Anti-ly6e antibody, antibody-drug conjugates and methods of use

Abstract

Antibody constructs that bind human lymphocyte antigen 6 complex, locus E (Ly6E) and antibody-drug conjugates (ADCs) comprising an anti-Ly6E antibody construct conjugated to a drug, such as a cytotoxin or immune modulator. The use of anti-Ly6E antibody constructs and ADCs comprising an anti-Ly6E antibody construct conjugated to a drug as therapeutics or diagnostics, for example, in the treatment of cancer or in the diagnosis or analysis of cancer.

Description

ANTI-LY6E ANTIBODY, ANTIBODY-DRUG CONJUGATES AND METHODS OF USE FIELD

[0001] The present disclosure relates to the field of immunotherapeutics, and in particular to antihuman lymphocyte antigen 6 complex, locus E (Ly6E) antibody, Ly6E antibody-drug conjugates and their methods of use.BACKGROUND

[0002] Lymphocyte antigen 6 complex, locus E (Ly6E), encoded by the Ly6E gene on human chromosome 8, is a member of the lymphostromal cell membrane Ly6 superfamily. Ly6E is a glycosyl-phosphatidylinositol (GPI) -anchored cell-surface protein that is expressed on B cells, immature and activated T cells, thymus stromal cells and macrophages and is likely related to immune cell maturation, oncogenesis and differentiation and self-renewal of erythroid progenitors. Ly6E is also involved in modulation of viral infection by coronaviruses, SARS-CoV, MERS-CoV and SARS-CoV-2. Ly6E is overexpressed and amplified in multiple cancers including ovarian, lung, breast, colon, pancreatic, kidney and gastric cancers, and is associated with poor prognosis in some indications. Ly6E overexpression is associated with poor overall survival of pancreatic ductal adenocarcinoma and renal papillary cell carcinoma. Increased expression of Ly6E is also detected in various gastrointestinal carcinomas, such as gastric, colorectal and pancreatic cancer.

[0003] Given the overexpression of Ly6E in various cancers, including in cancers with limited therapeutic options, it is an attractive target for treatment of cancer. However, there are no clinically approved therapeutics that target Ly6E for treatment of cancer. Antibody-drug conjugates (ADCs) combine the tumor-targeting of antibodies with the cytotoxicity of their payload drugs for treatment of cancer. ADCs represent one of the most rapidly expanding anticancer treatment modalities and have gained therapeutic success in clinic for both solid and hematological malignancies.

[0004] Camptothecin analogues have been developed as payloads for ADCs. Two such ADCs have been approved for treatment of cancer. Trastuzumab deruxtecan (Enhertu™) in which the camptothecin analogue, deruxtecan (Dxd), is conjugated to the anti-HER2 antibody, trastuzumab, via a cleavable tetrapeptide-based linker, and sacituzumab govitecan (Trodelvy™) in which the1IPTS / 200345621.2camptothecin analogue, SN-38, is conjugated to the anti-Trop-2 antibody, sacituzumab, via a hydrolysable, pH-sensitive linker.

[0005] Other camptothecin analogues and derivatives, as well as ADCs comprising them have been described. See, for example, International (PCT) Application Publication Nos. WO 2019 / 195665, WO 2019 / 236954, WO 2020 / 200880, WO 2020 / 219287 and WO 2022 / 246576.

[0006] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present disclosure available. No admission is necessarily intended, nor should it be construed, that any of the preceding information constitutes prior art against the claimed invention.SUMMARY

[0007] Described herein are anti-Ly6E antibody constructs, anti-Ly6E antibody-drug conjugates and their methods of use. One aspect of the present disclosure relates to an antibody construct comprising an antigen-binding domain that binds to human lymphocyte antigen 6 complex, locus E, protein (Ly6E), wherein the antigen-binding domain comprises complementarity determining region (CDR) sequences of a heavy chain variable domain (VH) comprising a sequence as set forth in any one of SEQ ID NOs: 4, 30, 31, 32, 33, 34, 35, 39, 40, 41 or 42, and CDR sequences of a light chain variable domain (VL) comprising a sequence as set forth in any one of SEQ ID NOs: 5, 36, 37 or 38.

[0008] Another aspect of the present disclosure relates to a polynucleotide or set of polynucleotides encoding an antibody construct that binds to human Ly6E as described herein.

[0009] Another aspect of the present disclosure relates to an expression vector or set of expression vectors comprising a polynucleotide or set of polynucleotides encoding an antibody construct that binds to human Ly6E as described herein.

[0010] Another aspect of the present disclosure relates to a host cell comprising a polynucleotide or set of polynucleotides encoding an antibody construct that binds to human Ly6E as described herein or an expression vector or set of expression vectors comprising the polynucleotide or set of polynucleotides.

[0011] Another aspect of the present disclosure relates to a method of preparing an antibody construct that binds to human Ly6E as described herein comprising transfecting a host cell with a polynucleotide or set of polynucleotides encoding the antibody construct or an expression vector2IPTS / 200345621.2or set of expression vectors comprising the polynucleotide or set of polynucleotides, and culturing the host cell under conditions suitable for expression of the antibody construct.

[0012] Another aspect of the present disclosure relates to an antibody-drug conjugate comprising an antibody construct that binds to human Ly6E as described herein conjugated to between 2 and about 8 drug moieties.

[0013] Another aspect of the present disclosure relates to an antibody-drug conjugate having the formula: A-(L-(D)m)n, wherein A is an antibody construct that binds to human Ly6E as described herein; L is a linker; D is a drug moiety; m is between 1 and about 8; and n is between 1 and about 12.

[0014] Another aspect of the present disclosure relates to a method of preparing an antibodydrug conjugate as described herein comprising conjugating drug-linker L-(D)mto an antibody construct that binds to human Ly6E as described herein.

[0015] Another aspect of the present disclosure relates to an antibody-drug conjugate having the structure:

[0016] wherein n is about 8; and A is an antibody construct comprising an antigen-binding domain that binds to human lymphocyte antigen 6 complex, locus E, protein (Ly6E), wherein the antigen-binding domain comprises complementarity determining region (CDR) sequences of a heavy chain variable domain (VH) comprising a sequence as set forth in SEQ ID NO: 42 and CDR sequences of a light chain variable domain (VL) comprising a sequence as set forth in SEQ ID NO: 38.

[0017] Another aspect of the present disclosure relates to a method of preparing an antibodydrug conjugate as described herein comprising conjugating a drug -linker having the structure:3IPTS / 200345621.2

[0018] to an antibody construct that binds to human Ly6E as described herein (A), wherein the drug-linker is conjugated to native cysteine residues of the antibody construct A.

[0019] Another aspect of the present disclosure relates to a pharmaceutical composition comprising an antibody construct that binds to human Ly6E as described herein, or an antibodydrug conjugate as described herein, and a pharmaceutically acceptable carrier or diluent.

[0020] Another aspect of the present disclosure relates to an antibody construct that binds to human Ly6E as described herein or an antibody-drug conjugate as described herein for use in therapy.

[0021] Another aspect of the present disclosure relates to a use of an antibody construct that binds to human Ly6E as described herein or an antibody-drug conjugate as described herein in the manufacture of a medicament for treatment of cancer.

[0022] Another aspect of the present disclosure relates to a method of inhibiting the growth of tumor cells comprising contacting the cells with an antibody construct that binds to human Ly6E as described herein or an antibody-drug conjugate as described herein.

[0023] Another aspect of the present disclosure relates to a method of treating a subject having a cancer comprising administering to the subject an effective amount of an antibody construct that binds to human Ly6E as described herein or an antibody-drug conjugate as described herein.

[0024] Another aspect of the present disclosure relates to a kit comprising an antibody construct that binds to human Ly6E as described herein or an antibody-drug conjugate as described herein, and a label and / or package insert containing instructions for use.

[0025] Another aspect of the present disclosure relates to a method of detecting human lymphocyte antigen 6 complex, locus E, protein (Ly6E) in a biological sample comprising contacting the biological sample with an antibody construct that binds to human Ly6E as described herein under conditions that allow for binding of the antibody construct to human Ly6E and4IPTS / 200345621.2detecting formation of a complex between the antibody construct and the human Ly6E in the biological sample.BRIEF DESCRIPTION OF DRAWINGS

[0026] FIG. 1A-C depict the binding of parental chimeric anti-Ly6E antibody variant v38866, benchmark antibody hu9B12.v12 (v33706) and palivizumab (v22277) to human Ly6E (FIG. 1A), cynomolgus monkey Ly6E (FIG. 1B) and mouse Ly6E (FIG. 1C).

[0027] FIG. 2A-B show the amino acid sequences of heavy chain CDRs (defined by AbM numbering) of the chimeric antibody v38866 ported onto a selected VH human germline framework (IGHV3-23*01) (SEQ ID NO: 30) (FIG. 2A) and light chain CDRs (defined by AbM numbering) of the chimeric antibody v38866 ported onto a selected VL human germline framework (IGKV1-39*O1) (SEQ ID NO: 36) (FIG. 2B).

[0028] FIG. 3 shows the Caliper electrophoresis profiles under non-reducing (-NR) conditions and reducing (-R) conditions for representative humanized anti-Ly6E antibody variants v38811, v38812 and v38820.

[0029] FIG. 4A-B depict the binding of parental chimeric anti-Ly6E antibody variant v38866, representative humanized anti-Ly6E antibody variants, benchmark antibody hu9B12.v12 (v33706) and palivizumab (v22277) to human Ly6E (FIG. 4A) and to cynomolgus monkey Ly6E (FIG.4B)

[0030] FIG.5A-B show the Caliper electrophoresis profiles under non-reducing (-NR) conditions and reducing (-R) conditions for humanized anti-Ly6E antibody variants v39333, v39334, v39335 and v39336 (FIG. 5A) and variants v39336 and v41252 (FIG. 5B).

[0031] FIG. 6A-D depict the binding of parental chimeric anti-Ly6E antibody variant v38866, representative humanized anti-Ly6E antibody variants, benchmark antibody hu9B12.v12 (v33706) and palivizumab (v22277 or v21995) to human Ly6E in transfected cells (FIG. 6A), to cynomolgus monkey Ly6E in transfected cells (FIG.6B), to human Ly6E in stable cell lines (FIG.6C), and to cynomolgus monkey Ly6E in stable cell lines (FIG. 6D).

[0032] FIG. 7A-E depict the binding of optimized, humanized anti-Ly6E antibody variants v39336 and v41252, benchmark antibody hu9B12.v12 (v33706) and palivizumab (v21995) to human Ly6E in HCC1569 (FIG. 7A), MX-1 (FIG. 7B), H1650 (FIG. 7C), H446 (FIG. 7D), and SK-CO-1 (FIG. 7E) cancer cell lines.5IPTS / 200345621.2

[0033] FIG. 8A-B depict the internalization of optimized, humanized anti-Ly6E antibody variants v39336 and v41252, benchmark antibody hu9B12.v12 (v33706) and palivizumab (v21995) in stably transfected HEK cells over-expressing human Ly6E or cynomolgus monkey Ly6E (FIG. 8A) and in Ly6E expressing cancer cell lines HCC1569 and NCI-H1650 (FIG. 8B).

[0034] FIG. 9A-B depict the N-curve analysis of binding of optimized, humanized anti-Ly6E antibody variant v39336 to human Ly6E over-expressed on HEK cells (FIG. 9A) and to cynomolgus monkey Ly6E over-expressed on HEK cells (FIG. 9B). For each panel, the left curve shows the data for 5nM constant binding partner, and the right curve shows the data for 0.5 nM constant binding partner.

[0035] FIG. 10A-C depict the spheroid penetration ability of optimized, humanized anti-Ly6E antibody variant v41252 (FIG. 10A), benchmark anti-Ly6E antibody hu9B12.v12 (v33706) (FIG.10B) and palivizumab (v21995) (FIG. 10C) in Ly6E expressing spheroid cancer cell line RT 112 / 84.

[0036] FIG. 11 shows the pharmacokinetic profile of optimized, humanized anti-Ly6E antibody variants v39333 and v39336, and benchmark antibody hu9B12.v12 (v33706) in hFcRn Tg32 mice (n=4).

[0037] FIG. 12A-S depict the cytotoxicity of anti-Ly6E ADCs v39336-DLl and v41252-DLl, benchmark antibody hu9B12.v12 ADC v33706-DL1 and non-targeted antibody palivizumab ADC v21995-DLl in 2D monolayer cultures of breast carcinoma cells lines MX1 (FIG. 12A) and DU4475 (FIG. 12B), colorectal adenocarcinoma cell line SK-CO-1 (FIG. 12C), gastric adenocarcinoma cell lines MKN74 (FIG. 12D), MKN7 (FIG. 12E), SNU-216 (FIG. 12F) and AGS (FIG. 12G), pancreatic adenocarcinoma cell lines BxPC3 (FIG. 12H), PK-8 (FIG. 12I), Panc 03.27 (FIG. 12J), SU.86.86 (FIG. 12K), PK-59 (FIG. 12L), HPAF-II (FIG. 12M), and Capan-1 (FIG. 12N), tongue carcinoma cell line CAL27 (FIG. 12O), head and neck squamous carcinoma cell lines SCC-9 (FIG. 12P), DETROIT-562 (FIG. 12Q), FaDu (FIG. 12R) and RPMI2650 (FIG. 12S).

[0038] FIG. 13A-R depict the cytotoxicity of anti-Ly6E ADCs v39336-DLl and v41252-DLl, benchmark antibody hu9B12.v12 ADC v33706-DL1 and non-targeted antibody palivizumab ADC v21995-DLl in 3D spheroids of HCC1569 breast carcinoma (FIG. 13A), MX-1 breast carcinoma (FIG. 13B), H1650 lung adenocarcinoma (FIG. 13C), DU4475 breast carcinoma (FIG. 13D), H446 lung carcinoma (FIG. 13E), SK-CO-1 colorectal adenocarcinoma (FIG. 13F), MKN746IPTS / 200345621.2gastric adenocarcinoma (FIG. 13G), N87 gastric carcinoma (FIG. 13H), AGS gastric adenocarcinoma (FIG. 13I), Panc 03.27 pancreatic adenocarcinoma (FIG. 13J), Capan-1 pancreatic adenocarcinoma (FIG. 13K), FaDu head and neck squamous carcinoma (FIG. 13L), RPMI2650 head and neck squamous carcinoma (FIG. 13M), RT112 / 84 bladder carcinoma (FIG.13N), Caov-3 ovarian adenocarcinoma (FIG. 13O), COV362 ovarian carcinoma (FIG. 13P) and TE-8 esophageal squamous carcinoma (FIG. 13Q) cell lines. FIG. 13R depicts pIC50 values of anti-Ly6E antibody-drug ADCs v39336-DLl, v41252-DLl and v39336-DL3, and benchmark antibody hu9B12.v12 ADCs v33706-DL1 and v33706-DL3 in various cancer cell 3D spheroids.

[0039] FIG. 14A-D depict the payload release of anti-Ly6E ADCs v39336-DL1 and v41252-DL1, and non-targeted antibody palivizumab ADC v21995-DL1 in HCC1569 breast carcinoma (FIG. 14A (intracellular) and FIG. 14C (extracellular)) and RT112 / 84 bladder carcinoma (FIG.14B (intracellular) and FIG. 14D (extracellular)) cell lines.

[0040] FIG. 15 depicts the bystander activity of anti-Ly6E ADCs v39336-DL1 and v41252-DL1 against a Ly6E-negative SK-CO-1-mCherry cell line. V39336-DL3 was used as a positive control. v21995-DLl and v21995-DL3 were used as non-targeted controls.

[0041] FIG. 16A-B depict the ability of anti-Ly6E antibody variants v39336 and v41252, and palivizumab (v21995) to mediate antibody-dependent cellular toxicity (ADCC) (FIG. 16A) and antibody-dependent cellular phagocytosis (ADCP) (FIG. 16B) in Ly6E expressing RT112 / 84 cancer cell line. FIG. 16C-D depict the ability of anti-Ly6E antibody variants v39336 and v41252, and palivizumab (v21995) to mediate complement-dependent cytotoxicity (CDC) in human Ly6E-overexpressing HEK cells in the presence of human serum (FIG. 16C) and in the absence of human serum (FIG. 16D).

[0042] FIG. 17A-D depict the anti-tumor activity of anti-Ly6E ADCs v39336-DL1 and v39336-DL3, benchmark antibody hu9B12.v12 ADC v33706-DL3 and non-targeted antibody palivizumab ADC v21995-DL1 in NCI-H446 (FIG. 17A), SW-900 (FIG. 17B), NCI-H1650 (FIG. 17C) and MX-1 (FIG. 17D) xenograft models in a first CDX study. FIG. 17E-F depict the anti-tumor activity of anti-Ly6E antibody-drug conjugate v39336-DLl in MX-1 (FIG. 17E) and NCI-H446 (FIG. 17F) xenograft models in a second CDX study.

[0043] FIG. 18A-C depict the pharmacokinetic profile of anti-Ly6E ADCs v39336-DL1 and v39336-DL3, benchmark antibody hu9B12.v12 ADC v33706-DL3 and non-targeted antibody palivizumab ADC v21995-DL1 in MX-1 (FIG. 18A), NCI-H446 (FIG. 18B) and NCI-H16507IPTS / 200345621.2(FIG. 18C) xenograft models in a first CDX study. FIG. 18D-E depict the pharmacokinetic profile of anti-Ly6E ADC v39336-DLl in MX-1 (FIG. 18D) and NCI-H446 (FIG. 18E) xenograft models in a second CDX study.

[0044] FIG. 19A and FIG. 19D depict the anti-tumor activity of anti-Ly6E ADC v41252-DL1 in DU4475 (FIG. 19A) and NCI-H228 (FIG. 19D) xenograft models in a third CDX study. FIG.19B-C and FIG. 19E depict the anti-tumor activity of anti-Ly6E ADCs v39336-DLl and v41252-DL1 in MX-1 (FIG. 19B), NCI-H446 (FIG. 19C) and NCI-H1650 (FIG. 19E) xenograft models in a third CDX study.

[0045] FIG. 20A-B depict the pharmacokinetic profile of anti-Ly6E ADCs v39336-DL1 and v41252-DL1 in MX-1 (FIG. 20A) and NCI-H446 (FIG. 20B) xenograft models in a third CDX study. FIG. 20C-D depict the pharmacokinetic profile of anti-Ly6E ADC v41252-DLl in NCI-H228 (FIG. 20C) and DU4475 (FIG. 20D) xenograft models in a third CDX study.

[0046] FIG. 21A-F depict the anti-tumor activity of anti-Ly6E ADC v41252-DL1, anti-Ly6E antibody variant v41252 and non-targeted antibody palivizumab ADC v43209-DL1 in BR1282 (FIG.21A), LU0876 (FIG.21B), LU2071 (FIG.21C), LU2512 (FIG.21D), BR0438 (FIG.21E) and LU6918 (FIG. 21F) xenograft models in a PDX study.

[0047] FIG. 22 depicts the total antibody concentration time-plot for anti-Ly6E ADC v41252-DL1 in cynomolgus monkeys.

[0048] FIG. 23 lists the CDR sequences of humanized VH and VL sequences and optimized, humanized VH sequences comprised by anti-Ly6E antibody variants disclosed herein.

[0049] FIG. 24A-B depict the binding of optimized humanized anti-Ly6E antibody variant v41252 and the ADC v41252-DLl (DAR 8) to human Ly6E in PK-8 (FIG. 24A) and SW480 (FIG. 24B) cancer cell lines.

[0050] FIG. 25 depicts the internalization of optimized humanized anti-Ly6E antibody variant v41252 and ADC v41252-DL1 (DAR 8) in Ly6E expressing cancer cell lines PK-8 and SW480. Non-targeted antibody palivizumab v43209 was used as a control.

[0051] FIG. 26A-D depict the spheroid penetration ability of optimized humanized anti-Ly6E antibody variant v41252 (FIG. 26A), ADC v41252-DL1 (DAR 8) (FIG. 26B) and palivizumab (v21995) (FIG. 26C) in Ly6E expressing spheroid cancer cell line RT112 / 84.

[0052] FIG. 27A-C depict the cytotoxicity of anti-Ly6E ADCs v41252-DL1 (DAR 8), v39336-DL3 (DAR 4), v33706-DL1 (DAR 8), v33706-DL3 (DAR 4), and non-targeted antibody8IPTS / 200345621.2palivizumab ADC v21995-DL1 (DAR 8) in 3D spheroids of HT-29 (FIG. 27A), SW480 (FIG.27B), and HCT116 (FIG. 27C) colorectal adenocarcinoma cells.

[0053] FIG. 28A-L depict the anti-tumor activity of anti-Ly6E ADC v41252-DL1, anti-Ly6E antibody variant v41252 and non-targeted antibody palivizumab ADC v43209-DL1 in BxPC3 (FIG. 28A), Capanl (FIG. 28B), Detroit562 (FIG. 28C), FaDu (FIG. 28D), HCT-116 (FIG.28E), HPAFII (FIG. 28F), MKN45 (FIG. 28G), NCI-N87 (FIG. 28H), HT-29 (FIG. 281), COLO205 (FIG. 28 J), KYSE-30 (FIG. 28K) and KYSE-150 (FIG. 28L) xenograft models in a CDX study.

[0054] FIG.29A-E depict the pharmacokinetic profile ofthe anti-Ly6E ADC v41252-DL1, anti-Ly6E antibody variant v41252 and non-targeted antibody palivizumab ADC v43209-DL1 in BxPC3 (FIG. 29A), Capanl (FIG. 29B), HPAFII (FIG. 29C), MKN45 (FIG. 29D), and NCI-N87 (FIG. 29E) CDX models.

[0055] FIG.30A-F depict the pharmacokinetic profile of the anti-Ly6E ADC v41252-DL1, anti-Ly6E antibody variant v41252 and non-targeted antibody palivizumab ADC v43209-DL1 in BR1282 (FIG. 30A), BR0438 (FIG. 30B), LU0876 (FIG. 30C), LU2071 (FIG. 30D), LU2512 (FIG. 30E), and LU6918 (FIG. 30F) PDX models.

[0056] FIG. 31A-B depict the unconjugated payload (Compound 1) (FIG. 31A) and antibody-conjugated payload (FIG. 31B) serum concentrations after administration of 30 or 60 mg / kg anti-Ly6E ADC v41252-DL1 (DAR 8) in cynomolgus monkeys.DETAILED DESCRIPTION

[0057] The present disclosure relates to antibody constructs that bind to Lymphocyte antigen 6 complex, locus E (Ly6E). In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure are capable of binding to human Ly6E. In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure are also capable of binding to cynomolgus monkey Ly6E.

[0058] The present disclosure also relates to antibody-drug conjugates (ADCs) comprising an anti-Ly6E antibody construct as described herein conjugated to a drug, such as a cytotoxin or an immune modulator. In certain embodiments, the ADCs of the present disclosure comprise an anti-Ly6E antibody construct conjugated to a camptothecin analogue of Formula (II) as described herein.9IPTS / 200345621.2

[0059] The anti-Ly6E antibody constructs and ADCs of the present disclosure may find use, for example, as therapeutics or diagnostics. Certain aspects of the present disclosure relate to therapeutic methods and uses of the anti-Ly6E antibody constructs and ADCs, for example, in the treatment of cancer. Some aspects relate to diagnostic methods and uses of the anti-Ly6E antibody constructs and ADCs, for example, in the diagnosis or analysis of cancer.Definitions

[0060] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0061] As used herein, the term “about” refers to an approximately + / - 10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

[0062] Where a range of values is provided herein, for example where a value is defined as being “between” an upper limit value and a lower limit value, it is understood that the range encompasses both the upper limit value and the lower limit value as well as each intervening value.

[0063] The use of the word “a” or “an” when used herein in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one” and “one or more than one.”

[0064] The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), and antibody fragments as long as they bind to the desired epitope or antigen.

[0065] The terms “anti-Ly6E antibody” or “an antibody that binds to Ly6E” refers to an antibody that is capable of binding to Ly6E with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting Ly6E.

[0066] The term “antibody construct” refers to a polypeptide or a set of polypeptides that comprises one or more antigen-binding domains, where each of the one or more antigen-binding domains specifically binds to an epitope or antigen. Where the antibody construct comprises two or more antigen-binding domains, each of the antigen-binding domains may bind the same epitope or antigen (i.e. the antibody construct is monospecific) or they may bind to different epitopes or antigens (i.e. the antibody construct is bispecific or multispecific). The antibody construct may10IPTS / 200345621.2further comprise a scaffold and the one or more antigen-binding domains can be fused or covalently attached to the scaffold, optionally via a linker, as described herein.

[0067] An “antibody that competes with” a reference antibody refers to an antibody that blocks binding of the reference antibody to its epitope in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its epitope in a competition assay by 50% or more.

[0068] The term “chimeric” antibody refers to an antibody which comprises at least one variable domain from a non-human source or species, such as a rabbit or rodent (for example, murine) antibody, and at least one constant domain from a human antibody. The human constant domain of a chimeric antibody need not be of the same isotype as the non-human constant domain it replaces. Chimeric antibodies are discussed, for example, in Morrison et al., 1984, Proc. Natl. Acad. Se USA, 81:6851-55, and U. S. Patent No. 4,816,567.

[0069] As used herein, the terms “comprising,” “having,” “including” and “containing,” and grammatical variations thereof, are inclusive or open-ended and do not exclude additional, unrecited elements and / or method steps. The term “consisting essentially of’ when used herein in connection with a composition, use or method, denotes that additional elements and / or method steps may be present, but that these additions do not materially affect the manner in which the recited composition, method or use functions. The term “consisting of’ when used herein in connection with a composition, use or method, excludes the presence of additional elements and / or method steps. A composition, use or method described herein as comprising certain elements and / or steps may also, in certain embodiments consist essentially of those elements and / or steps, and in other embodiments consist of those elements and / or steps, whether or not these embodiments are specifically referred to.

[0070] A “complementarity determining region” or “CDR” is an amino acid sequence that contributes to antigen-binding specificity and affinity. “Framework” regions (FR) can aid in maintaining the proper conformation of the CD Rs to promote binding between the antigen-binding region and an antigen. From N-terminus to C-terminus, both the light chain variable region (VL) and the heavy chain variable region (VH) of an antibody typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three heavy chain CD Rs are referred to herein as HCDR1, HCDR2, and HCDR3, and the three light chain CDRs are referred to herein as LCDR1, LCDR2, and LCDR3. CDRs provide the majority of contact residues for the binding of the11IPTS / 200345621.2antibody to the antigen or epitope. Often, the three heavy chain CDRs and the three light chain CDRs are required to bind antigen. However, in some instances, even a single variable domain can confer binding specificity to the antigen. Furthermore, as is known in the art, in some cases, antigen-binding may also occur through a combination of a minimum of one or more CDRs selected from the VH and / or VL domains, for example HCDR3.

[0071] A number of different definitions of the CDR sequences are in common use, including those described by Kabat et al., 1983, Sequences of Proteins of Immunological Interest, NIH Publication No. 369-847, Bethesda, MD, by Chothia et al., 1987, J Mol Biol, 196:901-917, as well as the IMGT (Lefranc et al., 1997, Immunol Today, 18:509), AbM (Abhinandan et al., 2008, A / o / . Immunol, 45:3832-3839) and Contact (MacCallum et al., 1996, J Mol Biol, 262(5):732-745) definitions. By way of example, CDR definitions according to Kabat, Chothia, IMGT, AbM and Contact are provided in Table 1. Accordingly, as would be readily apparent to one skilled in the art, the exact numbering and placement of CDRs may differ based on the numbering system employed. (Dondelinger etal., 2018, Front Immunol, 9:2278; Zhu etal., 2024, Antibodies, 13, 99). However, it is to be understood that the disclosure herein of a VH includes the disclosure of the associated (inherent) heavy chain CDRs (HCDRs) as defined by any of the known numbering systems. Similarly, the disclosure herein of a VL includes the disclosure of the associated (inherent) light chain CDRs (LCDRs) as defined by any of the known numbering systems.Table 1: Common CDR Definitions1Heavy Chain Light Chain DefinitionCDR12CDR2 CDR3 CDR1 CDR2 CDR3 Kabat H31-H35B H50-H65 H95-H102 L24-L34 L50-L56 L89-L97 H26-H32,Chothia H33 or H52-H56 H96-H101 L26-L32 L50-L52 L91-L96 H34H26- IMGT H51-H56 H93-H102 L27-L32 L50-L51 L89-L97 H35BAbM H26-H35B H50-H58 H95-H102 L24-L34 L50-L56 L89-L97 Contact H30-H35B H47-H58 H93-H101 L30-L36 L46-L55 L89-L961As would be readily apparent to one skilled in the art, some of these definitions (particularly for Chothia) vary depending on the individual publication examined.2The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop because the Kabat numbering scheme places insertions at H35A and H35B.

[0072] A “comparison window” refers to a segment of a sequence comprising contiguous amino acid or nucleotide positions which may be, for example, from about 10 to 600 contiguous amino acid or nucleotide positions, or from about 10 to about 200, or from about 10 to about 150 contiguous amino acid or nucleotide positions over which a test sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are known to those of ordinary skill in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, 1970, Adv. Appl. Math., 2:482c; by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol., 48:443; by the search for similarity method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85:2444, or by computerized implementations of these algorithms (for example, GAP, BESTFIT, FASTA or TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI), or by manual alignment and visual inspection (see, for example, Ausubel et al., 1995 supplement, Current Protocols in Molecular Biology, Cold Spring Harbor Laboratory Press). Examples of available algorithms suitable for determining percent sequence identity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1997, Nuc. Acids Res., 25:3389-3402, and Altschul et al., 1990, J. Mol. Biol., 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the website for the National Center for Biotechnology Information (NCBI).

[0073] A “Fab fragment” contains the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CHI) along with the variable domains of the light and heavy chains (VL and VH, respectively). Fab' fragments differ from Fab fragments by the addition of a few amino acid residues at the C-terminus of the heavy chain CHI domain, including one or more cysteines from the antibody hinge region. A Fab fragment may also be a single-chain Fab molecule, i.e. a Fab molecule in which the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain. For example, the C-terminus of the Fab light chain may be connected to the N-terminus of the Fab heavy chain in the single-chain Fab molecule.

[0074] The terms “Fc region,” “Fc” or “Fc domain” as used herein refer to a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The termincludes native sequence Fc regions and variant Fc regions, comprising one or more amino acid modifications relative to a corresponding native or wildtype Fc region. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD.

[0075] An “Fc polypeptide” in the context of a dimeric Fc refers to one of the two polypeptides forming the dimeric Fc domain, i. e. a polypeptide comprising one or more C-terminal constant regions of an immunoglobulin heavy chain that is capable of stable self-association. When referring to a dimeric Fc region, the terms “first Fc polypeptide” and “second Fc polypeptide” may be used interchangeably provided that the Fc region comprises one first Fc polypeptide and one second Fc polypeptide.

[0076] A “humanized antibody” refers to a type of chimeric antibody which comprises minimal amino acid residues derived from a non-human antibody. Generally, humanized antibodies are human immunoglobulins (recipient antibody) in which amino acid residues from a hypervariable region of the recipient are replaced by amino acid residues from a hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit or non-human primate, having the desired specificity and affinity for a target antigen. This technique for creating humanized antibodies is often referred to as “CDR grafting.” A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergone humanization.

[0077] The term “identical” in the context of two or more polynucleotide or polypeptide sequences, refers to two or more sequences or subsequences that are the same. Sequences are “substantially identical” if they have a percentage of amino acid residues or nucleotides that are the same (for example, about 80%, about 85%, about 90%, about 95%, or about 98% identity, over a specified region) when compared and aligned for maximum correspondence over a comparison window or over a designated region as measured using one of the commonly used sequence comparison algorithms as known to persons of ordinary skill in the art or by manual alignment and visual inspection. For sequence comparison, typically test sequences are compared to a designated reference sequence. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or14IPTS / 200345621.2alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0078] The terms “nucleic acid,” “nucleic acid molecule” and “polynucleotide” are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogues thereof. Non-limiting examples of polynucleotides include a gene, a gene fragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.

[0079] The term “operably linked,” as used herein, means that the components described are in a relationship permitting them to function in their intended manner.

[0080] The term “parenteral” as used herein includes subcutaneous injection, and intradermal, intra-articular, intravenous, intramuscular, intravascular, intrastemal or intrathecal injection or infusion.

[0081] An “scFv” includes a heavy chain variable domain (VH) and a light chain variable domain (VL) of an antibody in a single polypeptide chain. The scFv may optionally further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form a desired structure for antigen binding. For example, an scFv may include a VL connected from its C-terminus to the N-terminus of a VH by a polypeptide linker. Alternately, an scFv may comprise a VH connected through its C-terminus to the N-terminus of a VL by a polypeptide linker (see review in Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)).

[0082] An “sdAb” format refers to a single immunoglobulin domain. The sdAb may be, for example, of camelid origin. Camelid antibodies lack light chains and their antigen-binding sites consist of a single domain, termed a “VHH.” An sdAb comprises three CDR / hypervariable loops that form the antigen-binding site: CDR1, CDR2 and CDR3. sdAbs are fairly stable and easy to express, for example, as a fusion with the Fc chain of an antibody (see, for example, Harmsen & De Haard (2007, Appl. Microbiol Biotechnol., 71(1):13-22).

[0083] The term “specifically binds,” as used herein, refers to an affinity between an antibody, an antibody construct, or a fragment thereof and an antigen or epitope in which the dissociation constant (KD or Ka) value is 10'6M or less.15IPTS / 200345621.2

[0084] The term “subject,” as used herein, refers to an animal, in some embodiments a mammal, which is the object of treatment, observation or experiment. The animal may be a human, a nonhuman primate, a companion animal (for example, dog, cat, or the like), farm animal (for example, cow, sheep, pig, horse, or the like) or a laboratory animal (for example, rat, mouse, guinea pig, non-human primate, or the like). In certain embodiments, the subject is a human.

[0085] It is contemplated that any embodiment discussed herein can be implemented with respect to any method, use or composition disclosed herein, and vice versa.

[0086] Particular features, structures and / or characteristics described in connection with an embodiment disclosed herein may be combined with features, structures and / or characteristics described in connection with another embodiment disclosed herein in any suitable manner to provide one or more further embodiments.

[0087] It is also to be understood that the positive recitation of a feature in one embodiment, serves as a basis for excluding the feature in an alternative embodiment. For example, where a list of options is presented for a given embodiment or claim, it is to be understood that one or more option may be deleted from the list and the shortened list may form an alternative embodiment, whether or not such an alternative embodiment is specifically referred to.ANTI-LY6E ANTIBODY CONSTRUCTS

[0088] Certain embodiments of the present disclosure relate to antibody constructs that bind to Lymphocyte antigen 6 complex, locus E (Ly6E).

[0089] In accordance with the present disclosure, the anti-Ly6E antibody construct comprises at least one antigen-binding domain that specifically binds to human Ly6E. In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure may also be capable of binding to Ly6E from one or more non-human species. In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure are capable of binding to both human Ly6E and cynomolgus monkey Ly6E.

[0090] Human Ly6E is also known as Retinoic Acid Induced Gene E (RIGE), Stem Cell Antigen 2 (SCA-2) or Thymic Shared Antigen 1 (TSA-1). The protein sequences of Ly6E from various sources are known in the art and readily available from publicly accessible databases, such as GenBank or UniProtKB. An exemplary human Ly6E protein sequence is provided in Table 2 as SEQ ID NO: 1 (Uniprot Q16553). An exemplary cynomolgus monkey Ly6E protein sequence16IPTS / 200345621.2(SEQ ID NO: 2; Uniprot I7G3A4) and mouse Ly6E protein sequence (SEQ ID NO: 3; Uniprot Q64253) are also provided in Table 2.Table 2: Amino Acid Sequences of Human, Cynomolgus Monkey and Mouse Ly6E SEQ IDSpecies SequenceNO LMCFSCLNQKSNLYCLKPTICSDQDNYCVTVSASAGIGNLHuman VTFGHSLSKTCSPACPIPEGVNVGVASMGISCCQSFLCNFS 1 AADGGLRASVTLLGAGLLLSLLPALLRFGP LMCFSCLNQKSNLYCLKPTICSDQDNYCVTVSTSAGIGNL CynomolgusVTFGHSLSKTCSPACPLPEGINVGVASMGISCCQSFLCNFS 2 monkeyAADGGLRASATLLGAGLLLSLLPALLRFGP LMCFSCTDQKNNINCLWPVSCQEKDHYCITLSAAAGFGNMouse VNLGYTLNKGCSPICPSENVNLNLGVASVNSYCCQSSFCN 3FSAAGLGLRASIPLLGLGLLLSLLALLQLSP

[0091] Binding of an antigen-binding domain to a target antigen or epitope may be measured, for example, through an enzyme-linked immunosorbent assay (ELISA), a surface plasmon resonance (SPR) technique (employing, for example, a Biacore® system) (Liljeblad et al., 2000, Glyco J, 17:323-329), a solution-based kinetic exclusion assay (KinExA™), flow cytometry or a traditional binding assay (Heeley, 2002, Endocr Res, 28:217-229). In certain embodiments, the extent of binding of an anti-Ly6E antibody construct to a non-target protein (here, a non-Ly6E protein) is less than about 10% of the binding of the anti-Ly6E antibody construct to Ly6E as measured by commonly known methods in the art, such as ELISA or flow cytometry.

