EA042216B1 - PHARMACEUTICAL COMBINATIONS CONTAINING ANTIBODIES TO LY75 - Google Patents

Description

Introduction

The invention generally relates to the fields of immunology and molecular biology. More specifically, this document provides a pharmaceutical combination containing (A) antibodies or antigennegative parts directed against LY75, and (B) a second anticancer agent; the use of a pharmaceutical combination; and a method for treating diffuse large B-cell lymphoma (DLBCL).

Background of the invention

Types of leukemia and lymphoma belong to a large group of tumors that affect the blood, bone marrow and lymphoid system; they are known as tumors of the hematopoietic and lymphoid tissues.

Lymphoma is a group of blood cell tumors that develop from lymphocytes. Signs and symptoms may include swollen lymph nodes, fever, sweating, unintentional weight loss, itching, and feeling tired all the time. There are a number of subtypes of lymphoma: the two main groups of lymphomas are Hodgkin's lymphomas (HL) and non-Hodgkin's lymphomas (NHL). The World Health Organization (WHO) includes two other groups as types of lymphoma: multiple myeloma and immunoproliferative diseases. Approximately 90% of lymphomas are non-Hodgkin's lymphomas.

Leukemia is a group of cancers that usually start in the bone marrow and result in a large number of abnormal white blood cells. Symptoms may include bleeding and bruising problems, feeling tired, having a fever, and an increased risk of infections. These symptoms are due to a lack of normal blood cells. Diagnosis is usually made through blood tests or bone marrow biopsy. There are four main types of leukemia: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), as well as a number of less common types.

Treatment of types of leukemia and lymphoma may include one or more of chemotherapy, radiation therapy, targeted therapy, and surgery (and bone marrow transplantation in the case of types of leukemia). The success of leukemia treatment depends on the type of leukemia and the age of the patient. The outcome of treatment for lymphoma depends on the subtype, with some being curable and in most cases treatment increasing survival.

A number of chemotherapeutic agents have previously been used to treat types of leukemia, including prednisone, vincristine, anthracyclines, L-asparaginase, cyclophosphamide, methotrexate, 6-mercaptopurine, fludarabine, pentostatin, and cladribine. Chemotherapeutic agents for treating types of lymphoma include cyclophosphamide, hydroxydaunorubicin (also known as doxorubicin or adriamycin), oncovin (vincristine), prednisone, prednisolone, bleomycin, dacarbazine, etoposide, and procarbazine.

Combination chemotherapy involves treating a patient with two or more different drugs at the same time. Medicines may differ in their mechanism of action and side effects. The greatest advantage of this approach is to minimize the chances of developing resistance to any one agent. In addition, drugs can often be administered at lower doses, reducing toxicity. Combination therapies for the treatment of Hodgkin's disease include MOPP (mustargen, vincristine, procarbazine, prednisolone) and ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine). Types of combination therapy for the treatment of non-Hodgkin's lymphoma include CHOP (cyclophosphamide, doxorubicin, vincristine, prednisolone). In view of the number of drugs that are known due to use in the treatment of leukemia and lymphoma types, the number of permutations and combinations of possible drug treatments is definitely large. In addition, the aforementioned combination therapies do not involve antibodies.

There remains, however, a need for new treatments for the types of leukemia and lymphoma, and in particular for effective combination therapies.

Lymphocyte antigen 75 acts as an endocytic receptor, directing captured antigens from the extracellular space to a specialized antigen-processing compartment, and is believed to cause a decrease in B-lymphocyte proliferation. Expression of lymphocyte antigen 75 has been observed in pancreatic, ovarian, breast, colon and rectum, esophagus, skin, thyroid, and lung (non-small cell) cancers, as well as in multiple myeloma and many different subtypes of lymphoma and leukemia. WO 2009/061996 discloses isolated monoclonal antibodies that bind to human DEC-205 (LY75) and related antibody compositions and molecules. Also disclosed are pharmaceutical compositions containing antibodies, as well as therapeutic and diagnostic methods for using antibodies. WO 2008/104806 discloses affinity reagents capable of binding to LY75 for use in the treatment or prevention of cancer. WO 2015/052537 discloses specific isolated antibodies capable of binding to LY75 and their use in the treatment of various forms of cancer.

Rituximab is a monoclonal antibody to the CD20 protein, which is widely expressed on B cells (Oncogene (2003 Oct 20), Smith MR, Rituximab (monoclonal anti-CD20 antibody): mechanisms of action and resistance, 22(47):7359-68 ). Rituximab destroys both normal and malignant

- 1 042216 pure B cells that have CD20 on their surface and are therefore used to treat diseases that are characterized by the presence of too many B cells, overactive B cells or dysfunctional B cells. Rituximab has previously been used to treat a number of autoimmune diseases and some types of cancer, including rheumatoid arthritis, idiopathic thrombocytopenic purpura, pemphigus vulgaris, multiple sclerosis, systemic lupus erythematosus, non-Hodgkin's lymphoma, chronic inflammatory demyelinating polyneuropathy, chronic lymphocytic leukemia, and types of autoimmune anemia.

Ibrutinib (Imbravica) is a small molecule drug that permanently binds to Bruton's tyrosine kinase (BTK), which plays an important role in B cells. Ibrutinib has previously been used to treat B-cell cancers such as mantle cell lymphoma, chronic lymphocytic leukemia, and Waldenström's macroglobulinemia.

Combinations of (i) certain anti-LY75 antibodies with rituximab and (ii) certain anti-LY75 antibodies with ibrutinib have now been found to exhibit synergistic results in the treatment of types of lymphoma.

Brief description of the invention

In one aspect, the present invention provides a pharmaceutical combination comprising (A) (i) an anti-LY75 antibody or antigen-binding portion thereof, said antibody or portion comprising

a) a heavy chain variable region containing

i) a first vhCDR containing SEQ ID NO: 5;

ii) a second vhCDR containing SEQ ID NO: 6; and iii) a third vhCDR containing SEQ ID NO: 7; And

b) a light chain variable region containing

i) a first vlCDR containing SEQ ID NO: 8;

ii) a second vlCDR containing SEQ ID NO: 9; and iii) a third vlCDR containing SEQ ID NO: 10; or (ii) an anti-LY75 antibody or antigen-binding portion thereof, wherein said antibody or portion contains

a) a heavy chain variable region containing

i) a first vhCDR containing SEQ ID NO: 5;

ii) a second vhCDR containing SEQ ID NO: 6; and iii) a third vhCDR containing SEQ ID NO: 7; And

b) a light chain variable region containing

i) a first vlCDR containing SEQ ID NO: 8;

ii) a second vlCDR containing SEQ ID NO: 9; and iii) a third vlCDR comprising SEQ ID NO: 10, wherein any one or more of the above SEQ ID NOs independently comprise one or two amino acid substitutions, wherein the anti-LY75 antibody or antigen-binding portion thereof is covalently attached to a drug, wherein said drug is is maytansinoid drug 1 (DM1) or maytansinoid drug 4 (DM4); and (B) rituximab, wherein the pharmaceutical combination is in the form of a combined preparation suitable for simultaneous, separate or sequential use in the treatment of diffuse large B-cell lymphoma (DLBCL).

In one embodiment, the LY75 antibody, or antigen-binding portion thereof, 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 variable region. a chain containing 1, 2 or 3 CDRs selected from the group consisting of CDRs containing SEQ ID NOs: 8, 9 and 10.

In some embodiments, anti-LY75 antibodies bind to LY75 (SEQ ID NO: 15) and are capable of being internalized by a cell expressing LY75.

In another embodiment, an anti-LY75 antibody contains complementarity determining regions (CDRs) or variable regions (VRs) of the heavy and/or light chains of the specific antibody described herein (eg, referred to herein as LY75_A1). Accordingly, in one embodiment, the anti-LY75 antibody comprises the heavy chain variable region (VH) CDR1, CDR2 and CDR3 domains of the LY75_A1 antibody having the sequence shown under SEQ ID NO: 1 and/or the light chain variable region CDR1, CDR2 and CDR3 domains ( VL) LY75_A1 having the sequence shown under SEQ ID NO: 2.

In another embodiment, anti-LY75 antibodies bind to human LY75 and contain a heavy chain variable region comprising SEQ ID NO: 1 and/or conservative sequence modifications thereof. The antibody may further comprise a light chain variable region comprising SEQ ID NO: 2 and/or conservative sequence modifications thereof.

In a further embodiment, anti-LY75 antibodies bind to human LY75 and comprise a heavy chain variable region and a light chain variable region comprising the amino acid sequences shown under SEQ ID NO: 1 and/or 2, respectively, and conservative sequence modifications thereof.

Antibodies containing heavy and light chain variable regions characterized by 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 greater sequence identity with any from the above sequences are also included in the present invention. Intermediate ranges of the above values, such as heavy and light chain variable regions having at least 80-85%, 85-90%, 90-95% or 95-100% sequence identity with any of the above sequences, are also intended to be encompassed by the present invention.

In one embodiment, an anti-LY75 antibody comprises a heavy chain variable region comprising SEQ ID NO: 1 or a sequence of 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, the anti-LY75 antibody comprises a light chain variable region comprising SEQ ID NO: 2 or a sequence of at least 80%, at least 85%, at least 90%, at least 91%, at least 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 anti-LY75 antibody contains a heavy chain framework region containing an amino acid sequence of 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 heavy chain variable region framework of SEQ ID NO: 1 shown under SEQ ID NO: 16, 17, 18, and 19. In another embodiment, the anti-LY75 antibody comprises a light chain framework region containing an amino acid sequence by 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 light chain variable region framework of SEQ ID NO: 2 shown under SEQ ID NO : 20, 21, 22 and 23.

