino acids 1-26) and mature sequence are shown in SEQ ID NO: 23 and 28, respectively. The amino acid sequences of an exemplary mouse Ly6E precursor (having a signal sequence, amino acids 1-26) and mature sequence are shown in SEQ ID NO: 22 and 27, respectively.

[0071] The term "Ly6E-positive cancer" refers to a cancer that includes cells expressing Ly6E on its surface. For the purpose of determining whether a cell expresses Ly6E on its surface, Ly6E mRNA expression is considered to correlate with Ly6E expression on the cell surface. In some embodiments, the expression of Ly6E mRNA is determined by a method selected from in situ hybridization and RT-PCR (including quantitative RT-PCR). Alternatively, the expression of Ly6E on the cell surface can be determined using an antibody against Ly6E by methods such as immunohistochemistry, FACS, etc. In some embodiments, Ly6E-positive cancer means breast cancer, metastatic breast cancer (including Her2-negative breast cancer and / or triple-negative breast cancer), pancreatic cancer, colon cancer, colorectal cancer, melanoma, ovarian cancer, non-small cell lung cancer (either squamous and / or non-squamous), or gastric cancer, each showing a high level of Ly6E expression.

[0072] The term "Ly6E-positive cell" refers to a cancer cell that expresses Ly6E on its surface.

[0073] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for, e.g., naturally occurring mutations or variant antibodies that may arise during the production of a monoclonal antibody preparation, which variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody obtained from a substantially uniform collection of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be made by a variety of techniques including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals that contain all or part of the human immunoglobulin loci, and such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0074] "Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. A naked antibody may be present in a pharmaceutical formulation.

[0075] "Natural antibodies" refer to naturally occurring immunoglobulin molecules having various structures. For example, natural IgG antibodies are approximately 150,000 Dalton hetero-tetrameric glycoproteins composed of two identical light chains and two identical heavy chains linked by disulfide bonds. Each heavy chain from the N-terminus to the C-terminus has a variable region (VH), also called the variable heavy chain domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, each light chain from the N-terminus to the C-terminus has a variable region (VL), also called the variable light chain domain, followed by a constant light (CL) domain. The light chains of an antibody can be assigned to one of two types called kappa (κ) and lambda (λ) based on the amino acid sequence of their constant domains.

[0076] The term "package insert" is used to refer to the instructions customarily included in the commercial package of a therapeutic product that contain information about the indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings regarding its use.

[0077] The "percent amino acid sequence identity (%)" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of the reference polypeptide, after aligning the sequences and introducing gaps if necessary to obtain the maximum percent sequence identity, and assuming that any conservative substitutions are not part of the sequence identity. Alignments for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for aligning sequences, including any algorithm necessary to achieve the maximum alignment over the full length of the sequences being compared. However, for the purposes herein, the percent amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code has been filed with the U.S. Copyright Office, Washington D.C., 20559, together with user documentation, and is registered as U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California), or can be compiled from its source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In the context where ALIGN-2 is used for amino acid sequence comparison, the percent amino acid sequence identity of a given amino acid sequence A to, with, or against an amino acid sequence B (or, as described as a given amino acid sequence A having or including a particular percent amino acid sequence identity to, with, or against an amino acid sequence B) is calculated as follows: 100 × fraction X / Y In the formula, X is the number of amino acid residues scored as identical matches in the alignment of A and B by the array alignment program ALIGN-2, and Y is the total number of amino acid residues in B. When the length of amino acid sequence A is not equal to the length of amino acid sequence B, it will be understood that the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values used in this specification are obtained as described in the previous paragraph using the ALIGN-2 computer program.

[0078] The term "pharmaceutical preparation" refers to a preparation in a form such that the biological activity of the active ingredient contained therein is effective and which does not contain additional constituents that are unacceptably toxic to the subject to which the preparation is administered.

[0079] "Pharmaceutically acceptable carrier" refers to a component in a pharmaceutical preparation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0080] As used herein, "platinum complex" refers to anti-cancer chemotherapeutic agents such as, but not limited to, cisplatin, oxaliplatin, carboplatin, iproplatin, satraplatin, CI-973, AZ0473, DWA2114R, nedaplatin, and sprioplatin, which exert efficacy against tumors based on their ability to covalently bind to DNA.

[0081] As used herein, "treatment" (and its grammatical variants, e.g., "treat" or "treating") refers to a clinical intervention in an attempt to alter the natural course of an individual being treated, which can be performed for prophylaxis or during the course of clinical pathology. Desired effects of treatment include preventing the onset or recurrence of a disease, alleviating symptoms, attenuating any direct or indirect pathological consequence of the disease, preventing metastasis, decreasing the rate of disease progression, remission or palliation of the condition, and recovery or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of a disease or to slow the progression of a disease.

[0082] The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (VH and VL, respectively) generally have similar structures, and each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al. Kuby Immunology, 6 th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using the VH or VL domain of an antibody that binds the antigen to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0083] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is ligated. This term includes not only vectors as self-replicating nucleic acid structures but also vectors integrated into the genome of a host cell into which the vector has been introduced. Certain vectors can direct the expression of nucleic acids operably linked thereto. Such vectors are referred to herein as "expression vectors."

[0084] With respect to Formula A, the term "C 1-12 alkyl" as used in Formula A refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 12 carbon atoms, which may be aliphatic or alicyclic and which may be saturated or unsaturated (e.g., partially unsaturated, fully unsaturated), but not aromatic. Thus, the term "alkyl" includes subclasses such as alkenyl, alkynyl, cycloalkyl, etc. Examples of saturated alkyl groups can include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, and heptyl. Examples of saturated straight-chain alkyl groups can include, but are not limited to, methyl, ethyl, n-propyl (C 3 ), n-butyl (C 4 ), n-pentyl (amyl) (C 5 ), n-hexyl (C 6 ), and n-heptyl. Examples of saturated branched alkyl groups can include, but are not limited to, iso-propyl (C 3 ), iso-butyl (C 4 ), sec-butyl (C 4 ), tert-butyl (C 4 ), iso-pentyl (C 5 ), and neo-pentyl (C 5 ). As used herein with respect to Formula A, an alkenyl group refers to an alkyl group having one or more carbon-carbon double bonds. Examples of alkenyl groups can include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, isopropenyl, butenyl, pentenyl, and hexenyl. In some embodiments, the C 1-12 alkyl of Formula A is a saturated acyclic alkyl.

[0085] In other uses herein, "alkyl" contains normal, secondary, tertiary or cyclic carbon atoms and is a C 1 -C 12 hydrocarbon. Examples are methyl (Me, -CH 3 ), ethyl (Et, -CH 2 CH 3 ), 1-propyl (n-Pr, n-propyl, -CH 2 CH 2 CH 3 ), 2-propyl (i-Pr, i-propyl, -CH(CH 3 )) 2 ), 1-butyl (n-Bu, n-butyl, -CH 2 CH 2 CH 2 CH 3 ), 2-methyl-1-propyl (i-Bu, i-butyl, -CH 2 CH(CH 3 )) 2 ), 2-butyl (s-Bu, s-butyl, -CH(CH 3 )CH 2 CH 3 ), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH 3 )) 3 ), 1-pentyl (n-pentyl, -CH 2 CH 2 CH 2 CH 2 CH 3 ), 2-pentyl (-CH(CH 3 )CH 2 CH 2 CH 3 ), 3-pentyl (-CH(CH 2 CH 3 )) 2 ), 2-methyl-2-butyl (-C(CH 3 )) 2 CH 2 CH 3 ), 3-methyl-2-butyl (-CH(CH 3 )CH(CH 3 )) 2 ), 3-methyl-1-butyl (-CH 2 CH 2 CH(CH3 ) 2 )、2-methyl-1-butyl (-CH 2 CH(CH 3 )CH 2 CH 3 )、1-hexyl (-CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 )、2-hexyl (-CH(CH 3 )CH 2 CH 2 CH 2 CH 3 )、3-hexyl (-CH(CH 2 CH 3 )(CH 2 CH 2 CH 3 ))、2-methyl-2-pentyl (-C(CH 3 ) 2 CH 2 CH 2 CH 3 )、3-methyl-2-pentyl (-CH(CH 3 )CH(CH 3 )CH 2 CH 3 )、4-methyl-2-pentyl (-CH(CH 3 )CH 2 CH(CH 3 ) 2 )、3-methyl-3-pentyl (-C(CH 3 )(CH 2 CH 3 ) 2 )、2-methyl-3-pentyl (-CH(CH 2 CH 3 )CH(CH 3 ) 2 )、2,3-dimethyl-2-butyl (-C(CH 3 ) 2 CH(CH 3 ) 2 )、and 3,3-dimethyl-2-butyl (-CH(CH 3 )C(CH 3 ) 3 .

[0086] As used herein, particularly with respect to Formula A, the term "C 3-12 alkylene" refers to a bidentate moiety obtained by removing two hydrogen atoms from a hydrocarbon compound having 3 to 12 carbon atoms (unless otherwise specified), which may be aliphatic or cycloaliphatic and may be saturated, partially unsaturated, or fully unsaturated (but not aromatic). The two hydrogen atoms may be removed from the same carbon atom or one each from two different carbon atoms. Thus, the term "alkylene" includes subclasses such as alkenylene, alkynylene, cycloalkylene, etc. Examples of straight-chain saturated C 3-12 alkylene groups include -(CH 2 ) n - (wherein n is an integer from 3 to 12, for example, -CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 -, and -CH 2 CH 2 CH 2 CH-2CH 2 CH 2 CH 2 -), but are not limited thereto. Examples of branched saturated C 3-12 alkylene groups include -CH(CH 3 )CH 2 -, -CH(CH 3 )CH 2 CH 2 -, -CH(CH 3 )CH 2 CH 2 CH 2 -, -CH 2 CH(CH 3 )CH 2 -, -CH 2 CH(CH 3 )CH 2 CH 2 -, -CH(CH2 CH 3 )-, -CH(CH 2 CH 3 )CH 2 -, and -CH 2 CH(CH 2 CH 3 )CH 2 - may be included, but are not limited thereto. The straight-chain partially unsaturated C 3-12 alkylene group (C 3-12 alkenylene and alkynylene groups) examples include, -CH=CH-CH 2 -, -CH 2 -CH=CH 2 -, -CH=CH-CH 2 -CH 2 -, -CH=CH-CH 2 -CH 2 -CH 2 -, -CH=CH-CH=CH-, -CH=CH-CH=CH-CH 2 -, -CH=CH-CH=CH-CH 2 -CH 2 -, -CH=CH-CH 2 -CH=CH-, -CH=CH-CH 2 -CH 2 -CH=CH-, and -CH 2 -C≡C-CH 2 - may be included, but are not limited thereto. The branched partially unsaturated C 3-12 alkylene group (C 3-12 alkenylene group and alkynylene group) examples include, -C(CH 3 )=CH-, -C(CH 3 )=CH-CH 2 -, -CH=CH-CH(CH 3 )- and -CsC-CH(CH 3 )- may be included, but are not limited thereto. The alicyclic saturated C 3-12 alkylene group (C 3- - I2 cycloalkylene) examples may include cyclopentylene and cyclohexylene, but are not limited thereto. The alicyclic partially unsaturated C 3-12Examples of alkylene groups can include, but are not limited to, cyclopentenylene and cyclohexenylene. In some embodiments of Formula A, C 3-12 alkylene refers to a straight-chain saturated hydrocarbon group of the formula -(CH 2 ) 3-12 -, examples of which include propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decalene, undecalene, and dodecalene.

[0087] As used herein with respect to Formula A, the term "C 5-20 aryl" refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, the moiety having 3 to 20 ring atoms. In some embodiments, each aryl ring has 5 to 7 ring atoms. Prefixes such as C 3-20 , C 5-7 , C 5-6 , etc., refer to the number of ring atoms or the range of the number of ring atoms, regardless of whether the atoms are carbon atoms or heteroatoms. For example, as used herein, "C 5 - 6The term "aryl" refers to an aryl group having 5 or 6 ring atoms. In some embodiments, as in the case of a "carboaryl group", all ring atoms are carbon atoms. Such carboaryl groups can include, but are not limited to, those derived from benzene (i.e., phenyl), naphthalene, azulene, anthracene, phenanthrene, naphthacene, and pyrene. In some embodiments, aryl includes a fused ring containing at least one aromatic ring, such as a group derived from indane, indene, isoindene, tetralin, acenaphthene, fluorene, phenalene, acephenanthrene, and aceanthrene. In other embodiments, the ring atoms can include one or more heteroatoms, as in the case of a "heteroaryl group". Examples of monocyclic heteroaryl groups can include, but are not limited to, those derived from pyrrole, pyridine, and furan. Thiophene, oxazole, isoxazole, isoxazine, oxadiazole, thiazole, isothiazole, and triazole. In some embodiments, heteroaryl includes a fused ring, and at least one of the rings contains ring heteroatoms such as groups derived from benzofuran, isobenzofuran, isoindole, indolizine, indoline, isoindoline, purine, benzimidazole, indazole, and benzoxazole. In some embodiments, with respect to formula A, C 5-20 aryl is C 6 -C 20 a carboaryl group.

[0088] "Substituted alkyl", "substituted aryl", and "substituted arylalkyl" each mean an alkyl, aryl, and arylalkyl, respectively, in which one or more hydrogen atoms are each independently replaced by a substituent. Typical substituents include, but are not limited to, -X, -R, -O - , -OR, -SR, -S - , -NR 2 , -NR 3 , =NR, -CX 3 , -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO 2 , =N 2 , -N3 、 NC(=O)R, -C(=O)R, -C(=O)NR 2 、 -SO 3 - 、 -SO 3 H, -S(=O) 2 R, -OS(=O) 2 OR, -S(=O) 2 NR, -S(=O)R, -OP(=O)(OR) 2 、 -P(=O)(OR) 2 、 -PO - 3 、 -PO 3 H 2 、 -C(=O)R, -C(=O)X, -C(=S)R, -CO 2 R, -CO 2 - 、 -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NR 2 、 -C(=S)NR 2 、 -C(=NR)NR 2 is included, where each X is independently a halogen: F, Cl, Br, or I; and each R is independently -H, C 2 -C 18 alkyl, C 6 -C 20 aryl, C 3 -C 14 heterocyclyl, a protecting group or a prodrug moiety. The above-mentioned alkylene group, alkenylene group and alkynylene group may also be similarly substituted.

[0089] As used herein, the term "C 3-20 heterocyclyl" refers, particularly with respect to formula A, to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has 3 to 20 ring atoms, of which 1 to 10 are ring heteroatoms. In some embodiments, each ring has 3 to 7 ring atoms, of which 1 to 4 are ring heteroatoms. In some embodiments, each heteroatom is independently selected from the group consisting of O, N and S. When referring to heterocyclyl, a prefix (e.g., C 3-20 、 C 3-7 、 C 5-6etc.) indicates the number or range of ring atoms, regardless of whether they are carbon atoms or heteroatoms. For example, as used herein, "C 5 - 6 heterocyclyl" refers to a heterocyclyl group having 5 or 6 ring atoms, regardless of whether those ring atoms are carbon atoms or heteroatoms. Examples of monocyclic heterocyclyl groups can include, but are not limited to, those derived from aziridine, azetidine, pyrrolidine, pyrroline, piperidine, dihydropyridine, tetrahydropyridine, azepine, oxirane, oxetane, oxolane (tetrahydrofuran), oxole, oxane, dihydropyran, pyran, dioxane, imidazolidine, pyrazolidine, imidazoline and pyrazoline. In some embodiments, the heterocyclyl group for formula A is a saturated monocyclic or bicyclic ring containing 3 to 10 ring atoms, 1 to 3 of which are ring heteroatoms independently selected from the group consisting of O and N.