[0092] In certain embodiments, binding of an antibody construct to human Ly6E may be expressed by a dissociation constant (KD or Kd). KD between an antibody and its antigen can be determined using methods well established in the art. One method for determining such KD is by using surface plasmon resonance (SPR), typically using a biosensor system such as a Biacore® system. Isothermal titration calorimetry (ITC) is another method that can be used to measure KD. The Octet™ system and the KinExA™ system may also be used to measure the affinity of antibodies for a target antigen.

[0093] In certain embodiments, an antibody construct that binds to Ly6E has a dissociation constant (KD) of <500 nM, for example, <250 nM, <100 nM, <50 nM, <10 nM or <5 nM. In certain embodiments, an antibody construct that binds to Ly6E has a dissociation constant (KD) of 10'6Mor less, for example, 10'7M or less, or 10'8M or less, or 10'9M or less. The data provided in the Examples illustrate this general point. In certain embodiments, KD is determined by cellular binding. In certain embodiments, KD is determined in a cell-based assay. As is known in the art the numerical value of the KD obtained may vary depending on how it is tested. For example, the numerical value of KD when measured in a cell-based assay may be affected by the expression level of the target antigen (in this case, Ly6E) in the cell line, format of the antibody construct (i.e. monovalent or bivalent) and / or type of assay (for example, flow cytometry or KinExA™).

[0094] In some embodiments, the anti-Ly6E antibody constructs of the present disclosure have a KD that is lower than that of the benchmark antibody hu9B12.vl2 (see, U. S. Patent No.9,724,427; see also Asundi et al., 2015, Clin Cancer Res,' 21(14):3252-62; Tolaney et al., 2020 Clin Cancer Res,' 26(21): 5588-5597) when measured by flow cytometry in cells transfected with human or cynomolgus monkey Ly6E. Accordingly, in these embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an antigen-binding domain having an affinity for Ly6E that is higher than the benchmark antibody hu9B12.vl2. In some embodiments, the anti-Ly6E antibody constructs of the present disclosure have a KD that is lower than that of the benchmark antibody hu9B12.vl2, when measured by flow cytometry in human endogenous Ly6E-expressing cells (i.e., Ly6E-positive cells). Accordingly, in these embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an antigen-binding domain having an affinity for Ly6E that is higher than the benchmark antibody hu9B 12.vl2.

[0095] In certain embodiments, the anti-Ly6E constructs of the present disclosure exhibit greater internalization than the benchmark antibody hu9B12.vl2 in stably transfected cells overexpressing human Ly6E or over-expressing cyno Ly6E as well as in Ly6E-expressing cancer cell lines. Antibody internalization may be measured using methods known in the art, for example, by a direct internalization method according to the protocol detailed in Schmidt et al., 2008, Cancer Immunol. Immunother., 57:1879-1890, or using commercially available fluorescent dyes such as the pHAb Dyes (Promega Corporation, Madison, WI), pHrodo iFL, Deep Red Dyes, and Incucyte® Fabfluor-pH Antibody Labeling Reagent (Sartorius AG, Gottingen, Germany), or commercially available fluorescent antibody conjugates, such as anti-human IgG Fab fragment Alexa Fluor®488 conjugate (Jackson ImmunoRe search Labs) and analysis techniques such as microscopy, FACS, high content imaging or other plate-based assays.

[0096] In some embodiments, the fluorescently labeled anti-Ly6E constructs of the present disclosure display high signal within the Ly6E-expressing spheroid inner layers as well as the spheroid center of Ly6E-expressing spheroid cell lines, indicating efficient spheroid penetration. The fluorescently labeled benchmark anti-Ly6E antibody hu9B12.vl2 shows some signal at the spheroid outer layers but low signal at the inner layers and spheroid center of Ly6E-expressing spheroid cell lines, indicating poor spheroid penetration ability. Accordingly, in these embodiments, the anti-Ly6E constructs of the present disclosure displays superior spheroid penetration over the benchmark anti-Ly6E antibody hu9B12.vl2.

[0097] Ly6E expression varies depending on cell type as indicated throughout the disclosure and the level of Ly6E expression can be referred to herein as “high”, “mid,” “low” or “negative.” These terms are used for reference to describe levels of expression in general according to the designations shown in Table 8.1 in Example 8 and are not intended to be limited to specific numerical values for average Ly6E protein per cell. Alternatively, expression level of LyE6 in cells or tumors may be assessed by immunohistochemistry (IHC) according to methods known in the art. For example, IHC may be used to stain for Ly6E in tumor tissue samples from xenograft models, cell-derived (CDX) or patient-derived (PDX). Tissue samples may be examined, and an H-score may be calculated as known in the art. The higher the H-score, the higher the expression of Ly6E in the tissue sample.Antigen Binding Domains

[0098] The anti-Ly6E antibody constructs of the present disclosure comprise at least one antigenbinding domain that is capable of binding to human Ly6E. In certain embodiments, the at least one antigen-binding domain specifically binds to human Ly6E (an “anti-Ly6E antigen-binding domain”). The at least one anti-Ly6E antigen-binding domain typically is an immunoglobulin-based binding domain, such as an antigen-binding antibody fragment. Examples of an antigenbinding antibody fragment include, but are not limited to, a Fab fragment, a Fab’ fragment, a single chain Fab (scFab), a single chain Fv (scFv) and a single domain antibody (sdAb).

[0099] In those embodiments in which the anti-Ly6E antibody constructs comprise two or more antigen-binding domains, each additional antigen-binding domain may independently be an immunoglobulin-based domain, such as an antigen-binding antibody fragment, or a non-immunoglobulin-based domain, such as a non-immunoglobulin-based antibody mimetic, or other polypeptide or small molecule capable of specifically binding to its target, for example, a natural 19IPTS / 200345621.2or engineered ligand. Non-immunoglobulin-based antibody mimetic formats include, for example, anticalins, fynomers, affimers, alphabodies, DARPins and avimers.

[0100] The present disclosure describes herein the identification of a rabbit antibody that binds to human Ly6E and also binds to cynomolgus monkey Ly6E. A rabbit-human chimeric variant of this antibody, variant 38866, is described herein. The anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain derived from this rabbit-human chimeric antibody or humanized antibody variants of same. Representative humanized antibody variants (e.g., v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826, v38827, v39333, v39334, v39335, v39336 and v41252) of the rabbit anti-Ly6E antibody are also described (see Examples and Sequence Tables). In certain embodiments, the anti-Ly6E antibody constructs described herein specifically bind human Ly6E having the sequence as set forth in SEQ ID NO: 1.

[0101] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain comprising heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) of any one of variants v38866, v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826, v38827, v39333, v39334, v39335, v39336 or v41252 as defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems.

[0102] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) selected from the heavy chain CDR amino acid sequences of any one of variants v38866, v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826, v38827, v39333, v39334, v39335, v39336 or v41252 as defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems, and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) selected from the light chain CDR amino acid sequences of any one of variants v38866, v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826, v38827, v39333, v39334, v39335, v39336 or v41252 as defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems.20IPTS / 200345621.2

[0103] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain having:(i) an HCDR1 amino acid sequence selected from the HCDR1 amino acid sequences of any one of variants v38866, v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826, v38827, v39333, v39334, v39335, v39336 or v41252; an HCDR2 amino acid sequence selected from the HCDR2 amino acid sequences of any one of variants v38866, v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826, v38827, v39333, v39334, v39335, v39336 or v41252; and an HCDR3 amino acid sequence selected from the HCDR3 amino acid sequences of any one of variants v38866, v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826, v38827, v39333, v39334, v39335, v39336 or v41252, and(ii) an LCDR1 amino acid sequence selected from the LCDR1 amino acid sequences of any one of variants v38866, v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826, v38827, v39333, v39334, v39335, v39336 or v41252; an LCDR2 amino acid sequence selected from the LCDR2 amino acid sequences of any one of variants v38866, v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826, v38827, v39333, v39334, v39335, v39336 or v41252; and an LCDR3 amino acid sequence selected from the LCDR3 amino acid sequences of any one of variants v38866, v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826, v38827, v39333, v39334, v39335, v39336 or v41252, where the CDR amino acid sequences are as defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems (see, e.g., Table 4.1; Table 6.2; FIG. 23).

[0104] Analysis of the CDR sequences from the parental and humanized anti-Ly6E antibody variants described herein identified a consensus sequence for each CDR when defined by the AbM numbering systems (see Table 3a). In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain comprising an HCDR1 comprising the sequence as set forth in SEQ ID NO: 17; an HCDR2 comprising the sequence as set forth in SEQ ID NO: 94; a HCDR3 comprising the sequence as set forth in SEQ ID NO: 14;21IPTS / 200345621.2an LCDR1 comprising the sequence as set forth in SEQ ID NO: 95; an LCDR2 comprising the sequence as set forth in SEQ ID NO: 26, and an LCDR3 comprising the sequence as set forth in SEQ ID NO: 24.Table 3a: CDR Consensus Sequences by AbM NumberingCDR Sequence Variable (X) SEQ ID NOHeavy ChainHCDR1 GFDFSSNTIY 17HCDR2 XIYYGDGSTY X = C, S, V, T or A 94HCDR3 DFKL 14Light ChainLCDR1 XSSESVYNNNWLA X = Q or R 95LCDR2 MASILAS 26LCDR3 QGSYLSSGWYFT 24

[0105] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise at least one anti-Ly6E antigen-binding domain, where the antigen-binding domain comprises a set of CDRs based on the CDRs of parental chimeric antibody v38866 described herein. The CDR sequences of the parental chimeric antibody v38866 are shown in Table 4.1.

[0106] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise at least one anti-Ly6E antigen-binding domain, where the antigen-binding domain comprises a set of CDRs based on the CDRs of the humanized antibody v39336 described herein. The CDR sequences of the humanized antibody v39336 are shown in Table 6.2.

[0107] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 17, 44 and 14, and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 48, 26, and 24.

[0108] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 17, 18 and22IPTS / 200345621.214, and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 25, 26, and 24.

[0109] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 17, 18 and 14, and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 48, 26, and 24.

[0110] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 17, 73 and 14, and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 25, 26, and 24.

[0111] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 17, 73 and 14, and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 48, 26, and 24.

[0112] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 17, 81 and 14, and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 48, 26, and 24.

[0113] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 17, 85 and 14, and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 48, 26, and 24.

[0114] One skilled in the art will appreciate that a limited number of amino acid substitutions may be introduced into the CDR sequences or into the VH or VL sequences of known antibodies without the antibody losing its ability to bind its target. Candidate amino acid substitutions may be identified by computer modeling or by art-known techniques such as alanine scanning, with the23IPTS / 200345621.2resulting variants being tested for binding activity by standard techniques. Accordingly, in certain embodiments, the anti-Ly6E antibody constructs of the present disclosure may comprise an anti-Ly6E antigen-binding domain that comprises a variant of the set of CDR sequences (i.e. heavy chain HCDR1, HCDR2 and HCDR3, and light chain LCDR1, LCDR2 and LCDR3) that have 90% or greater, 95% or greater, 98% or greater, 99% or greater, or 100% sequence identity to a set of CDRs of any one of variants v38866, v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826, v38827, v39333, v39334, v39335, v39336 or v41252, where the % sequence identity is calculated across all six CDRs and where the antigen-binding domain retains the ability to bind human Ly6E.

[0115] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain that comprises a variant of the set of CDR sequences of any one of variants v38866, v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826, v38827, v39333, v39334, v39335, v39336 or v41252, where the variant comprises between 1 and 10 amino acid substitutions across the set of CDRs (i.e. the CDRs may be modified by up to 10 amino acid substitutions with any combination of the six CDRs being modified), and where the antigen-binding domain retains the ability to bind human Ly6E. In some embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain that comprises a variant of the set of CDR sequences of any one of variants v38866, v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826, v38827, v39333, v39334, v39335, v39336 or v41252, where the variant comprises between 1 and 9 amino acid substitutions, between 1 and 8 amino acid substitutions, between 1 and 7 amino acid substitutions, between 1 and 6 amino acid substitutions, between 1 and 5 amino acid substitutions, between 1 and 4 amino acid substitutions, between 1 and 3 amino acid substitutions, between 1 and 2 amino acid substitutions, or 1 amino acid substitution, across the set of CDRs, and where the antigen-binding domain retains the ability to bind Ly6E.

[0116] In some embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise the VH and VL sequences of any one of v38866, v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826, v38827, v39333, v39334, v39335, v39336 or v41252. The SEQ ID NOs: of theVH and VL sequences of these variants are provided in Table 3b. The sequences are provided in Table 1.1, Table 4.2 and Table 6.1 of the Examples, as well as in the Sequence Tables.Table 3b: VH and VL Sequences of Parental Chimeric and Humanized Anti-Ly6E Antibody ConstructsVariant VH Sequence VL Sequence Number (SEQ ID NO:) (SEQ ID NO:) v38866 4 5v38810 30 36v38811 30 37v38812 30 38v38813 31 36v38814 31 37v38815 31 38v38816 32 36v38817 32 37v38818 32 38v38819 33 36v38820 33 37v38821 33 38v38822 34 36v38823 34 37v38824 34 38v38825 35 36v38826 35 37v38827 35 38v39333 39 37v39334 40 38v39335 41 38v39336 42 38v41252 42 38

[0117] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain that comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence of any one of variants v38866, v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826, v38827, v39333, v39334, v39335, v39336 or v41252, where the antigen-binding domain retains the ability to bind human Ly6E. In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain that comprises a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence of any one of variants v38866, v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826, v38827, v39333, v39334, v39335, v39336 or v41252, where the antigen-binding domain retains the ability to bind human Ly6E.

[0118] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain comprising a VH amino acid sequence selected from the VH amino acid sequences of any one of variants v38866, v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826, v38827, v39333, v39334, v39335, v39336 or v41252. In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain comprising a VL amino acid sequence selected from the VL amino acid sequences of any one of variants v38866, v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826, v38827, v39333, v39334, v39335, v39336 or v41252.

[0119] In some embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain comprising the VH sequence and the VL sequence of v38866. In some embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain comprising the VH sequence and the VL sequence of v38812. In some embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain comprising the VH sequence and the VL sequenceof v39336. In some embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain comprising the VH sequence as set forth in SEQ ID NO: 42, and the VL sequence as set forth in SEQ ID NO: 38.

[0120] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain that comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence of v38866, and a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence of v38866, where the antigen-binding domain retains the ability to bind human Ly6E.

[0121] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain that comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence of v38812, and a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence of v38812, where the antigen-binding domain retains the ability to bind human Ly6E.

[0122] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain that comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence of v39336, and a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence of v39336, where the antigen-binding domain retains the ability to bind human Ly6E.

[0123] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain that comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set27IPTS / 200345621.2forth in SEQ ID NO: 42, and a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 38, where the antigen-binding domain retains the ability to bind human Ly6E.

[0124] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain comprising the CDR sequences of the VH sequence of any one of variants v38866, v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826, v38827, v39333, v39334, v39335, v39336 or v41252. In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an antigen-binding domain comprising the CDR sequences of the VL sequence of any one of variants v38866, v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826, v38827, v39333, v39334, v39335, v39336 orv41252.

[0125] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an anti-Ly6E antigen-binding domain comprising a) a VH sequence having the HCDR1, HCDR2 and HCDR3 sequences of v38866 and having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the VH sequence of v38866, and b) a VL sequence having the LCDR1, LCDR2 and LCDR3 sequences of v38866 and having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the VL sequence of v38866, wherein the HCDRs and LCDRs are defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems, and where the antigen-binding domain retains the ability to bind Ly6E.

[0126] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an antigen -binding domain comprising a) a VH sequence having the HCDR1, HCDR2 and HCDR3 sequences of v38812 and having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the VH sequence of v41252, and b) a VL sequence having the LCDR1, LCDR2 and LCDR3 sequences of v38812 and having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the VL sequence of v41252, wherein the HCDRs and LCDRs are defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems, and where the antigen-binding domain retains the ability to bind Ly6E.

[0127] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise an antigen -binding domain comprising a) a VH sequence having the HCDR1, HCDR2 and HCDR3 sequences of v39336 and having at least at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the VH sequence of v39336, and b) a VL sequence having the LCDR1, LCDR2 and LCDR3 sequences of v39336 and having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the VL sequence of v39336, wherein the HCDRs and LCDRs are defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems, and where the antigen-binding domain retains the ability to bind Ly6E.

[0128] In certain embodiments, the anti-Ly6E antibody construct of the present disclosure comprises two heavy chains having the amino acid sequence as set forth in SEQ ID NO: 87 and two light chains having the amino acid sequence as set forth in SEQ ID NO: 68.

[0129] In certain embodiments, the anti-Ly6E antibody construct of the present disclosure comprises two heavy chains having (a) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO: 87 and (b) two light chains having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO:68, where the anti-Ly6E antibody construct retains the ability to bind human Ly6E.

[0130] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure may comprise a silenced (or “knock out”) Fc region as described elsewhere herein (herein also referred to as anti-Ly6E Fc silenced antibody construct or as an anti-Ly6E Fc knock-out antibody construct). In some such embodiments, the anti-Ly6E antibody construct of the present disclosure comprises two heavy chains having the amino acid sequence as set forth in SEQ ID NO: 88 and two light chains having the amino acid sequence as set forth in SEQ ID NO:68.

[0131] In certain embodiments, the anti-Ly6E Fc silenced antibody construct of the present disclosure comprises two heavy chains having (a) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO:88 and (b) two light chains having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO: 68,29IPTS / 200345621.2where the anti-Ly6E Fc silenced antibody construct retains the ability to bind human Ly6E and has a reduced binding to all Fey receptors.Formats

[0132] The anti-Ly6E antibody constructs of the present disclosure may have various formats. The minimal component of the anti-Ly6E antibody construct is an antigen-binding domain that binds to human Ly6E. The anti-Ly6E antibody constructs may further optionally comprise one or more additional antigen-binding domains and / or a scaffold. In those embodiments in which the anti-Ly6E antibody construct comprises two or more antigen-binding domains, each additional antigen-binding domain may independently bind to the same epitope within human Ly6E, may bind to a different epitope within human Ly6E, or may bind to a different antigen. Thus, the anti-Ly6E antibody construct may be, for example, monospecific, biparatopic, bispecific or multispecific.

[0133] In certain embodiments, the anti-Ly6E antibody construct of the present disclosure comprises at least one antigen-binding domain that binds to human Ly6E and a scaffold, where the antigen-binding domain is operably linked to the scaffold. Examples of suitable scaffolds are described below.

[0134] In certain embodiments, the anti-Ly6E antibody construct of the present disclosure comprises two antigen-binding domains optionally operably linked to a scaffold. In some embodiments, the anti-Ly6E antibody construct of the present disclosure may comprise three or four antigen-binding domains and optionally a scaffold. In these formats, when comprising a scaffold, at least a first antigen-binding domain is operably linked to the scaffold and the remaining antigen-binding domain(s) may each independently be operably linked to the scaffold or to the first antigen-binding domain or, when more than two antigen-binding domains are present, to another antigen-binding domain.

[0135] The anti-Ly6E antibody constructs of the present disclosure that lack a scaffold may comprise a single antigen-binding domain in an appropriate format, such as an sdAb, or they may comprise two or more antigen-binding domains optionally operably linked by one or more linkers. In such anti-Ly6E antibody constructs, the antigen-binding domains may be in the form of scFvs, Fabs, sdAbs, or a combination thereof. For example, using scFvs as the antigen-binding domains, formats such as a tandem scFv ((scFv)2 or taFv) may be constructed, in which the scFvs are connected together by a flexible linker. scFvs may also be used to construct diabody formats,30IPTS / 200345621.2which comprise two scFvs connected by a short linker (usually about 5 amino acids in length). The restricted length of the linker results in dimerization of the scFvs in a head-to-tail manner. In any of the preceding formats, the scFvs may be further stabilized by inclusion of an interdomain disulfide bond. For example, a disulfide bond may be introduced between VL and VH through introduction of an additional cysteine residue in each chain (for example, at position 44 in VH and position lOO inVL) (see, for example, Fitzgerald etal., 1997, Protein Engineering, 10:1221-1225), or a disulfide bond may be introduced between two VHs to provide a construct having a DART format (see, for example, Johnson et al., 2010, J Mol. Biol., 399:436-449).

[0136] Similarly, formats comprising two sdAbs, such as VHs or VHHs, connected together through a suitable linker may be employed in some embodiments. Other examples of anti-Ly6E antibody construct formats that lack a scaffold include those based on Fab fragments, for example, Fab2 and F(ab’)2 formats, in which the Fab fragments are connected through a linker or an IgG hinge region.

[0137] Combinations of antigen-binding domains in different formats may also be employed to generate alternative scaffold-less formats. For example, an scFv or a sdAb may be fused to the C-terminus of either or both of the light and heavy chain of a Fab fragment resulting in a bivalent (Fab-scFv / sdAb) construct.

[0138] In certain embodiments, the anti-Ly6E antibody construct of the present disclosure may be in an antibody format that is based on an immunoglobulin (Ig). In certain embodiments, the anti-Ly6E antibody construct may be based on an IgG class immunoglobulin, for example, an IgGl, IgG2, IgG3 or IgG4 immunoglobulin. In some embodiments, the anti-Ly6E antibody construct may be based on an IgGl immunoglobulin. In the context of the present disclosure, when an anti-Ly6E antibody construct is based on a specified immunoglobulin isotype, it is meant that the anti-Ly6E antibody construct comprises all or a portion of the constant region of the specified immunoglobulin isotype. For example, an anti-Ly6E antibody construct based on a given Ig isotype may comprise at least one antigen-binding domain operably linked to an Ig scaffold, where the scaffold comprises an Fc region from the given isotype and optionally an Ig hinge region from the same or a different isotype. It is to be understood that the anti-Ly6E antibody constructs may also comprise hybrids of isotypes and / or subclasses in some embodiments. It is also to be understood that the Fc region and / or hinge region may optionally be modified to impart one or more desirable functional properties as is known in the art. Thus, in certain embodiments, the anti-Ly6E antibody construct of the present disclosure comprises a VH amino acid sequence fused to IgGl constant domain amino acid sequences (i.e., CHI, hinge, CH2, CH3 amino acid sequences) and a VL amino acid sequence fused to kappa or lambda constant domain amino acid sequences (i.e., CL amino acid sequences). Exemplary amino acid sequences are provided in the Examples and Sequence Tables.

[0139] In some embodiments, the anti-Ly6E antibody constructs of the present disclosure may be derived from two or more immunoglobulins that are from different species, for example, an anti-Ly6E antibody construct of the present disclosure may be a chimeric antibody or a humanized antibody. The terms “chimeric antibody” and “humanized antibody” both refer generally to antibodies that combine immunoglobulin regions or domains from more than one species.

[0140] In some instances, additional modifications may be made to a humanized antibody to further refine antibody performance. For example, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues, or the humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody. In general, a variable domain in a humanized antibody will comprise all or substantially all of the hypervariable regions from a non-human immunoglobulin and all or substantially all of the FRs from a human immunoglobulin sequence. Humanized antibodies are described in more detail in Jones etal., 1986, Nature, 321:522-525; Riechmann etal., 1988, Nature, 332:323-329, and Presta, 1992, Curr. Op. Struct. Biol., 2:593-596, for example.

[0141] A number of approaches are known in the art for selecting the most appropriate human frameworks in which to graft the non-human CDRs. Early approaches used a limited subset of well-characterised human antibodies, irrespective of the sequence identity to the non-human antibody providing the CDRs (the “fixed frameworks” approach). More recent approaches have employed variable regions with high amino acid sequence identity to the variable regions of the non-human antibody providing the CDRs (“homology matching” or “best-fit” approach). An alternative approach is to select fragments of the framework sequences within each light or heavy chain variable region from several different human antibodies. CDR-grafting may in some cases result in a partial or complete loss of affinity of the grafted molecule for its target antigen. In such cases, affinity can be restored by back-mutating some of the residues of human origin to the corresponding non-human ones. Methods for preparing humanized antibodies by these approaches are well-known in the art (see, for example, Humanization of Monoclonal Antibodies, Tsurushita32IPTS / 200345621.2& Vasquez, 2004, Molecular Biology of B Cells, 533-545, Elsevier Science USA); Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-329; Presta et al., 1997, Cancer Res, 57(20):4593-4599).

[0142] Alternatively, or in addition to, these traditional approaches, other technologies may be employed to further reduce the immunogenicity of a CDR-grafted humanized antibody. For example, frameworks based on human germline sequences or consensus sequences may be employed as acceptor human frameworks rather than human frameworks with somatic mutation(s). Another technique that aims to reduce the potential immunogenicity of non-human CDRs is to graft only specificity-determining residues (SDRs). In this approach, only the minimum CDR residues required for antigen-binding activity (the “SDRs”) are grafted into a human germline framework. This method improves the “humanness” (i.e. the similarity to human germline sequence) of the humanized antibody and thus may help reduce the risk of immunogenicity of the variable region. These techniques have been described in various publications (see, for example, Almagro & Fransson, 2008, Front Biosci, 13:1619-1633; Tan et al., 2002, J Immunol, 169: 1119-1125; Hwang et al., 2005, Methods, 36:35-42; Pelat et al., 2008, J Mol Biol, 384: 1400-1407; Tamura etal., 2000, J Immunol, 164:1432-1441; Gonzales etal., 2004, Mol Immunol, 1:863-872; and Kashmiri et al., 2005, Methods, 36:25-34).

[0143] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise humanized antibody sequences, for example, one or more humanized variable domains. In some embodiments, the anti-Ly6E antibody construct can be a humanized antibody. Nonlimiting examples of humanized antibody variants based on the anti-Ly6E antibody variant v38866 are described herein (see Examples and Sequence Tables for v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826, v38827, v39333, v39334, v39335, v39336 and v41252.)Scaffolds

[0144] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise one or more antigen-binding domains operably linked to a scaffold. The antigen-binding domain(s) may be in one or a combination of the formats described above (for example, scFvs, Fabs and / or sdAbs). Examples of suitable scaffolds are described in more detail below and include, but are not limited to, immunoglobulin Fc regions, albumin, albumin analogues and derivatives, heterodimerizing peptides (such as leucine zippers, heterodimer-forming “zipper” peptides derivedfrom Jun and Fos, IgG CHI and CL domains or bamase -barstar toxins), cytokines, chemokines or growth factors. Other examples include antibodies based on the DOCK-AND-LOCK™ (DNL™) technology developed by IBC Pharmaceuticals, Inc. and Immunomedics, Inc. (see, for example, Chang et al., 2007, Clin. Cancer Res., 13:5586s-5591s).

[0145] A scaffold may be a peptide, polypeptide, polymer, nanoparticle or other chemical entity. Where the scaffold is a polypeptide, each antigen-binding domain of the anti-Ly6E antibody construct may be linked to either the N- or C-terminus of the polypeptide scaffold. Anti-Ly6E antibody construct comprising a polypeptide scaffold in which one or more of the antigen-binding domains are linked to a region other than the N- or C-terminus, for example, via the side chain of an amino acid with or without a linker, are also contemplated in certain embodiments.

[0146] In embodiments where the anti-Ly6E antibody construct comprises a scaffold that is a peptide or polypeptide, the antigen-binding domain(s) may be linked to the scaffold by genetic fusion or chemical conjugation. Typically, when the scaffold is a peptide or polypeptide, the antigen-binding domain(s) are linked to the scaffold by genetic fusion. In some embodiments, where the scaffold is a polymer or nanoparticle, the antigen-binding domain(s) may be linked to the scaffold by chemical conjugation.

[0147] A number of protein domains are known in the art that comprise selective pairs of two different polypeptides and may be used to form a scaffold. An example is leucine zipper domains such as Fos and Jun that selectively pair together (Kostelny et al., 1992, J Immunol, 148: 1547-5; Wranik et al., 2012, J. Biol. Chem., 287:43331-43339). Other selectively pairing molecular pairs include, for example, the bamase-barstar pair (Deyev et al., 2003, Nat Biotechnol, 21: 1486-1492), DNA strand pairs (Chaudri et al., 1999, FEBS Letters, 450( 1— 2):23-26) and split fluorescent protein pairs (International Patent Application Publication No. WO 2011 / 135040).

[0148] Other examples of protein scaffolds include immunoglobulin Fc regions, albumin, albumin analogues and derivatives, toxins, cytokines, chemokines and growth factors. The use of protein scaffolds in combination with antigen-binding moieties has been described (see, for example, Muller et al., 2007, J. Biol. Chem., 282:12650-12660; McDonaugh et al., 2012, Mol. Cancer Ther., 11:582-593; Vallera et al., 2005, Clin. Cancer Res., 11:3879-3888; Song et al., 2006, Biotech. Appl. Biochem., 45:147-154, and U. S. Patent Application Publication No.2009 / 0285816.

[0149] For example, fusing antigen-binding moieties such as scFvs, diabodies or single chain diabodies to albumin has been shown to improve the serum half-life of the antigen-binding moieties (Muller et al., ibid.). Antigen-binding moieties may be fused at the N- and / or C-termini of albumin, optionally via a linker. Derivatives of albumin in the form of heteromultimers that comprise two transporter polypeptides obtained by segmentation of an albumin protein such that the transporter polypeptides self-assemble to form quasi-native albumin have been described (see International Patent Application Publication Nos. WO 2012 / 116453 and WO 2014 / 012082). As a result of the segmentation of albumin, the heteromultimer includes four termini and thus can be fused to up to four different antigen-binding moieties, optionally via linkers.

[0150] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure may comprise a protein scaffold. In some embodiments, the anti-Ly6E antibody constructs of the present disclosure may comprise a protein scaffold that is based on an immunoglobulin Fc region, an albumin or an albumin analogue or derivative. In some embodiments, the anti-Ly6E antibody constructs of the present disclosure may comprise a protein scaffold that is based on an immunoglobulin Fc region, for example, an IgG Fc region.Fc Regions

[0151] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure may comprise a scaffold that is based on an immunoglobulin Fc region (or Fc domain). The Fc region may be dimeric and composed of two Fc polypeptides or alternatively, the Fc region may be composed of a single polypeptide.

[0152] An Fc region may comprise a CH3 domain, or it may comprise both a CH3 and a CH2 domain. For example, in certain embodiments of the anti-Ly6E antibody constructs of the present disclosure, an Fc polypeptide of a dimeric IgG Fc region may comprise an IgG CH2 domain sequence and an IgG CH3 domain sequence. In such embodiments, the CH3 domain comprises two CH3 sequences, one from each of the two Fc polypeptides of the dimeric Fc region, and the CH2 domain comprises two CH2 sequences, one from each of the two Fc polypeptides of the dimeric Fc region.

[0153] In some embodiments, the anti-Ly6E antibody constructs of the present disclosure may comprise a scaffold that is based on an IgG Fc region. In some embodiments, the anti-Ly6E antibody construct may comprise a scaffold that is based on a human IgG Fc region. In some embodiments, the anti-Ly6E antibody construct may comprise a scaffold based on an IgG I Fc 35IPTS / 200345621.2region. In some embodiments, the anti-Ly6E antibody construct may comprise a scaffold based on a human IgGl Fc region.

[0154] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure may comprise a scaffold based on an IgG Fc region, which is a homodimeric Fc region, comprising a first Fc polypeptide and a second Fc polypeptide, each comprising a CH3 sequence, and optionally a CH2 sequence, and in which the amino acid sequences of the first and second Fc polypeptides are the same.

[0155] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure may comprise a scaffold based on an IgG Fc region, which is a heterodimeric Fc region, comprising a first Fc polypeptide and a second Fc polypeptide, each comprising a CH3 sequence, and optionally a CH2 sequence and in which the first and second Fc polypeptides are different. In some embodiments, the anti-Ly6E antibody constructs may comprise a scaffold based on an Fc region which comprises two CH3 sequences, at least one of which comprises one or more amino acid modifications. In some embodiments, the anti-Ly6E antibody constructs may comprise a scaffold based on an Fc region which comprises two CH3 sequences and two CH2 sequences, and at least one of the CH2 sequences comprising one or more amino acid modifications.

[0156] In some embodiments, the anti-Ly6E antibody construct may comprise a heterodimeric Fc region comprising a modified CH3 domain, where the modified CH3 domain is an asymmetrically modified CH3 domain comprising one or more asymmetric amino acid modifications. As used herein, an “asymmetric amino acid modification” refers to a modification, such as a substitution or an insertion, in which an amino acid at a specific position on a first CH3 or CH2 sequence is different to the amino acid on a second CH3 or CH2 sequence at the same position. These asymmetric amino acid modifications can be a result of modification of only one of the two amino acids at the same respective amino acid position on each sequence, or different modifications of both amino acids at the same respective position on each of the first and second CH3 or CH2 sequences. Each of the first and second CH3 or CH2 sequences of a heterodimeric Fc may comprise one or more than one asymmetric amino acid modification.

[0157] In some embodiments, the anti-Ly6E antibody constructs of the present disclosure may comprise a heterodimeric Fc region comprising a modified CH3 domain, where the modified CH3 domain comprises one or more amino acid modifications that promote formation of the36IPTS / 200345621.2heterodimeric Fc region over formation of a homodimeric Fc region. In some embodiments, one or more of the amino acid modifications are asymmetric amino acid modifications.

[0158] Amino acid modifications that may be made to the CH3 domain of an Fc in order to promote formation of a heterodimeric Fc are known in the art and include, for example, those described in International Publication No. WO 96 / 027011 (“knobs into holes”); Gunasekaran et al., 2010, J Biol Chem, 285, 19637-46 (“electrostatic steering”); Davis et al., 2010, Prot Eng Des Sei, 23(4): 195-202 (strand exchange engineered domain (SEED) technology) and Labrijn et al., 2013, Proc Natl Acad Sci USA, 110(13):5145-50 (Fab-arm exchange). Other examples include approaches combining positive and negative design strategies to produce stable asymmetrically modified Fc regions as described in International Publication Nos. WO 2012 / 058768 and WO 2013 / 063702. In certain embodiments, the anti-Ly6E antibody construct may comprise a scaffold based on a modified Fc region as described in International Publication No. WO 2012 / 058768 or WO 2013 / 063702.

[0159] Table 4 provides the amino acid sequence of the human IgGl Fc sequence (SEQ ID NO: 93), corresponding to amino acids 231 to 447 of the full-length human IgGl heavy chain. The CH3 sequence comprises amino acids 341-447 of the full-length human IgGl heavy chain. Also shown in Table 4 are CH3 domain amino acid modifications that promote formation of a heterodimeric Fc as described in in International Patent Application Publication Nos. WO 2012 / 058768 and WO 2013 / 063702.

[0160] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure may comprise a heterodimeric Fc scaffold having a modified CH3 domain comprising the modifications of any one of Variant 1, Variant 2, Variant 3, Variant 4 or Variant 5, as shown in Table 4.Table 4: Human IgGl Fc Sequence and CH3 Domain Amino Acid Modifications Promoting Heterodimer Formation1APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 93)Variant No Chain Mutations1 A L351Y_F405A_Y407V37IPTS / 200345621.2B T366L_K392M_T394WA L351Y_F405A_Y407V2B T366L_K392L_T394WA T350V_L351Y_F4O5A_Y4O7V3B T350V_T366L_K392L_T394WA T350V_L351Y_F4O5A_Y4O7V4B T350V_T366L_K392M_T394WA T350V_L351Y_S4OOE_F4O5A_Y4O7V5B T350V_T366L_N390R_K392M_T394W'Sequence from positions 231-447 (EU Numbering)

[0161] In some embodiments, the anti-Ly6E antibody construct may comprise a scaffold based on an Fc region comprising two CH3 sequences and two CH2 sequences, at least one of the CH2 sequences comprising one or more amino acid modifications. Modifications in the CH2 domain can affect the binding of Fc receptors (FcRs) to the Fc, such as receptors of the FcyRI, FcyRII and FcyRIII subclasses.

[0162] In some embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise a scaffold based on an IgG Fc having a modified CH2 domain, wherein the modification of the CH2 domain results in altered binding to one or more of the FcyRI, FcyRII and FcyRIII receptors.