In one embodiment, an anti-LY75 antibody competes for binding to LY75 with an antibody containing heavy and/or light chain variable regions containing the amino acid sequences respectively listed under SEQ ID NOs: 1 and 2, or amino acid sequences, by 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 them. In another embodiment, an anti-LY75 antibody competes for binding to LY75 with an antibody containing heavy and/or light chain variable regions containing the amino acid sequences shown under SEQ ID NOs: 1 and 2 (LY75_A1).

Other antibodies of the present invention bind to the same epitope or epitope on LY75 recognized by the antibodies described herein. In another specific embodiment, the antibody binds to an epitope on LY75 recognized by an antibody containing heavy and/or light chain variable regions containing amino acid sequences respectively listed under SEQ ID NOs: 1 and 2, or amino acid sequences, by at least 80%. identical to them. In another embodiment, the antibody binds to an epitope on LY75 recognized by an antibody containing heavy and/or light chain variable regions containing the amino acid sequences shown under SEQ ID NOs: 1 and 2 (LY75_A1).

In a further embodiment, anti-LY75 antibodies specifically bind to one or more, for example 2, 3, 4, 5, 6, 7, 8, 9, or 10, peptide(s) selected from the group consisting of SEQ ID nO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37, or fragments thereof, where these fragments contain 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 anti-LY75 antibodies 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, where said fragments contain 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 at least 10 contiguous amino acids. In a further embodiment, the epitope recognized by the antibodies

- 3 042216 to LY75, contains one or more peptides, for example two or three peptides selected from the group consisting of SEQ ID NO: 30, 36 and 37, or fragments thereof, where these fragments contain 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 an additional embodiment, the anti-LY75 antibodies contain different CDRs compared to the parent antibodies described herein. Thus, the present invention provides variants of antibodies containing variants of the variable regions of the original antibody, where the original antibody contains the first vhCDR containing SEQ ID NO: 5, the second vhCDR containing SEQ ID NO: 6, the third vhCDR containing SEQ ID NO: 7 , the first vlCDR containing SEQ ID nO: 8, the second vlCDR containing SEQ ID NO: 9 and the third vlCDR containing SEQ ID NO: 10, and where the antibody variant in the aggregate has 1, 2, 3, 4, 5 or 6 amino acids substitutions in a set of first vhCDR, second vhCDR, third vhCDR, first vlCDR, second vlCDR, and third vlCDR, where 1-4, 1-3, or 1-2 substitutions are particularly useful, and where the antibody retains specific binding to LY75.

All antibodies disclosed herein may be full length antibodies, for example, any of the following isotypes: IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgA secretory component, IgD, and IgE. Alternatively, antibodies may be fragments such as an antigen-binding portion or a single chain antibody (e.g., Fab, F(ab') ₂ , Fv, single chain Fv fragment, isolated complementarity determining region (CDR), or a combination of two or more isolated CDR). The antibodies can be any type of antibody, including but not limited to human, humanized, and chimeric antibodies.

In other embodiments, the anti-LY75 antibodies are in the form of an immunoconjugate (ie, additionally contain a covalently attached component).

In one embodiment, an anti-LY75 antibody comprises a heavy chain variable region and a light chain variable region encoded by nucleic acid sequences comprising SEQ ID NOs: 3 and 4, respectively, or nucleic acid sequences characterized by at least 85, 86, 87, 88, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the above nucleic acid sequences or sequences other than SEQ ID NOs: 3 and 4 due to the degeneracy of the genetic code.

In one embodiment, the anti-CD20 antibody is a chimeric mouse/human antibody, a humanized antibody, or a human antibody. Preferably, the anti-CD20 antibody is rituximab.

In another aspect of the present invention, there are sets of expression vectors containing nucleic acids encoding the variable regions of the heavy and/or light chains of the antibodies described herein, operably linked to one or more regulatory elements.

In a preferred embodiment, the host cell contains expression vectors containing nucleic acids encoding (i) the heavy chain of an antibody to LY75 or an antigen-binding portion thereof;

(ii) the light chain of an anti-LY75 antibody or antigen-binding portion thereof;

(iii) the heavy chain of an anti-CD20 antibody or antigen-binding portion thereof; and (iv) a light chain of an anti-CD20 antibody or antigen-binding portion thereof.

In a further aspect, a method for treating diffuse large B-cell lymphoma (DLBCL) in a patient is provided, comprising the simultaneous, sequential or separate administration to a patient in need thereof of a therapeutically effective amount of components (A) and (B) of a pharmaceutical combination of the present invention.

In a further aspect of the present invention, there is provided a pharmaceutical combination of the present invention for use in the treatment of diffuse large B-cell lymphoma (DLBCL).

Also provided is the use of components (A) and (B), as defined herein, in the manufacture of a pharmaceutical combination for simultaneous, separate or sequential use in the treatment of diffuse large B-cell lymphoma (DLBCL).

Other features and advantages of the present invention will become apparent from the following detailed description and claims.

Brief description of graphic materials

In FIG. 1 shows the heavy chain alignment of LY75_A1 (SEQ ID NO: 1), human VH 3-15 germline (SEQ ID NO: 11) and human JH4 germline (SEQ ID NO: 12). The LY75_A1 heavy chain CDR regions are underlined.

In FIG. 2 shows the light chain alignment of LY75_A1 (SEQ ID NO: 2), human VK 012 germline (SEQ ID NO: 13) and human JK4 germline (SEQ ID NO: 14). The LY75_A1 light chain CDR regions are underlined.

In FIG. 3a shows the cytotoxic activity of DM1-conjugated anti-LY75 monoclonal antibodies against HT-29 and shows that while most antibodies bind to

- 4 042216

LY75, only some of them are effective.

In FIG. 3b shows the cytotoxic activity of DM1 or DM4-conjugated anti-LY75 antibodies against HT-29.

In FIG. 3c shows the cytotoxic activity of DM1 or DM4-conjugated anti-LY75 antibodies against RAJI cells.

In FIG. 3d shows the cytotoxic activity of DM1 or DM4-conjugated anti-LY75 antibodies against Namalwa cells.

In FIG. 3e shows the cytotoxic activity of DM1 or DM4-conjugated anti-LY75 antibodies against Karpas 299 cells.

In FIG. 3f shows the cytotoxic activity of DM1 or DM4-conjugated anti-LY75 antibodies against BxPC3 cells.

In FIG. 3g shows the cytotoxic activity of DM1 or DM4-conjugated anti-LY75 antibodies against HupT4 cells.

In FIG. 3h shows the cytotoxic activity of DM1 or DM4-conjugated anti-LY75 antibodies against HPAFFII cells.

In FIG. 3i shows the cytotoxic activity of DM1 or DM4-conjugated anti-LY75 antibodies against EHEB cells.

In FIG. 3j shows the cytotoxic activity of DM1 or DM4-conjugated anti-LY75 antibodies against Mec-1 cells.

In FIG. 3k shows the cytotoxic activity of DM1 or DM4-conjugated anti-LY75 antibodies against AML-193 cells.

In FIG. 3l shows the cytotoxic activity of DM1 or DM4-conjugated anti-LY75 antibodies against HCC 70 cells.

In FIG. 3m shows the cytotoxic activity of DM1 or DM4-conjugated anti-LY75 antibodies against HCC 1806 cells.

In FIG. 3n shows the cytotoxic activity of DM1 or DM4-conjugated anti-LY75 antibodies against MDA-MB-468 cells.

In FIG. 3o shows the cytotoxic activity of DM1 or DM4-conjugated anti-LY75 antibodies against RT4 cells.

In FIG. 3p shows the cytotoxic activity of DM1 or DM4-conjugated anti-LY75 antibodies against 5637 cells.

In FIG. 3q shows the cytotoxic activity of DM1 or DM4-conjugated anti-LY75 antibodies against SW780 cells.

In FIG. 3r shows the cytotoxic activity of DM1 or DM4-conjugated anti-LY75 antibodies against SCC-9 cells.

In FIG. 3s shows the cytotoxic activity of DM1 or DM4-conjugated anti-LY75 antibodies against OE 19 cells.

In FIG. 3t shows the cytotoxic activity of DM1 or DM4-conjugated anti-LY75 antibodies against OVCAR-3 cells.

In FIG. 3u shows the cytotoxic activity of DM1 or DM4-conjugated anti-LY75 antibodies against SK-OV-3 cells.

In FIG. 3v shows the cytotoxic activity of DM1 or DM4-conjugated anti-LY75 antibodies against MOLP-8 cells.

In FIG. 3w shows the cytotoxic activity of DM1 or DM4-conjugated anti-LY75 antibodies against RPMI8226 cells.

In FIG. 4a shows the efficacy of DM1 or DM4-conjugated anti-LY75 antibodies against Burkitt's lymphoma Raji cells in a SCID mouse xenograft model.

In FIG. 4b shows the efficacy of anti-LY75 antibodies conjugated to either DM1 or DM4 on Namalwa cells of Burkitt's lymphoma in a xenograft model in mice with SCID.

In FIG. 4c shows the efficacy of DM1 or DM4-conjugated anti-LY75 antibodies against pancreatic adenocarcinoma HPAFII cells in a nude mouse xenograft model.

In FIG. 4d shows the efficacy of DM1 or DM4-conjugated anti-LY75 antibodies against human bladder carcinoma SW780 cells in a SCID mouse xenograft model.

In FIG. 4e shows the efficacy of DM1 or DM4-conjugated anti-LY75 antibodies against MDA-MB-468 cells in a nude mouse xenograft model.

In FIG. 4f shows the efficacy of DM1 or DM4-conjugated anti-LY75 antibodies against colorectal adenocarcinoma COLO205 cells in a nude mouse xenograft model.