[0090] As used herein, particularly with respect to formula A, the term "ether" refers to the group -OR, wherein R is an ether substituent, such as a C 1-7 alkyl group (which may also be referred to as a C 1-7 alkoxy group), a C 3-20 heterocyclyl group (which may also be referred to as a C 3-20 heterocyclyloxy group), or a C 5-20 aryl group (which may also be referred to as a C 5-20 aryloxy group). In some embodiments, the ether refers to the group -OR wherein R is a saturated C 1-7 alkyl group.

[0091] "Linker" refers to a chemical moiety that includes a covalent bond or a chain of atoms that covalently attaches an antibody to a drug moiety. In various embodiments, the linker includes a divalent radical such as alkyldiyl, aryldiyl, heteroaryldiyl, etc., for example: -(CR 2 ) n O(CR 2 ) n-, alkyloxy (e.g., polyethyleneoxy, PEG, polymethyleneoxy) and alkylamino (e.g., polyethyleneamino, repeating units of Jeffamine™; and diesters and amides of diacids including succinic acid esters, succinamic acids, diglycolic acid esters, malonic acid esters, and caproamides. In various embodiments, the linker can include one or more amino acid residues such as valine, phenylalanine, lysine, and homolysine).

[0092] The term "chiral" refers to a molecule having the property of non-superimposability of mirror image partners, while the term "achiral" refers to a molecule that can be superimposed on its mirror image partner.

[0093] The term "stereoisomer" refers to a compound having the same chemical constitution but differing in the arrangement of atoms or groups in space.

[0094] "Diastereomer" refers to stereoisomers having two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties such as, for example, melting point, boiling point, spectral characteristics, and reactivity. A mixture of diastereomers may be separated under high-resolution analytical procedures such as electrophoresis and chromatography.

[0095] "Enantiomer" refers to two stereoisomers of a compound that are non-superimposable mirror images of each other.

[0096] The stereochemical definitions and conventions used herein generally follow S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., New York. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. When describing optically active compounds, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center. The prefixes d and l or (+) and (-) are used to denote the sign of rotation of plane-polarized light by the compound, and (-) or l means that the compound is levorotatory. A compound with the prefix (+) or d is dextrorotatory. In a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Certain stereoisomers may also be called enantiomers, and a mixture of such isomers is often called a racemic mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur when there is no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species and are optically inactive.

[0097] A "leaving group" refers to a functional group that can be replaced by another functional group. Certain leaving groups are well known in the art and examples thereof include, but are not limited to, halides (e.g., chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate) and trifluoromethylsulfonate.

[0098] The term "protecting group" refers to a substituent that is commonly used to block or protect a particular functionality while reacting with other functional groups on a compound. For example, an "amino protecting group" is a substituent that binds to an amino group and blocks or protects the amino functionality of a compound. Suitable amino protecting groups include, but are not limited to, acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ), and 9-fluorenylmethylenoxycarbonyl (Fmoc). For a general description of protecting groups and their use, see T.W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991 or subsequent editions.

[0099] II. Compositions and Methods In one aspect, the invention is based, in part, on antibodies that bind to LY6E and immunoconjugates comprising such antibodies. The antibodies and immunoconjugates of the invention are useful, for example, for the diagnosis or treatment of LY6E-positive cancers.

[0100] A. Exemplary Anti-Ly6E Antibodies In some embodiments, the invention provides an isolated antibody that binds to LY6E. In certain embodiments, the anti-LY6E antibody has at least one or more of the following characteristics, in any combination.

[0101] Non-limiting exemplary antibodies of the invention are Ly6E and its humanized variants. In some embodiments, Ly6E is human Ly6E, e.g., human Ly6E of SEQ ID NO: 1. In some embodiments, Ly6E is selected from human, cynomolgus monkey, rhesus monkey, mouse or rat Ly6E.

[0102] In some such embodiments, the anti-Ly6E antibody binds Ly6E with an affinity of ≤25 nM, or ≤20 nM, or ≤15 nM, or ≤10 nM, or ≤9 nM, or ≤8 nM, or ≤7 nM, or ≤6 nM, or ≤5 nM, or ≤4 nM, or ≤3 nM, or ≤2 nM, or ≤1 nM, and optionally, ≥0.0001 nM, or ≥0.001 nM, or ≥0.01 nM, as measured by either surface plasmon resonance (SPR) or Scatchard analysis. In some such embodiments, the anti-Ly6E antibody binds Ly6E with an affinity of 0-25 nM, 0-20 nM, 0-10 nM, or 10-20 nM. In some embodiments, Ly6E is human Ly6E. In some embodiments, Ly6E is human Ly6E, mouse Ly6E, rat Ly6E, or cynomolgus monkey Ly6E. In some embodiments, the monovalent affinity for human Ly6E is 10-20 nM. In some embodiments, the divalent affinity for human Ly6E is approximately 3-fold more potent than the monovalent affinity.

[0103] In one aspect, the invention provides an anti-Ly6E antibody comprising at least 1, 2, 3, 4, 5, or 6 HVRS selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 11; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 12; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 8; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 9.

[0104] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 11; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 12. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 12. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 12 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 9. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 9, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 11. In a further embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 11; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 12.

[0105] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 8; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 9. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 8; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 9.

[0106] In another aspect, the antibody of the invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 12; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 9.

[0107] In another aspect, the invention provides an antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 11; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 12; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 8; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 9.

[0108] In any of the above embodiments, the anti-Ly6E antibody is humanized. In one embodiment, the anti-Ly6E antibody comprises HVRs as in any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0109] In another aspect, the anti-Ly6E antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 32. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-Ly6E antibody comprising such sequence retains the ability to bind Ly6E. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 32. In certain embodiments, the substitutions, insertions, or deletions occur within regions outside of the HVRs (i.e., within the FRs). Optionally, the anti-Ly6E antibody comprises the VH sequence of SEQ ID NO: 32, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 12.

[0110] In another aspect, an anti-Ly6E antibody is provided that comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 31. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity includes substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-Ly6E antibody comprising such a sequence retains the ability to bind Ly6E. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 31. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., within the FRs). Optionally, the anti-Ly6E antibody comprises the VL sequence of SEQ ID NO: 31, which includes post-translational modifications of the sequence. In certain embodiments, the VL comprises 1, 2, or 3 HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 9.

[0111] In another aspect, an anti-Ly6E antibody is provided that comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 32 and SEQ ID NO: 31, respectively, which include post-translational modifications of those sequences.

[0112] In yet another aspect, the invention provides an antibody that binds to the same epitope as an anti-Ly6E antibody provided herein. For example, in certain embodiments, an antibody is provided that binds to the same epitope as an anti-Ly6E antibody comprising the VH sequence of SEQ ID NO: 32 and the VL sequence of SEQ ID NO: 31.

[0113] In a further aspect, the anti-Ly6E antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric antibody, a humanized antibody, or a human antibody. In one embodiment, the anti-Ly6E antibody is an antibody fragment, e.g., Fv, Fab, Fab’, scFv, diabody, or F(ab’) 2 fragment. In another embodiment, the antibody is a full-length antibody, e.g., an intact IgG1 antibody or other antibody class or isotype as defined herein.

[0114] In another aspect, an anti-Ly6E antibody is provided, the antibody including an HC as in any of the embodiments provided above and a VH as in any of the embodiments provided above. In some embodiments, the antibody includes the HC of SEQ ID NO: 5 and the LC of SEQ ID NO: 3, SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 29, including any post-translational modification of those sequences. In one embodiment, the antibody includes the HC of SEQ ID NO: 5 and the LC of SEQ ID NO: 3, including any post-translational modification of these sequences.

[0115] In a further aspect, the anti-Ly6E antibody according to any of the above embodiments can incorporate any of the features, alone or in combination, as described below.

[0116] Assay The anti-Ly6E antibody binds with an affinity of "≤25 nM, or ≤20 nM, or ≤15 nM, or ≤10 nM, or ≤9 nM, or ≤8 nM, or ≤7 nM, or ≤6 nM, or ≤5 nM, or ≤4 nM, or ≤3 nM, or ≤2 nM, or ≤1 nM", or is "0-25 nM, 0-20 nM, 0-10 nM, or 10-20 nM", which in some embodiments is determined according to Scatchard analysis. Alternatively, the anti-Ly6E antibody affinity can be determined, for example, according to a BIAcore™ assay. Specifically, Kd is measured using a surface plasmon resonance assay using BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ). The BIAcore™ Research Grade CM5 chip is activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) reagents according to the supplier's instructions. Goat anti-human Fc IgG is coupled to the chip to achieve approximately 10,000 response units (RU) in each flow cell. Unreacted coupling groups are blocked with 1 M ethanolamine. For kinetic measurements, the anti-Ly6E antibody is captured to achieve approximately 300 RU. A two-fold serial dilution of human Ly6E is injected at a flow rate of 30 μl / min at 25°C in HBS-P buffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 0.005% surfactant P20). The association rate (k on ) and dissociation rate (k off ) are calculated using a 1:1 Langmuir binding model (BIAcore™ Evaluation Software version 3.2). The equilibrium dissociation constant (Kd) is calculated as the ratio of k off / k on . The on-rate by the above surface plasmon resonance assay is 10 6 M -1 s -1When exceeding, the on-rate can be determined using a fluorescence quenching technique that measures the increase or decrease in the fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab type) (pH 7.2) in PBS at 25°C in the presence of increasing antigen concentrations, when measured with a spectrophotometer such as a spectrophotometer with stop-flow (Aviv Instruments) or an 8000 series SLM-Aminco® spectrophotometer (ThermoSpectronic) equipped with a stirred cuvette.

[0117] In any of the above embodiments, the anti-Ly6E antibody is humanized. In one embodiment, the anti-Ly6E antibody contains HVRs as in any of the above embodiments and further contains a human acceptor framework, for example, a human immunoglobulin framework or a human consensus framework. In certain embodiments, the human acceptor framework is a human VL kappa IV consensus (VL KIV ) framework and / or VH framework VH 1 is.

[0118] In a further aspect, the anti-Ly6E antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric antibody, a humanized antibody, or a human antibody. In one embodiment, the anti-Ly6E antibody is an antibody fragment, for example, Fv, Fab, Fab’, scFv, diabody, or F(ab’) 2 fragment. In another embodiment, the antibody is a substantially full-length antibody, for example, an IgG1 antibody as defined herein, or another antibody class or isotype.

[0119] In a further aspect, the anti-Ly6E antibody according to any of the above embodiments can incorporate any of the features, alone or in combination, as described below.

[0120] In a further aspect, an anti-Ly6E antibody according to any of the above embodiments is a monoclonal antibody, including a human antibody. In one embodiment, the anti-Ly6E antibody is an antibody fragment, e.g., Fv, Fab, Fab’, scFv, diabody, or F(ab’) 2 fragment. In another embodiment, the antibody is a substantially full-length antibody, e.g., an IgG2a antibody as defined herein, or another antibody class or isotype.

[0121] In a further aspect, an anti-Ly6E antibody according to any of the above embodiments can incorporate any of the features, alone or in combination, as described below.

[0122] 1. Antibody affinity In certain embodiments, the antibodies provided herein have a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM, and optionally, ≧10 -13 M (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M).

[0123] In one embodiment, the Kd is measured by a radiolabeled antigen binding assay (RIA) performed using the Fab version of the antibody of interest and its antigen, as described by the following assay. The solution binding affinity of the Fab for the antigen is in the presence of a titration series of unlabeled antigen, at the minimum concentration of ( 125I) It is measured by equilibrating Fab with the labeled antigen and then capturing the bound antigen on a plate coated with an anti-Fab antibody (see, for example, Chen et al., J. Mol. Biol. 293:865 - 881 (1999)). To establish the assay conditions, a MICROTITER® multiwell plate (Thermo Scientific) is coated overnight with 5 μg / ml of the capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) bovine serum albumin in PBS for 2 - 5 hours at room temperature (approximately 23°C). In a non-adsorptive plate (Nunc number 269620), 100 pM or 26 pM of 125 I]-antigen is mixed with a serial dilution of the Fab of interest (consistent with, for example, the evaluation of anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593 - 4599 (1997)). The Fab of interest is then incubated overnight, although the incubation can be continued for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. The mixture is then transferred to the capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate is dry, 150 μl / well of scintillant (MICROSCINT-20™, Packard) is added, and the plate is counted on a TOPCOUNT® gamma counter (Packard) for 10 minutes. The concentration of each Fab that results in 20% or less of the maximum binding is selected for use in the competitive binding assay.

[0124] According to another embodiment, Kd is measured using Scatchard analysis. According to another embodiment, Kd is measured at 25° C. using a surface plasmon resonance assay using a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) with an immobilized antigen CM5 chip at approximately 10 response units (RU). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIAcore, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted with 10 mM sodium acetate (pH 4.8) to 5 μg / ml (˜0.2 μM) and then injected at a flow rate of 5 μl / min until a bound protein of approximately 10 response units (RU) is reached. After injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold dilutions of Fab (0.78 nM to 500 nM) are injected at 25° C. at a flow rate of approximately 25 μl / min into PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST). The association rate (kon) and dissociation rate (koff) are calculated by simultaneously fitting the association and dissociation sensorgrams using a simple 1:1 Langmuir binding model (BIAcoreTM™ Evaluation Software version 3.2). The equilibrium dissociation constant (Kd) is calculated as the koff / kon ratio. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999).When the association rate by the above surface plasmon resonance assay exceeds 106 M-1 s-1, the association rate can be determined using a fluorescence quenching technique that measures the increase or decrease in the fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab type) (pH 7.2) in PBS at 25 °C in the presence of increasing concentrations of antigen, when measured with a spectrometer such as a spectrophotometer with flow stop (Aviv Instruments) or an 8000 series SLM-AMINCO (trademark) spectrophotometer (ThermoSpectronic) equipped with a stirred cuvette.

[0125] 2. Antibody fragments In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab’, Fab’-SH, F(ab’) 2 , Fv, and scFv fragments, and other fragments described below. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994). See also International Publication No. 93 / 16185, and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a description of Fab and F(ab’) 2 fragments that contain salvage receptor binding epitope residues and have an increased in vivo half-life, see U.S. Pat. No. 5,869,046.

[0126] A diabody is an antibody fragment having two antigen-binding sites that can be bivalent or bispecific. See, e.g., EP 404,097; International Publication No. WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetra-bodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0127] A single-domain antibody is an antibody fragment that comprises all or part of the heavy-chain variable domain or all or part of the light-chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516).

