NZ718617B2 - Conjugated antibodies against ly75 for the treatment of cancer - Google Patents

Abstract

The invention provides antibodies which bind to LY75. Nucleic acid molecules encoding the antibodies, expression vectors, host cells and methods for expressing the antibodies are also provided. The antibodies may be used for the treatment of cancer, including pancreatic cancer, ovarian cancer, breast cancer, colorectal cancer, esophageal cancer, skin cancer, thyroid cancer, lung cancer, bladder cancer, multiple myeloma and lymphoma. In a particular embodiment, the antibody or antigen binding portion thereof comprises the heavy chain CDRs NAWMS, RIKSKTDGGTTDYAAPVQG, and FGVVSFDY; and the light chain CDRs RASQSISDYLS, AASNLKT and QQSYRSPWT. t cancer, colorectal cancer, esophageal cancer, skin cancer, thyroid cancer, lung cancer, bladder cancer, multiple myeloma and lymphoma. In a particular embodiment, the antibody or antigen binding portion thereof comprises the heavy chain CDRs NAWMS, RIKSKTDGGTTDYAAPVQG, and FGVVSFDY; and the light chain CDRs RASQSISDYLS, AASNLKT and QQSYRSPWT.

Description

Conjugated antibodies against LY75 for the treatment of cancer INTRODUCTION The present disclosure relates generally to the fields of immunology and molecular biology.

More specifically, provided herein are antibodies and other therapeutic ns directed against LY75, nucleic acids encoding such antibodies and therapeutic ns, methods for preparing monoclonal antibodies and other therapeutic proteins, and methods for the treatment of diseases, such as cancers mediated by LY75 expression/activity and/or associated with abnormal expression/activity of ligands therefore.

BACKGROUND Lymphocyte antigen 75 acts as an endocytic receptor to direct captured antigens from the extracellular space to a specialized antigen-processing compartment and is thought to cause a reduction in proliferation of B-lymphocytes. Expression of Lymphocyte antigen 75 has been observed in pancreatic, ovarian, breast, colorectal, esophageal, skin, d and lung mallcell ) cancers as well as Multiple Myeloma and many different subtypes of lymphomas and leukaemias.

WO2009/061996 discloses isolated monoclonal antibodies which bind to human 5 (LY75) and related antibody based itions and molecules. Also disclosed are pharmaceutical compositions comprising the antibodies, as well as eutic and diagnostic methods for using the antibodies.

WO2008/104806 discloses affinity reagents capable of binding to LY75 for use in the treatment or prophylaxis of cancer.

BRIEF SUMMARY OF THE INVENTION The present disclosure relates to dies directed t LY75, nucleic acids encoding such antibodies, host cells comprising such c acids encoding the antibodies of the disclosure, methods for preparing anti-LY75, and methods for the treatment of diseases, such as the LY75 mediated disorders, e.g. human cancers, including pancreatic cancer, ovarian , breast cancer, colorectal cancer, esophageal cancer, skin cancer, thyroid cancer, lung cancer, head and neck , bladder cancer, gastric cancer, leukaemia, multiple myeloma and lymphoma.

In one aspect, the disclosure relates to an antibody, or an antigen-binding portion thereof, which: (a) binds an epitope on LY75 which is ized by an antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the amino acid ce set forth in SEQ ID NO: 2, or (b) es for binding to LY75 with an dy comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1, and a light chain le region comprising the amino acid ce set forth in SEQ ID NO: 2. 18544345_1 (GHMatters) P41668NZ00 In one embodiment, the antibody or antigen-binding portion thereof binds to human LY75 and comprises a heavy chain variable region comprising 1, 2 or 3 CDRs selected from the group consisting of CDRs comprising SEQ ID NOs: 5, 6, and 7, and/or a light chain le region comprising 1, 2 or 3 CDRs selected from the group consisting of CDRs comprising SEQ ID NOs: 8, 9 and 10.

In preferred embodiments said antibodies are isolated antibodies.

In some embodiments, the antibodies of the disclosure bind to LY75 (SEQ ID No: 15) and are internalized by a cell sing LY75, elicit an antibody ent cellular cytotoxicity (ADCC) response in the presence of effector cells or elicit a cytotoxic T-Cell response in the presence of effector cells.

In another embodiment, the antibody comprises the heavy and/or light chain complementarity determining regions (CDRs) or le regions (VRs) of the particular antibody described herein (e.g., referred to herein as "LY75_A1"). Accordingly, in one embodiment, the antibody ses the CDR1, CDR2, and CDR3 domains of the heavy chain variable (VH) region of antibody 1 having the ce shown in SEQ ID NO:1, and/or the CDR1, CDR2 and CDR3 domains of the light chain variable (VL) region of LY75_A1 having the sequence shown in SEQ ID NO:2.

In another embodiment, the antibody comprises a heavy chain variable region comprising a first vhCDR comprising SEQ ID NO:5; a second vhCDR comprising SEQ ID NO:6; and a third vhCDR comprising SEQ ID NO:7; and/or a light chain variable region comprising a first vlCDR comprising SEQ ID NO:8; a second vlCDR comprising SEQ ID NO:9; and a third vlCDR comprising SEQ ID NO:10, optionally n any one or more of the CDRs independently comprise one, two, three, four or five amino acid substitutions, additions or deletions.

In another embodiment, the antibodies of the disclosure bind to human LY75 and include a heavy chain variable region comprising SEQ ID NO:1, and/or conservative sequence modifications thereof. The dy may further include a light chain variable region comprising SEQ ID NO:2, and/or conservative sequence modifications thereof.

In a r embodiment, the antibodies of the disclosure bind to human LY75 and e a heavy chain variable region and a light chain variable region including the amino acid sequences set forth in SEQ ID NOs:1 and/or 2, respectively, and conservative sequence modifications thereof.

Isolated antibodies which e heavy and light chain variable regions having at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or more sequence identity to any of the above sequences are also included in the present disclosure. Ranges intermediate to the above-recited , e.g., heavy and light chain variable regions having at least 80-85%, 85-90%, 90-95% or 95-100% sequence identity to any of the above sequences are also intended to be encompassed by the present disclosure. In one ment, the antibody comprises a heavy chain variable region comprising SEQ ID NO:1 or a 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% identical to SEQ ID NO: 1. In another embodiment, 18544345_1 (GHMatters) P41668NZ00 the antibody comprises a light chain variable region comprising SEQ ID NO:2 or a 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% identical to SEQ ID NO: 2. In another embodiment, the antibody comprises a heavy chain framework region comprising an amino acid 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% identical to the framework of the heavy chain le region of SEQ ID NO: 1 as shown in SEQ ID NOS: 16, 17, 18 and 19. In another embodiment, the antibody comprises a light chain framework region comprising an amino acid 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% identical to the framework of the light chain variable region of SEQ ID NO:2 as shown in SEQ ID NOS: 20, 21, 22 and 23.

Also encompassed by the present disclosure are antibodies which compete for binding to LY75 with the antibodies of the disclosure. In a particular embodiment, the antibody competes for binding to LY75 with an antibody comprising heavy and/or light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs:1 and 2, respectively, or amino acid sequences 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% cal thereto. In another embodiment, the antibody competes for binding to LY75 with an antibody sing heavy and/or light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs:1 and 2 (LY75_A1).

Other antibodies of the disclosure bind to the same epitope or an e on LY75 recognized by the dies described . In another particular embodiment, the dy binds to an epitope on LY75 ized by an antibody comprising heavy and/or light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs:1 and 2, respectively, or amino acid sequences at least 80% identical thereto. In r embodiment, the antibody binds to an epitope on LY75 recognized by an antibody comprising heavy and/or light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs:1 and 2 (LY75_A1).

In a further embodiment, the dies of the disclosure bind specifically to one or more, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10, peptide(s) selected from the group comprising SEQ ID NOs: 24, , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 or fragments thereof, wherein said fragments ses at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 contiguous amino acids. In a further embodiment, the epitope recognized by the antibodies of the present disclosure comprises one or more peptides, two or more or three or more peptides selected from the group consisting of SEQ ID NOs: 27, 29, 30, 34, 35, 36 or 37 or fragments thereof wherein said fragments comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 contiguous amino acids. In a further embodiment, the epitope recognized by the antibodies of the present disclosure ses one or more peptides, for example, two or three peptides selected from the group consisting of SEQ ID NOs: 30, 36 and 37 or fragments 18544345_1 (GHMatters) P41668NZ00 thereof wherein said fragments comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 contiguous amino acids.

In a further embodiment, the antibodies of the disclosure comprise variable CDRs as compared to the parent antibodies described herein. Thus, the disclosure relates to variant antibodies comprising variant variable regions of a parent antibody, wherein the parent antibody ses a first vhCDR comprising SEQ ID NO:5, a second vhCDR comprising SEQ ID NO: 6, a third vhCDR comprising SEQ ID NO:7, a first vlCDR comprising SEQ ID NO:8, a second vlCDR comprising SEQ ID NO:9 and a third vlCDR comprising a SEQ ID NO:10, and wherein the variant antibody has 1, 2, 3, 4, 5 or 6 amino acid substitutions collectively in the set of the first vhCDR, the second vhCDR, the third vhCDR, the first vlCDR, the second vlCDR and the third vlCDR, with from 1 to 4, 1 to 3 or 1 to 2 substitutions of ular use, and wherein the antibody retains specific binding to LY75.

The antibodies of the disclosure can either be full-length, for example, any of the following isotypes: IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, and IgE. Alternatively, the antibodies can be fragments such as an n-binding portion or a single chain antibody (e.g., a Fab, F(ab')2, Fv, a single chain Fv fragment, an isolated complementarity determining region (CDR) or a combination of two or more isolated CDRs). The antibodies can be any kind of antibody, ing, but not limited to, human, zed, and chimeric antibodies.

In other embodiments, the dies of the disclosure are in the form of an immunoconjugate (i.e., further e a covalently attached moiety). In a particular embodiment, the moiety is a drug, such as a sinoid, a dolastatin, an auristatin, a thecene, a calicheamicin, CC1065 or derivatives thereof. In a preferred embodiment, the drug moiety is DM1 or DM4 In other embodiments, the antibodies of the disclosure further encompass a bispecific molecule and as such can elicit an antibody dependent cellular cytotoxicity (ADCC) response in the presence of effector cells, thus killing LY75-expressing cells.

In other embodiments, the dies of the disclosure further encompass a bispecific molecule and as such can elicit a cytotoxic T-cell response in the presence of effector cells, thus killing LY75-expressing cells.

In another aspect, the disclosure relates to nucleic acids encoding the heavy and/or light chain variable regions of the antibodies of the disclosure. In one embodiment, there is disclosed a nucleic acid comprising a sequence encoding the heavy chain of the antibody of the disclosure or the antigen-binding portion thereof. In another embodiment there is disclosed a c acid comprising a sequence ng the light chain of the antibody of the disclosure or the antigenbinding n thereof. In a further embodiment there is sed a nucleic acid comprising a sequence ng the heavy and light chain variable regions of the antibodies of the disclosure.

In one embodiment, the disclosure relates to an isolated monoclonal antibody that binds human LY75, wherein the antibody comprises a heavy chain variable region and a light chain variable region d by nucleic acid sequences comprising SEQ ID NOs:3 and 4, respectively, 18544345_1 ters) P41668NZ00 or nucleic acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the aforementioned nucleic acid sequences or sequences which differ from SEQ ID NOs: 3 and 4 due to degeneracy of the genetic code.

In another aspect the disclosure relates to expression vectors comprising nucleic acids encoding heavy and/or light chain variable regions of the dies of the disclosure operably linked to one or more regulatory elements.

In another aspect, the disclosure relates to host cells containing nucleic acids encoding heavy and/or light chain variable regions or the antigen binding ns thereof of the foregoing antibodies.

Preferably, wherein the host cell ses said heavy and/or light chain variable regions or the antigen binding portions thereof when the host cell is grown under conditions wherein the nucleic acid(s) is expressed.

In a red embodiment the host cell comprises: (i) an expression vector according to the present disclosure; or (ii) a first expression vector comprising the nucleic acid sequence encoding the heavy chain of the dy of the disclosure or the antigen-binding portion thereof and a second expression vector comprising the nucleic acid sequence encoding the light chain of the antibody of the disclosure or the antigen-binding portion thereof.

In a further aspect, the disclosure relates to making an antibody or an antigen-binding portion f, comprising culturing a host cell ing to the present disclosure under conditions where the antibody or an antigen-binding portion thereof is sed and optionally isolating the antibody or an antigen-binding portion thereof.

In a further aspect there is provided a method of treating cancer sing administering to a t in need thereof an antibody or an antigen-binding portion thereof of according to the present sure wherein the antibody or antigen-binding portion thereof is internalized by a cell expressing LY75, said antibody or antigen-binding n comprising a covalently attached drug conjugate. It will be understood that the antibody or an antigen-binding portion thereof of the disclosure is one which binds to LY75 (SEQ ID No: 15). In one ment, the antibody comprises a heavy chain variable region comprising a first vhCDR comprising SEQ ID NO:5; a second vhCDR comprising SEQ ID NO:6; and a third vhCDR sing SEQ ID NO:7; and a light chain variable region comprising a first vlCDR comprising SEQ ID NO:8; a second vlCDR comprising SEQ ID NO: 9; and a third vlCDR comprising SEQ ID NO:10 and a covalently attached drug conjugate.

In a further , the disclosure relates to a method of treating cancer, wherein a patient in need thereof is administered an antibody or antibodies or an antigen-binding portion thereof of the disclosure and wherein such antibody or antibodies or an n-binding portion thereof of the disclosure elicit an ADCC response in the presence of effector cells. Preferably, the antibody or an antigen-binding portion thereof comprises a heavy chain le region comprising a first vhCDR comprising SEQ ID NO: 5; a second vhCDR comprising SEQ ID NO: 6; and a third vhCDR comprising SEQ ID NO: 7; and a light chain variable region comprising a first vlCDR comprising 18544345_1 (GHMatters) P41668NZ00 SEQ ID NO: 8; a second vlCDR comprising SEQ ID NO: 9; and a third vlCDR comprising SEQ ID NO: 10.

In a further aspect the disclosure relates to a method of treating cancer, n a patient in need thereof is stered an antibody or antibodies or an antigen-binding portion thereof of the disclosure and wherein such antibody or antibodies or an antigen-binding portion thereof of the disclosure elicit a cytotoxic T-cell response in the presence of effector cells. Preferably, the antibody comprises a heavy chain variable region comprising a first vhCDR comprising SEQ ID NO: 5; a second vhCDR sing SEQ ID NO: 6; and a third vhCDR comprising SEQ ID NO: 7; and a light chain variable region comprising a first vlCDR comprising SEQ ID NO: 8; a second vlCDR comprising SEQ ID NO: 9; and a third vlCDR comprising SEQ ID NO: 10.

In a further aspect , the disclosure relates to one or more antibodies of the disclosure for use in the treatment of cancer.

Also provided is the use of one or more antibodies of the disclosure in the manufacture of a medicament for the treatment of cancer.

In some embodiments, the cancer is selected from the group consisting of pancreatic cancer, kidney cancer, liver cancer, ovarian , breast , ctal cancer, esophageal cancer, head and neck cancer, skin , thyroid cancer, bladder cancer, gastric cancer, lung cancer, leukaemia, a, preferably multiple myeloma, and lymphoma. Particularly preferred cancers e non- Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), B-Cell Lymphoma, Follicular Lymphoma, Mantle Cell Lymphoma, Lymphoma of Mucosa-Associated Lymphoid Tissue (MALT), TCell /Histiocyte-Rich B-Cell Lymphoma, Burkitt’s ma, Lymphoplasmacytic Lymphoma, Small Lymphocytic Lymphoma, Marginal Zone Lymphoma, T Cell Lymphoma, Peripheral T-Cell Lymphoma, Anaplastic Large Cell Lymphoma and AngioImmunoblastic T-Cell Lymphoma, acute d leukaemia, chronic lymphocytic leukaemia, bladder , pancreatic cancer and triple- negative breast cancer.

According to a still r aspect the disclosure relates to a method of detecting, diagnosing and/or screening for or monitoring the progression of a cancer wherein LY75 is expressed in said , or of monitoring the effect of a cancer drug or therapy directed to said cancer, in a subject which ses ing the presence or level of antibodies capable of immunospecific binding to LY75, or one or more nts thereof.

Preferably, the cancer is selected from the group consisting of pancreatic cancer, kidney cancer, liver , ovarian cancer, breast cancer, colorectal cancer, esophageal cancer, head and neck cancer, skin cancer, thyroid cancer, bladder cancer, gastric , lung cancer, leukaemia, myeloma, preferably multiple myeloma, and lymphoma. Particularly preferred cancers e non- Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), B-Cell Lymphoma, Follicular Lymphoma, Mantle Cell Lymphoma, Lymphoma of Mucosa-Associated Lymphoid Tissue (MALT), TCell /Histiocyte-Rich B-Cell Lymphoma, Burkitt’s Lymphoma, Lymphoplasmacytic Lymphoma, Small Lymphocytic Lymphoma, Marginal Zone Lymphoma, T Cell Lymphoma, Peripheral T-Cell Lymphoma, Anaplastic Large Cell Lymphoma and AngioImmunoblastic T-Cell Lymphoma, acute 18544345_1 (GHMatters) P41668NZ00 myeloid leukaemia, chronic lymphocytic leukaemia, bladder cancer, pancreatic cancer and triplenegative breast cancer.

Also, within the scope of the disclosure are kits comprising the compositions (e.g., antibodies) of the disclosure and, optionally, instructions for use. The kit can further contain a least one onal reagent or one or more onal antibodies of the disclosure.

Other features and advantages of the instant disclosure will be apparent from the ing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 s the alignment of LY75_A1 heavy chain (SEQ ID NO:1), the human VH 3-15 Germline (SEQ ID NO:11) and the human JH4 Germline (SEQ ID NO:12). The CDR regio ns of LY75_A1 heavy chain are underlined.

Figure 2 depicts the alignment of LY75_A1 light chain (SEQ ID NO:2), the human VK O12 Germline (SEQ ID NO:13) and the human JK4 Germline (SEQ ID NO:14). The CDR regions of LY75_A1 light chain are underlined.

Figure 3a depicts cytotoxic activity of anti-LY75 monoclonal antibodies conjugated with DM1 in HT-29 and shows while most dies bind to LY75 only a few display efficacy.

Figure 3b depicts xic activity of anti-LY75 dies conjugated to either DM1 or DM4 in HT-29.

Figure 3c depicts cytotoxic activity of anti-LY75 antibodies conjugated to either DM1 or DM4 in RAJI cells.

Figure 3d depicts cytotoxic activity of Y75 antibodies conjugated to either DM1 or DM4 in Namalwa cells.

Figure 3e depicts cytotoxic activity of anti-LY75 dies conjugated to either DM1 or DM4 in Karpas 299cells.

Figure 3f depicts cytotoxic activity of anti-LY75 antibodies conjugated to either DM1 or DM4 in BxPC3 cells.

Figure 3g depicts cytotoxic activity of anti-LY75 antibodies conjugated to either DM1 or DM4 in HupT4 cells.

Figure 3h depicts cytotoxic ty of Y75 antibodies conjugated to either DM1 or DM4 in HPAFFII cells.

Figure 3i depicts cytotoxic activity of anti-LY75 antibodies conjugated to either DM1 or DM4 in EHEB cells.

Figure 3j depicts cytotoxic activity of anti-LY75 antibodies conjugated to either DM1 or DM4 in Mec-1 cells.

Figure 3k depicts cytotoxic activity of anti-LY75 antibodies conjugated to either DM1 or DM4 in AML-193 cells.

Figure 3l depicts cytotoxic activity of anti-LY75 antibodies conjugated to either DM1 or DM4 in HCC 70 cells. 18544345_1 (GHMatters) P41668NZ00 Figure 3m depicts cytotoxic activity of anti-LY75 antibodies conjugated to either DM1 or DM4 in HCC 1806 cells.

Figure 3n depicts cytotoxic activity of anti-LY75 antibodies conjugated to either DM1 or DM4 in MDA-MB-468 cells.

Figure 3o depicts cytotoxic activity of anti-LY75 antibodies ated to either DM1 or DM4 in RT4 cells.

Figure 3p depicts cytotoxic activity of anti-LY75 antibodies conjugated to either DM1 or DM4 in 5637 cells.

Figure 3q depicts xic activity of anti-LY75 antibodies conjugated to either DM1 or DM4 in SW780 cells.

Figure 3r depicts cytotoxic activity of anti-LY75 antibodies conjugated to either DM1 or DM4 in SCC-9 cells.

Figure 3s depicts cytotoxic activity of anti-LY75 antibodies ated to either DM1 or DM4 in OE 19 cells.

Figure 3t depicts cytotoxic activity of anti-LY75 antibodies conjugated to either DM1 or DM4 in 3 cells.

Figure 3u depicts cytotoxic ty of anti-LY75 dies conjugated to either DM1 or DM4 in SK-OV-3 cells.

Figure 3v depicts cytotoxic activity of anti-LY75 antibodies conjugated to either DM1 or DM4 in MOLP-8 cells.

Figure 3w depicts cytotoxic activity of anti-LY75 antibodies conjugated to either DM1 or DM4 in 26 cells.

Figure 4a depicts the efficacy of anti-LY75 dies conjugated to either DM1 or DM4 in Raji Burkitt’s lymphoma SCID mouse xenograft model.

Figure 4b s the efficacy of anti-LY75 antibodies conjugated to either DM1 or DM4 in Namalwa Burkitt’s lymphoma SCID mouse xenograft model.

Figure 4c depicts the efficacy of anti-LY75 antibodies conjugated to either DM1 or DM4 in HPAFII pancreatic adenocarcinoma athymic nude mousexenograft model.

Figure 4d s the efficacy of anti-LY75 antibodies conjugated to either DM1 or DM4 in SW780 human bladder carcinoma SCID mouse xenograft model.

Figure 4e depicts the efficacy of anti-LY75 antibodies conjugated to either DM1 or DM4 in MDA-MB-468 athymic nude mouse xenograft model.

Figure 4f depicts the efficacy of anti-LY75 antibodies conjugated to either DM1 or DM4 in COLO205 colorectal adenocarcinoma athymic nude mouse xenograft model.

Figure 5a shows competitive g of anti-LY75-mAb and an anti-LY75-mAb ated to MCC-DM1.

Figure 5b shows non-competitive binding of 1 and an anti-LY75-mAb conjugated to MCC-DM1. 18544345_1 (GHMatters) P41668NZ00 Figure 6a-6j show graphical entations of the binding of antibody 1 to LY75 peptides on a peptide rray.

Figure 7 shows an amino acid alignment of peptides bound by antibody LY75_A1 in both the peptide microarray assay and the peptide pull down assay. es highlighted are those likely to form the epitope recognized by antibody 1.

DETAILED DESCRIPTION OF THE INVENTION The present disclosure relates to isolated antibodies which bind to LY75 protein described in SEQ ID No: 15 as ed herein.

In addition, the LY75 antibodies of the present invention maybe a bispecific molecule and as such can elicit an antibody dependent cellular cytotoxicity (ADCC) response in the presence of effector cells, thus killing LY75-expressing cells.

In addition, the LY75 antibodies of the present invention maybe a bispecific le and as such can elicit a xic T-cell response in the presence of effector cells, thus killing LY75- expressing cells.

In addition, the LY75 antibodies of the present invention maybe alized when contacted with cells sing the LY75 receptor. As discussed herein, the LY75 receptor is overexpressed and/or differentially expressed on certain cancer cells, including but not limited to, kidney cancer, liver cancer, esophageal cancer, head and neck , skin cancer, thyroid cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, breast , ovarian cancer bladder cancer, leukaemia preferably acute myeloid leukaemia or chronic lymphocytic leukaemia, myeloma, preferably multiple myeloma, lymphoma, preferably DLBCL B-Cell Lymphoma, Follicular Lymphoma, Mantle Cell Lymphoma, Lymphoma of Mucosa-Associated Lymphoid Tissue (MALT), TCell ocyte-Rich B-Cell Lymphoma, Burkitt’s Lymphoma, Lymphoplasmacytic Lymphoma, Small Lymphocytic Lymphoma, Marginal Zone Lymphoma, T Cell ma, Peripheral T-Cell Lymphoma, Anaplastic Large Cell Lymphoma and AngioImmunoblastic T-Cell Lymphoma, and lung cancer.

As such, when the LY75 antibodies of the present invention are conjugated to drugs (sometimes referred to herein as "antibody-drug ates" or "ADCs"), the internalization of these ADC molecules into cancer cells s in cell death and thus tumor treatment.

The present invention provides antibodies that possess particular structural features such as CDR regions with particular amino acid sequences. Described herein, are a set of CDRs which can form an affinity reagent, e.g. an antibody, which exhibits binding to LY75.

