Anti-Ly6E antibodies, immunoconjugates and uses thereof

Abstract

The present invention provides anti-Ly6E antibodies and immunoconjugates and methods of using the same.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 337,945, filed on May 3, 2022, and U.S. Provisional Application No. 63 / 492,297, filed on March 27, 2023, each of which is incorporated herein by reference in its entirety for any purpose.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in XML format. The XML copy was 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 format of the sequence listing is incorporated herein by reference in its entirety. Technical Field

[0005] The present invention relates to anti-Ly6E antibodies and immunoconjugates and methods of using the same. Background Art

[0006] Lymphocyte antigen 6 complex, locus E (Ly6E), also known as retinoic acid-induced gene E (RIG-E) and stem cell antigen 2 (SCA-2). It is a GPI-linked protein of unknown function of 131 amino acids and about 8.4 kDa, with no known binding partners. It was originally identified as a transcript expressed in mouse immature thymocytes, thymic medullary epithelial cells. 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. USA 93: 5910-5914 (1996).

[0007] There is a need in the art for agents that target Ly6E for use in diagnosing and treating conditions associated with Ly6E, such as cancer. The present invention satisfies this need and provides other advantages. Summary of the invention

[0008] The present invention provides anti-Ly6E antibodies, immunoconjugates and methods of using the same.

[0009] In some embodiments, the present disclosure provides an isolated antibody that binds to Ly6E, wherein the antibody 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.

[0010] 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.

[0011] In some embodiments, the antibody comprises: (a) a VH sequence having at least 95% sequence identity to SEQ ID NO: 32; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 31; or (c) the VH sequence in (a) and the VL sequence 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.

[0012] In some embodiments, the present disclosure provides an isolated antibody comprising the VH sequence of SEQ ID NO:32 and the VL sequence of SEQ ID NO:31.

[0013] In some embodiments, the antibody is an IgG1 antibody, an IgG2a antibody, an IgG2b antibody, an IgG3 antibody, or an IgG4 antibody.

[0014] 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: 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 an amino acid sequence of SEQ ID NO: 5; and a light chain comprising an amino acid sequence of SEQ ID NO: 3. In some embodiments, the present disclosure provides an isolated antibody that binds to Ly6E, wherein the antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 5; and a light chain comprising an amino acid sequence of SEQ ID NO: 3.

[0015] 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 Ly6E and CD3.

[0016] In some embodiments, the present disclosure provides an isolated nucleic acid encoding an antibody. In some embodiments, the present disclosure provides an expression vector comprising a nucleic acid encoding an antibody. In some embodiments, the present disclosure provides a host cell comprising a nucleic acid or an expression vector. In some embodiments, the present disclosure provides a host cell expressing an antibody. In some embodiments, the present disclosure provides a method for producing an antibody, the method comprising culturing a host cell to produce an antibody.

[0017] In some embodiments, the present disclosure provides an immunoconjugate comprising an antibody and a cytotoxic agent. In some embodiments, the immunoconjugate has the formula Ab-(LD)p, wherein: (a) Ab is an antibody provided herein; (b) L is a linker; (c) D is a pyrrolobenzodiazepine and (d)p is in the range of 1 to 8. In some embodiments, D is a pyrrolobenzazepine of formula A

[0018] in:

[0019] R 2 Having formula II: Where A is C 5-7 Aryl group, X is selected from the group consisting of OH, SH, CO2H, COH, N=C=O, NHR N and (OC2H4) m OCH3, where R N Selected from the group consisting of: H and C 1-4 Alkyl, and wherein m is an integer from 1 to 3, and any of the following:

[0020] (i)Q 1 is a single bond, and Q 2 Selected from a single bond and -Z-(CH2) n -, wherein Z is a single bond, O, S or NH, and n is an integer from 1 to 3; or

[0021] (ii)Q 1 is -CH=CH-, and Q 2 is a single bond;

[0022] R 2' is C optionally substituted with one or more substituents selected from the group consisting of 5-10 Aryl groups: halo, nitro, cyano, ether, C 1-7Alkyl, C 3-7 Heterocyclyl and bis-oxy-C 1-3 Alkylene;

[0023] R 6 and R 9 Independently selected from: H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro,

[0024] Me3Sn and halogenation;

[0025] R 7 Selected from the group consisting of: H, R, OH, OR, SH, SR, NH2, NHR, NHRR', nitro, Me3Sn and halo;

[0026] wherein R and R' are independently selected from optionally substituted C 1-12 Alkyl, C 3-20 Heterocyclic and C 5-20 Aryl groups;

[0027] Any of the following:

[0028] (a)R 10 is H, and R 11 OH or OR A , where R A C 1-4 alkyl;

[0029] (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

[0030] (c)R 10 H and R 11 For SO Z M, wherein z is 2 or 3, and M is a monovalent pharmaceutically acceptable cation;

[0031] R" is C 3-12 an 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 or pyridine;

[0032] Y is O, S or NH;

[0033] R 6' , R 7' and R 9' Selected from R 6 , R 7 and R 9 The same group, and R 10' and R 11' respectively

[0034] With R 10 and R 11 Same, if R 11 and R 11' For SO Z M, then M may represent a divalent pharmaceutically acceptable cation; and

[0035] The connection point with connector L is through R 2 or R 2' .

[0036] In some embodiments, D has the following structure:

[0037]

[0038] The wavy line indicates the connection point with the connector L.

[0039] In some embodiments, (LD) has the following structure:

[0040]

[0041] The wavy lines indicate the points of attachment to the protein.

[0042] In some embodiments, p ranges from 1.5 to 5 or 1.5 to 6 or 1.5 to 4 or 2 to 3.

[0043] In some embodiments, the present disclosure provides a pharmaceutical preparation comprising an immunoconjugate and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical preparation further comprises an additional therapeutic agent.

[0044] In some embodiments, the present disclosure provides a method of treating an individual with a 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

[0045] In some embodiments, the method further comprises administering to the individual an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a platinum complex.

[0046] In some embodiments, the present disclosure provides a method for inhibiting the proliferation of Ly6E-positive cells, the method comprising exposing the cells to an immunoconjugate under conditions that allow the immunoconjugate to bind to Ly6E on the cell surface, thereby inhibiting the proliferation of the cells. In some embodiments, the cells are breast cancer, pancreatic cancer, colon cancer, colorectal cancer, melanoma, ovarian non-small cell lung cancer, or gastric cancer cells.

[0047] 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.

[0048] 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 allow the anti-Ly6E antibody to bind 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.

[0049] In some embodiments, the present disclosure provides a method for detecting Ly6E-positive cancer, the method comprising: (i) administering a labeled anti-Ly6E antibody to a subject having or suspected of having Ly6E-positive cancer, wherein the labeled anti-Ly6E antibody comprises an 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 a 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

[0050] Figure 1 The structure of (maleimide-sq-ala)-PBD linker-drug before conjugation to anti-Ly6E antibody is shown.

[0051] Figures 2A to 2C Binding of chimeric anti-Ly6E antibodies to cells is shown by flow cytometric analysis of chimeric anti-Ly6E and 9B12 binding to several cell lines known to express Ly6E ( Figure 2A Kuramochi cells, Figure 2B NCI-H1781, Figure 2C HCC1569x2).

[0052] Figure 3A and 3B Binding of chimeric and humanized anti-Ly6E antibodies to cells was shown by flow cytometric analysis of chimeric and humanized anti-Ly6E binding to several cell lines known to express Ly6E, as well as 9B12 and isotype controls.

[0053] FIG. 4A to FIG. 4C . Figure 4AThe peptide map of CDR-L3 of humanized anti-Ly6E (positions 61 to 102 of SEQ ID NO: 17) is shown. Figure 4B and 4C In , humanized anti-Ly6E was subjected to zero or two weeks of heat stress and then trypsinized before LC-MS / MS analysis. The peaks of native peptide, isomerized peptide and succinimidyl peptide were integrated at retention times of 30.6-30.7, 31.1 and 32-32.1 minutes, respectively.

[0054] Figure 5 Cellular binding of anti-Ly6E Fab variants is shown. Binding of humanized anti-Ly6E Fab and variants designed to remove the aspartate isomerization site to NCl-H1781 cells was analyzed by flow cytometry.

[0055] Figures 6A to 6B The in vitro activity of anti-Ly6E ADCs with seco-CBI-dimer payloads was shown. The anti-Ly6E-SN36325 conjugate had a stronger ability to induce cell death than non-targeted ADCs or 9B12 conjugates.

[0056] Figure 7 The in vitro activity of anti-Ly6E ADCs with PBD payloads was shown. The anti-Ly6E-SGD-1882 conjugate had a stronger ability to induce cell death than the 9B12 conjugate.

[0057] Fig. 8A and 8B The in vivo efficacy of anti-Ly6E ADC in HCC1569X2 tumor xenograft model is shown. The growth of breast model HCC1569X2 was assessed after a single administration of vehicle or ADC. The conjugate was administered once on day 0, and individual tumor volume and body weight changes were plotted. The numbers next to the trace indicate the dosage (in mg / kg) of each conjugate administered once IV on day 0. Tumor volumes fitted by cubic splines are plotted for each treatment group (n=5 / group). The cubic spline fits of the vehicle group (blue dashed line) and the treatment group (solid line) are included for reference.

[0058] Figures 9A to 9F The in vitro activity of humanized anti-Ly6E bispecific antibodies was shown. Anti-Ly6E / anti-CD3 bispecific T cell engagers induced cell death in multiple cell lines. Anti-Ly6E bispecific antibodies showed stronger activity than 9B12 bispecific antibodies.

[0059] Figures 10A to 10BIt was shown that chimeric (Ch)Ly6E4B10 and 3A3 showed >400-fold potency difference over non-target controls (e.g., isotype control anti-gD and anti-CD22) in the cell line NCl-H1781 after a 5-day antiproliferation assay, compared to only a 9-fold potency difference for 9B12. Fig. 10B In the table, TDC Ab-SN36325 refers to the ThioMab drug conjugate conjugated with Auckland SN36325 drug, and CNJ indicates the conjugation batch.

[0060] Figures 11A to 11B It was shown that chimeric (Ch)Ly6E 3A3 exhibited the highest potency against the cell line HCC1569x2, despite overall weak potency and partial killing in the 5-day antiproliferation assay.

[0061] Figures 12A to 12D The best antibody binders using imaging at 4°C for 1 hour and 4 hours are shown to have similar rankings to FACS binding. Fig. 12A 3A3 is shown to have the highest average intensity. Figures 12B to 12D Shown in Kuramochi( Fig. 12B )、NCl-H1781( Fig. 12C ) and HCC-1569x2( Fig.12D ) cell lines, the highest median fluorescence intensity (MFI) at each concentration (μg / ml) of 3A3 (3.A3.hlgG1).

[0062] Fig.13 It is shown that after 3 hours, 3A3 (3.A3.hlgG1) co-localized in lysosomes (per cell) to a significantly higher extent compared to antibodies 1D5.3, 2.5G6, 9B12 and negative control. PE - pulse exposure (antibody washed out), CE - continuous exposure.

[0063] Fig.14 The internalization rate of 9B12 (Ly6E9B12NKD) was shown to be faster than that of 3A3 (3.A3.hlgG1), but the higher binding of 3A3 resulted in sustained efficacy as seen in in vivo studies. PE-Pulse Exposure.

[0064] Fig.15 Different extracellular matrices (ECM) are shown (such as all ECM, (GIBCO TM ), periodontal ligament (PDL), fibronectin, and phosphate-buffered saline (PBS). DETAILED DESCRIPTION

[0065] I. Definitions

[0066] For the purposes of this article, "acceptor human framework" is a framework that includes an 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. The acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may include an amino acid sequence identical to the human immunoglobulin framework or a human consensus framework, or it may include amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical to the VL human immunoglobulin framework sequence or a human consensus framework sequence in sequence.

[0067] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (such as an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0068] An "affinity matured" antibody is one with one or more alterations in one or more hypervariable regions (HVRs) which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess such alterations.

[0069] The terms "anti-Ly6E antibody" and "antibody that binds to Ly6E" refer to an antibody that is capable of binding to Ly6E with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent targeting Ly6E. In one embodiment, the anti-Ly6E antibody binds to unrelated, non-Ly6E proteins to a lesser extent than that detected, for example, by radioimmunoassay (RIA) or by Scatchard analysis or by surface plasmon resonance (such as, for example, BIAcore). TM ) about 10% of the binding of the antibody to Ly6E as measured by . In certain embodiments, the dissociation constant (Kd) of the antibody binding to Ly6E is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13M). In certain embodiments, the anti-Ly6E antibody binds to an epitope of Ly6E that is conserved in Ly6E from different species.

[0070] The term "antibody" herein is used in the broadest sense and includes 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.

[0071] As used herein, the term "antibody drug conjugate" (ADC) is equivalent to the term "immunoconjugate".

[0072] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0073] An "antibody that binds to the same epitope as a reference antibody" is an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, whereas the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. An exemplary competition assay is provided herein.

[0074] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by uncontrolled cell growth / proliferation. Examples of cancer include, but are not limited to, malignancies, lymphomas (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastomas, sarcomas, and leukemias. More specific examples of such cancers include cancers that overexpress Ly6E, which may include, 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 (squamous and / or non-squamous), gastric cancer, squamous cell carcinoma, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, glioma, cervical cancer, liver cancer, bladder cancer, liver cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[0075] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a particular source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0076] The "class" of an antibody refers to the type of constant domain or constant region possessed by the heavy chain of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. In some aspects, the antibody is an IgG1 isotype. In some aspects, the antibody is an IgG1 isotype, which has P329G, L234A and L235A mutations to reduce Fc region effector functions. In other aspects, the antibody is an IgG2 isotype. In some aspects, the antibody is an IgG4 isotype, which comprises an S228P mutation in the hinge region to improve the stability of the IgG4 antibody. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ and μ, respectively. The light chains of antibodies can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0077] As used in the present application, the term "human-derived constant region" or "human constant region" refers to the constant heavy chain region and / or constant light chain kappa or lambda region of a human antibody of subclass IgG1, IgG2, IgG3 or IgG4. Such constant regions are well known in the prior art and are described, for example, by Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also, for example, Johnson, G. and Wu, TT, Nucleic Acids Res. 28 (2000) 214-218; Kabat, EA et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788). Unless otherwise indicated herein, the numbering of the amino acid regions in the constant region is according to the EU numbering system, also known as the EU index of Kabat, as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication No. 91-3242.