In FIG. 5a shows the competitive binding of an anti-LY75 mAb and an anti-LY75 mAb conjugated to MCC-DM1.

- 5 042216

In FIG. 5b shows non-competitive binding of LY75_A1 and anti-LY75 mAb conjugated to

MCC-DM1.

In FIG. 6a-6j show graphical representations of LY75_A1 antibody binding to peptides.

LY75 in a peptide microarray.

In FIG. 7 shows the amino acid sequence alignment of the peptides to which the LY75_A1 antibody binds in both microarray peptide and coprecipitation peptide assays. The isolated peptides likely form an epitope recognized by the LY75_A1 antibody.

In FIG. 8a and 8b show the antiproliferative effect of different (nM) doses of LY75_DM4 as a single treatment or in combination with rituximab on two different ABC-DLBCL cell lines (TMD8 and HBL1). (O=Chow-Talalai additivity index). Fig. 8a: median CI=0.27. Fig. 8b: median CI=0.57.

In FIG. 9 shows the antiproliferative effect of various (nM) doses of LY75_DM4 as a single treatment or in combination with ibrutinib on HBL-1 (ABC-DLBCL) cell lines. (O=Chow-Talalai additivity index). Median CI=0.24.

Detailed description of the invention

The present invention relates to pharmaceutical combinations containing components (A) and (B) as defined herein, where the pharmaceutical combination is in the form of a combined preparation for simultaneous, separate or sequential use. Component (A) refers to an antibody to LY75 as defined herein. Component (B) refers to either (i) an anti-CD20 antibody as defined herein, or (ii) ibrutinib or a pharmaceutically acceptable salt thereof.

One example of a LY75 protein is shown under SEQ ID NO: 15 herein. The terms anti-LY75 antibody and LY75 antibody are used interchangeably herein.

The LY75 antibodies disclosed herein can be internalized upon contact with cells expressing the LY75 receptor. As discussed herein, the LY75 receptor is overexpressed and/or differentially expressed in certain cancer cells, including but not limited to leukemia, preferably acute myeloid leukemia or chronic lymphocytic leukemia, lymphoma, preferably DLBCL, B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, mucosal lymphoid tissue (MALT) lymphoma, T-cell/histiocyte-rich B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, small lymphocytic lymphoma, marginal zone cell lymphoma, T-cell lymphoma, peripheral T-cell lymphoma , anaplastic large cell lymphoma, and angioimmunoblastic T-cell lymphoma.

Therefore, if the anti-LY75 antibodies disclosed herein are drug-conjugated (sometimes referred to herein as antibody-drug conjugates or ADCs), internalization of these ADC molecules by cancer cells results in cell death and thus tumor treatment.

Anti-LY75 antibodies have specific structural features, such as CDR regions with specific amino acid sequences. This document describes a set of CDRs that can form an affinity reagent, such as an antibody that is characterized by binding to LY75.

Thus, the present invention provides antibodies, preferably isolated antibodies (which, as summarized below, include a wide variety of well-known structures, derivatives, mimetics and conjugates of antibodies), nucleic acids encoding combinations of antibodies, host cells used to obtain combinations antibodies, methods for producing combinations of antibodies and pharmaceutical combinations containing antibodies and optionally a pharmaceutical carrier, methods of treatment, including the use of pharmaceutical combinations, and the use of pharmaceutical combinations for the treatment of forms of cancer.

Lymphocyte antigen 75 acts as an endocytic receptor, directing captured antigens from the extracellular space to a specialized antigen-processing compartment, and is believed to cause a decrease in B-lymphocyte proliferation.

According to SWISS-PROT, lymphocyte antigen 75 is expressed in the spleen, thymus, colon, and peripheral blood lymphocytes. It has been identified in myeloid cell lines and lymphoid B-cells. The isoforms, referred to herein as OGTA076b and OGTA076c, are expressed in malignant cells of Hodgkin's lymphoma called Hodgkin and Reed Sternberg (HRS) cells. LY75 acts as an endocytic receptor, directing captured antigens from the extracellular space to a specialized antigen processing compartment. It causes a decrease in the proliferation of B-lymphocytes.

LY75 expression has been observed in cancers of the pancreas, bladder, ovary, breast (including triple negative), colon and rectum, esophagus, skin, thyroid, and lung (non-small cell), as well as in multiple myeloma and many different

- 6 042216 subtypes of lymphoma (including DLBCL) and leukemia.

An anti-LY75 antibody can in some cases cross-react with LY75 from a non-human species. For example, to facilitate the conduct of a clinical trial, antibodies to LY75 can cross-react with LY75 molecules from mice or primates. Alternatively, in some embodiments, the antibodies may have full specificity for human LY75 and may not exhibit species or other types of cross-reactivity to non-human molecules.

The present invention relates to antibodies to LY75 and in some embodiments, the implementation of antibodies to CD20, usually therapeutic antibodies, as described in this document. Antibodies of use in the present invention may take a number of formats described herein, including traditional antibodies, as well as derivatives, fragments and mimics of antibodies described below. In one embodiment, the present invention provides antibody structures containing a set of 6 CDRs defined herein (containing a small number of amino acid changes described below).

An antibody as used herein includes a wide variety of structures understood by those skilled in the art, which in some embodiments comprise at least a set of the 6 CDRs defined herein; including, without limitation, conventional antibodies (including both monoclonal and polyclonal antibodies), humanized and/or chimeric antibodies, antibody fragments, engineered antibodies (e.g., having the amino acid modifications summarized below), polyspecific antibodies (including bispecific antibodies ) and other analogues known from the prior art.

The structural units of conventional antibodies typically include a tetramer. Each tetramer typically consists of two identical pairs of polypeptide chains, with each pair having one light chain (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 isotype of an antibody as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including, without limitation, IgM1 and IgM2. Thus, isotype, as used herein, means any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. Known isotypes of human immunoglobulins are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD and IgE. It should be understood that therapeutic antibodies may also include hybrids from any combination of isotypes and/or subclasses.

In many embodiments, IgG isotypes are used in the present invention, with IgG1 being of particular use in a number of applications.

The amino-terminal portion of each chain contains a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. In the variable region, in each of the heavy chain and light chain V domains, three loops are brought together to form an antigen-binding site. Each of the loops is referred to as a complementarity determining region (hereinafter referred to as a CDR) in which the amino acid sequence variability is most significant. Variable refers to the fact that certain segments of the variable region vary substantially in sequence among antibodies. The variability within the variable region is unevenly distributed. In fact, V regions are composed of relatively invariant regions called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called hypervariable regions, each of which is 9-15 amino acids or more in length.

Each VH and VL consists of three hypervariable regions (complementarity determining regions, CDRs) and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The hypervariable region typically spans amino acid residues from amino acid residues approximately 24-34 (LCDR1; L stands for light chain), 50-56 (LCDR2) and 89-97 (LCDR3) in the light chain variable region and approximately 31-35B (HCDR1; H means heavy chain), 5065 (HCDR2) and 95-102 (HCDR3) in the variable region of the heavy chain; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991), and/or 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 present invention are described below.

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

CDRs contribute to the formation of antigen-binding, or more specifically epitope-binding

- 7 042216 antibody region. 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 by both adjacent amino acids and non-adjacent amino acids located side by side due to the folding of the protein into a tertiary structure. Epitopes formed by contiguous amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost upon exposure to denaturing solvents. An epitope typically contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. The methods described herein for determining which epitopes a given antibody binds to (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays where overlapping or contiguous LY75 peptides are examined for reactivity with the specified antibody to LY75. Methods for determining the spatial conformation of epitopes include techniques known in the art and techniques described herein, such as X-ray crystallography and 2D nuclear magnetic resonance spectroscopy (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, vol. 66, G. E. Morris, ed. (1996)). The term epitope mapping refers to a method for identifying the molecular determinants of antigen recognition by an antibody.

The carboxy-terminal portion of each chain defines the constant regions that are primarily responsible for effector function. Kabat et al. collected data on a variety of primary sequences of the variable regions of heavy chains and light chains. Based on the degree of sequence conservation, they assigned individual primary sequences to CDRs and framework regions and compiled a list of them (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, no. 91-3242, E.A. Kabat et al.).

Within the IgG immunoglobulin subclass, there are several immunoglobulin domains in the heavy chain. An immunoglobulin (Ig) domain, as used herein, refers to an immunoglobulin region having a distinct tertiary structure. In the present invention, heavy chain domains, including heavy chain constant domains (CH) and heavy chain hinge domains, are of interest.

For IgG antibodies, each of the IgG isotypes has three CH regions. Accordingly, the CH domains for IgG are as follows: CH1 refers to positions 118-220 according to the Kabat EU index. CH2 refers to positions 237-340 of the Kabat EU index, and CH3 refers to positions 341-447 of the Kabat EU index.

Another type of Ig heavy chain domain is the hinge region. By a hinge region, or a hinge region, or an antibody hinge region, or an immunoglobulin hinge region, as used herein, is meant a flexible polypeptide containing 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 EU residue 237. Thus, for an IgG antibody, the hinge region is defined herein as comprising positions 221 (D221 in IgG1) to 236 (G236 in IgG1), where the numbering corresponds to the Kabat EU index. In some embodiments, for example in relation to the Fc region, a lower hinge portion is included, with the lower hinge portion typically at positions 226 or 230.