[0128] Antibody fragments can be made by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0129] 3. Chimeric and Humanized Antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switch" antibody in which the class or subclass has been changed from those of the parent antibody. A chimeric antibody includes its antigen-binding fragment.

[0130] In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce their immunogenicity in humans while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or a portion thereof) are derived from a non-human antibody and the FRs (or a portion thereof) are derived from human antibody sequences. A humanized antibody also optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues of the humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived) to, for example, restore or improve the specificity or affinity of the antibody.

[0131] Reviews of humanized antibodies and methods of making them are provided, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat’l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR (α-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "reshaping"); Dall’Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing "guided selection" approach to FR shuffling).

[0132] Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the "best fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatic mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0133] 4. Human Antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be made using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0134] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies having human variable regions in response to an antigen challenge. Such animals typically contain all or part of the human immunoglobulin locus that replaces the endogenous immunoglobulin locus, or exist extrachromosomally, or are randomly integrated into the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Also see, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Pat. No. 5,770,429, which describes HuMab® technology; U.S. Pat. No. 7,041,870, which describes K-M MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VelociMouse® technology. The human variable regions derived from intact antibodies produced by such animals can be further modified, for example, by combining them with different human constant regions.

[0135] In addition, human antibodies can be produced by methods using hybridomas. Human myeloma cell lines and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991).) Human antibodies produced using human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include, for example, U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies derived from hybridoma cell lines), and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (triooma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0136] Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0137] 5. Antibodies from Libraries The antibodies of the present invention can be isolated by screening a combinatorial library for antibodies having the desired activity or activities. For example, methods for producing phage display libraries and screening such libraries for antibodies having the desired binding characteristics are known in the art. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O’Brien et al., eds., Human Press, Totowa, NJ, 2001), and are further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0138] In certain phage display methods, the repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and then screened for antigen-binding phages as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments as either single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunization sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, as described in Griffiths et al., EMBO J, 12:725-734 (1993), naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self and self antigens without immunization. Finally, naive libraries can be synthetically generated by cloning unrearranged V gene segments from stem cells and achieving rearrangement in vitro using PCR primers containing random sequences to encode highly variable CDR3 regions, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Patent No. 5,750,373, as well as U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0139] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0140] 6. Multispecific Antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. A multispecific antibody is a monoclonal antibody having binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for LY6E and the other is for any other antigen. In certain embodiments, one of the binding specificities is for LY6E and the other is for CD3. See, e.g., U.S. Patent No. 5,821,337. In certain embodiments, the bispecific antibody can bind to two different epitopes of Ly6E. The bispecific antibody can also be used to localize a cytotoxic agent to cells expressing Ly6E. The bispecific antibody can be prepared as a full-length antibody or an antibody fragment.

[0141] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (Milstein and Cuello, Nature 305:537 (1983)), WO 93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Patent No. 5,731,168). Multispecific antibodies can also be made by engineering the electrostatic steering effect for making antibody Fc heterodimeric molecules (WO 2009 / 089004); cross-linking of two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); use of leucine zippers for making bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); use of "diabody" technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and can also be made, for example, by the preparation of trispecific antibodies as described in Tutt et al. J. Immunol. 147:60 (1991).

[0142] Also included herein are engineered antibodies having three or more functional antigen-binding sites, including "octopus antibodies" (see, e.g., US 2006 / 0025576).

[0143] Antibodies or fragments herein also include "dual action FAb" or "DAF" that includes an antigen-binding site that binds Ly6E as well as another different antigen (see, e.g., US 2008 / 0069820).

[0144] 7. Antibody Variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from residues within the amino acid sequence of the antibody and / or insertions into residues within the amino acid sequence of the antibody and / or substitutions of residues within the amino acid sequence of the antibody. Deletions, insertions, and substitutions can be arbitrarily combined to reach the final construct, provided that the final construct possesses the desired properties, such as antigen binding.

[0145] a) Substitution, insertion and deletion variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for introducing mutations by substitution include HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading "Preferred substitutions". More substantial changes are provided in Table 1 under the heading "Exemplary substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the product screened for the desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

Table 1

[0146] Amino acids can be classified according to common side chain properties. (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe

[0147] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0148] Certain types of substituted variants involve substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Generally, the resulting variant(s) selected for further study have a modification (e.g., improvement) of certain biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or have certain biological properties of the parent antibody that are substantially retained. Exemplary substituted variants are affinity matured antibodies and may be readily generated, for example, using phage display-based affinity maturation techniques as described herein. Briefly, one or more HVR residues are mutated, the variant antibody is displayed on a phage, and screened for a particular biological activity (e.g., binding affinity).

[0149] Modifications (e.g., substitutions) may be made, for example, in the HVRs to improve antibody affinity. Such modifications may be made in the “hot spots” of the HVRs, i.e., residues encoded by codons that mutate frequently during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in the SDR (a-CDR), and the resulting variant VH or VL is tested for binding affinity. Affinity maturation by constructing and then reselecting a secondary library is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa, NJ (2001)). In some embodiments of affinity maturation, diversity is introduced into the selected variable gene to be matured by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify antibody variants having the desired affinity. Another method of introducing diversity involves an HVR-directed approach in which several HVR residues (e.g., 4-6 residues at a time) are randomized. The HVR residues involved in antigen binding may be specifically identified, for example, using alanine-scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0150] In certain embodiments, substitutions, insertions, or deletions can occur within one or more of the HVRs so long as such modifications do not substantially reduce the ability of the antibody to bind the antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made within the HVRs. Such modifications may be outside of HVR “hot spots” or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unmodified or contains one, two, or three or fewer amino acid substitutions.

[0151] A useful method for identifying residues or regions of an antibody that can be targeted for mutagenesis is called “alanine scanning mutagenesis” as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, one residue or a group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at positions of amino acids that show functional sensitivity to the initial substitution. Alternatively, or in addition, the crystal structure of the antigen-antibody complex is used to identify the points of contact between the antibody and the antigen. Such contact residues and adjacent residues may be targeted as candidates for substitution or removed. Variants may be screened to determine whether they have the desired properties.

[0152] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides of 100 residues or more, as well as intrasequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of the antibody molecule include fusions of the antibody N- or C-terminus with an enzyme (e.g., in the case of ADEPT) or with a polypeptide that increases the serum half-life of the antibody.

[0153] b) Glycosylation variants In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree to which the antibody is glycosylated. Addition or deletion of glycosylation sites to the antibody can be readily accomplished by changing the amino acid sequence such that one or more glycosylation sites are created or removed.

[0154] If the antibody comprises an Fc region, the carbohydrates attached to the antibody may be altered. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally attached by N-linkage to Asn297 in the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modification of the oligosaccharides in the antibody may be performed to generate antibody variants with certain improved properties.

[0155] In one embodiment, an antibody variant is provided that has a carbohydrate structure lacking fucose (either directly or indirectly) attached to the Fc region. For example, the amount of fucose in such an antibody can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined, for example, as described in WO 2008 / 077546, by calculating the average amount of fucose in the sugar chain of Asn297 relative to the total of all sugar structures (e.g., complex, hybrid, and high-mannose structures) attached to Asn297 measured by MALDI-TOF mass spectrometry. Asn297 refers to the asparagine residue located at approximately position 297 of the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylation variants can have improved ADCC function. See, for example, US Patent Application Publication No. 2003 / 0157108 (Presta, L.); US Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications regarding "defucosylated" or "fucose-deficient" antibody variants include the following: US Patent Application Publication No. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US Patent Application Publication No. 2003 / 0115614; US Patent Application Publication No. 2002 / 0164328; US Patent Application Publication No. 2004 / 0093621; US Patent Application Publication No. 2004 / 0132140; US Patent Application Publication No. 2004 / 0110704; US Patent Application Publication No. 2004 / 0110282; US Patent Application Publication No. 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; US Patent Application Publication No. 2002 / 031140; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application Publication No. 2003 / 0157108, Presta, L; and International Publication No. 2004 / 056312, Adams et al., particularly Example 11), and knockout cell lines, such as the α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and International Publication No. 2003 / 085107).

[0156] For example, there is further provided an antibody variant having a bisected oligosaccharide in which the bisected oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in International Publication No. 2003 / 011878 (Jean-Mairet et al.); US Patent No. 6602684 (Umana et al.); and US Patent Application Publication No. 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in International Publication No. 1997 / 30087 (Patel et al.), International Publication No. 1998 / 58964 (Raju, S.), and International Publication No. 1999 / 22764 (Raju, S.).

[0157] c) Fc region variant In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibodies presented herein, thereby creating Fc region variants. The Fc region variants may include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) that contains an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0158] In certain embodiments, the present invention contemplates antibody variants that, while not all, have some effector functions and are thus desirable candidates for applications where the in vivo half-life of an antibody is important but certain effector functions (such as complement and ADCC) are unnecessary or harmful. To confirm the reduction / abolition of CDC and / or ADCC activity, in vitro and / or in vivo cytotoxicity assays can be performed. For example, an Fc receptor (FcR) binding assay can be carried out to confirm that the antibody lacks FcγR binding (and thus is likely to lack ADCC activity), but retains the ability to bind to FcRn. NK cells, which are the major cells mediating ADCC, express only Fc(RIII, while monocytes express Fc(RI, Fc(RII, and Fc(RIII. The expression of FcRs in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (for example, the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA), and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively or additionally, the ADCC activity of a molecule of interest can be evaluated in vivo, e.g., in an animal model as disclosed in Clynes et al., Proc. Nat’l Acad. Sci. USA 95:652-656 (1998). Also, a C1q binding assay may be performed to confirm that the antibody is unable to bind C1q and lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in International Publication Nos. 2006 / 029879 and 2005 / 100402. A CDC assay may be performed to evaluate complement activation (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al., Int’l. Immunol. 18(12):1759-1769 (2006)).

[0159] Examples of antibodies with reduced effector function include antibodies having one or more substitutions at residues 238, 265, 269, 270, 297, 327, and 329 of the Fc region (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0160] Certain antibody variants with improved or decreased binding to FcR are described. (See, e.g., U.S. Patent No. 6,737,056, International Publication No. 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0161] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 (EU numbering of residues) in the Fc region.

[0162] In some embodiments, modifications are made within the Fc region to effect a change (i.e., either an improvement or a decrease) in C1q binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, International Publication No. 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0163] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn) that is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) are described in US2005 / 0014934A1 (Hinton et al.). Those antibodies comprise an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, for example, substitutions at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0164] See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and International Publication No. 94 / 29351 for other examples of Fc region variants.

[0165] d) Cysteine-engineered antibody variants In certain embodiments, it may be desirable to create a cysteine-engineered antibody, such as a “thioMAb,” in which one or more residues of the antibody are replaced with cysteine residues. In certain embodiments, the replaced residues are present at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are positioned at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be replaced with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies can be generated, for example, as described in U.S. Patent No. 7,521,541.

[0166] e) Antibody derivatives In certain embodiments, the antibodies provided herein can be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. Suitable sites for derivatization of the antibody include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), copolymers of ethylene glycol / propropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof, but are not limited thereto. Polyethylene glycol propionaldehyde may be advantageous during production due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if multiple polymers are attached, they may be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative is to be used therapeutically under defined conditions, etc.

[0167] In another embodiment, conjugates of antibodies and non-protective sites that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells but heat the non-protective site to a temperature at which cells proximal to the antibody non-protective site die.

[0168] B. Recombinant Methods and Compositions Antibodies may be produced, for example, using the recombinant methods and compositions described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-LY6E antibody described herein is provided. Such a nucleic acid can encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In a further embodiment, a host cell comprising such a nucleic acid is provided. In such an embodiment, the host cell comprises (e.g., is transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a nucleic acid encoding an amino acid sequence comprising the VH of the antibody, or (2) a vector comprising a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphocyte cell (e.g., Y0, NS0, Sp20 cells). In one embodiment, a method of making an anti-LY6E antibody is provided, the method comprising culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or the host cell culture medium).

[0169] For the recombinant production of anti-LY6E antibodies, for example as described above, the nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody).

[0170] Suitable host cells for cloning or expressing the antibody-encoding vector include the prokaryotic or eukaryotic cells described herein. For example, the antibody may be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibodies of the invention may be isolated from the soluble fraction of the bacterial cell paste and further purified.

[0171] In addition to prokaryotes, eukaryotes such as filamentous fungi and yeast are suitable as cloning or expression hosts for vectors encoding antibodies, including strains and yeast strains in which the glycosylation pathway has been "humanized", resulting in the production of antibodies having a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0172] In addition, host cells suitable for expressing glycosylated antibodies are derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. A number of baculovirus strains have been identified and can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0173] Plant cell cultures can also be used as hosts. See, for example, U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429, which describe the PLANTIBODIES™ technique for generating antibodies in transgenic plants.

[0174] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are the monkey kidney CV1 line transformed by SV40 (COS-7); the 293 cells or 293 cells described in the human fetal kidney lineage (e.g., Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); dog kidney cells (MDCK; buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells described, for example, in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include (DHFR -Chinese hamster ovary (CHO) cells containing CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For an overview of specific mammalian host cell lines suitable for antibody production, see, for example, the following. Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0175] C. Assay The anti-LY6E antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0176] In one aspect, the antibodies of the invention are tested for their antigen-binding activity by known methods such as, for example, ELISA, BIACore™, FACS, or Western blot.

[0177] In another aspect, competitive assays can be used to identify antibodies that compete with any of the antibodies described herein for binding to LY6E. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) that is bound by the antibodies described herein. Detailed exemplary methods for mapping the epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0178] In an exemplary competitive assay, immobilized LY6E is incubated in a solution containing a first labeled antibody that binds to LY6E (e.g., any of the antibodies described herein), and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to LY6E. The second antibody may be present in a hybridoma supernatant. As a control, immobilized LY6E is incubated in a solution containing the first labeled antibody but no second unlabeled antibody. After incubation under conditions that permit binding of the first antibody to LY6E, excess unbound antibody is removed and the amount of label associated with the immobilized LY6E is measured. If the amount of label associated with the immobilized LY6E is substantially reduced in the test sample compared to the control sample, it is shown that the second antibody competes with the first antibody for binding to LY6E. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0179] D. Immunoconjugates The present invention also provides an immunoconjugate comprising an anti-LY6E antibody herein conjugated to one or more cytotoxic agents such as a chemotherapeutic agent or drug, a growth inhibitor, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioisotope (i.e., a radio-conjugate).

[0180] The immunoconjugate allows for targeting the delivery of the drug moiety to a tumor and, in some embodiments, enables intracellular accumulation thereof when systemic administration of the unconjugated drug would result in unacceptable levels of toxicity to normal cells (Polakis P. (2005) Current Opinion in Pharmacology 5:382 - 387).