[0163] A number of amino acid modifications to the CH2 domain that selectively alter the affinity of the Fc for different Fey receptors are known in the art. Amino acid modifications that result in increased binding and amino acid modifications that result in decreased binding can each be useful in certain indications. For example, increasing binding affinity of an Fc for FcyRIIIa (an activating receptor) may result in increased antibody dependent cell-mediated cytotoxicity (ADCC), which in turn results in increased lysis of the target cell. Decreased binding to FcyRIIb (an inhibitory receptor) likewise may be beneficial in some circumstances. In certain indications, a decrease in, or elimination of, ADCC and complement-mediated cytotoxicity (CDC) may be desirable. In such cases, modified CH2 domains comprising amino acid modifications that result in increased binding to FcyRIIb or amino acid modifications that decrease or eliminate binding of the Fc region to all of the Fey receptors (“knock-out” or “Fc silenced” variants) may be useful.38IPTS / 200345621.2

[0164] Examples of amino acid modifications to the CH2 domain that alter binding of the Fc to Fey receptors include, but are not limited to, the following: S298A / E333A / K334A and S298A / E333A / K334A / K326A (increased affinity for FcyRIIIa) (Lu et al., 2011, J Immunol Methods, 365(1-2): 132-41); F243L / R292P / Y300L / V305EP396L (increased affinity for FcyRIIIa) (Stavenhagen et al., 2007, Cancer Res, 67(18):8882-90); F243L / R292P / Y300L / L235V / P396L (increased affinity for FcyRIIIa) (Nordstrom et al., 2011, Breast Cancer Res, 13(6): R123); F243L (increased affinity for FcyRIIIa) (Stewart et al., 2011, Protein Eng Des Sei., 24(9):671-8); S298A / E333A / K334A (increased affinity for FcyRIIIa) (Shields et al., 2001, J Biol Chem, 276(9):6591-604); S239D / I332E / A330L and S239D / I332E (increased affinity for FcyRIIIa) (Lazar et al., 2006, Proc Natl Acad Sci USA, 103(11):4005-10), and S239D / S267E and S267E / L328F (increased affinity for FcyRIIb) (Chu et al., 2008, Mol Immunol, 45(15):3926-33). Various amino acid modifications to the CH2 domain that alter binding of the Fc by FcyRIIb are described in International Patent Application Publication No. WO 2021 / 232162. Additional modifications that affect Fc binding to Fey receptors are described in Strohl & Strohl (2012, Therapeutic Antibody Engineering, Woodhead Publishing series in Biomedicine No 11, ISBN 1 907568 37 9, page 283).

[0165] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure comprise a scaffold based on an IgG Fc having a modified CH2 domain, in which the modified CH2 domain comprises one or more amino acid modifications that result in decreased or eliminated binding of the Fc region to all of the Fey receptors (i.e. a “knock-out” variant).

[0166] Various publications describe strategies that have been used to engineer antibodies to produce “knock-out” variants (see, for example, Strohl, 2009, Curr Opin Biotech 20:685-691, and Strohl & Strohl, 2012, " Antibody Fc engineering for optimal antibody performance" in Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing, pp 225-249). These strategies include reduction of effector function through modification of glycosylation, use of IgG2 / IgG4 scaffolds, or the introduction of mutations in the hinge or CH2 domain of the Fc (see also, U. S. Patent Application Publication No. 2011 / 0212087, International Patent Application Publication No. WO 2006 / 105338, U. S. Patent Application Publication No. 2012 / 0225058, U. S. Patent Application Publication No. 2012 / 0251531 and Strop et al., 2012, Mol. Biol., 420: 204-219).

[0167] Examples of mutations that may be introduced into the hinge or CH2 domain to produce a “knock-out” or “Fc silenced” variant with a reduced effector function include, but are not limited to, the amino acid modifications L234A / L235A (LALA), and L234A / L235A / D265S (LALADS). LALA substitutions in IgGl antibodies have been shown to reduce binding of the antibody to FcyRs (Wilkinson et al., 2021, PLoS One; 16(12)), thus disrupting, but not completely knocking out, the ability of these antibodies to interact with certain cells in tissues. In general, antibodies having an Fc region comprising the LALADS modifications are Fc-silenced and have a reduced effector function; specifically, they exhibit reduced or no ADCC (antibody-dependent cellular cytotoxicity), ADCP (antibody-dependent cellular phagocytosis) and CDC (complementdependent cytotoxicity) activity as compared to antibody having a wild-type Fc. The LALADS modifications are described in International Patent Application Publication No. WO 2014 / 012085.

[0168] Effector function may also be increased or decreased by modifying glycosylation of an Fc, as is known in the art. For example, mutation of the conserved asparagine residue at position 297 to alanine, glutamine, lysine or histidine (i.e. N297A, Q, K or H) results in an aglycosylated Fc that lacks all effector function (Bolt et al., 1993, Eur. J. Immunol., 23:403-411; Tao & Morrison, 1989, J. Immunol., 143:2595-2601). Conversely, removal of fucose from heavy chain N297-linked oligosaccharides has been shown to enhance ADCC, based on improved binding to FcyRIIIa (see, for example, Shields et al., 2002, J Biol Chem., 277:26733 -26740, and Niwa et al., 2005, J. Immunol. Methods, 306:151-160). Such low fucose antibodies may be produced, for example in knockout Chinese hamster ovary (CHO) cells lacking fucosyltransferase (FUT8) (Yamane-Ohnuki et al., 2004, Biotechnol. Bioeng., 87:614-622); in the variant CHO cell line, Lee 13, that has a reduced ability to attach fucose to N297-linked carbohydrates (International Patent Application Publication No. WO 03 / 035835), or in other cells that generate afucosylated antibodies (see, for example, Li et al., 2006, Nat Biotechnol, 24:210-215; Shields et al., 2002, ibid, and Shinkawa et al., 2003, J. Biol. Chem., 278:3466-3473). In addition, International Patent Application Publication No. WO 2009 / 135181 describes the addition of fucose analogues to culture medium during antibody production to inhibit incorporation of fucose into the carbohydrate on the antibody. Other methods of producing antibodies with little or no fucose on the Fc glycosylation site (N297) are well known in the art. For example, the GlymaX® technology (ProBioGen AG) (see von Horsten etal., 2010, Glycobiology, 20(12): 1607-1618 and U. S. Patent No. 8,409,572).

[0169] Other glycosylation variants include those with bisected oligosaccharides, for example, variants in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by N-acetylglucosamine (GlcNAc). Such glycosylation variants may have reduced fucosylation and / or improved ADCC function (see, for example, International Patent Application Publication No. WO 2003 / 011878, U. S. Patent No. 6,602,684 and U. S. Patent Application Publication No. US 2005 / 0123546). Useful glycosylation variants also include those having at least one galactose residue in the oligosaccharide attached to the Fc region, which may have improved CDC function (see, for example, International Patent Application Publication Nos. WO 1997 / 030087, WO 1998 / 58964 and WO 1999 / 22764).

[0170] In certain embodiments, the anti-Uy6E antibody constructs of the present disclosure may comprise a scaffold based on an IgG Fc in which native glycosylation has been modified.

[0171] In certain embodiments, the anti-Uy6E antibody constructs of the present disclosure have the format of a full-size antibody (FSA). In some embodiments, the anti-Ly6E antibody constructs have the format of an IgG FSA, for example, an IgGl FSA. In some embodiments, the anti-Ly6E antibody construct is a FSA comprising a first heavy chain sequence (HC1), a second heavy chain sequence (HC2), a first light chain sequence (LC1) and a second light chain sequence (LC2). In some embodiments, the anti-Ly6E antibody construct is a monospecific FSA with a homodimeric Fc and comprises HC1, HC2, LC1 and LC2 sequences, where HC1 and HC2 have the same amino acid sequence, and LC1 and LC2 have the same amino acid sequence. In some embodiments, the anti-Ly6E antibody construct is a monospecific FSA with a heterodimeric Fc and comprises HC1, HC2, LC1 and LC2 sequences, where HC1 and HC2 have different amino acid sequences, and LC1 and LC2 have the same amino acid sequence. In some embodiments, the anti-Ly6E antibody construct is a bispecific or biparatopic FSA with a heterodimeric Fc and comprises HC1, HC2, LC1 and LC2 sequences, where HC1 and HC2 have different amino acid sequences, and LC1 and LC2 have different amino acid sequences.

[0172] In certain embodiments, the anti-Ly6E antibody construct of the present disclosure is a FSA having a set of HC1, HC2, LC1 and LC2 sequences comprising the HC1, HC2, LC1 and LC2 amino acid sequences as set forth in Sequence Tables A & B for any one of variants v38866, v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826, v38827, v39333, v39334, v39335, v39336 or v41252. As is known in the art, expression of antibody heavy chain sequences in certain cell linesor from certain expression vector may result in the inclusion of a C-terminal lysine residue on one or both of the heavy chains. Accordingly, certain embodiments of the present disclosure relate to anti-Ly6E antibody constructs that are FSAs having a set of HC1, HC2, LC1 and LC2 sequences comprising the HC1, HC2, LC1 and LC2 amino acid sequences as set forth in Sequence Tables A & B for any one of variants v38866, v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826, v38827, v39333, v39334, v39335, v39336 or v41252, in which one or both of the HC1 and HC2 sequences comprise a C-terminal lysine.Fc-Silenced Anti-Ly6E Antibody Constructs

[0173] In certain embodiments, the anti-Ly6E antibody construct of the present invention is an Fc-silenced anti-Ly6E antibody construct comprising amino acid modifications in the Fc region that reduce the Fc effector function. In certain embodiments, the anti-Ly6E antibody construct of the present disclosure is an Fc-silenced anti-Ly6E antibody construct comprising LALADS amino acid substitutions in the Fc region. In some embodiments, the anti-Ly6E antibody construct of the present disclosure is an Fc-silenced anti-Ly6E antibody construct comprising LALADS amino acid substitutions in the Fc region such that the Fc-silenced anti-Ly6E antibody construct has a reduced effector function compared to an anti-Ly6E antibody construct with wild-type Fc. In some embodiments, the Fc-silenced anti-Ly6E antibody construct comprising LALADS amino acid substitutions in the Fc region binds to Ly6E-expressing cancer cells, and / or is internalized by Ly6E-expressing cancer cells. In some embodiments, target-independent binding, i.e., the binding to cells that do not (or only minimally) express Ly6E, such as the binding of the Fc region to one or more Fey receptors of non-target cells (e.g., macrophages), of the Fc-silenced anti-Ly6E antibody constructs of the present disclosure is lower compared to the binding of comparable Fc WT anti-Ly6E antibody constructs. In some embodiments, the Fc-silenced anti-Ly6E antibody construct comprising LALADS amino acid substitutions in the Fc region exhibits reduced binding to one or more Fey receptors when compared to a reference anti-Ly6E antibody construct that comprises a wildtype Fc region (e.g., an Fc region that does not comprise LALADS amino acid substitutions). In some embodiments, the Fc-silenced anti-Ly6E antibody construct comprising LALADS amino acid substitutions in the Fc region exhibits reduced ADCC and / or ADCP when compared to a reference anti-Ly6E antibody construct that comprises a wildtype Fc region (e.g., an Fc region that does not comprise LALADS amino acid substitutions). Without being bound by 42IPTS / 200345621.2any theory, the reduced ADCC and / or ADCP as well as the reduced binding to Ly6E-negative cells via the Fc region of the Fc-silenced anti-Ly6E antibody construct comprising LALADS amino acid substitutions in the Fc region may indicate reduced off-target cell engagement and may translate into the potential for reduced toxicity in vivo. Hence, in some embodiments, Fc-silencing of an anti-Ly6E antibody construct of the present disclosure can have the potential to minimize toxicities driven by target-independent cellular uptake of the antibody construct via FcyRs.Preparation of Anti-Ly6E Antibody Constructs

[0174] The anti-Ly6E antibody constructs of the present disclosure may be produced using standard recombinant methods known in the art (see, for example, U. S. Patent No. 4,816,567 and “ Antibodies: A Laboratory Manual,” 2ndEdition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014).

[0175] Typically, for recombinant production of an antibody construct, a polynucleotide or set of polynucleotides encoding the anti-Ly6E antibody construct is generated and inserted into one or more vectors for further cloning and / or expression in a host cell. Polynucleotide(s) encoding the anti-Ly6E antibody construct may be produced by standard methods known in the art (see, for example, Ausubel et al., 1994 & update, Current Protocols in Molecular Biology, John Wiley & Sons, New York, and “ Antibodies: A Laboratory Manual,” 2ndEdition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014). As would be appreciated by one of skill in the art, the number of polynucleotides required for expression of the anti-Ly6E antibody construct will be dependent on the format of the construct, including whether or not the antibody construct comprises a scaffold. For example, when an anti-Ly6E antibody construct is in a monospecific mAb format with a homodimeric Fc, two polynucleotides each encoding a polypeptide chain will be required, whereas when an anti-Ly6E antibody construct is in a monospecific mAb format with a heterodimeric Fc, three polynucleotides each encoding a polypeptide chain will be required. When multiple polynucleotides are required, they may be incorporated into one vector or into more than one vector.

[0176] Generally, for expression, the polynucleotide or set of polynucleotides is incorporated into an expression vector or vectors together with one or more regulatory elements, such as transcriptional elements, which are required for efficient transcription of the polynucleotide. Examples of such regulatory elements include, but are not limited to, promoters, enhancers, terminators, and polyadenylation signals. One skilled in the art will appreciate that the choice ofregulatory elements is dependent on the host cell selected for expression of the antibody construct and that such regulatory elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian or insect genes. The expression vector may optionally further contain heterologous nucleic acid sequences that facilitate expression or purification of the expressed protein. Examples include, but are not limited to, signal peptides and affinity tags such as metalaffinity tags, histidine tags, avidin / streptavidin encoding sequences, glutathione-S-transferase (GST) encoding sequences and biotin encoding sequences. The expression vector may be an extrachromosomal vector or an integrating vector.

[0177] Suitable host cells for cloning or expression of the anti-Ly6E antibody constructs include various prokaryotic or eukaryotic cells as known in the art. Eukaryotic host cells include, for example, mammalian cells, plant cells, insect cells and yeast cells (such as Saccharomyces or Pichia cells). Prokaryotic host cells include, for example, E. coli, A. salmonicida or B. subtilis cells.

[0178] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed, as described for example in U. S. Patent Nos. 5,648,237; 5,789,199, and 5,840,523, and in Charlton (2003, Methods in Molecular Biology, Vol. 248, pp. 245-254, B. K. C. Lo, ed., Humana Press, Totowa, N. J.).

[0179] Eukaryotic microbes such as filamentous fungi or yeast may be suitable expression host cells in certain embodiments, in particular fungi and yeast strains whose glycosylation pathways have been “humanized” resulting in the production of an antibody construct with a partially or fully human glycosylation pattern (see, for example, Gemgross, 2004, Nat. Biotech. 22:1409- 1414, and (Li et al., 2006, Nat. Biotech. 24:210-215).

[0180] Suitable host cells for the expression of glycosylated anti-Ly6E antibody constructs of the present disclosure are usually eukaryotic cells. For example, U. S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 describe PLANTIBODIES™ technology for producing antigen-binding constructs in transgenic plants. Mammalian cell lines adapted to grow in suspension may be particularly useful for expression of antibody constructs. Examples include, but are not limited to, monkey kidney CV1 line transformed by SV40 (COS-7), human embryonic kidney (HEK) line 293 or 293 cells (see, for example, Graham et al.,Gen Virol., 36:59)), baby hamster kidney cells (BHK), mouse sertoli TM4 cells (see, for example, Mather (1980, BiolReprod, 23:243-251)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma (HeLa) cells, canine kidney cells (MDCK), buffalo rat liver cells (BRL 3 A), human lung cells (W 138), human liver cells (Hep G2), mouse mammary tumour (MMT 060562), TRI cells (see, for example, Mather et al., 1982, Annals N. Y. Acad Sci, 383:44-68), MRC 5 cells, FS4 cells, Chinese hamster ovary (CHO) cells (including DHFR CHO cells, see Urlaub et al., 1980, Proc Natl Acad Sci USA, 77:4216, and myeloma cell lines (such as Y0, NSO and Sp2 / 0)). Exemplary mammalian host cell lines suitable for production of antibody constructs are reviewed in Yazaki & Wu, 2003, Methods in Molecular Biology, Vol. 248, pp. 255-268, B. K. C. Lo, ed., Humana Press, Totowa, N. J..

[0181] In certain embodiments, the host cell may be a transient or stable higher eukaryotic cell line, such as a mammalian cell line. In some embodiments, the host cell may be a mammalian HEK293T, CHO, HeLa, NSO or COS cell line, or a cell line derived from any one of these cell lines. In some embodiments, the host cell may be a stable cell line that allows for mature glycosylation of the antibody construct.

[0182] The host cells comprising the expression vector(s) encoding the anti-Ly6E antibody constructs of the present disclosure may be cultured using routine methods to produce the anti-Ly6E antibody constructs. Alternatively, in some embodiments, host cells comprising the expression vector(s) encoding the anti-Ly6E antibody constructs of the present disclosure may be used therapeutically or prophylactically to deliver the anti-Ly6E antibody construct to a subject, or polynucleotides or expression vectors may be administered to a cell from a subject ex vivo and the cell then returned to the body of the subject.

[0183] Typically, the anti-Ly6E antibody constructs of the present disclosure are purified after expression. Proteins may be isolated or purified in a variety of ways known to those skilled in the art (see, for example, Scopes (1994, Protein Purification: Principles and Practice, 3rdEd., Springer-Verlag, NY)). Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reversephase, carried out at atmospheric pressure or at high pressure using systems such as FPLC and HPLC. Additional purification methods include electrophoretic, immunological, precipitation, dialysis and chromatofocusing techniques. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. As is well known in the art, a variety of natural proteins bind Fc and antibodies, and these proteins may be used for purification of certainantibody constructs. For example, the bacterial proteins A and G bind to the Fc region. Likewise, the bacterial protein L binds to the Fab region of some antibodies. Purification may also be enabled by a particular fusion partner. For example, antibodies may be purified using glutathione resin if a GST fusion is employed, Ni+2affinity chromatography if a His-tag is employed or immobilized anti-flag antibody if a flag-tag is used. The degree of purification necessary will vary depending on the use of the anti-Ly6E antibody constructs. In some instances, no purification may be necessary.

[0184] In certain embodiments, the anti-Ly6E antibody constructs of the present disclosure are substantially pure. The term “substantially pure” (or “substantially purified”) when used in the context an anti-Ly6E antibody construct of the present disclosure, indicates that the antibody construct is substantially or essentially free of components that normally accompany or interact with a protein as found in its naturally occurring environment, such as a native cell, or a host cell in the case of recombinantly produced construct. In certain embodiments, an anti-Ly6E antibody construct of the present disclosure that is substantially pure is a protein preparation having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% (by dry weight) of contaminating protein.

[0185] Certain embodiments of the present disclosure relate to a method of making an anti-Ly6E antibody construct comprising culturing a host cell into which one or more polynucleotides encoding the anti-Ly6E antibody construct, or one or more expression vectors encoding the anti-Ly6E antibody construct, have been introduced, under conditions suitable for expression of the anti-Ly6E antibody construct, and optionally recovering the anti-Ly6E antibody construct from the host cell (or from host cell culture medium).Post-Translation Modifications

[0186] In certain embodiments, the anti-Ly6E antibody constructs described herein may comprise one or more post-translational modifications. Such post-translational modifications may occur in vivo, or they be conducted in vitro after isolation of the anti-Ly6E antibody construct from the host cell.

[0187] Post-translational modifications include various modifications as are known in the art (see, for example, Proteins - Structure and Molecular Properties, T. E. Creighton, 1993, 2nd Ed., W. H. Freeman and Company, New York; Post-Translational Covalent Modification of Proteins, B. C. Johnson, 1983, Academic Press, New York, pgs. 1-12; Seifter eta / ., \99Q, Meth. Enzymol.,182:626-646, and Ratan et al., 1992, Ann. N. Y. Acad. Se, 663:48-62). In those embodiments in which an anti-Ly6E antibody construct of the present disclosure comprises one or more post-translational modifications, the construct may comprise the same type of modification at one or several sites, or it may comprise different modifications at different sites.

[0188] Examples of post-translational modifications include glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, formylation, oxidation, reduction, proteolytic cleavage or specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease or NaBH₄.

[0189] Other examples of post-translational modifications include, for example, addition or removal of N-linked or O-linked carbohydrate chains, chemical modifications of N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends, atachment of chemical moieties to the amino acid backbone, and addition or deletion of an N-terminal methionine residue resulting from prokaryotic host cell expression. Post-translational modifications may also include modification with a detectable label, such as an enzymatic, fluorescent, luminescent, isotopic or affinity label to allow for detection and isolation of the protein. Examples of suitable enzyme labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase and acetylcholinesterase. Examples of suitable prosthetic group complexes include, but are not limited to, streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin. Examples of luminescent materials include luminol, and bioluminescent materials such as luciferase, luciferin and aequorin. Examples of suitable radioactive materials include iodine, carbon, sulfur, tritium, indium, technetium, thallium, gallium, palladium, molybdenum, xenon and fluorine.

[0190] Additional examples of post-translational modifications include acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent atachment of a heme moiety, covalent atachment of a nucleotide or nucleotide derivative, covalent atachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, pegylation, prenylation, racemization, selenoylation, sulfation,47IPTS / 200345621.2transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.Polynucleotides, Vectors and Host Cells

[0191] Certain embodiments of the present disclosure relate to an isolated polynucleotide or a set of polynucleotides encoding an anti-Ly6E antibody construct described herein. A polynucleotide in this context may encode all or part (for example, a heavy chain or a light chain) of an anti-Ly6E antibody construct.

[0192] A polynucleotide that “encodes” a given polypeptide is a polynucleotide that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A transcription termination sequence may be located 3' to the coding sequence.

[0193] Certain embodiments of the present disclosure relate to vectors (such as expression vectors) comprising one or more polynucleotides encoding an anti-Ly6E antibody construct described herein. The polynucleotide(s) may be comprised by a single vector or by more than one vector. In some embodiments, the polynucleotides are comprised by a multicistronic vector.

[0194] Certain embodiments of the present disclosure relate to host cells comprising polynucleotide (s) encoding an anti-Ly6E antibody construct described herein or one or more vectors comprising the polynucleotide (s). In some embodiments, the host cell is eukaryotic, for example, a Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell (for example, a Y0, NSO, Sp20 cell).ANTIBODY-DRUG CONJUGATES

[0195] Certain embodiments of the present disclosure relate to antibody-drug conjugates (ADCs) comprising an anti-Ly6E antibody construct, as disclosed herein, conjugated to one or more drug moieties, such as cytotoxins or immune modulators.

[0196] Typically, in an ADC, the anti-Ly6E antibody construct of the present disclosure is conjugated to one or more drug moieties via a linker, which may be a cleavable or non-cleavable linker. The anti-Ly6E antibody construct may be conjugated to a single drug moiety, or it may be conjugated to multiple drug moieties. When the anti-Ly6E antibody construct is conjugated to48IPTS / 200345621.2multiple drug moieties, each drug moiety may be conjugated to the anti-Ly6E antibody construct by a separate linker or multiple drug moieties may be conjugated via a single (polyvalent) linker. The number of drug molecules conjugated to a single anti-Ly6E antibody construct is defined by the drug-to-antibody ratio (DAR). In certain embodiments, in the ADCs of the present disclosure, the DAR is in the range of from about 1 to about 12, or from about 2 to about 12, or from about 2 to about 8. When the ADC comprises multiple drug moieties (i.e. the DAR is > 2), the drug moieties may be the same or they may be different. In some embodiments, the ADCs of the present disclosure comprise an anti-Ly6E antibody construct conjugated to more than one drug moiety and the drug moieties are the same. In some embodiments, the ADCs of the present disclosure comprise an anti-Ly6E antibody construct conjugated to more than one drug moiety and the drug moieties are different.

[0197] In certain embodiments, the ADCs of the present disclosure have the general Formula (I):A-(L-(D)m)n(I)wherein:A is an anti-Ly6E antibody construct as described herein;L is a linker;D is a drug moiety;m is between 1 and 8, andn is between about 1 and about 12.

[0198] In certain embodiments, in conjugates of Formula (I), m is between 1 and 2. In some embodiments, m is 1. In some embodiments, in conjugates of Formula (I), n is between about 1 and about 8, for example, between about 2 and about 8. In some embodiments, n is between about 4 and about 8.

[0199] In certain embodiments, in conjugates of Formula (I), m is between 1 and 2, and n is between about 2 and about 8, or between about 4 and about 8. In some embodiments, in conjugates of Formula (I), m is 1, and n is between about 2 and about 8, or between about 4 and about 8. In some embodiments, in conjugates of Formula (I), m is between 1 and 2, and n is between about 2 and about 6. In some embodiments, in conjugates of Formula (I), m is 1, and n is between about 2 and about 6. In some embodiments, in conjugates of Formula (I), m is 1, and n is between about 349IPTS / 200345621.2and about 5. In some embodiments, in conjugates of Formula (I), m is 1, and n is about 4. In some embodiments, in conjugates of Formula (I), m is 1, and n is about 8.

[0200] Those skilled in the art will appreciate that, while any particular anti-Ly6E antibody construct A is conjugated to an integer number of drug moieties D, analysis of a preparation of the conjugate to determine the ratio of drug moieties to anti-Ly6E antibody construct may give a noninteger result, reflecting a statistical average. Accordingly, the ADCs of the present disclosure comprising an anti-Ly6E antibody construct of the present disclosure having non-integer DARs are intended to be encompassed by Formula (I), as described herein.Drug Moiety (D)

[0201] Various compounds known to be useful as cytotoxic or immunomodulatory ADC payloads may be employed as the drug moiety in the ADCs comprising the anti-Ly6E antibody constructs of the present disclosure. Examples include, but are not limited to, microtubule inhibitor payloads, such as, maytansinoids and maytansinoid analogues, auristatins and auristatin analogues, hemiasterlins and hemiasterlin analogues, eribulin and eribulin analogues, tubulysin and tubulysin analogues, cryptophycins and cryptophycin analogues, kinesins and kinesin analogues; DNA targeting payloads, such as, benzodiazepines and pyrrolobenzodiazepines, duocarmycins such as CC-1065 and analogues, anthracyclines and anthracycline analogues, enediynes and enediyne analogues, calicheamicins and calicheamicin analogues, camptothecins and camptothecin analogues; RNA targeting payloads, such as amatoxins and amatoxin analogues, thailanstatin and thailanstain analogues; immune-stimulating payloads, such as TLR agonists (such as agonists of TLR7 and / or TLR8), STING agonists, glucocorticoid receptor modulators; Bcl-xL inhibitors; NAMPT inhibitors; and proteosome inhibitors (such as carmaphycins). (Wang et al., 2023, Acta Pharmaceutica Sinica B, 13(10): 4025-4059).

[0202] In certain embodiments, the drug moiety comprised by the ADCs of the present disclosure is a microtubule inhibitor or a DNA-targeting agent.

[0203] In certain embodiments, the drug moiety comprised by the ADCs of the present disclosure is a microtubule inhibitor. In some embodiments, the microtubule inhibitor comprised by the ADCs of the present disclosure is an auristatin or auristatin analogue. In some embodiments, the microtubule inhibitor comprised by the ADCs of the present disclosure is MMAE or MMAF. In some embodiments, the auristatin analogue comprised by the ADCs of the present disclosure is an auristatin analogue as described in International Patent Application Publication No. WO 50IPTS / 200345621.22016 / 041082. In some embodiments, the microtubule inhibitor comprised by the ADCs of the present disclosure is a hemiasterlin or a hemiasterlin analogue. In some embodiments, the hemiasterlin analogue comprised by the ADCs of the present disclosure is a hemiasterlin analogue as described in International Patent Application Publication No. WO 2014 / 144871.

[0204] In certain embodiments, the drug moiety comprised by the ADCs of the present disclosure is a DNA-targeting agent. In certain embodiments, the DNA-targeting agent comprised by the ADCs of the present disclosure is a topoisomerase 1 inhibitor. In some embodiments, the topoisomerase I inhibitor is exatecan or Dxd. In some embodiments, the topoisomerase 1 inhibitor comprised by the ADCs of the present disclosure is a camptothecin analogue as described in International Patent Application Publication No. WO 2022 / 246576.

[0205] In certain embodiments, the drug moiety comprised by the ADCs of the present disclosure is a camptothecin analogue having Formula (II):wherein:Xk / X<n R99. NHR4is I;Xaand Xbare each O, andR9is selected from: -H, -Ci-Cg hydroxyalkyl, -Ci-Cg aminoalkyl, -aminoaryl and -(Ci-Cg alkyl)-aminoaryl.

[0206] In certain embodiments, in camptothecin analogues of Formula (II), R9is -Ci-Cg hydroxyalkyl.

[0207] In certain embodiments, the drug moiety comprised by the ADCs of the present disclosure is a camptothecin analogue, Compound 1, having the following structure:51IPTS / 200345621.2Compound 1

[0208] In certain embodiments, the drug moiety comprised by the ADCs of the present disclosure is Compound 1, where the drug moiety is attached to linker, L, as shown below:where * is the point of attachment to linker, L.Linker (L)

[0209] The ADCs of Formula (I) include a linker, L, which is a bifunctional or multifunctional moiety capable of linking one or more drug molecules, D, to the anti-Ly6E antibody construct, A, of the present disclosure. In some embodiments, the linker, L, may be bifunctional (or monovalent) such that it links a single drug molecule, D, to a single site on the anti-Ly6E antibody construct, A. In some embodiments, the linker, L, may be multifunctional (or polyvalent) such that it links more than one drug molecule, D, to a single site on the anti-Ly6E antibody construct, A. Multifunctional linkers, L, may also be used to link one drug molecule, D, to more than one site on the anti-Ly6E antibody construct, A, in some embodiments.

[0210] Attachment of a linker to an anti-Ly6E antibody construct can be accomplished in a variety of ways, such as through surface lysines, reductive-coupling to oxidized carbohydrates, or through cysteine residues liberated by reducing interchain disulfide linkages. Alternatively, attachment of a linker to an anti-Ly6E antibody construct may be achieved by modification of the antibody construct to include additional cysteine residues (see, for example, U. S. Patent Nos.52IPTS / 200345621.27,521,541; 8,455,622 and 9,000,130; International Patent Application Publication No. WO 2022 / 198335) or non-natural amino acids that provide reactive handles, such as selenomethionine, p-acetylphenylalanine, formylglycine or p-azidomethyl-L-phenylalanine to allow for site-specific conjugation (see, for example, Hofer et al., 2009, Biochemistry, 48:12047-12057; Axup et al., 2012, PNAS, 109: 16101-16106; Wu etal., 2009, PNAS, 106:3000-3005; Zimmerman eta / ., 2014, Bioconj. Chem., 25:351-361). A further option is the use of GlycoConnect™ technology (Synaffix BV, Nijmegen, Netherlands), which involves enzymatic remodelling of the antibody glycans to allow for attachment of a linker by metal-free click chemistry (see, for example, European Patent No. EP 2911 699).

[0211] Linker, L, typically includes a functional group capable of reacting with the target group or groups on the anti-Ly6E antibody construct, A, and one or more functional groups capable of reacting with a target group on the drug moiety, D. Suitable functional groups are known in the art and include those described, for example, in G. T. Hermanson, 2013, Bioconjugate Techniques, Academic Press. Functional groups on the anti-Ly6E antibody construct, A, and the drug moiety, D, that may serve as target groups for linker attachment include, but are not limited to, thiol, hydroxyl, carboxyl, amine, aldehyde and ketone groups.

[0212] Non-limiting examples of functional groups capable of reacting with thiols include maleimide, haloacetamide, haloacetyl, activated esters (such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters), anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. Also useful in this context are “self-stabilizing” maleimides such as those described in Lyon et al., 2014, Nat. Biotechnol., 32: 1059-1062.

[0213] Non-limiting examples of functional groups capable of reacting with amines include activated esters (such as N-hydroxy succinamide (NHS) esters and sulfo-NHS esters), imido esters (such as Trant’s reagent), isothiocyanates, aldehydes and acid anhydrides (such as diethylenetriaminepentaacetic anhydride (DTP A)). Other examples include the use of succinimido-l,l,3,3-tetra-methyluronium tetrafluoroborate (TSTU) or benzotriazol- 1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) to convert a carboxylic acid to an activated ester, which may then be reacted with an amine.

[0214] Non-limiting examples of functional groups capable of reacting with an electrophilic group (such as an aldehyde or ketone carbonyl group) include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate and arylhydrazide.

[0215] In certain embodiments, linker, L, may include a functional group that allows for bridging of two interchain cysteines on the antibody construct, such as a ThioBridge™ linker (Badescu et al., 2014, Bioconjug. Chem., 25:1124-1136), a dithiomaleimide (DTM) linker (Behrens et al., 2015, Mol. Pharm., 12:3986-3998), a dithioaryl(TCEP)pyridazinedione-based linker (Lee et al., 2016, Chem. Sci., 7:799-802) or a dibromopyridazinedione-based linker (Maruani et al., 2015, Nat. Commun., 6:6645).

[0216] Alternatively, the anti-Ly6E antibody construct, A, may be modified to include a nonnatural reactive group, such as an azide, that allows for conjugation to the linker via a complementary reactive group on the linker. For example, conjugation of the linker to the anti-Ly6E antibody construct may make use of click chemistry reactions (see, for example, Chio & Bane (2020, Methods Mol. Biol., 2078:83-97)), such as the azide-alkyne cycloaddition (AAC) reaction, which has been used successfully in the development of antibody-drug conjugates. The AAC reaction may be a copper-catalyzed AAC (CuAAC) reaction, which involves coupling of an azide with a linear alkyne, or a strain-promoted AAC (SPAAC) reaction, which involves coupling of an azide with a cyclooctyne.

[0217] A variety of linkers for linking drugs to antibodies are known in the art, including hydrazone-, disulfide- and peptide-based linkers. In some embodiments of the present disclosure, linker, L, may be cleavable or non-cleavable. A cleavable linker is typically susceptible to cleavage under intracellular conditions, for example, through lysosomal processes. Examples include linkers that are protease-sensitive, acid-sensitive or reduction-sensitive. Non-cleavable linkers by contrast, rely on the degradation of the antibody construct in the cell, which typically results in the release of an amino acid-linker-drug moiety.

[0218] Examples of cleavable linkers include, for example, linkers comprising an amino acid sequence that is a cleavage recognition sequence for a protease. Many such cleavage recognition sequences are known in the art. For ADCs that are not intended to be internalized by a cell, for example, an amino acid sequence that is recognized and cleaved by a protease present in the extracellular matrix in the vicinity of a target cell, such as a cancer cell, may be employed. Examples of extracellular tumor-associated proteases include, for example, plasmin, matrix metalloproteases (MMPs), elastase and kallikrein-related peptidases. For ADCs intended to be internalized by a cell a linker may comprise an amino acid sequence that is recognized and cleavedby an endosomal or lysosomal protease. Examples of such proteases include, for example, cathepsins B, C, D, H, L and S, and legumain.

[0219] Cleavage recognition sequences may be, for example, dipeptides, tripeptides or tetrapeptides. Non-limiting examples of dipeptide recognition sequences that may be included in cleavable linkers include, but are not limited to, Ala-(D)Asp, Ala-Lys, Ala-Phe, Asn-Lys, Asn-(D)Lys, Asp-Val, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, MesLys-Pro, Met-Lys, Met-(D)Lys, NorVal-(D)Asp, Phe-Arg, Phe-Cit, Phe-Lys, PhenylGly-(D)Lys, Pro-(D)Lys, Trp-Cit, Vai-Ala, Val-(D)Asp, Val-Cit, Val-Gly, Val-Gln and Val-Lys. Examples of tripeptide cleavage sequences include, but are not limited to, Ala-Ala-Asn, Ala-Val-Cit, (D)Ala-Phe-Lys, Asp-Val-Ala, Asp-Val-Cit, Gly-Cit-Val, Lys-Val-Ala, Lys-Val-Cit, Met-Cit-Val, (D)Phe-Phe-Lys and Asn-Pro-Val. Examples of tetrapeptide cleavage sequences include, but are not limited to, Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly and Gly-Phe-Gly-Gly.

[0220] Additional examples of cleavable linkers include disulfide-containing linkers and linkers hydrolysable at a specific pH or within a pH range, such as hydrazone linkers. Examples of disulfide-containing linkers include, but are not limited to, N-succinimydyl-4-(2-pyridyldithio) butanoate (SPDB) and N-succinimydyl-4-(2-pyridyldithio)-2-sulfo butanoate (sulfo-SPDB). Disulfide-containing linkers may optionally include additional groups to provide steric hindrance adjacent to the disulfide bond in order to improve the extracellular stability of the linker, for example, inclusion of a geminal dimethyl group. Linkers comprising combinations of these functionalities may also be useful, for example, linkers comprising both a hydrazone and a disulfide.