[0078] 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, but are not limited to, radioactive isotopes (e.g., At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, doxorubicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitors; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and various anti-tumor or anti-cancer drugs disclosed below.

[0079] "Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. 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 receptor); and B cell activation.

[0080] An "effective amount" of an agent (eg, a pharmaceutical formulation) refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0081] The term "epitope" refers to a specific site on an antigen molecule to which an antibody binds.

[0082] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0083] "Framework" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. The FR of the variable domain is usually composed of the following four FR domains: FR1, FR2, FR3 and FR4. Therefore, HVR and FR sequences usually appear in the following sequence in VH (or VL): FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

[0084] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0085] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived from the primary transformed cell, regardless of the number of passages. Progeny may not be identical to the nucleic acid content of the parent cell, but may contain mutations. Mutant progeny as screened or selected for the same function or biological activity as in the original transformed cell are included herein.

[0086] A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human antibody using a full repertoire of human antibodies or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0087] "Human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. In general, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. In general, the subgroup of sequences is a subgroup as described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one embodiment, for VL, the subgroup is subgroup κI as in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al., supra.

[0088] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will substantially comprise at least one, usually two, variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to HVRs of non-human antibodies, and all or substantially all FRs correspond to FRs of human antibodies. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. An antibody in a "humanized form," e.g., a non-human antibody, refers to an antibody that has been humanized.

[0089] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence and / or forms a structurally defined loop ("hypervariable loop"). Typically, a natural four-chain antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and / or from "complementarity determining regions" (CDRs), which have the highest sequence variability and / or are involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of L1, amino acid residues 50-56 of L2, amino acid residues 89-97 of L3, amino acid residues 31-35B of H1, amino acid residues 50-65 of H2, and amino acid residues 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).) In addition to CDR1 in VH, CDRs generally include amino acid residues that form hypervariable loops. CDRs also include "specificity determining residues" or "SDRs," which are residues that contact the antigen. SDR is contained in the CDR region called shortened CDR or a-CDR.Exemplary a-CDR (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2 and a-CDR-H3) occurs at amino acid residues 31-34 of L1, amino acid residues 50-55 of L2, amino acid residues 89-96 of L3, amino acid residues 31-35B of H1, amino acid residues 50-58 of H2, and amino acid residues 95-102 of H3. (See Almagro and Fransson, Front.Biosci.13:1619-1633 (2008).) Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domain are numbered herein according to Kabat et al., cited above.

[0090] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent. Immunoconjugate is equivalent to the term "antibody drug conjugate" (ADC).

[0091] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0092] An "isolated antibody" is an antibody that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99% by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0093] "Isolated nucleic acid" refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that normally 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.

[0094] "Isolated nucleic acid encoding an anti-Ly6E antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present in one or more locations in a host cell.

[0095] As used herein, the term "Ly6E" refers to any natural, mature Ly6E produced by processing of a Ly6E precursor protein in a cell. Unless otherwise indicated, the term includes Ly6E from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys or rhesus monkeys) and rodents (e.g., mice and rats). The term also includes naturally occurring variants of Ly6E, for example, splice variants or allelic variants. The amino acid sequence of an exemplary human Ly6E precursor protein (with a signal sequence, amino acids 1 to 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 to 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 sequence of an exemplary rat Ly6E precursor (with signal sequence, amino acids 1 to 26) and the mature sequence are shown in SEQ ID NOs: 23 and 28, respectively. The amino acid sequence of an exemplary mouse Ly6E precursor (with signal sequence, amino acids 1 to 26) and the mature sequence are shown in SEQ ID NOs: 22 and 27, respectively.

[0096] The term "Ly6E positive cells" refers to cancers comprising cells expressing Ly6E on their surfaces. In order to determine whether a cell expresses Ly6E on the surface, Ly6E mRNA expression is considered to be associated 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, antibodies against Ly6E can be used, for example, in methods such as immunohistochemistry, FACS, etc. to determine the expression of Ly6E on the cell surface. In some embodiments, Ly6E positive cancers refer to 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 (squamous and / or non-squamous) or gastric cancer, each of which shows high levels of Ly6E expression.

[0097] The term "Ly6E-positive cells" refers to cancer cells that express Ly6E on their surface.

[0098] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody preparations, such variants are usually present in small amounts), each antibody comprising the population is identical and / or binds to the same epitope. Contrary to the polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody in the monoclonal antibody preparation is directed to a single determinant on the antigen. Therefore, the modifier "monoclonal" represents that the feature of the antibody is obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring antibody to be produced by any ad hoc method. For example, the monoclonal antibody to be used according to the present invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0099] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in pharmaceutical preparations.

[0100] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains bonded by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also referred to as a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also referred to as a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain. The light chain of an antibody can be classified into one of two types based on the amino acid sequence of its constant domain, which are called kappa (κ) and lambda (λ).

[0101] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0102] " Amino acid sequence identity percentage (%) " with respect to a reference polypeptide sequence is defined as after comparing a candidate sequence with a reference polypeptide sequence and introducing a vacancy (if necessary) to achieve the maximum sequence identity percentage, and without considering any conservative substitution as a component of sequence identity, the percentage of the amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence. The comparison for determining the amino acid sequence identity percentage can be implemented in various ways within the technical scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment on the full length of the compared sequence. However, for the purposes of this article, the sequence comparison computer program ALIGN-2 is used to generate amino acid sequence identity%. The ALIGN-2 sequence comparison computer program is written by Genentech, Inc., and the source code has been submitted to US Copyright Office, Washington DC, 20559, with user documentation, where it is registered with U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and will not change. In the case of using ALIGN-2 for amino acid sequence comparison, the amino acid sequence identity % (which can be alternatively expressed as a given amino acid sequence A having or comprising a certain amino acid sequence identity % with a given amino acid sequence B) of a given amino acid sequence A and a given amino acid sequence B is calculated as follows:

[0103] Multiply 100 by the fraction X / Y

[0104] Where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be understood that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained as described in the preceding paragraph using the ALIGN-2 computer program.

[0105] The term "pharmaceutical formulation" refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the preparation would be administered.

[0106] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical preparation other than the active ingredient that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0107] As used herein, "platinum complex" refers to anti-cancer chemotherapy drugs, such as, for example, but not limited to, cisplatin, oxaliplatin, carboplatin, iproplatin, satraplatin, CI-973, AZ0473, DWA2114R, nedaplatin, and spiroplatin, which exert their anti-tumor effects based on their ability to covalently bind to DNA.

[0108] As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

[0109] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that participates in the binding of an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th edition, WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be separated using the VH or VL domains from antibodies that bind to the antigen, respectively, to screen libraries of complementary VL or VH domains. See, for example, Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0110] As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0111] With respect to Formula A, the term "C 1-12 "Alkyl" refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having 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 the subclasses alkenyl, alkynyl, cycloalkyl, and the like. Examples of saturated alkyl groups may include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, and heptyl. Examples of saturated straight-chain alkyl groups include, but are not limited to, methyl, ethyl, n-propyl (C3), The saturated branched alkyl groups include isopropyl (C3), isobutyl (C4), sec-butyl (C4), tert-butyl (C4), isopentyl (C5) and neopentyl (C5). With respect to formula A, alkenyl as used herein refers to an alkyl group having one or more carbon-carbon double bonds. Examples of alkenyl groups may include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, isopropenyl, butenyl, pentenyl and hexenyl. In some embodiments, the C of formula A is 1-12 The alkyl group is a saturated acyclic alkyl group.

[0112] As used herein, "alkyl" is a C1-C ... 12Hydrocarbons. Examples are methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH(CH3) CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl 1-Hexyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-C H(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2) and 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3.

[0113] As used herein and 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 alicyclic, 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 hydrogen atom may be removed from each of two different carbon atoms. Thus, the term "alkylene" includes the subclasses alkenylene, alkynylene, cycloalkylene, etc. Straight chain saturated C 3-12 Examples of alkylene groups may include, but are not limited to, -(CH2) n-, wherein n is an integer from 3 to 12, such as -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, and -CH2CH2CH2CH-2CH2CH2CH2-. Branched chain saturated C 3-12 Examples of alkylene groups may include, but are not limited to, -CH(CH3)CH2-, -CH(CH3)CH2CH2-, -CH(CH3)CH2CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH(CH3)CH2CH2-, -CH(CH2CH3)-, -CH(CH2CH3)CH2-, and -CH2CH(CH2CH3)CH2-. The linear chain portion is unsaturated. 3-12 The alkylene group (C 3-12 Examples of alkenylene and alkynylene groups) may include, but are not limited to, -CH=CH-CH2-, -CH2-CH=CH2-, -CH=CH-CH2-CH2-, -CH=CH-CH2-CH2-, -CH=CH-CH=CH-, -CH=CH-CH=CH-, -CH=CH-CH=CH-CH2-, -CH=CH-CH=CH-, -CH=CH-CH=CH-CH2-, -CH=CH-CH=CH-CH2-, -CH=CH-CH2-CH=CH-, -CH=CH-CH2-CH=CH-, and -CH2-C≡C-CH2-. The branched portion is unsaturated C 3-12 The alkylene group (C 3-12 Examples of alkenylene and alkynylene groups may include, but are not limited to, -C(CH3)=CH-, -C(CH3)=CH-CH2-, -CH=CH-CH(CH3)-, and -CsC-CH(CH3)-. Alicyclic saturated C 3-12 The alkylene group (C 3--I2 Examples of cycloalkylene include, but are not limited to, cyclopentylene and cyclohexylene. The alicyclic moiety is unsaturated C 3-12 Examples of alkylene groups may include, but are not limited to, cyclopentenylene and cyclohexenylene. 3-12 Alkylene refers to the formula -(CH2) 3-12 - is a straight-chain saturated hydrocarbon group, examples of which include propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene and dodecylene.

[0114] 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 from 3 to 20 ring atoms. In some embodiments, each aryl ring has from 5 to 7 ring atoms. The prefix, such as C 3-20 , C 5-7 , C 5-6, etc., refers to the number of ring atoms or a range of ring atoms, whether the ring atoms are carbon atoms or heteroatoms. For example, as used herein, the term "C 5-6 "Aryl" refers to an aromatic group having 5 or 6 ring atoms. In some embodiments, all ring atoms are carbon atoms, as in a "carboaryl group". Such carboaryl groups may include, but are not limited to, those derived from benzene (i.e., phenyl), naphthalene, azulene, anthracene, phenanthrene, tetracene, and pyrene. In some embodiments, the aryl group comprises fused rings, wherein at least one of the fused rings is an aromatic ring, such as, for example, groups derived from indane, indene, isoindene, tetralin, acenaphthene, fluorene, phenanthren, acetophenanthrene, and acetoanthracene. In other embodiments, the ring atoms may include one or more Multiple heteroatoms, such as the heteroatoms in a "heteroaryl group". Examples of monocyclic heteroaryl groups may include, but are not limited to, those derived from pyrrole, pyridine, furan. Thiophene, oxazole, isoxazole, isoxazine, oxadiazole, thiazole, isothiazole and triazole. In some embodiments, the heteroaryl group comprises fused rings, at least one of which comprises ring heteroatoms, such as groups derived from benzofuran, isobenzofuran, isoindole, indolizine, indoline, isoindolin, purine, benzimidazole, indazole and benzoxazole. In some embodiments, with respect to Formula A, C 5-20 Aryl is C6-C 20 Carboaryl groups.

[0115] "Substituted alkyl", "substituted aryl" and "substituted arylalkyl" refer to 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 - , -NR2, -NR3, =NR, -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, NC(=O)R, -C(=O)R, -C(=O)NR2, -SO3 - , -SO3H, -S(=O)2R, -OS(=O)2OR, -S(=O)2NR, -S(=O)R, -OP(=O)(OR)2, -P(=O)(OR)2, -PO - 3. -PO3H2, -C(=O)R, -C(=O)X, -C(=S)R, -CO2R, -CO2 - , -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NR2, -C(=S)NR2, -C(=NR)NR2, wherein each X is independently a halogen: F, Cl, Br or I; and each R is independently -H, C2-C 18 Alkyl, C6-C 20 Aryl, C3-C 14Heterocyclic groups, protecting groups or prodrug moieties.Alkylene, alkenylene and alkynylene groups as described above may also be similarly substituted.

[0116] As used herein, particularly with respect to Formula A, the term “C 3-20 "Heterocyclyl" refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, the moiety having 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 a heterocyclyl, the prefix (e.g., C 3-20 , C 3-7 , C 5-6 ) refers to the number of ring atoms or a range of ring atoms, whether the ring atoms are carbon atoms or heteroatoms. For example, as used herein, the term "C 5-6 "Heterocyclyl" refers to a heterocyclyl group having 5 or 6 ring atoms, whether those ring atoms are carbon or heteroatoms. Examples of monocyclic heterocyclyl groups may include, but are not limited to, those derived from aziridine, azetidine, pyrrolidine, pyrroline, piperidine, dihydropyridine, tetrahydropyridine, azepine, In some embodiments, the heterocyclyl group is a saturated monocyclic or bicyclic ring containing 3 to 10 ring atoms, wherein 1 to 3 ring atoms are independently selected from the group consisting of O and N.

[0117] As used herein, particularly with respect to Formula A, the term "ether" refers to a group -OR, wherein R is an ether substituent, for example, C 1-7 Alkyl groups (which may also be referred to as C 1-7 Alkoxy groups), C 3-20 The heterocyclyl group (which may also be referred to as C 3-20 heterocyclyloxy group) or C 5-20 Aryl groups (which may also be referred to as C 5-20 In some embodiments, ether refers to a group -OR, wherein R is a saturated C 1-7 Alkyl group.

[0118] "Linker" refers to a chemical moiety comprising a covalent bond or chain of atoms that covalently links the antibody to the drug moiety. In various embodiments, the linker includes a divalent group such as an alkyl diyl, an aryl diyl, a heteroaryl diyl, a moiety such as: -(CR2) n O(CR2) n-, alkoxy (e.g., polyethyleneoxy, PEG, polymethyleneoxy) and alkylamino (e.g., polyethyleneamino, Jeffamine TM ) repeating unit; and diacids and amides, including succinate, succinamide, diglycolate, malonate and caproamide. In various embodiments, the linker may include one or more amino acid residues, such as valine, phenylalanine, lysine and homolysine.

[0119] The term "chiral" refers to molecules that have the property of being non-superimposable on their mirror image partner, whereas the term "achiral" refers to molecules that are superimposable on their mirror image partner.