Of particular interest in the present invention are Fc regions. By Fc, or Fc region, or Fc domain, as used herein, is meant a polypeptide comprising an antibody constant region excluding the first domain of an immunoglobulin constant region and, in some cases, part of the hinge region. Thus, Fc refers to the last two domains of the IgA, IgD, and IgG constant region, the last three domains of the IgE and IgM constant region, and the flexible hinge located N-terminally from these domains. In the case of IgA and IgM, the Fc may contain a J chain. In the case of IgG, the Fc domain contains the immunoglobulin domains Cy2 and Cy3 (Cy2 and Cy3) and the lower hinge region between Cy1 (Cy1) and Cy2 (Cy2). Although the boundaries of the Fc region may vary, the Fc region of a human IgG heavy chain is generally defined as comprising residues from C226 or P230 to its carboxyl terminus, where numbering corresponds to Kabat's EU index. In some embodiments, as described more fully below, amino acid modifications are made in the Fc region, for example, to change binding to one or more FcyR receptors or to an FcRn receptor.

In some embodiments, the antibodies are full length antibodies. A full-length antibody is herein understood to mean a structure that is the natural biological form of an antibody containing variable and constant regions containing one or more of the modifications summarized herein.

Alternatively, antibodies can be a range of structures including, but not limited to, antibody fragments, monoclonal antibodies, bispecific antibodies, mini-antibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as antibody mimetics), chimeric antibodies, humanized antibodies, products antibody fusions (sometimes referred to herein as antibody conjugates) and, accordingly, fragments of each. Structures based on the use of a set of CDRs are included in the definition of an antibody.

In one embodiment, the antibody is an antibody fragment. Specific antibody fragments include, without limitation (i) a Fab fragment consisting of the VL, VH, CL and CH1 domains;

(ii) an Fd fragment consisting of VH and CH1 domains;

(iii) an Fv fragment consisting of the VL and VH domains of a single antibody;

(iv) a dAb fragment (Ward et al., 1989, Nature, 341:544-546, incorporated by reference in its entirety) consisting of a single variable region;

(v) dedicated CDR regions;

(vi) F(ab') ₂ fragments, bivalent fragments containing two linked Fab fragments;

(vii) single chain Fv molecules (scFv) wherein the VH domain and the VL domain are linked by a peptide linker allowing the two domains to join to form an antigen binding site (Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci., USA, 85:5879-5883, incorporated by reference in its entirety);

(viii) bispecific single chain Fvs (WO 03/11161, incorporated by reference in its entirety); and (ix) diabody or triabody, polyvalent or polyspecific fragments constructed by gene fusion (Tomlinson et. al., 2000, Methods Enzymol., 326:461-479; WO 94/13804; Holliger et al., 1993, Proc. Natl Acad Sci USA 90:6444-6448, all of which are incorporated by reference in their entirety).

In some embodiments, the antibody may be a combination from different species, such as a chimeric antibody and/or a humanized antibody. In other words, in the present invention, CDR sets can be used with framework and constant regions other than those specifically described herein in sequence.

In general, both chimeric antibodies and humanized antibodies refer to antibodies that combine regions from more than one species. For example, chimeric antibodies traditionally contain variable(s) region(s) from a mouse (or in some cases, a rat) and constant(s) region(s) from a human. Humanized antibodies generally refer to non-human antibodies in which the variable domain framework regions have been replaced with sequences found in human antibodies. Typically, in a humanized antibody, all of the antibody except for the CDR is encoded by a polynucleotide of human origin or, except for its CDRs, is identical to such an antibody. CDRs, some or all of which are encoded by nucleic acids derived from a non-human organism, are grafted into a beta-sheet framework in the variable region of a human antibody to produce an antibody whose specificity is determined by the grafted CDRs. The production of such antibodies is described, for example, in WO 92/11018, Jones, 1986, Nature, 321:522-525; Verhoeyen et al., 1988, Science, 239:1534-1536, all of which are incorporated by reference in their entirety. Backmutation to replace selected acceptor framework residues with the corresponding donor residues is often necessary to restore the affinity lost by the construct after initial transplantation (US 5530101; US 5585089; US 5693761; US 5693762; US 6180370; US 5859205; US 6407213, all of which are incorporated by reference in their entirety). The humanized antibody also optimally contains at least a portion of the constant region of an immunoglobulin, typically a human immunoglobulin, and thus typically contains a human Fc region. Humanized antibodies can also be generated using mice with genetically engineered immune systems. Roque et al., 2004, Biotechnol. Prog., 20:639-654, incorporated by reference in its entirety. A number of techniques and methods are well known in the art for humanization and reengineering of non-human antibodies (see Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA), and references mentioned there, all of which are incorporated by reference in their entirety). Humanization methods include, without limitation, those described in Jones et al., 1986, Nature, 321:522525; Riechmann 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 of which are incorporated by reference in their entirety. Humanization or other methods of reducing the immunogenicity of non-human antibody variable regions may include surface modification methods as described, for example, in Roguska et al., 1994, Proc. Natl. Acad. Sci., USA, 91:969973, incorporated by reference in its entirety. In one embodiment, the parent antibody has been subjected to affinity maturation as is known in the art. Structural methods such as those described in the USSN can be used for humanization and affinity maturation.

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11/004590. Selection-based methods can be used to humanize and/or affinity mature the antibody variable regions, including but not limited to those 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 of which are incorporated by reference in their entirety. Other humanization methods may include transplantation of only portions of the CDR, including, without limitation, the methods described in USSN 09/810510; Tan et al., 2002, J. Immunol., 169:1119-1125; De Pascalis et al., 2002, J. Immunol., 169:3076-3084, all of which are incorporated by reference in their entirety.

The antibodies disclosed herein may be isolated or recombinant. Isolated, used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated from and/or recovered from the cell or cell culture in which it was expressed. Thus, an isolated antibody is intended to mean an antibody substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to LY75 is substantially free of antibodies that specifically bind to antigens other than LY75). Thus, the isolated antibody is in a form not normally found in nature (eg, not naturally occurring). An isolated antibody as defined herein may, in one embodiment, contain at least one amino acid not found in a naturally occurring antibody. This amino acid may be introduced by addition or substitution. It will be appreciated that the amino acid administered may be a naturally occurring or non-naturally occurring amino acid. In some embodiments, the antibodies of the present invention are recombinant proteins, isolated proteins, or substantially pure proteins. The isolated protein is not accompanied by at least some of the material to which it is normally associated in its natural state, such as at least about 5% or at least about 50% by weight of the total protein in said sample. It is understood that the isolated protein may comprise from 5% to 99.9% by weight of the total protein content, depending on the circumstances. For example, a protein can be produced at a significantly higher concentration by using an inducible or highly expressed promoter, whereby the protein is produced at higher 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 known in the art, in which it is not produced naturally. Typically, an isolated polypeptide is obtained by at least one purification step. An isolated antibody refers to an antibody substantially free of other antibodies with different specificities for antigens. For example, an isolated antibody that specifically binds to LY75 is substantially free of antibodies that specifically bind to antigens other than LY75, with the exception of antibodies that bind to CD20.

Isolated monoclonal antibodies with different specificities can be combined into a well-defined composition. Thus, for example, an antibody of the present invention may optionally and individually be included or not included, as discussed further below.

Anti-LY75 antibodies of the present invention specifically bind to LY75 (eg, SEQ ID NO: 15). The anti-CD20 antibodies of the present invention bind specifically to CD20. Specific binding, or specifically binds to, or specific to a particular antigen or epitope, means a binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining the binding of a molecule in comparison with the binding of a control molecule, which is usually a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target.

Specific binding to a particular antigen or epitope may be manifested by, for example, an antibody having a KD for the antigen or epitope of at least about 10-4 M, at least about 10-5 M, at least 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 more, where KD refers to the dissociation rate constant for a particular interaction of antibody and antigen. An antibody that specifically binds to an antigen will typically have a KD that is 20, 50, 100, 500, 1000, 5000, 10,000 or more times greater for the control molecule compared to the antigen or epitope. However, in the present invention, when administering the anti-LY75 ADC of the present invention, it is important that the KD be sufficient to allow for internalization and hence cell death without significant side effects.

Specific binding to a particular antigen or epitope can also be manifested by, for example, an antibody having a KA or Ka for the antigen or epitope of at least 20, 50, 100, 500,

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1000, 5000, 10000 or more times greater for an epitope than a control, where KA or Ka refers to the association rate constant for a particular antibody-antigen interaction.

Standard assays to assess the ability of antibodies to bind to LY75 or CD20 can be performed at the protein or cellular level and are known in the art, including, for example, variations of ELISA, Western blotting, RIA, BIAcore® assays, and flow cytometry assays . Suitable assays are detailed in the Examples section. The binding kinetics (eg, binding affinity) of antibodies can also be assessed using standard assays known in the art, such as using the Biacore® system assay. To assess binding to Raji or Daudi cells of a B-cell tumor, Raji (ATCC deposit number CCL-86) or Daudi (ATCC deposit number CCL-213) cells can be obtained from publicly available sources such as the American Type Culture Collection and used in standard assays such as flow cytometric assays.

Anti-LY75 antibodies that bind to LY75 (SEQ ID NO: 15) can be internalized upon contact with cells expressing LY75 on the cell surface. These antibodies are referred to herein as antibodies that bind to LY75 or, for ease of description, antibodies to LY75. Both terms are used interchangeably in this document.

Anti-LY75 antibodies are internalized upon contact with cells, in particular tumor cells, on the surface of which LY75 is expressed. In other words, the anti-LY75 antibodies defined herein, which also contain conjugated drugs, are internalized by tumor cells, resulting in drug release and subsequent cell death, providing treatment for cancers characterized by LY75 expression. Internalization in this case can be measured in several ways. In one embodiment, anti-LY75 antibodies are contacted with cells, such as the cell line summarized herein, using standard assays, such as using MAbZap. It will be apparent to one of skill in the art that the MabZap assay illustrates the expected effect that can be observed for an antibody-drug conjugate (ADC). In the latter case, the ADC will be internalized, thus bringing the drug into the cell. A toxic drug will have the ability to kill the cell, ie. to kill the target cancer cell. Data from MabZap assays are readily accepted by those skilled in the art as illustrative of ADC assays (Kohls, M and Lappi, D. (2000), Biotechniques, vol. 28, no. 1, 162-165).