[0221] A further example of a cleavable linker is a linker comprising a [3-glucuronide, which is cleavable by [3-glucuronidase, an enzyme present in lysosomes and tumor interstitium (see, for example, De Graaf et al., 2002, Curr. Pharm. Des. 8:1391-1403, and International Patent Application Publication No. WO 2007 / 011968). [3-glucuronide may also function to improve the hydrophilicity of linker, L.

[0222] Another example of a linker that is cleaved internally within a cell and improves hydrophilicity is a linker comprising a pyrophosphate diester moiety (see, for example, Kern et al., 2016, J Am Chem Soc., 138:2430-1445).

[0223] In certain embodiments of the present disclosure, the linker, L, comprised by the ADC of Formula I is a cleavable linker. In some embodiments, linker, L, is a peptide-containing linker. Insome embodiments, linker, L, comprises a cleavage recognition sequence. In some embodiments, linker, L, is a protease-cleavable linker. In some embodiments, linker, L, may comprise an amino acid sequence that is recognized and cleaved by a lysosomal protease.

[0224] Cleavable linkers may optionally further comprise one or more additional functionalities such as self-immolative and self-elimination groups, stretchers, or hydrophilic moieties.

[0225] Self-immolative and self-elimination groups that find use in linkers include, for example, p-aminobenzyl (PAB) and p-aminobenzyloxycarbonyl (PABC) groups, methylated ethylene diamine (MED) and hemi-aminal groups. Other examples of self-immolative groups include, but are not limited to, aromatic compounds that are electronically similar to the PAB or PABC group such as heterocyclic derivatives, for example 2-aminoimidazol-5-methanol derivatives as described in U. S. Patent No. 7,375,078. Other examples include groups that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995, Chemistry Biology, 2:223-227) and 2-aminophenylpropionic acid amides (Amsberry et al., 1990, Org. Chem., 55:5867-5877). Self-immolative / self-elimination groups are typically attached to an amino or hydroxyl group on the compound, D. Self-immolative / self-elimination groups, alone or in combination are often included in peptide-based linkers, but may also be included in other types of linkers.

[0226] In some embodiments of the present disclosure, linker, L, comprised by the ADC of Formula (I) may include one or more self-immolative / self-elimination groups, for example, a PABC group, a p-aminobenzyl ether (PABE) group, or a combination of a PABC or PABE group and an MED.

[0227] Stretchers that find use in linkers for ADCs include, for example, alkylene groups and stretchers based on aliphatic acids, diacids, amines or diamines, such as diglycolate, malonate, caproate and caproamide. Other stretchers include, for example, glycine-based stretchers and polyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG) stretchers. PEG and mPEG stretchers can also function as hydrophilic moieties within a linker and may be particularly useful with hydrophobic drugs, although their use in linkers with other drugs is also contemplated in some embodiments. For example, PEG or mPEG may be included in a linker either “in-line” or as pendant groups to increase the hydrophilicity of the linker (see, for example, U. S. Patent Application Publication No. US 2016 / 0310612). Various PEG-containing linkers are commercially available from companies such as Quanta BioDesign, Ltd (Plain City, OH). Other56IPTS / 200345621.2hydrophilic groups that may optionally be incorporated into linker, L, include, for example, -glucuronide, sulfonate groups, carboxylate groups and pyrophosphate diesters.

[0228] In certain embodiments of the present disclosure, linker, L, may include a stretcher having one of the following structures:O O O— (CH2)t— C —. — (CH2CH2O)U— C —. — (CH2)t— (CH2CH2O)U— C —.o o— (CH2CH2O)U— (CH2)t— C -. — (CH2)t— (CH2CH2O)U— (CH2)t— C —O R OHRH— (CH2)t— C — N— (CH2)t— C —or— (CH2)t— C— N— (CH2CH2O)U— C —wherein:R is H or Ci-Ce alkyl;t is an integer between 2 and 10, andu is an integer between 1 and 10.

[0229] In some embodiments of the present disclosure, in ADCs of Formula (I), linker, L, is a cleavable linker having Formula (III):,-z st4qAAiAfl2lxt%(III)wherein:Z is a linking group that joins the linker to a target group on the anti-Ly6E antibody construct, A;Str is a stretcher;AAi and AA2 are each independently an amino acid, wherein AAi-[AA2]r forms a protease cleavage site;X is a self-immolative group;q is 0 or 1;ris 1, 2 or 3;s is 0, 1 or 2;# is the point of attachment of the anti-Ly6E antibody construct A, and% is the point of attachment of the drug moiety, D.

[0230] In some embodiments, in linkers of Formula (III), s is 0. In some embodiments, in linkers of Formula (III), s is 1.

[0231] In some embodiments of the present disclosure, in ADCs of Formula (I), linker, L, is a cleavable linker having Formula (Illa):._z-[str]-AA1_[AA2]-l.Y]_%(Hla)wherein:Z is a linking group that joins the linker to a target group on the anti-Ly6E antibody construct, A;Str is a stretcher;AAi and AA2 are each independently an amino acid, wherein AAi-[AA2]rforms a protease cleavage site;Y is -NH-CH2- or -NH-CH2-C(O)-;q is 0 or 1;ris 1, 2 or 3;v is 0 or 1;# is the point of attachment to the anti-Ly6E antibody construct, A, and% is the point of attachment to the drug moiety, D.

[0232] In some embodiments, in linkers of Formula (Illa), v is 1.

[0233] In some embodiments, in linkers of Formula (III) or Formula (Illa), q is 1.

[0234] In some embodiments, in linkers of Formula (III) or Formula (Illa):T N-*Zis O where # is the point of attachment to A, and * is the point of attachment to the reminder of the linker.

[0235] In some embodiments, in linkers of Formula (III) or Formula (Illa), Str is selected from:O O O— (CH2)t— C —. — (CH2CH2O)U— C —. — (CH2)t— (CH2CH2O)U— C —.58IPTS / 200345621.2O O— (CH2CH2O)U— (CH2)t— C —. — (CH2)t— (CH2CH2O)U— (CH2)t— C —O R O O R O— (CH2)t— C-N— (CH2)t— C— — (CH2)t— C-N— (CH2CH2O)U— C—wherein:R is H or Ci-Cg alkyl;t is an integer between 2 and 10, andu is an integer between 1 and 10.

[0236] In some embodiments, in linkers of Formula (III) or Formula (Illa), Str is:O O— (CH2)t— C—or— (CH2CH2O)U— (CH2)t— C —

[0237] In some embodiments, in linkers of Formula (III) or Formula (Illa), AAi-[AA2]r is a dipeptide (i.e. r = 1). In some embodiments, in linkers of Formula (III) or Formula (Illa), AAi-[AA2]r has a sequence selected from: Ala-(D)Asp, Ala-Lys, Ala-Phe, Asn-Lys, Asn-(D)Lys, Asp-Val, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, MesLys-Pro, Met-Lys, Met-(D)Lys, NorVal-(D)Asp, Phe-Arg, Phe-Cit, Phe-Lys, PhenylGly-(D)Lys, Pro-(D)Lys, Trp-Cit, Vai-Ala, Val-(D)Asp, Val-Cit, Val-Gly, Val-Gln and Val-Lys.

[0238] In some embodiments, in linkers of Formula (III) or Formula (Illa), AAi-[AA2]ris a tripeptide (i.e. r = 2). In some embodiments, in linkers of Formula (III) or Formula (Illa), AAi-[AA2]r has a sequence selected from: Ala-Ala-Asn, Ala-Val-Cit, (D)Ala-Phe-Lys, Asp-Val-Ala, Asp-Val-Cit, Gly-Cit-Val, Lys-Val-Ala, Lys-Val-Cit, Met-Cit-Val, (D)Phe-Phe-Lys, and Asn-Pro-Val.

[0239] In some embodiments, in linkers of Formula (III) or Formula (Illa), AAi-[AA2]r is a tetrapeptide (i.e. r = 3). In some embodiments, in linkers of Formula (III) or Formula (Illa), AAi-[AA2]r has a sequence selected from: Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly and Gly-Phe-Gly-Gly.

[0240] In some embodiments, ADCs of Formula (I) may comprise a disulfide-containing linker. In some embodiments, in ADCs of Formula (I), linker, L, is a cleavable linker having Formula (IV):59IPTS / 200345621.2R RXN', NR R(IV)wherein:Z is a linking group that joins the linker to a target group on the anti-Ly6E antibody construct, A;Q is is -(CH2)P- or -(CH2CH2O)q-, wherein p and q are each independently an integer between 1 and 10;each R is independently H or Ci-Cg alkyl;n is 1, 2 or 3;# is the point of attachment to the anti-Ly6E antibody construct, A, and% is the point of attachment to the drug moiety, D.

[0241] In some embodiments of the present disclosure, ADCs of Formula (I) may comprise a -glucuronide-containing linker.

[0242] Various non-cleavable linkers are known in the art for linking drugs to antibodies and may be useful in the ADCs of the present disclosure in certain embodiments. Examples of non-cleavable linkers include linkers having an N-succinimidyl ester or N-sulfosuccinimidyl ester moiety for reaction with the antibody construct, as well as a maleimido- or haloacetyl -based moiety for reaction with the drug moiety, or vice versa. An example of such a non-cleavable linker is based on sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-l -carboxylate (sulfo-SMCC). Sulfo-SMCC conjugation typically occurs via a maleimide group which reacts with sulfhydryls (thiols, — SH), while the sulfo-NHS ester is reactive toward primary amines. Other non-limiting examples of such linkers include those based on N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-l-carboxy-(6-amidocaproate) (“long chain” SMCC or LC-SMCC), K-maleimidoundecanoic acid N-succinimidyl ester (KMUA), y-maleimidobutyric acid N-succinimidyl ester (GMBS), s-maleimidocaproic acid N-hydroxy succinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(a-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-6-(p-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB) and N-(p-maleimidophenyl)isocyanate (PMPI). Other60IPTS / 200345621.2examples include those comprising a haloacetyl-based functional group such as N-succinimidyl- 4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA) and N-succinimidyl 3-(bromoacetamido)propionate (SBAP).

[0243] In certain embodiments, the drug-linker comprised by the ADCs of Formula (I) has the structure of any one of DL1, DL2, DL3 or DL4 shown below:(DL2),(DL3), or61IPTS / 200345621.2

[0244] In certain embodiments, the ADCs of Formula (I) comprise the drug-linker DL1, where the drug-linker is conjugated to anti-Ly6E antibody construct, A, via the maleimide group as shown below:where ** is the point of conjugation to the anti-Ly6E antibody construct, A.

[0245] In certain embodiments, the ADCs of Formula (I) have the following structure:wherein:A is an anti-Ly6E antibody construct as described herein;L is a linker, andn is between about 2 and about 8, for example, between about 4 and about 8.62IPTS / 200345621.2Preparation of ADCs

[0246] ADCs of Formula (I) comprising an anti-Ly6E antibody construct of the present disclosure may be prepared by one of several methods known in the art (see, for example, G. T. Hermanson, 2013, Bioconjugate Techniques, Academic Press). For example, conjugation may be achieved by (1) reaction of a functional group of an anti-Ly6E antibody construct, A, with a bivalent linker, L, to form antibody-linker intermediate A-L, via a covalent bond, followed by reaction of A-L with an activated drug moiety D; or (2) reaction of a functional group of a drug moiety, D, with a linker, L, to form drug-linker intermediate D-L, via a covalent bond, followed by reaction of D-L with a functional group of an anti-Ly6E antibody construct, A. Conjugation methods (1) and (2) may be employed with a variety of anti-Ly6E antibody constructs, drug moieties, and linkers to prepare the ADCs of the present disclosure. Alternatively, the drug -linker, D-L, may be synthesized as a unit from commercially available starting materials or intermediates and then conjugated to the anti-Ly6E antibody construct of the present disclosure.

[0247] Suitable functional groups on drug moiety, D, for attachment of linker, L, in either of the above approaches include, but are not limited to, thiol groups, amine groups, carboxylic acid groups and hydroxyl groups. In some embodiments of the present disclosure, linker, L, is attached to drug moiety, D, via a hydroxyl or amine group on the compound.

[0248] Suitable functional groups on the anti-Ly6E antibody construct, A, for attachment of linker, L, in either of the above approaches include sulfhydryl groups (for example, on the sidechain of cysteine residues), amino groups (for example, on the side-chain of lysine residues), carboxylic acid groups (for example, on the side-chains of aspartate or glutamate residues), and carbohydrate groups.

[0249] For example, the anti-Ly6E antibody construct, A, may comprise one or more naturally occurring sulfhydryl groups allowing the anti-Ly6E antibody construct, A, to bond to linker, L, via the sulfur atom of a sulfhydryl group. Alternatively, the anti-Ly6E antibody construct, A, may comprise one or more lysine residues that can be chemically modified to introduce one or more sulfhydryl groups. Reagents that can be used to modify lysine residues include, but are not limited to, A-succinimidyl S-acetylthioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate (“SPDP”) and 2-iminothiolane hydrochloride (Traut’s Reagent). Alternatively, the anti-Ly6E antibody construct, A, may comprise one or more carbohydrate groups that can be chemically modified to include one or more sulfhydryl groups.63IPTS / 200345621.2

[0250] Carbohydrate groups on the anti-Ly6E antibody construct, A, may also be oxidized to provide an aldehyde (-CH0) group (see, for example, Laguzza et al., 1989, J. Med. Chem.32(3):548-55), which could subsequently be reacted with linker, L, for example, via a hydrazine or hydroxylamine group on linker, L.

[0251] The anti-Ly6E antibody construct, A, may also be modified to include additional cysteine residues (see, for example, U. S. Patent Nos. 7,521,541; 8,455,622 and 9,000,130) or non-natural amino acids that provide reactive handles, such as selenomethionine, p-acetylphenylalanine, formylglycine or p-azidomethyl-L-phenylalanine (see, for example, Hofer et al., 2009, Biochemistry, 48:12047-12057; Axup et al., 2012, PNAS, 109:16101-16106; Wu et al., 2009, PNAS, 106:3000-3005; Zimmerman et al., 2014, Bioconj. Chem., 25:351-361), to allow for sitespecific conjugation. Alternatively, the anti-Ly6E antibody construct, A, may be modified to include a non-natural reactive group, such as an azide, that allows for conjugation to the linker via a complementary reactive group on the linker, for example, by click chemistry (see, for example, Chio & Bane, 2020, Methods Mol. Biol., 2078:83-97). A further option is the use of GlycoConnect™ technology (Synaffix BV, Nijmegen, Netherlands), which involves enzymatic remodelling of the antibody glycans to allow for attachment of a linker by metal-free click chemistry (see, for example, European Patent No. EP 2 911 699).

[0252] Other protocols for the modification of proteins for the attachment or association of linker, L, are known in the art and include those described in Current Protocols in Protein Science, Coligan et al., 2002, vol. 2, John Wiley & Sons.

[0253] Alternatively, ADCs may be prepared using the enzyme transglutaminase, in particular, bacterial transglutaminase (BTG) from Streptomyces moharaensis (see, for example, Jeger et al., 2010, Angew. Chem. Int. Ed., 49:9995-9997). BTG forms an amide bond between the side chain carboxamide of a glutamine (the amine acceptor, typically on the antibody) and an alkyleneamino group (the amine donor, typically on the drug-linker), which can be, for example, the s-amino group of a lysine or a 5-amino-n-pentyl group. Antibodies may also be modified to include a glutamine containing peptide, or “tag,” which allows BTG conjugation to be used to conjugate the antibody to a drug-linker (see, for example, U. S. Patent Application Publication No. US 2013 / 0230543 and International Patent Application Publication No. WO 2016 / 144608).

[0254] A similar conjugation approach utilizes the enzyme sortase A. In this approach, the antibody is typically modified to include the sortase A recognition motif (LPXTG, where X is anynatural amino acid) and the drug-linker is designed to include an oligoglycine motif (typically GGG) to allow for sortase A-mediated transpeptidation (see, for example, Beerli, et al., 2015, PLos One, 10:e0131177; Chen etal., 2016, Nature. 'Scientific Reports, 6:31899).

[0255] Various prepared linkers, linker components and drugs are commercially available or may be prepared using standard synthetic organic chemistry techniques (see, for example, March ’s Advanced Organic Chemistry, Smith & March, 2006, Sixth Ed., Wiley; Toki et al., 2002, J. Org. Chem., 67: 1866-1872; Frisch et a / ., 1997, Bioconj. Chem., 7:180-186; Bioconjugate Techniques, G. T. Hermanson, 2013, Academic Press, and Antibody-Drug Conjugates: Methods in Molecular Biology, Ducry (Ed.), 2013, Springer). In addition, a number of pre-formed drug -linkers suitable for reaction with a selected antibody construct are available commercially, for example, druglinkers comprising DM1, DM4, MMAE, MMAF or Duocarmycin SA are available from Creative Bio Labs (Shirley, NY). Various antibody drug conjugation services are also available commercially from companies such as Lonza Inc. (Allendale, NJ), Abzena PLC (Cambridge, UK), ADC Biotechnology (St. Asaph, UK), Baxter BioPharma Solutions (Baxter Healthcare Corporation, Deerfield, IL) and Piramal Pharma Solutions (Grangemouth, UK).

[0256] Certain embodiments of the present disclosure relate to methods of preparing an ADC of Formula (I) comprising conjugating drug-linker L-(D)mto antibody, A. Some embodiments relate to methods of preparing an ADC of Formula (I) comprising conjugating drug-linker L-(D)mto native cysteine residues of antibody, A. In some embodiments in the methods of preparing an ADC of Formula (I), in drug -linker L-(D)m, m is 1. In some embodiments, methods of preparing an ADC of Formula (I), drug-linker L-(D)mhas the structure:(DL2),(DL3),

[0257] The ADCs, once prepared, may be purified by standard techniques such as chromatography (for example, HPLC, size-exclusion, adsorption, ion exchange and / or affinity capture), dialysis and / or tangential flow filtration. The average DAR of the prepared ADC may optionally be determined by standard techniques such as UV / VIS spectroscopic analysis, ELISA-based techniques, chromatography techniques such as hydrophobic interaction chromatography (HIC), UV-MALDI mass spectrometry (MS) or MALDI-TOF MS. In addition, distribution of drug-linked forms (for example, the fraction of the anti-Ly6E antibody construct, A, containing zero, one, two, three, etc. conjugated drug moieties, D) may also optionally be analyzed, for example by MS (with or without an accompanying chromatographic separation step), hydrophobicinteraction chromatography, reverse-phase HPLC or iso-electric focusing gel electrophoresis (IEF) (see, for example, Wakankar etal., 2011, mAbs, 3:161-172).METHODS OF USE

[0258] Certain aspects of the present disclosure relate to the therapeutic or diagnostic use of the anti-Ly6E antibody constructs and ADCs comprising an anti-Ly6E antibody construct as described herein. Ly6E is expressed in a variety of cancers and certain embodiments of the present disclosure thus relate to the methods of using the anti-Ly6E antibody constructs and ADCs comprising an anti-Ly6E antibody construct in the treatment or diagnosis of a Ly6E-expressing cancer.

[0259] Certain embodiments of the present disclosure relate to methods of inhibiting abnormal cancer cell or tumor cell growth; inhibiting cancer cell or tumor cell proliferation, or treating cancer in a subject, comprising administering an anti-Ly6E antibody construct or an ADC described herein. In certain embodiments, the anti-Ly6E antibody constructs or the ADCs described herein may be used in the treatment of cancer. Some embodiments of the present disclosure thus relate to the use of the anti-Ly6E antibody constructs or the ADCs as anti -cancer agents.

[0260] Certain embodiments relate to methods of inhibiting the growth of Ly6E-expressing cancer or tumor cells comprising contacting the cells with an anti-Ly6E antibody construct or an ADC described herein, for example, an ADC of Formula (I). The cells may be in vitro or in vivo. In certain embodiments, the anti-Ly6E antibody constructs and ADCs comprising an anti-Ly6E construct may be used in methods of treating a Ly6E-expressing cancer or tumor in a subject. Some embodiments relate to a method of killing cancer or tumor cells comprising contacting the cells with an anti-Ly6E antibody construct or an ADC as described herein, for example, an ADC of Formula (I).

[0261] Some embodiments of the present disclosure relate to methods of treating a subject having a cancer by administering to the subject an anti-Ly6E antibody construct or an ADC as described herein, for example, an ADC of Formula (I). In this context, treating the subject may result in one or more of alleviation of symptoms, a reduction in the size of a tumor, the slowing or prevention of an increase in the size of a tumor, inhibiting growth of a tumor, an increase in the disease-free survival time between the disappearance or removal of a tumor and its reappearance, prevention of a subsequent occurrence of a tumor (for example, metastasis), an increase in the time to progression, reduction of one or more adverse symptom associated with a tumor, diminishing one67IPTS / 200345621.2or more direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, improving survival, increasing progression-free survival, remission, and / or improving prognosis.

[0262] Certain embodiments relate to the use of an anti-Ly6E antibody construct or an ADC as described herein, for example, an ADC of Formula (I), in a method of inhibiting tumor growth in a subject. Some embodiments relate to the use of an anti-Ly6E antibody construct or an ADC as described herein, for example, an ADC of Formula (I), in a method of inhibiting proliferation of and / or killing cancer cells in vitro. Some embodiments relate to the use of an anti-Ly6E antibody construct or an ADC as described herein, for example, an ADC of Formula (I), in a method of inhibiting proliferation of and / or killing cancer cells in vivo in a subject having a cancer.

[0263] Examples of cancers which may be treated in certain embodiments are carcinomas, including adenocarcinomas and squamous cell carcinomas; melanomas and sarcomas. Carcinomas and sarcomas are also frequently referred to as “solid tumors.” Various forms of lymphoma also may result in the formation of a solid tumor and, therefore, may also be considered to be solid tumors in certain situations. Ly6E-expressing cancers are typically solid tumors. Examples include, but are not limited to ovarian, colorectal, gastric, breast (including triple negative breast cancer), lung, bladder, brain and CNS, cervical, esophageal, head and neck, and pancreatic cancer. Certain embodiments of the present disclosure relate to methods of treating a Ly6E-expressing cancer with an anti-Ly6E antibody construct or ADC as described herein, for example an ADC of Formula (I), or the use of an anti-Ly6E antibody construct or ADC as described herein, for example an ADC of Formula (I), in a method of treating a Ly6E-expressing cancer, where the cancer is ovarian cancer, colorectal cancer, gastric cancer, breast cancer (including triple negative breast cancer), lung cancer, bladder cancer, brain and CNS cancer, cervical cancer, esophageal cancer, head and neck cancer or pancreatic cancer.

[0264] In certain embodiments of the present disclosure, when used in the treatment of cancer, the anti-Ly6E antibody constructs or ADCs as described herein, for example an ADC of Formula (I), may be administered systemically to the subject to be treated. In certain embodiments, when used in the treatment of cancer, the anti-Ly6E antibody constructs or ADCs as described herein, for example an ADC of Formula (I) may be administered to the subject locally at the site to be treated.68IPTS / 200345621.2

[0265] It is contemplated that the anti-Ly6E antibody constructs or ADCs comprising an anti-Ly6E antibody construct of the present disclosure may be used alone or in combination with one or more known chemotherapeutic or immunotherapeutic agents typically used in the treatment of cancer. Combinations of the anti-Ly6E antibody constructs or ADCs comprising an anti-Ly6 construct of the present disclosure with standard chemotherapeutics or immunotherapeutics may act to improve the efficacy of the chemotherapeutic or immunotherapeutic and, therefore, may improve standard cancer therapies. This application can be important in the treatment of drugresistant cancers which are not responsive to standard treatment. When used in conjunction with one or more known chemotherapeutic or immunotherapeutic agents, the anti-Ly6E antibody construct or ADC comprising an anti-Ly6E antibody construct of the present disclosure may be administered prior to, or after, administration of the chemotherapeutic or immunotherapeutic agents, or they may be administered concomitantly.

[0266] The dosage of the the anti-Ly6E antibody construct or ADC comprising an anti-Ly6E antibody construct of the present disclosure to be administered is not subject to defined limits, but it will be a therapeutically effective amount. A “therapeutically effective amount” refers to that amount of an anti-Ly6E antibody construct or ADC comprising an anti-Ly6 construct of the present disclosure which, when administered to a subject, is sufficient to effect a treatment of the particular indication. A therapeutically effective amount of anti-Ly6E antibody construct or ADC comprising an anti-Ly6E antibody construct of the present disclosure in respect of cancer treatment may, for example, have one or more of the following effects: reduce the number of cancer cells, reduce the tumor size, inhibit cancer cell infiltration into peripheral organs, inhibit tumor metastasis, inhibit tumor growth; increase survival time and / or relieve to some extent one or more of the symptoms associated with the cancer. For cancer therapy, efficacy may alternatively be measured, for example, by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

[0267] Certain aspects of the present disclosure relate to compositions for and methods of detecting the presence of Ly6E in a biological sample, such as a sample comprising cells or tissue, using an anti-Ly6E antibody construct of the present disclosure. In one embodiment, an anti-Ly6E antibody construct of the present disclosure is provided for use in a method of diagnosis or detection of presence of Ly6E. In some embodiments, in a method for detecting the presence of Ly6E in a biological sample, the biological sample may have been taken from a patient, for69IPTS / 200345621.2example, a patient known or suspected to have a cancer. Some embodiments relate to methods of detecting the presence of Ly6E in a biological sample that comprise contacting the sample with an anti-Ly6E antibody construct of the present disclosure. In some embodiments, the anti-Ly6E antibody constructs of the present disclosure are contacted with a biological sample under conditions that allow for binding of the anti-Ly6E antibody construct of the present disclosure to Ly6E present in the biological sample leading to formation of a complex and detecting the formation of such complex. Such method may be an in vitro method or in vivo method.

[0268] Certain aspects of the present disclosure relate to methods of diagnosing a disorder associated with increased expression of Ly6E, such as a cancer, using an anti-Ly6E antibody construct of the present disclosure. In some embodiments, the method of diagnosis may be an in vivo method in which the anti-Ly6E antibody construct of the present disclosure is administered to the subject, while in some embodiments it may be an in vitro method in which a sample taken from the subject is contacted with the anti-Ly6E antibody construct of the present disclosure. For in vivo methods, administration of the anti-Ly6E antibody construct may be systemic or local.

[0269] In certain embodiments, in methods of detecting the presence of Ly6E or diagnosing a disorder associated with increased expression of Ly6E, the anti-Ly6E antibody construct of the present disclosure may be labelled with a detectable label, such as a fluorescent, luminescent, chromophoric, chemiluminescent, radioactive, enzymatic or any other suitable label as is known in the art. In some embodiments, labelled anti-Ly6E antibody constructs are provided. The anti-Ly6E antibody construct of the present disclosure may be labelled with a detectable label, such as a fluorescent, luminescent, chromophoric, chemiluminescent, radioactive, electron-dense, enzymatic or any other suitable label as is known in the art.PHARMACEUTICAL COMPOSITIONS

[0270] For therapeutic use, the anti-Ly6E antibody constructs and ADCs as described herein, for example, an ADC of Formula (I), may be provided in the form of pharmaceutical compositions comprising the anti-Ly6E antibody construct or ADC as described herein, for example, an ADC of Formula (I) and a pharmaceutically acceptable excipient, for example, a carrier or diluent. Such pharmaceutical compositions may be prepared by procedures known in the art.

[0271] Pharmaceutical compositions may be formulated for administration to a subject by, for example, parenteral, oral (including, for example, buccal or sublingual), topical, rectal or vaginal70IPTS / 200345621.2routes, or by inhalation or spray. Parenteral administration may be subcutaneous injection, or intradermal, intra-articular, intravenous, intramuscular, intravascular, intrastemal or intrathecal injection or infusion. The pharmaceutical composition will typically be formulated in a format suitable for administration to the subject, for example, as a syrup, elixir, tablet, troche, lozenge, hard or soft capsule, pill, suppository, oily or aqueous suspension, dispersible powder or granule, emulsion, injectable or solution. Pharmaceutical compositions may be provided as unit dosage formulations.

[0272] In certain embodiments, pharmaceutical compositions comprising the anti-Ly6E antibody constructs or ADCs as described herein, for example, an ADC of Formula (I), may be formulated for parenteral administration by infusion or in a unit dosage injectable form, for example as lyophilized formulations or aqueous solutions.

[0273] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed. Examples of such carriers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkyl parabens (such as methyl or propyl paraben), catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin or gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes such as Zn-protein complexes, and non-ionic surfactants such as polyethylene glycol (PEG).

[0274] In certain embodiments, pharmaceutical compositions comprising the anti-Ly6E antibody constructs or ADCs as described herein, for example, an ADC of Formula (I), may be in the form of a sterile injectable aqueous or oleaginous solution or suspension. Such suspensions may be formulated using suitable dispersing or wetting agents and / or suspending agents that are known in the art. The sterile injectable solution or suspension may comprise the anti-Ly6E antibody construct or ADC as described herein, for example, an ADC of Formula (I), in a non-toxic parentally acceptable diluent or solvent. Acceptable diluents and solvents that may be employed71IPTS / 200345621.2include, for example, 1,3 -butanediol, water, Ringer’s solution or isotonic sodium chloride solution. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose, various bland fixed oils may be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Adjuvants such as local anesthetics, preservatives and / or buffering agents may also be included in the injectable solution or suspension.

[0275] In certain embodiments, pharmaceutical compositions comprising the anti-Ly6E antibody constructs or ADCs as described herein, for example, an ADC of Formula (I), may be formulated for intravenous administration to a subject, for example a human. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and / or a local anaesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0276] Other pharmaceutical compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in “ Remington: The Science and Practice of Pharmacy" (formerly “Remingtons Pharmaceutical Sciences”) Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).PHARMACEUTICAL KITS

[0277] Certain aspects of the present disclosure relate to pharmaceutical kits comprising an anti-Ly6E antibody construct or ADC as described herein, for example, an ADC of Formula (I).

[0278] In certain embodiments of the present disclosure, the kit typically will comprise a container holding the anti-Ly6E antibody construct or ADC as described herein, for example, an ADC of Formula (I) and a label and / or package insert on or associated with the container. The label or package insert contains instructions customarily included in commercial packages of therapeutic products, providing information about the indications, usage, dosage, administration,72IPTS / 200345621.2contraindications and / or warnings concerning the use of such therapeutic products. The label or package insert may further include a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, for use or sale for human or animal administration. In some embodiments, the container may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper that may be pierced by a hypodermic injection needle.

[0279] In addition to the container holding the anti-Ly6E antibody construct or ADC as described herein, for example, an ADC of Formula (I), the kit may optionally comprise one or more additional containers comprising other components of the kit. For example, a pharmaceutically acceptable buffer (such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer’s solution or dextrose solution), other buffers or diluents.

[0280] Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, and the like. The containers may be formed from a variety of materials such as glass or plastic. If appropriate, one or more components of the kit may be lyophilized or provided in a dry form, such as a powder or granules, and the kit can additionally contain a suitable solvent for reconstitution of the lyophilized or dried component(s).

[0281] The kit may further include other materials desirable from a commercial or user standpoint, such as fdters, needles, and syringes.CERTAIN EMBODIMENTS

[0282] The present disclosure also encompasses the following Embodiments 1-93.

[0283] Embodiment 1. An antibody construct comprising an antigen-binding domain that binds to human lymphocyte antigen 6 complex, locus E, protein (Ly6E), wherein the antigen-binding domain comprises complementarity determining region (CDR) sequences of a heavy chain variable domain (VH) comprising a sequence as set forth in any one of SEQ ID NOs: 4, 30, 31, 32, 33, 34, 35, 39, 40, 41 or 42, and CDR sequences of a light chain variable domain (VL) comprising a sequence as set forth in any one of SEQ ID NOs: 5, 36, 37 or 38.

[0284] Embodiment 2. The antibody construct according to Embodiment 1, wherein the antigen-binding domain comprises a heavy chain CDR 1 (HCDR1) comprising a sequence as set forth in SEQ ID NO: 17; a heavy chain CDR 2 (HCDR2) comprising a sequence as set forth in73IPTS / 200345621.2SEQ ID NO: 94; a heavy chain CDR 3 (HCDR3) comprising a sequence as set forth in SEQ ID NO: 14; a light chain CDR 1 (LCDR1) comprising a sequence as set forth in SEQ ID NO: 95; a light chain CDR 2 (LCDR2) comprising a sequence as set forth in SEQ ID NO: 26; and a light chain CDR 3 (LCDR3) comprising a sequence as set forth in SEQ ID NO: 24.

[0285] Embodiment 3. The antibody construct of Embodiment 1, wherein the antigen-binding domain comprises a heavy chain CDR 1 (HCDR1) comprising a sequence as set forth in any one of SEQ ID NOs: 9, 12, 15, 17 or 19; a heavy chain CDR 2 (HCDR2) comprising a sequence as set forth in any one of SEQ ID NOs: 10, 13, 16, 18, 20, 43, 44, 45, 63, 64, 72, 73, 74, 78, 80, 81, 82, 84, 85 or 86; a heavy chain CDR 3 (HCDR3) comprising a sequence as set forth in any one of SEQ ID NOs: 11, 14, 21, 47 or 46; a light chain CDR 1 (LCDR1) comprising a sequence as set forth in any one of SEQ ID NOs: 22, 25, 27, 48 or 66; a light chain CDR 2 (LCDR2) comprising a sequence as set forth in any one of SEQ ID NOs: 23, 26 or 28; and a light chain CDR 3 (LCDR3) comprising a sequence as set forth in any one of SEQ ID NOs: 24 or 29.

[0286] Embodiment 4. The antibody construct of Embodiment 1, wherein the antigen-binding domain comprises a heavy chain complementarity determining region (CDR) 1 (HCDR1) comprising the sequence as set forth in SEQ ID NO: 17, a heavy chain CDR 2 (HCDR2) comprising the sequence as set forth in SEQ ID NO: 18; a HCDR3 comprising the sequence as set forth in SEQ ID NO: 14; a light chain CDR 1 (LCDR1) comprising the sequence as set forth in SEQ ID NO: 25, a light chain CDR 2 (LCDR2) comprising the sequence as set forth in SEQ ID NO: 26, and a light chain CDR 3 (LCDR3) comprising the sequence as set forth in SEQ ID NO: 24.

[0287] Embodiment 5. The antibody construct of Embodiment 1, wherein the antigen-binding domain comprises a heavy chain complementarity determining region (CDR) 1 (HCDR1) comprising the sequence as set forth in SEQ ID NO: 17, a heavy chain CDR 2 (HCDR2) comprising the sequence as set forth in SEQ ID NO: 44; a HCDR3 comprising the sequence as set forth in SEQ ID NO: 14; a light chain CDR 1 (LCDR1) comprising the sequence as set forth in SEQ ID NO: 48, a light chain CDR 2 (LCDR2) comprising the sequence as set forth in SEQ ID NO: 26, and a light chain CDR 3 (LCDR3) comprising the sequence as set forth in SEQ ID NO: 24.74IPTS / 200345621.2

[0288] Embodiment 6. The antibody construct of any one of Embodiments 1 to 5, wherein the antigen-binding domain comprises a humanized version of the VH sequence as set forth in SEQ ID NO: 4 and a humanized version of the VL sequence as set forth in SEQ ID NO: 5.

[0289] Embodiment 7. The antibody construct of any one of Embodiments 1 to 6, wherein the antigen-binding domain comprises: (a) a VH sequence having at least 90% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 30, 31, 32, 33, 34, 35, 39, 40, 41 or 42; or (b) a VL sequence having at least 90% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 36, 37 or 38; or (c) a VH sequence as in (a) and a VL sequence as in (b).

[0290] Embodiment 8. The antibody construct of any one of Embodiments 1 to 7, wherein the antigen-binding domain comprises: (a) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 30 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 36; (b) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 30 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 37; (c) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 30 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 38; (d) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 31 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 36; (e) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 31 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 37; (f) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 31 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 38; (g) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 32 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 36; (h) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 32 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 37; (i) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 32 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 38; (j) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 33 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 36; (k) a VH sequence having at least 90% sequence identity to the sequence as set forth inSEQ ID NO: 33 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 37; (1) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 33 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 38; (m) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 34 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 36; (n) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 34 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 37; (o) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 34 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 38; (p) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 35 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 36; (q) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 35 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 37; (r) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 35 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 38; (s) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 39 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 37; (t) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 40 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 38; (u) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 41 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 38; or (v) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 42 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 38.

[0291] Embodiment 9. The antibody construct of any one of Embodiments 1 to 3, 5, 7 and 8, wherein the antigen-binding domain comprises a VH sequence as set forth in SEQ ID NO: 42.