[0120] The term "stereoisomers" refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

[0121] "Diastereomers" refers to stereoisomers with two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting points, boiling points, spectral characteristics, and reactivity. Mixtures of diastereomers can be separated under high resolution analytical procedures such as electrophoresis and chromatography.

[0122] "Enantiomers" refers to two stereoisomers of a compound that are non-superimposable mirror images of one another.

[0123] The stereochemical definitions and conventions used herein generally follow: SP 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 indicate the absolute configuration of the molecule around its chiral center. The prefixes d and l or (+) and (-) are used to indicate the sign of the rotation of the compound to plane polarized light, where (-) or 1 indicates that the compound is left-handed. Compounds with the (+) or d prefix are right-handed. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Specific stereoisomers may also be referred to as enantiomers, and mixtures of such isomers are often referred to as enantiomeric mixtures. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may occur without stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two optically inactive enantiomeric species.

[0124] "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 include, but are not limited to, halides (e.g., chlorides, bromides, iodides), methylsulfonyl (mesyl), p-toluenesulfonyl (tosyl), trifluoromethylsulfonyl (trifluoromethanesulfonate), and trifluoromethylsulfonate.

[0125] The term "protecting group" refers to a substituent that is generally used to block or protect a specific functional group while reacting with other functional groups on a compound. For example, an "amino protecting group" is a substituent attached to an amino group that blocks or protects the amino functional group in the compound. Suitable amino protecting groups include, but are not limited to, acetyl, trifluoroacetyl, tert-butyloxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and 9-fluorenylmethyleneoxycarbonyl (Fmoc). For general descriptions of protecting groups and their uses, see TW Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991 or later.

[0126] II. Compositions and Methods

[0127] 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 can be used, for example, to diagnose or treat LY6E-positive cancers.

[0128] A. Exemplary Anti-Ly6E Antibodies

[0129] In some embodiments, the invention provides isolated antibodies that bind to LY6E. In certain embodiments, the anti-LY6E antibody has at least one or more of the following features in any combination.

[0130] Non-limiting exemplary antibodies of the present invention are Ly6E and humanized variants thereof. In some embodiments, Ly6E is human Ly6E, such as human Ly6E of SEQ ID NO: 1. In some embodiments, Ly6E is selected from human, cynomolgus monkey, rhesus monkey, mouse or rat Ly6E.

[0131] In some such embodiments, the anti-Ly6E antibody binds to Ly6E with an affinity of ≤25nM, or ≤20nM, or ≤15nM, or ≤10nM, or ≤9nM, or ≤8nM, or ≤7nM, or ≤6nM, or ≤5nM, or ≤4nM, or ≤3nM, or ≤2nM, or ≤1nM and optionally ≥0.0001nM, or ≥0.001nM, or ≥0.01nM, as measured by surface plasmon resonance (SPR) or Scatchard analysis. In some such embodiments, the anti-Ly6E antibody binds to Ly6E with an affinity of 0nM to 25nM, 0nM to 20nM, 0nM to 10nM, or 10nM to 20nM. 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 nM to 20 nM. In some embodiments, the bivalent affinity for human Ly6E is about three times the monovalent affinity.

[0132] In one aspect, the present invention provides an anti-Ly6E antibody comprising at least one, two, three, four, five or six HVRs selected from the following: (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.

[0133] In one aspect, the present invention provides an antibody comprising at least one, at least two or all three VH HVR sequences selected from the following: (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.

[0134] In another aspect, the present invention provides an antibody comprising at least one, at least two or all three VL HVR sequences selected from the following: (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.

[0135] In another aspect, an antibody of the invention comprises: (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 11 and (iii) HVR-H3 comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 12; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 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.

[0136] On the other hand, the present 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 SEQID NO:8; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:9.

[0137] In any of the above embodiments, the anti-Ly6E antibody is humanized. In one embodiment, the anti-Ly6E antibody comprises the HVRs in any of the above embodiments, and further comprises an acceptor human framework, eg, a human immunoglobulin framework or a human consensus framework.

[0138] On the other hand, an 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 with 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%, 99% sequence identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-Ly6E antibody comprising the sequence retains the ability to bind to Ly6E. In certain embodiments, in SEQ ID NO: 32, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain embodiments, the substitution, insertion or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the anti-Ly6E antibody comprises the VH sequence shown in SEQ ID NO: 32, including post-translational modifications of the sequence. In a specific embodiment, VH comprises one, two or three HVRs selected from the following: (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.

[0139] On the other hand, an anti-Ly6E antibody is provided, wherein the antibody comprises: a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 31. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-Ly6E antibody comprising the sequence retains the ability to bind to Ly6E. In certain embodiments, in SEQ ID NO: 31, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain embodiments, the substitution, insertion or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the anti-Ly6E antibody comprises the VL sequence shown in SEQ ID NO: 31, including post-translational modifications of the sequence. In specific embodiments, VL comprises one, two or three HVRs selected from the following: (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.

[0140] In another aspect, an anti-Ly6E antibody is provided, wherein the antibody 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 sequence and the VL sequence in SEQ ID NO: 32 and SEQ ID NO: 31, respectively, including post-translational modifications of those sequences.

[0141] In another aspect, the present invention provides an antibody that binds to the same epitope as the anti-Ly6E antibody provided herein. For example, in certain embodiments, an antibody that binds to the same epitope as the anti-Ly6E antibody is provided, the antibody comprising a VH sequence of SEQ ID NO: 32 and a VL sequence of SEQ ID NO: 31.

[0142] In a further aspect, the anti-Ly6E antibody according to any one of the above embodiments is a monoclonal antibody, including chimeric, humanized or human antibodies. 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 full-length antibody, for example, a complete IgG1 antibody or other antibody class or isotype as defined herein.

[0143] In another aspect, an anti-Ly6E antibody is provided, wherein the antibody comprises a 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 comprises a HC in SEQ ID NO: 5 and a LC in SEQ ID NO: 3, SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 29, including any post-translational modifications of those sequences. In one embodiment, the antibody comprises a HC in SEQ ID NO: 5 and a LC of SEQ ID NO: 3, including any post-translational modifications of those sequences.

[0144] In a further aspect, the anti-Ly6E antibody according to any of the above embodiments may incorporate any of the features described below, alone or in combination.

[0145] Determination

[0146] In some embodiments, the anti-Ly6E antibody "binds with an affinity of ≤25nM, or ≤20nM, or ≤15nM, or ≤10nM, or ≤9nM, or ≤8nM, or ≤7nM, or ≤6nM, or ≤5nM, or ≤4nM, or ≤3nM, or ≤2nM, or ≤1nM" or "binds with an affinity of 0nM to 25nM, 0nM to 20nM, 0nM to 10nM, or 10nM to 20nM" is determined according to a Scatchard analysis. Alternatively, the anti-Ly6E antibody affinity can be determined according to, for example, BIAcore TM Specifically, BIAcore TM Kd was measured by surface plasmon resonance using a BIAcore-3000 (BIAcore, Inc., Piscataway, NJ). The BIAcore was activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) reagents according to the supplier's instructions. TM Research grade CM5 chip. Goat anti-human Fc IgG was coupled to the chip to achieve approximately 10,000 response units (RU) in each flow cell. Unreacted coupling groups were blocked with 1 M ethanolamine. For kinetic measurements, anti-Ly6E antibodies were captured to achieve approximately 300 RU. Two-fold serial dilutions of human Ly6E were injected into HBS-P buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% surfactant P20) at 25°C with a flow rate of 30 μl / min. A 1:1 Langmuir binding model (BIAcore TM Evaluation software version 3.2) to calculate the association rate (k on ) and dissociation rate (k off The equilibrium dissociation constant (Kd) is expressed as the ratio k off / k on If the association rate exceeds 10 6 M -1 s -1 If the association rate is quantified, the association rate can be determined by using the fluorescence quenching technique, i.e., in a spectrometer such as a spectrophotometer equipped with a stop flow device (Aviv Instruments) or a 8000 series The increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS pH 7.2 at 25°C was measured in a spectrophotometer (ThermoSpectronic) using a stirred cuvette in the presence of increasing concentrations of antigen.

[0147] In any of the above embodiments, the anti-Ly6E antibody is humanized. In one embodiment, the anti-Ly6E antibody comprises the HVRs in any of the above embodiments and further comprises a human acceptor framework, such as a human immunoglobulin framework or a human consensus framework. In certain embodiments, the human acceptor framework is a human VLκIV consensus (VL KIV ) frame and / or VH frame VH1.

[0148] In a further aspect, the anti-Ly6E antibody according to any one of the above embodiments is a monoclonal antibody, including chimeric, humanized or human antibodies. 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 or other antibody class or isotype as defined herein.

[0149] In a further aspect, the anti-Ly6E antibody according to any of the above embodiments may incorporate any of the features described below, alone or in combination.

[0150] In a further aspect, the anti-Ly6E antibody according to any one 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 or other antibody classes or isotypes as defined herein.

[0151] In a further aspect, the anti-Ly6E antibody according to any of the above embodiments may incorporate any of the features described below, alone or in combination.

[0152] 1. Antibody affinity

[0153] 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, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 M).

[0154] In one embodiment, Kd is measured by performing a radiolabeled antigen binding assay (RIA) with the Fab form of the antibody of interest and its antigen as described in the following assay. The Kd is measured by using a minimum concentration ( 125 The solution binding affinity of Fab for antigen was measured by equilibrating Fab with 1) labeled antigen and then capturing bound antigen with a plate coated with anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To determine the conditions for the assay, 5 μg / ml capture anti-Fab antibody (Cappel Labs) was coated in 50 mM sodium carbonate (pH 9.6). Multiwell plates (Thermo Scientific) were blocked overnight and then blocked with 2% (w / v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 I]-antigen is mixed with a serial dilution of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation can be continued for longer (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (e.g., one hour). The solution is then removed and washed with 0.1% polysorbate 20 in PBS. The plates were washed eight times. When the plates were dry, 150 μl / well of scintillant (MICROSCINT-20 TM ; Packard), and in TOPCOUNT TM The plates were counted for tens of minutes on a gamma counter (Packard).The concentration of each Fab that gave less than or equal to 20% of maximal binding was chosen for use in the competitive binding assay.

[0155] According to another embodiment, Kd is measured using Scatchard analysis. According to another embodiment, BIAcore was used at 25°C with an immobilized antigen CM5 chip at approximately 10 response units (RU). TM -2000 or BIAcore TM-3000 (BIAcore, Inc., Piscataway, NJ), Kd was measured by surface plasmon resonance assay. Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore, Inc.) were activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigens were diluted to 5 μg / ml (about 0.2 μM) with 10 mM sodium acetate pH 4.8 and then injected at a flow rate of 5 μl / min to obtain approximately 10 response units (RU) of coupled protein. After injection of antigen, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, 5% paraformaldehyde (PVA) was injected in a solution containing 0.05% polysorbate 20 (TWEEN-20 TM Two-fold serial dilutions (0.78 nM to 500 nM) of Fab in PBS containing PBST (PBST) were used. A simple one-to-one Langmuir binding model ( The association rate (kon) and dissociation rate (koff) are calculated using the ELISA software version 3.2. The equilibrium dissociation constant (Kd) is calculated as the ratio koff / kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the association rate exceeds 106 M-1 s-1 as determined by the surface plasmon resonance assay described above, the association rate can be determined by using a fluorescence quenching technique, i.e., in a spectrometer such as a spectrophotometer equipped with a stop flow device (Aviv Instruments) or a 8000 series SLM-AMINCO TM The increase or decrease in fluorescence emission intensity (excitation wavelength = 295 nm; emission wavelength = 340 nm, bandpass = 16 nm) of 20 nM anti-antigen antibody (Fab form) in PBS pH 7.2 at 25°C was measured in a spectrophotometer (ThermoSpectronic) using a stirred cuvette in the presence of increasing concentrations of antigen.

[0156] 2. Antibody fragments

[0157] In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthün, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, ed., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046.

[0158] Diabodies are antibody fragments with two antigen binding sites that can be bivalent or bispecific. See, e.g., EP 404,097; 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 tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0159] Single domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody.In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516 Bl).

[0160] Antibody fragments can be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phage), as described herein.

[0161] 3. Chimeric and humanized antibodies

[0162] In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984). In one example, a chimeric antibody includes a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate (such as a monkey)) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0163] In certain embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibody. Typically, humanized antibodies include one or more variable domains, wherein HVR, such as CDR (or a portion thereof) are derived from non-human antibodies, and FR (or a portion thereof) are derived from human antibody sequences. Humanized antibodies will optionally also include at least a portion of a human constant region. In certain embodiments, some FR residues in humanized antibodies are replaced by corresponding residues from non-human antibodies (e.g., antibodies from which HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0164] Humanized antibodies and methods for making them are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Natl. 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 (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "surface remodeling"); Dall'Acqua et al., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "surface remodeling"); Dall'Acqua et al., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); 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 the "guided selection" method for FR shuffling).

[0165] 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 the consensus sequence of human antibodies of a particular subgroup 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 (199 3)); human mature (somatically 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 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)).

[0166] 4. Human Antibodies

[0167] In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using various 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).

[0168] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic stimulation. Such animals typically contain all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus, or exists outside the chromosome of the animal or is randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus is typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, description of XENOMOUSE TM US Patent Nos. 6,075,181 and 6,150,584 for the technology; description US Patent No. 5,770,429 for technology; describes KM US Patent No. 7,041,870, and describes The human variable regions from intact antibodies produced by such animals can be further modified, for example by combining with different human constant regions.

[0169] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma 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 via human B cell hybridoma technology are also described in Li et al. Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Additional methods include, for example, those described in U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26 (4): 265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20 (3): 927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27 (3): 185-91 (2005).

[0170] Human antibodies can also be produced by isolating the Fv clone variable domain sequence selected from the human phage display library. Such variable domain sequences can then be combined with the expected human constant domains. The technology of selecting human antibodies from the antibody library is described below.

[0171] 5. Antibodies from the library

[0172] The antibodies of the present invention can be isolated by screening the combinatorial library for antibodies with one or more desired activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries to obtain antibodies with desired binding characteristics. Other methods are reviewed in, for example, Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further described in, for example, McCafferty et al., Nature 348: 552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248: 161-175 (Lo ed., Human Press, Totowa, NJ, 2001). 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).