In these embodiments of the in vitro assays, anti-LY75 antibodies are added together with an anti-LY75 antibody containing a toxin; for example, an anti-LY75 antibody may be murine or humanized, and an anti-LY75 antibody may be an anti-mouse or an anti-humanized antibody and contain a toxin such as saporin. Once the [anti-LY75 antibody]-[anti-LY75 antibody-drug conjugate] complex is formed, the complex is internalized and the drug (eg, saporin) is released, resulting in cell death. The drug is released only after internalization, and therefore the cells remain viable in the absence of internalization. As summarized below without being bound by theory, in therapeutic applications, an anti-LY75 antibody contains a toxin and upon internalization, the bond between the antibody and the toxin is cleaved to release the toxin and kill the cell.

In one embodiment, an anti-LY75 antibody contains complementarity determining regions (CDRs) or variable regions (VRs) of the heavy and light chains of the specific antibody described herein (eg, referred to herein as LY75_A1). Accordingly, in one embodiment, the antibody comprises the heavy chain variable region (VH) CDR1, CDR2 and CDR3 domains of the LY75_A1 antibody having the sequence shown under SEQ ID NO:1 and the light chain variable region (VL) CDR1, CDR2 and CDR3 domains of the LY75_A1 antibody. having the sequence shown under SEQ ID NO: 2.

In another embodiment, the LY75 antibody comprises a heavy chain variable region comprising a first vhCDR comprising SEQ ID NO: 5; a second vhCDR containing SEQ ID NO: 6; and a third vhCDR containing SEQ ID NO: 7; and the variable region of the light chain containing the first vlCDR containing SEQ ID NO: 8; a second vlCDR containing SEQ ID NO: 9; and a third vlCDR containing SEQ ID nO: 10.

In another embodiment, anti-LY75 antibodies bind to human LY75 and comprise a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NO: 1 and conservative sequence modifications thereof. The antibody may further comprise a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 2 and conservative sequence modifications thereof.

In a further embodiment, the anti-LY75 antibodies bind to human LY75 and comprise a heavy chain variable region and a light chain variable region comprising the amino acid sequences listed under SEQ ID NO: 1 and/or 2, respectively, and conservative sequence modifications thereof. As used herein, the term conservative sequence modification refers, for example, to replacing an amino acid with an amino acid having similar characteristics. It is generally well known to one of ordinary skill in the art which of these substitutions may be considered conservative. Other modifications that may be considered conservative sequence modifications include, for example, glycosylation.

Optionally, one or more of SEQ ID NOs: 5-10 independently contain one, two, three, four or five conservative amino acid substitutions; optionally, one or more of SEQ ID NOs: 5-10 independently contain one or two conservative amino acid substitutions.

Preferably, the term conservative sequence modifications is meant to include amino acid modifications that do not affect or significantly alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions or deletions. Modifications can be introduced into the antibody of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Such families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine, phenylalanine , tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of an antibody of the present invention can be replaced with other amino acid residues from the same side chain family, and the altered antibody can be examined for retention of function using the functional assays described herein.

Isolated antibodies containing heavy and light chain variable regions characterized by 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 greater sequence identity with any of the above sequences are also included in the present invention. Intermediate ranges of the above values, such as heavy and light chain variable regions having at least 80-85%, 85-90%, 90-95% or 95-100% sequence identity with any of the above sequences, are also intended to be encompassed by the present invention. In one embodiment, an anti-LY75 antibody comprises a heavy chain variable region comprising SEQ ID NO: 1 or a sequence of 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 anti-LY75 antibody comprises a lung variable region chain containing SEQ ID NO: 2 or sequence, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 2. In another embodiment, the anti-LY75 antibody comprises a heavy chain framework region containing an amino acid sequence of 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 heavy chain variable region framework of SEQ ID NO: I, comprising SEQ ID NO: 16, 17 and 18. In another embodiment, the anti-LY75 antibody comprises a light chain framework region containing an amino acid sequence of 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 region of the light chain variable region under SEQ ID NO: 2, containing SEQ ID NO: 19 , 20 and 21.

In one embodiment, the anti-LY75 antibody, herein referred to as the LY75_A1 antibody, contains the following CDRs, as well as variants thereof containing a small number of amino acid variants.

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A1 SEQ GO NO Heavy chain variable region CDR1 5 Heavy chain variable region CDR2 6 Heavy chain variable region CDR3 7 Light chain variable region CDR1 8 Light chain variable region CDR2 9 Light chain variable region CDR3 10

Also disclosed herein are heavy and light chain variable regions that comprise the CDR sets of the present invention, as well as full length heavy and light chains (eg, also containing constant regions). As will be appreciated by those skilled in the art, anti-LY75 antibody CDR sets can be incorporated into murine, humanized or human constant regions (including framework regions). Accordingly, the present invention provides heavy and light chain variable regions at least about 90-99% identical to the SEQ IDs disclosed herein, with all of 90, 91, 92, 93, 94, 95, 96, 97, 98 and 99% are used in the present invention.

In some embodiments, an anti-LY75 antibody is an antibody that competes for binding to human LY75 with an antibody comprising a heavy chain variable region comprising the amino acid sequence shown under SEQ ID NO: 1 and a light chain variable region containing the amino acid sequence shown under SEQ ID NO: 2. Antibodies competing for binding can be identified using standard techniques. Such techniques include, for example, an immunoassay that demonstrates the ability of one antibody to block the binding of another antibody to a target antigen, i. competitive binding analysis. Competitive binding is determined in an assay in which the immunoglobulin under test inhibits the specific binding of a reference antibody to a common antigen, such as LY75. Many types of competitive binding assays are known, such as solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), competitive sandwich assay (see Stahli et al., Methods in Enzymology, 9:242 (1983) ); solid phase direct EIA using a biotin-avidin complex (see Kirkland et al., J. Immunol., 137:3614 (1986)); labeled direct solid phase assay, labeled solid phase direct sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct RIA labeled using the I-125 label (see Morel et al., Mol. Immunol., 25(1):7 (1988)); solid phase direct EIA using biotin-avidin complex (Cheung et al., Virology, 176:546 (1990)) and labeled direct RIA (Moldenhauer et al., Scand. J. Immunol., 32:77 (1990)). Typically, such an assay involves the use of a purified antigen bound to a solid surface or cells bearing either of these, an unlabeled immunoglobulin of interest, and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of label associated with a solid surface or with cells in the presence of the investigated immunoglobulin. The immunoglobulin under test is usually present in excess. If the competing antibody is present in excess, it will typically inhibit the specific binding of the reference antibody to the common antigen 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.

Monoclonal antibodies can be characterized by binding to LY75 or CD20 using a number of known techniques. Typically, antibodies are first characterized by ELISA. Briefly, microtiter plates can be coated with purified LY75 or CD20 in PBS and then blocked with irrelevant proteins such as bovine serum albumin (BSA) diluted in PBS. Plasma dilutions from mice immunized with LY75 or CD20 are added to each well.

- 13 042216 and incubated for 1-2 hours at 37°C. The plates are washed with PBS/Tween 20 and then incubated with an alkaline phosphatase conjugated goat anti-human IgG polyclonal antibody reagent for 1 hour at 37°C. After washing, the plates are developed with ABTS substrate and analyzed at OD 405. Preferably, mice showing the highest titers will be used for fusion procedures.

The ELISA assay described above can be used to screen for antibodies, and hence hybridomas, that produce antibodies that show positive reactivity for the LY75/CD20 immunogen. Hybridomas that bind, preferably with high affinity, to LY75/CD20 can then be subcloned and further characterized. One clone from each hybridoma that retains the reactivity of the original cells (by ELISA) can then be selected for cell bank preparation and antibody purification.

To purify anti-LY75 antibodies or anti-CD20 antibodies, selected hybridomas can be grown in spinner flasks, two-liter spinner flasks, or other culture systems. Supernatant samples can be filtered and concentrated prior to Protein A affinity chromatography on Sepharose (Pharmacia, Piscataway, NJ) to purify the protein. After changing the buffer to PBS, the concentration can be determined by OD280 using an extinction coefficient of 1.43, or preferably by nephelometric analysis. IgG can be tested using gel electrophoresis and using an antigen-specific method.

To determine whether selected anti-LY75 monoclonal antibodies or anti-CD20 antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Binding of biotinylated mAbs can be detected using a streptavidin-labeled probe. To determine the isotype of purified antibodies, isotyping ELISA can be performed using techniques accepted in the art. For example, wells of microtiter plates can be coated with 10 μg/ml of anti-Ig antibody overnight at 4°C. After blocking with 5% BSA, the plates are reacted with 10 μg/ml monoclonal antibodies or purified isotype controls at ambient temperature for 2 hours. The wells can then be reacted with conjugated probes specific for IgG1 or other isotypes. The plates are developed and analyzed as described above.

Flow cytometry can be used to study the binding of monoclonal antibodies to live cells expressing LY75 or CD20. Briefly, cell lines and/or human PBMC expressing membrane-bound LY75 or CD20 (grown under standard growth conditions) are mixed with various concentrations of monoclonal antibodies in PBS containing 0.1% BSA at 4° C. for 1 h. After washing the cells are reacted with an antibody to IgG labeled with fluorescein under the same conditions as when staining with the primary antibody. Samples can be analyzed with a FACScan instrument using light scatter and side scatter properties to introduce a logical constraint on individual cells and determine the binding of labeled antibodies. You can use an alternative analysis using fluorescence microscopy in addition to analysis by the method of flow cytometry or instead. Cells can be stained exactly as described above and examined by fluorescence microscopy. This method provides visualization of individual cells, but may be characterized by reduced sensitivity depending on the density of the antigen.