[0181] Antibody-drug conjugates (ADCs) are targeted chemotherapy molecules that combine the properties of both antibodies and cytotoxic drugs by targeting potent cytotoxic drugs to antigen-expressing tumor cells (Teicher, B.A. (2009) Current Cancer Drug Targets 9:982-1004), thereby enhancing the therapeutic index by maximizing efficacy and minimizing off-target toxicity (Carter, P.J. and Senter P.D. (2008) The Cancer Jour. 14(3):154-169; Chari, R.V. (2008) Acc. Chem. Res. 41:98-107).

[0182] The ADC compounds of the present invention include those having anti-cancer activity. In some embodiments, the ADC compounds include an antibody conjugated, i.e., covalently bound, to a drug moiety. In some embodiments, the antibody is covalently bound to the drug moiety by a linker. The antibody-drug conjugates (ADCs) of the present invention selectively deliver an effective dose of the drug to tumor tissue, thereby achieving a higher selectivity, i.e., a lower effective dose, while increasing the therapeutic index (the "therapeutic concentration range").

[0183] The drug moiety (D) of an antibody-drug conjugate (ADC) may include any compound, moiety, or group having a cytotoxic or cytostatic effect. The drug moiety may confer its cytotoxic and cytostatic effects by mechanisms including, but not limited to, tubulin binding, DNA binding or intercalation, and inhibition of RNA polymerase, protein synthesis, and / or topoisomerase. Exemplary drug moieties include, but are not limited to, maytansinoids, dolastatin, auristatin, calicheamicin, pyrrolobenzodiazepine (PBD), nemorubicin and its derivatives, PNU-159682, anthracyclines, duocarmycin, vinca alkaloids, taxanes, trichothecene, CC1065, camptothecin, eribulin, and their stereoisomers, isosteres, analogs, and derivatives having cytotoxic activity. Non-limiting examples of such immunoconjugates are discussed in further detail below.

[0184] 1. Exemplary Antibody-Drug Conjugates Exemplary embodiments of an antibody-drug conjugate (ADC) compound include an antibody (Ab) that targets tumor cells, a drug moiety (D), and a linker moiety (L) that couples the Ab to the D. In some embodiments, the antibody is coupled to the linker moiety (L) via one or more amino acid groups (e.g., lysine and / or cysteine).

[0185] An exemplary ADC has the formula I: Ab-(L-D) p Formula I

[0186] having, wherein p is from 1 to about 20. In some embodiments, the number of drug moieties that can be conjugated to the antibody is limited by the number of free cysteine residues. In some embodiments, the free cysteine residues are introduced into the antibody amino acid sequence by the methods described herein. Exemplary ADCs of Formula I include, but are not limited to, antibodies having 1, 2, 3, or 4 engineered cysteine amino acids (Lyon, R. et al (2012) Methods in Enzym. 502:123-138). In some embodiments, one or more free cysteine residues are already present in the antibody without the use of engineering, in which case the existing free cysteine residues may be used to conjugate the antibody to the drug. In some embodiments, the antibody is exposed to reducing conditions prior to conjugation of the antibody to generate one or more free cysteine residues.

[0187] a) Exemplary linker A "linker" (L) is a bifunctional or polyfunctional moiety that can be used to link one or more drug moieties (D) to an antibody (Ab) to form an antibody-drug conjugate (ADC) of Formula I. In some embodiments, an antibody-drug conjugate (ADC) can be prepared using a linker having reactive functional groups for covalently binding to the drug and the antibody. For example, in some embodiments, the cysteine thiol of an antibody (Ab) can form a bond with a reactive functional group of the linker or a drug-linker intermediate to generate an ADC.

[0188] In one aspect, the linker has functionality that can react with a free cysteine present on the antibody to form a covalent bond. Non-limiting examples of such reactive functional groups include maleimide, haloacetamide, α-haloacetyl, succinimide ester, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester and other activated esters, anhydrides, acid chlorides, sulfonyl chloride, isocyanate, and isothiocyanate. See, for example, the conjugation method on page 766 of Klussman, et al (2004), Bioconjugate Chemistry 15(4):765-773, and the examples therein.

[0189] In some embodiments, the linker has functionality that can react with an electrophilic group present on the antibody. Exemplary such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some embodiments, the heteroatom of the reactive functional group of the linker can react with an electrophilic group on the antibody to form a covalent bond with the antibody unit. Non-limiting examples of such reactive functional groups include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide, but are not limited thereto.

[0190] The linker may include one or more linker components. Exemplary linker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-succinimidyl 4-(2-pyridylthio) valerate (“SPP”), and 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (“MCC”). Various linker components are known in the art, and some of them are described below.

[0191] The linker may be a "cleavable linker" that facilitates drug release. Non-limiting exemplary cleavable linkers include acid-labile linkers (e.g., including hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photosensitive linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52:127-131 (1992), U.S. Patent No. 5,208,020).

[0192] In certain embodiments, the linker has the following Formula II: TIFF2025517612000007.tif7170 Formula II

[0193] Wherein, A is a "spacer unit" and a is an integer from 0 to 1; W is an "amino acid unit" and w is an integer from 0 to 12; Y is a "spacer unit" and y is 0, 1, or 2; Ab, D, and p are defined as above for Formula I. Exemplary embodiments of such linkers are described in U.S. Patent No. 7,498,298, which is hereby expressly incorporated by reference herein.

[0194] In some embodiments, the linker component includes a "spacer unit" that links the antibody to another linker component or drug moiety. Non-limiting exemplary spacer units are shown below (where the wavy line indicates a site of covalent attachment to the antibody, drug, or additional linker component): TIFF2025517612000008.tif102170

[0195] In some embodiments, the linker component comprises "amino acid units". In some such embodiments, the amino acid units enable cleavage of the linker by proteases, thereby promoting release of the drug from the immunoconjugate upon exposure to intracellular proteases such as lysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21:778-784). Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include, but are not limited to, valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe), phenylalanine-lysine (fk or phe-lys), phenylalanine-homolysine (phe-homolys), and N-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include, but are not limited to, glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). The amino acid units may include naturally occurring amino acid residues, and / or trace amino acids, and / or non-naturally occurring amino acid analogs such as citrulline. The amino acid units can be designed and optimized for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases.

[0196] In some embodiments, the linker component includes a "spacer" unit that links the antibody directly to the drug moiety or via a stretcher unit and / or an amino acid unit. The spacer unit can be "self-destructive" or "non-self-destructive". A "non-self-destructive" spacer unit is one in which part or all of the spacer unit remains attached to the drug moiety upon cleavage of the ADC. Examples of non-self-destructive spacer units include, but are not limited to, glycine spacer units and glycine-glycine spacer units. In some embodiments, enzymatic cleavage of an ADC containing a glycine-glycine spacer unit by a tumor cell-associated protease results in the release of the glycine-glycine-drug moiety from the remainder of the ADC. In some such embodiments, the glycine-glycine-drug moiety is subjected to a hydrolysis step in the tumor cell and thus cleaves the glycine-glycine spacer unit from the drug moiety.

[0197] A "self-destructive" spacer unit enables the release of the drug moiety. In certain embodiments, the spacer unit of the linker includes a p-aminobenzyl unit. In some such embodiments, p-aminobenzyl alcohol is attached to the amino acid unit via an amide bond, and a carbamate, methylcarbamate, or carbonate is created between the benzyl alcohol and the drug (Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15:1087-1103). In some embodiments, the spacer unit is p-aminobenzyloxycarbonyl (PAB). In some embodiments, an ADC comprising a self-destructive linker has the following structure: TIFF2025517612000009.tif28170

[0198] Wherein Q is -C 1 -C 8 alkyl, -O-(C 1 -C 8is alkyl), - halogen, - nitro or - cyano; m is an integer in the range of 0 to 4; p is in the range of 1 to about 20. In some embodiments, p is in the range of 1 to 10, 1 to 7, 1 to 5, or 1 to 4.

[0199] Other examples of self - cleaving spacers include, but are not limited to, aromatic compounds electronically similar to the PAB group, such as 2 - aminoimidazole - 5 - methanol derivatives (U.S. Patent No. 7,375,078; Hay et al., (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho - or para - aminobenzyl acetals. In some embodiments, substituted and unsubstituted 4 - aminobutyric acid amides (Rodrigues et al., (1995) Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al., (1972) J. Amer. Chem. Soc. 94:5815) and spacers that undergo cyclization upon amide bond hydrolysis such as 2 - aminophenylpropionic acid amide (Amsberry et al., (1990) J. Org. Chem. 55:5867) can be used. Linking of the drug to the α - carbon of a glycine residue is another example of a self - cleaving spacer that may be useful in an ADC (Kingsbury et al., (1984) J. Med. Chem. 27:1447).

[0200] In some embodiments, linker L can be a dendritic type linker for covalently attaching more than one drug moiety to the antibody through a branched polyfunctional linker moiety (Sun et al., (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al., (2003) Bioorganic & Medicinal Chemistry 11:1761-1768). Dendritic linkers can increase the molar ratio of drug to antibody, i.e., the loading, which is related to the potency of the ADC. Thus, if the antibody has only one reactive cysteine thiol group, multiple drug moieties can be attached via the dendritic linker.

[0201] Non-limiting exemplary linkers are shown below in the context of the ADC of Formula I: TIFF2025517612000010.tif166170

[0202] Further non-limiting exemplary ADCs include the following structures where -S- is part of the antibody. TIFF2025517612000011.tif89170Wherein X is: TIFF2025517612000012.tif61170Y is: TIFF2025517612000013.tif16170Each R is, independently, H or C 1 -C 6 alkyl; and n is from 1 to 12.

[0203] Typically, peptide-type linkers can be prepared by forming peptide bonds between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, by liquid phase synthesis methods (e.g., E. Schroder and K. Lubke (1965), "The Peptides", volume 1, pp 76-136, Academic Press).

[0204] In some embodiments, the linker is substituted with groups that modulate solubility and / or reactivity. As non-limiting examples, charged substituents such as sulfonate (-SO 3 - ) or ammonium can increase the water solubility of the linker reagent and facilitate the coupling reaction of the linker reagent with the antibody and / or drug moiety, depending on the synthetic route used to prepare the ADC, or can facilitate the coupling reaction of Ab-L (antibody-linker intermediate) with D, or D-L (drug-linker intermediate) with Ab. In some embodiments, a portion of the linker is coupled to the antibody and a portion of the linker is coupled to the drug, and then Ab-(linker portion) a is coupled to drug-(linker portion) b to form the ADC of Formula I. In some such embodiments, the antibody comprises two or more (linker portion) a substituents such that two or more drugs are coupled to the antibody in the ADC of Formula I.

[0205] The compounds of the present invention specifically contemplate, without limitation, ADCs prepared using the following linker reagents: bis-maleimide-trioxyethylene glycol (BMPEO), N-(β-maleimidopropyloxy)-N-hydroxysuccinimide ester (BMPS), N-(ε-maleimidocaproyloxy) succinimide ester (EMCS), N-[γ-maleimidobutyryloxy] succinimide ester (GMBS), 1,6-hexane-bis-vinylsulfone (HBVS), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl) butyric acid hydrazide (MPBH), succinimidyl 3-(bromoacetamido) propionate (SBAP), succinimidyl iodoacetate (SIA), succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), N-succinimidyl-4-(2-pyridylthio) pentanoate (SPP), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), succinimidyl 4-(p-maleimidophenyl) butyrate (SMPB), succinimidyl 6-[(beta-maleimidopropionamido) hexanoate] (SMPH), iminothiolane (IT), sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and succinimidyl-(4-vinylsulfone) benzoate (SVSB), including bismaleimide reagents: dithiobismaleimide ethane (DTME), 1,4-bismaleimide butane (BMB), 1,4 bismaleimidyl-2,3-dihydroxybutane (BMDB), bismaleimide hexane (BMH), bismaleimide ethane (BMOE), BM(PEG) 2 (shown below), and BM(PEG) 3(shown below); bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). In some embodiments, the bismaleimide reagent enables the binding of the thiol group of cysteine in an antibody to a thiol-containing drug moiety, linker, or linker-drug intermediate. Other functional groups that are reactive with thiol groups include, but are not limited to, iodoacetamide, bromoacetamide, vinylpyridine, disulfide, pyridyldisulfide, isocyanate, and isothiocyanate. TIFF2025517612000014.tif32170

[0206] In some embodiments, the linker is an MC-sq-Ala linker. Examples of peptidomimetic linkers are available, for example, in WO 2015 / 095227.

[0207] Certain useful linker reagents can be obtained from a variety of commercial sources such as Pierce Biotechnology, Inc. (Rockford, IL), Molecular Biosciences Inc. (Boulder, CO), or can be synthesized according to procedures described in the art. For example, Toki et al., (2002) J. Org. Chem. 67:1866-1872; Dubowchik et al., (1997) Tetrahedron Letters, 38:5257-60; Walker, M.A. (1995) J. Org. Chem. 60:5352-5355; Frisch et al., (1996) Bioconjugate Chem. 7:180-186; US 6214345; WO 02 / 088172; US 2003130189; US2003096743; WO 03 / 026577; WO 03 / 043583; and WO 04 / 032828.

[0208] Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radio nucleotides to antibodies. See, for example, International Publication No. 94 / 11026.

[0209] b) Exemplary drug moieties Pyrrolobenzodiazepine In some embodiments, the ADC comprises a pyrrolobenzodiazepine (PBD). In some embodiments, the PDB dimer recognizes and binds to a specific DNA sequence. The PBD, a natural product anthramycin, was first reported in 1965 (Leimgruber et al., (1965) J. Am. Chem. Soc., 87:5793-5795; Leimgruber et al., (1965) J. Am. Chem. Soc., 87:5791-5793). Since then, numerous PBDs, both natural and analogs, have been reported (Thurston et al., (1994) Chem. Rev. 1994, 433-465 (US 6884799; US 7049311; US 7067511; US 7265105; US 7511032; US 7528126; US 7557099), including dimers of the tricyclic PBD skeleton). Without intending to be bound by any particular theory, it is believed that the dimer structure confers a three-dimensional shape appropriate for isohelicity with the minor groove of B-form DNA, thereby resulting in a snug fit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-VanDevanter, (1986) Acc. Chem. Res., 19:230-237). Dimeric PBD compounds having C2 aryl substituents have been shown to be useful as cytotoxic agents (Hartley et al., (2010) Cancer Res. 70(17):6849-6858; Antonow (2010) J. Med. Chem. 53(7):2927-2941; Howard et al., (2009) Bioorganic and Med. Chem. Letters 19(22):6463-6466).

[0210] In some embodiments, the PBD compounds can be used as prodrugs by protecting them with nitrogen protecting groups that are removable in vivo at the N10 position (WO 00 / 12507; WO 2005 / 023814).

[0211] The PBD dimer is conjugated to an antibody, and the resulting ADC has been shown to have anti-cancer properties (U.S. Patent Application Publication No. 2010 / 0203007). Non-limiting representative binding sites on the PBD dimer include a 5-membered pyrrolo ring, a tether between PBD units, and an N10-C11 imine group (International Publication No. 2009 / 016516, U.S. Patent Application Publication No. 2009 / 304710, U.S. Patent Application Publication No. 2010 / 047257, U.S. Patent Application Publication No. 2009 / 036431, U.S. Patent Application Publication No. 2011 / 0256157, International Publication No. 2011 / 130598).