[0292] Embodiment 10. The antibody construct of any one of Embodiments 1 to 3, 5 and 7 to 9, wherein the antigen-binding domain comprises a VL sequence as set forth in SEQ ID NO. 38.

[0293] Embodiment 11. The antibody construct according to any one of Embodiments 1 to 3, 5 and 7 to 10, wherein the antigen-binding domain comprises a heavy chain variable domain (VH)sequence as set forth in SEQ ID NO: 42 and a light chain variable domain (VL) sequence as set forth in SEQ ID NO: 38.

[0294] Embodiment 12. The antibody construct according to any one of Embodiments 1 to 11, wherein the antigen-binding domain is a Fab or an scFv.

[0295] Embodiment 13. The antibody construct according to any one of Embodiments 1 to 11, wherein the antigen-binding domain is a Fab.

[0296] Embodiment 14. The antibody construct of any one of Embodiments 1 to 13 further comprising a scaffold, wherein the antigen-binding domain is operably linked to the scaffold.

[0297] Embodiment 15. The antibody construct of Embodiment 14, wherein the scaffold comprises an IgG Fc region.

[0298] Embodiment 16. The antibody construct of Embodiment 14 or 15, wherein the scaffold comprises an IgGl Fc region.

[0299] Embodiment 17. The antibody construct of Embodiment 14 or 15, wherein the IgG Fc region comprises one or more amino acid modifications that reduce effector function.

[0300] Embodiment 18. The antibody construct of Embodiment 17, wherein the one or more amino acid modifications are L234A, L235A and D265S, wherein the numbering of residues is according to the EU numbering system.

[0301] Embodiment 19. The antibody construct of Embodiment 1, wherein the antibody construct comprises two heavy chains comprising the sequence as set forth in SEQ ID NO: 87 and two light chains comprising the sequence as set forth in SEQ ID NO: 68.

[0302] Embodiment 20. The antibody construct of Embodiment 1, wherein the antibody construct comprises two heavy chains comprising the sequence as set forth in SEQ ID NO: 88 and two light chains comprising the sequence as set forth in SEQ ID NO: 68.

[0303] Embodiment 21. The antibody construct of any one of Embodiments 1 to 18 further comprising a second antigen-binding domain.

[0304] Embodiment 22. The antibody construct of Embodiment 21, wherein the second antigen-binding domain binds to Ly6E.

[0305] Embodiment 23. The antibody construct of Embodiment 22, wherein both antigenbinding domains are the same.

[0306] Embodiment 24. The antibody construct of Embodiment 21, wherein the second antigen-binding domain binds to an antigen other than Ly6E.77IPTS / 200345621.2

[0307] Embodiment 25. The antibody construct of any one of Embodiments 1 to 24, wherein the antibody construct is internalized in a cell that expresses Ly6E upon binding to Ly6E.

[0308] Embodiment 26. A polynucleotide or set of polynucleotides encoding the antibody construct of any one of Embodiments 1 to 25.

[0309] Embodiment 27. An expression vector or set of expression vectors comprising the polynucleotide or set of polynucleotides of Embodiment 26.

[0310] Embodiment 28. A host cell comprising the polynucleotide or set of polynucleotides of Embodiment 26 or the expression vector or set of expression vectors of Embodiment 27.

[0311] Embodiment 29. A method of preparing the antibody construct of any one of Embodiments 1 to 25 comprising transfecting a host cell with the polynucleotide or set of polynucleotides according to claim 26 or the expression vector or set of expression vectors according to claim 27, and culturing the host cell under conditions suitable for expression of the antibody construct.

[0312] Embodiment 30. An antibody-drug conjugate comprising the antibody construct of any one of Embodiments 1 to 25 conjugated to one or more drug moieties.

[0313] Embodiment 31. The antibody-drug conjugate of Embodiment 30, wherein the antibody construct is conjugated to between 2 and about 8 drug moieties.

[0314] Embodiment 32. An antibody-drug conjugate having the formula A-(L-(D)m)n, wherein: A is the antibody construct according to any one of Embodiments 1 to 25; L is a linker; D is a drug moiety; m is between 1 and about 8; and n is between 1 and about 12.

[0315] Embodiment 33. The antibody-drug conjugate of Embodiment 32, wherein m is 1 or 2.

[0316] Embodiment 34. The antibody-drug conjugate of Embodiment 32 or 33, wherein n is between about 2 and about 8.

[0317] Embodiment 35. The antibody-drug conjugate of any one of Embodiments 32 to 34, wherein the drug moiety is a maytansinoid, maytansinoid analogue, benzodiazepine, pyrrolobenzodiazepine, duocarmycin, calicheamicin, calicheamicin analogue, auristatin, auristatin analogue, hemiasterlin, hemiasterlin analogue, tubulysin, tubulysin analogue, amatoxin, amatoxin analogue, camptothecin, camptothecin analogue, eribulin, TLR agonist or STING agonist.

[0318] Embodiment 36. The antibody-drug conjugate of any one of Embodiments 32 to 34, wherein the drug moiety is a camptothecin analogue.78IPTS / 200345621.2

[0319] Embodiment 37. The antibody-drug conjugate of any one of Embodiments 32 to 36, wherein the drug moiety has the formula:R4wherein R4is; Xaand Xbare each O, and R9is selected from: -H, -Ci-Cg hydroxyalkyl, -Ci-Cg aminoalkyl, -aminoaryl and -(Ci-Cg alkylj-aminoaryl.

[0320] Embodiment 38. The antibody construct of Embodiment 37, wherein R9is -Ci-Ce hydroxyalkyl.

[0321] Embodiment 39. The antibody-drug conjugate of any one of Embodiments 32 to 38, wherein the drug moiety has the structure:*oxO wherein * is the point of attachment to linker, L.

[0322] Embodiment 40. The antibody-drug conjugate of any one of Embodiments 32 to 39, wherein L is a cleavable linker.

[0323] Embodiment 41. The antibody-drug conjugate of Embodiment 40, wherein L is a protease cleavable linker.

[0324] Embodiment 42. The antibody-drug conjugate of Embodiment 41, wherein L comprises a dipeptide, tripeptide or tetrapeptide.

[0325] Embodiment 43. The antibody-drug conjugate of any one of Embodiments 40 to 42, wherein L has formula (III):79IPTS / 200345621.2(III)wherein Z is a linking group that joins the linker to a target group on the antibody construct, A; Str is a stretcher; AAi and AA2 are each independently an amino acid, wherein AAi-[AA2]rforms a protease cleavage site; X is a self-immolative group; q is 0 or 1; r is 1, 2 or 3; s is 0, 1 or 2; # is the point of attachment of the antibody construct A, and % is the point of attachment of the drug moiety, D.

[0326] Embodiment 44. The antibody-drug conjugate of any one of Embodiments 40 to 42, wherein L has formula (Illa):(Illa) wherein Z is a linking group that joins the linker to a target group on the anti-Ly6E antibody construct, A; Str is a stretcher; AAi and AA2 are each independently an amino acid, wherein AAi-[AA2]rforms a protease cleavage site; Y is -NH-CH2- or -NH-CH2-C(O)-; q is 0 or 1; r is 1, 2 or 3; v is 0 or 1; # is the point of attachment to the anti-Ly6E antibody construct, A, and % is the point of attachment to the drug moiety, D.

[0327] Embodiment 45. The antibody-drug conjugate of Embodiment 44, wherein v is 1.

[0328] Embodiment 46. The antibody-drug conjugate of Embodiment 44 or 45, wherein Y is -NH-CH2-.

[0329] Embodiment 47. The antibody-drug conjugate of any one of Embodiments 43 to 46,O Owherein Str is:(CH2)tcOr (CH2CH2°)u (CH2)t C

[0330] Embodiment 48. The antibody-drug conjugate of any one of Embodiments 32 to 34, wherein L-(D) is:80IPTS / 200345621.2wherein ** is the point of conjugation to the antibody construct A.

[0331] Embodiment 49. The antibody-drug conjugate of Embodiment 48, wherein m is 1.

[0332] Embodiment 50. The antibody-drug conjugate of Embodiment 48 or 49, wherein n is about 8.

[0333] Embodiment 51. A method of preparing the antibody-drug conjugate of any one of Embodiments 32 to 50 comprising conjugating drug-linker L-(D)mto the antibody construct A.

[0334] Embodiment 52. The method of Embodiment 51, wherein drug-linker L-(D)mis conjugated to native cysteine residues of the antibody construct A.

[0335] Embodiment 53. The method of Embodiment 51 or 52, wherein m is 1.

[0336] Embodiment 54. An antibody-drug conjugate having the structure:wherein n is about 8; and A is an antibody construct comprising an antigen-binding domain that binds to human lymphocyte antigen 6 complex, locus E, protein (Ly6E), wherein the antigen-binding domain comprises complementarity determining region (CDR) sequences of a heavy chain variable domain (VH) comprising a sequence as set forth in SEQ ID NO: 42 and CDR sequences of a light chain variable domain (VL) comprising a sequence as set forth in SEQ ID NO: 38.

[0337] Embodiment 55. The antibody-drug conjugate of Embodiment 54, wherein the antigenbinding domain comprises a heavy chain CDR 1 (HCDR1) comprising a sequence as set forth in SEQ ID NO: 17; a heavy chain CDR 2 (HCDR2) comprising a sequence as set forth in SEQ ID NO: 94; a heavy chain CDR 3 (HCDR3) comprising a sequence as set forth in SEQ ID NO: 14; a light chain CDR 1 (LCDR1) comprising a sequence as set forth in SEQ ID NO: 95; a light chain CDR 2 (LCDR2) comprising a sequence as set forth in SEQ ID NO: 26; and a light chain CDR 3 (LCDR3) comprising a sequence as set forth in SEQ ID NO: 24.

[0338] Embodiment 56. The antibody-drug conjugate of Embodiment 54, wherein the antigenbinding domain comprises a heavy chain CDR 1 (HCDR1) comprising the sequence as set forth in SEQ ID NO: 12, a heavy chain CDR 2 (HCDR2) comprising the sequence as set forth in SEQ ID NO: 43; a heavy chain CDR3 (HCDR3) comprising the sequence as set forth in SEQ ID NO: 14; a light chain CDR1 (LCDR1) comprising the sequence as set forth in SEQ ID NO: 48, a light chain CDR 2 (LCDR2) comprising the sequence as set forth in SEQ ID NO: 26, and a light chain CDR 3 (LCDR3) comprising the sequence as set forth in SEQ ID NO: 24.

[0339] Embodiment 57. The antibody-drug conjugate of any one of Embodiments 54 to 56, wherein the antigen-binding domain comprises a heavy chain variable domain (VH) sequence as set forth in SEQ ID NO: 42.

[0340] Embodiment 58. The antibody-drug conjugate of any one of Embodiments 54 to 57, wherein the antigen-binding domain comprises a light chain variable domain (VL) sequence as set forth in SEQ ID NO: 38.

[0341] Embodiment 59. The antibody-drug conjugate of any one of Embodiments 54 to 56, wherein the antigen-binding domain comprises a heavy chain variable domain (VH) sequence as set forth in SEQ ID NO: 42 and a light chain variable domain (VL) sequence as set forth in SEQ ID NO: 38.

[0342] Embodiment 60. The antibody-drug conjugate of any one of Embodiments 54 to 59, wherein the antigen-binding domain is a Fab.

[0343] Embodiment 61. The antibody-drug conjugate of any one of Embodiments 54 to 60, wherein the antibody construct further comprises an IgG Fc region.

[0344] Embodiment 62. The antibody-drug conjugate of Embodiment 61, wherein the IgG Fc region comprises one or more amino acid modifications that reduce effector function.82IPTS / 200345621.2

[0345] Embodiment 63. The antibody-drug conjugate of Embodiment 62, wherein the one or more amino acid modifications are L234A, L235A and D265S, wherein the numbering of residues is according to the EU numbering system.

[0346] Embodiment 64. The antibody-drug conjugate of any one of Embodiments 54 to 63 further comprising a second antigen-binding domain.

[0347] Embodiment 65. The antibody-drug conjugate of cl Embodiment 64, wherein the second antigen-binding domain binds to Ly6E.

[0348] Embodiment 66. The antibody-drug conjugate of Embodiment 65, wherein both antigen-binding domains are the same.

[0349] Embodiment 67. The antibody-drug conjugate of Embodiment 54, wherein the antibody construct comprises two heavy chains comprising the sequence as set forth in SEQ ID NO: 87 and two light chains comprising the sequence as set forth in SEQ ID NO: 68.

[0350] Embodiment 68. The antibody-drug conjugate of Embodiment 54, wherein the antibody construct A comprises two heavy chains comprising the sequence as set forth in SEQ ID NO: 88 and two light chains comprising the sequence as set forth in SEQ ID NO: 68.

[0351] Embodiment 69. A method of preparing the antibody-drug conjugate of any one of Embodiments 54 to 68 comprising conjugating a drug-linker having the structure:construct A.

[0352] Embodiment 70. The method of Embodiment 69, wherein the drug -linker is conjugated to native cysteine residues of the antibody construct A.

[0353] Embodiment 71. A pharmaceutical composition comprising the antibody construct of any one of Embodiments 1 to 31, and a pharmaceutically acceptable carrier or diluent.83IPTS / 200345621.2

[0354] Embodiment 72. An antibody construct of any one of Embodiments 1 to 31 for use in therapy.

[0355] Embodiment 73. The antibody construct for use according to Embodiment 72, wherein the therapy comprises treatment of cancer.

[0356] Embodiment 74. Use of an antibody construct of any one of Embodiments 1 to 31 in the manufacture of a medicament for treatment of cancer.

[0357] Embodiment 75. A method of inhibiting the growth of tumor cells comprising contacting the cells with an antibody construct of any one of Embodiments 1 to 31.

[0358] Embodiment 76. A method of treating a subject having a cancer comprising administering to the subject an effective amount of the antibody construct of any one of Embodiments 1 to 31.

[0359] Embodiment 77. The method of Embodiment 76, wherein the cancer is a Ly6E-positive cancer.

[0360] Embodiment 78. The method of Embodiment 77, wherein the Ly6E-positive cancer is selected from ovarian cancer, colon cancer, colorectal cancer, gastric cancer, breast cancer, lung cancer, bladder cancer, brain and CNS cancer, cervical cancer, esophageal cancer, head and neck cancer, tongue cancer or pancreatic cancer.

[0361] Embodiment 79. The method of any one of Embodiments 76 to 78 comprising administering an additional therapeutic agent to the subject.

[0362] Embodiment 80. A kit comprising the antibody construct of any one of Embodiments 1 to 31, and a label and / or package insert containing instructions for use.

[0363] Embodiment 81. A method of detecting human lymphocyte antigen 6 complex, locus E, protein (Ly6E) in a biological sample comprising contacting the biological sample with the antibody construct of any one of Embodiments 1 to 31 under conditions that allow for binding of the antibody construct to human Ly6E and detecting formation of a complex between the antibody construct and the human Ly6E in the biological sample.

[0364] Embodiment 82. The method of Embodiment 81, wherein the biological sample is an ovarian cancer sample, colon cancer sample, colorectal cancer sample, gastric cancer sample, breast cancer sample, lung cancer sample, bladder cancer sample, brain and CNS cancer sample, cervical cancer sample, esophageal cancer sample, head and neck cancer sample, or pancreatic cancer sample.84IPTS / 200345621.2

[0365] Embodiment 83. A method for detecting a Ly6E-positive cancer comprising administering an antibody construct of any one of Embodiments 1 to 31 labelled with a suitable label to a subject and detecting the labelled antibody construct in the subject, wherein the detection of the labelled antibody construct indicates a Ly6E-positive cancer in the subject.

[0366] Embodiment 84. A pharmaceutical composition comprising the antibody-drug conjugate of any one of Embodiments 30 to 50 and 54 to 68, and a pharmaceutically acceptable carrier or diluent.

[0367] Embodiment 85. An antibody-drug conjugate of any one of Embodiments 30 to 50 and 54 to 68 for use in therapy.

[0368] Embodiment 86. The antibody-drug conjugate for use according to Embodiment 85, wherein the therapy comprises treatment of cancer.

[0369] Embodiment 87. Use of an antibody-drug conjugate of any one of Embodiments 30 to 50 and 54 to 68 in the manufacture of a medicament for treatment of cancer.

[0370] Embodiment 88. A method of inhibiting the growth of tumor cells comprising contacting the cells with an antibody-drug conjugate of any one of Embodiments 30 to 50 and 54 to 68.

[0371] Embodiment 89. A method of treating a subject having a cancer comprising administering to the subject an effective amount of the antibody-drug conjugate of any one of Embodiments 30 to 50 and 54 to 68.

[0372] Embodiment 90. The method of Embodiment 89, wherein the cancer is a Ly6E-positive cancer.

[0373] Embodiment 91. The method of Embodiment 90, wherein the Ly6E-positive cancer is selected from ovarian cancer, colon cancer, colorectal cancer, gastric cancer, breast cancer, lung cancer, bladder cancer, brain and CNS cancer, cervical cancer, esophageal cancer, head and neck cancer, or pancreatic cancer.

[0374] Embodiment 92. The method of any one of Embodiments 89 to 91 comprising administering an additional therapeutic agent to the subject.

[0375] Embodiment 93. A kit comprising the antibody-drug conjugate of any one of Embodiments 30 to 50 and 54 to 68, and a label and / or package insert containing instructions of use.

[0376] The following Examples are provided for illustrative purposes and are not intended to limit the scope of the claimed invention in any way.85IPTS / 200345621.2EXAMPLESEXAMPLE 1: PREPARATION OF ANTI-LY6E ANTIBODIES

[0377] Antibodies that specifically bind Ly6E were generated by immunizing rabbits with human cells over-expressing Ly6E as summarized below.

[0378] HEK293-6E cells (National Research Council of Canada) were transiently transfected with pCMV3 based expression plasmid encoding human Ly6E (Sino Biological, Inc., Wayne, PA, expressing the sequences of Ly6E as set forth in SEQ ID NO: 1), according to manufacturer’s instructions for Lipofectamine 2000™ (Thermo Fisher Scientific Inc., Waltham, MA). Two New Zealand White rabbits were subcutaneously immunized with transfected HEK293-6E cells over 63 days, after which blood was drawn and spleens harvested.

[0379] Anti-human Ly6E antibody titers were determined by flow cytometry using streptavidin beads (Spherotech Inc., Green Oaks, IL) coated with rhuLy6E antigen (Abeam Inc., Cambridge, UK). Terminal bleed sera mounted a significant response against human Ly6E.

[0380] Immunized rabbits were sacrificed, and the spleens harvested and pooled. Splenocytes were used for B cell enrichment and sorted on a FACSAria™ cell sorter (Becton, Dickinson & Co., Franklin Lakes, NJ) into wells containing lysis buffer with a modified protocol based on the Selected Lymphocyte Antibody Method (SLAM) (Babcook et al., 1996, Proc Natl Acad Sci USA, 93(15):7843-7848).

[0381] Total RNA from wells containing a single B cell was used as template with SuperScript™ III (Thermo Fisher Scientific) and oligo-dT20 (Integrated DNA Technologies, Inc., Coralville, IA) to transcribe cDNA from mRNA. Initial PCR of heavy and light chain antibody-coding sequences was performed using primers and methods modified from Babcook et al., ibid.,' von Boehmer et al., 2016, NatProtoc., 11(10): 1908 and Peng etal., 2017, J. Mol. Biol, 429(19):2954-2973, with cDNA as the nucleic acid template. A subsequent PCR reaction was then performed on these unique sequences using V-segment family and J-segment family-specific primers and the resulting amplicons were cloned into pTT5-based expression plasmids (National Research Council of Canada). Unique heavy chain sequences and light chain sequences emerging from a single well sample were co-expressed in Expi293F™ cells (Thermo Fisher Scientific, Cat# A14527). Heavy and light chain PCR amplicons were sequenced using next generation sequencing (NGS)-based Amplicon-EZ and analyzed for unique antibody-coding sequences.86IPTS / 200345621.2

[0382] Cell supernatants containing secreted antibodies were assessed for human and cynomolgus monkey (cyno) Ly6E specificity by binding on CHO-S cells transiently transfected with a pCMV3 based plasmid encoding human or cyno Ly6E (Sino Biological). The anti-Ly6E antibody 10E02 was selected for further evaluation. Sequences of the rabbit heavy chain variable domain (VH) and rabbit light chain variable domain (VL) are provided in Table 1.1.Table 1.1: VH and VL Sequences for Anti-Ly6E Antibody 10E02 and Chimeric Antibody Variant v38866Description Sequence SEQ ID NO Rabbit VH QEQLEESGGDLVQPEGSLTLTCTA 4SGFDFSSNTIYWVRQAPGKGLEWI ACIYYGDGSTYYATWAKGRFTISK TSSTTVTLQMTSLTAADTATYFCA RDFKLWGPGTLVTISSRabbit VL GQVLTQTPSSVSAAVGGTVTINCQ 5SSESVYNNNWLAWFQQKPGQPPK LLIYMASILASGVPSRFSGSGSGTQ FTLTISGVQSDDAATYYCQGSYLS SGWYFTFGGGTEWVK

[0383] A rabbit-human chimeric IgGl / kappa anti-Ly6E antibody (also referred to herein as the “chimeric antibody” or the “chimeric anti-Ly6E antibody”), variant v38866, was generated as follows. Rabbit VH domain sequence (SEQ ID NO: 4) was appended to the human CHl-hinge-CH2-CH3 domain sequence of IGHGl*01 (SEQ ID NO: 6; see Table 4.3) to provide a rabbithuman chimeric full heavy chain sequence. Rabbit VL domain sequence (SEQ ID NO: 5) was appended to the human kappa CL sequence of IGKC*01 (SEQ ID NO: 7; see Table 4.3) to provide a rabbit-human chimeric light chain sequence. All sequences were reverse translated to DNA, codon optimized for mammalian expression and gene synthesized.

[0384] Heavy chain vector inserts comprising a signal peptide (artificially designed sequence: MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO: 8) (Barash et al., 2002, Biochem and Biophys Res. Comm., 294:835-842) and the heavy chain clone terminating at residue G446 (EU numbering) of the CH3 domain were ligated into apTT5 vector to produce heavy chain expression vectors. Light chain vector inserts comprising the same signal peptide were ligated into a pTT5 vector to produce light chain expression vectors. The resulting heavy and light chain expression vectors were sequenced to confirm correct reading frame and sequence of the coding DNA.87IPTS / 200345621.2

[0385] The full-size antibody, comprising two heavy chains and two light chains was expressed in ExpiCHO™ system. ExpiCHO™ cells were cultured at 37°C in ExpiCHO™ expression medium (Thermo Fisher Scientific) on an orbital shaker rotating at 120 rpm in a humidified atmosphere of 8% CO2. 100mL expression volumes were used. Each 1 mL of cells at a density of 6 x 106cells / mL was transfected with a total of 0.8 pg DNA. Prior to transfection, the DNA was diluted in 76.8 pL OptiPRO™ SFM (Thermo Fisher Scientific), after which 3.2 pL of ExpiFectamine™ CHO reagent (Thermo Fisher Scientific) was directly added to make a total volume of 80 pL. After incubation for 1-5 minutes, the DNA-ExpiFectamine™ CHO Reagent complex was added to the cell culture (80 µL complex per 1 mL of cell culture) and incubated in a 90 or 120 rpm shaking incubator at 37°C and 8% CO2. Following incubation at 37°C for 18-22 hours, 6 pL of ExpiCHO™ Enhancer and 240 pL of ExpiCHO™ Feed (Thermo Fisher Scientific) were added per ImL of culture. Cells were maintained in culture at 37°C for a total of 8 days, after which each culture was harvested by transferring into appropriately sized centrifuge tubes and centrifuging at 4200 rpm for 15 minutes. Supernatants were filtered using a 0.2 mm polyethersulfone membrane (Thermo Fisher Scientific), then analyzed by non-reducing SDS-PAGE and Octet™ (Sartorius Stedim Biotech., Germany).

[0386] Protein purification was performed in either batch mode or with the use of an AKTA Pure™ purification system (Cytiva, Marlborough, MA). In batch mode, supernatants from transient transfections were applied to slurries containing 50% MabSelect SuRe™ resin (Cytiva) and incubated at room temperature for 1 hr on an orbital shaker at 150 rpm. The slurries were transferred into chromatography columns and supernatants were allowed to flow through while resins remained in the column. The resins were then washed with at least 5 Bed Volumes (BV) of resin Equilibration Buffer (Gibco™ PBS, pH7.4). To elute the targeted proteins, 2.5 BV of Elution Buffer (100 mM sodium citrate buffer pH 3.5) was added to the columns and collected. Elutions were then neutralized by adding 16% (v / v) 1 M Tris pH 9.0 to reach a final pH of 5.5. In AKTA Pure™ purification mode, supernatants from transient transfections were loaded onto HiTrap MabSelect SuRE™ LX columns (Cytiva) that were pre -equilibrated with 5 Column Volume (CV) of PBS. After the proteins were captured, the columns were then washed with 10 CV of PBS. The captured proteins were eluted with 5 CV of Elution Buffer (100 mM sodium citrate buffer pH 3.5) in fractions. Pooled fractions were neutralized with 16% (v / v) if 1 M Tris pH 9.0. Samples werethen buffer exchanged into PBS buffer. The protein content of samples was determined by 280 nm absorbance measurement using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific).

[0387] The purity of protein samples was assessed by non-reducing and reducing LabChip™ capillary electrophoresis (CE)-SDS using LabChip™ GXII Touch (Perkin Elmer, Waltham, MA). Analysis was carried out according to Protein Express Assay User Guide (PerkinElmer), with the following modifications. Samples at a concentration range of 5-2000 ng / pL were added to separate wells in 96 well plates along with 7 pL of HT Protein Express Sample Buffer (Perkin Elmer; Cat. No. CLS920003,) and denatured at 90°C for 5 mins. The LabChip™ instrument was operated using the LabChip™ HT Protein Express Chip (Perkin Elmer; Cat. No. 760528) with HT Protein Express 200 assay setting.

[0388] Species homogeneity of the antibodies was assessed by UPLC-SEC after protein-A purification. UPLC-SEC was performed using an Agilent Technologies AdvanceBio™ SEC300A SEC column (7.8 x 150 mm, 1.7 pm particles) (Agilent Technologies Inc., Santa Clara, CA) set to 25°C and mounted on an Agilent Technologies 1260 Infinity II™ system with a diode array detector (DAD). Run times consisted of 7 min and a total volume per injection of 5 uL with a running buffer of PBS pH 7.4. Elution was monitored by UV absorbance in the range 190-400 nm, and chromatograms were extracted at 280 nm. Peak integration was performed using OpenLAB™ ChemStation™ (Agilent Technologies) chromatography data software.

[0389] The yield for v38866 was 29.6 mg with 95.2% purity by UPLC-SEC and 97.8% by Caliper (non-reducing, NR), reflective of high species homogeneity.EXAMPLE 2: FUNCTIONAL CHARACTERIZATION OF CHIMERIC ANTI-LY6E ANTIBODY - BINDING TO HUMAN, CYNOMOLGUS MONKEY AND MOUSE LY6E

[0390] The ability of chimeric anti-Ly6E antibody variant v38866 to bind to human, cynomolgus monkey and mouse Ly6E was assessed by flow cytometry using transfected CHO-S cells or LL / 2 cells (ATCC) as described below. LL / 2 cells express endogenous mouse Ly6E. hu9B12.vl2 (U. S. Patent No. 9,724,427) (v33706) was used as the benchmark antibody (positive control), and palivizumab (anti-RSV) (v22277) was used as a negative control. (In certain Examples, as disclosed herein, palivizumab variant v21995 is used instead of ll Jl. Variant v22277 includes a heterodimeric Fc, while v21995 includes a homodimeric Fc. For the purposes of this disclosure, as used herein, the two variants do not differ in their activity.)

[0391] Briefly, CHO-S cells were transfected for ~24 hours to transiently express human Ly6E, or cynomolgus monkey Ly6E, 0.5 ug DNA per 1 million cells, using the Neon™ Transfection System (Thermo Fisher Scientific). Cells were seeded at 50,000 cells / well in V-bottom 96-well plates and treated with antibody for 1 hour at 4°C to prevent internalization. Following incubation, cells were washed and stained with anti-human IgG Fc AF647 conjugate (Jackson Immuno Research Labs, West Grove, PA; Cat. No. 109-605-098) at 4°C for 30 min. Following incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) with 1,000 minimum events collected per well. The AF647 / APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-human AF647 binding) was calculated for the live singlet cell population for each primary antibody using FlowJo™ v8 software (BD Biosciences). The apparent KD of each primary antibody was calculated using GraphPad Prism Version 9 (GraphPad Software, San Diego, CA).

[0392] The results are shown in Table 2.1 and FIG. 1A (human Ly6E), FIG. IB (cynomolgus Ly6E), and FIG. 1C (mouse Ly6E). Variant v38866 showed binding to human and cynomolgus Ly6E on transfected CHO-S cells, and no binding to mouse Ly6E expressing LL / 2 cells. hu9B12.vl2 (v33706) displayed minimal binding to human and cynomolgus Ly6E and no binding to mouse Ly6E expressing LL / 2 cells. Further, the apparent KD value of variant v38866 was superior to that of hu9B12.vl2 (v33706) for both human and cynomolgus monkey Ly6E binding. No binding by negative control palivizumab (v22277) was observed on any species tested, as expected.Table 2.1: Binding to Human, Cynomolgus Monkey and Mouse Ly6EBmax (APC-A Geomean) Apparent KD (nM) Variant LL / 2 LL / 2 CHO-S Hu CHO-S Cyno CHO-S Hu CHO-S Cyno Mouse Mouse Ly6E Ly6E Ly6E Ly6ELy6E Ly6E v38866 2350 1215 IC 1.5 5.2 NB v33706 956 374 IC 78.6 17.3 NB v22277 NB NB NB NB NB NBIC = Incomplete CurveNB = No BindingEXAMPLE 3: FUNCTIONAL CHARACTERIZATION OF CHIMERIC ANTI-LY6E ANTIBODY - COMPETITION BINDING

[0393] To characterize the binding of chimeric anti-Ly6E antibody variant v38866 to Ly6E, competition binding (epitope binning) assays were carried out against the anti-Ly6E benchmark antibody hu9B12.v12 (v33706). Binding was assessed by flow cytometry using COV362 cells as described below. COV362 cells express endogenous human Ly6E at high levels.

[0394] Anti-Ly6E chimeric antibody variant v38866, the benchmark antibody hu9B12.v12 (v33706) and the negative control palivizumab (v22277) were conjugated with AF647 fluorophores using Zenon™ Human IgG labeling kit (ThermoFisher Scientific; Cat. No. Z25408). Each well of a V-bottom 96-well plate was seeded with 100,000 cells and incubated with 100 pg / mL of unlabeled competitor anti-Ly6E antibody for an hour on ice. Post incubation, 2.5pg / mL of AF647-conjugated anti-Ly6E detection antibody was spiked in and incubated for an hour on ice. Following staining and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences) with 10,000 minimum events collected per well. The AF647 / APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-human AF647 binding) was calculated using FlowJo™ Version 10.8.1 (BD Biosciences). Percentage competition was calculated using the following formula: / Competitor mAb Geomean% Competition = 100 — - - - - —— - x 100\ Irrelevant mAb Geomean

[0395] The results are shown in Table 3.1. Chimeric anti-Ly6E antibody variant v38866 competed against itself (96% inhibition, see data in bold text) as expected. No competition binding against negative control palivizumab (v22277) was observed.Table 3.1: Competition Binding - Percentage Inhibition (vs. Negative Control Antibody) % Inhibition vs. Negative Labeled Detection Antibody (2.5 pg / mL) Control Ab v38866 v33706 v22277v38866 96% 52% -2% UnlabeledCompetitor v33706 31% 70% 35% Antibody v22277 0% 0% 0%(100 pg / mL)No mAb -1% 14% 35%EXAMPLE 4: HUMANIZATION OF ANTI-LY6E ANTIBODY

[0396] The chimeric anti-human Ly6E antibody variant v38866, generated as described in Example 1, was humanized as described below. The CDR sequences of variant v38866 are provided in Table 4.1, and the rabbit VH and VL sequences are provided in Table 1.1.Table 4.1: CDR Sequences of Anti-Ly6E Antibody Variant v38866Numbering Heavy Chain SEQ Heavy Chain SEQ Heavy Chain SEQ System CDR1 ID NO CDR2 ID NO CDR3 ID NO IMGT GFDFSSNT 9 IYYGDGST 10 ARDFKL 11CIYYGDGSTYKabat SNTIY 12 13 DFKL 14YATWAKGChothia GFDFSSN 15 YYGDGS 16 DFKL 14 AbM GFDFS SNTIY 17 CIYYGDGSTY 18 DFKL 14WIACIYYGDGContact S SNTIY 19 20 ARDFK 21STYLight Chain SEQ Light Chain SEQ Light Chain SEQ CDR1 ID NO CDR2 ID NO CDR3 ID NO QGSYLSSG IMGT ESVYNNNW 22 MAS 23 24WYFT QSSESVYNN QGSYLSSGKabat 25 MASILAS 26 24NWLA WYFTChothia QSSESVYNN MASILAS QGSYLSSG25 26 24 NWLA WYFTAbM QSSESVYNN MASILAS QGSYLSSG25 26 24 NWLA WYFTContact YNNNWLA LLIYMASILA QGSYLSSG27 28 29 WF WYF4.1 Humanization

[0397] Sequence alignment of the rabbit VH and VL sequences of v38866 to respective human germline sequences identified IGHV3-23*01 and IGKV1-39*O1 as the closest, as well as most frequent, human germline sequences. Respectively, IGHJ4*01 and IGKJ4*01 joining region germline sequences were also selected. CDR sequences according to the AbM definition (see Table 4.1) were ported onto the framework of these selected human germline sequences as shown in FIG. 2. Back mutations to rabbit residues in the resultant sequences at positions judged likely to be important for the retention of binding affinity to antigen, Ly6E, were included creating92IPTS / 200345621.2several humanized sequences in which generated sequences built on the previous sequence, and where the first humanized sequence contained no back mutations.

[0398] This process resulted in six variable heavy chain humanized sequences (H1-H6) and three variable light chain humanized sequences (L1-L3). Sequences of the humanized heavy chain variable domains (VH) and humanized light chain variable domains (VL) are provided in Table 4.2. The CDR sequences as defined by the AbM numbering system for most of the humanized VH and VL sequences (FIG. 23), except as discussed herein, remained the same as the parental chimeric variant v38866 CDR sequences listed in Table 4.1. The humanized sequences L2 and L3 include a substitution of the first residue of CDR1 of the parental chimeric variant v38866 (as defined by the AbM numbering), glutamine to arginine, to mimic the most common residue in this position in human antibody sequences. The humanized sequence H4 includes a substitution of the first residue of CDR2 of the parental chimeric variant v38866 (as defined by the AbM numbering), cysteine to serine, to remove potential liability. When defined by other numbering systems, certain CDR sequences showed some differences to the parental chimeric variant v38866. The CDR sequences for each of the variable heavy chain humanized sequences and variable light chain humanized sequences shown in Table 4.2 are listed in FIG. 23.Table 4.2: Amino Acid Sequences of Humanized VH and VL DomainsChain Amino Acid Sequence SEQ ID NO EVQLLESGGGLVQPGGSLRLSCAASGFDFSSNTIYWVRQAPHl GKGLEWVSCIYYGDGSTYYADSVKGRFTISRDNSKNTLYLQ 30 MNSLRAEDTAVYYCAKDFKLWGQGTLVTVSS EVQLLESGGGLVQPGGSLRLSCAASGFDFSSNTIYWVRQAPH2 GKGLEWVSCIYYGDGSTYYADSVKGRFTISKDNSKNTVYL 31 QMNSLRAEDTAVYYCARDFKLWGQGTLVTVSS EVQLLESGGGLVQPGGSLRLSCAASGFDFSSNTIYWVRQAPH3 GKGLEWIACIYYGDGSTYYADSVKGRFTISKDNSKNTVYLQ 32 MNSLRAEDTAVYYCARDFKLWGQGTLVTVSS EVQLLESGGGLVQPGGSLRLSCAASGFDFSSNTIYWVRQAPH4 GKGLEWIASIYYGDGSTYYADSVKGRFTISKDNSKNTVYLQ 33 MNSLRAEDTAVYYCARDFKLWGQGTLVTVSS EEQLLESGGGLVQPGGSLRLSCAASGFDFSSNTIYWVRQAPH5 GKGLEWIACIYYGDGSTYYATWAKGRFTISKDSSKNTVYL 34QMNSLRAEDTAVYYCARDFKLWGQGTLVTISS93IPTS / 200345621.2Chain Amino Acid Sequence SEQ ID NO EVQLLESGGGLVQPGGSLRLSCAASGFDFSSNTIYWVRQAPH6 GKGLEWIACIYYGDGSTYYADSVKGRFTISKDSSNTVYLQM 35 NSLRAEDTAVYYCARDFKLWGQGTLVTVSS DIQMTQSPSSLSASVGDRVTITCQSSESVYNNNWLAWYQQ LI KPGKAPKLLIYMASILASGVPSRFSGSGSGTDFTLTISSLQPE 36 DFATYYCQGSYLSSGWYFTFGGGTKVEIK DIQLTQSPSSLSASVGDRVTITCRSSESVYNNNWLAWFQQKL2 PGKAPKLLIYMASILASGVPSRFSGSGSGTDFTLTISSLQPED 37 FATYYCQGSYLSSGWYFTFGGGTKVEIK GQQLTQSPSSVSASVGDRVTITCRSSESVYNNNWLAWFQQL3 KPGKAPKLLIYMASILASGVPSRFSGSGSGTDFTLTISSVQPE 38DAATYYCQGSYLSSGWYFTFGGGTKVEVK4.2 Production of Humanized Antibody Constructs

[0399] Full heavy chain sequences containing humanized heavy chain variable domain (VH) and hlgGl heavy chain constant domains (CHI, hinge, CH2, CH3), and full light chain sequence containing humanized light chain variable domain (VL) and human kappa light chain constant domain (kappa CL) were assembled. Monoclonal antibody (mAb) variants were then assembled such that each of the humanized heavy chains was paired with each of the humanized light chains to provide 18 humanized variants to be evaluated experimentally (v38810, v38811, v38812, v38813, v38814, v38815, v38816, v38817, v38818, v38819, v38820, v38821, v38822, v38823, v38824, v38825, v38826 and v38827). Each of the 18 humanized antibody constructs was produced in full-size antibody (FSA) format containing two identical full-length heavy chains and two identical kappa light chains. The VH and VL chain composition of each of the humanized variants is shown in Table 5.1 (Example 5). The CDR sequences of humanized VH and VL sequences are depicted in FIG. 23. Heavy and light chain sequences, as well as VH, VL and CDR sequences, for each of the 18 humanized variants are provided in Sequence Tables (see Table A, Table B).