Thus, the disclosure provides antibodies, ably isolated antibodies , as outlined below, includes a wide variety of well-known antibody structures, derivatives, mimetics and conjugates), nucleic acids encoding these antibodies, host cells used to make the antibodies, s of making the antibodies, and pharmaceutical compositions comprising the antibodies and optionally a pharmaceutical carrier, methods of treatment and diagnosis comprising the use of the antibodies and the use of the antibodies for the treatment of cancers. 18544345_1 (GHMatters) P41668NZ00 LY75 Proteins Lymphocyte n 75 acts as an endocytic receptor to direct captured antigens from the extracellular space to a specialized antigen-processing compartment and is thought to cause a reduction in proliferation of B-lymphocytes.

According to SWISS-PROT, Lymphocyte antigen 75 is expressed in spleen, thymus, colon and peripheral blood lymphocytes. It has been detected in myeloid and B lymphoid cell lines.

Isoforms designated herein OGTA076b and OGTA076c are expressed in malignant Hodgkin's lymphoma cells called Hodgkin's and Reed-Sternberg (HRS) cells. LY75 acts as an endocytic receptor to direct captured antigens from the extracellular space to a specialized antigen-processing compartment. It causes d proliferation of hocytes.

Expression of LY75 has been observed in pancreatic, bladder, ovarian, breast (including triple negative), colorectal, esophageal, skin, thyroid and lung (non-small-cell) cancers as well as Multiple Myeloma and many different subtypes of lymphomas (including DLBCL) and leukaemias.

The antibody of the invention may, in certain cases, cross-react with the LY75 from species other than human. For example, to tate clinical testing, the antibodies of the invention may cross react with murine or primate LY75 molecules. atively, in certain embodiments, the dies may be completely specific for human LY75 and may not exhibit species or other types of non-human cross-reactivity.

Antibodies The present invention provides Y75 antibodies, generally eutic and/or diagnostic antibodies as described . Antibodies that find use in the present invention can take on a number of formats as described herein, including traditional antibodies as well as dy derivatives, fragments and mimetics, described below. In one embodiment, the ion provides antibody structures that n a set of 6 CDRs as defined herein (including small numbers of amino acid changes as bed below).

"Antibody" as used herein includes a wide variety of structures, as will be appreciated by those in the art, that in some embodiments contain at a minimum a set of 6 CDRs as defined herein; including, but not limited to traditional antibodies (including both monoclonal and polyclonal dies), humanized and/or chimeric antibodies, antibody fragments, engineered antibodies (e.g. with amino acid modifications as outlined below), multispecific antibodies (including bispecific antibodies), and other analogs known in the art.

Traditional antibody structural units typically se a tetramer. Each tetramer is lly composed of two identical pairs of polypeptide chains, each pair having one "light" (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several sses, including, but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has sses, including, but not limited to, IgM1 and IgM2. Thus, "isotype" as used herein is meant any of the subclasses of immunoglobulins defined by the chemical 18544345_1 (GHMatters) P41668NZ00 and antigenic characteristics of their constant regions. The known human globulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE. It should be understood that therapeutic antibodies can also comprise s of any combination of isotypes and/or subclasses.

In many embodiments, IgG isotypes are used in the t invention, with IgG1 finding ular use in a number of applications.

The terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen ition. In the variable region, three loops are gathered for each of the V domains of the heavy chain and light chain to form an antigen-binding site. Each of the loops is referred to as a complementarity-determining region (hereinafter ed to as a "CDR"), in which the variation in the amino acid sequence is most significant. "Variable" refers to the fact that certain segments of the variable region differ extensively in sequence among antibodies. Variability within the variable region is not evenly distributed. Instead, the V s consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme ility called "hypervariable regions" that are each 9-15 amino acids long or longer.

Each VH and VL is composed of three ariable regions ("complementary determining s," "CDRs") and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The hypervariable region generally encompasses amino acid residues from about amino acid residues 24-34 (LCDR1; "L" denotes light chain), 50-56 (LCDR2) and 89-97 (LCDR3) in the light chain variable region and around about 31-35B (HCDR1; "H" denotes heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable region; Kabat et al., CES OF PROTEINS OF IMMUNOLOGICAL ST, 5th Ed. Public Health e, National Institutes of Health, Bethesda, Md. (1991) and/or those residues forming a hypervariable loop (e.g. residues 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and 26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain variable region; Chothia and Lesk (1987) J.

Mol. Biol. 196:901-917. Specific CDRs of the invention are described below.

Throughout the present specification, the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately, residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region) (e.g, Kabat et al., supra (1991)).

The CDRs contribute to the ion of the antigen-binding, or more specifically, epitope binding site of antibodies. The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary g are typically lost on treatment with denaturing solvents. An epitope lly es at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. As described herein, methods for determining what 18544345_1 (GHMatters) P41668NZ00 epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and e, for example, immunoblotting and immunoprecipitation assays, n overlapping or contiguous peptides from LY75 are tested for reactivity with the given anti-LY75 antibody. Methods of determining spatial conformation of epitopes include techniques in the art and those described herein, for example, x-ray crystallography and 2-dimensional r magnetic resonance (see, e.g., Epitope Mapping ols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)). The term "epitope mapping" refers to the process of identification of the molecular determinants for antibody-antigen recognition.

The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector on. Kabat et al. collected numerous primary sequences of the variable s of heavy chains and light chains. Based on the degree of conservation of the sequences, they classified individual primary ces into the CDR and the framework and made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No. 2, E.A.

Kabat et al.).

In the IgG subclass of immunoglobulins, there are several immunoglobulin domains in the heavy chain. By "immunoglobulin (Ig) domain" herein is meant a region of an immunoglobulin having a distinct tertiary ure. Of interest in the present invention are the heavy chain domains, including, the nt heavy (CH) domains and the hinge domains. In the context of IgG antibodies, the IgG isotypes each have three CH regions. Accordingly, "CH" domains in the context of IgG are as follows: "CH1" refers to positions 118-220 according to the EU index as in Kabat. "CH2" refers to positions 237-340 according to the EU index as in Kabat, and "CH3" refers to positions 341-447 ing to the EU index as in Kabat. r type of Ig domain of the heavy chain is the hinge region. By "hinge" or "hinge region" or "antibody hinge region" or "immunoglobulin hinge region" herein is meant the flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody.

Structurally, the IgG CH1 domain ends at EU position 220, and the IgG CH2 domain begins at residue EU position 237. Thus for IgG the dy hinge is herein defined to include ons 221 (D221 in IgG1) to 236 (G236 in IgG1), wherein the numbering is according to the EU index as in Kabat. In some embodiments, for example in the context of an Fc region, the lower hinge is included, with the "lower hinge" generally referring to positions 226 or 230.

Of particular interest in the present invention are the Fc s. By "Fc" or "Fc region" or "Fc domain" as used herein is meant the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain and in some cases, part of the hinge.

Thus Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may include the J chain. For IgG, the Fc domain comprises immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3) and the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to e residues C226 or P230 to its carboxyl-terminus, wherein 18544345_1 (GHMatters) P41668NZ00 the numbering is according to the EU index as in Kabat. In some embodiments, as is more fully described below, amino acid modifications are made to the Fc , for example to alter binding to one or more FcγR receptors or to the FcRn receptor.

In some embodiments, the dies are full . By "full length antibody" herein is meant the structure that constitutes the natural biological form of an antibody, including variable and nt regions, ing one or more modifications as outlined herein.

Alternatively, the antibodies can be a variety of structures, including, but not limited to, dy fragments, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, dy fusions (sometimes referred to as "antibody conjugates"), and nts of each, respectively. Structures that rely on the use of a set of CDRs are included within the definition of "antibody".

In one embodiment, the antibody is an antibody fragment. Specific antibody fragments include, but are not limited to, (i) the Fab nt consisting of VL, VH, CL and CH1 domains, (ii) the Fd fragment consisting of the VH and CH1 s, (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward et al., 1989, Nature 341:544-546, ly incorporated by reference) which consists of a single variable region, (v) isolated CDR regions, (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al., 1988, Science 3-426, Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883, entirely incorporated by reference), (viii) bispecific single chain Fv (WO 03/11161, hereby incorporated by reference) and (ix) "diabodies" or "triabodies", multivalent or multispecific fragments constructed by gene fusion (Tomlinson et. al., 2000, s Enzymol. 326:461-479; WO94/13804; Holliger et al., 1993, Proc. Natl. Acad. Sci.

U.S.A. 90:6444-6448, all entirely orated by reference).

Chimeric and Humanized Antibodies In some embodiments, the antibody can be a mixture from ent species, e.g. a chimeric antibody and/or a humanized antibody. That is, in the present invention, the CDR sets can be used with framework and constant regions other than those specifically described by sequence herein.

In general, both ric antibodies" and "humanized antibodies" refer to antibodies that combine regions from more than one species. For example, "chimeric antibodies" ionally comprise variable region(s) from a mouse (or rat, in some cases) and the constant region(s) from a human. "Humanized antibodies" generally refer to non-human antibodies that have had the variable- domain ork regions swapped for sequences found in human antibodies. Generally, in a humanized antibody, the entire antibody, except the CDRs, is encoded by a polynucleotide of human origin or is cal to such an antibody except within its CDRs. The CDRs, some or all of which are encoded by nucleic acids originating in a non-human organism, are grafted into the beta-sheet framework of a human antibody variable region to create an antibody, the specificity of which is determined by the engrafted CDRs. The creation of such antibodies is bed in, e.g., WO 18544345_1 (GHMatters) P41668NZ00 92/11018, Jones, 1986, Nature 321:522-525, Verhoeyen et al., 1988, Science 239:1534-1536, all entirely incorporated by reference. "Backmutation" of selected acceptor framework residues to the corresponding donor residues is often required to regain affinity that is lost in the initial grafted construct (US 5530101; US 5585089; US 5693761; US 5693762; US 6180370; US 5859205; US 5821337; US 6054297; US 6407213, all entirely incorporated by reference). The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region, lly that of a human immunoglobulin, and thus will lly comprise a human Fc region. zed antibodies can also be generated using mice with a genetically engineered immune system. Roque et al., 2004, Biotechnol. Prog. 20:639-654, entirely incorporated by reference. A variety of techniques and methods for humanizing and reshaping non-human antibodies are well known in the art (See Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA), and references cited therein, all entirely incorporated by reference). Humanization methods include but are not d to methods described in Jones et al., 1986, Nature 321:522-525; ann et al.,1988; Nature 332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA 86:10029-33; He et al., 1998, J. Immunol. 160: 1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA 89:4285-9, Presta et al., 1997, Cancer Res. 57(20):4593-9; Gorman et al., 1991, Proc. Natl.

Acad. Sci. USA 88:4181-4185; O’Connor et al., 1998, Protein Eng 11:321-8, all entirely incorporated by reference. Humanization or other methods of reducing the immunogenicity of nonhuman antibody variable regions may include resurfacing s, as described for example in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973, entirely orated by nce. In one embodiment, the parent antibody has been affinity matured, as is known in the art. ure-based methods may be employed for humanization and affinity tion, for example as described in USSN 11/004,590. ion based methods may be employed to humanize and/or affinity mature antibody variable regions, including but not limited to methods described in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol. Chem. 271(37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering 16(10):753-759, all entirely orated by reference. Other humanization methods may involve the grafting of only parts of the CDRs, including but not limited to methods described in USSN 09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis et al., 2002, J. Immunol. 169:3076-3084, all entirely incorporated by nce.

In one embodiment, the antibodies of the invention can be multispecific antibodies, and y bispecific dies, also sometimes referred to as "diabodies". These are antibodies that bind to two (or more) different antigens, or ent epitopes on the same antigen. Diabodies can be manufactured in a variety of ways known in the art (Holliger and , 1993, Current Opinion hnol. 4:446-449, entirely incorporated by reference), e.g., prepared chemically or from hybrid hybridomas.

In one embodiment, the antibody is a minibody. Minibodies are minimized antibody-like ns comprising a scFv joined to a CH3 domain. Hu et al., 1996, Cancer Res. 56:3055-3061, 18544345_1 ters) P41668NZ00 entirely incorporated by nce. In some cases, the scFv can be joined to the Fc region, and may e some or the entire hinge region. It should be noted that minibodies are included within the definition of "antibody" despite the fact it does not have a full set of CDRs.

The antibodies of the present invention are generally isolated or recombinant. "Isolated," when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated and/or red from a cell or cell culture from which it was expressed. Thus an isolated antibody is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g. an ed antibody that specifically binds to the LY75 is substantially free of antibodies that specifically bind antigens other than the LY75). Thus, an "isolated" antibody is one found in a form not normally found in nature (e.g. nonnaturally occurring). An ed antibody as defined herein may, in one embodiment, include at least one amino acid which does not occur in the "naturally" occurring antibody. This amino acid may be introduced by way of an addition or a substitution. It will be understood that the introduced amino acid may be a naturally occurring or non-naturally occurring amino acid. In some embodiments, the antibodies of the invention are recombinant proteins, isolated proteins or ntially pure proteins.

An "isolated" protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, for example constituting at least about 5%, or at least about 50% by weight of the total protein in a given . It is understood that the isolated protein may constitute from 5 to 99.9% by weight of the total n content depending on the circumstances. For example, the protein may be made at a significantly higher concentration through the use of an inducible promoter or high expression er, such that the protein is made at increased concentration levels. In the case of recombinant proteins, the definition includes the production of an antibody in a wide variety of organisms and/or host cells that are known in the art in which it is not naturally produced. Ordinarily, an isolated polypeptide will be prepared by at least one purification step. An "isolated antibody," refers to an antibody which is substantially free of other antibodies having ent antigenic specificities. For instance, an isolated antibody that specifically binds to LY75 is substantially free of antibodies that ically bind antigens other than LY75.

Isolated monoclonal antibodies, having different specificities, can be combined in a welldefined composition. Thus for example, the dy of the invention can optionally and individually be included or excluded in a formulation, as is further discussed below.

The anti-LY75 antibodies of the present invention specifically bind LY75 (e.g. SEQ ID No: 15). fic binding" or "specifically binds to" or is fic for" a particular antigen or an epitope means g that is ably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a l le that is similar to the target.

Specific binding for a ular antigen or an epitope can be exhibited, for example, by an antibody having a KD for an antigen or e of at least about 10-4 M, at least about 10-5 M, at least 18544345_1 (GHMatters) P41668NZ00 about 10-6 M, at least about 10-7 M, at least about 10-8 M, at least about 10-9 M, alternatively at least about 10-10 M, at least about 10-11 M, at least about 10-12 M, or r, where KD refers to a dissociation rate of a particular antibody-antigen interaction. Typically, an antibody that specifically binds an antigen will have a KD that is 20-, 50-, 100-, 500-, 1000-, 5,000-, - or more times greater for a l molecule relative to the antigen or epitope. However, in the present invention, when administering ADCs of the LY75 antibodies of the invention, what is ant is that the KD is sufficient to allow internalization and thus cell death without significant side effects.

Also, specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a KA or Ka for an antigen or epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for the e relative to a control, where KA or Ka refers to an association rate of a particular antibody-antigen ction.

Standard assays to evaluate the binding ability of the antibodies toward LY75 can be done on the protein or cellular level and are known in the art, including for example, ELISAs, Western blots, RIAs, BIAcore® assays and flow cytometry analysis. Suitable assays are bed in detail in the Examples. The binding kinetics (e.g. binding affinity) of the antibodies also can be assessed by standard assays known in the art, such as by Biacore® system analysis. To assess binding to Raji or Daudi B cell tumor cells, Raji (ATCC Deposit No. CCL-86) or Daudi (ATCC Deposit No. CCL-213) cells can be obtained from publicly available sources, such as the American Type Culture Collection, and used in rd , such as flow cytometric analysis.

LY75 Antibodies The present invention provides LY75 antibodies that bind to LY75 (SEQ ID No: 15) and maybe internalized when ted with cells expressing LY75 on the cell surface or may elicit an ADCC se in the presence of effector cells or elicit a cytotoxic T-cell response in the ce of effector cells. These antibodies are ed to herein either as "anti-LY75" antibodies or, for ease of description, "LY75 antibodies".

The LY75 antibodies are internalized upon contact with cells, particularly tumor cells, which express LY75 on the surface. That is, LY75 antibodies as defined herein that also comprise drug conjugates are internalized by tumor cells, resulting in the release of the drug and subsequent cell death, allowing for treatment of cancers that exhibit LY75 expression. Internalization in this context can be measured in several ways. In one embodiment, the LY75 antibodies of the ion are contacted with cells, such as a cell line as outlined herein, using standard assays such as MAbZap. It would be clear to the skilled person that the MabZap assay is representative of the effect that would be expected to be seen with an antibody-drug conjugate (ADC). In the latter case, the ADC would be internalized, thus taking the drug into the cell. A toxic drug would have the capacity to kill the cell, i.e. to kill the targeted cancer cell. Data from MabZap assays are readily accepted by persons of skill in the art to be representative of ADC assays (Kohls, M and Lappi, D., [2000] Biotechniques, vol. 28, no. 1, 162-165).

In these in vitro assay embodiments, the LY75 antibodies of the invention are added, along with an anti-LY75 antibody sing a toxin; for e, the LY75 antibody may be murine or 18544345_1 (GHMatters) P41668NZ00 humanized and the anti-LY75 antibody can be anti-murine or anti-humanized and contain a toxin such as saporin. Upon formation of the [LY75 antibody of the ion]-[anti-LY75 antibody-drug conjugate] complex, the complex is internalized and the drug (e.g. saporin) is released, ing in cell death. Only upon internalization does the drug get released, and thus cells remain viable in the absence of internalization. As outlined below, without being bound by theory, in therapeutic applications, the anti-LY75 antibody contains the toxin, and upon internalization, the bond between the antibody and the toxin is d, releasing the toxin and killing the cell.

In addition, the LY75 antibodies elicit an ADCC response in the presence of or cells, particularly tumor cells, that s LY75 on the surface.

In one embodiment, the antibody comprises the heavy and light chain complementarity determining regions (CDRs) or variable s (VRs) of the particular antibody described herein (e.g., referred to herein as "LY75_A1"). Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the heavy chain variable (VH) region of dy LY75_A1 having the sequence shown in SEQ ID NO:1, and the CDR1, CDR2 and CDR3 domains of the light chain variable (VL) region of antibody LY75_A1 having the sequence shown in SEQ ID NO:2.

In r embodiment, the antibody comprises a heavy chain variable region comprising a first vhCDR comprising SEQ ID NO: 5; a second vhCDR sing SEQ ID NO: 6; and a third vhCDR comprising SEQ ID NO:7; and a light chain variable region comprising a first vlCDR comprising SEQ ID NO:8; a second vlCDR comprising SEQ ID NO: 9; and a third vlCDR comprising SEQ ID NO:10.

In another embodiment, the antibodies of the invention bind to human LY75 and include a heavy chain variable region sing an amino acid sequence comprising SEQ ID NO:1, and vative sequence modifications f. The antibody may further include a light chain variable region comprising an amino acid sequence comprising SEQ ID NO:2, and conservative sequence cations thereof.

In a further embodiment, the antibodies of the invention bind to human LY75 and include a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs:1 and/or 2, respectively, and conservative sequence modifications thereof.

As used herein, the term conservative sequence modification refers to, for example, the substitution of an amino acid with an amino acid having similar characteristics. It is common general knowledge for one skilled in the art what such substitutions may be considered conservative. Other modifications which can be considered to be conservative sequence modifications include, for example, glycosylation.

Isolated antibodies which e heavy and light chain variable regions having at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or more sequence identity to any of the above sequences are also included in the t invention. Ranges intermediate to the above-recited values, e.g., heavy and light chain le regions having at least 80-85%, 85-90%, 90-95% or 95-100% sequence identity to any of the above ces are also 18544345_1 (GHMatters) P41668NZ00 intended to be encompassed by the present invention. In one embodiment, the antibody comprises a heavy chain variable region comprising SEQ ID NO:1 or a sequence that is 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% identical to SEQ ID NO: 1. In another embodiment, the antibody comprises a light chain variable region comprising SEQ ID NO:2 or a sequence that is 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% identical to SEQ ID NO: 2. In r ment, the antibody comprises a heavy chain framework region comprising an amino acid sequence that is 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% identical to the ork of the heavy chain variable region of SEQ ID NO: 1 sing SEQ ID NOs: 16, 17 and 18. In another embodiment, the antibody ses a light chain framework region comprising an amino acid sequence that is 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% identical to the framework of the light chain variable region of SEQ ID NO:2 comprising SEQ ID , 20 and 21.

In one embodiment, the antibody of the invention is an anti-LY75 antibody (referred to herein as "LY75_A1 antibody") comprising the following CDRs, as well as ts containing a limited number of amino acid variants: A1 SEQ ID NOs variable heavy CDR1 5 variable heavy CDR2 6 variable heavy CDR3 7 variable light CDR1 8 variable light CDR2 9 variable light CDR3 10 Disclosed herein are also variable heavy and light chains that comprise the CDR sets of the invention, as well as full length heavy and light chains (e.g. comprising constant regions as well). As will be appreciated by those in the art, the CDR sets of the ion can be incorporated into murine, humanized or human constant regions ding ork regions). Accordingly, the present invention provides variable heavy and light chains that are at least about 90%-99% identical to the SEQ IDs disclosed herein, with 90, 91, 92, 93, 94, 95, 96, 97, 98 and 99% all finding use in the present invention.

Antibodies that Bind to the Same Epitope as the LY75 Antibodies of the Invention In another embodiment, the invention provides antibodies that bind to the same epitope on the human LY75 as any of the LY75 onal antibodies of the invention. The term "binds to the same epitope" with reference to two or more antibodies means that the antibodies compete for binding to an antigen and bind to the same, overlapping or encompassing continuous or discontinuous segments of amino acids. Those of skill in the art understand that the phrase "binds 18544345_1 (GHMatters) P41668NZ00 to the same epitope" does not necessarily mean that the antibodies bind to exactly the same amino acids, gh in one embodiment it can be defined as such. In another embodiment, the precise amino acids to which the antibodies bind can differ. For example, a first antibody can bind to a segment of amino acids that is completely encompassed by the segment of amino acids bound by a second dy. In another example, a first antibody binds one or more segments of amino acids that icantly overlap the one or more segments bound by the second antibody. For the purposes herein, such antibodies are considered to "bind to the same epitope." Accordingly, also, encompassed by the present invention in one embodiment are antibodies that bind to an epitope on LY75 which comprises all or a portion of an epitope ized by the particular antibodies described herein (e.g., the same or an pping region or a region between or spanning the region). Also encompassed by the present invention, are antibodies that bind specifically to at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10, peptide(s) selected from the group comprising SEQ ID NOs: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 or a fragment thereof, wherein said fragment comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 contiguous amino acids. In a further embodiment, the epitope recognized by the antibodies of the present invention comprises at least one peptide, at least two or at least three peptides selected from the group consisting of SEQ ID NOs: 27, 29, 30, 34, 35, 36 or 37 or fragments thereof wherein said fragments comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 contiguous amino acids. In a further embodiment, the epitope recognized by the antibodies of the present invention comprises at least one es, for example, one, two or three peptides selected from the group consisting of SEQ ID NOs: 30, 36 and 37 or fragments thereof, wherein said fragments comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 contiguous amino acids.

Also encompassed by the t invention are dies that bind the same epitope and/or antibodies that compete for binding to human LY75 with the antibodies described . dies that recognize the same epitope or compete for binding can be fied using routine techniques.

Such techniques include, for example, an immunoassay, which shows the y of one antibody to block the binding of another antibody to a target antigen, i.e., a competitive binding assay. itive binding is determined in an assay in which the immunoglobulin under test inhibits specific binding of a nce antibody to a common antigen, such as LY75. Numerous types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology 9:242 ); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phase direct d assay, solid phase direct labeled sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct label RIA using I-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, such an assay 18544345_1 ters) P41668NZ00 involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test immunoglobulin and a d reference immunoglobulin. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Usually the test immunoglobulin is present in excess. Usually, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common n by at least 50-55%, 55-60%, 60-65%, 65-70% 70-75% 75-80% 80-85% 85-90% 90-95% 95-99% or more.

Other techniques include, for example, epitope mapping methods, such as, x-ray analyses of crystals of antigen:antibody complexes which provides atomic resolution of the epitope. Other methods monitor the binding of the antibody to antigen fragments or mutated variations of the n where loss of binding due to a modification of an amino acid e within the antigen sequence is often considered an indication of an epitope component. In addition, computational combinatorial methods for e mapping can also be used. These methods rely on the ability of the dy of interest to affinity isolate specific short peptides from combinatorial phage y peptide libraries. The peptides are then regarded as leads for the definition of the epitope corresponding to the antibody used to screen the e library. For epitope mapping, computational thms have also been developed which have been shown to map conformational discontinuous epitopes.

In a ular embodiment, the antibody competes for binding to LY75 with an antibody comprising heavy and/or light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs:1 and 2, respectively, or amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto. In another embodiment, the antibody competes for g to LY75 with an antibody comprising heavy and/or light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs:1 and 2 (LY75_A1).