[0212] In some embodiments of the ADCs provided herein, the drug moiety (e.g., drug D) is a compound of Formula A: TIFF2025517612000015.tif28170 wherein, R 2 is of Formula II: TIFF2025517612000016.tif9170 wherein A is a C 5-7 aryl group, X is OH, SH, CO 2 H, COH, N=C=O, NHR N , and (OC 2 H 4 ) m OCH 3 selected from the group consisting of, wherein R N is selected from the group consisting of H and C 1-4 alkyl, m is an integer from 1 to 3, (i) Q 1 is a single bond and Q 2 is selected from a single bond and -Z-(CH 2 ) n -, wherein Z is a single bond, O, S, or NH, and n is an integer from 1 to 3; or (ii) Q 1 is -CH=CH- and Q 2 is a single bond; R 2’ is halo, nitro, cyano, ether, C1-7 Alkyl, C 3-7 Heterocyclyl, and bis-oxy-C 1-3 An aryl group optionally substituted by one or more substituents selected from the group consisting of alkylene; 5-10 is; R 6 and R 9 are, independently, H, R, OH, OR, SH, SR, NH 2 , NHR, NRR’, nitro, Me 3 Sn and halo; R 7 is H, R, OH, OR, SH, SR, NH 2 , NHR, NHRR’, nitro, Me 3 Sn and halo; wherein R and R’ are independently optionally substituted C 1-12 alkyl, C 3-20 heterocyclyl and C 5-20 aryl groups; any of the following: (a) R 10 is H, and R 11 is OH or OR A wherein R A is C 1-4 alkyl; or (b) R 10 and R 11 form a nitrogen-carbon double bond between the nitrogen atom and the carbon atom to which they are attached; or (c) R 10 is H, and R 11 is SO Z M, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; R” is a C 3-12 alkylene group, the chain of which may be interrupted by one or more heteroatoms independently selected from the group consisting of O, S and NH, and / or one or more aromatic rings independently selected from the group consisting of benzene and pyridine; Y is O, S or NH; R6’ , R 7’ and R 9’ are each selected from the same groups as R 6 , R 7 and R 9 and are the same as R 10’ and R 11’ are each the same as R 10 and R 11 and when R 11 and R 11’ is SO Z M, M may represent a divalent pharmaceutically acceptable cation; and the point of attachment to linker L is via R 2 or R 2’ .

[0213] In some embodiments of formula (A), R 2 is of formula II: TIFF2025517612000017.tif9170wherein A is phenyl and X is selected from the group consisting of OH, SH, CO 2 H, COH, N=C=O, NHR N , and (OC 2 H 4 ) m OCH 3 , where R N is selected from the group consisting of H and C 1-4 alkyl, m is an integer from 1 to 3, and (i) Q 1 is a single bond and Q 2 is a single bond; R 2’ is a phenyl group optionally substituted by one or more substituents selected from the group consisting of halo, nitro, cyano and -OR, where R is a saturated C 1-7 alkyl group; R 6 and R 9 are independently selected from H, R, OH, OR, SH, SR, NH 2 , NHR, NRR’, nitro, Me 3 Sn and halo; R7 is selected from the group consisting of H, R, OH, OR, SH, SR, NH 2 , NHR, NHRR’, nitro, Me 3 Sn and halo; wherein R and R’ are independently selected from unsubstituted C 1-12 saturated alkyl; R 10 and R 11 form a nitrogen-carbon double bond between the nitrogen atom and the carbon atom to which they are attached; R” is a C 3-12 saturated alkylene group, the chain of which may be interrupted by one or two heteroatoms independently selected from the group consisting of O, S and NH; Y is O, S or NH; R 6’ , R 7’ and R 9’ are each selected from the same groups as R 6 , R 7 and R 9 and are the same as R 10’ and R 11’ respectively; and 10 and R 11 are the same as R and the point of attachment to linker L is via R 2 or R 2’ .

[0214] Exemplary PDB moieties of ADCs include, but are not limited to, the following (the wavy line indicates the site of covalent attachment to the linker): TIFF2025517612000018.tif28170.

[0215] Non-limiting exemplary linker-PBDs have the following structures. TIFF2025517612000019.tif28170.

[0216] Non-limiting exemplary linker-PBD moieties of ADCs include, but are not limited to, the following. TIFF2025517612000020.tif30170 wherein the wavy line indicates the covalent binding site to the antibody.

[0217] PBD and ADCs containing PBD can be prepared according to methods known in the art. For example, WO 2009 / 016516; US 2009 / 304710; US 2010 / 047257; US 2009 / 036431; US 2011 / 0256157; WO 2011 / 130598; and WO2010 / 043880.

[0218] c) Drug loading Drug loading is represented by p, which is the average number of drug moieties per antibody in the molecule of Formula I. The drug loading can range from 1 to 20 drug moieties (D) per antibody. The ADC of Formula I comprises a collection of antibodies conjugated with drug moieties in the range of 1 to 20. The average number of drug moieties per antibody in the preparation of the ADC from the conjugation reaction can be characterized by conventional means such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of the ADC with respect to p can also be determined. In some examples, the isolation, purification, and characterization of a homogeneous ADC in which p is a specific value from ADCs having other drug loadings can be achieved by means such as reverse phase HPLC or electrophoresis.

[0219] In some antibody-drug conjugates, p can be limited by the number of binding sites on the antibody. For example, as in the above specific exemplary embodiments, when the binding is a cysteine thiol, the antibody can have only one or several cysteine thiol groups, or can have only one or several sufficiently reactive thiol groups, whereby the linker can be attached. In certain embodiments, as the drug load increases, for example, when p exceeds 5, aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates can be caused. In certain embodiments, the average drug load of the ADC ranges from 1 to about 8, i.e., about 2 to about 6, or about 3 to about 5. In fact, in certain ADCs, the optimal ratio of drug moieties per antibody can be less than 8 and has been shown to be about 2 to about 5 (U.S. Patent No. 7,498,298).

[0220] In certain embodiments, fewer drug moieties than the theoretical maximum are conjugated to the antibody during the conjugation reaction. The antibody can contain, for example, lysine residues that do not react with the drug-linker intermediate or the linker reagent, as discussed below. Generally, the antibody does not contain many free and reactive cysteine thiol groups that can be linked to the drug moiety. In fact, most cysteine thiol residues in the antibody exist as disulfide bridges. In certain embodiments, the antibody can be reduced under partial or complete reduction conditions with a reducing agent such as dithiothreitol (DTT) or tricarbonyl ethylphosphine (TCEP) to generate reactive cysteine thiol groups. In certain embodiments, the present antibody is subjected to denaturing conditions to expose reactive nucleophilic groups such as lysine or cysteine.

[0221] The load of the ADC (drug / antibody ratio) can be controlled in different ways, for example, (i) limiting the molar excess of the drug-linker intermediate or the linker reagent compared to the antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limited reduction conditions for cysteine thiol modification.

[0222] When two or more nucleophilic groups react with a drug-linker intermediate or a linker reagent, it should be understood that the resulting product is a mixture of ADC compounds having the distribution of one or more drug moieties attached to the antibody. The average number of drugs per antibody can be calculated from the mixture by a dual ELISA antibody assay specific for the antibody and the drug. Individual ADC molecules can be identified in the mixture by mass spectrometry and separated, for example, by HPLC, such as hydrophobic interaction chromatography (see, e.g., McDonagh et al., (2006) Prot. Engr. Design & Selection 19(7):299-307; Hamblett et al., (2004) Clin. Cancer Res. 10:7063-7070; Hamblett et al., “Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate,” Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004; Alley et al., “Controlling the location of drug attachment in antibody-drug conjugates,” Abstract No. 627, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). In certain embodiments, homogeneous ADCs having a single loading value may be isolated from the conjugate mixture by electrophoresis or chromatography.

[0223] d) Specific methods for preparing immunoconjugates The ADCs of Formula I can be prepared by several routes using organic chemical reactions, conditions, and reagents known to those of skill in the art, including (1) reacting a nucleophilic group of the antibody with a bivalent linker reagent to form Ab-L by covalent bond, and then reacting with the drug moiety D, and (2) reacting a nucleophilic group of the drug moiety with a bivalent linker reagent to form D-L by covalent bond, and then reacting with the nucleophilic group of the antibody. An exemplary method for preparing an ADC of Formula I via the latter route is described in U.S. Patent No. 7,498,298, which is hereby expressly incorporated by reference.

[0224] Nucleophilic groups on the antibody include, but are not limited to, (i) the N-terminal amine group, (ii) side-chain amine groups such as lysine, (iii) side-chain thiol groups such as cysteine, and (iv) sugar hydroxyl or amino groups to which the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and can react with electrophilic groups on linker moieties and linker reagents including (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides, (ii) alkyl and benzyl halides such as haloacetamides, and (iii) aldehyde, ketone, carboxyl, and maleimide groups to form covalent bonds. Certain antibodies have reducible interchain disulfides, i.e., cysteine bridges. The antibody can be reacted with respect to conjugation with the linker reagent by treatment with a reducing agent such as DTT (dithiothreitol) or tricarbonyl ethylphosphine (TCEP) so as to be completely or partially reduced. Thus, each cysteine bridge will theoretically form two reactive thiol nucleophilic reagents. Additional nucleophilic groups can be introduced into the antibody by modification of lysine residues, for example, by reacting a lysine residue with 2-iminothiolane (Traut's reagent) to convert the amine to a thiol. Also, reactive thiol groups can be introduced into the antibody by introducing one, two, three, four, or more cysteine residues (e.g., by preparing a variant antibody containing one or more non-natural cysteine amino acid residues).

[0225] The antibody-drug conjugates of the present invention may also be produced by the reaction between an electrophilic group on the antibody such as an aldehyde or ketone carbonyl group and a nucleophilic group on the linker reagent or drug. Useful nucleophilic groups on the linker reagent include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide. In one embodiment, the antibody is modified to introduce an electrophilic moiety capable of reacting with a nucleophilic substituent on the linker reagent or drug. In another embodiment, the sugar of the glycosylated antibody may be oxidized with, for example, a periodate oxidation reagent to form an aldehyde group or a ketone group capable of reacting with the amine group of the linker reagent or drug moiety. The resulting imine Schiff base groups may form stable bonds or may be reduced, for example, by a borohydride reagent to form stable amine bonds. In one embodiment, reacting the carbohydrate portion of the glycosylated antibody with either galactose oxidase or sodium metaperiodate can result in the formation of carbonyl (aldehyde and ketone) groups in the antibody that are capable of reacting with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, an antibody containing an N-terminal serine or threonine residue can react with sodium metaperiodate, thereby generating an aldehyde in place of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146; U.S. Patent No. 5,362,852). Such aldehydes can react with the drug moiety or linker nucleophile.

[0226] Exemplary nucleophilic groups on the drug moiety include, but are not limited to, (i) activated esters such as NHS esters, HOBt esters, haloformates, and acid halides, (ii) alkyl and benzyl halides such as haloacetamides, and (iii) amines, thiols, hydroxyls, hydrazides, oximes, hydrazines, thiosemicarbazones, hydrazine carboxylates, and arylhydrazide groups that can react with electrophilic groups on linker moieties and linker reagents containing aldehyde, ketone, carboxyl, and maleimide groups to form covalent bonds.

[0227] Non-limiting exemplary cross-linking reagent examples that can be used to prepare ADCs are described in the section titled "Exemplary Linkers" herein. Methods of using such cross-linking reagent examples to link two moieties, including a proteinaceous moiety and a chemical moiety, are known in the art. In some embodiments, fusion proteins comprising an antibody and a cytotoxic agent can be made, for example, by recombinant techniques or peptide synthesis. The recombinant DNA molecule can contain a region encoding an antibody and a region encoding the cytotoxic portion of the conjugate, either adjacent to each other or separated by a region encoding a linker peptide that does not disrupt the desired properties of the conjugate.

[0228] In yet another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for use in pre-targeting of tumors, in which case the antibody-receptor conjugate is administered to the patient, followed by using a scavenger to remove unbound conjugate from the blood circulation, and then a "ligand" (such as avidin) conjugated to a cytotoxic agent (such as a drug or a radioactive nucleotide) is administered.

[0229] E. Methods and Compositions for Diagnosis and Detection In certain embodiments, any of the anti-LY6E antibodies provided herein are useful for detecting the presence of LY6E in a biological sample. As used herein, the term "detecting" encompasses quantitative or qualitative detection. A "biological sample" includes, for example, cells or tissues (e.g., biopsy material including cancerous or potentially cancerous colon, colorectal, endometrial, pancreatic, or ovarian tissue).

[0230] In one embodiment, an anti-LY6E antibody for use in a method of diagnosis or detection is provided. In a further aspect, a method of detecting the presence of LY6E in a biological sample is provided. In certain embodiments, the method comprises contacting a biological sample with an anti-LY6E antibody described herein under conditions that permit binding of the anti-LY6E antibody to LY6E, and detecting whether a complex has formed between the anti-LY6E antibody and LY6E in the biological sample. Such a method may be an in vitro or in vivo method. In one embodiment, for example, when LY6E is a biomarker for patient selection, an anti-LY6E antibody is used to select a subject eligible for treatment with the anti-LY6E antibody. In further embodiments, the biological sample is a cell or tissue (e.g., biopsy material including cancerous or potentially cancerous colon, colorectal, endometrial, pancreatic, or ovarian tissue).

[0231] In a further embodiment, the anti-LY6E antibody is used in vivo for the purpose of, for example, diagnosing cancer, determining the prognosis of cancer or staging cancer, determining an appropriate course of treatment, or monitoring the response of cancer to a treatment method, and detecting LY6E-positive cancer in a subject, for example, by in vivo imaging methods. One method known in the art for in vivo detection is, for example, the immuno-positron emission tomography (immunoPET) described in van Dongen et al., The Oncologist 12:1379-1389 (2007) and Verel et al., J. Nucl. Med. 44:1271-1281 (2003). In such an embodiment, a method for detecting LY6E-positive cancer in a subject, comprising administering a labeled anti-LY6E antibody to a subject having or suspected of having LY6E-positive cancer, and detecting the labeled anti-LY6E antibody in the subject, provides a method for indicating LY6E-positive cancer in the subject. In certain such embodiments, the labeled anti-LY6E antibody comprises an anti-LY6E antibody conjugated to a positron emitter such as 68 Ga, 18 F, 64 Cu, 86 Y, 76 Br, 89 Zr, and 124 I. In certain embodiments, the positron emitter is 89 Zr.

[0232] In a further embodiment, a diagnostic or detection method comprises contacting a first anti-LY6E antibody immobilized on a substrate with a biological sample to be tested for the presence of LY6E, exposing the substrate to a second anti-LY6E antibody, and detecting whether the second anti-LY6E antibody binds to a complex between the first anti-LY6E antibody and LY6E in the biological sample. The substrate may be any support medium, such as glass, metal, ceramic, polymeric beads, slides, chips, and other substrates. In certain embodiments, the biological sample comprises cells or tissue (e.g., biopsy material including cancerous or potentially cancerous colorectal, endometrial, pancreatic, or ovarian tissue). In certain embodiments, the first or second anti-LY6E antibody is any of the antibodies described herein. In such embodiments, the second anti-LY6E antibody can be 6D3 or 7C9; or an antibody derived from 6D3 or 7C9 described herein.