You can further examine the reactivity of IgG antibodies to LY75 against the LY75 antigen using Western blotting; the same can be done with anti-CD20 antibodies. Briefly, cell extracts can be obtained from cells expressing LY75/CD20 and subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. After electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked with 20% mouse serum and probed with the monoclonal antibodies to be tested. IgG binding can be detected with an alkaline phosphatase-conjugated anti-IgG antibody and visualized with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, MO).

Methods for analyzing the binding affinity, cross-reactivity, and binding kinetics of various anti-LY75 antibodies include standard assays known in the art, such as the Biacore™ Surface Plasmon Resonance (SPR) assay using the Biacore™ 2000 SPR instrument (Biacore AB, Uppsala, Sweden) .

In one embodiment, the antibody specifically binds to human LY75 comprising SEQ ID NO: 15. Preferably, the anti-LY75 antibody binds to human LY75 with high affinity.

Preferably, the anti-LY75 antibody binds to the LY75 protein with a K _D of 5x10'8 M or less, binds to the LY75 protein with a K _D of 2x10'8 M or less, binds to the LY75 protein with a K _D of 5x10'9 M or less, binds to the LY75 protein with K _D 4x10'9 M or less, binds to LY75 protein with K _D 3x10'9 M or less, binds to LY75 protein with K _D 2x10'9 M or less, binds to LY75 protein with K _D 1x10'9 M or less, binds to LY75 protein with K _D 5x10' ¹⁰ M or less, or binds to LY75 protein with K _D

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1x10 ^-10 M or less.

In one embodiment, anti-LY75 antibodies compete (eg, cross-compete) for binding to LY75 with specific anti-LY75 antibodies described herein (eg, LY75_A1). Such competing antibodies can be identified based on their ability to competitively inhibit the binding of one or more mAbs to LY75 in standard LY75 binding assays. For example, standard ELISA assays can be used in which recombinant human LY75 protein is immobilized on a plate, one of the antibodies is fluorescently labeled, and the ability of the unlabeled antibody to eliminate the labeled antibody from binding competition is assessed. Additionally or alternatively, a BIAcore assay can be used to assess the ability of antibodies to cross-compete. The ability of the test antibody to inhibit the binding of the anti-LY75 antibody of the present invention to human LY75 demonstrates that the test antibody can compete 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 specific anti-LY75 monoclonal antibodies described herein (eg, LY75_A1). Standard epitope mapping techniques such as X-ray crystallography and 2D nuclear magnetic resonance spectroscopy can be used to determine if an antibody binds to the same epitope as a reference antibody (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology , vol. 66, G. E. Morris, ed. (1996)).

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

Once one parent anti-LY75 mAb having the desired properties described herein has been isolated, other mAbs with similar properties, such as those having the same epitope, can be generated. For example, one can immunize mice with LY75 as described herein, generate hybridomas, and screen the resulting mAbs for their ability to compete with the parent mAb for binding to LY75. Mice can also be immunized with a smaller LY75 fragment containing the epitope to which the parent mAb binds. Epitope localization can be determined by, for example, screening for binding to a number of overlapping peptides spanning LY75. Alternatively, the method of Jespers et al., Biotechnology, 12:899, 1994, can be used to guide the selection of mAbs having the same epitope and therefore similar properties compared to the parent mAb. Using phage display, the parent antibody heavy chain is first paired with a set of (preferably human) light chains to select for LY75-binding mAbs, and then the new light chain is matched with a set of (preferably human) heavy chains for selection of (preferably human) mAbs that bind to LY75 having the same epitope as the parent mAb. Alternatively, variants of the parent mAb can be generated by mutagenesis of the cDNA encoding the heavy and light chains of the antibody.

To assess the level of competition between two antibodies, for example, radioimmunoassays or assays using other antibody labels can be used. For example, a LY75 antigen can be incubated with a saturating amount of a first anti-LY75 antibody or antigen-binding fragment thereof conjugated to a label compound (e.g., ³ H, ¹²⁵ I, biotin, or rubidium) in the presence of an equal amount of a second unlabeled anti-LY75 antibody. The amount of labeled antibody binding to the antigen in the presence of an unlabeled blocking antibody is then assessed and compared with binding in the absence of an unlabeled blocking antibody. Competition is determined by the percentage change in binding signals in the presence of an unlabeled blocking antibody compared to no blocking antibody. Thus, if there is 50% inhibition of labeled antibody binding in the presence of a blocking antibody compared to binding in the absence of a blocking antibody, then there is 50% competition between the two antibodies. Thus, referring to competition between the first and second antibodies 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 the binding of the second antibody (or vice versa) to the antigen by 50, 60, 70, 71, 72, 73, 74, 75, 80, 85, 90 , 95, 96, 97, 98, 99% or more (compared to binding to the antigen of the second antibody in the absence of the first antibody). Thus, inhibition of the binding of the first antibody to the antigen by the second antibody of 50%, 60%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more means that two antibodies bind to the same epitope.

In some embodiments, component (B) of the pharmaceutical combination is an anti-CD20 antibody as defined herein.

The B-lymphocyte antigen CD20 or CD20 is a glycosylation-activated phosphoprotein expressed on the surface of all B cells starting from the pro-B phase (CD45R+, CD117+) and gradually increasing until maturity (Hardy, Richard (2008). Chapter 7 : B Lymphocyte Development and Biology, B Paul, William, Fundamental Immunology (Book) (6th ed.), Philadelphia: Lippincott Williams & Wilkins, p. 237-269, ISBN 0-7817-6519-6).

- 15 042216

CD20 is expressed at all stages of B-cell development except for the first and last; it is present from the late pro-B cell stage to the memory cell stage, but is absent from early pro-3 cells or plasmablasts and plasma cells. It is found on cancer stem cells in B-cell lymphoma, hairy cell leukemia, B-cell chronic lymphocytic leukemia, and melanoma. CD20 expression is regulated by chemokine signaling through the CXCR4/SDF1 axis.

Immunohistochemistry can be used to determine the presence of CD20 on cells in histological tissue sections.

In humans, CD20 is encoded by the MS4A1 gene. This gene encodes a member of the 4A transmembrane protein gene family.

In one embodiment, component (B) of the pharmaceutical combination comprises an anti-CD20 antibody or an antigen-binding portion thereof, said antibody comprising

a) heavy chain variable region containing

i) a first vhCDR containing SEQ ID NO: 40;

ii) a second vhCDR containing SEQ ID NO: 41; and iii) a third vhCDR containing SEQ ID NO: 42; And

b) a light chain variable region containing

i) a first vlCDR containing SEQ ID NO: 43;

ii) a second vlCDR containing SEQ ID NO: 44; and iii) a third vlCDR comprising SEQ ID NO: 45, where optionally any one or more of the above SEQ ID NOs independently contain one, two, three, four or five amino acid substitutions, additions or deletions.

Optionally, one or more of the above SEQ ID NOs: 40-45 independently contain one, two, three, four or five conservative amino acid substitutions; more preferably, any one or more of the above SEQ ID NOs independently contain one or two conservative amino acid substitutions.

The anti-CD-20 antibody is preferably a monoclonal antibody, more preferably a human monoclonal antibody, and even more preferably a fully human IgG1 monoclonal antibody.

In a particularly preferred embodiment of the present invention, the anti-CD20 antibody is rituximab. Rituximab is a monoclonal antibody to the CD20 protein, which is widely expressed on B cells (Oncogene (2003 Oct 20), Smith M.R., Rituximab (monoclonal anti-CD20 antibody): mechanisms of action and resistance, 22(47):7359-68 ). Rituximab destroys both normal and malignant B cells that have CD20 on their surface and is therefore used to treat diseases characterized by the presence of too many B cells, overactive B cells, or dysfunctional B cells. Rituximab has previously been used to treat a number of autoimmune diseases and some types of cancer, including rheumatoid arthritis, idiopathic thrombocytopenic purpura, pemphigus vulgaris, multiple sclerosis, systemic lupus erythematosus, non-Hodgkin's lymphoma, chronic inflammatory demyelinating polyneuropathy, chronic lymphocytic leukemia, and types of autoimmune anemia. Rituximab is sold under the trade name Rituxan® by Roche.

Preferably, the heavy and/or light chain of the rituximab antibody comprises or consists of the amino acid sequence shown under SEQ ID NOs: 38 and 39, respectively.

The present invention encompasses antibody variants, sometimes also referred to as antibody derivatives or antibody analogs. In other words, there are a number of modifications that can be made to the antibodies disclosed herein, including, but not limited to, CDR (affinity maturation) amino acid modifications, framework region amino acid modifications, Fc region amino acid modifications, glycosylation variants, other types of covalent modifications (e.g. , for attaching conjugated drugs, etc.).

By variant is meant herein a polypeptide sequence that differs from that of the parent polypeptide by at least one amino acid modification. In this case, the parent polypeptide is a full length heavy or light chain variable region such as listed under SEQ ID NOs: 1 or 2, respectively, or heavy and light chain CDRs or frameworks listed under SEQ ID NOs 5-10 and 16-21 for LY75. Amino acid modifications may 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 non-conservative substitution, with conservative substitutions being preferred. Additionally, said substitution may be a substitution of a naturally occurring or non-naturally occurring amino acid.