Other antibodies of the invention bind to an epitope on LY75 recognized by the antibodies described herein. In r particular embodiment, the antibody binds to an epitope on LY75 ized by an antibody comprising heavy and/or light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs:1 and 2, respectively, or amino acid ces at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto. In another embodiment, the antibody binds to an epitope on LY75 recognized by an antibody comprising heavy and/or light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs:1 and 2 (LY75).

Characterization of Monoclonal Antibodies to LY75 Monoclonal antibodies of the invention can be terized for binding to LY75 using a y of known techniques. Generally, the antibodies are initially characterized by ELISA. Briefly, microtiter plates can be coated with purified LY75 in PBS, and then blocked with irrelevant proteins such as bovine serum n (BSA) diluted in PBS. Dilutions of plasma from mmunized mice are added to each well and incubated for 1-2 hours at 37oC. The plates are washed with 18544345_1 (GHMatters) P41668NZ00 PBS/Tween 20 and then incubated with a goat-anti-human IgG Fc-specific polyclonal reagent conjugated to alkaline phosphatase for 1 hour at 37oC. After washing, the plates are developed with ABTS substrate, and analyzed at OD of 405. Preferably, mice which develop the highest titers will be used for fusions.

An ELISA assay as described above can be used to screen for antibodies and, thus, hybridomas that produce antibodies that show positive reactivity with the LY75 gen.

Hybridomas that bind, preferably with high affinity, to LY75 can then be sub cloned and further characterized. One clone from each hybridoma, which retains the reactivity of the parent cells (by ELISA), can then be chosen for making a cell bank, and for antibody purification.

To purify anti-LY75 antibodies, selected hybridomas can be grown in roller bottles, ter r-flasks or other culture systems. Supernatants can be filtered and concentrated before affinity chromatography with protein A-Sepharose (Pharmacia, Piscataway, NJ) to purify the protein.

After buffer exchange to PBS, the concentration can be determined by OD280 using 1.43 extinction coefficient or preferably by nephelometric analysis. IgG can be d by gel electrophoresis and by antigen specific method.

To determine if the ed anti-LY75 monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL).

Biotinylated MAb binding can be detected with a streptavidin labeled probe. To determine the isotype of purified antibodies, e ELISAs can be performed using art recognized techniques.

For example, wells of microtiter plates can be coated with 10 g/ml of anti- Ig overnight at 4°C. After blocking with 5% BSA, the plates are reacted with 10 g/ml of monoclonal antibodies or purified isotype controls, at ambient temperature for two hours. The wells can then be d with either IgGl or other isotype specific conjugated probes. Plates are developed and ed as described above.

To test the binding of monoclonal antibodies to live cells expressing LY75, flow cytometry can be used. Briefly, cell lines and/or human PBMCs expressing membrane-bound LY75 (grown under standard growth ions) are mixed with various trations of monoclonal antibodies in PBS ning 0.1% BSA at 4°C for 1 hour. After washing, the cells are reacted with Fluoresceinlabeled anti- IgG antibody under the same conditions as the y antibody staining. The samples can be analyzed by FACScan instrument using light and side scatter properties to gate on single cells and binding of the d antibodies is determined. An alternative assay using fluorescence microscopy may be used (in on to or instead of) the flow cytometry assay. Cells can be stained exactly as described above and examined by fluorescence microscopy. This method allows visualization of individual cells, but may have diminished sensitivity depending on the y of the antigen.

Anti-LY75 IgGs can be further tested for reactivity with the LY75 n by Western blotting.

Briefly, cell extracts from cells expressing LY75 can be prepared and ted to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens will be transferred to nitrocellulose membranes, blocked with 20% mouse serum, and probed with the 18544345_1 (GHMatters) P41668NZ00 monoclonal antibodies to be tested. IgG binding can be detected using anti- IgG alkaline phosphatase and developed with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, MO).

Methods for analyzing binding affinity, cross-reactivity, and binding kinetics of various anti- LY75 antibodies include standard assays known in the art, for example, BiacoreTM surface n resonance (SPR) analysis using a BiacoreTM 2000 SPR ment (Biacore AB, Uppsala, Sweden.

In one embodiment, the antibody specifically binds to human LY75 comprising SEQ ID NO:15) Preferably, an antibody of the invention binds to human LY75 with high affinity.

Preferably, an antibody of the ion binds to a LY75 protein with a KD of 5 x 10-8 M or less, binds to a LY75 protein with a KD of 2 x 10-8 M or less, binds to a LY75 protein with a KD of 5 x 10-9 M or less, binds to a LY75 protein with a KD of 4 x 10-9 M or less, binds to a LY75 protein with a KD of 3 x 10-9 M or less, binds to a LY75 n with a KD of 2 x 10-9 M or less, binds to a LY75 protein with a KD of 1 x 10-9 M or less, binds to a LY75 protein with a KD of 5 x 10-10 M or less, or binds to a LY75 protein with a KD of 1 x 10-10 M or less.

In one embodiment, antibodies of the invention compete (e.g., cross-compete) for binding to LY75 with the particular anti-LY75 antibodies described herein (e.g.,_LY75_A1). Such competing antibodies can be identified based on their ability to competitively t binding to LY75 of one or more of mAbs in standard LY75 binding assays. For example, standard ELISA assays can be used in which a recombinant human LY75 n is immobilized on the plate, one of the antibodies is fluorescently labeled and the ability of non-labeled antibodies to compete off the binding of the labeled antibody is evaluated. Additionally or alternatively, BIAcore analysis can be used to assess the ability of the antibodies to cross-compete. The ability of a test antibody to inhibit the binding of an anti-LY75 antibody of the ion to human LY75 demonstrates that the test antibody can e with the antibody for binding to human LY75.

In one embodiment, the competing antibody is an antibody that binds to the same epitope on human LY75 as the particular anti-LY75 monoclonal antibodies described herein (e.g., LY75_A1).

Standard epitope mapping ques, such as x-ray crystallography and 2-dimensional r magnetic resonance, can be used to determine whether an antibody binds to the same epitope as a reference antibody (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).

In one embodiment, the antibody that competes for g to LY75 and/or binds to the same epitope on human LY75 is a human antibody.

Once a single, archetypal anti-LY75 mAb has been isolated that has the desired properties described , other mAbs with similar properties, e.g., having the same epitope may be generated. For e, mice may be immunized with LY75 as bed herein, omas produced, and the resulting mAbs screened for the ability to compete with the ypal mAb for binding to LY75. Mice can also be immunized with a smaller fragment of LY75 containing the epitope to which the archetypal mAb binds. The epitope can be zed by, e.g., screening for binding to a series of overlapping peptides spanning LY75. Alternatively, the method of Jespers et al., Biotechnology 12:899, 1994 may be used to guide the selection of mAbs having the same epitope 45_1 (GHMatters) P41668NZ00 and therefore similar properties to the archetypal mAb. Using phage display, first the heavy chain of the archetypal antibody is paired with a repertoire of (preferably human) light chains to select a LY75-binding mAb, and then the new light chain is paired with a repertoire of (preferably human) heavy chains to select a (preferably human) LY75-binding mAb having the same epitope as the archetypal mAb. Alternatively variants of the archetypal mAb can be obtained by mutagenesis of cDNA encoding the heavy and light chains of the antibody.

Epitope mapping, e.g., as described in Champe et al. (1995) J. Biol. Chem. 88-1394, can be med to determine whether the antibody binds an epitope of st. "Alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science 244: 1081-1085, or some other form of point mutagenesis of amino acid residues in human LY75 may also be used to determine the onal epitope for an anti-LY75 antibody of the present invention. nesis studies, however, may also reveal amino acid residues that are crucial to the overall imensional structure of LY75 but that are not directly involved in antibody-antigen contacts, and thus other methods may be necessary to confirm a functional epitope ined using this method.

The epitope bound by a specific antibody may also be determined by assessing binding of the antibody to peptides comprising fragments of human LY75. A series of overlapping peptides encompassing the ce of LY75 may be synthesized and screened for binding, e.g. in a direct ELISA, a competitive ELISA (where the peptide is assessed for its ability to prevent binding of an antibody to LY75 bound to a well of a microtiter plate), or on a chip. Such peptide screening methods may not be e of detecting some discontinuous functional epitopes, i.e. functional epitopes that involve amino acid es that are not contiguous along the primary sequence of the LY75 polypeptide chain.

The epitope bound by antibodies of the present invention may also be determined by structural methods, such as X-ray crystal structure determination (e.g., /044853), molecular modeling and nuclear ic resonance (NMR) spectroscopy, including NMR ination of the H-D exchange rates of labile amide hydrogens in LY75 when free and when bound in a complex with an antibody of interest Justin et al. (1992) Biochemistry 31, 11335-11347; Zinn-Justin et al. (1993) Biochemistry 32, 6884-6891).

With regard to X-ray crystallography, crystallization may be accomplished using any of the known methods in the art (e.g. Giege et al. (1994) Acta Crystallogr. D50:339-350; McPherson (1990) Eur. J. Biochem. 189:1-23), including microbatch (e.g. Chayen (1997) Structure 5:1269-1274), hanging-drop vapor diffusion (e.g. McPherson (1976) J. Biol. Chem. 251:6300-6303), seeding and dialysis. It is desirable to use a protein preparation having a tration of at least about 1 mg/mL and preferably about 10 mg/mL to about 20 mg/mL. Crystallization may be best achieved in a precipitant on containing polyethylene glycol 1000-20,000 (PEG; average molecular weight ranging from about 1000 to about 20,000 Da), preferably about 5000 to about 7000 Da, more preferably about 6000 Da, with concentrations ranging from about 10% to about 30% (w/v). It may also be desirable to include a protein izing agent, e.g. glycerol at a concentration g from about 0.5% to about 20%. A suitable salt, such as sodium chloride, lithium chloride or sodium citrate 18544345_1 (GHMatters) P41668NZ00 may also be desirable in the precipitant solution, preferably in a concentration ranging from about 1 mM to about 1000 mM. The itant is preferably buffered to a pH of from about 3.0 to about 5.0, preferably about 4.0. Specific buffers useful in the precipitant solution may vary and are well-known in the art (Scopes, Protein Purification: Principles and Practice, Third ed., (1994) Springer-Verlag, New York). Examples of useful buffers include, but are not limited to, HEPES, Tris, MES and acetate. Crystals may be grow at a wide range of temperatures, ing 2° C, 4° C, 8° C and 26° Antibody:antigen crystals may be studied using well-known X-ray diffraction techniques and may be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see e.g. ll & Johnson (1985) Meth. Enzymol. 114 & 115, H. W. f et al., eds., ic Press; U.S. Patent Application Publication No. 014194), and BUSTER (Bricogne (1993) Acta Cryst. D49:37-60; Bricogne (1997) Meth. Enzymol. 276A:361-423, Carter & Sweet, eds.; Roversi et al. (2000) Acta Cryst. D56:1313-1323), the disclosures of which are hereby incorporated by reference in their entireties.

Antibody competition assays, as described herein, can be used to determine whether an antibody "binds to the same epitope" as another antibody. Typically, competition of 50% or more, 60% or more, 70% or more, such as 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more, of an antibody known to interact with the epitope by a second antibody under conditions in which the second antibody is in excess and the first saturates all sites, is indicative that the antibodies "bind to the same epitope." To assess the level of competition between two antibodies, for example, radioimmunoassays or assays using other labels for the antibodies, can be used. For e, a LY75 antigen can be incubated with a saturating amount of a first anti- LY75 antibody or n-binding fragment thereof conjugated to a labeled compound (e.g., 3H, 125I, biotin, or rubidium) in the presence the same amount of a second unlabeled anti-LY75 antibody.

The amount of labeled dy that is bound to the antigen in the presence of the unlabeled blocking antibody is then assessed and compared to binding in the e of the unlabeled blocking antibody. Competition is determined by the percentage change in binding signals in the presence of the unlabeled blocking antibody compared to the e of the blocking antibody.

Thus, if there is a 50% inhibition of binding of the labeled dy in the presence of the blocking antibody compared to binding in the absence of the ng antibody, then there is ition between the two antibodies of 50%. Thus, reference to ition between a first and second antibody of 50% or more, 60% or more, 70% or more, such as 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more, means that the first antibody inhibits binding of the second antibody (or vice versa) to the n by 50%, 60%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (compared to binding of the antigen by the second antibody in the absence of the first antibody). Thus, inhibition of g of a first antibody to an antigen by a second antibody of 50%, 605, 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more indicates that the two antibodies bind to the same epitope. 18544345_1 (GHMatters) P41668NZ00 Antibody Modifications The present invention further provides variant antibodies, sometimes referred to as "antibody derivatives" or "antibody analogs" as well. That is, there are a number of modifications that can be made to the antibodies of the invention, ing, but not limited to, amino acid modifications in the CDRs (affinity maturation), amino acid modifications in the framework regions, amino acid modifications in the Fc region, glycosylation variants, covalent modifications of other types (e.g. for attachment of drug conjugates, etc.).

By "variant" herein is meant a polypeptide ce that differs from that of a parent ptide by virtue of at least one amino acid modification. In this case, the parent polypeptide is either the full length variable heavy or light chains, listed in SEQ ID Nos: 1 or 2, respectively or the CDR regions or the framework regions of the heavy and light chains listed in SEQ ID NOs 5-10 and 16-21. Amino acid modifications can include substitutions, insertions and deletions, with the former being preferred in many cases. It will be understood that an amino acid substitution may be a conservative or nservative substitution with conservative substitutions being preferred.

Further said substitution may be a substitution with either a naturally or non-naturally occurring amino acid.

In general, variants can include any number of modifications, as long as the on of the antibody is still present, as described herein. That is, LY75_A1, for example, the antibody should still specifically bind to human LY75. Similarly, if amino acid ts are generated with the Fc region, for example, the variant antibodies should maintain the required receptor binding functions for the particular ation or indication of the antibody.

"Variants" in this case can be made in either the listed CDR sequences, the ork or Fc regions of the antibody.

However, in general, from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions are generally utilized as often the goal is to alter function with a minimal number of cations. In some cases, there are from 1 to 5 cations (e.g. individual amino acid substitutions, insertions or deletions), with from 1-2, 1-3 and 1-4 also g use in many embodiments. The number of modifications can depend on the size of the region being modified; for example, in general, fewer modifications are desired in CDR regions. It will be understood by the skilled person that even within the CDR regions the location of the modification can significantly alter the effect. In one embodiment, the modifications can be made in any of CDR1, CDR2 or CDR3 of the heavy and/or light chains. In a further ment, the modifications are made in any of CDR1 or CDR2 of the heavy and/or light chains. In a still further embodiment, the modifications are located in CDR1 of the heavy and/or light It should be noted that the number of amino acid modifications may be within functional domains: for example, it may be desirable to have from 1-5 modifications in the Fc region of wildtype or engineered proteins, as well as from 1 to 5 cations in the Fv region, for example. A variant polypeptide sequence will preferably possess at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the parent sequences (e.g. the variable regions, 18544345_1 (GHMatters) P41668NZ00 the constant regions, and/or the heavy and light chain sequences and/or the CDRs of LY75_A1). It should be noted that depending on the size of the ce, the percent identity will depend on the number of amino acids.

By "amino acid substitution" or "substitution" herein is meant the replacement of an amino acid at a particular position in a parent ptide sequence with another amino acid which may be a natural or turally occurring amino acid. For example, the substitution S100A refers to a t polypeptide in which the serine at position 100 is replaced with alanine. By "amino acid insertion" or "insertion" as used herein is meant the addition of an amino acid at a particular position in a parent polypeptide sequence. By "amino acid deletion" or ion" as used herein is meant the removal of an amino acid at a particular position in a parent polypeptide sequence.

By "parent polypeptide", "parent n", "precursor polypeptide", or "precursor protein" as used herein is meant an unmodified polypeptide that is subsequently ed to generate a variant.

In general, the parent polypeptides herein are LY75_A1. Accordingly, by "parent antibody" as used herein is meant an antibody that is modified to generate a variant antibody.

By "wild type" or "WT" or "native" herein is meant an amino acid sequence or a tide sequence that is found in nature, including allelic variations. A WT n, polypeptide, antibody, immunoglobulin, IgG, etc. has an amino acid sequence or a nucleotide sequence that has not been ionally modified.

By "variant Fc region" herein is meant an Fc sequence that differs from that of a wild-type Fc sequence by virtue of at least one amino acid modification. Fc variant may refer to the Fc ptide itself, compositions comprising the Fc variant polypeptide, or the amino acid sequence.

In some embodiments, one or more amino acid modifications are made in one or more of the CDRs of LY75_A1. In general, only 1 or 2 or 3 amino acids are substituted in any single CDR, and generally no more than from 4, 5, 6, 7, 8 9 or 10 changes are made within a set of 6 CDRs.

However, it should be appreciated that any combination of no tutions, 1, 2 or 3 substitutions in any CDR can be independently and optionally combined with any other substitution. It will be apparent that substitutions can be made in any of the 6 CDRs. In one embodiment, substitutions are made in CDR1 of the heavy and/or light chains.

In some cases, amino acid modifications in the CDRs are referred to as "affinity maturation".

An "affinity matured" antibody is one having one or more alteration(s) in one or more CDRs which results in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). In some cases, although rare, it may be desirable to decrease the affinity of an antibody to its antigen, but this is generally not red.

Affinity maturation can be done to increase the binding affinity of the antibody for the antigen by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%or more, or 1, 2, 3, 4 to 5 fold as compared to the "parent" antibody. Preferred affinity d antibodies will have nanomolar or even lar affinities for the target antigen. Affinity matured dies are produced by known procedures. See, for e, Marks et al., 1992, Biotechnology 18544345_1 (GHMatters) P41668NZ00 :779-783 that describes affinity maturation by variable heavy chain (VH) and variable light chain (VL) domain shuffling. Random mutagenesis of CDR and/or framework residues is described in: Barbas, et al. 1994, Proc. Nat. Acad. Sci, USA 91:3809-3813; Shier et al., 1995, Gene 169:147-155; Yelton et al., 1995, J. Immunol. 155:1994-2004; Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al, 1992, J. Mol. Biol. 226:889-896, for example. atively, amino acid modifications can be made in one or more of the CDRs of the antibodies of the invention that are "silent", e.g. that do not significantly alter the affinity of the antibody for the antigen. These can be made for a number of reasons, including optimizing expression (as can be done for the nucleic acids encoding the dies of the invention).

Thus, included within the definition of the CDRs and antibodies of the invention are variant CDRs and antibodies; that is, the dies of the invention can e amino acid modifications in one or more of the CDRs of LY75_A1. In addition, as outlined below, amino acid modifications can also independently and optionally be made in any region outside the CDRs, including ork and constant regions as described herein.

In some embodiments, the anti-LY75 dies of the invention are composed of a variant Fc domain. As is known in the art, the Fc region of an dy interacts with a number of Fc receptors and ligands, imparting an array of important functional capabilities referred to as effector ons.

These Fc receptors include, but are not limited to, (in humans) FcγRI (CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa ding allotypes V158 and F158, correlated to antibody-dependent cell cytotoxicity (ADCC)) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), FcRn (the neonatal receptor), C1q (complement protein involved in complement dependent cytotoxicity (CDC)) and FcRn (the neonatal receptor involved in serum half-life). Suitable modifications can be made at one or more positions as is generally outlined, for e in US Patent Application ,654 and references cited therein, US 2004/013210, US 2005/0054832, US 2006/0024298, US 2006/0121032, US 2006/0235208, US 2007/0148170, USSN 12/341,769, US Patent No. 6,737,056, US Patent No. 7,670,600, US Patent No. 6,086,875 all of which are sly orated by reference in their entirety, and in particular for specific amino acid substitutions that se binding to Fc receptors.

In addition to the modifications outlined above, other modifications can be made. For e, the molecules may be stabilized by the incorporation of hide bridges linking the VH and VL domains r et al., 1996, Nature h. 14:1239-1245, entirely incorporated by reference).

In addition, modifications at nes are particularly useful in antibody-drug conjugate (ADC) applications, further described below. In some embodiments, the constant region of the dies can be engineered to contain one or more cysteines that are particularly "thiol reactive", so as to allow more specific and controlled placement of the drug moiety. See for example US Patent No. 7,521,541, incorporated by reference in its entirety herein. 18544345_1 (GHMatters) P41668NZ00 In addition, there are a y of covalent modifications of antibodies that can be made as outlined below.

Covalent modifications of antibodies are included within the scope of this invention, and are generally, but not always, done post-translationally. For example, several types of covalent modifications of the antibody are introduced into the molecule by reacting specific amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with acetates (and ponding amines), such as chloroacetic acid or acetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues may also be derivatized by reaction with bromotrifluoroacetone, o-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N- alkylmaleimides, 3-nitropyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2- chloromercurinitrophenol, or nitrobenzooxa-1,3-diazole and the like.

Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl e also is useful; the reaction is preferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl nimidate; pyridoxal phosphate; pyridoxal; borohydride; trinitrobenzenesulfonic acid; ylisourea; 2,4-pentanedione; and transaminase-catalyzed reaction with glyoxylate.

Arginyl residues are modified by reaction with one or several conventional reagents, among them glyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of ne residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine onal group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

The specific modification of tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or itromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O- acetyl l species and 3-nitro derivatives, respectively. Tyrosyl residues are ted using 125I or 131I to prepare labeled proteins for use in radioimmunoassay, the mine T method described above being suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'—N=C=N--R'), where R and R' are optionally different alkyl , such as 1- cyclohexyl(2-morpholinylethyl) iimide or 1-ethyl(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. 18544345_1 (GHMatters) P41668NZ00 Derivatization with bifunctional agents is useful for crosslinking antibodies to a water-insoluble t matrix or surface for use in a variety of methods, in addition to methods described below.

Commonly used crosslinking agents include, e.g., 1,1-bis(diazoacetyl)phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for e, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis (succinimidylpropionate), and bifunctional ides such as bis-N-maleimido-1,8-octane.

Derivatizing agents such as methyl[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cynomolgusogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 440, all entirely incorporated by reference, are employed for protein immobilization. inyl and asparaginyl es are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of , arginine, and histidine side chains (T. E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983], entirely incorporated by reference), acetylation of the N- terminal amine, and amidation of any C-terminal carboxyl group.

In addition, as will be appreciated by those in the art, labels (including fluorescent, enzymatic, magnetic, ctive, etc. can all be added to the antibodies (as well as the other compositions of the ion).

Bispecific Molecules In another aspect, the t invention features bispecific molecules comprising an anti- LY75 dy, or a fragment thereof, of the invention. An dy of the ion, or antigenbinding portions thereof, can be derivatized or linked to another functional molecule, e.g. another peptide or protein (e.g. another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibody of the invention may in fact be derivatized or linked to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also intended to be assed by the term "bispecific molecule" as used herein. To create a bispecific molecule of the invention, an antibody of the invention can be functionally linked (e.g. by al coupling, genetic fusion, noncovalent association or otherwise) to one or more other g molecules, such as another antibody, antibody fragment, peptide or binding c, such that a bispecific molecule results.

Accordingly, the present invention includes bispecific molecules comprising at least one first g icity for a first target epitope (i.e. LY75) and a second binding specificity for a second target epitope. The second target epitope maybe t on the same target protein as that bound 18544345_1 (GHMatters) P41668NZ00 by the first binding specificity; or the second target epitope may be present of a different target protein to that bound by the first protein to that bound by the first binding specificity. The second target epitope may be present on the same cell as the first target epitope (i.e. LY75); or the second target epitope may be present on a target which is not yed by the cell which displays the first target epitope. As used herein, the term ‘binding specificity’ refers to a moiety comprising at least one dy variable domain.

In a one embodiment of the invention, the second target epitope is an Fc receptor, e.g. human FcRI (CD64) or a human Fcα receptor (CD89). Therefore, the invention includes bispecific molecules capable of binding both to FcR or FcαR expressing effector cells (e.g. monocytes, hages or rphonuclear cells (PMNs), and to target cells sing LY75. These bispecific molecules target LY75 expressing cells to effector cell and trigger Fc receptor-mediated or cell activities, such as phagocytosis of LY75 expressing cells, antibody dependent diated cytotoxicity (ADCC), cytokine release, or generation of superoxide anion.

In another embodiment of the invention, the second target epitope is CD3 or CD5. Therefore, the invention includes bispecific molecules capable of g both to CD3 or CD5 expressing effector cells (e.g. CD3 or CD5 sing cytotoxic T cells), and to target cells expressing LY75.

These bispecific molecules target LY75 expressing cells to effector cell and trigger CD3 or CD5- mediated effector cell activities, such as T cell clonal expansion and T cell xicity. In this embodiment, the bispecific antibody of the invention may have a total of either two or three antibody variable domains, wherein first portion of the bispecific antibody is capable of recruiting the ty of a human immune effector cell by specifically binding to an or antigen located on the human immune effector cell, in which the effector antigen is the human CD3 antigen or the human CD5 antigen, said first portion consisting of one antibody variable domain, and a second portion of the bispecific dy is capable of specifically binding to a target antigen other than the effector antigen e.g. LY75, said target antigen being located on a target cell other than said human immune effector cell, and said second portion sing one or two antibody variable domains.