[0233] Exemplary disorders that can be diagnosed or detected according to any of the above embodiments include LY6E-positive cancers, such as LY6E-positive colorectal cancer (including adenocarcinoma), LY6E-positive ovarian cancer (including ovarian serous adenocarcinoma), LY6E-positive pancreatic cancer (including pancreatic ductal adenocarcinoma), and LY6E-positive endometrial cancer. In some embodiments, an LY6E-positive cancer is a cancer that receives an anti-LY6E immunohistochemistry (IHC) or in situ hybridization (ISH) score greater than "0", which corresponds to very weak or no staining in more than 90% of tumor cells. In another embodiment, an LY6E-positive cancer expresses LY6E at a 1+, 2+, or 3+ level. In some embodiments, an LY6E-positive cancer is a cancer that expresses LY6E according to a reverse transcriptase PCR (RT-PCR) assay that detects LY6E mRNA. In some embodiments, the RT-PCR is quantitative RT-PCR.

[0234] In certain embodiments, labeled anti-LY6E antibodies are provided. Labels include, but are not limited to, labels or moieties that are directly detectable (e.g., fluorescence, chromophores, electron-dense, chemiluminescence, and radiolabels), and moieties that are indirectly detectable (e.g., enzymes or ligands) by, for example, enzymatic reactions or molecular interactions. Exemplary labels include radioisotopes 32 P, 14 C, 125 I, 3 H, and 131 I, rare earth chelates or fluorophores such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferase, luciferases such as firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, enzymes that utilize hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, heterocyclic oxidases such as uricase and xanthine oxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, etc., but are not limited thereto. In another embodiment, the label is a positron emitter. Positron emitters include, but are not limited to, 68 Ga, 18 F, 64 Cu, 86 Y, 76 Br, 89 Zr, and 124 I. In certain embodiments, the positron emitter is 89 Zr.

[0235] F. Pharmaceutical Formulations The pharmaceutical formulations of the anti-LY6E antibodies or immunoconjugates described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing such antibodies or immunoconjugates having the desired purity with one or more optional pharmaceutically acceptable carriers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzetonium chloride, phenol, butyl, or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol, etc.), low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or nonionic surfactants such as polyethylene glycol (PEG), but are not limited thereto. Exemplary pharmaceutically acceptable carriers herein further include intervening drug dispersants such as soluble neutral active hyaluronidase glycoproteins (sHASEGP), such as human soluble PH-20 hyaluronidase glycoproteins such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Specific exemplary sHASEGP and methods of use including rHuPH20 are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases (e.g., chondroitinase).

[0236] Exemplary lyophilized antibody or immunoconjugate formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody or immunoconjugate formulations include those described in U.S. Patent No. 6,171,586 and International Publication No. 2006 / 044908, the latter formulation including histidine - acetate buffer.

[0237] The formulations herein may also contain two or more active ingredients as needed for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. For example, in some instances, it may be desirable to further provide a platinum complex for the treatment of LY6E - positive cancers such as, for example, LY6E - positive breast cancer, or LY6E - positive pancreatic cancer, or LY6E - positive colon cancer, or LY6E - positive colorectal cancer, or LY6E - positive melanoma cancer, or LY6E - positive ovarian cancer, or LY6E - positive non - small cell lung cancer, or LY6E - positive gastric cancer.

[0238] The active ingredient can also be incorporated within colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions by, for example, coacervation techniques or by interfacial polymerization, e.g., with hydroxy - methylcellulose or gelatin microcapsules and poly - (methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed., (1980).

[0239] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include a semipermeable matrix of a solid hydrophobic polymer containing an antibody or immunoconjugate, and this matrix is in the form of a shaped article, such as a film or microcapsule.

[0240] Formulations for in vivo administration are generally sterilized. Sterility can be readily achieved, for example, by filtering through a sterile filtration membrane.

[0241] G. Treatment methods and compositions Any of the anti-LY6E antibodies or immunoconjugates provided herein can be used in a method, for example, a treatment method.

[0242] In one aspect, the anti-LY6E antibodies or immunoconjugates provided herein are used in a method of inhibiting the proliferation of LY6E-positive cells, the method comprising exposing the cells to the anti-LY6E antibody or immunoconjugate under conditions that permit binding of the anti-LY6E antibody or immunoconjugate to LY6E on the surface of the cells, thereby inhibiting the proliferation of the cells. In certain embodiments, the method is an in vitro method or an in vivo method. In further embodiments, the cells are breast cancer cells or pancreatic cancer cells or colon cancer cells or colorectal cancer cells or melanoma cancer cells or ovarian cancer cells or non-small cell lung cancer cells or gastric cancer cells.

[0243] Inhibition of cell proliferation in vitro may be assayed using the CellTiter-Glo™ Luminescent Cell Viability assay, commercially available from Promega (Madison, WI). The assay determines the number of viable cells in a culture based on the quantification of ATP present, which indicates metabolically active cells. See Crouch et al., (1993) J. Immunol. Meth. 160:81-88, U.S. Patent No. 6,602,677. The assay may be performed in 96-well or 384-well formats suitable for automated high-throughput screening (HTS). See Cree et al., (1995) AntiCancer Drugs 6:398-404. The assay procedure involves directly adding a single reagent (CellTiter-Glo® Reagent) to the cultured cells. This results in the generation of a luminescence signal produced by cell lysis and the luciferase reaction. The luminescence signal is proportional to the amount of ATP present, which is directly proportional to the number of viable cells present in the culture. Data can be recorded by a luminometer or a CCD camera imaging device. Luminescence output is expressed as relative light units (RLU).

[0244] In another aspect, an anti-LY6E antibody or immunoconjugate for use as a medicament is provided. In a further aspect, an anti-LY6E antibody or immunoconjugate for use in a method of treatment is provided. In certain embodiments, an anti-LY6E antibody or immunoconjugate for use in the treatment of LY6E-positive cancer is provided. In certain embodiments, the present invention provides an anti-LY6E antibody or immunoconjugate for use in a method of treating an individual having LY6E-positive cancer, the method comprising administering to the individual an effective amount of the anti-LY6E antibody or immunoconjugate. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as, for example, as described below.

[0245] In a further aspect, the invention provides the use of an anti-LY6E antibody or immunoconjugate in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treating LY6E-positive cancer. In a further embodiment, the medicament is for use in a method of treating LY6E-positive cancer, the method comprising administering to an individual having LY6E-positive cancer an effective amount of the medicament. In such an embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent as described, for example, below.

[0246] In a further aspect, the invention provides a method for treating LY6E-positive cancer. In one embodiment, the method comprises administering to an individual having such LY6E-positive cancer an effective amount of an anti-LY6E antibody or immunoconjugate. In such an embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent as described below.

[0247] The LY6E-positive cancer according to any of the above embodiments can be, for example, LY6E-positive breast cancer, or LY6E-positive pancreatic cancer, or LY6E-positive colon cancer, or LY6E-positive colorectal cancer, or LY6E-positive melanoma cancer, or LY6E-positive ovarian cancer, or LY6E-positive non-small cell lung cancer, or LY6E-positive gastric cancer. In some embodiments, the LY6E-positive cancer is a cancer that receives an anti-LY6E immunohistochemistry (IHC) or in situ hybridization (ISH) score greater than "0", which corresponds to very weak or no staining in more than 90% of the tumor cells under the conditions described herein. In another embodiment, the LY6E-positive cancer expresses LY6E at a 1+, 2+ or 3+ level as defined under the conditions described herein. In some embodiments, the LY6E-positive cancer is a cancer that expresses LY6E according to a reverse transcriptase PCR (RT-PCR) assay that detects LY6E mRNA. In some embodiments, the RT-PCR is quantitative RT-PCR.

[0248] The "individual" according to any of the above embodiments can be a human.

[0249] In a further aspect, the present invention provides a pharmaceutical formulation comprising any of the anti-LY6E antibodies or immunoconjugates provided herein for use, for example, in any of the above-described treatment methods. In one embodiment, the pharmaceutical formulation comprises any of the anti-LY6E antibodies or immunoconjugates provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-LY6E antibodies or immunoconjugates provided herein and, for example, at least one additional therapeutic agent described below.

[0250] The antibodies or immunoconjugates of the present invention can be used alone or in combination with other agents in therapy. For example, the antibodies or immunoconjugates of the present invention may be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is, for example, a platinum complex for the treatment of LY6E-positive cancers such as LY6E-positive breast cancer, or LY6E-positive pancreatic cancer, or LY6E-positive colon cancer, or LY6E-positive colorectal cancer, or LY6E-positive melanoma cancer, or LY6E-positive ovarian cancer, or LY6E-positive non-small cell lung cancer, or LY6E-positive gastric cancer.

[0251] Such combination therapies as described above include co-administration (where two or more therapeutic agents are included in the same or separate formulations) and separate administration, in which case the administration of the antibodies or immunoconjugates of the present invention can be performed before, simultaneously with, and / or after the administration of the additional therapeutic agent and / or adjuvant. The antibodies or immunoconjugates of the present invention can also be used in combination with radiation therapy.

[0252] The antibodies or immunoconjugates of the present invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral administration, intralung administration, and intranasal administration, and, if desired in local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. The dosage can be by any suitable route, such as by injection, e.g., intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules are contemplated herein, including single or multiple administrations at various times, bolus dosing, and pulse infusion, but are not limited thereto.

[0253] The antibodies or immunoconjugates of the present invention will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site to which the agent is to be delivered, the method of administration, the dosing schedule, and other factors known to the medical practitioner. The antibodies or immunoconjugates can optionally, although not necessarily, be formulated with one or more of the agents currently being used to prevent or treat the disorder in question. The effective amount of such other agents will be determined by the amount of antibody or immunoconjugate present in the formulation, the type of disorder or treatment, and the other factors discussed above. These are generally used at the same dosage and by the same route as those described herein, or at about 1 to 99% of the dosage described herein, or at any dosage and by any route that is empirically / clinically determined to be appropriate.

[0254] For the prevention or treatment of a disease, the appropriate dosage of the antibody or immunoconjugate of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will be determined by the type of disease being treated, the type of antibody or immunoconjugate, the severity and course of the disease, whether the antibody or immunoconjugate is administered for prophylactic or therapeutic purposes, previous treatment methods, the patient's medical history, the response to the antibody or immunoconjugate, and the discretion of the attending physician. The antibody or immunoconjugate is preferably administered to the patient either as a single dose or over a series of treatments. Depending on the type and severity of the disease, for example, whether by one or more separate administrations or by continuous infusion, an antibody or immunoconjugate in the range of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) can be an initial candidate dosage for administration to the patient. A typical daily dosage may range from about 1 μg / kg to 100 mg / kg depending on the factors described above. For repeated administration over several days or more, the treatment is generally continued until the desired suppression of the disease symptoms occurs, depending on the condition of the disease. One exemplary dosage of the antibody or immunoconjugate is in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more dosages of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) can be administered to the patient. Such dosages can be administered intermittently, for example, once a week or once every three weeks (e.g., such that the patient receives about 2 to about 20 doses, or for example, about 6 doses of the antibody). An initial higher loading dose followed by one or more lower doses can also be administered. However, other dosing regimens may also be useful. The progress of this treatment can be easily monitored by conventional techniques and assays.

[0255] It is understood that any of the above formulations or treatment methods can be carried out using both the immunoconjugate of the present invention and the anti-LY6E antibody.

[0256] H. Manufactured Article In another aspect of the invention, there is provided a manufactured article comprising a material useful for treating, preventing and / or diagnosing the above-described disorders. The manufactured article comprises a container and a label or package insert inserted into or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container may be formed from various materials such as glass or plastic. The container holds a composition that is used alone or in combination with another composition effective to treat, prevent, and / or diagnose a disorder and may have a sterile access port (e.g., the container may be a vial having a stopper pierceable by an intravenous solution bag or a hypodermic needle). At least one active agent in the composition is an antibody or immunoconjugate of the invention. The label or package insert indicates that the composition is used to treat a selected symptom. Further, the manufactured article may comprise (a) a first container containing therein a composition (the composition comprising an antibody or immunoconjugate of the invention), and (b) a second container containing therein a composition (the composition comprising a further cytotoxic or therapeutic agent). The manufactured article in this embodiment of the invention may further comprise a package insert indicating that the composition can be used to treat a specific symptom. Alternatively, or additionally, the manufactured article may further comprise a second (or third) container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, or dextrose solution. It may further comprise other buffers, diluents, filters, needles, syringes, and other materials desirable from a commercial and user perspective.

[0257] I. Sequences In another aspect, there are provided the following sequences useful for treating, preventing and / or diagnosing the disorders described herein. [Table] TIFF2025517612000021.tif254170TIFF2025517612000022.tif225170TIFF2025517612000023.tif173170TIFF2025517612000024.tif202170TIFF2025517612000025.tif241170TIFF2025517612000026.tif246170TIFF2025517612000027.tif149170

Example

[0258] III. Example The following are examples of the methods and compositions of the present invention. It is understood that various other embodiments may be practiced in view of the general description given above.

[0259] Example 1 - Generation and Characterization of Virus-Like Particles (VLPs) Expi293 cells were co-transfected with a mammalian expression construct encoding full-length Ly6E and a mammalian expression construct encoding MLGag. Seven days after transfection, VLPs were purified from the supernatant using ultracentrifugation as described above (see, for example, Thery C., et al., “Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids,” Curr Protoc Cell Biology 2006;30:3.22.1-3.22.29, and WO2021 / 247457). VLP concentration was measured using the Bradford assay. Incorporation of Ly6E was confirmed by ELISA. Nunc Maxisorp ELISA plates (Thermo Scientific) were coated overnight at 4 °C with various concentrations of VLPs diluted in coating buffer (50 mM carbonate, pH 9.6). The plates were washed with wash buffer (1× PBS containing 0.05% Tween20) and then blocked for 1 hour at room temperature with ELISA assay diluent (PBS / 0.5% BSA / 0.05% polysorbate 20). The plates were then incubated for 1 hour at room temperature with anti-Ly6E (9B12; 1 μg / ml diluted in ELISA assay diluent). The plates were washed with wash buffer and the bound antibody was detected with a goat anti-rat horseradish peroxidase secondary antibody (Jackson Immunol Lab). The plates were incubated for 30 minutes at room temperature, washed with wash buffer, and developed with TMB solution (Surmodics, USA). The plates were read at 630 nm.

[0260] Example 2 - VLP Immunization The animals used in these studies were maintained in an AAALAC-accredited animal facility. All experiments were conducted in accordance with the Genentech Institutional Animal Care and Use Committee and OLAW Guidelines. Approval of the study design was obtained from the Genentech Institutional Animal Care and Use Committee prior to the start of this work.