[0173] In certain phage display methods, the repertoire of VH and VL genes is cloned individually by polymerase chain reaction (PCR) and randomly recombined in a phage library, from which antigen-binding phage can then be screened, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, the initial repertoire (e.g., from humans) can be cloned to provide a single source of antibodies to a wide range of non-self antigens and self antigens without any immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, natural libraries can also be made by cloning unrearranged V gene segments from stem cells; and using PCR primers containing random sequences to encode highly variable CDR3 regions and perform in vitro rearrangement, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and U.S. Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0174] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0175] 6. Multispecific Antibodies

[0176] In certain embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies with binding specificity to 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, for example, U.S. Patent No. 5,821,337. In certain embodiments, bispecific antibodies can bind to two different epitopes of Ly6E. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing Ly6E. Bispecific antibodies can be made into full-length antibodies or antibody fragments.

[0177] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs of different specificities (see 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. Pat. No. 5,731,168). Multispecific antibodies can also be made by the following techniques: engineering electrostatic manipulation effects to make antibody Fc-heterodimer molecules (WO 2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science 229:81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)); using "diabody" technology to make bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol. 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., by Tutt et al., J. Immunol. 147:60 (1991).

[0178] Engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies," are also included herein (see, e.g., US 2006 / 0025576 A1).

[0179] The antibodies or fragments herein also include "dual-acting FAbs" or "DAFs," which comprise an antigen binding site that binds to Ly6E as well as other different antigens (see, e.g., US 2008 / 0069820).

[0180] 7. Antibody variants

[0181] In certain embodiments, amino acid sequence variants of antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. The amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, the disappearance of residues in the antibody amino acid sequence, and / or insertion and / or substitution. Any combination of deletion, insertion and substitution can be performed to achieve the final construct, provided that the final construct has a desired feature, for example, antigen binding.

[0182] a) Substitution, insertion and deletion variants

[0183] In certain embodiments, antibody variants with one or more amino acid substitutions are provided. The target sites for substitution mutations include HVR and FR. Conservative substitutions are shown under the heading "Preferred Substitutions" in Table 1. More substantial changes are provided under the heading "Exemplary Substitutions" in Table 1, and are further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the target antibody, and the product can be screened for desired activity (e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

[0184] Table 1

[0185]

[0186]

[0187] Amino acids can be grouped according to common side chain properties:

[0188] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0189] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0190] (3) Acidic: Asp, Glu;

[0191] (4) Basic: His, Lys, Arg;

[0192] (5) Residues that affect chain orientation: Gly, Pro;

[0193] (6) Aromatic: Trp, Tyr, Phe.

[0194] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0195] One type of substitution variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Typically, relative to the parent antibody, one or more resulting variants selected for further study will have changes (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) and / or will substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity matured antibodies, which can be conveniently generated, for example, using affinity maturation techniques based on phage display such as those described herein. In short, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for specific biological activity (e.g., binding affinity).

[0196] For example, HVRs can be altered (e.g., substituted) to improve antibody affinity. Such changes can occur in HVR "hot spots", i.e., residues encoded by codons that undergo high frequency mutations during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)) and / or SDRs (a-CDRs) (detecting the binding affinity of the resulting variant VH or VL). Affinity maturation achieved by construction and reselection from a secondary library has been described, for example, by Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into variable genes selected for maturation purposes 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 any antibody variants with 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 can be specifically identified, for example, using alanine scanning mutagenesis or modeling. Specifically, CDR-H3 and CDR-L3 are often targeted.

[0197] In certain embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the antigen binding ability of the antibody. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such changes may be outside HVR "hot spots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR remains unchanged, or contains no more than one, two or three amino acid substitutions.

[0198] The method that can be used to identify antibody residues or regions 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, a residue or a group of target residues (e.g., charged residues such as arg, asp, his, lys and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Other substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues that are candidates for substitution can be targeted or eliminated. Variants can be screened to determine whether they have the desired properties.

[0199] Amino acid sequence insertion includes amino and / or carboxyl terminal fusions ranging in length from one residue to a polypeptide containing one hundred or more residues, and intrasequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions to the N-terminal or C-terminal ends of an antibody that increases the serum half-life of an antibody (e.g., for ADEPT) or a polypeptide.

[0200] b) Glycosylation variants

[0201] In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent of antibody glycosylation. Glycosylation sites can be conveniently added or deleted to an antibody by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0202] When antibody comprises Fc district, the carbohydrate attached thereto can be changed.Native antibodies produced by mammalian cells usually comprise the biantennary oligosaccharide of side chain, and this biantennary oligosaccharide is usually connected to the Asn297 of CH2 domain in Fc district by N key.See, for example, Wright et al. TIBTECH 15:26-32 (1997).Oligosaccharide can include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and the fucose of GlcNAc in the " trunk " of biantennary oligosaccharide structure.In certain embodiments, the oligosaccharide in antibody can be modified, so as to produce antibody variants with some improved characteristics.

[0203] In one embodiment, an antibody variant is provided, which has a carbohydrate structure lacking fucose connected (directly or indirectly) to the Fc region. For example, the content of fucose in such an antibody can be 1% to 80%, 1% to 65%, 5% to 65% or 20% to 40%. The content of fucose is determined by calculating the average content of fucose in the sugar chain on Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid and high mannose structures) attached to Asn 297 determined by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 in the Fc region (Eu numbering of Fc region residues); however, due to minor sequence changes in antibodies, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, US Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Related "defucosylated" or "fucose-deficient" antibody variants include: US2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US2003 / 0115614; US2002 / 0164328; US 2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 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 deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and knockout cell lines, such as CHO cells in which the α-1,6-fucosyltransferase gene FUT8 is knocked out (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0204] Further provided are antibody variants with bisected oligosaccharides, for example, wherein 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 in, for example, WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US2005 / 0123546 (Umana et al.). Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described in, for example, WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0205] c) Fc region variants

[0206] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (such as a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (such as, substitution) at one or more amino acid positions.

[0207] In certain embodiments, the present invention contemplates antibody variants that have some but not all effector functions, making them ideal candidates for applications where the half-life of the antibody in vivo is important, but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. Primary cells that mediate ADCC NK cells express only Fc(RIII), while monocytes express Fc(RI, Fc(RII, and Fc(RIII). FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of target molecules are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)). 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, for example, ACTI for flow cytometry). TM Nonradioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, CA); and CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the target molecule can be assessed in vivo, for example, in an animal model such as disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

[0208] Antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include those with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant, in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581).

[0209] Certain antibody variants with improved or decreased binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).)

[0210] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, eg, substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0211] In some embodiments, alterations are made in the Fc region resulting in altered (ie, improved or reduced) CIq binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0212] Antibodies with extended half-life and improved binding to the neonatal Fc receptor (FcRn) are described in US2005 / 0014934A1 (Hinton et al.), which is responsible for 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)). Those antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution to Fc region residue 434 (U.S. Pat. No. 7,371,826).

[0213] For other examples of Fc region variants, see also: Duncan and Winter, Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351.

[0214] d) Cysteine ​​engineered antibody variants

[0215] In certain embodiments, it is desirable to produce antibodies engineered with cysteine, such as "thioMAbs", in which one or more residues of an antibody are replaced with cysteine ​​residues. In a particular embodiment, the substituted residues are present at accessible sites of the antibody. As further described herein, by replacing those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties (such as drug moieties or linker-drug moieties) to produce immunoconjugates. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) in the heavy chain Fc region. Cysteine-engineered antibodies may be generated as described in, for example, U.S. Patent No. 7,521,541.

[0216] e) Antibody derivatives

[0217] In certain embodiments, the antibodies provided herein can be further modified to include other non-proteinaceous parts known in the art and easily available. Suitable parts for antibody derivatization include but are not limited to water-soluble polymers. Non-limiting examples of water-soluble polymers include but are not limited to polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) and dextran or poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylene polyols (e.g., glycerol), polyvinyl alcohol and mixtures thereof. Due to its stability in water, polyethylene glycol propionaldehyde can have advantages in manufacturing. Polymer can have any molecular weight, and can have side chains or not. The number of polymers attached to the antibody is variable, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, etc.

[0218] In another embodiment, a conjugate of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can have any wavelength, and includes but is not limited to wavelengths that are not harmful to normal cells, but heat the non-protein moiety to a temperature at which cells proximal to the antibody-non-protein moiety are killed.

[0219] B. Recombinant Methods and Compositions

[0220] Recombinant methods and compositions can be used to produce antibodies, for example, as described in U.S. Patent No. 4816567. In one embodiment, an isolated nucleic acid encoding an anti-LY6E antibody described herein is provided. Such nucleic acid encoding can constitute the amino acid sequence of the VL of the antibody and / or the amino acid sequence of the VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises the following (e.g., transformed with the following): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody; or (2) a first vector and a second vector, the first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody, and the 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, for example, a Chinese hamster ovary (CHO) cell or a lymphocyte (e.g., a Y0, NS0, Sp20 cell). In one embodiment, a method for preparing an anti-rabbit LY6E antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expressing the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0221] For recombinant production of anti-LY6E antibodies, nucleic acids encoding antibodies, such as those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be easily isolated and sequenced using routine procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding heavy and light chains of antibodies).

[0222] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. Regarding expressing antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in Escherichia coli.) Antibodies can be separated from bacterial cell paste in a soluble fraction after expression, and can be further purified.

[0223] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding antibodies, including fungi and yeast strains whose glycosylation pathways have been "humanized" so that antibodies with partially or fully human glycosylation patterns are produced. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004) and Li et al., Nat. Biotech. 24: 210-215 (2006).

[0224] Suitable host cells for expressing glycosylated antibodies also come from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that can be used with insect cells, particularly for transfecting Spodoptera frugiperda cells.

[0225] Plant cell cultures can also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES for producing antibodies in transgenic plants). TM technology).

[0226] 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 monkey kidney CV1 line (COS-7) transformed by SV40; human embryonic kidney line (293 or 293 cells, as described, for example, in Graham et al. J. Gen Virol. 36: 59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, in Mather Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse breast tumor cells (MMT 060562); TRI cells (as described, for example, in Mather et al., Annals NY Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, which include DHFR -CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0227] C. Determination

[0228] The physical / chemical properties and / or biological activities of the anti-LY6E antibodies provided herein can be identified, screened or characterized by various assays known in the art.

[0229] In one aspect, the antigen binding activity of the antibodies of the invention is measured by, for example, ELISA, BIACore TM , FACS or Western blotting.

[0230] On the other hand, competition 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 are bound to the same epitope (e.g., linear or conformational epitope) bound by the antibodies described herein. Detailed exemplary methods for mapping the epitope bound by antibodies are provided in Morris (1996) "Epitope Mapping Protocols" in Volume 66 of Methods in Molecular Biology (Humana Press, Totowa, NJ).

[0231] In an exemplary competitive assay, immobilized LY6E is incubated in a solution comprising a first labeled antibody (e.g., any antibody described herein) that binds to LY6E and a second unlabeled antibody whose ability to compete with the first antibody for binding to LY6E is being tested. The second antibody may be present in a hybridoma supernatant. As a control, immobilized LY6E is incubated in a solution comprising the first labeled antibody rather than the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to LY6E, excess unbound antibody is removed, and the amount of label associated with immobilized LY6E is measured. If the amount of label associated with immobilized LY6E is substantially reduced in the test sample relative to the control sample, it indicates that the second antibody competes with the first antibody for binding to LY6E. See Harlow and Lane (1988) Antibodies: A Laboratory Manual Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0232] D. Immunoconjugates

[0233] The present invention also provides immunoconjugates comprising an anti-LY6E antibody of the present invention conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins of bacterial, fungal, plant or animal origin, enzymatically active toxins, or fragments thereof) or radioactive isotopes (i.e., radioconjugates).

[0234] Immunoconjugates allow targeted delivery of drug moieties to tumors and, in some embodiments, intracellular accumulation therein, where systemic administration of the unconjugated drug may result in unacceptable levels of toxicity to normal cells (Polakis P. (2005) Current Opinion in Pharmacology 5:382-387).

[0235] Antibody-drug conjugates (ADCs) target chemotherapeutic molecules by combining the properties of both antibodies and cytotoxic drugs by targeting potent cytotoxic drugs to tumor cells expressing the antigen (Teicher, BA (2009) Current Cancer Drug Targets 9:982-1004), thereby improving the therapeutic index by maximizing efficacy and minimizing off-target toxicity (Carter, PJ and Senter PD (2008) The Cancer Jour. 14(3):154-169; Chari, RV (2008) Acc. Chem. Res. 41:98-107).

[0236] The ADC compounds of the present invention include those with anticancer activity. In certain embodiments, the ADC compound comprises an antibody conjugated (i.e., covalently attached) to a drug moiety. In certain embodiments, the antibody is covalently attached to the drug moiety via a linker. The antibody-drug conjugate (ADC) of the present invention can selectively deliver an effective dose of drug to tumor tissue, wherein greater selectivity, i.e., a lower effective dose, can be achieved while increasing the therapeutic index ("therapeutic window").

[0237] The drug portion (D) of the antibody-drug conjugate (ADC) may comprise any compound, moiety or group that has a cytotoxic or cytostatic effect. The drug portion may impart cytotoxic and cytostatic effects to the drug portion through mechanisms including, but not limited to, tubulin binding, DNA binding or embedding, and inhibition of RNA polymerase, protein synthesis, and / or topoisomerase. Exemplary drug portions include, but are not limited to, maytansinoids, dolastatins, auristatins, calicheamicin, pyrrolobenzodiazepines (PBD), nemorubicin and its derivatives, PNU-159682, anthracyclines, ducathiamycin, vinca alkaloids, taxanes, trichothecenes, CC1065, camptothecin, elinefand and their stereoisomers, isosteres, analogs and derivatives having cytotoxic activity. Non-limiting examples of such immunoconjugates are discussed in further detail below.

[0238] 1. Exemplary Antibody-Drug Conjugates

[0239] Exemplary embodiments of antibody-drug conjugate (ADC) compounds include an antibody (Ab) that targets tumor cells, a drug moiety (D), and a linker moiety (L) that attaches Ab to D. In some embodiments, the antibody is attached to the linker moiety (L) through one or more amino acid residues, such as lysine and / or cysteine.