In an embodiment of the invention in which the bispecific molecule is multispecific, the molecule can r include a third binding specificity, in addition to an anti-Fc binding specificity or anti-CD3 or anti-CD5 g specificity and an anti-LY75 binding specificity. In one embodiment, the third binding specificity is an nhancement factor (EF) portion, e.g. a molecule which binds to a surface protein involved in cytotoxic activity and thereby increases the immune response against the target cell. The "anti-enhancement factor portion" can be an antibody, functional antibody fragment or a ligand that binds to a given molecule, e.g. an antigen or a receptor, and thereby results in an enhancement of the effect of the binding inants for the Fc receptor or target cell antigen. The "anti-enhancement factor portion" can bind an Fc receptor or a target cell antigen. Alternatively, the anti-enhancement factor portion can bind to an entity that is different from the entity to which the first and second binding specificities bind. For example, the anti-enhancement factor portion can bind a cytotoxic T-cell (e.g. via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell that s in an increased immune response against the target cell). 18544345_1 (GHMatters) P41668NZ00 In one ment, the ific molecules of the ion comprise as a binding specificity at least one antibody, or an antibody fragment f, ing, e.g. an Fab, Fab', F(ab')2, Fv, Fd, dAb or a single chain Fv. The antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof such as an Fv or a single chain construct as bed in US Patent No. 4,946,778, the contents of which is expressly incorporated by reference.

In one embodiment, the binding specificity for an Fcγ or is provided by a monoclonal antibody, the binding of which is not blocked by human immunoglobulin G (IgG). As used herein, the term "IgG receptor" refers to any of the eight -chain genes located on some 1. These genes encode a total of twelve transmembrane or soluble receptor isoforms which are grouped into three Fc receptor classes: FcRI (CD64), FcRII(CD32), and FcRIII (CD16). In one preferred embodiment, the Fcγ receptor is a human high affinity FcRI. The human FcRI is a 72 kDa molecule, which shows high affinity for monomeric IgG 09 M-1).

The production and characterization of certain preferred anti-Fcγ monoclonal antibodies are described in PCT Publication WO 88/00052 and in US Patent No. 4,954,617, the teachings of which are fully incorporated by reference herein. These dies bind to an epitope of FcRI, FcRII or Fc RIII at a site which is distinct from the Fc binding site of the receptor and, thus, their binding is not blocked substantially by physiological levels of IgG. Specific anti-FcRI dies useful in this invention are mAb 22, mAb 32, mAb 44, mAb 62 and mAb 197. The hybridoma producing mAb 32 is available from the American Type Culture Collection, ATCC Accession No. HB9469. In other embodiments, the anti-Fc receptor dy is a humanized form of monoclonal antibody 22 (H22).

The production and characterization of the H22 antibody is described in Graziano, R.F. et al. (1995) J. Immunol 155 (10): 4996-5002 and PCT Publication WO 94/10332. The H22 antibody producing cell line was deposited at the American Type Culture Collection under the designation HA022CL1 and has the accession no. CRL 11177.

In still other preferred embodiments, the binding specificity for an Fc receptor is provided by an antibody that binds to a human IgA receptor, e.g. an Fc-alpha receptor [FcRI (CD89)], the g of which is preferably not blocked by human immunoglobulin A (IgA). The term "IgA receptor" is intended to include the gene product of one -gene (FcRI) located on chromosome 19.

This gene is known to encode several alternatively spliced transmembrane ms of 55 to 110 kDa. FcRI (CD89) is constitutively expressed on monocytes/macrophages, eosinophilic and neutrophilic granulocytes, but not on non-effector cell populations. FcRI has medium affinity ( 5  107 M-1) for both IgA1 and IgA2, which is increased upon re to nes such as G-CSF or GM-CSF [Morton, H.C. et al. (1996) Critical Reviews in logy 16:423-440]. Four FcRI- specific monoclonal antibodies, identified as A3, A59, A62 and A77, which bind FcRI outside the IgA ligand binding domain, have been described [Monteiro, R.C. et al. (1992) J. Immunol. 148:1764].

FcRI and FcRI are preferred trigger receptors for use in the bispecific molecules of the invention because they are (1) expressed primarily on immune effector cells, e.g. tes, PMNs, macrophages and dendritic cells; (2) sed at high levels (e.g. 5,000-100,000 per cell); (3) 18544345_1 (GHMatters) P41668NZ00 mediators of cytotoxic activities (e.g. ADCC, phagocytosis); and (4) mediate enhanced antigen presentation of antigens, including self-antigens, targeted to them.

Antibodies which can be employed in the ific molecules of the invention are murine, human, chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present invention can be ed by conjugating the constituent binding specificities, e.g. the anti-FcR, anti-CD3 anti-CD5 and anti-LY75 binding specificities, using methods known in the art. For example, the binding specificity of each bispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents e protein A, carbodiimide, N- succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), ophenylenedimaleimide (oPDM), N-succinimidyl(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxanecarboxylate (sulfo-SMCC) [see e.g.

Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648]. Other methods e those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118- 132; Brennan et al. (1985) Science 229:81-83, and Glennie et al. (1987) J. l. 139: 2367- 2375. Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL)].

When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonding of the C-terminus hinge regions of the two heavy . In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation. atively, both binding specificities can be encoded in the same vector and expressed and led in the same host cell. This method is particularly useful where the bispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab')2 or ligand x Fab fusion protein. A bispecific molecule of the invention can be a single chain le sing one single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants.

Bispecific molecules may comprise at least two single chain molecules. s for preparing bispecific molecules are described for example in US Patent Numbers 203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786; 653; 5,258,498; and 5,482,858, all of which are expressly incorporated herein by reference.

The antibodies of the invention may be Bi-specific T-cell engagers (BiTEs). BiTEs are a class of artificial bispecific monoclonal antibodies. They direct a host's immune system, more specifically the T cells' cytotoxic ty, t cancer cells. BiTEs are generally fusion proteins consisting of two single-chain variable fragments ) of ent antibodies, or amino acid ces from four different genes, on a single peptide chain, preferably of about 55 kilodaltons.

Preferably, one of the scFvs binds to T cells via the CD3 receptor, and the other to a tumour cell via a tumour-specific molecule. 18544345_1 (GHMatters) P41668NZ00 g of the ific molecules to their specific targets can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g. growth inhibition), or Western Blot assay. Each of these assays generally detects the presence of n-antibody complexes of particular interest by employing a labeled reagent (e.g. an antibody) specific for the complex of interest. For e, the FcR-antibody complexes can be detected using e.g. an enzyme-linked antibody or antibody fragment which recognizes and specifically binds to the antibody-FcR complexes. Alternatively, the complexes can be detected using any of a variety of other immunoassays. For example, the dy can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The ine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by such means as the use of a γ counter or a scintillation counter or by autoradiography. ylation Another type of covalent cation is alterations in glycosylation. In some embodiments, the antibodies disclosed herein can be modified to include one or more engineered glycoforms. By "engineered glycoform" as used herein is meant a carbohydrate composition that is covalently attached to the antibody, wherein the carbohydrate ition differs chemically from that of a parent antibody. Engineered glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing effector function. For example, an aglycoslated dy can be made (i.e. the antibody that lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the dy for antigen. Such ydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the dy sequence. For example, one or more amino acid substitutions can be made that result in ation of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for n. Such an approach is described in further detail in US Patent Nos. 5,714,350 and 6,350,861 by Co et al., and can be accomplished by ng the asparagine at position 297.

A preferred form of engineered glycoform is afucosylation, which has been shown to be correlated to an increase in ADCC function, ably through tighter binding to the FcγRIIIa receptor. In this context, sylation" means that the majority of the antibody produced in the host cells is substantially devoid of , e.g. 9098% of the generated antibodies do not have appreciable fucose as a component of the carbohydrate moiety of the antibody (generally attached at N297 in the Fc region). Defined onally, afucosylated antibodies generally exhibit at least a 50% or higher affinity to the FcγRIIIa receptor.

Engineered glycoforms may be generated by a variety of methods known in the art (Umaña et al., 1999, Nat Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473; US 6,602,684; USSN 10/277,370; USSN 10/113,929; PCT WO 00/61739A1; PCT WO 01/29246A1; PCT WO 02/31140A1; PCT WO 02/30954A1, all entirely incorporated by nce; 18544345_1 (GHMatters) P41668NZ00 (POTELLIGENT® technology [Biowa, Inc., Princeton, NJ]; GlycoMAb® glycosylation engineering technology [Glycart hnology AG, Zürich, Switzerland]). Many of these techniques are based on controlling the level of lated and/or bisecting oligosaccharides that are ntly attached to the Fc region, for example by expressing an IgG in various organisms or cell lines, engineered or ise (for example Lec-13 CHO cells or rat hybridoma YB2/0 cells, by regulating enzymes involved in the glycosylation pathway (for example FUT8 [α1,6-fucosyltranserase] and/or β1 N- acetylglucosaminyltransferase III [GnTIII]), or by modifying carbohydrate(s) after the IgG has been expressed. For example, the "sugar engineered antibody" or "SEA technology" of Seattle Genetics functions by adding modified saccharides that inhibit fucosylation during tion; see for e US2009/0317869, hereby incorporated by reference in its entirety. "Engineered glycoform "typically refers to the different carbohydrate or oligosaccharide as ed to the antibody made in the absence of the glycosylation technology; thus an antibody can include an engineered glycoform.

Alternatively, engineered glycoform may refer to the IgG variant that comprises the different carbohydrate or oligosaccharide. As is known in the art, ylation patterns can depend on both the sequence of the protein (e.g., the presence or e of particular glycosylation amino acid residues, discussed below), or the host cell or organism in which the protein is produced. Particular expression s are discussed below.

Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tri-peptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tri-peptide sequences in a polypeptide creates a ial glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose, to a yamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tri-peptide sequences (for N-linked glycosylation sites). The alteration may also be made by the on of, or substitution by, one or more serine or ine residues to the starting sequence (for ed ylation sites). For ease, the dy amino acid sequence is preferably altered through changes at the DNA level, particularly by mutating the DNA encoding the target polypeptide at preselected bases such that codons are generated that will ate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on the antibody is by chemical or enzymatic coupling of glycosides to the protein. These procedures are ageous in that they do not require production of the protein in a host cell that has glycosylation capabilities for N- and O-linked glycosylation. Depending on the ng mode used, the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. 18544345_1 (GHMatters) P41668NZ00 These methods are described in WO 87/05330 and in Aplin and Wriston, 1981, CRC Crit. Rev.

Biochem., pp. 6, both entirely incorporated by nce.

Removal of carbohydrate moieties present on the starting antibody (e.g. post-translationally) may be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid, or an lent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the polypeptide intact. Chemical deglycosylation is described by Hakimuddin et al., 1987, Arch. m. s. 259:52 and by Edge et al., 1981, Anal. Biochem. 118:131, both entirely incorporated by reference. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exoglycosidases as described by Thotakura et al., 1987, Meth. Enzymol. 0, entirely incorporated by reference, including removal of fucose residues using a fucosidase enzyme as is known in the art. Glycosylation at potential glycosylation sites may be prevented by the use of the compound tunicamycin as described by Duskin et al., 1982, J. Biol. Chem. 257:3105, entirely incorporated by reference. Tunicamycin blocks the formation of protein-N-glycoside linkages. r type of covalent modification of the antibody comprises linking the antibody to various oteinaceous polymers, including, but not limited to, various polyols such as polyethylene glycol, polypropylene glycol or polyoxyalkylenes, in the manner set forth in, for example, 2005-2006 PEG Catalog from Nektar Therapeutics (available at the Nektar website) US s 4,640,835; 4,496,689; 144; 4,670,417; 4,791,192 or 4,179,337, all entirely incorporated by reference. In addition, as is known in the art, amino acid substitutions may be made in various positions within the antibody to facilitate the on of polymers such as PEG. See for example, U.S. Publication No. 2005/0114037A1, entirely incorporated by reference.

In additional embodiments, for example in the use of the antibodies of the invention for stic or detection purposes, the antibodies may comprise a label. By ed" herein is meant that a compound has at least one element, isotope or chemical compound attached to enable the detection of the nd. In general, labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic, electrical, thermal; and c) d or luminescent dyes; although labels e enzymes and particles such as magnetic particles as well. Preferred labels e, but are not limited to, fluorescent lanthanide complexes (including those of Europium and Terbium), and fluorescent labels ing, but not limited to, quantum dots, fluorescein, ine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, ne, Lucifer Yellow, Cascade Blue, Texas Red, the Alexa dyes, the Cy dyes, and others described in the 6th Edition of the Molecular Probes Handbook by Richard P. Haugland, hereby expressly incorporated by reference.

Antibody-Drug Conjugates In some embodiments, the Y75 dies of the invention are conjugated with drugs to form antibody-drug conjugates (ADCs). In general, ADCs are used in oncology applications, where the use of antibody-drug conjugates for the local delivery of cytotoxic or cytostatic agents allows for 18544345_1 (GHMatters) P41668NZ00 the targeted delivery of the drug moiety to tumors, which can allow higher efficacy, lower toxicity, etc.

An overview of this technology is ed in Ducry et al., Bioconjugate Chem., 21:5-13 (2010), Carter et al., Cancer J. 14(3):154 (2008) and , Current Opin. Chem. Biol. 13:235-244 (2009), all of which are hereby incorporated by reference in their entirety.

Thus the invention provides anti-LY75 dies conjugated to drugs. Generally, conjugation is done by covalent attachment to the antibody, as further described below, and generally relies on a linker, often a peptide linkage (which, as described below, may be ed to be sensitive to cleavage by proteases at the target site or not). In addition, as described above, linkage of the linker-drug unit (LU-D) can be done by attachment to cysteines within the antibody. As will be appreciated by those in the art, the number of drug moieties per dy can change, depending on the conditions of the reaction, and can vary from 1:1 to 10:1 drug:antibody. As will be appreciated by those in the art, the actual number is an average.

Thus the invention provides anti-LY75 antibodies conjugated to drugs. As described below, the drug of the ADC can be any number of agents, including but not limited to cytotoxic agents such as chemotherapeutic agents, growth inhibitory agents, toxins (for example, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (that is, a radioconjugate) are provided. In other embodiments, the invention further provides methods of using the ADCs.

Drugs for use in the present invention include cytotoxic drugs, ularly those which are used for cancer therapy. Such drugs include, in general, DNA damaging agents, anti-metabolites, natural products and their analogs. Exemplary classes of cytotoxic agents e the enzyme inhibitors such as dihydrofolate reductase inhibitors, and thymidylate synthase inhibitors, DNA alators, DNA cleavers, topoisomerase tors, the cycline family of drugs, the vinca drugs, the cins, the cins, the cytotoxic nucleosides, the pteridine family of drugs, es, the yllotoxins, dolastatins, maytansinoids, entiation inducers, and taxols.

Members of these classes include, for example, taxol, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxanes including taxol, taxotere retinoic acid, butyric acid, N8-acetyl spermidine, thecin, calicheamicin, micin, ene-diynes, duocarmycin A, duocarmycin SA, eamicin, camptothecin, hemiasterlins, maytansinoids (including DM1), thylauristatin E (MMAE), monomethylauristatin F (MMAF), and maytansinoids (DM4) and their analogues.

Toxins may be used as antibody-toxin conjugates and include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al (2000) J. Nat. Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem.

Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al 18544345_1 (GHMatters) P41668NZ00 (1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342), hemiasterlins (WO2004/026293; Zask et al., (2004) J. Med. Chem, 47: 4774-4786). Toxins may exert their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

Conjugates of an anti-LY75 antibody and one or more small molecule toxins, such as a maytansinoids, dolastatins, auristatins, a trichothecene, calicheamicin, and CC1065, and the derivatives of these toxins that have toxin activity, may also be used.

Maytansinoids Maytansine compounds suitable for use as maytansinoid drug moieties are well known in the art, and can be isolated from l sources according to known methods, produced using genetic engineering ques (see Yu et al (2002) PNAS 99:7968-7973), or maytansinol and maytansinol analogues prepared synthetically according to known methods. As described below, drugs may be modified by the incorporation of a functionally active group such as a thiol or amine group for conjugation to the antibody.

Exemplary sinoid drug moieties include those having a modified aromatic ring, such as: echloro (U.S. Pat. No. 4,256,746) (prepared by lithium um hydride reduction of ansamytocin P2); Chydroxy (or Cdemethyl) +/-Cdechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared by demethylation using omyces or Actinomyces or dechlorination using LAH); and Cdemethoxy, cyloxy (--OCOR), +/-dechloro (U.S. Pat. No. 4,294,757) (prepared by acylation using acyl des) and those having modifications at other positions.

Exemplary maytansinoid drug moieties also include those having modifications such as: C SH (U.S. Pat. No. 4,424,219) (prepared by the reaction of maytansinol with H2S or P2S5); C alkoxymethyl(demethoxy/CH2OR) (U.S. Pat. No. 4,331,598); Chydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat. No. 254) (prepared from Nocardia); Chydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared by the conversion of maytansinol by Streptomyces); C methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated from Trewia nudlflora); CN- demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared by the demethylation of maytansinol by Streptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by the titanium trichloride/LAH reduction of maytansinol).

Of particular use are DM1 (disclosed in US Patent No. 5,208,020, incorporated by reference) and DM4 (disclosed in US Patent No. 7,276,497, incorporated by reference). See also a number of onal maytansinoid derivatives and methods in 5,416,064, WO/01/24763, 7,303,749, 7,601,354, USSN 12/631,508, WO02/098883, 6,441,163, 7,368,565, WO02/16368 and WO04/1033272, all of which are sly incorporated by reference in their entirety.

ADCs containing maytansinoids, s of making same, and their therapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020; 5,416,064; 163 and an Patent EP 0 425 235 B1, the disclosures of which are hereby sly incorporated by reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described ADCs comprising a maytansinoid designated DM1 linked to the onal antibody C242 directed against human colorectal cancer. 45_1 ters) P41668NZ00 The conjugate was found to be highly cytotoxic towards cultured colon cancer cells, and showed antitumor activity in an in vivo tumor growth assay.

Chari et al., Cancer Research 52:127-131 (1992) describe ADCs in which a maytansinoid was conjugated via a disulfide linker to the murine antibody A7 g to an antigen on human colon cancer cell lines, or to r murine monoclonal antibody TA.1 that binds the HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansonoid ate was tested in vitro on the human breast cancer cell line SK-BR-3, which expresses 3x105 HER-2 surface ns per cell. The drug conjugate achieved a degree of xicity similar to the free maytansinoid drug, which could be increased by increasing the number of maytansinoid molecules per antibody molecule. The A7- maytansinoid ate showed low systemic cytotoxicity in mice.

Auristatins and Dolastatins In some embodiments, the ADC comprises an anti-LY75 antibody conjugated to dolastatins or dolostatin peptidic analogs and derivatives, the auristatins (U.S. Pat. Nos. 483; 5,780,588).

Dolastatins and atins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al (2001) Antimicrob. Agents and her. 45(12):3580-3584) and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin or auristatin drug moiety may be ed to the antibody h the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linked monomethylauristatin drug moieties DE and DF, disclosed in "Senter et al, Proceedings of the American Association for Cancer Research, Volume 45, Abstract Number 623, presented Mar. 28, 2004 and described in United States Patent Publication No. 2005/0238648, the disclosure of which is expressly incorporated by reference in its ty.

An ary auristatin embodiment is MMAE (shown in Figure 10 wherein the wavy line indicates the covalent attachment to a linker (L) of an antibody drug conjugate; see US Patent No. 6,884,869 expressly incorporated by reference in its entirety).

Another exemplary auristatin embodiment is MMAF, shown in Figure 10 n the wavy line indicates the covalent attachment to a linker (L) of an dy drug ate (US 2005/0238649, 5,767,237 and 6,124,431, expressly incorporated by reference in their entirety): onal exemplary embodiments comprising MMAE or MMAF and various linker components (described further herein) have the following structures and abbreviations (wherein Ab means antibody and p is 1 to about 8): Typically, peptide-based drug moieties can be prepared by forming a e bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method (see E. Schroder and K. Lubke, "The Peptides", volume 1, pp 76-136, 1965, Academic Press) that is well known in the field of peptide chemistry. The auristatin/dolastatin drug moieties may be prepared according to the methods of: U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463- 18544345_1 (GHMatters) P41668NZ00 5465; Pettit et al (1998) ancer Drug Design -277; Pettit, G. R., et al. Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc. Perkin Trans. 1 5:859-863; and Doronina (2003) Nat Biotechnol 21(7):778-784.

Calicheamicin In other embodiments, the ADC comprises an antibody of the invention conjugated to one or more calicheamicin les. For example, Mylotarg is the first cial ADC drug and utilizes calicheamicin γ1 as the payload (see US Patent No. 198, orated by reference in its entirety). Additional calicheamicin derivatives are described in US Patent Nos. 5,264,586, 5,384,412, ,550,246, 5,739,116, 5,773,001, 5,767,285 and 5,877,296, all expressly incorporated by reference.

The calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at subpicomolar concentrations. For the preparation of conjugates of the calicheamicin family, see U.S.

Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 (all to American id Company). Structural analogues of calicheamicin which may be used include, but are not limited to, γ1I, α2I, α2I, N-acetyl- γ1I, PSAG and θI1 (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patents to American Cyanamid). Another anti-tumor drug that the antibody can be conjugated is QFA which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, ar uptake of these agents through antibody mediated internalization greatly enhances their cytotoxic effects.

Duocarmycins CC-1065 (see 4,169,888, incorporated by reference) and duocarmycins are members of a family of antitumor antibiotics utilized in ADCs. These antibiotics appear to work through ceselectively alkylating DNA at the N3 of adenine in the minor groove, which initiates a cascade of events that result in apoptosis.

Important members of the duocarmycins include duocarmycin A (US Patent No. 4,923,990, incorporated by reference) and mycin SA (U.S. Pat. No. 5,101,038, incorporated by nce), and a large number of analogues as described in US Patent Nos. 7,517,903, 7,691,962, ,101,038; 5,641,780; 186; 5,070,092; 5,070,092; 5,641,780; 038; 468, 092, 5,585,499, 5,846,545, WO2007/089149, WO2009/017394A1, 5,703,080, 6,989,452, 7,087,600, 7,129,261, 7,498,302, and 7,507,420, all of which are expressly incorporated by reference.

Pyrrolobenzodiazepines Pyrrolobenzodiazepine (PBD) (Journal of Medicinal Chemistry 2001, 44, 737-748) are DNA- interactive agents with significant cytotoxicity. Thirteen structures of this family have been isolate d, including compounds such as anthramycin, mazethramycin, porothramycin, prothracarcin, sibanomycin, tomaymycin, sibiromycin, chicamycin A, neothramycin A, B, and DC-81 (Medicinal Chemistry and Drug , ISBN: 978518, Ahmed Kamal et al. DOI: .5772/38869). Other analogues of this family have been prepared and have been conjugated to 18544345_1 (GHMatters) P41668NZ00 antibodies as described in Patent Nos. WO2011/130598 and WO2011/130616, which are expressly orated by reference.

Other Cytotoxic Agents Other antitumor agents that can be conjugated to the antibodies of the invention include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents known collectively LLE33288 complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S.

Pat. No. 5,877,296).

Enzymatically active toxins and fragments f which can be used include diphtheria A chain, ding active fragments of diphtheria toxin, exotoxin A chain (from monas nosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, aca americana proteins (PAPI, PAPII, and , momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, llin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for e, WO 93/21232 published Oct. 28, 1993.

The present invention further contemplates an ADC formed between an antibody and a nd with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radio-conjugated antibodies.

Examples include At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu.

The radio- or other labels may be incorporated in the conjugate in known ways. For example, the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid sors involving, for example, fluorine-19 in place of hydrogen. Labels such as Tc99m or I123, Re186, Re188 and In111 can be attached via a cysteine residue in the peptide.

Yttrium-90 can be attached via a lysine e. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporate Iodine-123. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail.

For compositions comprising a plurality of antibodies, the drug loading is represented by p, the average number of drug molecules per Antibody. Drug loading may range from 1 to 20 drugs (D) per Antibody. The average number of drugs per antibody in preparation of conjugation reactions may be characterized by conventional means such as mass spectroscopy, ELISA assay, and HPLC. The quantitative distribution of dy-Drug-Conjugates in terms of p may also be determined.

In some instances, separation, cation, and characterization of homogeneous Antibody- Drug-conjugates where p is a certain value from Antibody-Drug-Conjugates with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis. In ary embodiments, p is 2, 3, 4, 5, 6, 7, or 8 or a fraction thereof.

The generation of Antibody-drug conjugate compounds can be accomplished by any que known to the skilled artisan. Briefly, the dy-drug conjugate compounds can include 18544345_1 (GHMatters) P41668NZ00 an anti-LY75 antibody as the Antibody unit, a drug, and optionally a linker that joins the drug and the g agent.