[0400] The full-length heavy chain contained the human CHl-hinge-CH2-CH3 domain sequence of IGHGl*01 (SEQ ID NO: 6) and the light chain contained human kappa CL sequence of IGKC*01 (SEQ ID NO: 7) are provided in Table 4.3.Table 4.3: Human Constant Heavy and Light Chain Sequences94IPTS / 200345621.2Sequence SEQ ID NO CHl-hinge- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSCH2-CH3 WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT (IGHGl*01) YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN 6 GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG CL (kappa) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ (IGKC*01) WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE 7 KHKVYACEVTHQGLS SPVTKSFNRGEC

[0401] Each of the humanized VH domain sequences was appended to the human CHl-hinge-CH2-CH3 domain sequence of IGHGl*01 to provide six humanized full heavy chain sequences. Each of the humanized VL domain sequences was appended to the human kappa CL sequence of IGKC*01 to provide three humanized light chain sequences. All sequences were reverse translated to DNA, codon optimized for mammalian expression and gene synthesized.

[0402] Heavy chain vector inserts comprising a signal peptide (artificially designed sequence: MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO: 8) (Barash et al., 2002, Biochem and Biophys Res. Comm., 294:835-842) and the heavy chain clone terminating at residue G446 (EU numbering) of the CH3 domain were ligated into apTT5 vector to produce heavy chain expression vectors. Light chain vector inserts comprising the same signal peptide were ligated into a pTT5 vector to produce light chain expression vectors. The resulting heavy and light chain expression vectors were sequenced to confirm correct reading frame and sequence of the coding DNA.

[0403] The eighteen humanized antibodies were expressed in Expi293™ Expression System (Thermo Fisher Scientific). Expi293™ cells were cultured at 37°C in Expi293™ Expression Medium on an orbital shaker rotating at 120 rpm in a humidified atmosphere and 8% CO2.Expression was performed at 0.8 mL volume in a 96 deep well plate. Each 1 mb of cells at a density of 3 x 106cells / mL was transfected with a total of 1 pg DNA. Prior to transfection, the DNA was diluted to 0.1 ug / uL with distilled water, then in 60 pL Opti-MEM™ I Reduced Serum Medium (Thermo Fisher Scientific). In a volume of 56.8 pL Opti-MEM™ I Reduced Serum Medium, 3.2 pL of ExpiFectamine™ 293 Reagent (Thermo Fisher Scientific) was diluted and, after incubation for 3-5 minutes, combined with the diluted DNA to a total volume of 120 pL.95IPTS / 200345621.2After incubation for 10-20 minutes, the DNA-ExpiFectamine™ 293 Reagent complex was added to the cell culture (120 uL complex per 1 mL of cell culture) and incubated in a shaking incubator at 900 rpm, at 37°C and 8% CO2. 18-22 hours post transfection, 6 pL of ExpiFectamine™ 293 Transfection Enhancer 1 (Thermo Fisher Scientific) and 60 pL of ExpiFectamine™ 293 Transfection Enhancer 2 (Thermo Fisher Scientific) were added to the culture per 1 mL of the culture. Cells were maintained in culture at 37°C for 6 days following which supernatants were harvested by centrifuging the 96-deep well plate at 3500 rpm for 15 minutes. Following centrifugation, supernatants were aspirated off the cell pellets and transferred to a new plate. Analysis was performed with Octet™ (Sartorius Stedium Biotech). The titers ranged from ~40-330 mg / L for the supernatant material of 18 humanized antibody variants.

[0404] Selected variants v38811, v38812 and v38820 were further expressed in ExpiCHO™ system, purified and assessed by Caliper and UPLC-SEC. The same procedure as outlined in Example 1 was used with the difference that either 50 or 200ml volume of expression was used. Production and purification data is provided in Table 4.4. Caliper non-reduced (NR) profile (FIG.3) reflected predominantly single species corresponding to full-size antibody in the case ofv38812 and v38820 with lower yield in the case of v38811, and Caliper reduced ® profile (FIG. 3) reflected intact heavy and light chains for all three variants. Purity by UPLC-SEC as reported in Table 4.4 reflected high species homogeneity for all three variants.Table 4.4: Yield and Purity by UPLC-SECExpression pA UPLC-SEC Purity Variant Name pA Yield (mg)Volume (mL) (% monomer) v38811 10E02 H1 L2 200 0.19 100.0 v38812 10E02 H1 L3 200 3.12 100.0v38820 10E02 H4 L2 50 11.75 97.7EXAMPLE 5: FUNCTIONAL CHARACTERIZATION OF HUMANIZED ANTLLY6E ANTIBODY - BINDING TO HUMAN AND CYNOMOLGUS LY6E

[0405] The ability of humanized anti-Ly6E antibody variants from Example 4 to bind to human Ly6E and cynomolgus monkey Ly6E was assessed by flow cytometry using transfected CHO-S cells as described below. Benchmark antibody hu9B12.vl2 (v33706) was used as a positive control, and palivizumab (anti-RSV) (v22277) was used as a negative control.

[0406] CHO-S cells were transfected for ~24 hours to transiently express human Ly6E or cynomolgus monkey (cyno) Ly6E using 0.5 ug DNA per 1 million cells, using the Neon™ Transfection System (Thermo Fisher Scientific). Transfected cells were seeded at 50,000 cells / well in V-bottom 96-well plates and treated with antibody for 1 hour at 4°C to prevent internalization. Following incubation, cells were washed and stained with anti-human IgG Fc AF647 conjugate (Jackson Immuno Research Labs, West Grove, PA; Cat. No. 109-605-098) at 4°C for 30 min. Following incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences) with 1,000 minimum events collected per well. The AF647 / APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-Human AF647 binding) was calculated for the live singlet cell population for each primary antibody using FlowJo™ v8 software (BD Biosciences). The apparent Ka of each primary antibody was calculated using GraphPad Prism Version 9 (GraphPad Software, San Diego, CA).

[0407] The results of the single point data at 1 pg / ml are shown in Table 5.1. The benchmark antibody hu9B12.v12 (v33706) showed minimal binding to human Ly6E and cyno Ly6E on transfected CHO-S cells, with Geomean APC signals of 277 and 143, respectively. All humanized variants showed better binding to human Ly6E than the benchmark antibody hu9B 12.vl2 (v33706) and all humanized variants except v38810 and v38819 showed better binding to cyno Ly6E than the benchmark antibody. No binding by negative control palivizumab (v22277) was observed. Table 5.1: Single Point Binding of Chimeric and Humanized Anti-Ly6E Antibody Variants to Human and Cynomolgus Monkey Ly6EGeomean APCHeavy and Light (@ 1 pg / ml)Antibody VariantChain Combination CHO-S CHO-SHu Ly6E Cyno Ly6E v38866 Parental 3614 1208 v38810 Hl LI 1654 103 v38811 Hl L2 2917 697 v38812 Hl L3 3254 860 v38813 H2 L1 1528 242 v38814 H2 L2 1955 708 v38815 H2 L3 2396 860Geomean APCHeavy and Light (@ 1 ug / ml)Antibody VariantChain Combination CHO-S CHO-SHu Ly6E Cyno Ly6E v38816 H3 LI 1348 276 v38817 H3 L2 2597 742 v38818 H3 L3 2657 769 v38819 H4 L1 454 69 v38820 H4 L2 1656 410 v38821 H4 L3 1805 241 v38822 H5 LI 1522 341 v38823 H5 L2 2736 886 v38824 H5 L3 2882 841 v38825 H6 L1 1184 210 v38826 H6 L2 2737 790 v38827 H6 L3 2634 884 v33706 hu9B12.vl2 277 144

[0408] The dose-response binding for selected humanized anti-Ly6E antibody variants is shown in Table 5.2, and FIG. 4A (human Ly6E) and FIG. 4B (cynomolgus monkey Ly6E). The benchmark antibody hu9B12.v12 (v33706), used as apositive control, showed minimal binding to human Ly6E and cynomolgus monkey Ly6E on transfected CHO-S cells. No binding by negative control palivizumab (v22777) was observed. Humanized variants v38811 and v38812 showed similar binding to parental chimera v38866, yielding KD values within 2-fold and comparable Bmax values.Table 5.2: Dose-Response Binding of Chimeric and Humanized Anti-Ly6E Antibody Variants to Human and Cynomolgus Monkey Ly6EHeavy and Light Bmax KD (nM) Antibody ChainVariant Combination CHO-S Hu CHO-S Cyno CHO-S Hu CHO-S Cyno Ly6E Ly6E Ly6E Ly6E v38866 Parental 10577 5698 0.25 1.92 v38811 Hl L2 11876 4027 0.52 3.4898IPTS / 200345621.2Heavy and Light Bmax KD (nM) Antibody ChainVariant Combination CHO-S Hu CHO-S Cyno CHO-S Hu CHO-S Cyno Ly6E Ly6E Ly6E Ly6E v38812 Hl L3 11201 4907 0.24 2.50 v38820 H4 L2 6897 3360 1.38 32.43 v33706 hu9B12.vl2 IC IC IC IC v22277 palivizumab NB NB NB NBIC = Incomplete CurveNB = No BindingEXAMPLE 6: OPTIMIZATION OF HUMANIZED ANTI-LY6E ANTIBODY

[0409] As the presence of cysteine as the first residue of CDR2 in the humanized variable heavy chains H1-H3, H5 and H6 (see Table 4.1 and Table 4.2) creates a potential liability, even though this cysteine residue is mostly buried, additional humanized variable heavy chain sequences were generated based on the Hl sequence in which this cysteine was substituted with an alternative residue. Valine, threonine and alanine were each utilized to generate three optimized humanized variable heavy chains, Hl.l, Hl.2 and Hl.3, respectively. An additional modified humanized variable heavy chain sequence Hl.0 was generated by substitution of cysteine to serine at the first residue of CDR2 in the Hl sequence to serve as a comparator to the previously evaluated H4 sequence which also comprises a serine substitution at this position (see Table 4.1 and Table 4.2; see Example 4). The sequences of the resultant optimized (modified) humanized VH domains are provided in Table 6.1 with the cysteine substitution shown in bold and underline.Table 6.1: Amino Acid Sequences of Optimized Humanized VH DomainsHeavyAmino Acid Sequence of VH SEQ ID NO ChainH1.0 EVQLLESGGGLVQPGGSLRLSCAASGFDFSSNTIYWVRQ APGKGLEWVSSIYYGDGSTYYADSVKGRFTISRDNSKNT 39 (Cys to Ser) LYLQMNSLRAEDTAVYYCAKDFKLWGQGTLVTVS SHl.l EVQLLESGGGLVQPGGSLRLSCAASGFDFSSNTIYWVRQ APGKGLEWVSVIYYGDGSTYYADSVKGRFTISRDNSKNT 40 (Cys to Vai) LYLQMNSLRAEDTAVYYCAKDFKLWGQGTLVTVS S99IPTS / 200345621.2HeavyAmino Acid Sequence of VH SEQ ID NO ChainHl.2 EVQLLESGGGLVQPGGSLRLSCAASGFDFSSNTIYWVRQ APGKGLEWVSTIYYGDGSTYYADSVKGRFTISRDNSKNT 41 (Cys to Thr) LYLQMNSLRAEDTAVYYCAKDFKLWGQGTLVTVS SHl.3 EVQLLESGGGLVQPGGSLRLSCAASGFDFSSNTIYWVRQ APGKGLEWVSAIYYGDGSTYYADSVKGRFTISRDNSKNT 42 (Cys to Ala) LYLQMNSLRAEDTAVYYCAKDFKLWGQGTLVTVS S

[0410] Optimized humanized full-length heavy chains were assembled as previously described in Example 4. The antibody variant v38812 comprising the H1L3 combination was selected as the lead humanized variant, and so each of the three optimized humanized heavy chains (comprising the humanized variable heavy chains Hl.l, Hl.2 and Hl.3) was paired with a full- length kappa light chain comprising the humanized variable light chain L3 (SEQ ID NO: 38; see Table 4.2) to generate three optimized humanized antibody variants, v39334 (Hl.l L3), v39335 (Hl.2 L3) and v39336 (Hl.3 L3). Modified humanized variable heavy chain H1.0 was paired with L2 (SEQ ID NO: 37; see Table 4.2) to generate a comparator variant, v39333 (H1.0 L2), to that of the humanized H4L2 variant and used as the control for the optimized humanized variants. The CDR sequences for the optimized heavy chains Hl.l, Hl.2 and Hl.3, as well as the CDR sequences for the modified heavy chain H1.0, are shown in FIG. 23. Complete heavy and light chain CDR sequences for v39336 (Hl.3 L3) are shown in Table 6.2.Table 6.2: CDR Sequences of Optimized Humanized Anti-Ly6E Antibody Variant v39336 Numbering Heavy Chain SEQ Heavy Chain SEQ Heavy Chain SEQ System CDR1 ID NO CDR2 ID NO CDR3 ID NO IMGT GFDFSSNT 9 IYYGDGST 10 AKDFKL 47AIYYGDGSTYKabat SNTIY 12 43 DFKL 14YADSVKGChothia GFDFSSN 15 YYGDGS 16 DFKL 14 AbM GFDFS SNTIY 17 AIYYGDGSTY 44 DFKL 14WVSAIYYGDGContact S SNTIY 19 45 AKDFK 46STYLight Chain SEQ Light Chain SEQ Light Chain SEQ CDR1 ID NO CDR2 ID NO CDR3 ID NO100IPTS / 200345621.2QGSYLSSGIMGT ESVYNNNW 22 MAS 23 24 WYFT RSSESVYNN QGSYLSSGKabat 48 MASILAS 26 24 NWLA WYFTChothia RSSESVYNN MASILAS QGSYLSSG48 26 24 NWLA WYFTAbM RSSESVYNN MASILAS QGSYLSSG48 26 24 NWLA WYFT YNNNWLA QGSYLSSGContact 27 LLIYMASILA 28 29 WF WYF

[0411] An additional optimized humanized anti-Ly6E antibody variant, v41252, was generated with Fc knockout mutations at positions L234A, L235A and D265S (EU numbering) of the Fc region (referred to herein as “LALADS Fc”). Particularly, the optimized humanized full length heavy chain “Hl.3 LALADS” was assembled by appending optimized humanized Hl.3 VH to the human CHl-hinge-CH2-CH3 domain sequence of IGHGl*01 containing mutations L234A, L235A and D265S (see Table 6.3). The resultant heavy chain was paired with the full-length kappa light chain comprising L3 (as previously described in Example 4) to generate Fc-silent antibody variant, v41252.Table 6.3: Human Constant Heavy Chain Sequence with Mutations L234A, L235A and D265SName Sequence SEQ ID NO CH 1 -hinge - ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSCH2-CH3 WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT (IGHG1*O1) YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGwith PSVFLFPPKPKDTLMISRTPEVTCVWSVSHEDPEVKFNWYL234A, VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG 49 L235A, KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDED265S LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV mutations LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0412] The humanized anti-Ly6E antibody variants v39333, v39334, v39335, v39336 and v41252 were produced as described previously in Example 1, with expression volume of 200 ml or in the case of v41252 with expression volume of 750 ml. They were purified and assessed by Caliper and UPLC-SEC as described previously (see Example 1). Production and purification data101IPTS / 200345621.2are provided in Table 6.4. Caliper NR profile reflected single species corresponding to full-size antibody and Caliper R profile reflected intact heavy and light chains in the case of all variants (FIG. 5A and FIG. 5B). Purity by UPLC-SEC reflected high species homogeneity (see Table 6.4).Table 6.4: Yield and Purity by UPLC-SECAntibody Heavy and Light Expression pA Yield pA UPLC-SEC Variant Chain Combination Volume (mL) (mg) Purity (% monomer) v39333 H1.0 L2 200 20.19 100.0 v39334 Hl.l L2 200 27.34 100.0 v39335 Hl.2 L3 200 27.75 100.0 v39336 Hl. 3 L3 200 25.23 100.0 v41252 Hl.3 L3 LALADS 750 93 97.4EXAMPLE 7: FUNCTIONAL CHARACTERIZATION OF OPTIMIZED HUMANIZED ANTI-LY6E ANTIBODIES - BINDING TO HUMAN AND CYNOMOLGUS LY6E

[0413] The ability of optimized humanized anti-Ly6E antibody variants from Example 6 to bind to human and cynomolgus monkey Ly6E was assessed by flow cytometry using transfected CHO-S cells and stable cell lines as described below. Benchmark antibody hu9B12.vl2 (v33706) was used as a positive control and palivizumab (anti-RSV) (v21995 or v22277) was used as a negative control.

[0414] CHO-S cells were transfected for ~24 hours to transiently express human Ly6E or cynomolgus monkey Ly6E using 0.5 pg DNA per 1 million cells, using the Neon™ Transfection System (Thermo Fisher Scientific). Separately, stable cell lines were generated in HEK293-6e cells using lentiviral vector containing human Ly6E or cynomolgus monkey Ly6E with puromycin selection. Lentiviral vectors for human Ly6E (Vector ID: VB230830-1669qbc) and cynomolgus monkey Ly6E (Vector ID: VB230827-1519mrk) sequences were generated, and lentivirus particles were produced at VectorBuilder™ (VectorBuilder Inc., Germany). For stable expression of human or cynomolgus monkey Ly6E, HEK293-6E cells (National Research Council of Canada) were plated in media containing 8 pg / mL polybrene and transduced with the corresponding lentivirus at a multiplicity of infection (MOI) < 0.6. Virus was removed 24 hours post-infectionand Ly6E-expressing cells were selected with media supplemented with 4 pg / mL puromycin for 48 hours.

[0415] Cells were seeded at 50,000 cells / well in V-bottom 96-well plates and treated with antibody for 1 hour at 4°C to prevent internalization. Following incubation, cells were washed and stained with anti-human IgG Fc AF647 conjugate and the AF647 / APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-Human AF647 binding) and apparent KD of each primary antibody were calculated as described in Example 5.

[0416] The results are shown in Table 7.1 and FIG.6A (hu Ly6E CHO-S), FIG. 6B (cyno Ly6E CHO-S), FIG. 6C (hu Ly6E HEK293-6e), and FIG. 6D (10E02; cyno Ly6E HEK293-6e). The benchmark antibody variant v33706 showed minimal binding to human Ly6E and cynomolgus monkey Ly6E on both cell lines. No binding by control v22277 or v21995 was observed. (v21995 was used for CHO-S studies, while v22277 was used for HEK293-6e studies.) Optimized humanized antibody variants bound similarly to human Ly6E compared to the parental chimeric antibody variant v38866, yielding KD values within 2-fold. Humanized variant v39336 bound similarly to cynomolgus Ly6E compared to the parental chimeric antibody variant v38866, yielding KD values within 2-fold. Variant v41252 bound similarly to human and cynomolgus Ly6E compared to variant v39336, yielding comparable KD and Bmax values.Table 7.1: Single Point Binding of Chimeric and Optimized Humanized Anti-Ly6E Antibody Variants to Human and Cynomolgus Monkey Ly6EHeavy and Transient Transfection (CHO-S) Stable Cell Line (HEK293-6e) Antibody Light Chain Hu Cyno Hu Cyno Hu Cyno Hu Cyno Variant Combination Ly6E Ly6E Ly6E Ly6E Ly6E Ly6E Ly6E Ly6E Bmax Bmax KD(nM) KD(nM) Bmax Bmax KD(nM) KD(UM) v38866 Parental 4332 2986 0.10 1.10 NT NT NT NT v39333 H1.0 L2 2458 1358 0.15 12.29 NT NT NT NT v39335 Hl.2 L3 3036 1958 0.22 15.38 NT NT NT NT v39336 Hl.3 L3 3437 2527 0.05 0.44 37671 28392 4.78 3.17 Hl.3 L3v41252 NT NT NT NT 37921 29273 3.17 2.23 LALADSv33706 hu9B12.vl2 IC IC IC IC IC IC IC ICv21995or palivizumab IC IC IC IC IC IC IC IC v21995NT = Not TestedIC = Incomplete CurveEXAMPLE 8: QUANTIFICATION OF SURFACE LY6E ON CANCER CELL LINES

[0417] Surface Ly6E protein was measured on various tumor cell lines by quantitative flow cytometry using a set of beads with known levels of antibody binding capacity (ABC) as described below. A high-affinity anti-Ly6E chimeric antibody conjugated to Alexa Fluor® AF647 and control antibody palivizumab (anti-RSV) (v21995) conjugated to Alexa Fluor® AF647 were used to fluorescently label tumor cells and beads.

[0418] Conjugations of a high-affinity anti-Ly6E chimeric antibody and variant v21995 to Alexa Fluor® AF647 were performed as follows: high-affinity anti-Ly6E chimeric antibody and variant v21995 were each reacted with 8 equivalents of NHS-AF647 (Thermo Fisher Scientific; Cat. No. A20006, 10 mM) in PBS. The reaction was protected from light at room temperature and was allowed to proceed for 200 and 150 minutes, respectively. Following incubation, the reactions were purified through four and two rounds of purification, respectively, using a 40 kDa Zeba™ column (Thermo Fisher Scientific) pre-equilibrated with PBS, pH 7.4. Confirmation of conjugation and quantification of unconjugated NHS-AF647 were measured by SEC chromatography (Ex: 650 nm, Em: 665 nm).

[0419] Briefly, cells were detached from culture vessels using Cell Dissociation Buffer (Invitrogen, Waltham, MA) and seeded at 50,000 cells / well in conical-bottom 96-well plates in triplicate. Cells and anti-Human Quantum™ Simply Cellular® beads (Bangs Laboratories, Inc., Fishers, IN) were stained with high-affinity anti-Ly6E chimeric antibody-AF647 with a predetermined excess level of conjugated antibody or with the negative control v21995-AF647 at the same concentration and incubated for 30 minutes at 4°C. Following incubation, cells and beads were washed in FACS buffer and analyzed on the BD™ Fortessa HTS and processed using FLOWJOTMV8 software (BD Biosciences).

[0420] The median AF647 fluorescence intensity for all bead populations were plotted against relevant ABC values using Bangs Laboratories QuickCal® v 2.3 calibration line template for Quantum™ Simply Cellular® anti-Human IgG (Lot # 14490).

[0421] Surface receptor protein expression for cell lines was calculated based on a monovalent binding model and therefore equivalent to the background subtracted ABC (SABC). The median fluorescence intensity of v21995-AF647-stained cells from each of the respective cell lines was used as a background value for determination of SABC.

[0422] Tumor cell lines were designated as having high, medium, low, or negative expression of Ly6E based on the average number of Ly6E proteins detected per cell. Results are shown in Table 8.1.Table 8.1 Surface Ly6E Quantification on Tumor Cell LinesCell Line Tissue and Disease Designation ABC-1 Lung adenocarcinoma High HCC1569 Breast carcinoma High MX-1 Breast carcinoma High PK-8 Pancreatic adenocarcinoma High SW-900 Lung carcinoma Mid MKN74 Gastric adenocarcinoma Mid MKN7 Gastric adenocarcinoma Mid SNU-216 Gastric adenocarcinoma Mid BxPC3 Pancreatic adenocarcinoma Mid Pane 03.27 Pancreatic adenocarcinoma Mid SU.86.86 Pancreatic adenocarcinoma Mid PK-59 Pancreatic adenocarcinoma Mid HPAF-II Pancreatic adenocarcinoma Mid RT112 / 84 Bladder carcinoma Mid NCI-H1650 Lung Adenocarcinoma MidCAL27 Tongue carcinoma Mid SCC-9 Head and neck squamous cell carcinoma Mid DETROIT-562 Head and neck squamous cell carcinoma MidNCI-H2228 Lung Adenocarcinoma LowDU4475 Breast carcinoma Low NCI-H446 Ovarian adenocarcinoma Low105IPTS / 200345621.2Cell Line Tissue and Disease DesignationAGS Gastric adenocarcinoma Low Capan-1 Pancreatic adenocarcinoma Low N87 Gastric carcinoma Low FaDu Head and neck squamous carcinoma Low RPMI2650 Head and neck squamous carcinoma LowSK-CO-1 Colorectal adenocarcinoma NegativeEXAMPLE 9: FUNCTIONAL CHARACTERIZATION OF ANTLLY6E ANTIBODY VARIANTS - CELL BINDING BY FLOW CYTOMETRY

[0423] The ability of the optimized, humanized antibody variants v39336 (with wild-type Fc) and v41252 (with LALADS Fc) to bind to Ly6E expressed on cancer cell lines was assessed on the endogenous Ly6E-expressing cell lines HCC1569, MX-1, NCI-H1650, NCI-H446 and Ly6E-negative cell line SK-CO-1 by flow cytometry.

[0424] Briefly, cells were seeded at 50,000 cells / well in conical -bottom 96-well plates and treated with test antibody for 24 hours at 4°C to prevent internalization. Following incubation, cells were washed and stained with anti-Human IgG Fc AF647 conjugate and the AF647 / APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-Human AF647 binding) was calculated as described in Example 5.

[0425] Results are shown in Table 9.1, and FIG. 7A (HCC1569), FIG. 7B (MX-1), FIG. 7C (NCI-H1650), FIG. 7D (NCI-H446) and FIG. 7E (SK-CO-1, Ly6E-negative). The optimized, humanized anti-Ly6E variants v39336 and v41252 bound to cells similarly yielding comparable KD and Bmax values in all Ly6E-expressing cancer cell lines, and did not bind to the Ly6E-negative cell line SK-CO-1, indicating Ly6E-specific binding of v39336 and v41252. Benchmark antibody hu9B12.vl2 (v33706) showed higher KD values and lower Bmax values in all Ly6E-expressing cell lines compared to variants v39336 and v41252. These results demonstrated superiority in binding capability of variants v39336 and v41252 when compared to the benchmark. Negative control palivizumab (v21995) showed no cellular binding, as expected.Table 9.1: Cellular Binding of Optimized, Humanized Anti-Ly6E Antibody VariantsCancer cell line Test Antibodyv39336 v41252 v33706 v21995 (Ly6E expression#) VariantBmax 22,677 25,659 6,204 NB HCC1569KD(nM) 1.53 1.93 6.03 NB (High)Curve Hill Slope (h) 1.0 1.5 0.7 NB Bmax 7,790 7,807 4,013 NB MX-1KD(nM) 0.20 0.27 >100* NB (High)Curve Hill Slope (h) 1.0 1.0 0.5 NB Bmax 6,480 6,817 3,089 NB H1650KD (nM) 0.24 0.32 39.49 NB (Mid)Curve Hill Slope (h) 1.2 1.3 0.6 NB Bmax 3,652 3,710 NB NB H446KD (nM) 0.11 0.16 >100* NB (Low)Curve Hill Slope (h) 0.9 0.8 0.4 NB Bmax 16 20 NT NB SK-CO-1KD (nM) >100* >100* NT NB (Negative)Curve Hill Slope (h) NB NB NT NB*Apparent Kd value greater than the highest antibody testing concentration (>100 nM)#Determined as described in Example 8NB = No Binding; NT = Not TestedEXAMPLE 10: FUNCTIONAL CHARACTERIZATION OF ANTI-LY6E ANTIBODY VARIANTS - INTERNALIZATION

[0426] Internalization of the optimized, humanized anti-Ly6E antibody variants v33936 (with wild-type Fc) and v41252 (with LALADS Fc) in stably transfected HEK cells over-expressing human Ly6E (huLy6E OE HEK) or over-expressing cyno Ly6E (cynoLy6E OE HEK) as well as Ly6E-expressing cancer cell lines (HCC1569 and NCI-H1650) was determined by flow cytometry107IPTS / 200345621.2as described below. The Ly6E-targeting antibody hu9B12.v 12 (v33706) was used as a comparator, and palivizumab (anti-RSV) (v21995) was used as a negative control.

[0427] Briefly, antibodies were fluorescently labeled by coupling to an anti-human IgG Fab fragment AF488 conjugate (Jackson ImmunoResearch Labs; Cat. No. 109-547-008) at a 1: 1 molar ratio in PBS pH 7.4 (Thermo Fisher Scientific; Cat. No. 10010-023), for 24 hours at 4°C. Cells were seeded at 50,000 cells / well in Freestyle 293 (Thermo Fisher Scientific) supplemented with 1% fetal bovine serum (Thermo Fisher Scientific) and 1% Penicillin Streptomycin (Thermo Fisher Scientific) (huLy6E OE HEK and cynoLy6E OE HEK) or RPMI 1640, ATCC modification (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (HCC1569 and NCI-H1650) in 48-well plates and incubated overnight under standard culturing conditions (37°C / 5% CO2) to allow attachment. Coupled antibodies were added to cells the following day at 10 nM and incubated under standard culturing conditions for 24 hours to allow for internalization. Following incubation, cells were dissociated, washed, and surface AF488 fluorescence was quenched using an anti-AF488 antibody (Life Technologies, Carlsbad, CA; Cat. No. A-11094) at 100 nM for 30 minutes at 4°C. Quenched AF488 fluorescence (internalized fluorescence) was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences) with 1,000 minimum events collected per well. The AF488 / FITC-A GeoMean (fluorescence signal geometric mean, proportional to anti -Human Fab AF488 labelling) was calculated for the live single cell population using FlowJo™ Version 10.8.1 (BD Biosciences) and plotted using GraphPad Prism Version 10 (GraphPad Software).

[0428] The results are shown in Table 10.1 and FIG. 8A (Human and Cyno Ly6E OE HEK) and FIG.8B (HCC1569 and NCI-H1650). Optimized, humanized anti-Ly6E antibody variants v39336 and v41252 demonstrated comparable levels of internalization and much greater levels of internalization than comparator antibody hu9B12.vl2 (v33706) at 10 nM antibody treatment for both huLy6E and cynoLy6E OE HEK and for both Ly6E-expressing cancer cell lines HCC1569 and NCI-H1650. As shown in Table 10.1, following a 24-hour incubation with HCC1569, v39336 and v41252 showed 15.8 and 16.0-fold increase in internalized fluorescence compared to negative control palivizumab (v21995), respectively; while hu9B12.vl2 (v33706) showed only 1.8-fold increase in internalized fluorescence compared to negative control palivizumab (v21995).Table 10.1: Internalization of Anti-Ly6E Antibody VariantsInternalized Fluorescence Fold-Over Palivizumab Variant (10 nM) HuLy6E OE Cyno Ly6E OEHCC1569 H1650 HEK HEKv39336 130.9 92.2 15.8 10.8 v41252 132.7 85.1 16.0 13.6 v33706 (hu9B12.vl2) 8.3 1.9 1.8 1.7 v21995 (palivizumab) 1.0 1.0 1.0 1.0EXAMPLE 11: FUNCTIONAL CHARACTERIZATION OF ANTLLY6E ANTIBODY VARIANTS - CELL BINDING BY KINEXA

[0429] The binding affinity of optimized, humanized anti-Ly6E antibody variant v39336 was assessed by KinExA™ in stably transfected human and cyno Ly6E over-expressing HEK-293e cells (huLy6E OE HEK and cynoLy6E OE HEK) as described below.

[0430] HuLy6E OE HEK cells and cynoLy6E OE HEK cells were cultured in Freestyle 293 (Thermo Fisher Scientific) supplemented with 1% FBS (Thermo Fisher Scientific) and 1% Penicillin Streptomycin (Thermo Fisher Scientific) in T 175 culture flasks (Coming, Coming, NY) and incubated at 37°C with 5 % CO2 until achieving 80% confluency. Cells were harvested and maintained on ice until use. Cells were counted using the Vi-Cell™ XR Cell Viability Analyzer (Beckman Coulter, Brea, California).

[0431] The solid phase was prepared by coating one vial of PMMA (polymethyl methacrylate) beads (Sapidyne, Boise, Idaho) with 1 mL of 20 pg / mL BSA-biotin (Sigma-Aldrich, St. Louis, Missouri) in PBS pH 7.4. The beads were incubated for 2 hours at room temperature with gentle rotation. Beads were settled, supernatant was removed, and beads were rinsed five times with PBS pH 7.4. The beads were then coated with 1 mL of 100 pg / mL of streptavidin (Jackson Immuno Research, West Grove, PA) in PBS pH 7.4 with 10 mg / mL BSA (Sigma-Aldrich, St. Louis, Missouri) with rotation at room temperature for 1 hour. The beads were settled, supernatant was removed, and beads were rinsed five times with PBS pH7.4. In the final step of the solid phase preparation, beads were coated with 30 pg / mL of biotin goat anti-human IgG (Jackson ImmunoResearch) for 1 hour with rotation at room temperature.109IPTS / 200345621.2

[0432] The cell-binding assay was set up using the antibody variants as constant binding partner at two different concentrations of 0.5 nM and 5 nM. For the titration curve with antibody variants fixed at 0.5 nM, at least 50 million huLy6E OE HEK or cynoLy6E OE HEK cells were used as titrant. For the titration curve with antibody variants fixed at 5 nM, at least 30 million cells were used as titrant. The antibody variants and cells were mixed in PBS pH 7.4, 1 mg / mL BSA, 0.2 % NaN₃ and incubated at 4°C for 6 days with gentle rotation until equilibrium was reached. After incubation, the mixture of antibody variants and cells was centrifuged to separate the cells from the unbound free antibody variants. The free antibody variants were loaded onto the KinExA™ 3200 (Sapidyne, Boise, Idaho) with biotinylated anti-human IgG PMMA as solid phase and 0.5 pg / mL of Alexa 647 goat anti-human IgG (Jackson ImmunoResearch) as the detection antibody.