[0240] An exemplary ADC has Formula I:

[0241] Ab-(LD) p Formula I

[0242] Wherein p is 1 to about 20. In some embodiments, the number of drug moieties that can be conjugated to an antibody is limited to the number of free cysteine ​​residues. In some embodiments, free cysteine ​​residues are introduced into the antibody amino acid sequence by methods described herein. The exemplary ADC of Formula I includes but is not limited to antibodies (Lyon, R. et al. (2012) Methods in Enzym.502:123-138) with 1, 2, 3 or 4 engineered cysteine ​​amino acids. In some embodiments, without using engineering, one or more free cysteine ​​residues are already present in the antibody, in which case, existing free cysteine ​​residues can be used to conjugate the antibody to a drug. In some embodiments, before the antibody is conjugated, the antibody is exposed to reducing conditions to generate one or more free cysteine ​​residues.

[0243] a) Exemplary Linking Groups

[0244] "Linker" (L) is a bifunctional or multifunctional 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, antibody-drug conjugates (ADCs) can be prepared using linkers with reactive functional groups for covalent attachment to the drug and the antibody. For example, in some embodiments, a cysteine ​​thiol of an antibody (Ab) can form a bond with a reactive functional group of a linker or a drug-linker intermediate to prepare an ADC.

[0245] In one aspect, the linker has a functional group that can react with the free cysteine ​​present on the antibody to form a covalent bond. Non-limiting exemplary reactive functional groups of this type include: maleimide, haloacetamide, α-haloacetyl, activated esters such as succinimidyl esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acyl chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. See, for example, Klussman et al. (2004), Bioconjugate Chemistry 15 (4): 765-773, page 766 of the conjugation method, and examples herein.

[0246] In some embodiments, the linker has a functional group that can react with an electrophilic group present on the antibody. Exemplary such electrophilic groups include, but are not limited to, aldehyde and keto carbonyl groups. In some embodiments, the heteroatom of the reactive functional group of the linker can react with the electrophilic group on the antibody and form a covalent bond with the antibody unit. Non-limiting exemplary such reactive functional groups include, but are not limited to: hydrazide, oxime, amino, hydrazine, thiosemicarbazide, benzyl hydrazinecarboxylate, and aryl hydrazide.

[0247] The joint can include one or more joint components. Exemplary joint components include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("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 joint components are known in the art, some of which are described below.

[0248] The linker can be a "cleavable linker" that facilitates drug release. Non-limiting exemplary cleavable linkers include acid-labile linkers (e.g., containing hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52: 127-131 (1992); US 5208020).

[0249] In certain embodiments, the linker has the following Formula II:

[0250] -A a -W w -Y y -

[0251] Formula II

[0252] wherein A is a "Stretcher 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; and Ab, D and p are as defined above for Formula I. Exemplary embodiments of such linkers are described in U.S. Pat. No. 7,498,298, which is expressly incorporated herein by reference.

[0253] In some embodiments, the linker component comprises a "stretching unit" that connects the antibody to another linker component or a drug moiety. Non-limiting exemplary stretching units are shown below (wherein the wavy line indicates the site of covalent attachment to the antibody, drug or other linker component):

[0254]

[0255]

[0256] In some embodiments, the linker component comprises an "amino acid unit". In some such embodiments, the amino acid unit allows protease cleavage of the linker, thereby facilitating release of the drug from the immunoconjugate after 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, amino acids, 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 unit may include naturally occurring amino acid residues and / or minor 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 (eg, tumor-associated proteases, cathepsins B, C and D, or plasmin proteases).

[0257] In some embodiments, the linker component comprises a "spacer" unit that directly or indirectly connects the antibody to the drug portion through a stretcher unit and / or an amino acid unit. The spacer unit may be "self-eliminating" or "non-self-eliminating". A "non-self-eliminating" spacer unit is a spacer unit in which a portion or all of the spacer unit remains bound to the drug portion after cleavage of the ADC. Examples of non-self-eliminating spacers include, but are not limited to, glycine spacers and glycine-glycine spacers. In some embodiments, an ADC comprising a glycine-glycine spacer unit is cleaved by a tumor cell-associated protease, resulting in the release of the glycine-glycine-drug portion from the rest of the ADC. In some such embodiments, the glycine-glycine-drug portion undergoes a hydrolysis step in the tumor cell, thereby cleaving the glycine-glycine spacer unit from the drug portion.

[0258] The "self-eliminating" spacer can release the drug moiety. In certain embodiments, the spacer of the linker comprises 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 formed between the benzyl alcohol and the drug (Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15: 1087-1103). In some embodiments, the spacer is p-aminobenzyloxycarbonyl (PAB). In some embodiments, the ADC comprising a self-eliminating linker has the following structure:

[0259]

[0260] wherein Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano; m is an integer in the range of 0 to 4; and 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.

[0261] Other examples of self-eliminating spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (U.S. Pat. No. 7,375,078; Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and o- or p-aminobenzyl acetals. In some embodiments, spacers that undergo cyclization upon amide bond hydrolysis may be used, such as 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 2-aminophenylpropionic acid amides (Amsberry et al. (1990) J. Org. Chem. 55:5867). Attachment of the drug to the α-carbon of a glycine residue is another example of a self-immolative spacer that can be used in an ADC (Kingsbury et al. (1984) J. Med. Chem. 27: 1447).

[0262] In some embodiments, the linker L may be a dendritic linker (Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12: 2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11: 1761-1768) for covalently attaching more than one drug moiety to an antibody via a branched multifunctional linker moiety. Dendritic linkers can increase the molar ratio of drug to antibody, i.e., loading, which is related to the efficacy of the ADC. Thus, in the case where an antibody carries only one reactive cysteine ​​thiol group, a large number of drug moieties can be attached via a dendritic linker.

[0263] Non-limiting exemplary linkers are shown below in the context of an ADC of Formula I:

[0264]

[0265] Other non-limiting exemplary ADCs include the following structures, wherein -S- is part of an antibody:

[0266]

[0267]

[0268] Where X is:

[0269] —(CH2) n -,-(CH2CH2O) n -,

[0270]

[0271] Y is:

[0272]

[0273] Each R is independently H or C1-C6 alkyl; n is 1 to 12.

[0274] Typically, peptide linkers can be prepared by forming a peptide bond between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to liquid phase synthesis methods (e.g., E. and K. Lübke (1965), "The Peptides", Vol. 1, pp. 76-136, Academic Press).

[0275] In some embodiments, the linker is substituted with a group that modulates solubility and / or reactivity. As a non-limiting example, a charged substituent such as sulfonate (-SO3-) or ammonium can increase the water solubility of the linker reagent and promote the coupling reaction of the linker reagent with the antibody and / or drug portion, or promote the coupling reaction of Ab-L (antibody-linker intermediate) with D, or DL ​​(drug-linker intermediate) with Ab, depending on the synthetic route used to prepare the ADC. 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) is coupled to the antibody. a With drug-(linker part) b conjugated to form an ADC of Formula I. In some such embodiments, the antibody comprises more than one (linker moiety) a substituent, such that more than one drug is conjugated to the antibody in the ADC of Formula I.

[0276] The compounds of the present invention specifically contemplate, but are not limited to, ADCs prepared with the following linker reagents: bis-maleimido-trioxyethylene glycol (BMPEO), N-(β-maleimidopropoxy)-N-hydroxysuccinimide ester (BMPS), N-(ε-maleimidocaproyloxy)succinimide ester (EMCS), N-[γ-maleimidobutyryloxy]succinimide ester (GMBS), 1,6-hexane-bis-vinyl sulfone (HBVS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidobenzoyl)-N-hydroxysuccinimide ester (MBS), succinimidyl)butyric acid hydrazide (MPBH), succinimidyl 3-(bromoacetylamino) 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-[(β-maleimidopropionamide) hexanoate] (SMPH), iminotetrahydro Thiophene (IT), sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB and succinimidyl-(4-vinyl sulfone) benzoate (SVSB), and including bismaleimide reagents: dithiobismaleimidoethane (DTME), 1,4-bismaleimidobutane (BMB), 1,4-bismaleimido-2,3-dihydroxybutane (BMDB), bismaleimidohexane (BMH), bismaleimidoethane (BMOE), BM(PEG)2 (as shown below) and BM(PEG)3 (as shown below); difunctional derivatives of imide esters (such as dimethyl hexadecene); The invention relates to bis-maleimide reagents, for example, ...

[0277]

[0278] In some embodiments, the linker is a MC-sq-Ala linker. Examples of peptidomimetic linkers can be found in, for example, WO2015 / 095227A2.

[0279] Certain useful linker reagents can be obtained from various commercial sources such as Pierce Biotechnology, Inc. (Rockford, IL), Molecular Biosciences Inc. (Boulder, CO), or synthesized according to procedures described in the art; for example, as described in the following literature: Toki et al., (2002) J. Org. Chem. 67: 1866-1872; Dubowchik et al., (1997) Tetrahedron Letters, 38: 5257-60; Walker, MA (1995) J. Org. Chem. 60: 5352-5355; Frisch et al., (1996) Bioconjugate Chem. 7: 180-186; US 6214345; WO 02 / 088172; US2003130189; US2003096743; WO 03 / 026577; WO 03 / 043583; and WO 04 / 032828.

[0280] Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See, for example, WO94 / 11026.

[0281] b) Exemplary Drug Moieties

[0282] Pyrrolobenzodiazepine

[0283] In some embodiments, the ADC comprises a pyrrolobenzodiazepine (PBD). In some embodiments, the PDB dimer recognizes and binds to a specific DNA sequence. The natural product anthramycin (PBD) 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, many PBDs (both naturally occurring PBDs and analogs) have been reported (Thurston et al., (1994) Chem. Rev. 1994, 433-465, including dimers of tricyclic PBD scaffolds (US 6884799; US 7049311; US ​​7067511; US ​​7265105; US 7511032; US 7528126; US 7557099)). Without wishing to be bound by any particular theory, it is believed that the dimeric structure confers the appropriate three-dimensional shape with the helicity of the minor groove of B-form DNA, resulting in a tight fit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-Van Devanter, (1986) Acc. Chem. Res., 19: 230-237). Dimeric PBD compounds bearing 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).

[0284] In some embodiments, PBD compounds can be used as prodrugs by protecting them at the N10 position with an in vivo removable nitrogen protecting group (WO 00 / 12507; WO 2005 / 023814).

[0285] PBD dimers have been conjugated to antibodies, and the resulting ADCs have been shown to have anticancer properties (US 2010 / 0203007). Non-limiting exemplary attachment sites on PBD dimers include five-membered pyrrole rings, linker arms between PBD units, and N10-C11 imine groups (WO 2009 / 016516; US 2009 / 304710; US2010 / 047257; US2009 / 036431; US2011 / 0256157; WO 2011 / 130598).

[0286] In some embodiments of the ADCs provided herein, the drug moiety (e.g., Drug D) is a compound of Formula A:

[0287]

[0288] in:

[0289] R 2 Having formula II: Where A is C 5-7 Aryl group, X is selected from the group consisting of OH, SH, CO2H, COH, N=C=O, NHR N and (OC2H4) m OCH3, where R N Selected from the group consisting of: H and C 1-4 Alkyl, and wherein m is an integer from 1 to 3, and any of the following:

[0290] (i)Q 1 is a single bond, and Q 2 Selected from a single bond and -Z-(CH2) n -, wherein Z is a single bond, O, S or NH, and n is an integer from 1 to 3; or

[0291] (ii)Q 1 is -CH=CH-, and Q 2 is a single bond;

[0292] R 2 ' is C optionally substituted by one or more substituents selected from the group consisting of the following 5-10 Aryl groups: halo, nitro, cyano, ether, C 1-7 Alkyl, C 3-7 Heterocyclyl and bis-oxy-C 1-3 Alkylene;

[0293] R 6 and R 9 Independently selected from: H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro,

[0294] Me3Sn and halogenation;

[0295] R 7 Selected from the group consisting of: H, R, OH, OR, SH, SR, NH2, NHR, NHRR', nitro, Me3Sn and halo;

[0296] R and R' are independently selected from optionally substituted C 1-12 Alkyl, C 3-20 Heterocyclic and C5-20 Aryl groups;

[0297] Any of the following:

[0298] (a)R 10 is H, and R 11 OH or OR A , where R A C 1-4 alkyl;

[0299] (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

[0300] (c)R 10 H and R 11 For SO Z M, wherein z is 2 or 3, and M is a monovalent pharmaceutically acceptable cation;

[0301] R" is C 3-12 The alkylene group, whose chain can be one or more independently selected from O, S and NH

[0302] heteroatoms from the group consisting of and / or one or more aromatic ring interruptions independently selected from the group consisting of benzene or pyridine;

[0303] Y is O, S or NH;

[0304] R 6' , R 7' and R 9' Selected from R 6 , R 7 and R 9 The same group, and R 10' and R 11' R 10 and R 11 Same, if R 11 and R 11' For SO Z M, then M may represent a divalent pharmaceutically acceptable cation; and

[0305] The connection point with connector L is through R 2 or R 2' .

[0306] In some embodiments of Formula (A):

[0307] R 2 Having formula II: Wherein A is phenyl, and X is selected from the group consisting of OH, SH, CO2H, COH, N=C=O, NHR Nand (OC2H4) m OCH3, where R N Selected from the group consisting of: H and C 1-4 alkyl, and wherein m is an integer from 1 to 3, and:

[0308] (i)Q 1 is a single bond, and Q 2 is a single bond;

[0309] R 2' is a phenyl group optionally substituted with one or more substituents selected from the group consisting of halo, nitro, cyano and -OR, wherein R is a saturated C 1-7 Alkyl groups;

[0310] R 6 and R 9 independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro, Me3Sn and halo;

[0311] R 7 is selected from the group consisting of: H, R, OH, OR, SH, SR, NH2, NHR, NHRR', nitro, Me3Sn, and halo;

[0312] R and R' are independently selected from unsubstituted C 1-12 Saturated alkyl;

[0313] R 10 and R 11 A nitrogen-carbon double bond is formed between the nitrogen atom and the carbon atom to which they are bound;

[0314] R" is C 3-12 a 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;

[0315] Y is O, S or NH;

[0316] R 6' , R 7' and R 9' Selected from R 6 , R 7 and R 9 The same group, and R 10' and R 11' R 10 and R 11 are the same; and

[0317] The connection point with connector L is through R 2 or R 2' .

[0318] Exemplary PDB portions of ADCs include, but are not limited to (wherein the wavy line indicates the site of covalent attachment to the linker):

[0319]

[0320] A non-limiting exemplary linker-PBD has the following structure:

[0321]

[0322] Non-limiting exemplary linker-PBD moieties of ADCs include, but are not limited to: The wavy line indicates the site of covalent attachment to the antibody.