A number of ent reactions are ble for covalent attachment of drugs and/or linkers to binding agents. This is can be accomplished by reaction of the amino acid residues of the binding agent, for e, antibody molecule, including the amine groups of lysine, the free carboxylic acid groups of glutamic and aspartic acid, the sulfhydryl groups of cysteine and the various moieties of the aromatic amino acids. A commonly used non-specific methods of covalent attachment is the carbodiimide reaction to link a carboxy (or amino) group of a compound to amino (or carboxy) groups of the dy. Additionally, bifunctional agents such as dialdehydes or imidoesters have been used to link the amino group of a compound to amino groups of an antibody le.

Also available for attachment of drugs to binding agents is the Schiff base reaction. This method involves the periodate oxidation of a drug that contains glycol or hydroxy groups, thus g an aldehyde which is then reacted with the g agent. Attachment occurs via formation of a Schiff base with amino groups of the binding agent. Isothiocyanates can also be used as coupling agents for covalently attaching drugs to binding agents. Other techniques are known to the skilled artisan and within the scope of the present invention.

In some embodiments, an intermediate, which is the precursor of the linker, is reacted with the drug under appropriate conditions. In other embodiments, reactive groups are used on the drug and/or the intermediate. The product of the on between the drug and the intermediate, or the derivatized drug, is subsequently reacted with an anti-LY75 antibody of the ion under appropriate ions.

It will be understood that al modifications may also be made to the desired compound in order to make reactions of that nd more convenient for purposes of preparing ates of the invention. For example a functional group e.g. amine, hydroxyl, or sulfhydryl, may be appended to the drug at a position which has minimal or an acceptable effect on the activity or other properties of the drug.

Linker Units Typically, the antibody-drug conjugate compounds comprise a Linker unit between the drug unit and the antibody unit. In some embodiments, the linker is cleavable under intracellular or extracellular conditions, such that cleavage of the linker releases the drug unit from the antibody in the appropriate environment. For example, solid tumors that secrete certain proteases may serve as the target of the cleavable linker; in other embodiments, it is the intracellular proteases that are utilized. In yet other embodiments, the linker unit is not cleavable and the drug is released, for example, by antibody degradation in lysosomes.

In some embodiments, the linker is cleavable by a ng agent that is present in the ellular environment (for example, within a lysosome or endosome or ea). The linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long or more. 18544345_1 (GHMatters) P41668NZ00 Cleaving agents can include, without limitation, cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in the e of active drug inside target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Peptidyl linkers that are cleavable by enzymes that are present in xpressing cells. For example, a peptidyl linker that is cleavable by the thiol-dependent se cathepsin-B, which is highly expressed in ous tissue, can be used (e.g., a Phe-Leu or a Gly-Phe-Leu-Gly linker (SEQ ID NO: X)). Other examples of such linkers are bed, e.g., in U.S. Pat. No. 6,214,345, incorporated herein by reference in its entirety and for all purposes.

In some embodiments, the peptidyl linker cleavable by an intracellular se is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with the val-cit linker).

In other embodiments, the cleavable linker is pH-sensitive, that is, sensitive to hydrolysis at certain pH values. Typically, the pH-sensitive linker hydrolyzable under acidic conditions. For example, an acid-labile linker that is hydrolyzable in the me (for example, a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, ster, acetal, ketal, or the like) may be used. (See, e.g., U.S. Pat. Nos. 5,122,368; 805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661.) Such linkers are relatively stable under neutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or 5.0, the approximate pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (such as, e.g., a thioether attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929).

In yet other embodiments, the linker is cleavable under reducing ions (for example, a disulfide linker). A variety of disulfide linkers are known in the art, including, for e, those that can be formed using SATA (N-succinimidylacetylthioacetate), SPDP (N-succinimidyl(2- pyridyldithio)propionate), SPDB (N-succinimidyl(2-pyridyldithio)butyrate) and SMPT (N- succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)- , SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody ates in Radioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935).

In other embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. :1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

In yet other embodiments, the linker unit is not cleavable and the drug is released by antibody degradation. (See U.S. Publication No. 238649 incorporated by reference herein in its entirety and for all purposes).

In many embodiments, the linker is self-immolative. As used herein, the term immolative Spacer" refers to a bifunctional chemical moiety that is capable of covalently linking together two spaced chemical es into a stable tripartite le. It will spontaneously separate from the second al moiety if its bond to the first moiety is cleaved. See for example, WO 18544345_1 (GHMatters) P41668NZ00 2007/059404A2, WO06/110476A2, WO05/112919A2, WO2010/062171, WO09/017394, WO07/089149, WO 07/018431, WO04/043493 and 83180, which are directed to drugcleavable substrate conjugates where the drug and cleavable substrate are ally linked through a self-immolative linker and which are all sly incorporated by reference.

Often the linker is not substantially sensitive to the extracellular environment. As used herein, "not substantially sensitive to the extracellular environment," in the context of a linker, means that no more than about 20%, 15%, 10%, 5%, 3%, or no more than about 1% of the linkers, in a sample of antibody-drug conjugate compound, are cleaved when the dy-drug conjugate compound ts in an extracellular environment (for example, in plasma). r a linker is not substantially sensitive to the ellular environment can be determined, for e, by incubating with plasma the antibody-drug conjugate compound for a predetermined time period (for example, 2, 4, 8, 16, or 24 hours) and then quantitating the amount of free drug present in the plasma.

In other, non-mutually exclusive embodiments, the linker promotes cellular internalization. In certain embodiments, the linker promotes cellular internalization when conjugated to the therapeutic agent (that is, in the milieu of the linker-therapeutic agent moiety of the antibody-drug conjugate compound as described herein). In yet other embodiments, the linker promotes cellular internalization when conjugated to both the auristatin compound and the anti-LY75 antibodies of the invention.

A variety of exemplary linkers that can be used with the t compositions and methods are described in 20050238649, and U.S. Publication No. 2006/0024317 (each of which is incorporated by reference herein in its entirety and for all purposes).

Drug Loading Drug loading is represented by p and is the average number of Drug moieties per antibody in a molecule. Drug loading ("p") may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more moieties (D) per antibody, although frequently the average number is a fraction or a decimal. lly, drug loading of from 1 to 4 is frequently useful, and from 1 to 2 is also useful.

ADCs of the invention e collections of antibodies conjugated with a range of drug moieties, from 1 to 20, for example, 1-15, 1-10, 2-9, 3-8, 4-7, 5-6. The average number of drug moieties per dy in ations of ADC from conjugation reactions may be characterized by conventional means such as mass spectroscopy and, ELISA assay.

The quantitative distribution of ADC in terms of p may also be determined. In some instances, tion, purification, and characterization of homogeneous ADC where p is a certain value from ADC with other drug loadings may be achieved by means such as electrophoresis.

For some dy-drug conjugates, p may be limited by the number of attachment sites on the dy. For example, where the attachment is a cysteine thiol, as in the exemplary embodiments above, an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker may be attached. In 18544345_1 (GHMatters) P41668NZ00 certain embodiments, higher drug loading, e.g. p>5, may cause aggregation, bility, toxicity, or loss of ar permeability of certain antibody-drug conjugates. In certain embodiments, the drug loading for an ADC of the invention ranges from 1 to about 8; from about 2 to about 6; from about 3 to about 5; from about 3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8; from about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3 to about 3.8; or from about 3.3 to about 3.7. Indeed, it has been shown that for certain ADCs, the optimal ratio of drug moieties per antibody may be less than 8, and may be about 2 to about 5. See US 2005/0238649 A1 (herein incorporated by nce in its entirety).

In certain embodiments, fewer than the tical maximum of drug moieties are conjugated to an antibody during a conjugation reaction. An antibody may contain, for example, lysine residues that do not react with the drug-linker intermediate or linker reagent, as discussed below. Generally, antibodies do not contain many free and reactive cysteine thiol groups which may be linked to a drug ; indeed most cysteine thiol residues in antibodies exist as disulfide bridges. In certain embodiments, an antibody may be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partial or total reducing conditions, to generate ve cysteine thiol groups. In certain embodiments, an antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or ne.

The loading (drug/antibody ratio) of an ADC may be controlled in ent ways, e.g., by: (i) ng the molar excess of drug-linker intermediate or linker reagent relative to dy, (ii) ng the conjugation on time or temperature, (iii) partial or limiting reductive conditions for cysteine thiol modification, (iv) engineering by recombinant techniques the amino acid sequence of the antibody such that the number and position of cysteine residues is modified for control of the number and/or on of linker-drug attachments (such as thioMab or b prepared as disclosed herein and in WO2006/034488 (herein incorporated by reference in its entirety)).

It is to be understood that where more than one nucleophilic group reacts with a drug-linker intermediate or linker reagent ed by drug moiety t, then the resulting product is a mixture of ADC compounds with a distribution of one or more drug moieties attached to an antibody.

The average number of drugs per antibody may be calculated from the mixture by a dual ELISA antibody assay, which is specific for antibody and specific for the drug. Individual ADC molecules may be identified in the mixture by mass spectroscopy and separated by HPLC, e.g. hobic interaction chromatography.

In some embodiments, a homogeneous ADC with a single loading value may be isolated from the ation mixture by electrophoresis or chromatography.

Methods of Determining Cytotoxic Effect of ADCs Methods of determining whether a Drug or Antibody-Drug conjugate exerts a cytostatic and/or cytotoxic effect on a cell are known. Generally, the xic or cytostatic activity of an Antibody Drug conjugate can be measured by: exposing mammalian cells expressing a target protein of the Antibody Drug conjugate in a cell culture medium; culturing the cells for a period from about 6 hours to about 5 days; and measuring cell viability. Cell-based in vitro assays can be used to measure 18544345_1 (GHMatters) P41668NZ00 viability feration), cytotoxicity, and induction of sis (caspase activation) of the Antibody Drug ate.

For determining whether an Antibody Drug conjugate exerts a atic effect, a thymidine oration assay may be used. For example, cancer cells expressing a target antigen at a density of 5,000 cells/well of a 96-well plated can be cultured for a 72-hour period and exposed to 0.5 μCi of 3H-thymidine during the final 8 hours of the 72-hour period. The incorporation of 3H-thymidine into cells of the culture is ed in the presence and absence of the Antibody Drug conjugate.

For determining xicity, necrosis or apoptosis ammed cell death) can be measured.

Necrosis is typically accompanied by increased permeability of the plasma membrane; swelling of the cell, and rupture of the plasma membrane. Apoptosis is typically characterized by membrane blebbing, condensation of cytoplasm, and the activation of endogenous endonucleases.

Determination of any of these effects on cancer cells indicates that an Antibody Drug conjugate is useful in the treatment of cancers.

Cell viability can be measured by determining in a cell the uptake of a dye such as neutral red, trypan blue, or ALAMAR™ blue (see, e.g., Page et al., 1993, Intl. J. Oncology 3:473-476). In such an assay, the cells are incubated in media containing the dye, the cells are washed, and the remaining dye, reflecting cellular uptake of the dye, is ed spectrophotometrically. The protein-binding dye sulforhodamine B (SRB) can also be used to measure cytoxicity (Skehan et al., 1990, J. Natl.

Cancer Inst. 7-12).

Alternatively, a tetrazolium salt, such as MTT, is used in a quantitative colorimetric assay for mammalian cell survival and proliferation by detecting living, but not dead, cells (see, e.g., n, 1983, J. Immunol. Methods 65:55-63).

Apoptosis can be quantitated by measuring, for example, DNA fragmentation. Commercial photometric methods for the quantitative in vitro determination of DNA fragmentation are available.

Examples of such assays, including TUNEL (which detects incorporation of labeled nucleotides in fragmented DNA) and ELISA-based assays, are described in Biochemica, 1999, no. 2, pp. 34-37 (Roche Molecular Biochemicals).

Apoptosis can also be determined by measuring morphological changes in a cell. For example, as with necrosis, loss of plasma membrane integrity can be ined by measuring uptake of certain dyes (e.g., a fluorescent dye such as, for example, acridine orange or ethidium bromide). A method for measuring apoptotic cell number has been described by Duke and Cohen, Current Protocols in Immunology an et al. eds., 1992, pp. 3.17.1-3.17.16). Cells also can be labeled with a DNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide) and the cells ed for chromatin sation and margination along the inner nuclear membrane. Other morphological changes that can be measured to determine sis include, e.g., cytoplasmic condensation, increased membrane blebbing, and cellular age.

The presence of apoptotic cells can be measured in both the attached and "floating" compartments of the cultures. For example, both compartments can be collected by removing the supernatant, trypsinizing the attached cells, combining the preparations ing a centrifugation 18544345_1 ters) P41668NZ00 wash step (e.g., 10 minutes at 2000 rpm), and detecting apoptosis (e.g., by measuring DNA ntation). (See, e.g., Piazza et al., 1995, Cancer Research 55:3110-16).

In vivo, the effect of a therapeutic composition of the anti-LY75 dy of the ion can be evaluated in a suitable animal model. For example, xenogenic cancer models can be used, wherein cancer explants or passaged xenograft s are introduced into immune compromised animals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine 3: 402-408). Efficacy can be measured using assays that measure tion of tumor formation, tumor regression or metastasis, and the like.

The therapeutic compositions used in the practice of the foregoing methods can be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method.

Suitable rs include any material that when ed with the therapeutic composition retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the t's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's ceutical Sciences 16th Edition, A. Osal., Ed., 1980).

Methods for ing the antibodies of the invention The present invention further provides methods for producing the disclosed anti-LY75 antibodies. These methods encompass culturing a host cell ning isolated nucleic acid(s) encoding the antibodies of the invention. As will be appreciated by those in the art, this can be done in a variety of ways, depending on the nature of the antibody. In some embodiments, in the case where the antibodies of the invention are full length ional antibodies, for e, a heavy chain variable region and a light chain variable region under ions such that an antibody is produced and can be isolated.

The variable heavy and light chains of LY75_A1 are disclosed herein (both protein and nucleic acid sequences); as will be appreciated in the art, these can be easily ted to produce full length heavy and light chains. That is, having provided the DNA fragments encoding VH and VK segments as outlined herein, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes, or to a scFv gene. In these lations, a VK- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operatively ", as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule ng heavy chain constant regions (CH1, CH2 and CH3). The sequences of murine heavy chain constant region genes are known in the art [see e.g. Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 2] and DNA fragments encompassing these regions can be obtained by standard PCR amplification. 18544345_1 (GHMatters) P41668NZ00 The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably is an IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CH1 nt .

The isolated DNA encoding the VL / VK region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of murine light chain constant region genes are known in the art [see, e.g. Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth n, US Department of Health and Human Services, NIH Publication No. 91-3242] and DNA fragments encompassing these regions can be obtained by standard PCR amplification. In preferred embodiments, the light chain constant region can be a kappa or lambda constant region.

To create a scFv gene, the VH- and VL / VK-encoding DNA fragments are operatively linked to another fragment ng a flexible linker, e.g. ng the amino acid sequence (Gly4 -Ser)3, such that the VH and VL / VK sequences can be expressed as a contiguous single-chain protein, with the VL / VK and VH regions joined by the flexible linker [see e.g. Bird et al. (1988) e 242:423- 426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554].

In general, nucleic acids are provided which encode the dies of the invention. Such polynucleotides encode for both the variable and constant regions of each of the heavy and light chains, although other combinations are also contemplated by the present invention in accordance with the compositions bed herein. The present invention also contemplates ucleotide fragments derived from the disclosed polynucleotides and nucleic acid sequences mentary to these polynucleotides.

The polynucleotides can be in the form of RNA or DNA. Polynucleotides in the form of DNA, cDNA, genomic DNA, nucleic acid analogs, and synthetic DNA are within the scope of the present invention. The DNA may be double-stranded or single-stranded, and if single stranded, may be the coding (sense) strand or non-coding (anti-sense) strand. The coding sequence that encodes the polypeptide may be identical to the coding sequence provided herein or may be a different coding sequence, which sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptides as the DNA provided herein.

In some embodiments, nucleic acid(s) encoding the antibodies of the invention are incorporated into expression vectors, which can be extrachromosomal or ed to integrate into the genome of the host cell into which it is introduced. sion vectors can contain any number of appropriate regulatory sequences ding, but not limited to, transcriptional and translational control sequences, promoters, ribosomal binding sites, enhancers, origins of ation, etc.) or other components tion genes, etc.), all of which are operably linked as is well known in the art.

In some cases two nucleic acids are used and each put into a different expression vector (e.g. heavy chain in a first expression vector, light chain in a second expression vector), or alternatively they can 45_1 (GHMatters) P41668NZ00 be put in the same expression vector. It will be appreciated by those skilled in the art that the design of the expression vector(s), including the ion of regulatory sequences may depend on such factors as the choice of the host cell, the level of expression of protein desired, etc.

In general, the nucleic acids and/or expression can be uced into a suitable host cell to create a recombinant host cell using any method appropriate to the host cell selected (e.g., transformation, transfection, oporation, ion), such that the nucleic acid molecule(s) are operably linked to one or more expression control elements (e.g., in a , in a construct created by processes in the cell, integrated into the host cell genome). The resulting recombinant host cell can be maintained under ions suitable for expression (e.g. in the presence of an inducer, in a suitable non-human animal, in suitable culture media supplemented with appropriate salts, growth factors, antibiotics, nutritional supplements, etc.), whereby the encoded polypeptide(s) are produced.

In some cases, the heavy chains are produced in one cell and the light chain in another.

Mammalian cell lines available as hosts for expression are known in the art and e many alized cell lines available from the American Type Culture Collection (ATCC), Manassas, VA including but not limited to Chinese hamster ovary (CHO) cells, HEK 293 cells, NSO cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a number of other cell lines. Non-mammalian cells including but not limited to bacterial, yeast, insect, and plants can also be used to express recombinant antibodies. In some embodiments, the antibodies can be produced in enic animals such as cows or chickens.

General methods for antibody molecular biology, expression, purification, and screening are well known, for example, see US Patent Nos. 4,816,567, 4,816,397, 6,331,415 and 7,923,221, as well as Antibody Engineering, edited by Kontermann & Dubel, Springer, Heidelberg, 2001 and 2010 Hayhurst & Georgiou, 2001, Curr Opin Chem Biol 5:683-689; Maynard & Georgiou, 2000, Annu Rev Biomed Eng 2:339-76; and Morrison, S. (1985) Science 229:1202.

Pharmaceutical Compositions In another aspect, the t invention provides a composition, e.g. a pharmaceutical composition, containing one or a ation of LY75 antibodies, or antigen-binding portion(s) thereof, of the present invention, formulated together with a pharmaceutically acceptable carrier.

Such compositions may include one or a combination of (e.g. two or more different) dies, or conjugates or ific molecules of the ion. For example, a ceutical composition of the invention can comprise a combination of antibodies (or conjugates or bispecifics) that bind to different epitopes on the target antigen or that have mentary activities.

Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e. combined with other agents. For example, the combination therapy can include an antiantibody of the present invention combined with at least one other anti-tumor agent, or an antiinflammatory or suppressant agent. Examples of therapeutic agents that can be used in 18544345_1 (GHMatters) P41668NZ00 combination therapy are described in greater detail below in the section on uses of the antibodies of the invention.

As used herein, aceutically able carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, ic and absorption ng agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, eral, spinal or epidermal administration (e.g. by injection or infusion). Depending on the route of administration, the active compound, i.e. antibody, immunoconjugate, or bispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural ions that may vate the compound.

The pharmaceutical compounds of the invention may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects [see, e.g. Berge, S.M., et al. (1977) J. Pharm. Sci. 66:1-19]. Examples of such salts e acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic c amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical ition of the invention also may include a pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically able antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hloride, sodium ate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated yanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Examples of suitable aqueous and non-aqueous rs that may be ed in the pharmaceutical itions of the ion include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of rganisms may be d both by ization procedures, supra, and by the ion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the 18544345_1 (GHMatters) P41668NZ00 compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any tional media or agent is incompatible with the active compound, use thereof in the pharmaceutical itions of the invention is contemplated. mentary active compounds can also be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered ure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures f. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, ol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the active nd in the required amount in an appropriate t with one or a combination of ients enumerated above, as ed, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable ons, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional d ingredient from a previously sterile-filtered solution f.

The amount of active ingredient which can be combined with a carrier al to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of 100 per cent, this amount will range from about 0.01 per cent to about 99 per cent of active ingredient, preferably from about 0.1 per cent to about 70 per cent, most preferably from about 1 per cent to about 30 per cent of active ingredient in combination with a pharmaceutically acceptable r.

Dosage regimens are adjusted to e the optimum desired response (e.g. a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as ted by the 18544345_1 (GHMatters) P41668NZ00 exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of . Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in ation with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique teristics of the active compound and the particular therapeutic effect to be achieved, and (b) the tions inherent in the art of compounding such an active nd for the treatment of sensitivity in individuals.

For administration of the antibody, the dosage ranges from about 0.0001 to 100 mg/kg, for example, 0.001 to 50mg/kg, 0.005 to 20mg/kg, 0.01 to 10mg/kg and more usually 0.01 to 5 mg/kg, of the host body weight. For example dosages can be 0.05 mg/kg body weight, 0.1 mg/kg body weight, 0.3 mg/kg body weight, 0.3 mg/kg body weight, 0.5 mg/kg body weight, 1 mg/kg body , 2 mg/kg body weight, 3 mg/kg body weight, 4 mg/kg body weight, 5 mg/kg body weight 6 mg/kg body weight, 7 mg/kg body weight, 8 mg/kg body weight, 9 mg/kg body , 10 mg/kg body weight, 12 mg/kg body weight, 15 mg/kg body weight, 20 mg/kg body weight, 25 mg/kg body weight, 30 mg/kg body weight, or within the range of 0.1-20 mg/kg, 0.5-15 mg/kg, 1-10 mg/kg, 2-8 mg/kg, 3-7 mg/kg, 4-6 mg/kg. An exemplary treatment regime entails administration once per day, once every 2 days, once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 6 weeks, once every 3 months or once every three to 6 . red dosage regimens for an anti-LY75 antibody of the invention include 1 mg/kg body weight or 3 mg/kg body weight via intravenous administration, with the antibody being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks.

In some methods, two or more monoclonal antibodies with different binding specificities are stered simultaneously, in which case the dosage of each antibody stered falls within the ranges indicated. Antibody is usually administered on multiple occasions. Intervals between single dosages can be, for example, daily, twice weekly, weekly, monthly, every three months, every six months, or yearly. Intervals can also be irregular as indicated by measuring blood levels of dy to the target antigen in the patient. In some methods, dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 μg /ml, 5-750 μg /ml, 10-600 μg /ml, 15-500 μg /ml, -400 μg /ml and in some methods about 25-300 μg /ml.

Alternatively, antibody can be administered as a sustained release formulation, in which case less nt administration is required. Dosage and frequency vary ing on the half-life of the antibody in the patient. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and nonhuman dies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent als over a long period of time. Some patients continue to receive treatment for the rest of their lives. In 18544345_1 (GHMatters) P41668NZ00 therapeutic applications, a relatively high dosage at vely short intervals is sometimes ed until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete ration of symptoms of disease. fter, the patient can be stered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of stration, without being toxic to the patient. The selected dosage level will depend upon a y of pharmacokinetic factors ing the activity of the ular compositions of the present ion employed, or the ester, salt or amide f, the route of stration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A "therapeutically effective dosage" of an anti-LY75 antibody of the invention preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free s, or a prevention of impairment or lity due to the disease affliction. For example, for the treatment of the LY75 mediated tumors, a "therapeutically effective dosage" preferably inhibits cell growth or tumor growth by at least about 20%, at least about 30%, more preferably by at least about 40%, at least about 50% even more preferably by at least about 60%, at least about 70% and still more preferably by at least about 80% or at least about 90%, relative to untreated subjects. The ability of a compound to inhibit tumor growth can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit cell growth, such inhibition can be measured in vitro by assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject.

One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.

A composition of the present invention can be administered via one or more routes of administration using one or more of a variety of s known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary ing upon the desired results. Preferred routes of administration for antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by ion or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, apsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, 18544345_1 (GHMatters) P41668NZ00 subcuticular, intraarticular, sular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. atively, an antibody of the invention can be administered via a renteral route, such as a topical, mal or mucosal route of administration, for example, intranasally, orally, vaginally, ly, sublingually or lly.

The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery s. Biodegradable, biocompatible rs can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, en, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or lly known to those skilled in the art [see, e.g. Sustained and Controlled Release Drug Delivery Systems (1978) J.R. Robinson, ed., Marcel Dekker, Inc., N.Y].

Therapeutic compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in US Patent Nos. ,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of wellknown implants and modules useful in the present invention include: US Patent No. 4,487,603, which discloses an implantable infusion pump for dispensing medication at a controlled rate; US Patent No. 4,486,194, which discloses a therapeutic device for stering medicaments through the skin; US Patent No. 4,447,233, which discloses a medication on pump for delivering medication at a precise infusion rate; US Patent No. 4,447,224, which discloses a variable flow implantable infusion tus for continuous drug delivery; US Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and US Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those d in the art.