In general, variants may contain any number of modifications, as long as the function of the antibody is still present, as described herein. In other words, in the case of LY75_A1, for example, the antibody must still specifically bind to human LY75. Similarly, anti-CD20 antibodies should still bind specifically to human CD20. If the amino acid variants are made with an Fc region, for example, then the antibody variants must retain the receptor binding functions required for the particular use or indication of the antibody.

Variants in this case can be obtained in the following CDR sequences, framework or Fc regions of the antibody.

However, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions are generally used, since the goal is often to change function with as few modifications as possible. In some instances, there are 1 to 5 modifications (eg, single amino acid substitutions, insertions, or deletions), with 1-2, 1-3, and 1-4 also finding use in many embodiments. The number of modifications may depend on the size of the region being modified; for example, fewer modifications are generally desirable in CDR regions. One skilled in the art will appreciate that even in CDR regions, the location of the modification can significantly alter the effect. In one embodiment, modifications can be made to any of the heavy and/or light chain CDR1, CDR2, or CDR3. In a further embodiment, modifications are made to any of the heavy and/or light chain CDR1 or CDR2. In yet another additional embodiment, the modifications are located in the CDR1 of the heavy and/or light chains.

It should be noted that a number of amino acid modifications may be in the functional domains: for example, there may be a need for 1-5 modifications in the Fc region of wild-type or engineered proteins, and 1 to 5 modifications in the Fv region, for example. The polypeptide sequence variant will preferably have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the original sequences (e.g., variable regions, constant regions, and/or heavy and light chains and/or CDR LY75_A1 or rituximab). It should be noted that depending on sequence size, percent identity will depend on the number of amino acids.

By amino acid substitution or substitution, as used herein, is meant the substitution of an amino acid at a particular position in the parent polypeptide sequence with another amino acid, which may or may not be a naturally occurring amino acid. For example, substitution S100A refers to a polypeptide variant in which serine at position 100 is replaced by alanine. By amino acid insertion or insertion as used herein is meant the addition of an amino acid at a specific position in the original polypeptide sequence. By amino acid deletion or deletion as used herein is meant the removal of an amino acid at a specific position in the original polypeptide sequence.

By parent polypeptide, parent protein, precursor polypeptide, or precursor protein as used herein, is meant an unmodified polypeptide that is subsequently modified to produce a variant. Typically, parent polypeptides herein are LY75_A1 and rituximab. Accordingly, parent antibody as used herein refers to an antibody that has been modified to produce a variant antibody.

By wild type, or WT, or native, as used herein, is meant an amino acid sequence or nucleotide sequence found in nature, including allelic variants. Protein, polypeptide, antibody, immunoglobulin, IgG, and the like. WTs have an amino acid sequence or a nucleotide sequence that has not been intentionally modified.

By variant Fc region is meant herein an Fc sequence that differs from the wild-type Fc sequence by at least one amino acid modification. An Fc variant may refer to an Fc polypeptide as such, to compositions containing a variant Fc polypeptide, or to an amino acid sequence.

In some embodiments, one or more amino acid modifications are made in one or more CDRs of LY75_A1 or rituximab. Typically, only 1, or 2, or 3 amino acids are changed in any single CDR, and no more than 4, 5, 6, 7, 8, 9, or 10 changes are typically made in a set of 6 CDRs. However, it should be understood that any combination of no substitutions, 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, the substitutions are made in the heavy and/or light chain CDR1s.

In some cases, amino acid modifications in the CDR are referred to as affinity maturation. An affinity matured antibody has one or more changes in one or more CDRs that result in an improvement in the affinity of the antibody for the antigen compared to the parent antibody not having the change(s). In some cases, although rare, it may be desirable to reduce the affinity of an antibody for its antigen, but this is usually not preferred.

Affinity maturation can be performed to increase the binding affinity of an antibody to an antigen by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,

- 17 042216 about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150% or more, or 1, 2, 3, 4 to 5 times the parent antibody . Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are prepared using known procedures. See, for example, Marks et al., 1992, Biotechnology, 10:779-783, which describes affinity maturation by shuffling the heavy chain variable region (VH) and light chain variable region (VL) domains. Random mutagenesis of CDR residues and/or framework regions 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.

Alternatively, amino acid modifications can be made in one or more CDRs of the antibodies of the present invention which are silent, eg which do not significantly alter the affinity of the antibody for the antigen. They can be produced for a number of reasons, including expression optimization (which can be done for nucleic acids encoding antibodies of the present invention).

Thus, CDR and antibody variants are included in the definition of CDRs and antibodies disclosed herein; in other words, the antibodies may contain amino acid modifications in one or more CDRs of LY75_A1 or rituximab. In addition, as summarized below, amino acid modifications can also be independently and optionally made in any region outside of the CDR, including the framework and constant regions described herein.

In some embodiments, anti-LY75 antibodies and/or anti-CD20 antibodies disclosed herein consist of a variant Fc domain. As is known in the art, the Fc region of an antibody interacts with a number of Fc receptors and ligands to confer a number of important functionalities referred to as effector functions. These Fc receptors include, but are not limited to (in humans) FcyRI (CD64), including the FcyRIa, FcyRIb and FcyRIc isoforms; FcyRII (CD32), including isoforms FcyRIIa (including allotypes H131 and R131), FcyRIIb (including FcyRIIb-1 and FcyRIIb-2) and FcyRIIc; and FcyRIII (CD16), including FcyRIIIa isoforms (including V158 and F158 allotypes associated with antibody-dependent cellular cytotoxicity (ADCC)) and FcyRIIIb (including FcyRIIIb-NA1 and FcyRIIIb-NA2 allotypes), FcRn (neonatal receptor) , C1q (a protein of the complement system involved in complement dependent cytotoxicity (CDC)) and FcRn (a neonatal receptor involved in the regulation of serum half-life). Suitable modifications can be made in one or more positions, as generally set forth, for example, in US patent application 11/841654 and references mentioned there, US 2004/013210, US 2005/0054832, US 2006/0024298, US 2006/ 0121032, US 2006/0235208, US 2007/0148170, USSN 12/341769, US Patent No. 6737056, US Patent No. 7670600, US Patent No. 6086875, all of which are expressly incorporated by reference in their entirety, in particular with respect to specific amino acid substitutions that enhance binding to Fc receptors.

In addition to the modifications outlined above, other modifications can be made. For example, molecules can be stabilized by including disulfide bridges linking VH and VL domains (Reiter et al., 1996, Nature Biotech., 14:1239-1245, incorporated by reference in its entirety).

In addition, modifications to cysteine residues are particularly useful in the routes of administration of antibody-drug conjugates (ADCs), further described below. In some embodiments, an antibody constant region can be engineered to contain one or more particularly thiol-reactive cysteine residues in order to allow for more specific and controlled placement of the drug component. See, for example, US Pat. No. 7,521,541, incorporated herein by reference in its entirety.

In addition, there are a number of covalent modifications of antibodies that can be made, as summarized below.

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

Cysteinyl residues are most commonly reacted with α-haloacetates (and the corresponding amines) such as chloroacetic acid or chloroacetamide to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues can also be derivatized by reaction with bromotrifluoroacetone, a-bromo-b-(5-imidazolyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl-2-pyridyl disulfide, p-chloromercurobenzoate, 2- chloromercuro-4-nitrophenol or chloro-7-nitrobenzo-2-oxa-1,3-diazole

- 18 042216 etc.

Histidyl residues are derivatized by reaction with diethyl pyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Also useful is para-bromophenacyl bromide; the reaction is preferably carried out in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino-terminal residues are reacted with anhydrides of succinic or other carboxylic acids. Derivatization by these means has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing residues containing alpha-amino groups include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione and glyoxylate in a reaction catalyzed by transaminase.

Arginyl residues are modified by reaction with one or more conventional reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be carried out under alkaline conditions due to the high pKa of the guanidine functional group. In addition, these reagents can react with lysine groups as well as the arginine epsilon-amino group.

Specific modifications of tyrosyl residues can be made, with particular interest being the introduction of spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. For the formation of O-acetyltyrosyl molecules and 3-nitro derivatives, N-acetylimidazole and tetranitromethane are most often used, respectively. Tyrosyl residues are iodinated with 125I or 131I to produce labeled proteins for use in radioimmunoassay, with the chloramine T method described above being preferred.

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 groups such as 1-cyclohexyl-3-( 2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4azonium-4,4-dimethylpentyl)carbodiimide. In addition, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for cross-linking antibodies to a water-insoluble support matrix or surface for use in a number of ways in addition to the methods described below. Commonly used crosslinking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, 4-azidosalicylic acid esters, homobifunctional imidoesters, including disuccinimidyl esters, such as 3,3'-dithiobis(succinimidyl propionate), and bifunctional maleimides such as bis-M-maleimido-1,8-octane. Derivatizing agents such as methyl 3-[(p-azidophenyl)dithio]propioimidate provide photoactivated intermediates capable of crosslinking in the presence of light. Alternatively, reactive water-insoluble matrices are used to immobilize proteins, such as cyanogen bromide-activated carbohydrates and reactive substrates as described in US Pat. Nos. 3,969,287; 3691016; 4195128; 4247642; 4229537 and 4330440, all of which are incorporated by reference in their entirety.

Glutaminyl and asparaginyl residues are often deamidated to give the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Any form of these residues is within the scope of the present invention.

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

In addition, as will be understood by those skilled in the art, any label (including fluorescent, enzymatic, magnetic, radioactive, etc.) can be added to antibodies (as well as other compositions of the present invention).

Another type of covalent modification is glycosylation changes. In some embodiments, the antibodies disclosed herein may be fully or partially aglycosylated, such as afucosylated.