[0323] PBD and ADC comprising PBD can be prepared according to methods known in the art. See, for example, WO 2009 / 016516; US 2009 / 304710; US 2010 / 047257; US 2009 / 036431; US ​​2011 / 0256157; WO 2011 / 130598; and WO 2010 / 043880.

[0324] c) Drug loading

[0325] Drug loading is represented by p, i.e., the average number of drug moieties per antibody in the molecule shown in Formula I. The drug loading range for each antibody is 1 to 20 drug moieties (D). The ADC shown in Formula I includes a collection of antibodies coupled to drug moieties in the range of 1 to 20. The average number of drug moieties per antibody in the ADC preparation obtained by the coupling reaction can be characterized by conventional methods such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of the ADC represented by p can also be determined. In some cases, the separation, purification, and characterization of homogeneous ADCs in which p is a specific value obtained by ADCs with other drug loadings can be achieved by methods such as reversed-phase HPLC or electrophoresis.

[0326] For some antibody drug conjugates, p may be limited by the number of binding sites on the antibody. For example, in the case of attachment to cysteine ​​thiol, as described in some exemplary embodiments above, the antibody may have only one or more cysteine ​​thiol groups, or may have only one or more thiol groups with sufficiently high reactivity, through which the linker can be attached. In certain embodiments, a higher drug loading, such as p>5, may result in aggregation, insolubility, toxicity or cell permeability of some antibody drug conjugates. In certain embodiments, the average drug loading of ADC ranges from 1 to about 8; from about 2 to about 6; or from about 3 to about 5. In fact, it has been shown that for some ADCs, the optimal ratio of the drug portion of each antibody may be less than 8, and may be from about 2 to about 5 (US 7498298).

[0327] In certain embodiments, during the coupling reaction, the number of drug moieties bound to the antibody is less than the theoretical maximum. The antibody may contain, for example, lysine residues that do not react with the drug-linker intermediate or linker reagent, as described below. Typically, antibodies do not contain many free and reactive cysteine ​​thiol groups that may be connected to the drug moiety; in fact, most cysteine ​​thiol residues in antibodies exist in the form of disulfide bonds. In certain embodiments, antibodies may be reduced under partial or full reduction conditions with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to generate reactive cysteine ​​thiol groups. In certain embodiments, antibodies are denatured to display reactive nucleophilic groups such as lysine or cysteine.

[0328] The drug loading (drug / antibody ratio) of ADCs can be controlled by various means, such as: (i) limiting the molar excess of drug-linker intermediates or linker reagents relative to the antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limited reducing conditions for cysteine ​​thiol modification.

[0329] It will be appreciated that when more than one nucleophilic group reacts with a drug-linker intermediate or linker reagent, the resulting product is a mixture of ADC compounds having a 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, which is specific for the antibody and specific for the drug. Individual ADC molecules in a mixture can be identified by mass spectrometry and can be separated 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, Vol. 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, Vol. 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 Meeting, March 27-31, 2004, Proceedings of the AACR, Vol. 45, March 2004). In certain embodiments, homogeneous ADCs having a single drug loading can be separated from the conjugation mixture by electrophoresis or chromatography.

[0330] d) Certain methods of preparing immunoconjugates

[0331] The ADC of Formula I can be prepared by a variety of routes using organic chemical reactions, conditions and reagents known to those skilled in the art, including: (1) reacting the nucleophilic group of the antibody with a divalent linker reagent to form Ab-L via a covalent bond, which is then reacted with the drug moiety D; and (2) reacting the nucleophilic group of the drug moiety with a divalent linker reagent to form DL via a covalent bond, which is then reacted with the nucleophilic group of the antibody. An exemplary method for preparing the ADC of Formula I via the latter route is described in US 7498298, which is expressly incorporated herein by reference.

[0332] Nucleophilic groups on antibodies include, but are not limited to: (i) N-terminal amino groups; (ii) side chain amino groups, such as lysine; (iii) side chain thiol groups, such as cysteine; and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated. Amino, thiol and hydroxyl groups are nucleophilic groups that can react with electrophilic groups on linker moieties and linker reagents to form covalent bonds, and the electrophilic groups include: (i) active esters, such as NHS esters, HOBt esters, haloformates and acyl halides; (ii) alkyl and benzyl halides, such as haloacetamides; and (iii) aldehydes, ketones, carboxyl and maleamido groups. Certain antibodies have reducible interchain disulfide bonds, i.e., cysteine ​​bridges. Antibodies can be reacted with linker reagents by treatment with reducing agents such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP), so that the antibodies are fully or partially reduced. Therefore, each cysteine ​​bridge will theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies by modifying lysine residues, for example, by reacting lysine residues with 2-iminothiolane (Traut's reagent) to convert amines into thiols. Reactive sulfhydryl groups can also be introduced into antibodies by introducing one, two, three, four or more cysteine ​​residues (e.g., by preparing variant antibodies comprising one or more non-natural cysteine ​​amino acid residues).

[0333] The antibody-drug conjugates of the present invention can also be prepared by the reaction between an electrophilic group on the antibody, such as, for example, an aldehyde or ketone carbonyl group, and a nucleophilic group on a linker reagent or a drug. Available nucleophilic groups on linker reagents include, but are not limited to, hydrazides, oximes, amino groups, hydrazines, thiosemicarbazides, hydrazine carboxylates, and aryl hydrazides. In one embodiment, the antibody is modified to introduce an electrophilic moiety that can react with a nucleophilic substituent on a linker reagent or a drug. In another embodiment, the sugar of the glycosylated antibody can be oxidized, for example, with a periodate oxidizing agent, to form an aldehyde or ketone group, which can react with an amine group of a linker reagent or a drug moiety. The resulting imine Schiff base group can form a stable connection, or can be reduced, for example, by a borohydride reagent to form a stable amine bond. In one embodiment, the reaction of the carbohydrate moiety of the glycosylated antibody with galactose oxidase or sodium metaperiodate can generate carbonyl (aldehyde and ketone) groups in the antibody, which can react with appropriate groups of the drug (Hermanson, Bioconjugate Techniques). In another embodiment, antibodies containing N-terminal serine or threonine residues can be reacted with sodium metaperiodate to generate an aldehyde in place of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3: 138-146; US 5362852). Such aldehydes can react with drug moieties or linker nucleophiles.

[0334] Exemplary nucleophilic groups on the drug moiety include, but are not limited to, amine, sulfhydryl, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazide, hydrazine carboxylate, and arylhydrazide groups, which are capable of reacting to form covalent bonds with electrophilic groups on the linker moiety and linker reagents, including: (i) active esters, such as NHS esters, HOBt esters, haloformates, and acyl halides; (ii) alkyl and benzyl halides, such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleamido groups.

[0335] Non-limiting exemplary cross-linking agents that can be used to prepare ADCs are described in the section entitled "Exemplary Linkers" herein. Methods of using such cross-linking agents to connect two moieties (including protein moieties and chemical moieties) are known in the art. In some embodiments, a fusion protein comprising an antibody and a cytotoxic agent can be prepared, for example, by recombinant techniques or peptide synthesis. A recombinant DNA molecule may comprise regions encoding the antibody and the cytotoxic moiety of the conjugate, which are adjacent to each other or separated by regions encoding linker peptides that do not destroy the desired properties of the conjugate.

[0336] In yet another embodiment, the antibody can be conjugated to a "receptor" (such as streptavidin) for tumor pretargeting, where the antibody-receptor conjugate is administered to the patient, followed by the use of a clearing agent to remove unbound conjugate from the circulation, followed by administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a drug or radionucleotide).

[0337] E Methods and compositions for diagnosis and detection

[0338] In certain embodiments, any of the anti-LY6E antibodies provided herein can be used to detect the presence of LY6E in a biological sample. As used herein, the term "detection" encompasses quantitative or qualitative detection. "Biological sample" includes, for example, cells or tissues (e.g., biopsy material, including cancerous or potentially cancerous colon, colorectal, endometrial, pancreatic or ovarian tissue).

[0339] In one embodiment, an anti-LY6E antibody for use in a diagnostic or detection method is provided. In a further aspect, a method for detecting the presence of LY6E in a biological sample is provided. In certain embodiments, the method includes contacting a biological sample with an anti-LY6E antibody as described herein under conditions that allow the anti-LY6E antibody and LY6E to bind, and detecting whether a complex is formed between the anti-LY6E antibody and LY6E in the biological sample. Such methods can be in vitro or in vivo methods. In one embodiment, an anti-LY6E antibody is used to select a subject who can be treated with an anti-LY6E antibody, for example, wherein LY6E is a biomarker for selecting a patient. In a further embodiment, the biological sample is a cell or tissue (e.g., a biopsy material, including cancerous or potentially cancerous colon, colorectal, endometrial, pancreatic or ovarian tissue).

[0340] In further embodiments, the anti-LY6E antibody is used in vivo to detect (e.g., by in vivo imaging) LY6E-positive cancer in a subject, for example, for the purpose of diagnosing, prognosing, or staging cancer, determining an appropriate course of treatment, or monitoring the response of cancer to therapy. One method known in the art for in vivo detection is immunopositron emission tomography (immunoPET), as described, for example, in van Dongen et al., The Oncologies 12:1379-1389 (2007) and Verel et al., J. Nucl. Med. 44:1271-1281 (2003). In such embodiments, a method for detecting LY6E-positive cancer in a subject is provided, the method 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, wherein detection of the labeled anti-LY6E antibody indicates a 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.

[0341] In a further embodiment, the diagnostic or detection method includes contacting a first anti-LY6E antibody fixed to 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 binds to a complex between the first anti-LY6E antibody and LY6E in the biological sample. The substrate can be any supporting medium, such as glass, metal, ceramic, polymer beads, slides, chips, and other substrates. In certain embodiments, the biological sample includes cells or tissues (e.g., biopsy materials, including cancerous or potentially cancerous colorectal, endometrial, pancreatic or ovarian tissues). 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 as described herein.

[0342] Exemplary diseases 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, 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 >90% of tumor cells. In another embodiment, LY6E-positive cancer expresses LY6E at a level of 1+, 2+ or 3+. In some embodiments, 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, RT-PCR is quantitative RT-PCR.

[0343] In certain embodiments, labeled anti-LY6E antibodies are provided. Labels include, but are not limited to, directly detected labels or moieties (such as fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and indirectly detected moieties (such as enzymes or ligands) (e.g., by enzymatic reactions or molecular interactions). Exemplary labels include, but are not limited to, radioisotopes. 32 P. 14 C. 125 I. 3 H and 131 I; fluorophores, such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone; luciferase, such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456); luciferin, 2,3-dihydrophthalazinone, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme; sugar oxidases, such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase; heterocyclic oxidases, such as urate oxidase and xanthine oxidase; coupled to enzymes (such as HRP, lactoperoxidase, or microperoxidase) that use hydrogen peroxide to oxidize dye precursors; biotin / avidin, spinning labels, phage labels, stable free radicals, etc. 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.

[0344] F Pharmaceutical preparations

[0345] Pharmaceutical preparations of anti-LY6E antibodies or immunoconjugates as described herein are prepared by mixing such antibodies or immunoconjugates having the desired purity with one or more optional pharmaceutical carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of lyophilized preparations or aqueous solutions. Pharmaceutical carriers are generally non-toxic to recipients at the doses and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) ) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutical carriers herein also include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0346] Exemplary lyophilized antibody or immunoconjugate formulations are described in US Pat. No. 6,267,958. Aqueous antibody or immunoconjugates include those described in US Pat. No. 6,171,586 and WO 2006 / 044908, the formulations in the latter comprising a histidine-acetate buffer.

[0347] The formulations herein may also contain more than one active ingredient essential for the particular indication being treated, preferably active ingredients with complementary activities that do not adversely affect each other. For example, in some cases, it may be desirable to further provide a platinum complex, for example, 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.

[0348] The active ingredient can be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively); embedded in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules); or embedded in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980).

[0349] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody or immunoconjugate, which matrices are in the form of shaped articles, e.g., films or microcapsules.

[0350] Preparations for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through sterile filtration membranes.

[0351] G. Methods of treatment and compositions

[0352] Any of the anti-LY6E antibodies or immunoconjugates provided herein can be used in methods, such as methods of treatment.

[0353] In one aspect, the anti-LY6E antibody or immunoconjugate provided herein is used in a method for inhibiting the proliferation of LY6E-positive cells, the method comprising exposing the cell to the anti-LY6E antibody or immunoconjugate under conditions that allow the anti-LY6E antibody or immunoconjugate to bind to LY6E on the cell surface, thereby inhibiting the proliferation of the cell. In certain embodiments, the method is an in vitro or in vivo method. In a further embodiment, the cell is a breast cancer cell, or a pancreatic cancer cell, or a colon cancer cell, or a colorectal cancer cell, or a melanoma cancer cell, or an ovarian cancer cell, or a non-small cell lung cancer cell, or a gastric cancer cell.

[0354] Inhibition of cell proliferation in vitro can be performed using CellTiter-Glo® commercially available from Promega (Madison, WI). TMThe assay is determined by a luminescent cell viability assay. The assay determines the number of viable cells in 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. Pat. No. 6,602,677. The assay can be performed in a 96-well or 384-well format, making it suitable for automated high-throughput screening (HTS). See Cree et al., (1995) AntiCancer Drugs 6:398-404. The assay procedure involves adding a single reagent ( The luciferase activity is measured by adding a luminescent reagent (e.g., a luciferase reagent) directly to the cultured cells. This causes cell lysis and generates a luminescent signal produced by the luciferase reaction. The luminescent signal is proportional to the amount of ATP present, which in turn is proportional to the number of viable cells present in the culture. The data can be recorded by a photometer or CCD camera imaging device. The luminescent output is expressed in relative light units (RLU).

[0355] On the other hand, an anti-LY6E antibody or immunoconjugate is provided for use as a medicament. In a further aspect, an anti-LY6E antibody or immunoconjugate is provided for use in a method of treatment. In certain embodiments, an anti-LY6E antibody or immunoconjugate is provided for use in the treatment of LY6E-positive cancers. In certain embodiments, the present invention provides an anti-LY6E antibody or immunoconjugate for use in a method of treating an individual with LY6E-positive cancer, the method comprising administering an effective amount of an anti-LY6E antibody or immunoconjugate to the individual. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as a therapeutic agent as described below.

[0356] In a further aspect, the present 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 used to treat LY6E-positive cancer. In a further embodiment, the medicament is used in a method for treating LY6E-positive cancer, the method comprising administering an effective amount of the medicament to an individual suffering from LY6E-positive cancer. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as a therapeutic agent as described below.