[0433] Results are shown in Table 11.1. N-curve analysis was used to calculate the affinity and receptor expression level with the concentrations of antibody variant as reference point. Narrow 95% confidence intervals were obtained for both the affinity and receptor expression level and % error of the fit was less than 4.5%. The N-curve analysis is shown in FIG. 9A (huLy6E OE HEK) and FIG. 9B (cynoLy6E OE HEK). For each panel, the right curve shows the data for 5 nM constant binding partner and the left curve shows the data for 0.5nM constant binding partner. The antibody variant v39336 demonstrated high affinity towards Ly6E in both huLy6E OE HEK and cynoLy6E OE HEK. The KD value was about 3.3 fold less for huLy6E than for cynoLy6E. Table 11.1: Binding of Anti-Ly6E Antibody to huLy6E OE HEK and cynoLy6E OE HEK Antibody Ly6E OE KD Expression % Error Variant HEK Level0.129 nMHuman 4.244E+6 2.99 (0.069 nM - 0.206 nM)*v393360.429 nMCyno 5.40E+6 3.51(0.307 nM- 0.587 nM)**95% confidence intervalEXAMPLE 12: DEVELOP ABILITY ASSESSMENT OF ANTLLY6E ANTIBODY VARIANTS

[0434] The isoelectric point, propensity for self-aggregation and thermal stability of certain optimized, humanized anti-Ly6E antibody variants were determined and compared to those for benchmark antibody hu9B12.v12 (v33706). Assessment of these properties provides a preliminary indication of the developability of antibodies and a means for ranking the variants. The isoelectricpoint was measured by capillary isoelectric focusing (cIEF), the propensity for self-aggregation was measured by affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS), and thermal stability was determined by differential scanning calorimetry (DSC), as described below.Capillary Isoelectric Focusing (cIEF)

[0435] cIEF was carried out using the Maurice™ C system, System Suitability Kit and Method Development Kit (ProteinSimple Inc., San Jose, CA). System suitability standard, fluorescence calibration standard, cartridge and samples were prepared according to manufacturer’s recommendations. The capillary was automatically calibrated with a fluorescence standard preconditioned with Maurice™ cIEF System Suitability Kit to ensure the capillary was functioning properly. The antibody samples were diluted to a concentration of 0.5 mg / mL in a final volume of 40 pL in Gibco™ distilled water and mixed Maurice™ cIEF Method Development Kit samples. The samples were then vortexed, centrifuged and the supernatant was pipetted into individual wells of a 96-well plate. All electropherograms were detected with UV absorbance at 280 nm. All data analyses were performed using manufacturer’s software Compass for imaged Capillary Electrophoresis (iCE) (ProteinSimple). Compass software aligns each electropherogram using pl markers so that the x-axis is displayed as a normalized pl for each injection.

[0436] The pl values were determined for the main isoform of the optimized humanized anti-Ly6E antibody variants (see Example 6) and compared to the benchmark antibody hu9B12.v12 (v33706).Affinity-Capture Self-Interaction Nanoparticle Spectroscopy (AC-SINS) Assay

[0437] AC-SINS was carried out in a 384-well plate format. Initially, 20 nm gold nanoparticles (Ted Pella, Inc., Redding, CA; Cat. No. 15705) washed with 0.22 pm filtered Gibco™ Distilled Water were coated with a mixture of capture antibody, 80% AffiniPure™ Goat Anti-Human IgG (H+L) (Jackson ImmunoResearch; Cat. No. 109-005-088), and the non-capture antibody, 20% ChromPure™ Goat IgG, whole molecule (Jackson ImmunoResearch; Cat. No. 005-000-003), that were initially buffer exchanged into 20 mM sodium acetate pH 4.3 and diluted to 0.4 mg / mL. The mixture of gold nanoparticles, capture antibody and non-capture antibody was incubated in the dark for 18h at room temperature. Sites unoccupied on the gold nanoparticles were blocked with 1 pM thiolated polyethylene glycol (2 kD) in 20 mM sodium acetate, pH 4.3, to a final concentration of 0.1 pM, followed by Ih incubation at room temperature. The coated nanoparticles were then concentrated by centrifugation at 21,000 g for 7 min, at 8°C. 95% of the supernatantwas removed and the gold pellet was resuspended in the remaining buffer. 5 pL of concentrated nanoparticles were added to 45 pL of antibody at 0.05 mg / mL in Gibco™ PBS pH 7.4 in a 384-well plate. The coated nanoparticles were incubated with the test antibody for 4h at room temperature in the dark. The absorbance was read from 450-700nm at 1 nm increments, and a Microsoft Excel macro was used to identify the max absorbance, smooth the data, and fit the data using a second-order polynomial.

[0438] The Alambda (nm) was calculated based on the smoothed max absorbance of the average blank (PBS alone) subtracted from the smoothed max absorbance of the antibody sample to determine the antibody AC-SINS score. Antibody-antibody interactions directly correlate with the shift in maximum absorbance wavelength of gold nanoparticles coated with the antibody of interest. The cutoff of Alambda for high self-aggregation propensity of the antibody was set at lOnm, based on the literature. (See, for example, Bailly, M. et al., 2020, mAbs, 12(1); Jain, T. et al., 2017, Proc. Natl. Acad. Sci. U. S. A., 114 (5) 944-949).

[0439] The results of cIEF and AC-SINS assays are shown in Table 12.1.Table 12.1 cIEF and AC-SINS Results for Optimized, Humanized Anti-Ly6E Antibodies Variant cIEF (pl) AC-SINS (Alambda, nm) v39333 8.46 4.5 v39335 8.71 3.5 v39336 8.63 7.0 v41252 8.78 6.0 v33706 (hu9B12.vl2) 8.70 4.5Thermal Stability by DSC

[0440] 400 µL of purified samples at concentrations of 0.4 mg / mL in PBS were used for DSC analysis with a VP-Capillary DSC system (Malvern Panalytical Inc., Westborough, MA). At the start of each DSC run, 5 buffer blank injections were performed to stabilize the baseline, and a buffer injection was placed before each sample injection for referencing. Each sample was scanned from 20°C to 100°C at a 60°C / hr rate, with low feedback, 8 sec filter, 3 min pre-scan thermostat, and 70 psi nitrogen pressure. The resulting thermograms were referenced and analyzed using Origin 7 software (OriginLab Corporation, Northampton, MA) to determine melting temperature (Tm) as an indicator of thermal stability.

[0441] The Fab Tm values determined for the optimized, humanized variants are shown in Table 12.2. The values observed were ~84°C and higher and were much greater than the Fab Tm values for the benchmark antibody hu9B12.v12 (v33706) and trastuzumab (about 80.9°C; trastuzumab is routinely used as a reference as a very stable antibody). These values indicated high Fab thermal stability of the optimized humanized anti-Ly6E variants.Table 12.2: Fab Thermal Stability of Optimized Humanized Anti-Ly6E Antibody Variants Variant Fab Tm (°C)v33706 (hu9B12.vl2) 79.6v39333 85.6v39335 87.9v39336 89.5v41252 84.0EXAMPLE 13: FUNCTIONAL CHARACTERIZATION OF ANTLLY6E ANTIBODY VARIANTS - SPHEROID PENETRATION

[0442] Spheroid penetration ability of the optimized humanized anti-Ly6E antibody variant v41252 in Ly6E-expressing spheroid cell line RT112 / 84 was determined by fluorescence imaging as described below. The benchmark anti-Ly6E antibody hu9B12.vl2 (v33706) was used as a comparator, and palivizumab (anti-RSV) (v21995) was used as a negative control.

[0443] Briefly, antibodies were fluorescently labeled by coupling a Fab fragment AF488 conjugate anti-human IgG Fc (Jackson ImmunoResearch Labs) at a 1: 1 molar ratio in PBS pH 7.4 (Thermo Fisher Scientific) for 24 hours at 4°C. Cells were seeded at 3,000 cells / well in RPMI medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) into Coming Ultra-Low Attachment 96-well round bottom plate (Sigma-Aldrich) and incubated for 2 days at 37 °C / 5% CO2 to allow spheroid formation. Coupled antibodies were added to the cells after spheroid formation at 50 nM and incubated under standard culturing conditions for 24 hours. Following incubation, spheroids were washed to remove unbound antibodies and cell nuclei were stained with Hoescht 33342 dye (Thermo Fisher Scientific) at 1,000 nM and surface AF488 fluorescence was quenched using an anti-AF488 antibody (Life Technologies) at 100 nM for 2 hours at 37°C / 5% CO2. Spheroids were then imaged using theOpereta™ CLS High Content Analyzer (Perkin Elmer, HH 16000020). Image analysis was performed using Harmony® 4.5 software (Perkin Elmer). Two-dimensional analysis was performed on the central slice of each spheroid, by dividing the spheroid object into subregions of concentric bands, each representing 20% area of the spheroid region (“layer”). Mean AF488 fluorescence within each subregion band was quantified, corrected by subtracting the spheroid center mean AF488 fluorescence of v21995 -treated spheroid, and ploted using GraphPad Prism Version 10 (GraphPad Software).

[0444] Results are shown in Table 13.1 and FIG. 10A (v41252), FIG. 10B (v33706) and FIG.10C (v21995). The optimized humanized anti-Ly6E antibody variant v41252 coupled to AF488 displayed high signal within the Ly6E-expressing spheroid inner layers as well as the spheroid center, which indicated efficient spheroid penetration. The benchmark anti-Ly6E antibody hu9B12.vl2 (v33706) showed some signal at the spheroid outer layers but low signal at the inner layers and spheroid center, which indicated poor spheroid penetration ability. These results demonstrated superiority in spheroid penetration of v41252 over the benchmark. Negative control palivizumab (v21995) showed limited signal at the outer layers of the spheroid, which indicated limited binding and spheroid penetration.Table 13.1: Spheroid Penetration of Anti-Ly6E Antibody Variants (50 nM)v41252 v33706 v21995 SpheroidLayer Fluorescence Distribution Fluorescence Distribution Fluorescence Distribution signal (MFI) (%) signal (MFI) (%) signal (MFI) (%) Layer 1 272 8.2 143 26.9 72 42.1 Layer 2 598 18.0 121 227 53 31.0 Layer 3 704 21.1 97 18.2 30 17.5 Layer 4 890 26.7 98 18.4 16 9.4 Spheroid867 26 73 13.7 0 0.0 centerEXAMPLE 14: STABILITY OF ANTLLY6E ANTIBODY VARIANTS IN MOUSE PLASMA, PBS AND FORMULATION BUFFERS

[0445] The stability of optimized humanized anti-Ly6E antibody variants v39333, v39336 and v41252 after incubation in mouse plasma or PBS pH 7.4 at 37° C, and the stability of v39336 andv41252 in the formulation buffers 20 mM Histidine pH 5.0, 6% w / v sucrose (His5Su) or 20 mM Sodium Succinate pH 4.5, 6% w / v sucrose (Succ5Su) was evaluated.

[0446] For mouse plasma stability studies, antibody variants v39333, v39336 and v41252 were each diluted into either mouse plasma or PBS to a final concentration of 0.5 mg / ml or 1 mg / ml, respectively, and incubated at 37° C. Samples were removed after 0, 7 or 14 days and stored at -80° C until characterization.

[0447] To assess the thermal stability of humanized anti-Ly6E antibody in two formulation buffers, variants v39336 and v41252 were buffer exchanged into either His5Su or Succ5Su. After buffer exchange, the samples were normalized to a concentration of 1 mg / ml in their corresponding buffer. Day 0 controls were frozen immediately after concentration adjustment, and the rest of the samples were incubated at 40° C. Incubated samples were removed after 7 or 14 days and stored at -80 °C until characterization.

[0448] For characterization of variants v39333 and v39336 incubated in mouse plasma, samples were thawed at room temperature and incubated with 1.5 pg of recombinant Endo S endoglycosidase for one hour at room temperature. An immunoprecipitation slurry was generated by a 45 min incubation of 95 pL / sample of magnetic Sepharose streptavidin-coated beads (Mag Sepharose™, Cytiva; Cat. No. 28985799) with 15 pg / sample of biotinylated goat anti-Human IgG Fc capture antibody (Jackson ImmunoResearch; Cat. No. 109-065-098), followed by 4 washes with PBS-T pH 7.4 with the aid of a DynaMag™-2 magnet (Invitrogen). After deglycosylation, plasma-incubated samples were mixed with 95 pL of the immunoprecipitation slurry for 1.5 hrs at room temperature. Then, the slurry underwent 6 washes with PBS pH 7.4 and 2 washes with LC-MS-grade water with the aid of a DynaMag™-2 magnet (Invitrogen™). A final wash with PBS pH 7.4 was performed before elution. Antibody variants were eluted by incubating beads with 35 pL of LC-MS grade water with 20% acetonitrile and 0.1% formic acid for one hour at room temperature.

[0449] For variant v41252 incubated in mouse plasma, samples were deglycosylated with recombinant Endo S endoglycosidase (1 ug / 10 ug protein) for 1 hour at 25 °C. Immunoprecipitation was performed using an AssayMAP Bravo™ platform (Agilent Technologies). In brief, 5 pL streptavidin cartridges (SA-W, Agilent Technologies; Cat. No. G5496-60010) were primed with 1.0% v / v formic acid, with subsequent washing and equilibrium steps in PBS. The primed cartridges were then coupled to 50 pL of 0.25 mg / mL biotinylated goatanti-Human IgG Fc capture antibody (Jackson ImmunResearch; Cat. No. 109-065-098) in PBS using 96-well low-binding PCR plates. Mouse plasma samples (100 pL) were loaded onto each cartridge and eluted with 20 pL of a solution containing 20% v / v acetonitrile and 1.0% v / v formic acid into a 96-well low-binding PCR plate containing 20 pL of 500 mM ammonium acetate.

[0450] Immunoprecipitated antibody variants were characterized by intact protein reversed phase liquid chromatography coupled to mass spectrometry (RPLC-MS). Antibody variants incubated in formulation buffers or PBS did not undergo immunoprecipitation and were characterized by analytical size exclusion chromatography (analytical SEC) and intact protein RPLC-MS.

[0451] For RPLC-MS, immunoprecipitated or buffer-incubated samples were injected onto an Agilent™ 1290 Infinity II LC coupled with an Agilent™ 6545 Quadrupole Time of Flight (Q-TOF) mass spectrometer (Agilent Technologies). Protein species were separated using a BioSuite™ pPhenyl RPC column (1000A, 10 pM, 75 x 2.0 mm; Waters Corp., Milford, MA) maintained at 70 °C, at a flow rate of 0.3 mL / min with the gradient shown in Table 14.1. Mobile Phases consisted of A: LC-MS grade water with 0.1% v / v formic acid, 0.025% v / v trifluoroacetic acid and 10% v / v isopropyl alcohol, and B: acetonitrile with 0.1% v / v formic acid and 10% v / v isopropyl alcohol.Table 14.1: Gradient Applied for Protein Separation in RPLC-MS Method% Mobile Phase % Mobile PhaseTime (min)A B0 90 105 40 607 10 907.1 1 998.5 1 99Post-run time of 2 min 90 10

[0452] Electrospray ionization (ESI) for MS analysis was performed in a Dual AJS™ ESI source (Agilent Technologies) in positive mode with 5kV of capillary voltage, 300 °C gas temperature, 170 V fragmentor voltage, 13L / min gas flow, 45 psig nebuliser, 400 °C sheath gas temperature and 12 L / min sheath gas flow. Data was acquired at 1 spectra / sec, with a m / z range from 500 to 7000.

[0453] Peak integration, MS deconvolution and mass assignments were performed in Protein Metrics Byos® (Protein Metrics Inc., Cupertino, CA). For all time points, the highest intensity deconvoluted mass was assigned as the reference mass of variant v39333, v39336 or v41252. The reference mass was defined as the average mass of variant v39333, v39336 or v41252 with two 2-acetamido-2-deoxy-beta-D-glucopyranose-(l-4)-[alpha-L-fucopyranose-(l-6)] stubs arising from EndoS activity on N-glycans, 16 disulfide bonds and the formation of pyroglutamic acid, if applicable, at the N-terminus. Other variant proteoform adducts assigned based on mass shifts relative to the reference mass were: variant reference mass with a phosphoric acid adduct, variant reference mass with the loss of one fucose unit, variant reference mass with the addition of a hexose unit. Apparent purity was calculated as the ratio of all variant proteoforms deconvoluted peak intensities divided by all observed deconvoluted peak intensities. Mouse plasma proteins, if identified, were not considered in the apparent purity calculations. The results of RPLC-MS are provided in Table 14.2. Deconvoluted peak broadening attributed to non-covalent adduct formation did not allow estimation of purity of variant v41252 in PBS by RPLC-MS.Table 14.2: Stability of Anti-Ly6E Antibody Variants Incubated in Mouse Plasma or PBS at 37°CVariant v39333 v39336 v41252 Timepoint(days) PBS Mouse Plasma PBS Mouse Plasma PBS Mouse Plasma 0 95% 100% 97.6% 100% N / A 88.3% 7 95.2% 99.4% 92.2% 97.6% N / A 87.3% 14 92.3% 96.5% 90.3% 88.4% N / A N / AN / A = No data available

[0454] For variants v39333 and v39336 incubated in PBS at 37 °C, analytical SEC was performed using an Agilent™ Infinity II 1290 HPLC with an Agilent™ Advance Bio SEC column (300 A, 2.7 pm, 7.8 x 150 mm; Agilent Technologies) equilibrated with 5 column volumes of Mobile Phase A (200 mM KPO4, 200 mM KC1, pH 7.0) at room temperature. 5 pL of incubated sample was injected and eluted isostatically for 7 mins at 1 mL / min with absorbance monitored at A280.

[0455] For v39336 and v41252 incubated in PBS, His5Su or Succ5Su at 40° C, analytical SEC was performed using an Agilent™ Infinity II 1290 HPLC with an Acquity™ UPLC (R) ProteinBEH SEC column (200 A, 1.7 pm, 2.1 x 150 mm, Waters) equilibrated with 5 column volumes of Mobile Phase A (200 mM KPO4, 200 mM KC1, pH 7.0) at room temperature. 1 pl of incubated sample was injected and eluted isostatically for 7 mins at 1 mL / min with absorbance monitored at A280.

[0456] Chromatograms were exported and integrated in Protein Metrics Byos® (Protein Metrics), to provide complete, baseline-to-baseline integration of each peak. The peak corresponding to the major component for IgG (approximate peak apex time 3.5 min, integration window from 3.1 to 4.2 min) was reported as the monomer based on the SEC profde of control trastuzumab. A time window from 2 to 3.1 min was designated as high molecular weight species (HMWS), and a time window from 4.2 to 5.0 min was designated as low molecular weight species (LMWS), excluding solvent peaks (over 5.0 min).

[0457] The results of UPLC-SEC are provided in Table 14.3. Analytical size exclusion chromatography showed that all tested His5Su and Succ5Su thermal stability samples had >95% monomer after incubation at 40°C.Table 14.3: Accelerated Thermal Buffer Stability (40°C) of Anti-Ly6E antibodies in PBS, His5Su or Succ5Suv39333 v39336 v41252 Time(days) PBS PBS His5Su Succ5Su PBS His5Su Succ5Su 37°C 40°C 40°C 40°C 40°C 40°C 40°C 0 100% 92.0% 97.1% 97.6% 91.4% 97.0% 97.5% 7 100% N / A 96.4% 96.9% N / A 96.3% 96.5% 14 100% 90.5% 95.8% 95.1% 87.5% 95.5% 95.8%N / A = No data availableEXAMPLE 15: PHARMACOKINETIC STUDY OF ANTI-LY6E ANTIBODY VARIANTS IN Tg32 MICE

[0458] Pharmacokinetics (PK) of optimized humanized anti-Ly6E antibody variants v39333 and v39336 were assessed in humanized FcRn Tg32 mice as described below. PK of the benchmark antibody hu9B12.v12 (v33706) was also assessed for comparison. The Tg32 mouse model is used to predict pharmacokinetics of a drug in humans (see Avery et al., 2016, mAbs, 8:6, 1064-1078).

[0459] All test antibody variants were administered at 5 mg / kg to hFcRn Tg32 mice (The Jackson Laboratory, Sacramento, CA; Cat. No. 014565) by intravenous injection. For each test antibodyvariant, blood was collected from n=4 animals by retro-orbital bleed at 1 hour, 6 hours, and 1, 3, 7, 10, 14, and 21 days post-dose. Blood was processed to serum and stored frozen at -80°C in screw-cap tubes prior to pharmacokinetic analysis.

[0460] Test antibody variant concentrations were measured in mouse serum by sandwich ELISA on a 384-well plate coated with goat anti -Human IgG Fc antibody (Jackson ImmunoResearch; Cat. No. 109-005-098). The total IgG was detected with HRP-conjugated goat anti-Human IgG F(ab’)2 antibody (Jackson ImmunoResearch; Cat. No. 109-035-097). Absorbance at 450 nm was measured using a Synergy™ Hl Hybrid Multi-Mode Plate Reader (BioTek Instruments, Winooski, VT). Sample data were analyzed using SoftMax® Pro 7.1 (Molecular Devices, San Jose, CA). Pharmacokinetic parameters were calculated using non-compartmental analysis using Phoenix WinNonlin™ software (Certara, Princeton, NJ).

[0461] The results of pharmacokinetic study are shown in FIG 11 and demonstrate that the total IgG PK profiles of both anti-Ly6E antibody variants, v39333 and v39336, were comparable, with typical antibody-like prolonged exposures. Table 15.1 provides a summary of various pharmacokinetic parameters determined in the hFcRn Tg32 study. Elimination half-life of variants v39333 and v39336 was 12.2 days and 8.1 days, respectively. Elimination half-life of the benchmark antibody hu9B12.v12 (v33706) was 8.3 days. The Cmax for variants v39333 and v39336 was 117.4 and 110.8 pg / mL, respectively. The Cmax for the benchmark antibody hu9B12.v12 (v33706) was 135.2 pg / mL.Table 15.1: Pharmacokinetic Profile of Anti-Ly6E Antibody Variants in Tg32 Mice Total IgG Volume ofDose Cmax AUC Clearance Variant Half-life distribution(mg / kg) (pg / mL)( (day* pg / mL) (mL / day / kg) Days) (mL / kg)v39333 5 117.4 12.2 1206.8 72.8 4.1 v39336 5 110.8 8.1 783.8 74.9 6.4 v33706 5 135.2 8.3 942.6 63.5 5.3EXAMPLE 16: PREPARATION OF ANTI-LY6E ANTIBODY-DRUG CONJUGATES

[0462] Antibody-drug conjugates (ADCs) comprising chimeric or optimized, humanized anti-Ly6E antibody variants (v38866, v39333, v39335, v39336 and v41252) conjugated to a topoisomerase 1 inhibitor (Compound 1) drug-linker (DL1), an ADC comprising v39336conjugated to a monomethyl auristatin E (MMAE) drug-linker (DL3) and ADCs comprising benchmark anti-Ly6E antibody hu9B12.vl2 (v33706) conjugated to DL1, to deruxtecan (DL2) or to DL3 were prepared as described below. The structures of Compound 1 and the drug-linkers DL1, DL2 and DL3 are shown below.Compound 1:DL3:I NI H N 016.1 Benchmark ADC: v33706-DL3 (DAR 4)

[0463] The benchmark antibody hu9B12.v12 (v33706) (1-10 mg / mL in phosphate buffered saline, pH 7.4) was reduced with TCEP (1-10 mM in dH2O) (2.9-3.4 eq.) in the presence of 1 mM DTPA. The solution was mixed thoroughly and incubated at 37 °C for 90 min before cooling on ice. The reduced antibody solution was then buffer exchanged into PBS, pH 7.4 by passage over a Zeba™ Spin Desalting Column (Thermo Fisher Scientific; Cat. No. 87773). To the reduced protein solution was added the maleimide functionalized drug-linker (10 mM in DMSO) (12-20 eq.). In some instances, DMSO (10 percent v / v%) and / or propylene glycol (24 percent v / v%) was added to the reduced protein solution prior to the addition of drug -linker. The conjugation reaction was immediately mixed thoroughly by pipetting and conjugation was allowed to proceed at room temperature for 60-120 min while mixing. In some instances where the ADC was produced at >5 mg scale, the conjugation reaction was followed by quenching of excess drug-linker with 10-100 mM aqueous stock of N-acetyl cysteine (6-8 eq.) for 30-60 mins. Mixing was achieved by rotating the reaction tubes on atube revolver (Thermo Fisher Scientific; Cat. No. 88881001).16.2 Benchmark ADCs: v33706-DL2 (DAR 4 & 8), v33706-DLl (DAR 8)

[0464] The ADCs v33706-DL2 (DAR 4 & 8) and v33706-DLl (DAR 8) were prepared as described in Section 16.1 with the following changes. TCEP (1-10 mM in dH2O) was used at 3.0-12.0 eq., the solution was incubated at 37 °C for 90-180 min before cooling on ice, and the reduced antibody solution was buffer exchanged into 10 mM sodium acetate buffer, pH 4.5 by passage over a Zeba™ Spin Desalting Column. In those instances where the ADC was produced at >5 mg scale, the conjugation reaction was followed by quenching of excess drug -linker with 10 mM aqueous stock of N-acetyl cysteine (8 eq.) for 30-60 mins.16.3 Parental Chimera and Optimized Humanized Anti-Ly6E Antibody ADCs

[0465] The ADCs v38866-DLl (DAR 8), v39333-DLl (DAR 8), v39335-DLl (DAR 8), V39336-DL1 (DAR 4, 6 & 8), v41252-DLl (DAR 8) and v39336-DL3 (DAR 4) were prepared as121IPTS / 200345621.2described in Section 16.1 with the following changes. TCEP (1-10 mM in dH2O) was used at 2.4-12 eq., the solution was incubated at 37 °C for 180 min before cooling on ice, and the reduced antibody solution was buffer exchanged into 10 mM sodium acetate buffer, pH 4.5 (pH 7.4 for v39336-DL3 ADC) by passage over a Zeba™ Spin Desalting Column.EXAMPLE 17: PURIFICATION AND BIOPHYSICAL CHARACTERIZATION OF ANTI-LY6E ANTIBODY-DRUG CONJUGATES

[0466] Purification of the anti-Ly6E antibody-drug conjugates (ADCs), as prepared under Example 16, from small molecules was performed by passage over a Zeba™ Spin Desalting Column (Thermo Fisher Scientific; Cat. No. 87773) into PBS pH 7.4, 150 mM sodium chloride in 10 mM sodium acetate pH 4.5 or 50 mM sodium acetate pH 5.5. Where the ADC was produced at >5 mg scale, purification was performed on an AKTA Pure™ FPLC operated by Unicom 7 software (Cytiva; Cat. No. 29018224). A pre-packed HiPrep™ 26 / 10 desalting column (Cytiva; Cat. No. 17508701) with Sephadex G-25 resin was used as per the manufacturer’s standard and the protein was eluted with 150 mM sodium chloride in 10 mM sodium acetate pH 4.5 or 50 mM sodium acetate pH 5.5. Selected fractions based on their UV absorbance and area were pooled for analysis. The pooled and purified conjugates were sterile filtered (0.22 mm), stored at 4 °C and analyzed for total protein content by spectrophotometric protein quantification at 280 nm or bicinchoninic acid assay (Pierce micro-BCA protocol; Thermo Fisher Scientific; Cat. No.23225).

[0467] Characterization of the ADCs was performed by HPLC-hydrophobic interaction chromatography (HIC), HPLC-SEC, CE-SDS and RP-HPLC-MS. The average drug-to-antibody ratio (DAR) and drug distribution were derived from interpretation of HIC and / or LC-MS data as described below. Endotoxin levels were assessed using Endosafe® LAL test cartridges (Charles River Laboratories, Wilmington, MA; Cat. No. PTS20005F), with a sensitivity of 0.005 EU / mL on an Endosafe® nexgen-PTS™ testing system. Residual free compound and drug-linker levels (unconjugated payload species, UPS) were assessed by RP-UPLC-MS or RP-UPLC with fluorescence detection (FLD), with a threshold set at 1% ((free compound + drug-linker) / (conjugated drug-linker)). The results are shown in Table 17.9.17.1 DAR Determination by HIC

[0468] The average DAR by HIC was assessed as described in Ducry, L. (2013, Antibody Drug Conjugates, Methods in Molecular Biology, vol. 1045, pp. 275-284). The experiments were performed on an Agilent™ Infinity II 1290 HPLC system (Agilent Technologies) using a TSKgel® Butyl-NPR column (2.5pm, 4.6 x 35mm; TOSOH Bioscience GmbH, Germany) preequilibrated with 5 column volumes of Buffer A (1.5 M (NH4)2SO4, 25 mM NaH2PO4, pH 6.95) and at room temperature. Usually, 10-30 pg of sample at 2-3 mg / mL concentration was loaded on the column with 95% Mobile Phase A (MPA) and 5% Mobile Phase B (MPB) (75% 25 mM NaH2PO4 plus 25% isopropanol, pH 6.95) and run at 0.5 mL / min using the gradient of 95 / 5% MPA / MPB to 5 / 95% MPA / MPB over a period of 12 minutes. HIC chromatograms were integrated using 25 appropriate parameters that provided complete, baseline-to-baseline integration of each peak, followed by integration of each peak showing reasonable separation. As a reference, unconjugated naked antibodies were run on the same gradient to obtain the HIC retention time of DAR = 0 species.17.2 DAR Determination by RP-HPLC-MS

[0469] ADC samples were deglycosylated using recombinant EndoS endoglycosidase for 1 hour at 25° C, followed by reduction with tris(2-carboxyethyl)phosphine (TCEP) at a final concentration of 50 mM for 1 hour at 25° C. Samples were injected onto an Agilent™ 1290 Infinity II LC system coupled with an Agilent™ 6545 Quadrupole Time of Flight (Q-TOF) mass spectrometer (Agilent Technologies). Protein species were separated using a BioSuite™ pPhenyl RPC column (1000A, 10 pM, 75 x 2.0 mm; Waters) maintained at 70 °C, at a flow rate of 0.3 mL / min. with the gradient shown in Table 17.1. For conjugates of variants v33706, v38866, v39333, v39335 and v39336, Mobile Phase A consisted of 0.1% v / v formic acid (FA), 0.025% v / v trifluoroacetic acid (except for v39336, with no trifluoroacetic acid) and 10% v / v isopropyl alcohol in water, whereas Mobile Phase B consisted of 0.1 % v / v formic acid and 10% v / v isopropyl alcohol in acetonitrile. For antibody-drug conjugate v41252-DLl, Mobile Phase A consisted of 0.1% v / v formic acid, 0.025% v / v trifluoroacetic acid in water, whereas Mobile Phase B consisted of 0.1% v / v formic acid and 0.025% v / v trifluoroacetic acid in acetonitrile.Table 17.1: RP-HPLC-MS GradientTime (min) % Mobile phase A % Mobile phase B0 90 105 40 607 10 907.1 1 998.5 1 99Post-run time of 2 min 90 10

[0470] ESI was performed in a Dual AJS™ ESI source (Agilent Technologies) in positive mode. For conjugates of variants v33706, v38866, v39333, v39335 and v39336, source conditions were 5kV of capillary voltage, 300 °C gas temperature, 170 V fragmentor voltage, 13L / min gas flow, 45 psig nebuliser, 400 °C sheath gas temperature and 12 L / min sheath gas flow. For conjugate v41252-DLl, source conditions were 3.5kV of capillary voltage, 325 °C gas temperature, 175 V fragmentor voltage, 7 L / min gas flow, 35 psig nebuliser, 200 °C sheath gas temperature and 4.5 L / min sheath gas flow. Data was acquired at 1 spectra / sec, with a m / z range from 500 to 7000.

[0471] Peak integration, MS deconvolution and mass assignments were performed in Protein Metrics Byos® (Protein Metrics). Reference masses were defined as the average mass of the light chain or heavy chains of antibody variants with one 2-acetamido-2-deoxy-beta-D-glucopyranose-(l-4)-[alpha-L-fucopyranose-(l-6)] stub per heavy chain arising from EndoS activity on N-glycans, 0 disulfide bonds and the formation of pyroglutamic acid, if applicable, at the N-terminus. Drug-conjugated chains were assigned based on mass differences relative to reference masses corresponding to up to 3 units of DL-1 with a mass tolerance of ±10 Da. Average DAR was the calculated from the deconvoluted spectrum using the following equations:Z n Drug loadi ■ peak intensityi - - -; - Total peak intensity1 = 0 ADC DAR = (2 * average DAR of light chain) + (2 * average DAR of heavy chain)17.3 Unconjugated Payload Species Analysis by RP-UPLC-MS or RP-UPLC-UV

[0472] For unconjugated payload species (UPS) analysis, samples were first diluted with 3 volumes of acetonitrile with 0.1% formic acid (FA) before incubating at 4 °C for a minimum of 30 minutes. Samples were then centrifuged at 17 k r.c.f. for 10 minutes to precipitate proteins, and the supernatant was removed for analysis. Samples were injected onto an Agilent™ 1290 InfinityII LC system coupled with an Agilent™ 6470A Triple Quadrupole (QQQ) mass spectrometer for MRM-based methods (Agilent Technologies) or coupled with an Agilent™ 6545 Quadrupole Time of Flight (Q-TOF) mass spectrometer for UV-based methods. Payload species were separated using a Zorbax™ Eclipse Plus C18 column (95 A, 2.1 x 50 mm, 1.8 pm, Agilent Technologies) maintained at 30 °C using water with 0.1% FA as Mobile Phase A and acetonitrile with 0.1% FA as Mobile Phase B.

[0473] For conjugates v33706-DLl and v33706-DL2, UPS were detected by UV absorption at 380 nM and quantified by comparison to calibration curves of authentic standards. The compounds were separated using the gradient shown in Table 17.2 with a flow rate of 0.5 m.Table 17.2: LC Gradient for UPS Analysis of Conjugates v33706-DLl and v33706-DL2 Time (min) Mobile Phase A (%) Mobile Phase B (%)0.0 80 200.25 80 204.24 58 424.75 3 975.50 3 975.51 80 206.50 80 20

[0474] All other compounds were detected and quantified by multiple reaction monitoring (MRM) or single ion monitoring (SIM) mass spectrometry techniques, using ESI with the source parameters listed in Table 17.3.Table 17.3: ESI-MS Source Parameters for MS-Based UPS AnalysisParameter ValueGas Temp (°C) 325Gas Flow (1 / min) 10Nebulizer (psi) 20Sheath Gas Temp (°C) 400Sheath Gas Flow (1 / min) 11Capillary Voltage (V) 3700

[0475] For conjugates v33706-DL3 and v41252-DLl, UPS were detected by MRM (transitions listed in Table 17.4) and quantified by comparison to calibration curves of authentic standards. For conjugate v33706-DL3, the compounds were separated using the gradient shown in Table 17.5 with a flow rate of 0.5 mL / min. For v41252-DLl, the compounds were separated using the gradient shown in Table 17.6 with a flow rate of 0.5 mL / min.Table 17.4: MRM Transitions for UPS Analysis of Conjugates v33706-DL3 and v41252-DL1Cell Precursor Product Fragmentor CollisionCompound Accelerator Polarity Ion (m / z) Ion (m / z) (V) Energy (V)Voltage (V)DL3 1317.1 293.4 200 35 5 Positive DL3 payload 718.7 686.9 170 30 5 Positive DL1 TCEP adduct 1289.47 763.3 276 54 5 Positive DL1 N-acetyl- 1223.4 726.2 288 50 5 Positive cysteine adductDL1 1038.4 120 144 86 5 Positive DL1 payload 498.1 454.1 150 30 1 PositiveTable 17.5: LC Gradient for UPS Analysis of Conjugate v33706-DL3Time (min) Mobile Phase A (%) Mobile Phase B (%)0.0 90 100.25 90 103.25 3 974.0 3 974.05 90 104.50 90 10Table 17.6: LC Gradient for UPS Analysis of Conjugate v41252-DLlTime (min) Mobile Phase A (%) Mobile Phase B (%)0.0 75 251.5 75 254.0 65 356.24 50 506.25 3 977.0 3 977.01 75 257.75 75 25

[0476] For conjugates v38866-DLl, v39333-DLl, v39335-DLl, and v39336-DLl, UPS were detected by single ion monitoring (SIM - transitions listed in Table 17.7) and quantified by comparison to calibration curves of authentic standards. The compounds were separated using the gradient shown in Table 17.8 with a flow rate of 0.5 mL / min.Table 17.7: SIM Transitions for UPS Analysis of Conjugates v38866-DLl, v39333-DLl, V39335-DL1, and v39336-DLlCellFragmentorCompound Mass (m / z) Accelerator Polarity (V) Voltage (V)DU-1 N-Acetyl Cysteine1223.3 135 5 Positive adductDU1 1060.3 135 5 Positive DU1 payload 498.1 135 5 Positive DU1 cysteine adduct 655.7 135 5 PositiveTable 17.8: LC Gradient for UPS Analysis of Conjugates v38866-DLl, v39333-DLl, V39335-DL1 and v39336-DLlTime (min) Mobile Phase A (%) Mobile Phase B (%)0.0 75 252.0 65 356.24 50 506.25 3 977.0 3 97127IPTS / 200345621.2Time (min) Mobile Phase A (%) Mobile Phase B (%) 7.01 75 258.0 75 25

[0477] %UPS was calculated by comparing the sum of the calculated concentrations of the unconjugated payload containing species (drug-linker (DL), DL payload, and DL adducts) to the calculated concentration of payload conjugated to ADC in the sample (ADC concentration multiplied by the average DAR).17.4 SEC-HPLC Analysis of ADCs

[0478] Analytical SEC was performed using an Agilent™ Infinity II 1260 HPLC system (Agilent Technologies) with Advance Bio SEC column (300 A, 2.7 pm, 7.8 x 150 mm or 300 A, 2.7 pm, 7.8 x 300 mm; Agilent Technologies) equilibrated with 5 column volumes of Buffer (150 mM Na2PC>4, pH 6.95) at room temperature. Usually, 10 pg of sample at 1-3 mg / mL concentration was eluted isostatically for 7-12 mins at 1 mL / min and absorbance monitored at A280. Chromatograms were integrated to provide complete, baseline-to-baseline integration of each peak, with reasonably placed separation between partially resolved peaks. The peak corresponding to the major component for IgG (approximate retention time 3.3-7.1 min) was reported as the monomer based on the SEC profile of unmodified antibodies. Any peak occurring prior to 3.3-7.1 min was designated as high molecular weight species (HMWS), and any peak occurring after 3.3-7.1 min was designated as low molecular weight species (LMWS), excluding solvent peaks (over 5.2-10 min).17.5 CE-SDS Analysis of ADCs

[0479] Initially, all samples were diluted to Img / mL before preparing the samples in a 96-well PCR plate following manufacturer’s protocol (Protein Express Assay LabChip™; PerkinElmer, Inc., Waltham, MA). Briefly, 2 pg of ADC was mixed with 7 pL Protein Express buffer in the presence (reducing) or absence (non-reducing) of 34 mM dithiothreitol (DTT), followed by heat denaturation at 95°C for 5 minutes. Samples were then diluted in dH2O at a 9:44 ratio before data acquisition using the HT Protein Express Chip (PerkinElmer; Cat. No. 760499) and the HT Protein Express 200 Assay Setting. After each CE-SDS run, the electropherogram and corresponding gel were analyzed using LabChip™ Reviewer (PerkinElmer). The percentage purity was determined by the sum of partially or fully reduced species.Table 17.9: Biophysical Properties of ADCsADC Target Reported Monomer %UPS Endotoxin Purity DAR DAR % (mol% / DAR) (EU / mg) by CE- SDS HPLC LC- (HPLC- -HIC MS SEC) (%) v33706- 4.0 4.2 4.2 98 <1% ND 97.8 DL3v33706- 4.0 ND 2.8 98 ND ND 93.2 DL2*v33706- 8.0 8.0 7.9 100 <1% ND 99.7 DL2v33706- 8.0 8.0 8.0 100 <1% ND 99.6 DL1v38866- 8.0 7.4 8.0 98 <1% ND 97.7 DL1*v39333- 8.0 7.8 8.0 100 <1% ND 99.0 DL1*v39335- 8.0 7.8 8.0 100 <1% ND 98.5 DL1*v39336- 8.0 8.0 8.3 100 <1% 0.07 99.7 DL1v39336- 4.0 4.3 3.8 100 <1% 0.18 99.0 DL1v39336- 6.0 5.8 6.2 100 <1% 0.11 99.3 DL1v41252- 8.0 8.0 8.0 100 <1% 0.25 99.5 DL1ND = Not Determined* Small scale preparation, endotoxin and / or % UPS not determinedEXAMPLE 18: STABILITY OF ANTIBODY-DRUG CONJUGATES IN MOUSE PLASMA, CYNOMOLGUS MONKEY PLASMA AND FORMULATION BUFFERS

[0480] The stability of antibody-drug conjugates v39336-DLl (DAR 8) and v41252-DLl (DAR 8) after incubation in mouse or cynomolgus monkey plasma at 37° C, and the thermal stability of these antibody-drug conjugates in the formulation buffers 20 mM Histidine pH 5.0, 6% w / v sucrose (His5Su) or 20 mM Sodium Succinate pH 4.5, 6% w / v sucrose (Succ5Su) was evaluated.