[0261] Sprague Dawley rats (Charles River Laboratories, Hollister, CA) were immunized with Ly6E VLPs in PBS together with Ribi adjuvant (Sigma). Subsequently, the rats were boosted three times at two-week intervals with additional Ly6E VLPs. This was followed by four injections over a two-week period of a plasmid encoding Ly6E cDNA via a Genegun. The DNA / gold particle bullets were prepared essentially as described previously. See, for example, Tang et al., Nature 1992; 356:152-4, and Hansen et al., Sci Rep-uk 2016; 6:21925.

[0262] Each bullet of DNA was prepared to contain a total of 1 μg of DNA coated onto 0.5 mg of gold particles (BioRad, catalog number #1652264). The bullets were stored at 4 o C in the dark in the presence of desiccant pellets.

[0263] pAb was purified by protein A and assayed by FACS to identify antibodies that bind to cell surface Ly6E. Hybridoma fusion was performed as previously described, except that the myeloma partner SP2ab, which enables surface display of IgG cells, was used to generate monoclonal antibodies. See, for example, Price et al., J Immunol Methods 2009;343:28-41. Vij et al., Sci Rep-uk 2018;8:7136. Hybridoma supernatants were harvested and IgG was purified from the supernatants using MabSelect SuRe (GE Healthcare, Piscataway, NJ, USA). Anti-Ly6E hybridomas were identified by ELISA and FACS screening. The variable light and variable heavy chain sequences of the anti-Ly6E hybridomas were determined using 5’ RACE followed by sequencing of the PCR amplification products. Clones showing promising binding were cloned and expressed as rat-human chimeric antibodies (SEQ ID NOs: 4 and 6). The purified proteins were used for further analysis.

[0264] Example 3 - Characterization of anti-Ly6E The chimeric anti-Ly6E was compared with the previously described anti-Ly6E antibody (9B12) (Asundi et al., Clin. Canc. Res. 2015, 3252-3262) for binding to cells expressing Ly6E. Cell lines were cultured as recommended by ATCC. Prior to staining, adherent strains were released by Accutase treatment, all cells were filtered, and equilibrated in BD staining buffer (BSA). Cells were stained with the indicated concentration of primary antibody for 1 hour at 4°C, washed three times, stained with secondary Ab (Jackson ImmunoResearch 109-606-003) (1:100) for 1 hour at 4°C, washed, stained with Fixable Viability Dye eFluor 780 (1:1000) for 1 hour at 4°C, and washed twice. Samples were fixed in 1% PFA (PBS) prior to analysis. Samples were evaluated on a BD LSRFortessa. After exclusion of dead cells, singlets were evaluated for antibody binding using FlowJo software (Figures 2A-2C). In all three test cell lines, anti-Ly6E showed enhanced binding compared to 9B12.

[0265] Example 4 - Ly6E Humanization and Reformatting The Ly6E antibody was humanized by grafting the HC CDR and LC CDR into human germline frameworks (IGHV3-73*01 / IGHJ1*01 and IGKV1-33*01 / IGKJ2*01, respectively) and iterating between human and rat residues at Vernier positions to maximize binding while minimizing non-human residues. The binding of the humanized anti-Ly6E (including SEQ ID NOs: 3 and 5) was compared to that of the chimeric anti-Ly6E (including SEQ ID NOs: 4 and 6), 9B12, and isotype controls (Figures 3A-3B). For two cell lines, the humanized version showed similar binding to the chimeric anti-Ly6E and enhanced binding to 9B12. To enable production with THIOMAB® antibodies, cysteine residues were incorporated at previously described positions (Ohri et al., Bioconj Chem. 2018, 473-485). The humanized VH was cloned into a knob-into-hole scaffold to generate bispecific antibodies (Ridgeway et al., Prot. Eng. 1996, 617-621).

[0266] Example 5 - Characterization and Optimization of Anti-Ly6E Stability To assess potential manufacturing liability, anti-Ly6E was incubated at 40 °C for 2 weeks in low ionic strength histidine–acetic acid buffer pH 5.5 and analyzed by peptide mapping essentially as previously described (Xu et al. Mol. Pharmaceutics 2018, 4529–4537). This heat stress resulted in a 6.2% increase in the amount of the isomerized light chain (LC) D92 (Figs. 4A–4C). Mutations were incorporated to remove the problematic aspartic acid. LC D92S (SEQ ID NO: 29) and LC D92E (SEQ ID NO: 3) variants were generated, proteolyzed to Fab to generate monovalent binders, and evaluated for binding to NCI-H1781 cells by flow cytometry as described above (Fig. 5). Both variants showed similar binding to the humanized anti-Ly6E. In particular, the Fv charge of the variant was 7.5 for LC D92S and 6.5 for LC D92E. D92E was selected as the preferred mutant because its charge profile better matched the prediction method for normal PK (Hotzel et al., mAbs 2012, 753–760).

[0267] Example 6—Generation of Anti-Ly6E Antibody–Drug Conjugates Briefly, cysteine residues were engineered at desired positions in the heavy and / or light chains of antibodies targeting Her2, Ly6E, and CD33 antigens to generate their THIOMAB variants. As previously described, the THIOMAB antibodies were conjugated to linker drugs. See Junutula et al., Nat Biotechnol 2008, 26:925-932. Briefly, the antibodies were reduced overnight in the presence of 100-fold molar excess of DTT (Calbiochem). The reducing agent and cysteine or glutathione blocks were removed by purification using a HiTrap SP-HP column (GE Healthcare). The antibody was re-oxidized for 2.5 hours in the presence of 15-fold molar excess of dhAA (MP Biomedical). Formation of inter-chain disulfide bonds was monitored by LC / MS. A 10-fold molar excess of linker drug with respect to the protein was incubated with the activated THIOMAB antibody for 2 or 3 hours in the presence of 15% DMF. The antibody-drug conjugate was purified by a HiTrap SP-HP column (GE Healthcare) to remove the excess linker drug. If more than 5% aggregation still remained by analytical SEC, it was purified by a Hi Load Superdex 200pg 16 / 600 column (GE Healthcare) using 20 mM histidine-acetate, 150 mM NaCl, pH 5.5 as the running buffer. The number of conjugated linker drug molecules per THIOMAB antibody was quantified by LC / MS analysis. Purity was evaluated by size exclusion chromatography.

[0268] Mass spectrometry LC / MS analysis was performed using a 6530 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) LC / MS (Agilent Technologies). The sample was chromatographed on a PRLP-S, 1000 Å, 8 μm (50 mm × 2.1 mm, Agilent Technologies) heated to 80 °C. A linear gradient of 30 - 60% B over 4.3 minutes (solvent A, 0.05% TFA in water; solvent B, 0.04% TFA in acetonitrile) was used, and the eluate was directly ionized using an electrospray source. Data were collected and deconvoluted using Agilent Mass Hunter qualitative analysis software. Prior to LC / MS analysis, the antibody-drug conjugate was treated with DTT for 30 minutes at 15 mM, pH 8.0, and 37 °C to generate the HC and LC moieties for easier analysis. The drug-to-antibody ratio (DAR) was calculated using the abundance of the ions present in the LC / MS deconvolution results. Peaks were identified using LC / MS.

[0269] LC / MS analysis of the reduced conjugate showed that the antibody-drug conjugate had a range of 1.8 - 2 drugs per antibody for all conjugates.

[0270] Characterization of anti-Ly6E as an Example 7 - SN36325 antibody-drug conjugate Cells were seeded in 384-well plates, grown for 24 hours, and treated with the indicated ADCs containing isotype controls (anti-gD and anti-CD22) and conjugates to 9B12. After 5 days of continuous ADC incubation, cell viability was determined using the Promega CellTiter-Glo® luminescent reagent. Luminescence intensity was measured using a PerkinElmer EnVision® reader (Figures 6A - 6B). Relative cell viability was calculated by normalizing to non-drug-treated controls. The anti-Ly6E conjugate shows more potent killing against both the HCC1569x2 and SW900 cell lines than the isotype controls and the 9B12 conjugate. Minimal differences were seen between the chimeric and humanized versions of anti-Ly6E.

[0271] Example 8 - Synthesis of (maleimide-sq-ala)-PBD linker-drug (maleimide-sq-ala)-PBD linker-drug synthesis scheme: TIFF2025517612000028.tif111170

[0272] The maleimide-sq-ala linker is conjugated to the PBD payload in two steps, first introducing alanine and then conjugating maleimide-sq via INT5, an activated maleimide-sq-ester. The linker in this example has a 5-carbon spacer between maleimide and sq.

[0273] INT5-precursor synthesis scheme: TIFF2025517612000029.tif74170

[0274] General procedure for preparing Compound 3: TIFF2025517612000030.tif19170

[0275] To a solution of Compound 2 (386 mg, 1.24 mmol) in DCM (20 mL) and methanol (2 mL) was added EEDQ (306 mg, 1.24 mmol). The mixture was stirred at 20 °C for 5 minutes. Then, Compound 1 (300.0 mg, 0.41 mmol; commercially available, for example, BOC Sciences "PBD dimer-CAS 1222490-34-7" at www.bocsci.com / product / pbd-dimer-cas-1222490-34-7-477053.html) was added to the above solution. The reaction mixture was stirred at 20 o °C for 12 hours. The mixture was concentrated and methyl tert-butyl ether (50 mL) was added. The solid was collected and washed with methyl tert-butyl ether (50 mL × 2) to obtain crude Product Compound 3 (400 mg, 60%) as a yellow solid, which was used directly in the next step. LCMS (5-95, AB, 1.5 min): R T = 0.838 min, m / z = 1019.6 [M+H] + .

[0276] General procedure for preparing Compound 4: TIFF2025517612000031.tif17170

[0277] To a solution of Compound 3 (70.0 mg, 0.07 mmol) in DMF (2 mL) was added piperidine (0.01 mL, 0.14 mmol). The mixture was stirred at 20 °C for 30 minutes. The mixture was concentrated and methyl tert-butyl ether (40 mL) was added. The mixture was filtered to obtain a solid, which was washed with methyl tert-butyl ether (40 mL × 2) to obtain crude Compound 4 (40 mg, 73%) as a yellow solid. LCMS (5-95, AB, 1.5 min): RT = 0.765 min, m / z = 797.4 [M+H] + .

[0278] General procedure for coupling the INT5 precursor to the ala-PBD payload: TIFF2025517612000032.tif70170

[0279] To a solution of compound 5 (47.6 mg, 0.1 mmol) in DMF (2 mL) was added compound 4 (40.0 mg, 0.05 mmol) and N,N - diisopropylethylamine (0.02 mL, 0.1 mmol). The reaction mixture was stirred at 20 °C for 2 h. The mixture was purified by Pre - HPLC (acetonitrile: 40% - 70% / 0.225% FA in water) to afford the product linker - payload X (25 mg, 46%) as a yellow solid. LCMS (5 - 95, AB, 1.5 min): RT = 0.838 min, m / z = [M + H] + 1087.5。

[0280] General procedure for preparing compound 8: TIFF2025517612000033.tif31170

[0281] To a mixture of cyclobutane - 1,1 - dicarboxylic acid (0.82 g, 5.72 mmol) in DMF (10 mL) were added HATU (2.17 g, 5.72 mmol) and N,N - diisopropylethylamine (2.22 g, 17.15 mmol), followed by the addition of 1 - (5 - aminopentyl)pyrrole - 2,5 - dione hydrochloride (1.0 g, 4.57 mmol) at 0 °C. The mixture was stirred at 0 o °C for 1 h. LCMS indicated that the reaction was complete. The solvent was removed in vacuo, dissolved in EtOAc (50 mL), and then washed with aqueous HCl (60 mL, 1 mol / L) and brine (30 mL). The organic phase was dried over Na2SO4, filtered, and concentrated. The residue was purified by chromatography on silica eluting with 0 - 5% methanol in DCM to afford 1 - [5 - (2,5 - dioxopyrrol - 1 - yl)pentylcarbamoyl]cyclobutanecarboxylic acid (0.90 g, 64%) as a white solid. LCMS: (5 - 95, AB, 1.5 min), RT = 0.688 min, m / z = 308.9 [M + H] + ; 1 H NMR(400MHz,CDCl 3): δ 6.71 (s, 2H), 6.37 (br s, 1H), 3.53 (t, J = 6.8 Hz, 2H), 3.34 - 3.31 (m, 2H), 2.68 - 2.66 (m, 2H), 2.54 - 2.51 (m, 2H), 2.11 - 1.98 (m, 2H), 1.64 - 1.57 (m, 4H), 1.32 - 1.28 (m, 2H).

[0282] General procedure for preparing Compound 5: TIFF2025517612000034.tif34170

[0283] Compound 8 (37.0 mg, 0.12 mmol) was added to a solution of pentafluorophenol (33 mg, 0.18 mmol) and DIC (22.7 mg, 0.18 mmol) in DCM (5 mL) at 0 °C. The mixture was brought to 20 °C and stirred for 4 h. The solvent was then removed under vacuum, EtOAc (1.5 mL) was added to the mixture, the resulting precipitate was removed by filtration, and the filtrate was concentrated to give Compound 5 (50 mg, 69%) as a colorless oil. LCMS (5 - 95, AB, 1.5 min): RT = 0.785 min, m / z = 475.1 [M + H]+.

[0284] Example 9 - In vitro characterization of anti - Ly6E as an SGD - 1882 antibody - drug conjugate Anti - Ly6E was conjugated to SGD - 1882 essentially as described above using a maleimide - sq - ala linker having a 5 - carbon spacer between maleimide and sq. All conjugates had less than 5% aggregates and were produced with a DAR > 1.8. Cells were seeded in 384 - well plates, grown for 24 h, and treated with the indicated ADCs containing the previously described anti - Ly6E antibody (9B12). After 5 days of continuous ADC incubation, cell viability was determined using Promega CellTiter - Glo® luminescence reagent. Luminescence intensity was measured using a PerkinElmer EnVision® reader (Figure 7). Relative cell viability was calculated by normalizing to a non - drug - treated control. Anti - Ly6E shows more potent killing against the SW900 cell line than 9B12.

[0285] Example 9 - In Vivo Characterization of Anti-Ly6E as an SGD-1882 Antibody-Drug Conjugate (ADC) The efficacy of the Ly6E antibody-drug conjugate was examined in the human breast cancer xenograft model HCC1569X2. The HCC1569X2 cell line was derived at Genentech from the parental HCC1569 cells (ATCC) to provide optimal tumor growth in mice. This cell line was authenticated by short tandem repeat (STR) profiling using the Promega PowerPlex® 16 System to determine the cell line's ancestry compared to the external STR profile of the cell line. Animal studies using this cell line were conducted at Genentech in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals and were approved by Genentech's Institutional Animal Care and Use Committee (IACUC). To establish the xenograft model, 5 million tumor cells (suspended in 0.2 mL of HBSS containing Matrigel) were inoculated into the mammary fat pad of the chest of female C.B-17 SCID-beige mice (Charles River Laboratory; Hollister, CA).