In certain embodiments, the monoclonal antibodies of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the ion cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g. US Patents 811; 5,374,548; and 5,399,331. The liposomes may se one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery [see, e.g. V.V. Ranade (1989) J. Clin. Pharmacol. 29:685]. Exemplary targeting moieties include folate or biotin (see, e.g. US Patent 016.); mannosides [Umezawa et al. (1988) Biochem. Biophys. Res. Commun. 153:1038]; antibodies [P.G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180]; surfactant protein A receptor [Briscoe et al. (1995) Am. J. Physiol. 1233:134]; p120 [Schreier et al. (1994) J.

Biol. Chem. 269:9090]; see also K. en; M.L. Laukkanen (1994) FEBS Lett. 346:123; J.J.

Killion; I.J. Fidler (1994) Immunomethods 4:273. 18544345_1 (GHMatters) P41668NZ00 Uses and Methods The antibodies, dy compositions and methods of the present invention have us in vitro and in vivo diagnostic and therapeutic utilities involving the diagnosis and treatment of LY75 mediated disorders.

In some embodiments, these molecules can be administered to cells in culture, in vitro or ex vivo, or to human subjects, e.g. in vivo, to treat, prevent and to diagnose a variety of disorders. As used herein, the term "subject" is intended to include human and non-human animals. Non-human animals include all vertebrates, e.g. mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles. Preferred subjects include human patients having disorders mediated by LY75 activity. The methods are particularly suitable for treating human patients having a disorder associated with the aberrant LY75 expression. When antibodies to LY75 are administered together with another agent, the two can be administered in either order or simultaneously.

Given the specific binding of the antibodies of the invention for LY75, the antibodies of the invention can be used to specifically detect LY75 expression on the surface of cells and, moreover, can be used to purify LY75 via immunoaffinity purification.

Furthermore, given the expression of LY75 on tumor cells, the dies, antibody itions and methods of the present invention can be used to treat a subject with a tumorigenic disorder, e.g. a disorder characterized by the presence of tumor cells expressing LY75or in the manufacture of a medicament for the ent of such a disorder including, for e gastric cancer, kidney cancer, thyroid cancer, ageal cancer, head and neck cancer, skin cancer, liver cancer, atic cancer, colorectal cancer, bladder cancer, prostate cancer, breast cancer including triple ve breast cancer, ovarian cancer, lung cancer, myeloma, leukaemia, ing chronic lymphocytic leukaemia and acute myeloid mia, non-Hodgkin’s lymphoma, including DLBCL, B-Cell Lymphoma, Follicular Lymphoma, Mantle Cell Lymphoma, Lymphoma of Mucosa-Associated Lymphoid Tissue (MALT), T-Cell/Histiocyte-Rich B-Cell Lymphoma, Burkitt’s Lymphoma, Lymphoplasmacytic Lymphoma, Small Lymphocytic Lymphoma, al Zone Lymphoma, T Cell Lymphoma, Peripheral T-Cell Lymphoma, Anaplastic Large Cell Lymphoma and AngioImmunoblastic T-Cell Lymphoma. LY75 has been demonstrated to be internalised on antibody g as illustrated in Examples 5 and 7 below, thus enabling the antibodies of the invention to be used in any payload ism of action e.g. an ADC approach, radioimmunoconjugate, or ADEPT approach.

In one embodiment, the antibodies (e.g. onal antibodies, antibody fragments, Nanobody®, multispecific and bispecific les and compositions, etc.) of the invention can be used to detect levels of LY75, or levels of cells which contain LY75 on their membrane surface, which levels can then be linked to n disease symptoms. Alternatively, the antibodies, lly stered as ADCs, can be used to inhibit or block LY75 function which, in turn, can be linked to the prevention or amelioration of certain disease symptoms, thereby implicating the LY75 as a mediator of the disease. This can be ed by contacting a sample and a control sample with the 18544345_1 (GHMatters) P41668NZ00 anti-LY75 antibody under conditions that allow for the formation of a complex between the antibody and LY75. Any complexes formed between the antibody and the LY75 are detected and compared in the sample and the control.

In another embodiment, the dies (e.g. monoclonal antibodies, multispecific and bispecific molecules and compositions) of the invention can be initially tested for binding activity associated with therapeutic or diagnostic use in vitro. For example, compositions of the invention can be tested using the flow cytometric assays described in the Examples below.

The antibodies (e.g. monoclonal antibodies, multispecific and bispecific molecules, immunoconjugates and compositions) of the invention have additional utility in therapy and diagnosis of LY75 related es. For example, the monoclonal antibodies, the pecific or bispecific les and the immunoconjugates can be used to elicit in vivo or in vitro one or more of the following biological ties: to t the growth of and/or kill a cell expressing LY75; to mediate phagocytosis or ADCC of a cell expressing LY75 in the presence of human effector cells, or to block LY75 ligand binding to LY75.

In a particular ment, the antibodies (e.g. monoclonal dies, pecific and bispecific molecules and compositions) are used in vivo to treat, prevent or diagnose a variety of LY75-related diseases. Examples of elated diseases include, among others, human cancer s representing gastric cancer, colorectal cancer, prostate , breast cancer, ovarian cancer kidney cancer, thyroid cancer, oesophageal cancer, head and neck cancer, skin cancer, liver , atic cancer, bladder cancer, myeloma, leukaemia, including chronic lymphocytic leukaemia, acute myeloid leukaemia, non-Hodgkin’s lymphoma, including DLBCL, B-Cell Lymphoma, Follicular Lymphoma, Mantle Cell ma, Lymphoma of Mucosa-Associated Lymphoid Tissue (MALT), TCell /Histiocyte-Rich B-Cell Lymphoma, Burkitt’s Lymphoma, Lymphoplasmacytic Lymphoma, Small Lymphocytic Lymphoma, Marginal Zone ma, T Cell ma, Peripheral T-Cell ma, Anaplastic Large Cell Lymphoma and AngioImmunoblastic T-Cell Lymphoma and lung cancer.

Suitable routes of administering the dy compositions (e.g. monoclonal antibodies, multispecific and bispecific molecules and immunoconjugates) of the invention in vivo and in vitro are well known in the art and can be selected by those of ordinary skill. For example, the dy compositions can be administered by injection (e.g. intravenous or aneous). Suitable dosages of the molecules used will depend on the age and weight of the subject and the concentration and/or formulation of the antibody composition.

As previously described, the anti-LY75 antibodies of the invention can be co-administered with one or other more therapeutic agents, e.g. a cytotoxic agent, a radiotoxic agent or an immunosuppressive agent. The antibody can be linked to the agent (as an immunocomplex) or can be administered separate from the agent. In the latter case (separate administration), the antibody can be administered before, after or concurrently with the agent or can be co-administered with other known therapies, e.g. an anti-cancer therapy, e.g. radiation. Such therapeutic agents include, among others, anti-neoplastic agents such as doxorubicin (adriamycin), cisplatin bleomycin sulfate, 18544345_1 (GHMatters) P41668NZ00 carmustine, chlorambucil, and cyclophosphamide yurea which, by themselves, are only effective at levels which are toxic or subtoxic to a patient. Cisplatin is intravenously administered as a 100 mg/kg dose once every four weeks and adriamycin is intravenously administered as a 60-75 mg/ml dose once every 21 days. Other agents suitable for co-administration with the antibodies of the invention include other agents used for the treatment of cancers, e.g. gastric cancer, ctal cancer, prostate cancer, breast cancer, ovarian cancer or lung cancer, such as Avastin®, 5FU and gemcitabine. Co-administration of the anti-LY75 antibodies or antigen g fragments f, of the present invention with chemotherapeutic agents es two anti-cancer agents which operate via different mechanisms which yield a cytotoxic effect to human tumor cells. Such co-administration can solve ms due to pment of resistance to drugs or a change in the antigenicity of the tumor cells which would render them unreactive with the antibody.

Target-specific effector cells, e.g. effector cells linked to compositions (e.g. monoclonal antibodies, multispecific and ific molecules) of the invention can also be used as therapeutic agents. Effector cells for targeting can be human leukocytes such as macrophages, neutrophils or monocytes. Other cells include eosinophils, natural killer cells and other IgG- or IgA-receptor bearing cells. If d, effector cells can be obtained from the subject to be treated. The target-specific effector cells can be administered as a suspension of cells in a physiologically acceptable solution.

The number of cells stered can be in the order of 108-109, but will vary depending on the therapeutic purpose. In general, the amount will be sufficient to obtain localization at the target cell, e.g. a tumor cell expressing LY75, and to affect cell killing by, e.g. phagocytosis. Routes of administration can also vary.

Therapy with -specific effector cells can be performed in conjunction with other techniques for removal of targeted cells. For example, anti-tumor y using the compositions (e.g. monoclonal antibodies, pecific and bispecific molecules) of the invention and/or effector cells armed with these compositions can be used in conjunction with chemotherapy. Additionally, combination immunotherapy may be used to direct two ct cytotoxic effector populations toward tumor cell rejection. For example, anti-LY75 antibodies linked to c-gamma RI or anti-CD3 may be used in conjunction with IgG- or IgA-receptor specific binding agents.

Bispecific and multispecific molecules of the invention can also be used to modulate FcγR or FcγR levels on effector cells, such as by capping and elimination of receptors on the cell surface.

Mixtures of anti-Fc receptors can also be used for this purpose.

The itions (e.g. monoclonal antibodies, multispecific and bispecific molecules and immunoconjugates) of the invention which have complement binding sites, such as portions from IgG1, -2, or -3 or IgM which bind complement, can also be used in the presence of ment. In one embodiment, ex vivo treatment of a population of cells comprising target cells with a binding agent of the invention and appropriate or cells can be supplemented by the addition of complement or serum containing complement. Phagocytosis of target cells coated with a binding agent of the invention can be improved by binding of complement proteins. In another embodiment target cells coated with the itions (e.g. monoclonal antibodies, multispecific and bispecific 18544345_1 (GHMatters) P41668NZ00 molecules) of the invention can also be lysed by complement. In yet another embodiment, the compositions of the invention do not activate complement.

The compositions (e.g. monoclonal dies, multispecific and bispecific molecules and immunoconjugates) of the invention can also be administered er with complement. In n embodiments, the instant disclosure provides compositions comprising dies, pecific or bispecific molecules and serum or complement. These compositions can be ageous when the complement is located in close proximity to the antibodies, multispecific or bispecific molecules. atively, the antibodies, multispecific or bispecific molecules of the invention and the complement or serum can be administered separately.

Also within the scope of the present invention are kits comprising the antibody compositions of the invention (e.g. monoclonal antibodies, bispecific or multispecific molecules, or immunoconjugates) and instructions for use. The kit can further contain one or more additional reagents, such as an immunosuppressive reagent, a xic agent or a radiotoxic agent, or one or more additional antibodies of the invention (e.g. an antibody having a complementary activity which binds to an epitope in the LY75 antigen distinct from the first antibody).

Accordingly, patients treated with antibody compositions of the ion can be additionally stered (prior to, simultaneously with, or following stration of an dy of the invention) with another therapeutic agent, such as a cytotoxic or radiotoxic agent, which enhances or augments the therapeutic effect of the antibodies.

In other embodiments, the subject can be additionally treated with an agent that modulates, e.g. es or inhibits, the expression or activity of Fcγ or Fcγ receptors by, for example, treating the subject with a cytokine. red cytokines for administration during treatment with the multispecific molecule include of granulocyte colony-stimulating factor (G-CSF), granulocytemacrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), and tumor is factor (TNF).

The compositions (e.g. antibodies, multispecific and bispecific molecules) of the invention can also be used to target cells expressing FcγR or LY75, for example, for labeling such cells. For such use, the binding agent can be linked to a molecule that can be detected. Thus, the invention provides methods for localizing ex vivo or in vitro cells expressing Fc receptors, such as FcγR, or LY75. The detectable label can be, e.g. a radioisotope, a fluorescent nd, an enzyme, or an enzyme co-factor.

In a particular embodiment, the invention provides methods for detecting the presence of the LY75 antigen in a sample, or ing the amount of the LY75 antigen, comprising contacting the sample, and a control sample, with a monoclonal antibody, or an n binding portion thereof, which specifically binds to LY75, under conditions that allow for formation of a complex between the antibody or portion thereof and LY75. The formation of a complex is then detected, wherein a difference complex ion between the sample ed to the control sample is indicative the presence of the LY75 antigen in the sample. 18544345_1 (GHMatters) P41668NZ00 In other embodiments, the invention provides methods for treating a LY75 mediated er in a subject, e.g. human cancers, including gastric cancer, kidney cancer, thyroid cancer, oesophageal cancer, head and neck cancer, skin , liver cancer, pancreatic cancer, colorectal cancer, bladder cancer, prostate cancer, breast cancer including triple negative breast cancer, n cancer, lung cancer, a, leukaemia, including chronic lymphocytic leukaemia and acute myeloid leukaemia, and non-Hodgkin’s lymphoma, including DLBCL, B-Cell Lymphoma, Follicular Lymphoma, Mantle Cell Lymphoma, Lymphoma of Mucosa-Associated Lymphoid Tissue (MALT), T-Cell/Histiocyte-Rich B-Cell Lymphoma, Burkitt’s Lymphoma, Lymphoplasmacytic Lymphoma, Small Lymphocytic Lymphoma, Marginal Zone Lymphoma, T Cell Lymphoma, Peripheral T-Cell Lymphoma, stic Large Cell Lymphoma and AngioImmunoblastic T-Cell Lymphoma.

In all embodiments of the ion, preferred cancers include non-Hodgkin's lymphoma, acute myeloid leukaemia, chronic lymphocytic leukaemia and triple-negative breast cancer bladder cancer and pancreatic .

All nces cited in this specification, including without limitation all papers, publications, patents, patent ations, presentations, texts, reports, manuscripts, brochures, books, internet postings, journal es, periodicals, product fact sheets, and the like, one hereby incorporated by reference into this specification in their ties. The discussion of the references herein is intended to merely summarize the assertions made by their authors and no admission is made that any reference constitutes prior art and Applicants’ reserve the right to challenge the accuracy and pertinence of the cited nces.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ry skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the dependent claims.

The present invention is further illustrated by the following es which should not be construed as further limiting.

Example 1: Generation of Human Monoclonal Antibodies Against LY75-Antigen Following standard procedures, mice (xenomouse IgG1) were immunized with CHO cells transfected with full length LY75.

The specificity of antibodies raised against the LY75 was tested by flow cytometry on HEK293 cells transfected with LY75 and subsequently on LY75-expressing HT29 cells. To test the y of the antibodies to bind to the cell surface LY75 protein, the antibodies were incubated with the LY75- sing cells. Cells were washed in FACS buffer (DPBS, 2% FBS), centrifuged and resuspended in 100µl of the diluted primary LY75 antibody (also diluted in FACS buffer). The antibody-cell line complex was ted on ice for 60 min and then washed twice with FACS buffer as described above. The cell-antibody pellet was resuspended in 100µl of the diluted secondary antibody (also d in FACS ) and incubated on ice for 60 min on ice. The pellet was washed as before and 18544345_1 (GHMatters) P41668NZ00 resuspended in 200µl FACS buffer. The samples were loaded onto the BD FACScanto II flow cytometer and the data analyzed using the BD FACSdiva software (results not shown).

Example 2: Structural Characterization of Monoclonal Antibodies to LY75 The cDNA sequences encoding the heavy and light chain variable regions of the LY75_A1 monoclonal antibody were ed using standard PCR techniques and were sequenced using standard DNA sequencing techniques.

The antibody sequences may be nized to revert back to germline residues at one or more residues.

The nucleotide and amino acid sequences of the heavy chain variable region of LY75_A1 are shown in SEQ ID NO: 3 and 1, respectively.

The nucleotide and amino acid sequences of the light chain variable region of LY75_A1 are shown in SEQ ID NO: 4 and 2, tively.

Comparison of the LY75_A1 heavy chain globulin sequence to the known human ne immunoglobulin heavy chain sequences demonstrated that the LY75_A1 heavy chain utilizes a VH segment from human germline VH 3-15 and a JH segment from human germline JH JH4.

Further analysis of the LY75_A1 VH ce using the Kabat system of CDR region determination led to the delineation of the heavy chain CDR1, CDR2 and CDR3 regions as shown in SEQ ID NOs: , 6 and 7, respectively. The alignments of the LY75_A1 CDR1, CDR2 and CDR3 VH sequences to the germline VH 3-15 and germline JH JH4 sequence are shown in Figure 1.

Comparison of the 1 light chain immunoglobulin sequence to the known human germline immunoglobulin light chain sequences demonstrated that the LY75_A1 light chain utilizes a VK segment from human germline VK O12 and a JK segment from human germline JK JK4. Further analysis of the LY75_A1 VK sequence using the Kabat system of CDR region ination led to the delineation of the light chain CDR1, CDR2 and CDR3 regions as shown in SEQ ID NOs:8, 9 and , respectively. The alignments of the LY75_A1 CDR1, CDR2 and CDR3 VK sequences to the germline VK O12 and germline JK JK4 sequences are shown in Figure 2.

Example 3: Immunohistochemistry Using Monoclonal Antibody to LY75 Using the human monoclonal antibodies specific to LY75, immunohistochemistry was performed on FFPE HT-29 and A549 cell pellets, FFPE non-Hodgkin’s lymphoma and pancreatic cancer arrays, and fresh frozen lymphoma/leukaemia tumours, ovarian cancer, atic , and breast cancer sections and a normal tissue array.

Materials and methods Materials Xylenes (X5P-1gal) from Fisher Scientific, PA, USA. rep 100% l (HC1GAL) from Fisher Scientific, PA, USA. 10x Citrate buffer for heat induced e retrieval (AP9003125) from Thermo Scientific, MA, USA. 18544345_1 (GHMatters) P41668NZ00 Thermo Scientific* Pierce* Peroxidase Suppressor ) from Thermo Scientific, MA, USA.

Serum free protein block ) from Dako, CA, USA Secondary antibody: goat anti-human IgG TC conjugated 97-003) from Jackson Immunoresearch, PA, USA Chrome pure Human IgG, whole molecule (09003) from Jackson Immunoresearch, PA, USA Tertiary antibody: mouse anti-FITC (ab10257) from Abcam, MA, USA Purified human IgG isotype control (1-001A) from R&D Systems, MN, USA Tween-20 (BP337-100) from Fisher Scientific, PA, USA Acetone (BP2403-4) from Fisher Scientific, PA, USA Dual Link EnVision+ HRP-conjugated polymer, Mouse and Rabbit ) from Dako, CA, USA.

DAB 2-solution kit (882014) from ogen, NY, USA.

Harris Hematoxylin (23677) from Fisher Scientific, PA, USA.

Faramount mounting media (S302580) from Dako, CA, USA.

Tissue sections and arrays were purchased from US Biomax Inc., MD, USA or Origene, MD, Preparation of FFPE slides: Deparaffinisation and Rehydration FFPE slides were deparaffinised in xylene (2 x 3 minutes) then ated through 1:1 xylene: 100% ethanol (1 x 3 minutes), 100% ethanol (2 x 3 minutes), 95% ethanol (1 x 3 minutes), 70% l (1 x 3 minutes), 50% ethanol (1 x 3 minutes), and tap water (1 x 3 minutes).

Preparation of FFPE slides: Antigen Retrieval (Microwave).

The LY75 antigen was retrieved using microwave heat, high power until boiling then low power for 10 minutes in 50 mL 1x citrate buffer in a Coplin jar. Slides were then left to cool to room temperature for a further 15 min, then washed in tap water, 3 minutes. Circles were drawn around each tissue n/TMA with a hydrophobic barrier pen and slides were then washed 3 times in PBS, 3 minutes each wash.

Preparation of FF slides Slides were removed from storage at -80C and d to dry at room temperature in the fume hood for 20-30 minutes. The slides were fixed for 10 min in ice cold acetone at -20C, then allowed to dry for 20 min in the fume hood at room temperature. Slides were washed and rehydrated in PBS, 3 washes for 3 min each. Sections were outlined with a hydrophobic barrier pen.

Preparation of antibody complexes The primary anti-LY75 antibody was diluted in serum free protein block (SFPB) to obtain a solution with a concentration 20-fold greater than the final desired concentration (20 µg/mL for 1 µg/mL final). The secondary antibody, goat anti-human immunoglobulin G (IgG) antigen-binding nt (Fab), was ed similarly in SFPB to create a solution of equal concentration. 18544345_1 (GHMatters) P41668NZ00 Equal volumes of primary and secondary dies were combined in a ed tube, gently mixed, and incubated for 3 minutes at room temperature, resulting in a primary antibody concentration 10-fold greater than the desired final concentration (10 µg/mL for 1 µg/mL final). This mixture was diluted 1:5 with SFPB, gently mixed, and incubated for 30 minutes at room temperature, resulting in a primary antibody concentration twice that of the d final concentration (2 µg/mL for 1 µg/mL final).

To produce the final staining complexes, a 1% (10 µg/µL) solution of human IgG was prepared in SFPB and equal volume added to the primary/secondary antibody mixture. This combination was gently mixed and incubated at room temperature for 30 minutes, diluting by half the primary antibody tration of the primary/secondary antibody mixture and resulting in the desired final primary antibody concentration (1 µg/mL). staining Meanwhile, endogenous tissue peroxidase activity was blocked by ting tissues with peroxidase suppressor for 5-10 minutes at RT in a humidified chamber. Slides were then washed in PBS 3 x 3 minutes each wash. Tissues were incubated in SFPB for 30 minutes at room temperature in a humidified chamber. Final ng complexes were applied to each tissue section and/or microarray, and the slides were incubated for 30 min at room temperature in a fied chamber.

Slides were then washed once in PBS and once in PBST (PBS+0.125% Tween-20), 3 s each wash. The tertiary antibody mouse anti-FITC, was applied at 2 µg/mL concentration for 30 min, room temperature, in a humidified chamber. Sections were then washed once in PBS and once in PBST, 3 min each wash. Dual Link EnVision+ anti-mouse/rabbit-HRP-conjugated polymer was then applied to the tissues and the slides were incubated for 30 min at room ature in a humidified chamber. Slides were then washed once in PBS, once in PBST, 3 minutes each wash. Tissues were incubated in DAB solution prepared according to the manufacturer’s instructions at room temperature for 10 min. Slides were then washed once in running tap water for 2 minutes and once in PBS for 3 minutes. The slides were counterstained with Hematoxylin for 30 seconds at room temperature, and washed with running tap water. The slides were dried at room temperature for 30 minutes and coverslips were then d onto the slides using Faramount mounting media.

Results LY75_A1 showed positivity in FFPE Triple Negative breast cancer samples, where 77% of the sections showed positive staining and 55% exhibited robust (+++) staining.

Staining for LY75 in FF normal tissues was generally absent to low. Ductal epithelium of the breast, salivary gland, and pancreas exhibited marked low to te staining, and the spleen stained low ve. Therefore antibodies directed to LY75 may have utility as therapeutics and stics in some of the tested cancers and ly other cancer types showing expression of LY75.

Example 4: Efficacy of DM1-Conjugated Anti-LY75 Monoclonal Antibodies in HT-29 Cells 18544345_1 (GHMatters) P41668NZ00 Materials Cell stripper (Non-enzymatic cell dissociation) (MT056CI) from Fisher Scientific, PA, USA.

PBS pH 7.4 (1X) 28LS) from Fisher Scientific, PA, USA.

RPMI 1640 Media (MT041-CM) from Fisher Scientific, PA, USA.

Cell Titer Glo (G7572) from Promega, WI, USA.

Method Cells were iated using cell stripper and counted. 5e3 cells/well were spun down into a pellet (for suspension cells, more can be used depending on the doubling time of the cells, such as 10e3 cells/well). The pellet was resuspended in culture media to a concentration of 1e5 cells/mL. 50ul/well cell suspension was added to wells of a 96-well white sided, clear bottomed plate.

Antibodies were diluted and titrated to 8 points (3-fold ions) corresponding to concentrations between 0-20 nM (twice the test concentrations). Diluted dies or media (for untreated samples) (50ul/well) were added to the appropriate wells. Excess media (200ul/well) was added to the outside rows and columns of the plate to prevent evaporation. The plate was ted for 72h at 37C.

The plate was removed from the incubator and incubated at room temperature for 30 minutes. Meanwhile Cell Titer Glo solution was thawed. The plate was flicked and washed 1x with 100ul/well PBS (for suspension cells, plate is centrifuged first to pellet cells). 100ul/well PBS and 100ul Cell titer glo was added to each well and ated to mix. The plate was ted in the dark at room temperature for 15 minutes and visualized by microscopy to ensure efficient cell lysis occurred. The plate was then read on a Glomax luminometer.

Results The results depicted in Figure 3a show a subpopulation of antibodies, know to bind to LY75, which can induce cell kill of HT-29 cells. This suggests while dies can bind to LY75 only a few y efficacy when conjugated to DM1. Antibodies where then chosen from the subpopulation for further cytotoxic activity analysis.

Example 5: Efficacy of DM1-Conjugated and DM4-Conjugated Anti-LY75 Monoclonal Antibodies in Colorectal Cancer Cells Materials Cell stripper nzymatic cell iation) (MT056CI) from Fisher Scientific, PA, USA.

PBS pH 7.4 (1X) (SH30028LS) from Fisher ific, PA, USA.