[0481] Antibody-drug conjugates v39336-DLl and v41252-DLl were each diluted with mouse or cynomolgus monkey plasma to a final concentration of 0.5 mg / ml (mouse plasma), or buffer exchanged into H5Su or Succ5Su and concentrated to 1 mg / ml. Plasma-incubated samples were removed after 0, 1, 2, 3 or 7 days, whereas buffer-incubated samples were removed after 0, 7 or 14 days. Removed samples were stored at -80° C before processing.

[0482] Samples incubated in mouse plasma were deglycosylated with recombinant Endo S endoglycosidase (1 pg / 10 pg protein) for 1 hour at 25 °C. Immunoprecipitation was then performed using an AssayMAP Bravo™ platform (Agilent Technologies). In brief, 5 pL streptavidin cartridges (SA-W, Agilent Technologies; Cat. No. G5496-60010) were primed with 1.0% v / v formic acid, with subsequent washing and equilibrium steps in PBS. The primed cartridges were then coupled to 50 pL of 0.25 mg / mL anti-Fc goat biotinylated Ab in PBS (Jackson ImmunoResearch, Cat. No. 109-065-098) using 96-well low-binding PCR plates. Mouse plasma samples (100 pL) were loaded onto each cartridge and eluted with 20 pL of a solution containing 20% v / v acetonitrile and 1.0% v / v formic acid onto a 96-well low-binding PCR plate containing 20 pL of 500 mM ammonium acetate.

[0483] Samples incubated in cynomolgus monkey plasma were thawed at room temperature and 50 pg of each was incubated with 5 pg of recombinant Endo S endoglycosidase for one hour at room temperature. An immunoprecipitation slurry was generated by a 45 min incubation of 95 pL / sample of magnetic Sepharose streptavidin-coated beads with 15 pg / sample of biotinylated goat anti-Human IgG Fc capture antibody (Jackson ImmunoResearch, Cat. No. 109-065-098), followed by 4 washes with PBS pH 7.4 with the aid of a DynaMag™-2 magnet (Invitrogen). After deglycosylation, plasma-incubated samples were mixed with 95 pL of the immunoprecipitation slurry for 1.5 hrs at room temperature. Then, the slurry underwent 6 washes with PBS pH 7.4 and 2 washes with LC-MS-grade water with the aid of a DynaMag™-2 magnet (Invitrogen). ADCs were eluted by incubating beads with 35 pL of LC-MS grade water with 20% acetonitrile and 0.1% formic acid for one hour at room temperature.

[0484] Immunoprecipitated ADCs from mouse and cynomolgus monkey plasma were characterized by intact protein reversed phase liquid chromatography coupled to mass spectrometry (RPLC-MS). ADCs incubated in formulation buffers did not undergo immunoprecipitation and were incubated with lpg / 10 pg of protein of recombinant Endo S endoglycosidase for one hour at room temperature, followed by reduction in 50 mM tris(2-carboxyethyl)phosphine (TCEP) at 25 °C for one hour. Buffer-incubated samples were characterized by analytical size exclusion chromatography (analytical SEC) and intact protein RPLC-MS.

[0485] For each immunoprecipitated sample, a 5 pL eluate aliquot was diluted with 5 pL 20% v / v acetonitrile, 0.1% v / v formic acid in water and then reduced by incubation in 50 mM TCEP at 25 °C for one hour. Samples were then injected onto an Agilent™ 1290 Infinity II LC system coupled with an Agilent 6545 Quadrupole Time of Flight (Q-TOF) mass spectrometer (Agilent Technologies). Protein species were separated using a BioSuite™ pPhenyl RPC column (lOOOA, 10 pM, 75 x 2.0 mm; Waters) maintained at 70 °C, at a flow rate of 0.3 mL / min with the gradient shown in Table 18.1. Mobile Phase A: 0.1% v / v formic acid, 0.025% v / v trifluoroacetic acid in water. Mobile Phase B: 0.1% v / v formic acid, 0.025% v / v trifluoroacetic acid in acetonitrile. Table 18.1: Gradient Applied for Protein Separation in LC-MS MethodTime (min) % Mobile phase A % Mobile phase B0 90 105 40 607 10 907.1 1 998.5 1 99Post-run time of 2 min 90 10

[0486] ESI was performed in a Dual AJS™ ESI source (Agilent Technologies) in positive mode with 3.5kV of capillary voltage, 325 °C gas temperature, 175 V fragmentor voltage, 7 L / min gas flow, 35 psig nebuliser, 200 °C sheath gas temperature and 4.5 L / min sheath gas flow. Data was acquired at 1 spectra / sec, with am / z range from 500 to 7000.

[0487] Peak integration, MS deconvolution and mass assignments were performed in Protein Metrics Byos®. Reference masses were defined as the average mass of the light chain or heavy chains of antibody variants v39336 or v41252 with one 2-acetamido-2-deoxy-beta-D-glucopyranose-(I-4)-[alpha-L-fucopyranose-(I-6)] stub per heavy chain arising from Endo S activity on N-glycans, 0 disulfide bonds and the formation of pyroglutamic acid, if applicable, at the N-terminus. Drug-conjugated chains were assigned based on mass differences relative to reference masses corresponding to up to 3 units of DL1 with a mass tolerance of ±10 Da. Up toone addition of 18 Da per DL1 was also considered in the drug -conjugated chain assignment to account for thio succinimide ring hydrolysis.

[0488] Average DAR was calculated from the deconvoluted spectrum using the following equations:Z n Drug loadi ■ peak intensityi -; -; - Total peak intensityADC DAR = (2 * average DAR of light chain) + (2 * average DAR of heavy chain)

[0489] Percent average DAR of conjugates v39336-DLl and v41252-DLl relative to day 0 controls after incubation in mouse or cynomolgus monkey plasma is shown in Table 18.2. Percent average DAR of conjugates v39336-DLl and v41252-DLl relative to day 0 was >90% in all formulation buffers tested for incubations at 40 °C (Table 18.3). Monomer percent calculated by analytical SEC after conjugates v39336-DLl and v41252-DLl were incubated in formulation buffers was >90% for His5Su and Succ5Su, as shown in Table 18.4.Table 18.2: Stability of Antibody-Drug Conjugates in Mouse Plasma or Cynomolgus Monkey Plasma (Percent Average DAR Relative to Day 0 Determined by RPLC-MS)V39336-DL1 V41252-DL1Time Cynomolgus CynomolgusMouse Mousemonkey monkey(days) plasma plasmaplasma plasma0 100% 100% 100% 100% 1 77.6% 69.4% 82.9% 73.2% 2 68.3% 59.9% 73.1% 65.9% 3 49.5% 53.0% 67.6% 54.7% 4 50% 48.3% 62.7% 49.6% 7 27.5% 39.9% 54.4% 36.7%Table 18.3: Accelerated Thermal Buffer Stability of Antibody-Drug Conjugates in PBS and Formulation Buffers (Percent Average DAR Relative to Day 0 Determined by RPLC-MS)132IPTS / 200345621.2v39336-DLl V41252-DL1 Time(days) PBS His5Su Succ5Su PBS His5Su Succ5Su 40°C 40°C 40°C 40°C 40°C 40°C0 100% 100% 100% 100% 100% 100% 7 96% 100% 100% 100% 100% 100% 14 93% 100% 100% 100% 100% 100%Table 18.4: Accelerated Thermal Buffer Stability of Antibody-Drug Conjugates in PBS. His5Su or Scc5Su. (Monomer % Determined by Analytical Size Exclusion Chromatography)V39336-DL1 V41252-DL1Time(days) PBS His5Su Succ5Su PBS His5Su Succ5Su 40°C 40°C 40°C 40°C 40°C 40°C0 92.4% 96.6% 97.2% 93.6% 96.4% 97.0% 7 81.9% 95.4% 93.4% 86.0% 94.0% 95.4% 14 82.5% 94.7% 91.2% 86.9% 92.5% 92.0%EXAMPLE 19: IN VITRO CYTOTOXICITY OF ANTIBODY-DRUG CONJUGATES - 2D MONOLAYER

[0490] The cell growth inhibition (cytotoxicity) capabilities of the humanized anti-Ly6E antibody variant v39336 conjugated to DL1 at DAR 8, 6 or 4, as well as humanized anti-Ly6E antibody variant v41252 conjugated to DL1 at DAR 8, were assessed in a panel of Ly6E-expressing cell lines as described below. Cell lines used were: breast carcinoma cell lines MX-1 and Du4475; gastric adenocarcinoma cell lines MKN74, MKN7, SNU-216 and AGS; pancreatic adenocarcinoma cell lines BxPC3, PK-8, Pane 03.27, SU.86.86, PK-59, HPAF-II and Capan-1; tongue carcinoma cell line CAL27; head and neck squamous cell carcinoma cell lines SCC-9, DETROIT-562, FaDu and RPMI2650, and colorectal adenocarcinoma cell line SK-CO-1 ( Ly6E-negative). ADC with benchmark antibody hu9B12.vl2, v33706-DLl (DAR 8), was used as comparator. ADC with palivizumab (anti -RS V), v21995-DLl (DAR 8), was used as non-targeted control.

[0491] Briefly, cells were seeded in 384-well plates and treated with a titration of test ADC prepared in cell growth medium. Cells were incubated for 6 days under standard culturing conditions. After incubation, CellTiter-Glo® reagent (Promega Corporation, Madison, WI) was 133IPTS / 200345621.2spiked in all wells and luminescence corresponding to ATP present in each well was measured using a Synergy ™H1 plate reader (BioTek Instruments, Winooski, VT). Percent cytotoxicity values were calculated based on blank wells (no ADC added), and plotted against ADC concentration using GraphPad Prism 10 software (GraphPad Software). EC50 values were calculated based on a non-linear regression log(agonist) versus response, variable slope (four parameters) by GraphPad Prism 10.

[0492] The results are shown in Table 19.1 and Table 19.2 and representative curves are plotted in FIG. 12A (MX-1), FIG. 12B (Du4475), FIG. 12C (SK-CO-1), FIG. 12D (MKN74), FIG. 12E (MKN7), FIG. 12F (SNU-216), FIG. 12G (AGS), FIG. 12H (BxPC3), FIG. 121 (PK-8), FIG.12J (Pane 03.27), FIG. 12K (SU.86.86), FIG. 12L (PK-59), FIG. 12M (HPAF-II), FIG. 12N (Capan-1), FIG. 120 (CAL27), FIG. 12P (SCC-9), FIG. 12Q (DETROIT-562), FIG. 12R (FaDu) and FIG. 12S (RPMI2650). The anti-Ly6E ADCs v39336-DLl DAR 8, DAR 6 and DAR 4, and v41252-DLl DAR 8 displayed significant cytotoxicity in Ly6E expressing cell lines MX-1 (except v39336-DLl DAR 4), and Du4475, yielding single-digit nanomolar or lower EC50 values after 6-day treatment. ADCs with DAR 8, v39336-DLl DAR 8 and v41252-DLl DAR 8, showed greater potency compared to ADCs with DAR 6 and DAR 4. The anti-Ly6E benchmark ADC, v33706-DLl DAR 8, displayed at least 10-fold higher EC50 values in MX-1 and Du4475 than ADCs v39336-DLl DAR 8 and DAR 6 and v41252-DLl DAR 8, indicating an inferior potency of the benchmark ADC. In Ly6E-negative cell line, ADCs v39336-DLl and v41252-DLl yielded potency comparable to non-targeted control palivizumab ADC.

[0493] The anti-Ly6E ADCs v39336-DLl DAR 8 and v41252-DLl DAR 8 displayed significant cytotoxicity in all tested gastric and pancreatic Ly6E expressing cell lines, yielding double-digit nanomolar or lower EC50 values after 6-day treatment in MKN74, MKN7, CAL27 and DETROIT-562 cell lines and single-digit nanomolar or lower EC50 values after 6-day treatment in SNU-216, AGS, BxPC3, PK-8, Pane 03.27, SU.86.86, PK-59, HPAF-II, SCC-9, FaDu and RPMI2650 cell lines. The anti-Ly6E benchmark ADC v33706-DLl DAR 8 displayed at least 5-fold higher EC50 values in all tested cell lines, except DETROIT-562, which indicates inferior potency compared to v39336-DLl and v41252-DLl.Table 19.1: EC50 Values for ADCs for Breast and Colorectal Cancer Cell Lines134IPTS / 200345621.2EC50 (nM)ADC DAR MX-1 Du4475 SK-CO-1V39336-DL1 8.0 0.64 0.38 ICV39336-DL1 6.0 5.59 0.69 ICV39336-DL1 4.0 IC 3.92 ICV41252-DL1 8.0 1.53 0.37 ICV33706-DL1 8.0 27.60 13.31 18.69 V21995-DL1 8.0 IC 24.46 ICIC = Incomplete CurveTable 19.2: EC50 Values for ADCs in Gastric, Pancreatic and Head and Neck Cancer Cell LinesADC V39336-DL1 V41252-DL1 V33706-DL1 V21995-DL1 DAR 8.0 8.0 8.0 8.0 MKN74 18.54 23.09 IC IC MKN7 8.52 11.76 63.01 IC SNU-216 0.5 0.63 IC IC AGS 2.41 3.73 18.77 IC BxPC3 1.26 1.35 53.88 36.15 PK-8 0.94 0.79 IC IC Pane 03.27 0.62 0.6 13.43 IC SU.86.86 2.5 2.71 70.5 IC EC50 (nM)PK-59 1.66 1.18 37.03 IC HPAF-II 4.59 9.11 46.62 IC Capan-1 4.31 Not tested 48.06 53.27 CAL27 40.0 40.0 IC IC SCC-9 0.55 0.65 87.08 IC DETROIT 562 35.5 32.19 58.98 IC FaDu 0.88 1.06 31.12 IC RPMI2650 6.22 8.84 38.81 ICIC = Incomplete CurveEXAMPLE 20: IN VITRO CYTOTOXICITY OF ANTIBODY-DRUG CONJUGATES -3D SPHEROIDS

[0494] The cytotoxicity capabilities of the anti-Ly6E optimized, humanized antibody variant v39936 conjugated to DL1 at DAR 8, 6 or 4, or to DL3 at DAR 4, and of the anti-Ly6E optimized, humanized antibody variant v41252 conjugated to DL1 at DAR 8 were assessed in a panel of Ly6E-expressing cell spheroids as described below. Cell lines used were: breast carcinoma cell lines HCC1569, MX-1, Du4475 and BT-20; lung adenocarcinoma cell lines NCI-H1650 and NCI-H441; lung carcinoma cell lines NCI-H446, NCI-H358 and ABC-1; gastric adenocarcinoma cell lines MKN74, N87, AGS, MKN45 and FU97; gastric carcinoma cell line SNU16; pancreatic adenocarcinoma cell lines Pane 03.27, Capan-1, Capan-2 and HPAC; head and neck squamous cell carcinoma cell lines FaDu and RPMI2650; esophageal adenocarcinoma cell line OE-19; esophageal carcinoma cell lines TE-8, TE-14 and KYSE-70; ovarian carcinoma cell lines COV362 and Caov-3, bladder carcinoma cell line RT112 / 84, and colorectal adenocarcinoma cell line SK-CO-1 (Ly6E-negative). Benchmark antibody hu9B12.vl2 ADCs v33706-DLl (DAR 8) and v33706-DL3 (DAR 4) were used as comparators. ADC with palivizumab (anti-RSV), v21995-DL1 (DAR 8), was used as a non-targeted control.

[0495] Briefly, cells were seeded in ultra-low attachment 384-well plates at 1,000 cells / well, centrifuged, and incubated for 2 days under standard culturing conditions to allow for spheroid formation and growth. Monoculture cell line spheroids were then treated with a titration of test ADC, generated in cell growth medium. Spheroids were incubated for 6 days under standard culturing conditions. After incubation, CellTiter-Glo® 3D reagent (Promega) was spiked in all wells. Plates were incubated in the dark at room temperature for 1 hour and luminescence was quantified using a BioTek Cytation™ 5 Cell Imaging Multi-Mode Reader (Agilent Technologies, Inc., Santa Clara, CA). Percent cytotoxicity values were calculated based on blank wells (no test article added), and plotted against test article concentration using GraphPad Prism 10 software (GraphPad Software, San Diego, CA). ECso values were calculated based on a non-linear regression log(agonist) versus response, variable slope (four parameters) by GraphPad Prism 10 (GraphPad Software).

[0496] The results are shown in Table 20.1 and Table 20.2 and FIG. 13A-Q. All ADCs comprising anti-Ly6E antibody variants v39336 or v41252 conjugated to DL1 showed targeted killing against Ly6E-expressing spheroids with sub-nanomolar to single-digit nanomolar ECso 136IPTS / 200345621.2values, except ADCs v39336-DLl (DAR 4) in H1650. ADCs v39336-DLl and v41252-DLl at DAR 8 showed greater potency than at DAR 6 or DAR 4 (see Table 20.1.) The benchmark ADC, v33706-DLl (DAR 8) displayed significantly higher ECso values in all Ly6E-expressing cell spheroids evaluated than ADCs v39336-DLl (DAR 8 or DAR 6) or v41252-DLl (DAR 8), indicating an inferior potency of the benchmark ADC. In Ly6E-negative cell line, ADCs v39336-DL1 and v41252-DLl yielded potency comparable to the non-targeted control palivizumab ADC (v21995-DLl).Table 20.1: ECso Values for Anti-Ly6E ADC v39336-DLl (DAR 4 or 6) in Breast, Lung and Colorectal Cancer Cell LinesECso (nM)ADC DAR HCC1569 MX-1 H1650 Du4475 H446 SK-CO-1 V39336-DL1 6.0 0.17 0.49 6.08 0.87 0.32 IC V39336-DL1 4.0 0.26 1.56 10.94 2.81 1.30 ICIC = Incomplete CurveTable 20.2: ECso Values for Anti-Ly6E ADCs (DAR 8) in Various Cancer Cell Lines ADCV39336-DL1 V41252-DL1 V33706-DL1 V21995-DL1 MKN74 3.59 3.61 34.68 IC N87 5.27 5.28 IC IC AGS 0.80 0.48 20.0 IC ECso (nM) Pane 03.27 0.71 0.35 4.95 IC Capan-1 0.46 NA 42.14 78.57 FaDu 3.07 3.03 29.15 IC RPMI2650 0.18 0.24 10.0 IC HCC1569 0.10 0.19 2.60 41.58 MX-1 0.56 0.46 13.26 32.5 H1650 3.09 3.45 29.71 IC Du4475 0.32 0.55 14.15 38.55 H446 0.23 0.26 9.64 33.16137IPTS / 200345621.2ADCV39336-DL1 V41252-DL1 V33706-DL1 V21995-DL1 RT112 / 84 5.09 NA 39.49 IC Caov-3 0.98 NA ...

Claims

WE CLAIM:

1. An antibody construct comprising an antigen-binding domain that binds to human lymphocyte antigen 6 complex, locus E, protein (Ly6E), wherein the antigen-binding domain comprises complementarity determining region (CDR) sequences of a heavy chain variable domain (VH) comprising a sequence as set forth in any one of SEQ ID NOs: 4, 30, 31, 32, 33, 34, 35, 39, 40, 41 or 42, and CDR sequences of a light chain variable domain (VL) comprising a sequence as set forth in any one of SEQ ID NOs: 5, 36, 37 or 38.

2. The antibody construct according to claim 1, wherein the antigen-binding domain comprises a heavy chain CDR 1 (HCDR1) comprising a sequence as set forth in SEQ ID NO: 17; a heavy chain CDR 2 (HCDR2) comprising a sequence as set forth in SEQ ID NO: 94; a heavy chain CDR 3 (HCDR3) comprising a sequence as set forth in SEQ ID NO: 14; a light chain CDR 1 (LCDR1) comprising a sequence as set forth in SEQ ID NO: 95; a light chain CDR 2 (LCDR2) comprising a sequence as set forth in SEQ ID NO: 26; and a light chain CDR 3 (LCDR3) comprising a sequence as set forth in SEQ ID NO: 24.

3. The antibody construct of claim 1, wherein the antigen-binding domain comprises a heavy chain complementarity determining region (CDR) 1 (HCDR1) comprising the sequence as set forth in SEQ ID NO: 17, a heavy chain CDR 2 (HCDR2) comprising the sequence as set forth in SEQ ID NO: 44; a HCDR3 comprising the sequence as set forth in SEQ ID NO: 14; a light chain CDR 1 (LCDR1) comprising the sequence as set forth in SEQ ID NO: 48, a light chain CDR 2 (LCDR2) comprising the sequence as set forth in SEQ ID NO: 26, and a light chain CDR 3 (LCDR3) comprising the sequence as set forth in SEQ ID NO: 24.

4. The antibody construct of any one of claims 1 to 3, wherein the antigen-binding domain comprises:(a) a VH sequence having at least 90% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 30, 31, 32, 33, 34, 35, 39, 40, 41 or 42; or(b) a VL sequence having at least 90% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 36, 37 or 38; or(c) a VH sequence as in (a) and a VL sequence as in (b).

5. The antibody construct of any one of claims 1 to 4, wherein the antigen-binding domain comprises:(a) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 30 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 36;(b) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 30 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 37;(c) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 30 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 38;(d) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 31 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 36;(e) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 31 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 37;(f) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 31 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 38;(g) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 32 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 36;(h) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 32 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 37;(i) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 32 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 38;(j) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 33 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 36;(k) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 33 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 37;(l) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 33 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 38;(m) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 34 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 36;(n) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 34 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 37;(o) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 34 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 38;(p) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 35 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 36;(q) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 35 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 37;(r) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 35 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 38;(s) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 39 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 37;(t) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 40 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 38;(u) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 41 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 38; or(v) a VH sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 42 and a VL having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 38.

6. The antibody construct of any one of claims 1 to 5, wherein the antigen-binding domain comprises:(a) a VH sequence as set forth in SEQ ID NO: 42;(b) a VL sequence as set forth in SEQ ID NO. 38; or(c) a heavy chain variable domain (VH) sequence as set forth in SEQ ID NO: 42 and a light chain variable domain (VL) sequence as set forth in SEQ ID NO: 38.

7. The antibody construct according to any one of claims 1 to 6, wherein the antigen-binding domain is a Fab.

8. The antibody construct of any one of claims 1 to 7 further comprising a scaffold, wherein the antigen-binding domain is operably linked to the scaffold, and wherein the scaffold comprises an IgG Fc region.

9. The antibody construct of claim 8, wherein the scaffold comprises an IgGl Fc region.

10. The antibody construct of claim 8 or 9, wherein the IgG Fc region comprises one or more amino acid modifications that reduce effector function.

11. The antibody construct of claim 10, wherein the one or more amino acid modifications are L234A, L235A and D265S, wherein the numbering of residues is according to the EU numbering system.

12. The antibody construct of claim 1, wherein the antibody construct comprises:(a) two heavy chains comprising the sequence as set forth in SEQ ID NO: 87 and two light chains comprising the sequence as set forth in SEQ ID NO: 68, or(b) two heavy chains comprising the sequence as set forth in SEQ ID NO: 88 and two light chains comprising the sequence as set forth in SEQ ID NO: 68.

13. The antibody construct of any one of claims 1 to 11 further comprising a second antigenbinding domain.

14. The antibody construct of claim 13, wherein the second antigen-binding domain binds to Ly6E.

15. The antibody construct of claim 14, wherein both antigen-binding domains are the same.

16. A polynucleotide or set of polynucleotides encoding the antibody construct of any one of claims 1 to 15.

17. An expression vector or set of expression vectors comprising the polynucleotide or set of polynucleotides of claim 16.

18. A host cell comprising the polynucleotide or set of polynucleotides of claim 16 or the expression vector or set of expression vectors of claim 17.

19. A method of preparing the antibody construct of any one of claims 1 to 15 comprising transfecting a host cell with the polynucleotide or set of polynucleotides according to claim 16 or the expression vector or set of expression vectors according to claim 17, and culturing the host cell under conditions suitable for expression of the antibody construct.

20. An antibody-drug conjugate comprising the antibody construct of any one of claims 1 to 15 conjugated to between 2 and about 8 drug moieties.

21. An antibody-drug conjugate having the formulaA-(L-(D)m)n,wherein:A is the antibody construct according to any one of claims 1 to 25;L is a linker;D is a drug moiety;m is between 1 and about 8; andn is between 1 and about 12.

22. The antibody-drug conjugate of claim 21, wherein(a) m is 1 or 2, and / or(b) n is between about 2 and about 8.

23. The antibody-drug conjugate of claim 21 or claim 22, wherein the drug moiety is a maytansinoid, maytansinoid analogue, benzodiazepine, pyrrolobenzodiazepine, duocarmycin, calicheamicin, calicheamicin analogue, auristatin, auristatin analogue, hemiasterlin, hemiasterlin analogue, tubulysin, tubulysin analogue, amatoxin, amatoxin analogue, camptothecin, camptothecin analogue, eribulin, TLR agonist or STING agonist.

24. The antibody-drug conjugate of claim 21 or claim 22, wherein the drug moiety is a camptothecin analogue.

25. The antibody-drug conjugate of claim 21 or claim 22, wherein the drug moiety has the formula:wherein:X'k ^X1’R9NHR4is J;Xaand Xbare each O, andR9is selected from: -H, -Ci-Cg hydroxyalkyl, -Ci-Cg aminoalkyl, -aminoaryl and -(Ci- Ce alkylj-aminoaryl.

26. The antibody construct of claim 25, wherein R9is -Ci-Ce hydroxyalkyl.

27. The antibody-drug conjugate of claim 21 or claim 22, wherein the drug moiety has the structure:wherein * is the point of attachment to linker, L.

28. The antibody-drug conjugate of any one of claims 21 to 27, wherein Lis a cleavable linker.

29. The antibody-drug conjugate of claim 28, wherein L is a protease cleavable linker and comprises a dipeptide, tripeptide or tetrapeptide.

30. The antibody-drug conjugate of any one of claims 28 to 29, wherein:(a) L has formula (III):■-z4s,rVA'“tAA2Ux]r"'’an)wherein:Z is a linking group that joins the linker to a target group on the antibody construct, A; Str is a stretcher;AAi and AA2 are each independently an amino acid, wherein AAi-[AA2]r forms a protease cleavage site;X is a self-immolative group;q is 0 or 1;ris 1, 2 or 3;s is 0, 1 or 2;# is the point of attachment of the antibody construct A, and% is the point of attachment of the drug moiety, D, or(b) L has formula (Illa):’-z4s,qAA'4AA^UYt%(Hla)wherein:Z is a linking group that joins the linker to a target group on the anti-Ly6E antibody construct, A;Str is a stretcher;AAi and AA2 are each independently an amino acid, wherein AAi-[AA2]rforms a protease cleavage site;Y is -NH-CH2- or -NH-CH2-C(O)-;q is 0 or 1;ris 1, 2 or 3;v is 0 or 1;# is the point of attachment to the anti-Ly6E antibody construct, A, and% is the point of attachment to the drug moiety, D.

31. The antibody-drug conjugate of claim 30, wherein L has formula (Illa) and wherein:(a) v is 1, and / or(b) Y is -NH-CH2-.

32. The antibody-drug conjugate of claim 30 or claim 31, wherein Str is:O O— (CH2)t— C—or— (CH2CH2O)U— (CH2)t— C—33. The antibody-drug conjugate of claim 21, wherein L-(D) is:wherein ** is the point of conjugation to the antibody construct A.

34. The antibody-drug conjugate of claim 33, wherein:(a) m is 1, and / or(b) n is about 8.

35. A method of preparing the antibody-drug conjugate of any one of claims 21 to 34 comprising conjugating drug-linker L-(D)mto the antibody construct A.

36. The method of claim 35, wherein drug-linker L-(D)mis conjugated to native cysteine residues of the antibody construct A.

37. The method of claim 35 or claim 36, wherein m is 1.

38. An antibody-drug conjugate having the structure:wherein n is about 8; andA is an antibody construct comprising an antigen-binding domain that binds to human lymphocyte antigen 6 complex, locus E, protein (Ly6E), wherein the antigen-bindingdomain comprises complementarity determining region (CDR) sequences of a heavy chain variable domain (VH) comprising a sequence as set forth in SEQ ID NO: 42 and CDR sequences of a light chain variable domain (VL) comprising a sequence as set forth in SEQ ID NO: 38.

39. The antibody-drug conjugate of claim 38, wherein the antigen-binding domain comprises a heavy chain CDR 1 (HCDR1) comprising a sequence as set forth in SEQ ID NO: 17; a heavy chain CDR 2 (HCDR2) comprising a sequence as set forth in SEQ ID NO: 94; a heavy chain CDR 3 (HCDR3) comprising a sequence as set forth in SEQ ID NO: 14; a light chain CDR 1 (LCDR1) comprising a sequence as set forth in SEQ ID NO: 95; a light chain CDR 2 (LCDR2) comprising a sequence as set forth in SEQ ID NO: 26; and a light chain CDR 3 (LCDR3) comprising a sequence as set forth in SEQ ID NO: 24.

40. The antibody-drug conjugate of claim 38, wherein the antigen-binding domain comprises a heavy chain CDR 1 (HCDR1) comprising the sequence as set forth in SEQ ID NO: 12, a heavy chain CDR 2 (HCDR2) comprising the sequence as set forth in SEQ ID NO: 43; a heavy chain CDR3 (HCDR3) comprising the sequence as set forth in SEQ ID NO: 14; a light chain CDR1 (LCDR1) comprising the sequence as set forth in SEQ ID NO: 48, a light chain CDR 2 (LCDR2) comprising the sequence as set forth in SEQ ID NO: 26, and a light chain CDR 3 (LCDR3) comprising the sequence as set forth in SEQ ID NO: 24.

41. The antibody-drug conjugate of any one of claims 38 to 40, wherein the antigen-binding domain comprises:(a) a heavy chain variable domain (VH) sequence as set forth in SEQ ID NO: 42; or (b) a light chain variable domain (VL) sequence as set forth in SEQ ID NO: 38, or (c) a heavy chain variable domain (VH) sequence as set forth in SEQ ID NO: 42 and a light chain variable domain (VL) sequence as set forth in SEQ ID NO: 38.

42. The antibody-drug conjugate of any one of claims 38 to 41, wherein the antigen-binding domain is a Fab.

43. The antibody-drug conjugate of any one of claims 38 to 42, wherein the antibody construct further comprises an IgG Fc region.

44. The antibody-drug conjugate of claim 43, wherein the IgG Fc region comprises one or more amino acid modifications that reduce effector function.

45. The antibody-drug conjugate of claim 44, wherein the one or more amino acid modifications are L234A, L235A and D265S, wherein the numbering of residues is according to the EU numbering system.

46. The antibody-drug conjugate of any one of claims 38 to 45 further comprising a second antigen-binding domain.

47. The antibody-drug conjugate of claim 46, wherein the second antigen-binding domain binds to Ly6E.

48. The antibody-drug conjugate of claim 47, wherein both antigen-binding domains are the same.

49. The antibody-drug conjugate of claim 38, wherein the antibody construct comprises:(a) two heavy chains comprising the sequence as set forth in SEQ ID NO: 87 and two light chains comprising the sequence as set forth in SEQ ID NO: 68, or(b) two heavy chains comprising the sequence as set forth in SEQ ID NO: 88 and two light chains comprising the sequence as set forth in SEQ ID NO: 68.

50. A method of preparing the antibody-drug conjugate of any one of claims 38 to 49 comprising conjugating a drug-linker having the structure:to the antibody construct A, wherein the drug-linker is conjugated to native cysteine residues of the antibody construct A.

51. A pharmaceutical composition comprising the antibody construct of any one of claims 1 to 15, or the antibody-drug conjugate of any one of claims 20 to 34 and 38 to 49, and a pharmaceutically acceptable carrier or diluent.

52. An antibody construct of any one of claims 1 to 15 or the antibody-drug conjugate of any one of claims 20 to 34 and 38 to 49 for use in therapy.

53. The antibody construct or antibody-drug conjugate for use according to claim 52, wherein the therapy comprises treatment of cancer.

54. Use of an antibody construct of any one of claims 1 to 15 or the antibody-drug conjugate of any one of claims 20 to 34 and 38 to 49 in the manufacture of a medicament for treatment of cancer.

55. A method of inhibiting the growth of tumor cells comprising contacting the cells with an antibody construct of any one of claims 1 to 15 or the antibody-drug conjugate of any one of claims 20 to 34 and 38 to 49.

56. A method of treating a subject having a cancer comprising administering to the subject an effective amount of the antibody construct of any one of claims 1 to 15 or the antibody-drug conjugate of any one of claims 20 to 34 and 38 to 49.

57. The method of claim 56, wherein the cancer is a Ly6E-positive cancer.

58. The method of claim 57, wherein the Ly6E-positive cancer is selected from ovarian cancer, colon cancer, colorectal cancer, gastric cancer, breast cancer, lung cancer, bladder cancer, brain and CNS cancer, cervical cancer, esophageal cancer, head and neck cancer, tongue cancer or pancreatic cancer.

59. A kit comprising the antibody construct of any one of claims 1 to 31, or the antibody-drug conjugate of any one of claims 20 to 34 and 38 to 49, and a label and / or package insert containing instructions for use.

60. A method of detecting human lymphocyte antigen 6 complex, locus E, protein (Ly6E) in a biological sample comprising contacting the biological sample with the antibody construct of any one of claims 1 to 15 under conditions that allow for binding of the antibody construct to human Ly6E and detecting formation of a complex between the antibody construct and the human Ly6E in the biological sample.

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