[0286] Once the tumors reached the desired volume (approx. 250 mm 3 ), the animals were divided into n = 5 groups with similar distributions of tumor volume and received an intravenous dose of vehicle (20 mM histidine acetate, 240 mM sucrose, 0.02% polysorbate-20, pH 5.5) or antibody-drug conjugate (ADC) via the tail vein. Treatment information was not blinded during the measurement. Tumors were measured in two dimensions (length and width) using calipers, and the tumor volume was calculated using the following formula. Tumor size (mm3) = 0.5 × (length × width × width). Changes in body weight were reported as a percentage of the starting body weight. Tumor size and mouse body weight were recorded twice a week over the course of the study. When the tumor volume reached 2000 mm 3Mice that exceeded or had a weight loss of 20% of their starting weight were immediately euthanized in accordance with IACUC guidelines.

[0287] Data were analyzed using the R statistical software system (R Foundation for Statistical Computing; Vienna, Austria), and mixed modeling was fitted within R using the nlme package (Pinheiro et al., (2013) Nlme: Linear and Nonlinear Mixed-Effects Models. R Package Version 31-110.3.1-113). Cubic regression splines were used to fit non-linear profiles to the large tree changes or the time course of log2 tumor volume at each dose level. These non-linear profiles were then associated with the dose within the mixed mode. This approach addresses both repeated measurements and minor dropout due to removal of animals unrelated to treatment before the end of the study. Results were plotted on the natural scale as the fitted weight change or tumor volume over time for each group (Figures 8A–8B).

[0288] Example 10 - Characterization of anti-Ly6E as a T cell involved in bispecificity Bispecific antibodies targeting Ly6E and CD3 with high-affinity or low-affinity arms were expressed using knob-into-hole essentially as described. See Mandikian et al., Molecular cancer therapeutics 2018, 17:776-85; Atwell et al., J Mol Biol 1997, 270:26-35; and Ridgway et al., Protein Eng Des Sel 1996, 9:617-21. Cell death induced by the bispecific antibodies was evaluated essentially as previously described (e.g., see Junttila et al., Canc.Res. 2014, 74:5561-5571) using a 4:1 effector-to-target ratio of CD8+ T cells and 3-day co-culture of the cells (Figures 9A-9F). As expected, the higher-affinity anti-CD3 arm corresponded to higher activity. Anti-Ly6E was demonstrated to be superior to 9B12 across all cell lines tested.

[0289] Example 11 - Further Characterization of Anti-Ly6E as an SN36325 Antibody-Drug Conjugate Cells were seeded in 384-well plates, grown for 24 hours, and treated with the indicated ADCs containing isotype controls (anti-gD and anti-CD22) and conjugates to 9B12. After 5 days of continuous ADC incubation, cell viability was determined using the Promega CellTiter-Glo® luminescent reagent. Luminescence intensity was measured using a PerkinElmer EnVision® reader. Relative cell viability was calculated by normalizing to non-drug-treated controls. Anti-Ly6E conjugates (3A3 and 4B10) showed more potent killing against both the NCl-H1781 (Figures 10A - 10B) and HCC1569x2 (Figures 11A - 11B) cell lines compared to isotype controls, 9B12, and other ADC conjugates. For example, Figures 10A - 10B show that the chimeric anti-Ly6E 4B10 and 3A3 show a >400-fold potency difference from non-targeted controls (e.g., isotype control anti-gD and anti-CD22), compared to only 9-fold with 9B12 in the NCl-H1781 cell line. Figures 11A - 11B show that the chimeric anti-Ly6E 3A3 shows the highest potency against the HCC1569x2 cell line compared to other ADCs and control conjugates, despite overall weaker potency with partial killing.

[0290] Example 12 - Characterization of 3A3 Binding On day 0, Kuramochi cells were seeded in 384-well Cell Carrier plates (Perkin Elmer) and allowed to adhere overnight to obtain 80% cell confluence. The next day, cells were treated with non-conjugated antibodies at 2 μg / mL and 20 μg / mL using cold medium containing 10 μg / mL leupeptin and 5 μM pepstatin protease inhibitors. Antibodies were allowed to bind for 1 hour on ice. Pulse-treated cells were washed with cold medium containing protease inhibitors, while continuously treated cells were left alone. Cells were incubated at 37°C, 5% humidified CO 2They were incubated for 1 hour and 3 hours. After incubation, the cells were fixed with 4% paraformaldehyde (PFA) / 4% sucrose in phosphate-buffered saline (PBS) for 10 minutes at room temperature (RT), and then the cells were washed 6 times with 1×PBS. The cells were blocked and permeabilized with 2% donkey serum containing 0.05% saponin (blocking buffer) for 1 hour. The blocking buffer was removed, and then rabbit anti-LAMP1 (Sigma Aldrich, L1418) primary antibody in the blocking buffer was added and incubated overnight. The next day, the cells were washed 6 times with PBS containing 0.05% saponin and then washed with secondary antibody donkey anti-human IgG-488. Only anti-Ly6E was demonstrated to show substantially enhanced binding to Ly6E overexpressing cells. See Figure 12A.

[0291] Furthermore, direct binding of antigen-binding fragment (Fab)-luciferase fusions to three different Ly6E-expressing cells was evaluated after 4-hour incubation at 4°C. Figures 12B-12D show that 3A3 (anti-ly6E) has substantially enhanced binding in the Kuramochi cell line (Figure 12B), NCl-H1781 cell line (Figure 12C), and HCC-1569x2 cell line (Figure 12D) where Ly6E expression was confirmed by Western blot. The dots represent the average of two replicate samples. BIAcore™ Fab binding (Kd = nM) was also shown for 3A3 (218Kd) > 2.2C2 (168Kd) > 2.2C5 (63Kd) > 1D5.3 (20Kd) > 2.5G6 (14Kd) > 1D5.2 (12Kd).

[0292] Example 13 - Co-localization Kuramochi cells were exposed to each antibody at 2 μg / mL for 1 hour at 4°C, and the antibody was washed for pulse exposure (PE) or not washed for continuous exposure (CE), and then co-localization and internalization (intracellular intensity / intracellular intensity + extracellular intensity) were measured at 37°C for 0, 1, and 3 hours. Figure 13 shows that the amount of 3A3 co-localization is significantly higher compared to antibodies 1D5.3, 2.5G6, 9B12, and the control.

[0293] Example 14 - Internalization To evaluate the internalization rate, a pulse-chase experiment was performed in which the antibody was incubated with Kuramochi cells at 2 μg / mL for 1 hour at 4°C, washed, and internalization was monitored over time (0, 1, 3 hours). Figure 14 shows that the internalization rate of 9B12 was faster compared to 3A3, but the higher binding of 3A3, as seen in the in vivo killing study, results in more sustained efficacy.

[0294] The above invention has been described in some detail by way of explanation and examples for the purpose of clarity of understanding, but the explanation and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific documents cited herein are hereby expressly incorporated by reference in their entirety.

Claims

1. An isolated antibody that binds to Ly6E, comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 8; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 9; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 11; and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO:

12.

2. The antibody according to claim 1, which is a monoclonal antibody.

3. The antibody according to claim 1 or 2, which is a humanized or chimeric antibody.

4. The antibody according to any one of claims 1 to 3, which is an antibody fragment.

5. (a) A VH sequence having at least 95% sequence identity with SEQ ID NO: 32; (b) A VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 31; or (c) A VH sequence as in (a) and a VL sequence as in (b), the antibody according to any one of claims 1 to 4.

6. The antibody according to claim 5, comprising the VH sequence of SEQ ID NO: 32 and the VL sequence of SEQ ID NO:

31.

7. An isolated antibody comprising the VH sequence of SEQ ID NO: 32 and the VL sequence of SEQ ID NO:

31.

8. The antibody according to any one of claims 1 to 7, which is IgG1, IgG2a, IgG2b, IgG3 or IgG4.

9. A heavy chain comprising an amino acid sequence selected from SEQ ID NO: 5 and a light chain comprising an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO: 29, the antibody according to any one of claims 1 to 8.

10. The antibody according to claim 9, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a light chain comprising the amino acid sequence of SEQ ID NO:

3.

11. An isolated antibody that binds to Ly6E, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a light chain comprising the amino acid sequence of SEQ ID NO:

3.

12. The antibody according to any one of claims 1 to 11, wherein the antibody is a multispecific antibody.

13. The antibody according to claim 12, wherein the multispecific antibody is a bispecific antibody.

14. The antibody according to claim 12 or 13, wherein the multispecific antibody binds to Ly6E and CD3.

15. An isolated nucleic acid encoding the antibody according to any one of claims 1 to 14.

16. An expression vector comprising the nucleic acid according to claim 15.

17. A host cell comprising the nucleic acid according to claim 14 or the expression vector according to claim 15.

18. A host cell that expresses the antibody according to any one of claims 1 to 14.

19. A method for producing an antibody, comprising culturing the host cell according to claim 17 or claim 18 so that the antibody is produced.

20. An immunoconjugate comprising the antibody according to any one of claims 1 to 14 and a cytotoxic agent.

21. The immunoconjugate according to claim 20, having the formula Ab-(L-D)p, wherein (a) Ab is the antibody according to any one of claims 1 to 14; (b) L is a linker; (c) D is pyrrolobenzodiazepine; and (d) p ranges from 1 to 8.

22. The immunoconjugate according to claim 21, wherein D is a pyrrolobenzodiazepine of formula A: wherein R ２ is of formula II: and in the formula, A is C ５－７ an aryl group, X is OH, SH, CO ２ H, COH, N=C=O, NHR Ｎ , and (OC ２ H ４ ) ｍ OCH ３ selected from the group consisting of, where R Ｎ is selected from the group consisting of H and C １－４ alkyl, and m is an integer from 1 to 3 (i) Q １ is a single bond, and Q ２ is selected from a single bond and -Z-(CH ２ ) ｎ -, where Z is a single bond, O, S, or NH, n is an integer from 1 to 3; or (ii) Q １ is -CH=CH-, and Q ２ is a single bond; R ２’ is an optionally substituted C １－７ alkyl, C ３－７ heterocyclyl, and bis-oxy-C １－３ alkylene, by one or more substituents selected from the group consisting of halo, nitro, cyano, ether, C ５－１０ aryl group; R ６ and R ９ are, independently, selected from H, R, OH, OR, SH, SR, NH ２ , NHR, NR R', nitro, Me ３ Sn and halo; R ７ is selected from the group consisting of H, R, OH, OR, SH, SR, NH ２ , NHR, NHRR', nitro, Me ３ Sn and halo; Here, R and R' are independently optionally substituted C １－１２ alkyl, C ３－２０ heterocyclyl and C ５－２０ selected from aryl groups; any of the following: (a) R １０ is H, and R １１ is OH or OR Ａ where R Ａ is C １－４ alkyl; or (b) R １０ and R １１ form a nitrogen-carbon double bond between the nitrogen atom and the carbon atom to which they are attached; or (c) R １０ is H, and R １１ is SO Ｚ M, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; "R” is C ３－１２ an alkylene group, and this chain may be interrupted by one or more heteroatoms independently selected from the group consisting of O, S and NH, and / or by one or more aromatic rings independently selected from the group consisting of benzene or pyridine; Y is O, S or NH; R ６’ 、 R ７’ and R ９’ are each selected from the same group as R ６ 、 R ７ and R ９ and are the same as R １０’ 、 R １１’ and R １０ respectively; when R １１ and R １１ are SO １１’ M, M may represent a divalent pharmaceutically acceptable cation; and Ｚ ​ The binding point with the linker L is R ２ or R ２’ via an immunoconjugate.

23. The immunoconjugate according to claim 22, wherein D has the following structure: wherein the wavy line indicates the binding point to the linker L.

24. The immunoconjugate according to any one of claims 21 to 23, wherein (L-D) has the following structure: having the formula, wherein the wavy line indicates the binding point to the protein.

25. The immunoconjugate according to any one of claims 21 to 24, wherein p ranges from 1.5 to 5 or 1.5 to 6 or 1.5 to 4 or 2 to 3.

26. The immunoconjugate according to any one of claims 20 to 25, comprising the antibody according to claim 7.

27. The immunoconjugate according to any one of claims 20 to 26, comprising the antibody according to claim 11.

28. A pharmaceutical preparation comprising the immunoconjugate according to any one of claims 20 to 27 and a pharmaceutically acceptable carrier.

29. The pharmaceutical preparation according to claim 28, further comprising an additional therapeutic agent.

30. A method for treating an individual having Ly6E-positive cancer, comprising administering to the individual an effective amount of the immunoconjugate according to any one of claims 20 to 27 or the pharmaceutical preparation according to claim 28 or claim 29.

31. The method according to claim 30, wherein the Ly6E-positive cancer is selected from breast cancer, pancreatic cancer, colon cancer, colorectal cancer, melanoma, ovarian cancer, non-small cell lung cancer, or gastric cancer.

32. The method according to claim 30 or claim 31, further comprising administering an additional therapeutic agent to the individual.

33. The method according to claim 32, wherein the additional therapeutic agent is a platinum complex.

34. A method of inhibiting the proliferation of Ly6E-positive cells, the method comprising exposing the cells to an immunoconjugate according to any one of claims 20 to 27 under conditions that permit binding of the immunoconjugate to Ly6E on the surface of the cells, thereby inhibiting the proliferation of the cells.

35. The method according to claim 34, wherein the cells are breast cancer cells, pancreatic cancer cells, colon cancer cells, colorectal cancer cells, melanoma cells, ovarian cancer cells, non-small cell lung cancer cells, or gastric cancer cells.

36. An antibody according to any one of claims 1 to 14 conjugated to a label.

37. The antibody according to claim 36, wherein the label is a positron emitter.

38. The positron emitter is ８９ Zr, and the antibody according to claim 37.

39. A method of detecting human Ly6E in a biological sample, the method comprising contacting the biological sample with an anti-Ly6E antibody according to any one of claims 1 to 14 under conditions that permit binding of the anti-Ly6E antibody to naturally occurring human Ly6E, and detecting whether a complex has formed between the anti-Ly6E antibody and naturally occurring human Ly6E in the biological sample.

40. The method according to claim 39, wherein the anti-Ly6E antibody is the antibody according to claim 7 or claim 11.

41. The method according to claim 39 or claim 40, wherein the biological sample is a breast cancer sample, a pancreatic cancer sample, a colon cancer sample, a colorectal cancer sample, a melanoma cancer sample, an ovarian cancer sample, a non-small cell lung cancer sample, or a gastric cancer sample.

42. A method for detecting Ly6E-positive cancer, the method comprising: (i) administering to a subject having or suspected of having Ly6E-positive cancer a labeled anti-Ly6E antibody comprising an anti-Ly6E antibody according to any one of claims 1 to 14; and (ii) detecting the labeled anti-Ly6E antibody in the subject, wherein detection of the labeled anti-Ly6E antibody indicates Ly6E-positive cancer in the subject. Claim 43 The method according to claim 42, wherein the labeled anti-Ly6E antibody is the antibody according to claim 7 or claim 11 which is labeled. Claim 44 The method according to claim 42 or claim 43, wherein the labeled anti-Ly6E antibody comprises an anti-Ly6E antibody conjugated to a positron emitter. Claim 45 wherein the positron emitter is ８９ Zr, the method according to claim 44.

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