RPMI 1640 Media (MT041-CM) from Fisher Scientific, PA, USA.

Cell Titer Glo (G7572) from Promega, WI, USA.

Method Cells were dissociated using cell stripper and counted. 5e3 cells/well were spun down into a pellet (for suspension cells, more can be used depending on the doubling time of the cells, such as 10e3 cells/well). The pellet was resuspended in culture media to a concentration of 1e5 cells/mL. 18544345_1 (GHMatters) P41668NZ00 50ul/well cell suspension was added to wells of a 96-well white sided, clear bottomed plate.

Antibodies were diluted and titrated to 8 points (3-fold titrations) corresponding to concentrations between 0-20 nM (twice the test concentrations). Diluted antibodies or media (for untreated samples) (50ul/well) were added to the appropriate wells. Excess media (200ul/well) was added to the outside rows and columns of the plate to prevent evaporation. The plate was incubated for 72h at 37C.

The plate was d from the incubator and incubated at room temperature for 30 minutes. Meanwhile Cell Titer Glo solution was thawed. The plate was flicked and washed 1x with 100ul/well PBS (for suspension cells, plate is centrifuged first to pellet cells). 100ul/well PBS and 100ul Cell titer glo was added to each well and triturated to mix. The plate was incubated in the dark at room temperature for 15 minutes and visualized by microscopy to ensure efficient cell lysis occurred. The plate was then read on a Glomax meter.

Results Figure 3b shows the cytotoxic ty of anti-LY75 antibodies conjugated to DM1 and DM4 towards HT-29 cells These results demonstrate an increase in cytotoxic activity proportional to antibody concentration and other anti-LY75 antibodies conjugated to a toxin (selected from Example Example 6: Efficacy of DM1-Conjugated and DM4-Conjugated Anti-LY75 Monoclonal Antibodies in ma Cell Lines Materials Cell stripper (Non-enzymatic cell dissociation) (MT056CI) from Fisher Scientific, PA, USA.

PBS pH 7.4 (1X) (SH30028LS) from Fisher Scientific, PA, USA.

RPMI 1640 Media CM) from Fisher Scientific, PA, USA.

Cell Titer Glo (G7572) from Promega, WI, USA.

Method Cells were dissociated using cell stripper and counted. 5e3 cells/well were spun down into a pellet (for suspension cells, more can be used depending on the doubling time of the cells, such as 10e3 cells/well). The pellet was ended in e media to a concentration of 1e5 mL. 50ul/well cell sion was added to wells of a 96-well white sided, clear bottomed plate.

Antibodies were diluted and ed to 8 points (3-fold titrations) corresponding to concentrations between 0-20 nM (twice the test trations). Diluted antibodies or media (for ted s) (50ul/well) were added to the appropriate wells. Excess media (200ul/well) was added to the outside rows and columns of the plate to prevent evaporation. The plate was incubated for 72h at 37C.

The plate was removed from the incubator and incubated at room temperature for 30 minutes. Meanwhile Cell Titer Glo solution was thawed. The plate was flicked and washed 1x with 100ul/well PBS (for suspension cells, plate is centrifuged first to pellet cells). 100ul/well PBS and 100ul Cell titer glo was added to each well and triturated to mix. The plate was ted in the dark 18544345_1 (GHMatters) P41668NZ00 at room temperature for 15 minutes and visualized by microscopy to ensure efficient cell lysis occurred. The plate was then read on a Glomax luminometer.

Results Figure 3c shows the cytotoxic activity of anti-LY75 dies conjugated to DM1 and DM4 towards RAJI cells. Figure 3d shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 towards Namalwa cells. Figure 3e shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 towards Karpas 299 cells. These results demonstrates an increase in cytotoxic ty proportional to antibody concentration and other anti-LY75 antibodies conjugated to DM1 and DM4 (selected from Example 1).

Example 7: cy of njugated and DM4-Conjugated Anti-LY75 onal Antibodies in Pancreatic Cancer Cell Lines Cell stripper (Non-enzymatic cell dissociation) (MT056CI) from Fisher Scientific, PA, USA.

PBS pH 7.4 (1X) 28LS) from Fisher Scientific, PA, USA.

RPMI 1640 Media (MT041-CM) from Fisher Scientific, PA, USA.

Cell Titer Glo (G7572) from Promega, WI, USA.

Method Cells were dissociated using cell stripper and counted. 5e3 cells/well were spun down into a pellet (for suspension cells, more can be used depending on the doubling time of the cells, such as 10e3 cells/well). The pellet was resuspended in culture media to a concentration of 1e5 cells/mL. 50ul/well cell sion was added to wells of a 96-well white sided, clear bottomed plate.

Antibodies were diluted and titrated to 8 points (3-fold ions) corresponding to trations between 0-20 nM (twice the test trations). Diluted antibodies or media (for untreated samples) (50ul/well) were added to the appropriate wells. Excess media (200ul/well) was added to the outside rows and columns of the plate to prevent evaporation. The plate was incubated for 72h at 37C.

The plate was removed from the incubator and incubated at room temperature for 30 minutes. Meanwhile Cell Titer Glo solution was thawed. The plate was flicked and washed 1x with 100ul/well PBS (for suspension cells, plate is centrifuged first to pellet cells). 100ul/well PBS and 100ul Cell titer glo was added to each well and triturated to mix. The plate was incubated in the dark at room temperature for 15 minutes and visualized by microscopy to ensure efficient cell lysis occurred. The plate was then read on a Glomax luminometer.

Results Figure 3f shows the xic activity of anti-LY75 antibodies conjugated to DM1 and DM4 towards BxPC3 cells. Figure 3g shows the cytotoxic activity of anti-LY75 antibodies ated to DM1 and DM4 towards HupT4 cells. Figure 3h shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 towards HPAFFII cells. These results demonstrates an increase in 18544345_1 (GHMatters) P41668NZ00 cytotoxic activity proportional to antibody concentration and other Y75 antibodies conjugated to DM1 and DM4 (selected from e 1). e 8: Efficacy of DM1-Conjugated and DM4-Conjugated Anti-LY75 Monoclonal Antibodies in Chronic Lymphocytic Leukaemia Cell Lines Materials Cell stripper (Non-enzymatic cell dissociation) (MT056CI) from Fisher Scientific, PA, USA.

PBS pH 7.4 (1X) (SH30028LS) from Fisher Scientific, PA, USA.

RPMI 1640 Media (MT041-CM) from Fisher Scientific, PA, USA.

Cell Titer Glo (G7572) from Promega, WI, USA.

Method Cells were dissociated using cell er and counted. 5e3 cells/well were spun down into a pellet (for suspension cells, more can be used depending on the ng time of the cells, such as 10e3 well). The pellet was resuspended in culture media to a concentration of 1e5 cells/mL. 50ul/well cell suspension was added to wells of a 96-well white sided, clear bottomed plate.

Antibodies were diluted and titrated to 8 points d titrations) corresponding to concentrations between 0-20 nM (twice the test concentrations). Diluted dies or media (for untreated samples) (50ul/well) were added to the appropriate wells. Excess media (200ul/well) was added to the outside rows and columns of the plate to prevent evaporation. The plate was incubated for 72h at 37C.

The plate was removed from the incubator and incubated at room temperature for 30 minutes. Meanwhile Cell Titer Glo solution was thawed. The plate was flicked and washed 1x with 100ul/well PBS (for suspension cells, plate is centrifuged first to pellet cells). 100ul/well PBS and 100ul Cell titer glo was added to each well and ated to mix. The plate was incubated in the dark at room temperature for 15 minutes and visualized by copy to ensure efficient cell lysis occurred. The plate was then read on a Glomax luminometer.

Results Figure 3i shows the xic activity of anti-LY75 antibodies conjugated to DM1 and DM4 towards EHEB cells. Figure 3j shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 towards Mec-1 cells. These results trates an increase in cytotoxic activity tional to antibody concentration and other anti-LY75 antibodies conjugated to DM1 and DM4 (selected from Example 1). e 9: Efficacy of DM1 -Conjugated and DM4-Conjugated Anti-LY75 Monoclonal Antibodies in Acute Monocytic Leukaemia Cell Lines Materials Cell stripper (Non-enzymatic cell dissociation) (MT056CI) from Fisher Scientific, PA, USA.

PBS pH 7.4 (1X) (SH30028LS) from Fisher Scientific, PA, USA.

RPMI 1640 Media (MT041-CM) from Fisher Scientific, PA, USA. 18544345_1 (GHMatters) P41668NZ00 Cell Titer Glo (G7572) from Promega, WI, USA.

Method Cells were dissociated using cell stripper and counted. 5e3 cells/well were spun down into a pellet (for suspension cells, more can be used depending on the doubling time of the cells, such as 10e3 cells/well). The pellet was resuspended in culture media to a tration of 1e5 cells/mL. 50ul/well cell suspension was added to wells of a l white sided, clear bottomed plate.

Antibodies were diluted and titrated to 8 points (3-fold titrations) corresponding to trations between 0-20 nM (twice the test concentrations). Diluted antibodies or media (for untreated samples) (50ul/well) were added to the appropriate wells. Excess media (200ul/well) was added to the outside rows and columns of the plate to prevent evaporation. The plate was incubated for 72h at 37C.

The plate was removed from the tor and incubated at room ature for 30 minutes. Meanwhile Cell Titer Glo solution was thawed. The plate was flicked and washed 1x with 100ul/well PBS (for suspension cells, plate is centrifuged first to pellet cells). 100ul/well PBS and 100ul Cell titer glo was added to each well and triturated to mix. The plate was incubated in the dark at room ature for 15 minutes and visualized by microscopy to ensure efficient cell lysis occurred. The plate was then read on a Glomax luminometer.

Results Figure 3k shows the cytotoxic activity of anti-LY75 dies conjugated to DM1 and DM4 towards AML-193 cells. These results demonstrates an increase in cytotoxic ty proportional to antibody concentration and other anti-LY75 antibodies conjugated to DM1 and DM4 (selected from Example 1).

Example 10: Efficacy of DM1-Conjugated and DM4-Conjugated Y75 Monoclonal Antibodies in Breast Cancer Cell Lines Materials Cell stripper (Non-enzymatic cell iation) -056CI) from Fisher Scientific, PA, USA.

PBS pH 7.4 (1X) (SH30028LS) from Fisher Scientific, PA, USA.

RPMI 1640 Media (MT041-CM) from Fisher Scientific, PA, USA.

Cell Titer Glo (G7572) from Promega, WI, USA.

Method Cells were dissociated using cell stripper and counted. 5e3 cells/well were spun down into a pellet (for suspension cells, more can be used depending on the doubling time of the cells, such as 10e3 cells/well). The pellet was resuspended in culture media to a concentration of 1e5 cells/mL. ell cell suspension was added to wells of a 96-well white sided, clear bottomed plate.

Antibodies were diluted and titrated to 8 points (3-fold titrations) corresponding to concentrations between 0-20 nM (twice the test trations). Diluted antibodies or media (for untreated samples) (50ul/well) were added to the appropriate wells. Excess media (200ul/well) was added to 18544345_1 (GHMatters) P41668NZ00 the outside rows and columns of the plate to prevent evaporation. The plate was incubated for 72h at 37C.

The plate was removed from the incubator and incubated at room temperature for 30 s. Meanwhile Cell Titer Glo on was thawed. The plate was flicked and washed 1x with 100ul/well PBS (for suspension cells, plate is centrifuged first to pellet cells). 100ul/well PBS and 100ul Cell titer glo was added to each well and triturated to mix. The plate was incubated in the dark at room ature for 15 s and visualized by microscopy to ensure efficient cell lysis occurred. The plate was then read on a Glomax luminometer.

Results Figure 3l shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 towards HCC 70 (ER ve, PR ve and Her2 negative) cells. Figure 3m shows the cytotoxic activity of anti-LY75 dies conjugated to DM1 and DM4 towards HCC 1806 (ER negative, PR negative and Her2 negative) cells. Figure 3n shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 towards MDA-MB-468 cells. These results demonstrates an increase in cytotoxic ty proportional to dy concentration.

Example 11: Efficacy of DM1-Conjugated and DM4-Conjugated Anti-LY75 Monoclonal Antibodies in Bladder Cancer Cell Lines Materials Cell er (Non-enzymatic cell dissociation) (MT056CI) from Fisher Scientific, PA, USA.

PBS pH 7.4 (1X) 28LS) from Fisher Scientific, PA, USA.

RPMI 1640 Media (MT041-CM) from Fisher Scientific, PA, USA.

Cell Titer Glo (G7572) from Promega, WI, USA.

Method Cells were iated using cell stripper and counted. 5e3 cells/well were spun down into a pellet (for suspension cells, more can be used depending on the doubling time of the cells, such as 10e3 cells/well). The pellet was resuspended in culture media to a concentration of 1e5 cells/mL. 50ul/well cell suspension was added to wells of a 96-well white sided, clear bottomed plate.

Antibodies were diluted and titrated to 8 points (3-fold titrations) corresponding to concentrations between 0-20 nM (twice the test trations). Diluted antibodies or media (for untreated samples) (50ul/well) were added to the appropriate wells. Excess media (200ul/well) was added to the outside rows and columns of the plate to prevent evaporation. The plate was incubated for 72h at 37C.

The plate was removed from the incubator and incubated at room temperature for 30 minutes. Meanwhile Cell Titer Glo solution was thawed. The plate was flicked and washed 1x with 100ul/well PBS (for suspension cells, plate is centrifuged first to pellet cells). 100ul/well PBS and 100ul Cell titer glo was added to each well and triturated to mix. The plate was incubated in the dark at room temperature for 15 minutes and ized by microscopy to ensure efficient cell lysis occurred. The plate was then read on a Glomax luminometer. 18544345_1 (GHMatters) P41668NZ00 Results Figure 3o shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 towards RT4 cells. Figure 3p shows the cytotoxic activity of anti-LY75 dies conjugated to DM1 and DM4 towards 5637 cells. Figure 3q shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 towards SW780 cells. These results demonstrate an increase in cytotoxic ty proportional to antibody tration.

Example 12: Efficacy of DM1-Conjugated and DM4-Conjugated Y75 Monoclonal Antibodies in Head and Neck Cancer Cell Lines Materials Cell stripper (Non-enzymatic cell dissociation) (MT056CI) from Fisher Scientific, PA, USA.

PBS pH 7.4 (1X) (SH30028LS) from Fisher ific, PA, USA.

RPMI 1640 Media (MT041-CM) from Fisher Scientific, PA, USA.

Cell Titer Glo (G7572) from Promega, WI, USA.

Method Cells were dissociated using cell stripper and counted. 5e3 cells/well were spun down into a pellet (for suspension cells, more can be used depending on the doubling time of the cells, such as 10e3 well). The pellet was ended in culture media to a concentration of 1e5 cells/mL. 50ul/well cell suspension was added to wells of a 96-well white sided, clear bottomed plate.

Antibodies were diluted and titrated to 8 points (3-fold titrations) corresponding to concentrations n 0-20 nM (twice the test concentrations). Diluted antibodies or media (for untreated samples) (50ul/well) were added to the appropriate wells. Excess media (200ul/well) was added to the outside rows and columns of the plate to prevent evaporation. The plate was incubated for 72h at 37C.

The plate was removed from the incubator and ted at room temperature for 30 minutes. Meanwhile Cell Titer Glo solution was thawed. The plate was d and washed 1x with 100ul/well PBS (for suspension cells, plate is centrifuged first to pellet cells). 100ul/well PBS and 100ul Cell titer glo was added to each well and ated to mix. The plate was incubated in the dark at room temperature for 15 s and visualized by microscopy to ensure efficient cell lysis occurred. The plate was then read on a Glomax luminometer.

Results Figure 3r shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 towards SCC-9 cells. These results demonstrate an increase in cytotoxic activity proportional to antibody concentration.

Example 13: Efficacy of DM1-Conjugated and DM4-Conjugated Anti-LY75 Monoclonal Antibodies in Oesophageal Cancer Cell Lines Materials Cell er (Non-enzymatic cell dissociation) (MT056CI) from Fisher Scientific, PA, USA. 18544345_1 (GHMatters) P41668NZ00 PBS pH 7.4 (1X) (SH30028LS) from Fisher Scientific, PA, USA.

RPMI 1640 Media (MT041-CM) from Fisher Scientific, PA, USA.

Cell Titer Glo (G7572) from Promega, WI, USA.

Method Cells were dissociated using cell stripper and counted. 5e3 cells/well were spun down into a pellet (for suspension cells, more can be used depending on the doubling time of the cells, such as 10e3 well). The pellet was resuspended in culture media to a concentration of 1e5 mL. 50ul/well cell suspension was added to wells of a 96-well white sided, clear bottomed plate.

Antibodies were d and titrated to 8 points (3-fold titrations) corresponding to trations between 0-20 nM (twice the test concentrations). Diluted antibodies or media (for untreated samples) (50ul/well) were added to the appropriate wells. Excess media (200ul/well) was added to the outside rows and columns of the plate to t evaporation. The plate was incubated for 72h at 37C.

The plate was removed from the incubator and incubated at room temperature for 30 minutes. Meanwhile Cell Titer Glo solution was . The plate was flicked and washed 1x with 100ul/well PBS (for suspension cells, plate is centrifuged first to pellet cells). 100ul/well PBS and 100ul Cell titer glo was added to each well and triturated to mix. The plate was incubated in the dark at room temperature for 15 minutes and visualized by microscopy to ensure efficient cell lysis occurred. The plate was then read on a Glomax luminometer.

Results Figure 3s shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 s OE 19 cells. These results demonstrate an increase in cytotoxic activity proportional to antibody tration.

Example 14: Efficacy of DM1-Conjugated and DM4-Conjugated Anti-LY75 onal dies in Ovarian Cancer Cell Lines Materials Cell stripper (Non-enzymatic cell iation) (MT056CI) from Fisher Scientific, PA, USA.

PBS pH 7.4 (1X) (SH30028LS) from Fisher Scientific, PA, USA.

RPMI 1640 Media (MT041-CM) from Fisher Scientific, PA, USA.

Cell Titer Glo (G7572) from a, WI, USA.

Method Cells were dissociated using cell stripper and counted. 5e3 cells/well were spun down into a pellet (for suspension cells, more can be used depending on the doubling time of the cells, such as 10e3 cells/well). The pellet was resuspended in culture media to a concentration of 1e5 cells/mL. 50ul/well cell sion was added to wells of a 96-well white sided, clear bottomed plate.

Antibodies were diluted and titrated to 8 points (3-fold titrations) corresponding to concentrations between 0-20 nM (twice the test concentrations). Diluted antibodies or media (for untreated samples) (50ul/well) were added to the appropriate wells. Excess media (200ul/well) was added to 18544345_1 (GHMatters) P41668NZ00 the outside rows and columns of the plate to prevent evaporation. The plate was incubated for 72h at 37C.

The plate was removed from the incubator and ted at room temperature for 30 minutes. Meanwhile Cell Titer Glo solution was thawed. The plate was flicked and washed 1x with 100ul/well PBS (for suspension cells, plate is centrifuged first to pellet . 100ul/well PBS and 100ul Cell titer glo was added to each well and triturated to mix. The plate was incubated in the dark at room temperature for 15 minutes and visualized by microscopy to ensure efficient cell lysis occurred. The plate was then read on a Glomax luminometer.

Results Figure 3t shows the cytotoxic activity of anti-LY75 antibodies ated to DM1 and DM4 towards OVCAR-3 cells. Figure 3u shows the xic activity of anti-LY75 antibodies ated to DM1 and DM4 towards SK-OV-3 cells. These results demonstrate an increase in cytotoxic activity proportional to antibody concentration.

Example 15: Efficacy of DM1-Conjugated and njugated Anti-LY75 Monoclonal Antibodies in Multiple Myeloma Cell Lines Materials Cell stripper (Non-enzymatic cell dissociation) (MT056CI) from Fisher Scientific, PA, USA.

PBS pH 7.4 (1X) (SH30028LS) from Fisher Scientific, PA, USA.

RPMI 1640 Media (MT041-CM) from Fisher Scientific, PA, USA.

Cell Titer Glo (G7572) from a, WI, USA.

Method Cells were dissociated using cell er and counted. 5e3 cells/well were spun down into a pellet (for suspension cells, more can be used depending on the doubling time of the cells, such as 10e3 cells/well). The pellet was resuspended in culture media to a concentration of 1e5 cells/mL. 50ul/well cell suspension was added to wells of a 96-well white sided, clear bottomed plate.

Antibodies were d and titrated to 8 points (3-fold titrations) corresponding to trations between 0-20 nM (twice the test concentrations). Diluted antibodies or media (for untreated samples) (50ul/well) were added to the riate wells. Excess media (200ul/well) was added to the outside rows and s of the plate to prevent evaporation. The plate was incubated for 72h at 37C.

The plate was removed from the incubator and incubated at room temperature for 30 minutes. Meanwhile Cell Titer Glo solution was . The plate was flicked and washed 1x with 100ul/well PBS (for suspension cells, plate is centrifuged first to pellet cells). 100ul/well PBS and 100ul Cell titer glo was added to each well and triturated to mix. The plate was incubated in the dark at room temperature for 15 minutes and visualized by microscopy to ensure efficient cell lysis occurred. The plate was then read on a Glomax luminometer.

Results 18544345_1 (GHMatters) P41668NZ00 Figure 3v shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 towards MOLP-8 cells. Figure 3w shows the xic activity of anti-LY75 antibodies conjugated to DM1 and DM4 towards RPMI8226 cells. These results demonstrate an se in cytotoxic activity proportional to antibody concentration.

Example 16: Efficacy of DM1-Conjugated and DM4-Conjugated Anti-LY75 Monoclonal Antibodies in Raji aft Models The efficacy of LY75_DM1 and LY75_DM4 were tested in subcutaneous Raji t’s lymphoma SCID mouse xenograft model.

Immunodeficient SCID mice were inoculated subcutaneously with Raji (human t’s lymphoma) tumour cells. Tumours were allowed to establish and mice were sorted into five treatment groups of 3-6 mice per group. When the mean tumour volume reached an e size of 129-132 mm3 per group, each group was treated with one of the following compounds, administered intravenously at the indicated dosages: Group 1 (Vehicle; phosphate buffered saline (PBS)); Group 2 (LY75_DM1; 10 mg/kg), Group 3 (Isotype control-DM1; 10 mg/kg), Group 4 (LY75_DM4; 5 mg/kg), Group 5 (isotype control-SPBDDM4; 5 mg/kg). A second dose was stered one week later.

Body weights (BW) were monitored, the mice were examined frequently for health and adverse side effects, and tumours were measured twice weekly. Mice were euthanized when their tumours d the tumour volume endpoint of 2000 mm3 or after 60 days, whichever came first. Efficacy was determined from tumour growth delay (TGD), the increase in median time-to-endpoint (TTE) and from logrank analysis of differences in Kaplan Meier survival curves in ADC-treated versus PBS- treat mice. The first five vehicle-treated control mice to reach endpoint were sampled for tumours that were processed by formalin fixation and paraffin embedded.

Results Figure 4a shows LY75_DM1 and LY75_DM4 each demonstrated significant anti-tumour activity and significantly extended survival in the Raji Burkitt’s ma SCID mouse xenograft model compared to controls; however, the 5 mg/kg LY75_DM4 doses were significantly more effective than the 10 mg/kg doses of LY75_DM1, resulting in 5 of 6 mice with complete but transient tumour regression. All treatments were well-tolerated and no clinical signs of ty were ed.

These data suggest the potential for ADCs directed towards LY75, for example LY75_DM1 and LY75_DM4, to provide clinical benefit in the treatment of human non-Hodgkin lymphoma cancer patients.

Example 17: Efficacy of njugated and DM4-Conjugated Anti-LY75 onal Antibodies in Namalwa Xenograft Models The cy of M1 and LY75_DM4 were tested in subcutaneous Namalwa Burkitt’s lymphoma SCID mouse aft model.

Immunodeficient SCID mice were inoculated subcutaneously with Namalwa (human t’s lymphoma) tumour cells. Tumours were allowed to establish and mice were sorted into five 18544345_1 (GHMatters) P41668NZ00 treatment groups of 6 mice per group. When the mean tumour volume reached an average size of 114 mm3 per group, each group was treated with one of the following compounds, administered intravenously at the ted dosages: Group 1 (Vehicle; phosphate buffered saline ; Group 2 (LY75_DM1; 10 mg/kg), Group 3 (Isotype control-DM1; 10 mg/kg), Group 4 (LY75_DM4; 5 mg/kg), Group 5 (isotype control-SPBDDM4; 5 mg/kg). Body weights (BW) were monitored, the mice were examined ntly for health and adverse side effects, and tumours were measured twice .

Mice were euthanized when their tumours reached the tumour volume endpoint of 2000 mm3 or after 60 days, whichever came first. Efficacy was ined from tumour growth delay (TGD), the increase in median time-to-endpoint (TTE), and from log rank analysis of differences in Kaplan Meier survival curves in ADC-treated versus PBS-treated mice. The first five vehicle-treated control mice to reach endpoint were d for s that were processed by formalin on and paraffin embedded.