[0357] In a further aspect, the invention provides a method for treating a LY6E-positive cancer. In one embodiment, the method comprises administering an effective amount of an anti-LY6E antibody or immunoconjugate to an individual suffering from such a LY6E-positive cancer. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent as described below.

[0358] According to any of the above embodiments, the LY6E-positive cancer 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, LY6E-positive cancer is a cancer that receives an anti-LY6E immunohistochemistry (IHC) or in situ hybridization (ISH) score greater than "0" under the conditions described herein, which corresponds to very weak or no staining in >90% of tumor cells. In another embodiment, LY6E-positive cancer expresses LY6E at a defined 1+, 2+, or 3+ level under the conditions described herein. In some embodiments, LY6E-positive cancer is a cancer that expresses LY6E according to a reverse transcriptase PCR (RT-PCR) assay for detecting LY6E mRNA. In some embodiments, RT-PCR is quantitative RT-PCR.

[0359] An "individual" according to any of the above embodiments may be a human.

[0360] In a further aspect, the present invention provides a pharmaceutical preparation comprising any of the anti-LY6E antibodies or immunoconjugates provided herein, for example, for use in any of the above-mentioned treatment methods. In one embodiment, the pharmaceutical preparation comprises any of the anti-LY6E antibodies or immunoconjugates provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical preparation comprises any of the anti-LY6E antibodies or immunoconjugates provided herein, and at least one additional therapeutic agent, for example, as described below.

[0361] The antibodies or immunoconjugates of the invention can be used alone or in combination with other agents for therapy. For example, the antibodies or immunoconjugates of the invention can be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is a platinum complex, which is used, for example, to treat 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.

[0362] Such combination therapies described above encompass combined administration (wherein two or more therapeutic agents are included in the same or separate preparations) and separate administration, in which case the administration of the antibody or immunoconjugate of the invention can be performed before, simultaneously with, and / or after the administration of the additional therapeutic agent and / or adjuvant. The antibody or immunoconjugate of the invention can also be used in combination with radiotherapy.

[0363] The antibody or immunoconjugate of the present invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and if necessary for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be carried out by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-lived or long-term. Various dosing schedules are contemplated herein, including but not limited to single or multiple administrations, push injection administrations, and pulse infusions at various time points.

[0364] The antibodies or immunoconjugates of the present invention will be formulated, dosed and applied in a manner consistent with good medical practice. Factors to be considered in this case include the specific illness being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the illness, the delivery site of the medicament, the method of administration, the time arrangement of administration, and other factors known to practitioners. Antibodies or immunoconjugates are not necessary, but are optionally co-formulated with one or more medicaments currently used to prevent or treat the illness in question. The effective amount of such other agents depends on the amount of the antibody or immunoconjugate present in the preparation, the type of illness or treatment, and other factors discussed above. These are usually used with the same dosage and route of administration as described herein, or with about 1% to 99% of the dosage described herein, or with any dosage and any route determined to be suitable by experience / clinically.

[0365] In order to prevent or treat a disease, the appropriate dose of the antibody or immunoconjugate of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody or immunoconjugate, the severity and progression of the disease, whether the antibody or immunoconjugate is used for prevention or treatment purposes, previous therapy, the patient's medical history and the response to the antibody or immunoconjugate, and the discretion of the attending physician. The antibody or immunoconjugate is appropriately administered to the patient once or in a series of treatments. Depending on the type and severity of the disease, an antibody or immunoconjugate of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) can be, for example, an initial candidate dose administered to the patient by one or more separate administrations or by continuous infusion. Depending on the above factors, a typical daily dose can range from about 1 μg / kg to 100 mg / kg or more. For repeated administration for several days or longer, depending on the condition, treatment will generally continue until desired disease symptom suppression occurs. An exemplary dosage of an antibody or immunoconjugate ranges from about 0.05 mg / kg to about 10 mg / kg. Therefore, 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, weekly or every three weeks (for example, so that the patient receives about two to about twenty or, for example, about six dosages of the antibody). An initial higher loading dose can be administered, followed by one or more lower doses. However, other dosage regimens may be useful. The progress of the therapy can be easily monitored by conventional techniques and determinations.

[0366] It should be understood that any of the above-described formulations or treatment methods can be performed using both the immunoconjugates of the invention and anti-LY6E antibodies.

[0367] H Products

[0368] In another aspect of the present invention, a product is provided, which contains a substance that can be used to treat, prevent and / or diagnose the above-mentioned illness. The product includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed of a variety of materials such as glass or plastic. The container holds a composition, which itself or in combination with another composition can effectively treat, prevent and / or diagnose the illness, and the container can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle). At least one active agent in the composition is an antibody or immunoconjugate of the present invention. The label or package insert indicates that the composition is used to treat the selected condition. In addition, the product may include (a) a first container containing a composition, wherein the composition comprises an antibody or immunoconjugate of the present invention; and (b) a second container containing a composition, wherein the composition comprises a further cytotoxic agent or other therapeutic agent. The article of manufacture in this embodiment of the invention may further include a package insert indicating that these compositions can be used to treat a particular condition. Alternatively or additionally, the article of manufacture may further include a second (or third) container containing a pharmaceutical buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, or dextrose solution. The article of manufacture may also include other substances required from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0369] I. Sequence

[0370] In another aspect, provided are sequences that can be used to treat, prevent and / or diagnose the disorders described herein.

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383] III. Examples

[0384] The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced given the general description provided above.

[0385] Example 1 - Virus-like particle (VLP) generation and characterization

[0386] 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 previously (see, e.g., 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 concentrations were 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 different concentrations of VLPs diluted in coating buffer (50 mM carbonate, pH 9.6). The plates were washed with wash buffer (1xPBS, containing 0.05% Tween20) and then blocked with ELISA assay dilution (PBS / 0.5% BSA / 0.05% polysorbate 20) for 1 hour at room temperature. The plates were then incubated with anti-Ly6E (9B12; diluted to 1 μg / ml in ELISA assay diluent) at room temperature for 1 hour. The plates were washed with wash buffer and bound antibodies were detected with goat anti-rat horseradish peroxidase secondary antibody (Jackson Immunol Lab). The plates were incubated at room temperature for 30 minutes, washed with wash buffer and developed with TMB solution (Surmodics, USA). The plates were read at 630 nm.

[0387] Example 2 - VLP Immunization

[0388] The animals used in these studies were maintained in an AAALAC-accredited animal facility. All experiments were performed in accordance with the Genentech Institutional Animal Care and Use Committee and OLAW guidelines. The study design was approved by the Genentech Institutional Animal Care and Use Committee before this work began.

[0389] Sprague Dawley rats (Charles River Laboratories, Hollister, CA) were immunized with Ly6E VLPs in PBS and Ribi adjuvant (Sigma). Rats were then boosted three times every 2 weeks with additional Ly6E VLPs. Plasmid encoding Ly6E cDNA was subsequently injected four times via Genegun over 2 weeks. DNA / gold particle bullets were prepared essentially as described above. See, e.g., Tang et al., Nature 1992; 356: 152–4 and Hansen et al., Sci Rep-uk 2016; 6: 21925.

[0390] Each DNA bullet was prepared to contain a total of 1 μg DNA, which was coated onto 0.5 mg gold particles (BioRad, Cat. # 1652264). The bullets were stored at 4°C in the dark in the presence of desiccant particles.

[0391] pAb was purified by protein A and determined by FACS to identify antibodies that bind to cell surface Ly6E. To generate monoclonal antibodies, hybridoma fusions were performed as described above, except that the myeloma partner SP2ab of IgG cells was enabled to be displayed on the surface. 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 supernatant 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 anti-Ly6E hybridomas were determined using 5'RACE, and then the PCR amplification products were sequenced. Clones showing promising binding were cloned and expressed as rat-human chimeric antibodies (SEQ ID NO. 4 and 6). The purified protein was used for further analysis.

[0392] Example 3 - Characterization of Anti-Ly6E

[0393] The chimeric anti-Ly6E was compared with a 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 according to ATCC recommendations. Prior to staining, adherent lines were released by Accutase treatment, and 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 3 times, stained with secondary antibody (Jackson ImmunoResearch 109-606-003) (1:100) for 1 hour at 4°C, washed, stained with the fixable viability dye eFluor780 (1:1000) for 1 hour at 4°C, and washed 2 times. Samples were fixed in 1% PFA (PBS) before analysis. Samples were evaluated on a BD LSRFortessa. After excluding dead cells, antibody binding of individual cells was evaluated using FlowJo software ( Figures 2A to 2C ). Anti-Ly6E showed enhanced binding compared to 9B12 in all three cell lines tested.

[0394] Example 4—Ly6E humanization and reformatting

[0395] The Ly6E antibody was humanized by grafting the HC and LC CDRs into human germline frameworks (IGHV3-73*01 / IGHJ1*01 and IGKV1-33*01 / IGKJ2*01, respectively) and iterating between human and rat residues at the vernier positions to maximize binding while minimizing non-human residues. The binding of humanized anti-Ly6E (comprising SEQ ID NOs. 3 and 5) was compared to chimeric anti-Ly6E (comprising SEQ ID NOs. 4 and 6), 9B12, and isotype controls ( Figures 3A to 3B ). The humanized version showed similar binding to chimeric anti-Ly6E and enhanced binding compared to 9B12 against both cell lines. Cysteine ​​residues were incorporated at the previously described positions to enable Humanized VH was cloned into a knob-in-hole framework to generate bispecific antibodies (Ridgeway et al., Prot. Eng. 1996, 617-621).

[0396] Example 5 – Characterization and optimization of anti-Ly6E stability

[0397] To assess potential manufacturing defects, anti-Ly6E was incubated at 40 degrees Celsius in low ionic strength histidine acetate buffer pH 5.5 for 2 weeks and analyzed by peptide mapping essentially as described previously (Xu et al. Mol. Pharmaceutics 2018, 4529-4537). This thermal stress increased the amount of isomerized light chain (LC) D92 by 6.2% ( Figures 4A to 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, proteolytically cleaved to Fab to generate monovalent binders, and binding to NCI-H1781 cells was assessed by flow cytometry as described above ( Figure 5 ). Both variants showed similar binding to humanized anti-Ly6E. Notably, for LC D92S, the variant had an Fv charge of 7.5, and for LC D92E, the variant had an Fv charge of 6.5. D92E was selected as the preferred mutant because its charge profile was more consistent with the prediction method of normal PK (Hotzel et al., mAbs 2012, 753-760).

[0398] Example 6 - Generation of anti-Ly6E antibody-drug conjugates

[0399] In short, cysteine ​​residues are engineered at the desired positions of the heavy and / or light chains of antibodies targeting Her2, Ly6E and CD33 antigens to produce their THIOMAB variants. As previously described, THIOMAB antibodies are conjugated to linker drugs. See Junutula et al., Nat Biotechnol 2008, 26: 925-932. In short, the antibody is reduced overnight in the presence of a hundred-fold molar excess of DTT (Calbiochem). The reducing agent and cysteine ​​and glutathione blocks are purified using HiTrap SP-HP columns (GE Healthcare). The antibody is reoxidized for 2.5 hours in the presence of a fifteen-fold molar excess of dhAA (MP Biomedical). The formation of interchain disulfide bonds is monitored by LC / MS. The linker drug in a ten-fold molar excess relative to the protein is incubated with the activated THIOMAB antibody in the presence of 15% DMF for 2 or 3 hours. The antibody drug conjugate is purified on a HiTrap SP-HP column (GE Healthcare) to remove excess linker drugs. If more than 5% aggregation was still present by analytical SEC, it was purified on a Hi Load Superdex 200pg 16 / 600 column (GE Healthcare) using 20 mM histidine acetate, 150 mM NaCl (pH 5.5) as running buffer. The number of conjugated linker drug molecules per THIOMAB antibody was quantified by LC / MS analysis. Purity was assessed by size exclusion chromatography.

[0400] Mass spectrometry analysis

[0401] LC / MS analysis was performed on a 6530 accurate mass quadrupole time-of-flight (Q-TOF) LC / MS (Agilent Technologies). The samples were heated to 80°C in a PRLP-S. 8μm (50mm 2.1mm, AgilentTechnologies) on the chromatographic analysis.Use 4.3 minutes from 30% to 60% B linear gradient (solvent A, 0.05% TFA / water; solvent B, 0.04% TFA / acetonitrile), and use electrospray source direct ionization eluent.Collect data and use Agilent Mass Hunter qualitative analysis software deconvolution.Before LC / MS analysis, antibody drug conjugates were treated with DTT at 15mM (pH 8.0) and 37°C for 30 minutes to produce HC and LC parts for analysis.The drug to antibody ratio (DAR) was calculated using the ion abundance present in the LC / MS deconvolution results.Use LC / MS to identify the peak.

[0402] LC / MS analysis of the reduced conjugates showed that the antibody drug conjugates had drugs ranging from 1.8 to 2 drugs per antibody for all conjugates.

[0403] Example 7 - Characterization of Anti-Ly6E as SN36325 Antibody-Drug Conjugate

[0404] Cells were seeded in 384-well plates, grown for 24 h, and treated with the indicated ADCs, including conjugates with isotype controls (anti-gD and anti-CD22) and 9B12. After 5 days of continuous ADC incubation, the cells were plated and plated using Promega Cell viability was determined using a luminescent reagent from PerkinElmer The reader measures the luminescence intensity ( Figures 6A to 6B ). Relative cell viability was calculated by normalization to non-drug treated controls. Anti-Ly6E conjugates showed more potent killing of both HCC1569x2 and SW900 cell lines compared to isotype control conjugates and 9B12 conjugates. Minimal differences were observed between the chimeric and humanized versions of anti-Ly6E.

[0405] Example 8 - Synthesis of (maleimide-sq-ala)-PBD linker-drug

[0406] Synthesis scheme of (maleimide-sq-ala)-PBD linker-drug:

[0407]

[0408] The maleimide-sq-ala linker was conjugated to the PBD payload in two steps, first installing the alanine, and then conjugating the maleimide-sq via INT5 (activated maleimide-sq-ester). The linker in this example has a 5-carbon spacer between the maleimide and sq.

[0409] Synthesis scheme of INT5-precursor:

[0410]

[0411] General procedure for the preparation of compound 3:

[0412]

[0413] 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. Compound 1 (300.0 mg, 0.41 mmol; commercially available, for example, BOC Sciences "PBD Dimer-CAS1222490-34-7", www.bocsci.com / product / pbd-dimer-cas-1222490-34-7-477053.html) was then added to the above solution. The reaction mixture was stirred at 20 °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 x 2) to give the 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 minutes): R T = 0.838 min, m / z = 1019.6 [M+H] + .

[0414] General procedure for the preparation of compound 4:

[0415]

[0416] 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 give a solid, which was washed with methyl tert-butyl ether (40 mL x 2) to give a crude compound 4 (40 mg, 73%) as a yellow solid. LCMS (5-95, AB, 1.5 minutes): RT = 0.765 minutes, m / z = 797.4 [M + H] + .

[0417] General procedure for conjugation of INT5 precursor with ala-PBD payload:

[0418]

[0419] Compound 4 (40.0 mg, 0.05 mmol) and N,N-diisopropylethylamine (0.02 mL, 0.1 mmol) were added to a solution of compound 5 (47.6 mg, 0.1 mmol) in DMF (2 mL). The reaction mixture was stirred at 20 ° C for 2 hours. The mixture was purified by preparative HPLC (acetonitrile: 40%-70% / 0.225% FA in water) to give the product linker-payload X (25 mg, 46%) as a yellow solid. LCMS (5-95, AB, 1.5 minutes): RT = 0.838 minutes, m / z = [M + H] + 1087.5.

[0420] General procedure for the preparation of compound 8:

[0421]

[0422] To a mixture of cyclobutane-1,1-dicarboxylic acid (0.82 g, 5.72 mmol) in DMF (10 mL) was added HATU (2.17 g, 5.72 mmol) and N,N-diisopropylethylamine (2.22 g, 17.15 mmol), followed by 1-(5-aminopentyl)pyrrole-2,5-dione hydrochloride (1.0 g, 4.57 mmol) at 0 °C. The mixture was stirred at 0 °C for 1 hour. LCMS showed that the reaction was complete. The solvent was removed in vacuo and dissolved in EtOAc (50 mL), followed by washing 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 silica chromatography eluting with 0 to 5% methanol / DCM to give 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 HNMR(400MHz, CDCl3): δ6.71(s,2H),6.37(br s,1H),3.53(t,J=6.8Hz,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

[0423] -1.57(m,4H),1.32-1.28(m,2H).

[0424] General procedure for the preparation of compound 5:

[0425]

[0426] 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 warmed to 20 ° C and stirred for 4 hours. The solvent was then removed under vacuum, and EtOAc (1.5 mL) was added to the mixture, and 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 minutes): RT = 0.785 minutes, m / z = 475.1 [M + H] +.

[0427] Example 9 - In vitro characterization of anti-Ly6E as SGD-1882 antibody-drug conjugate

[0428] Anti-Ly6E was conjugated to SGD-1882 essentially as described above using a maleimide-sq-ala linker with a 5-carbon spacer between the maleimide and sq. All conjugates were generated with less than 5% aggregates and DAR>1.8. Cells were seeded in 384-well plates, grown for 24 hours, and treated with the indicated ADCs, including the previously described anti-Ly6E antibody (9B12). After 5 days of continuous ADC incubation, the cells were plated using Promega Cell viability was determined using a luminescent reagent from PerkinElmer The reader measures the luminescence intensity ( Figure 7 ). Relative cell viability was calculated by normalization to non-drug treated controls. Anti-Ly6E showed more effective killing of SW900 cell line compared to 9B12.

[0429] Example 9 - In vivo characterization of anti-Ly6E as SGD-1882 antibody-drug conjugate (ADC)

[0430] The efficacy of Ly6E antibody drug conjugates was investigated in the human breast cancer xenograft model HCC1569X2. The HCC1569X2 cell line was derived from the parental HCC1569 cells (ATCC) from Genentech to provide optimal tumor growth in mice. Promega 16 The cell line was validated by short tandem repeat (STR) profiling and compared with external STR profiles of the cell line to determine the ancestry of the cell line. Animal studies using this cell line were conducted at Genentech in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Genentech Institutional Animal Care and Use Committee (IACUC). To establish a xenograft model, five million tumor cells (suspended in 0.2 mL of HBSS containing Matrigel) were inoculated into the chest mammary fat pad of female CB-17 SCID-beige mice (Charles River Laboratory; Hollister, CA).

[0431] When the tumor reaches the desired volume (about 250 mm 3 ), animals were divided into n=5 groups with similar tumor volume distribution and received intravenous doses 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 measurements. Tumors were measured in two dimensions (length and width) using a caliper, and tumor volume was calculated using the following formula: Tumor size (mm3) = 0.5 x (length x width x width). Body weight changes are reported as a percentage of starting weight. During the study, tumor size and mouse weight were recorded twice a week. According to IACUC guidelines, tumor volumes exceeding 2000 mm 3 Mice that lost 20% of their starting weight were immediately euthanized.

[0432] Data were analyzed using the R statistical software system (R Foundation for Statistical Computing; Vienna, Austria), and mixed models were fitted in R using the nlme package (Pinheiro et al., (2013) Nlme: Linear and Nonlinear Mixed-Effects Models. R Package Version 31-110.3.1-113). Nonlinear curves were fitted to the time course of weight change or log2 tumor volume at each dose level using cubic regression splines. These nonlinear curves were then dose-dependent in the mixed model. This approach addressed both repeated measurements and the moderate withdrawal caused by the removal of any non-treatment-related animals before the end of the study. The results were plotted in natural proportions as the fitted weight change or tumor volume ( Figures 8A to 8B ).

[0433] Example 10 - Characterization of anti-Ly6E as a T cell engaging bispecific antibody

[0434] Essentially as described, a pestle and mortar was used to express bispecific antibodies targeting Ly6E and CD3 with high or low affinity arms. 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. Essentially as described previously (see, e.g., Junttila et al., Canc. Res. 2014, 74: 5561-5571), a 4: 1 effector to target ratio was used to assess bispecific antibody-induced cell killing by CD8+ T cells and co-culture cells for 3 days ( Figures 9A to 9F As expected, the higher affinity anti-CD3 arm corresponds to higher activity. Anti-Ly6E was shown to be superior to 9B12 in all cell lines tested.

[0435] Example 11 - Further Characterization of Anti-Ly6E as SN36325 Antibody-Drug Conjugate

[0436] Cells were seeded in 384-well plates, grown for 24 h, and treated with the indicated ADCs, including conjugates with isotype controls (anti-gD and anti-CD22) and 9B12. After 5 days of continuous ADC incubation, the cells were plated and plated using Promega Cell viability was determined using a luminescent reagent from PerkinElmer The reader measures the luminescence intensity. Relative cell viability was calculated by normalization to the non-drug treated control. Compared with the isotype control, 9B12 and other ADC conjugates, the anti-Ly6E conjugates (3A3 and 4B10) showed significant effects on NCl-H1781 ( Figures 10A to 10B ) and HCC1569x2( Figures 11A to 11B ) cell lines showed more effective killing. For example, Figures 10A to 10B It was shown that in the cell line NCl-H1781, chimeric anti-Ly6E 4B10 and 3A3 showed >400-fold difference in potency over non-target controls (e.g., isotype controls anti-gD and anti-CD22), while 9B12 showed only a 9-fold difference in potency. Figures 11A to 11B It was shown that chimeric anti-Ly6E 3A3 showed the highest potency against the cell line HCC1569x2, despite weaker overall potency and partial killing compared to other ADCs and control conjugates.

[0437] Example 12 - Characterization of 3A3 Binding

[0438] On the 0th day, Kuramochi cells were inoculated into 384-well cell carrier plates (Perkin Elmer) and allowed to adhere overnight to reach 80% confluence. The next day, cold culture medium containing 10 μg / mL leupeptin and 5 μM pepstatin protease inhibitors was used to treat cells with unconjugated antibodies of 2 μg / mL and 20 μg / mL. Antibodies were allowed to bind on ice for 1 hour. Pulse-treated cells were washed with cold culture medium containing protease inhibitors, while cells treated continuously were placed separately. Cells were incubated at 37°C, 5% humidified CO2 for 1 hour and 3 hours to carry out antibody internalization. After incubation, cells were fixed with 4% paraformaldehyde (PFA) / 4% sucrose in phosphate buffered saline (PBS) for 10 minutes at room temperature (RT), and then washed six times with 1x PBS. Cells were blocked and permeabilized for 1 hour with 2% donkey serum (blocking buffer) containing 0.05% saponin. The blocking buffer was removed, and then the rabbit anti-LAMP1 (Sigma Aldrich, L1418) primary antibody in blocking buffer was added and incubated overnight. The next day, the cells were washed six times with PBS containing 0.05% saponin, and then washed with the secondary antibody donkey anti-human IgG-488. It was demonstrated that only anti-Ly6E showed significantly enhanced binding to Ly6E overexpressing cells. See Fig. 12A .

[0439] In addition, direct binding of antigen-binding fragment (Fab)-luciferase fusions to three different Ly6E-expressing cells was assessed after incubation for 4 hours at 4°C. Figures 12B to 12D 3A3 (anti-ly6E) was shown in Kuramochi ( Fig. 12B )、NCl-H1781( Fig. 12C ) and HCC-1569x2( Fig.12D ) cell lines that were verified to express Ly6E by Western blotting. Points represent the mean of two replicates. BIAcore TM Fab binding (Kd = nM) also showed 3A3 (218 Kd) > 2.2C2 (168 Kd) > 2.2C5 (63 Kd) > 1D5.3 (20 Kd) > 2.5G6 (14 Kd) > 1D5.2 (12 Kd).

[0440] Example 13 - Colocalization

[0441] Kuramochi cells were exposed to 2 μg / mL of each antibody for 1 hour at 4°C, and the antibodies were washed for pulse exposure (PE) or not washed for continuous exposure (CE), and then colocalization and internalization (intensity in cells / intensity in cells + outside cells) were measured at 37°C for 0, 1, and 3 hours. Fig.13It was shown that the amount of co-localization of 3A3 was significantly higher compared to antibodies 1D5.3, 2.5G6, 9B12 and the control.

[0442] Example 14 - Internalization

[0443] To assess internalization rate, pulse-chase experiments were performed in which antibodies were incubated with Kuramochi cells at 2 μg / mL for 1 hour at 4°C, washed, and internalization monitored over time (0, 1, and 3 hours). Fig.14 showed that the internalization rate of 9B12 was faster compared to 3A3, but the higher binding of 3A3 resulted in sustained efficacy as seen in the in vivo killing studies.

[0444] Although the present invention has been previously described in considerable detail by way of illustration and example for purposes of clarity of understanding, these descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

1. An isolated antibody that binds to Ly6E, wherein the antibody 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. The antibody according to claim 1 , which is a monoclonal antibody.

3. The antibody according to claim 1 or claim 2, which is a humanized antibody or a chimeric antibody. The antibody according to any one of claims 1 to 3, which is an antibody fragment.

5. The antibody according to any one of claims 1 to 4, comprising: (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) the VH sequence in (a) and the VL sequence in (b). 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. The antibody according to any one of claims 1 to 8, wherein 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 the following SEQ ID NOs: SEQ ID NO: 3, SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO:

29. 10 . The antibody of claim 9 , wherein 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.

11. An isolated antibody that binds to Ly6E, wherein 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.

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

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

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

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

19. A method of producing an antibody, the method comprising culturing the host cell according to claim 17 or claim 18 so as to produce the antibody.

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

21. The immunoconjugate of claim 20, having the formula Ab-(LD)p, wherein: (a) Ab is an 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 of claim 21, wherein D is a pyrrolobenzodiazepine of formula A in: R 2 Having formula II: Where A is C 5-7 Aryl group, X is selected from the group consisting of OH, SH, CO2H, COH, N=C=O, NHR N and (OC2H4) m OCH3, where R N Selected from the group consisting of: H and C 1-4 Alkyl, and wherein m is an integer from 1 to 3, and any of the following: (i)Q 1 is a single bond, and Q 2 Selected from a single bond and -Z-(CH2) 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 C optionally substituted by one or more substituents selected from the group consisting of the following 5-10 Aryl groups: halo, nitro, cyano, ether, C 1-7 Alkyl, C 3-7 Heterocyclyl and bis-oxy-C 1-3 Alkylene; R 6 and R 9 Independently selected from: H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro, Me3Sn and halo; R 7 Selected from the group consisting of: H, R, OH, OR, SH, SR, NH2, NHR, NHRR', nitro, Me3Sn and halo; wherein R and R' are independently selected from optionally substituted C 1-12 Alkyl, C 3-20 Heterocyclic and C 5-20 Aryl groups; Any of the following: (a)R 10 is H, and R 11 OH or OR A , where R A C 1-4 alkyl; (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 H and R 11 For SO Z M, wherein z is 2 or 3, and M is a monovalent pharmaceutically acceptable cation; R" is C 3-12 an 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 or pyridine; Y is O, S or NH; R 6 '、R 7 ' and R 9 ' respectively selected from 6 , R 7 and R 9 The same group, and R 10 ' and R 11 'Respectively with R 10 and R 11 Same, if R 11 and R 11 'For SO Z M, then M may represent a divalent pharmaceutically acceptable cation; and The connection point with connector L is through R 2 or R 2 '.

23. The immunoconjugate of claim 22, wherein D has the following structure: The wavy line indicates the connection point with the connector L.

24. The immunoconjugate of any one of claims 21 to 23, wherein (LD) has the following structure: The wavy lines indicate the points of attachment to the protein.

25. The immunoconjugate of 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 formulation of claim 28, further comprising an additional therapeutic agent.

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

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

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

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

35. The method of claim 34, wherein the cell is a breast, pancreatic, colon, colorectal, melanoma, ovarian cancer cell, non-small cell lung, or gastric cancer cell.

36. The antibody of any one of claims 1 to 14, which is conjugated to a label.

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

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

39. A method for 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 allow the anti-Ly6E antibody to bind 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.

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

41. The method of 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 a labeled anti-Ly6E antibody to a subject having or suspected of having a Ly6E-positive cancer, wherein the labeled anti-Ly6E antibody comprises the 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 a Ly6E-positive cancer in the subject.

43. The method of claim 42, wherein the labeled anti-Ly6E antibody is a labeled antibody of claim 7 or claim 11.

44. The method of claim 42 or claim 43, wherein the labeled anti-Ly6E antibody comprises an anti-Ly6E antibody conjugated to a positron emitter.

45. The method of claim 44, wherein the positron emitter is 89 Zr.

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