Results Figure 4b shows LY75_DM1 and LY75_DM4 each demonstrated significant anti-tumour activity and survival extension in the a t’s lymphoma SCID mouse aft model ed to controls; however, the 5 mg/kg LY75_DM4 dose was significantly more effective than the 10 mg/kg dose of LY75_DM1, causing a brief ion in tumour . All treatments were well-tolerated and no clinical signs of toxicity were observed. These data suggest the potential for ADCs directed towards LY75, for example LY75_DM1 and LY75_DM4, to provide clinical benefit in the treatment of human non-Hodgkin lymphoma cancer patients.

Example 18: Efficacy of DM1-Conjugated and DM4-Conjugated Anti-LY75 Monoclonal Antibodies in Pancreatic Cancer Xenograft Models The efficacy of LY75_DM1 and LY75_DM4 were tested in the subcutaneous HPAFII pancreatic adenocarcinoma athymic nude mousexenograft model.

Immunodeficient athymic nude mice were inoculated subcutaneously with HPAFII (human pancreatic adenocarcinoma) tumor cells. Tumors were allowed to establish and mice were sorted into five treatment groups of 6 mice per group. When the mean tumor volume reached an average size of ~114 mm3/group, each group was treated with one of the ing nds, administered intravenously at the indicated dosages: Group 1 (Vehicle; phosphate ed saline (PBS)); Group 2 (LY75_DM1; 10 mg/kg), Group 3 (Isotype control-DM1; 10 mg/kg), Group 4 (LY75_DM4; 5 mg/kg), Group 5 (isotype control-SPBDDM4; 5 mg/kg). Body weights (BW) were monitored, the mice were examined frequently for health and adverse side effects, and tumors were measured thrice weekly.

Mice were euthanized when their tumors reached the tumor volume endpoint of 2000 mm3 or after 90 days, whichever came first. Efficacy was determined from the effect of treatment on tumor volume and from log rank analysis of differences in Kaplan-Meier survival curves in ADC-treated or PBS-treated mice. The tumors were sampled from vehicle-treated control mice and processed by formalin on and paraffin embedded.

Results 18544345_1 (GHMatters) P41668NZ00 Figure 4c shows LY75_DM1 and LY75_DM4 displayed significant and similarly potent antitumor activity and survival extension in the HPAFII nude mouse xenograft model compared to controls. All treatments were well-tolerated and no clinical signs of toxicity were observed. These data suggest the potential for ADCs directed towards LY75, for example M1 and LY75_DM4, to e clinical benefit in the treatment of human pancreatic cancer patients. e 19: Efficacy of DM1-Conjugated and DM4-Conjugated Anti-LY75 Monoclonal dies in Bladder Cancer Xenograft Models The efficacy of LY75_DM1 and LY75_DM4 were tested in the aneous SW780 human bladder carcinoma SCID mouse xenograft model. deficient c nude mice were inoculated subcutaneously with HPAFII (human pancreatic adenocarcinoma) tumor cells. Tumors were allowed to establish and mice were sorted into five treatment groups of 6 mice per group. When the mean tumor volume reached an average size of ~114 mm3/group, each group was treated with one of the following compounds, administered intravenously at the indicated dosages: Group 1 (Vehicle; phosphate buffered saline ; Group 2 (LY75_DM1; 1 mg/kg), Group 3 (LY75_DM1; 2.5 mg/kg), Group 4 (LY75_DM1; 5 mg/kg), Group 5 (LY75_DM4; 1 mg/kg) ), Group 6 (LY75_DM4; 2.5 mg/kg) ), Group 7 (LY75_DM4; 5 mg/kg) ), Group 8 (isotype control-SPBDDM4; 5 mg/kg). Body weights (BW) were monitored, the mice were examined frequently for health and adverse side effects, and tumors were measured thrice weekly.

Mice were euthanized when their tumors reached the tumor volume endpoint of 2000 mm3 or after 90 days, ver came first. Efficacy was determined from the effect of treatment on tumor volume and from log rank analysis of differences in Kaplan-Meier survival curves in ADC-treated or PBS-treated mice. The tumors were sampled from vehicle-treated control mice and processed by formalin fixation and paraffin ed.

Results Figure 4d shows LY75_DM1 and LY75_DM4 displayed significant and similarly potent antitumor activity and survival extension in the SW780 nude mouse xenograft model compared to controls. All ents were well-tolerated and no clinical signs of toxicity were observed. These data suggest the potential for ADCs directed towards LY75, for example M1 and LY75_DM4, to provide al benefit in the treatment of human bladder cancer patients.

Example 20: Efficacy of DM1-Conjugated and DM4-Conjugated Anti-LY75 Monoclonal Antibodies in Breast Cancer Xenograft Models The efficacy of M1 and M4 were tested in the subcutaneous MDA-MB-468 athymic nude mouse xenograft model.

Immunodeficient athymic nude mice were ated subcutaneously with -468 (human triple negative breast adenocarcinoma) tumour cells. Tumours were allowed to establish and mice were sorted into seven treatment groups of 10 mice per group. When the mean tumour volume reached an average size of 167 mm3 per group, each group was treated with one of the following 18544345_1 (GHMatters) P41668NZ00 nds, administered intravenously at the indicated dosages: Group 1 (Vehicle; 20 mM sodium succinate, pH 5.0, 6% ose, 0.04% polysorbate); Group 2 DM1; 5 mg/kg), Group 3 (LY75_DM1; 10 , Group 4 (LY75_DM4; 5 mg/kg), Group 5 (LY75_DM4; 2.5 mg/kg), Group 6 DM4; 1 , Group 7 (Isotype control-DM4; 5 mg/kg). Body weights (BW) were monitored, the mice were examined frequently for health and adverse side effects, and tumours were measured twice weekly. Mice were ized 82 days after tumour ation. Efficacy was determined from anti-tumour activity (mean tumour size in treatment group/mean tumour size in control group x 100) and the increase in mean time-to-endpoint (TTE) in ADC-treated versus eated mice. The five largest tumours in vehicle-treated control mice on day 71 post inoculation were sampled processed by formalin fixation and paraffin embedded.

Results Figure 4e shows LY75_DM1 and LY75_DM4 each demonstrated dramatic anti-tumour activity in the MDA-MB-468 nude mouse aft model compared to controls. Dose dependent activity was observed with LY75_DM4, where 2.5 and 5 mg/kg were much more potent than 1 mg/kg. At 5 mg/kg, LY75_DM1 and LY75_DM4 were similarly ive. Sustained regressions in mean tumour volume were observed for LY75_DM1 at 10 and 5 mg/kg and LY75_DM4 at 5 and 2.5 mg/kg. All treatments were well-tolerated and no clinical signs of toxicity were observed. These data suggest the potential for ADCs directed towards LY75, for example LY75_DM1 and LY75_DM4, to provide clinical benefit in the treatment of human triple ve breast cancer patients.

Example 21: Efficacy of DM1-Conjugated and DM4-Conjugated Anti-LY75 Monoclonal Antibodies in Colorectal Cancer Xenograft Models The efficacy of LY75_DM1 and LY75_DM4 were tested in the subcutaneous COLO205 colorectal adenocarcinoma athymic nude mouse xenograft model.

Immunodeficient athymic nude mice were inoculated subcutaneously with COLO205 (human colorectal adenocarcinoma) tumor cells. Tumors were allowed to establish and mice were sorted into five treatment groups of 6 mice per group. When the mean tumor volume reached an average size of 117 mm3 per group, each group was treated with one of the ing compounds, administered intravenously at the indicated dosages: Group 1 (Vehicle; phosphate buffered saline (PBS)); Group 2 LY75_DM1; 10 mg/kg), Group 3 (Isotype control-DM1; 10mg/kg), Group 4 (LY75_DM4; 5 mg/kg), Group 5 (Isotype control-DM4; 5 mg/kg). A second dose was administered twelve days after the first.

Body s (BW) were monitored, the mice were ed frequently for health and adverse side effects, and tumors were measured twice weekly. Mice were euthanized when their tumors reached the tumor volume endpoint of 1000 mm3 or after 60 days, whichever came first. Efficacy was determined from tumor growth delay (TGD), the increase in median time-to-endpoint (TTE) and from log rank analysis of differences in Kaplan Meier survival curves in ADC-treated versus PBS-treated mice. The first five vehicle-treated control mice to reach endpoint were sampled for tumors that were processed by in fixation and paraffin embedded. 18544345_1 (GHMatters) P41668NZ00 Figure 4f shows LY75_DM1 and LY75_DM4 exhibited similar modest anti-tumor activity and survival ion in the COLO205 colorectal adenocarcinoma nude mouse aft model compared to controls. All treatments were well-tolerated and no clinical signs of toxicity were observed. These data suggest the potential for ADCs directed towards LY75, for example M1 and LY75_DM4, to provide clinical benefit in the treatment of human colorectal cancer patients.

Example 22: Toxicity of DM1-Conjugated and njugated Anti-LY75 Monoclonal Antibodies in Cynomolgus Monkeys Six male monkeys were assigned to the study with 2 monkeys/group. Either vehicle (PBS), LY75_DM4 (cleavable) or LY75_DM1 (non-cleavable) was administered twice (on Day 1 and Day 29) by a 15-minute intravenous on at 0 mg/kg/dose (PBS, vehicle), 5 mg/kg/dose (LY75_DM4, cleavable) or 10 dose (LY75_DM1, non-cleavable). Blood samples were collected for toxicokinetic evaluations prior to dose initiation (Day 1), and 1, 2, 3, 7, 14, 21 and 28 days post each dose. Blood samples for al pathology analyses were collected prior to dose initiation (Day 1), and 1, 3, 7, 14, 21 and 28 days post each dose (28 days post the 1st dose was also served as the pre-dose time point for the 2nd dose). All study animals were euthanized and necropsied following the final blood collection on Day 57. The plasma separated from each blood draw was isolated, frozen and shipped to Oxford BioTherapeutics, Inc. to be ed for ADC concentration by ELISA.

Treatment-related clinical pathology findings included a mild regenerative anemia and transient decreases in the blood leukocyte profile most notably in phils counts. Anemia was observed in both animals treated with 5 mg/kg LY75_DM4 and in one of the two animals treated with mg/kg M1. Severe neutropenia with a nadir at one-week post dose and a rapid recovery in counts was observed in all s; the nadir in absolute neutrophil count was lower in M4 treated animals. There were no test article-related effects on the APTT and PT coagulation parameters. Serum chemistry changes included transient increases in AST, CK, LDH (in 1 of 2animals in each ent group) and globulin ing administration of 5 mg/kg LY75_DM4 and mg/kg LY75_DM1. In addition, a transient increase in the liver specific enzyme ALT was ed only in the LY75_DM4 treated animals. The short duration of and/or the magnitude of the increases in serum chemistry parameters suggest they were not adverse. There were no test-article related urinalysis gs. Upon examination at necropsy following a 4-week recovery period there were no treatment related gross pathology findings or changes in absolute and relative organ weights. Histopathology findings only in the thyroid gland (an alteration in the colloid morphology in follicles) and kidney (dilated tubules in the outer cortex), were graded as minimal severity; not associated with s in other study parameters; and, not adverse and of minimal toxicological significance. Conclusion: Repeated dose treatment with two doses of 5 mg/kg LY75_DM4 or 10 mg/kg LY75_DM1 was well tolerated in cynomologus monkeys. All treatment-related toxicity findings were reversible following a 4-week recovery period. 18544345_1 (GHMatters) P41668NZ00 Example 23: Epitope characterisation of LY75_A1 by competitive Fluorescence Activated Cell Sorting (FACS) g analysis Method COLO205 cells (ATCC, catalog # CCL-222) were detached from tissue culture flasks with Cell Stripper (Cellgro, catalog # MT056CI). Cells were washed and resuspended in FACS buffer (PBS + 2%FBS), neutralized with growth media, and counted. Cells were plated at 50,000 cells per well in a V Bottom 96-well plate. Cells were washed once with FACS buffer (PBS (Fisher, catalog # SH30028-03) + 2% FBS). An anti-LY75-mAb (Selected from Example 1) or LY75_A1 was added to wells starting at 250 nM and d serially 3 fold and applied to the relevant wells for 45 minutes on ice. Test wells that required single or multiple staining steps were left in FACS buffer as appropriate to ensure the final staining was completed aneously for all conditions tested. Two wells were left unstained in FACS buffer as controls.

After the incubation with blocking antibody, cells were washed twice in FACS buffer. The cells were resuspended in FACS buffer containing the anti-LY75-mAb ated to MCC-DM1 (1 nM) and ted on ice for 45 minutes. The cells were washed as above and resuspended in FACS buffer plus 1 ug/ml mouse anti-maytansine antibody and incubated in ice for 45 minutes. The cells were washed as above and resuspended in FACS buffer containing 2 ug/ml goat anti-mouse kappa RPE. The cells were incubated on ice for 45 s then washed as above. The cells were resuspended in FACS buffer at 200 ul per well. Mean fluorescence intensity of each sample was determined using a Guava EasyCyte Plus HT Flow Cytometer (96 well plate formats) and the raw data was ed using the Guava Cytosoft.

Results Figure 5a shows blocking with the anti-LY75-mAb-MCC-DM1 reduced the g of anti- LY75-mAb. Analysis of the binding of LY75_A1 to COLO205 cells showed that LY75_A1 is unable to block binding of anti-LY75-mAb-MCC-DM1 (see Figure 5b). It can therefore be determined that the Y75-mAb and LY75_A1 are non-competing antibodies and LY75_A1 recognizes a different and unique epitope of LY75 to that of other Y75 antibodies.

Example 24 Epitope characterization of LY75_A1 by peptide micro array assay.

Method The peptide microarray analysis was performed by LC Sciences, Houston TX, in brief the method comprised the following steps:- Contiguous 8mer peptides of LY75 protein having one amino acid overlap spanning residues 216 to 1666 of the full length LY75 protein were synthesized and lized on a rray chip. The chip comprised three panels such that the experiment was performed in in triplicate. The microarray was sed with LY75_A1 to identify the peptides to which the dy bound. The binding assay was performed under the following conditions:- The rray comprising the contiguous peptides in triplicate was washed with 1 mL of binding buffer at 4 ºC for 20 min. It was then incubated with 1 g/mL LY75_A1 in binding buffer (pH 7.0) at 4 ºC for 2 hrs. The array was again washed with 0.5 mL of washing buffer at 4 ºC for 30 min then 18544345_1 (GHMatters) P41668NZ00 ted with 25 ng/mL anti-human IgG Alexa 647 conjugate in binding buffer (pH 7.0) at 4 ºC for 1 hr. The array was again washed with 0.5 mL of washing buffer at 4 ºC for 30 min.

The array was then Scanned at 635 nm and PMT 500 and the signal Intensity was recorded. The e was classed as detectable if it was present in at least 2/3 legal duplicates. The average signal intensity of the replicates was reported as the final signal intensity.

As can be seen from Figure 6 antibody 1 showed specific g to a number of peptides located on the array. The maximum signal seen for LY75_A1 binding was 25000 (scale 1- 65535), with the average signal for all spots on the array being about 885. A signal intensity of 3000 was set as the background cut off point for non-specific binding. Based on the level of antibody binding signal intensity seen potential sequences g the epitope for LY75_A1 were identified.

These regions are shown in Figures 6a – 6j and as SEQ ID NOs: 22-31.

Example 25 LY75_A1 Peptide pull down assay Method 1.1 Pull Down Assay Recombinant LY75 protein was digested by on-bead tryptic proteolysis (Promega, US). The resulting digest peptides were recovered using a C18 capture column (Thermo Fisher Scientific). Purified peptides were then incubated with 200 µl of n A beads cross-linked with LY75A1 antibody overnight at 4oC. Next day the unbound peptides were collected and the beads were washed with 1 ml of PBS twice. The antibody bound peptides were eluted from beads by heating them at 90oC in 100 µl of PBS for 5 minutes. This elution step was repeated. 1.2 Mass spectrometry s were analysed by liquid chromatography-mass spectrometry using a Waters nanoACQUITY UPLC System fitted with a QUITY UPLC BEH 130 C18 column, 75 µm x 250mm (186003545) and a LTQ ap Velos (Thermo Fisher Scientific). Peptides were eluted with a 300nl/min gradient increasing from 3% to 35% acetonitrile over 120 min. Full-scan mass spectra were acquired at 60000 resolving power n 00 m/z mass range in the Orbitrap.

In each cycle, the twenty most intense peptides were selected for CID MS/MS scans in the linear ion trap with nanospray ion source fitted on the instrument. 1.3 Amino acid sequence analysis of peptide The raw data generated from the LTQ Orbitrap Velos was processed h the Mascot software (Matrix Science) which uses the Mowse algorithm (Curr Biol. 1993 Jun 1;3(6):327-3) to infer amino acids sequences from the peak lists by searching against a sequence database consisting of Ensembl (http://www.ensembl.org/index.html), IPI (www.ebi.ac.uk/IPI/IPIhuman.html) and rot (http://www.uniprot.org) along with contaminant protein sequences. Criteria for peptide identification included trypsin digestion, up to 2 missed cleavage sites and various biological 18544345_1 (GHMatters) P41668NZ00 and chemical modifications (oxidized methionine, cysteine modification by MMTS or iodoacetamide and phosphorylation of , threonine and tyrosine). Peptides ranked 1 with an expectation value of 0.05% or less, an ion score of 28 or higher were loaded into our OGAP database. 1.4 Discrimination of LY75 associated peptides The process to identify LY75 used the peptide sequences ed experimentally by mass spectrometry, as described above, of naturally occurring human proteins to identify and organize coding exons in the published human genome sequence. These mentally determined sequences were ed with the OGAP® database which was compiled by processing and ation of peptide masses, peptide signatures, ESTs and Public Domain Genomic Sequence Data as described in ational Patent Application WO2009/087462.

Results The results of the peptide pull down assay using antibody LY75_A1 are shown in Table 1 below and in Figure 7. Peptides which were fied in both peptide elutions 1a and 1b in the pull down assay and in the microarray assay were considered to be the most likely candidates for forming the epitope.

Table 1 ison of e microarray and peptide pull down experiments.

Peptide Identified by Microarray Assay Peptide Identified by Pull Down Assay Region 1 (aa609-618) - Region 2 (aa651-662) - Region 3 (aa761-780) GWHFYDDR (765-772) Region 4 (aa883-901) ISEWPIDDHFTYSR(877 to 890) FPVTFGEECLYMSAK(896-910) Region 5 (aa1029-1040) ELTYSNFHPLLVSGR(1030-1044) Region 6 (aa1077-1093) HFVSLCQK (1084-1091) Region 7 (aa1107-1118) QTLQNASETVK (1099-1109) Region 8 (aa1368-1378) - Region 9 (aa1518-1528) - Region 10 (aa1535-1554) - Table 1 shows that a number of overlapping LY75 peptide regions were identified in both the Peptide Microarray assay and in both elutions 1a and 1b the Peptide pull down assay. These regions are considered to be the most likely to contain the e ized by antibody LY75_A1 as they are bound by LY75_A1 tested by both techniques employed. 18544345_1 (GHMatters) P41668NZ00 ey Docket No. 069801-5011 OB00037 and OB00045

Claims (24)

1. An isolated antibody or an antigen-binding portion thereof that binds to LY75, said antibody comprising: a) a heavy chain variable region comprising: i) a first vhCDR sing SEQ ID NO: 5; ii) a second vhCDR comprising SEQ ID NO: 6; and iii) a third vhCDR comprising SEQ ID NO: 7; and b) a light chain variable region comprising: i) a first vlCDR comprising SEQ ID NO: 8; ii) a second vlCDR comprising SEQ ID NO: 9; and iii) a third vlCDR sing SEQ ID NO: 10.

2. The isolated antibody or an antigen-binding portion thereof according to claim 1, comprising a heavy chain having at least 80%, 85%, 90%, 95% or 99% amino acid sequence identity to SEQ ID NO: 1 and a light chain having at least 80%, 85%, 90%, 95% or 99% amino acid sequence ty to SEQ ID NO: 2.

3. The isolated dy or an antigen-binding portion thereof according to claim 1 or claim 2, further comprising a covalently-attached moiety.

4. The isolated antibody or an antigen-binding portion thereof according to claim 3, wherein said moiety is a drug.

5. The isolated antibody or an antigen-binding portion thereof according to claim 4, wherein said drug is selected from the group consisting of a maytansinoid, a dolastatin, a hemiasterlin, an auristatin, a trichothecene, a eamicin, CC1065 and tives thereof.

6. The isolated antibody or an antigen-binding portion thereof according to claim 4 or claim 5, wherein said drug is a maytansinoid selected from the group ting of DM4 and DM1.

7. The isolated antibody according to claim 1 or claim 2, wherein said antibody induces antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC). DB2/ 22752268.1 79 18544345_1 ters) P41668NZ00

8. The isolated antibody according to claim 7, wherein said antibody is an engineered antibody having increased binding to Fc receptors and/or increased potency for ADCC, and/or a bispecific antibody.

9. A pharmaceutical composition sing an dy or antigen binding portion thereof ing to any one of claims 1 to 8, together with one or more pharmaceutically-acceptable diluents, excipients or carriers.

10. A nucleic acid encoding a heavy chain variable region of the dy or an antigenbinding portion thereof according to any one of the claims to 1 to 8.

11. A nucleic acid encoding a light chain variable region of the antibody or an antigen-binding portion thereof according to any one of claims 1 to 8.

12. An expression vector comprising the nucleic acid according to claim 10 or claim 11 operably linked to one or more regulatory elements.

13. An expression vector comprising a nucleic acid ng a heavy chain variable region operably linked to one or more regulatory elements and a nucleic acid encoding a light chain variable region operably linked to one or more regulatory elements, wherein the heavy chain variable region and the light chain variable region are each of the antibody or an antigen-binding portion f according to any one of the claims to 1 to 8.

14. An isolated or non-human host cell comprising an expression vector according to claim

15. An ed or non-human host cell comprising a first expression vector sing a nucleic acid encoding a heavy chain variable region operably linked to one or more regulatory elements, and a second expression vector comprising a nucleic acid encoding a light chain variable region operably linked to one or more regulatory elements wherein the heavy chain variable region and the light chain variable region are each of the antibody or an n-binding portion thereof according to any one of the claims to 1 to 8.

16. A method of making an antibody or an antigen-binding n thereof, comprising culturing a host cell according to claim 14 or 15 under conditions where the antibody or an antigen-binding portion thereof is expressed and optionally isolating the dy or an antigenbinding portion thereof. 18544345_1 (GHMatters) P41668NZ00

17. Use of the antibody or an antigen-binding portion thereof ing to any one of claims 1 to 8 in the manufacture of a medicament for treating cancer.

18. The use ing to claim 17, wherein the antibody or antigen-binding portion thereof is internalized by a cell expressing LY75.

19. The use according to claim 17 or 18, wherein the antibody or antigen-binding portion comprises a covalently attached drug conjugate.

20. The use according to claim 19, wherein the covalently ed drug conjugate is a maytansinoid, preferably DM4.

21. The use according to claim 17, wherein the antibody induces antibody-dependent cellmediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC).

22. The use ing to any one of claims 17 to 21, wherein said cancer is ed from the group consisting of pancreatic cancer, ovarian cancer, breast cancer, colorectal cancer, esophageal cancer, skin cancer, thyroid cancer, lung cancer, kidney cancer, liver cancer, head and neck cancer, bladder cancer, c cancer, leukaemia, myeloma, and lymphoma.

23. The use according to claim 22, wherein said mia is acute myeloid leukaemia or c lymphocytic leukaemia, said myeloma is multiple myeloma and said lymphoma is diffuse large B-cell lymphoma (DLBCL), B-Cell Lymphoma, Follicular Lymphoma, Mantle Cell Lymphoma, Lymphoma of Mucosa-Associated Lymphoid Tissue , T-Cell/Histiocyte-Rich B-Cell Lymphoma, Burkitt’s Lymphoma, Lymphoplasmacytic Lymphoma, Small Lymphocytic Lymphoma, Marginal Zone Lymphoma, T Cell Lymphoma, Peripheral T-Cell ma, Anaplastic Large Cell Lymphoma and mmunoblastic T-Cell Lymphoma.

24. The use according to claim 22 or 23, wherein said cancer is selected from the group consisting of r cancer, pancreatic cancer, triple-negative breast cancer and DLBCL. 18544345_1 (GHMatters) P41668NZ00 SEQ ID No: 11 EVQAVESGGGLVKPGGSLRESCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTT SEQ ID NO: 1 EVQAVESGGGLVKPGGSLRESCAASGFTYSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTT SEQ ID No: 12 777777777777777777777777777777777777777777777777777777777777 ****************************:************k**************k*** SEQ ID No: 11 KGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTTTVT777777777777777 SEQ ID No: 1 DYAAPVQGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTIFGVVSFDYWGQGTLVTVSS SEQ ID No: 12 77777777777777777777777777777777777777777777YFDYWGQGTLVTVSS ******:******************************** ***********k**