Another type of covalent modification of an antibody involves binding the antibody to various non-protein polymers, including, but not limited to, various polyols such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, as set forth, for example, in the PEG 2005-2006 catalog from Nektar Therapeutics (available from the Nektar Therapeutics website). Nektar), US Pat. Nos. 4,640,835; 4496689; 4301144; 4670417; 4,791,192 or 4,179,337, all of which are incorporated by reference in their entirety. In addition, as is known in the art, amino acid substitutions can be made in

- 19 042216 different positions in the antibody to facilitate the addition of polymers such as PEG. See, for example, US Patent Application Publication No. 2005/0114037 A1, incorporated by reference in its entirety.

In additional embodiments, the implementation of the antibodies may contain a label. By labeled herein is meant that a compound has at least one element, isotope, or chemical compound attached to enable detection of the compound. As a rule, labels are divided into three classes:

a) isotopic labels, which may be radioactive or heavy isotopes;

b) magnetic, electrical, temperature marks; And

c) colored or luminescent dyes, although labels also include enzymes and particles such as magnetic particles. Preferred labels include, but are not limited to, fluorescent lanthanide complexes (including europium and terbium) and fluorescent labels, including but not limited to quantum dots, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methylcoumarins, pyrene, malachite green, stilbene, lucifer yellow, Cascade blue, Texas red, Alexa dyes, cyan dyes, and others described in the 6th edition of the Molecular Probes Handbook, edited by Richard P. Haugland, expressly incorporated herein by reference.

Antibody-Drug Conjugates.

In some embodiments, anti-LY75 antibodies disclosed herein are conjugated with drugs to form antibody-drug conjugates (ADCs). Typically, ADCs are used in oncology applications where the use of antibody drug conjugates for local delivery of cytotoxic or cytostatic agents provides targeted delivery of the drug functional moiety to the tumor, which can provide higher efficacy, lower toxicity, etc. P. For a review of this technology, see Ducry et al., Bioconjugate Chem., 21:5-13 (2010), Carter et al., Cancer J. 14(3):154 (2008); and Senter, Current Opin. Chem. Biol., 13:235-244 (2009), all of which are incorporated herein by reference in their entirety.

Thus, the present invention provides pharmaceutical combinations containing, inter alia, drug-conjugated anti-LY75 antibodies. Typically, conjugation is performed by covalent attachment to the antibody, as detailed below, and is usually based on the use of a linker, often a peptide bond (which, as described below, can be provided to be sensitive or insensitive to protease cleavage at the target site). In addition, as described above, linker-drug (LU-D) linkage can be accomplished by attachment to cysteine residues in an antibody. As will be understood by those skilled in the art, the number of drug functional moieties per antibody may vary depending on the reaction conditions, and the drug:antibody ratio may vary from 1:1 to 10:1. As will be understood by those skilled in the art, the actual amount is an average.

Thus, anti-LY75 antibodies can be drug-conjugated. As described below, the ADC drug may be any number of agents, including, without limitation, the present cytotoxic agents, such as chemotherapeutic agents, growth inhibitors, toxins (e.g., enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), or radioactive isotopes (in other words, in a radioconjugate). In other embodiments, the present invention further provides methods for using ADC.

Drugs for use in the present invention include cytotoxic drugs, in particular those used for cancer therapy. Such drugs generally include DNA damaging agents, antimetabolites, natural products and their analogues. Illustrative classes of cytotoxic agents include enzyme inhibitors such as dihydrofolate reductase inhibitors and thymidylate synthase inhibitors, DNA intercalators, DNA cleaving agents, topoisomerase inhibitors, anthracycline family drugs, vinca drugs, mitomycins, bleomycins, cytotoxic nucleosides, pteridine family drugs, diinenes, podophyllotoxins, dolastatins, maytansinoids, differentiation inducers and taxols.

Members of these classes include, for example, taxol, methotrexate, metopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leirosine, leurosidein, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, carminomycin, aminopterin, thallisomycin, podophyllotoxin and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxanes, including taxol, taxotere, retinoic acid, butyric acid, K8-acetylspermidine, camptothecin, calicheamicin, esperamycin, endiins, duocarmycin A, duocarmycin SA , calicheamicin, camptothecin, hemiasterlins, maytansinoids (including DM1), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF) and maytansinoids (DM4) and their

- 20 042216 analogues.

Toxins can be used in 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. (1998), Cancer Res., 58:2928; Hinman et al. (1993), Cancer Res., 53:3336-3342), hemiasterlins (WO 2004/026293; Zask et al. (2004), J. Med. Chem., 47:4774-4786). Toxins may exert their cytotoxic and cytostatic effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

Conjugates of an anti-LY75 antibody and one or more small molecular weight toxins such as maytansinoids, dolastatins, auristatins, trichothecin, calicheamicin, duocarmycins, pyrrolebenzodiazepines, and CC1065, and derivatives of these toxins having toxin activity, can also be used.

The anti-LY75 antibody is preferably conjugated to DM1 or DM4, most preferably to DM4. Maytansine compounds suitable for use as maytansinoid drug functional moieties are well known in the art and can be isolated from natural sources according to known methods, be obtained using genetic engineering techniques (see Yu et al. (2002), PNAS 99:7968-7973), or maytansinol and maytansinol analogs obtained synthetically according to known methods. As described below, drugs can be modified by including a functionally active group, such as a thiol group or an amino group, for conjugation with an antibody.

Exemplary maytansinoid functional moieties that are drugs include components that have a modified aromatic ring, such as C-19-dechlorinated derivatives (US patent No. 4256746) (obtained by reduction of ansamitocin P2 lithium aluminum hydride); C-20-hydroxy- (or C-20-demethyl-) +/-C-19-dechlorinated derivatives (US Pat. -demethoxy, C-20-acyloxy (--OCOR), +/-dechloro derivatives (US patent No. 4294757) (obtained by acylation with acid chlorides), as well as components that have modifications in other positions.

Exemplary drug maytansinoid functional moieties also include components that have modifications such as C-9-SH (US Pat. No. 4,424,219) (obtained by reacting maytansinol with H2S or P2S5); C-14-alkoxymethyl(demethoxy/CH2OR) (US Pat. No. 4,331,598); C-14-hydroxymethyl or acyloxymethyl (CH2OH or _CH2OAc ) (US Pat. No. 4,450,254) (obtained from Nocardia); C-15-hydroxy/acyloxy (US patent No. 4364866) (obtained by the conversion of maytansinol under the action of Streptomyces); C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated from Trewia nudlflora); C-18-N-demethyl (US Pat. Nos. 4,362,663 and 4,322,348) (obtained by demethylation of maytansinol by Streptomyces) and 4,5-deoxy (US Pat. No. 4,371,533) (obtained by reduction of maytansinol with titanium trichloride/LAH).

Of particular applicability are DM1 (disclosed in US Pat. No. 5,208,020, incorporated by reference) and DM4 (disclosed in US Pat. No. 7,276,497, incorporated by reference).

See also a number of additional maytansinoid derivatives and methods in 5416064, WO 01/24763, 7303749, 7601354, USSN 12/631508, WO 02/098883, 6441163, 7368565, WO 02/16368 and WO 04/1033272, all of which explicitly incorporated by reference in their entirety.

Maytansinoid-containing ADCs, methods for their preparation, and their therapeutic uses are disclosed, for example, in US Pat. Nos. 5,208,020; 5416064; 6441163 and European patent EP 0425235 B1, the disclosures of which are expressly incorporated herein by reference. B Liu et al., Proc. Natl. Acad. Sci., USA, 93:8618-8623 (1996), describe ADCs containing a maytansinoid, designated DM1, linked to the C242 monoclonal antibody against human colon cancer. The conjugate was found to be highly cytotoxic to cultured colon cancer cells and exhibited antitumor activity in an in vivo tumor growth assay.

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

- 21 042216

For compositions containing multiple antibodies, the drug load is represented as p, the average number of drug molecules per antibody. Drug loading can range from 1 to 20 drug molecules (D) per antibody. The average number of drug molecules per antibody in the preparation obtained by conjugation reactions can be characterized using conventional methods such as mass spectroscopy, ELISA and HPLC analysis. It is also possible to determine the quantitative distribution of antibody-drug conjugates by p.

In some cases, separation, purification of homogeneous antibody-drug conjugates, where p is a certain value, from antibody-drug conjugates with a different drug load, and their characterization can be carried out using methods such as reverse phase HPLC or electrophoresis. In exemplary embodiments, p is 2, 3, 4, 5, 6, 7, or 8, or a fraction thereof.

The preparation of antibody-drug conjugate compounds can be carried out according to any technique known to the person skilled in the art. Briefly, antibody drug conjugate compounds may contain an anti-LY75 antibody as the antibody structural unit, a drug, and optionally a linker that connects the drug and the binding agent.

Many different reactions are available for the covalent attachment of drugs and/or linkers to binding agents. This can be done by reacting with amino acid residues of a binding agent, for example an antibody molecule, including lysine amino groups, free glutamic and aspartic acid carboxylic acid groups, cysteine sulfhydryl groups, and various aromatic amino acid functional moieties. A widely used non-specific method of covalent attachment is the carbodiimide reaction of binding the carboxyl (or amino) group of the compound with the amino (or carboxyl) groups of the antibody. Additionally, bifunctional agents such as dialdehydes or imidoesters have been used to link the amino group of the compound to the amino groups of the antibody molecule.

A Schiff base formation reaction is also available for the attachment of drugs to binders. This method involves periodate oxidation of a drug containing glycol groups or hydroxy groups, thereby forming an aldehyde, which is then reacted with a coupling agent. The attachment takes place via the formation of a Schiff base with the amino groups of the coupling agent. Isothiocyanates can also be used as coupling agents for the covalent attachment of drugs to binding agents. Other techniques are known to the person skilled in the art and are within the scope of the present invention.

In some embodiments, the linker precursor intermediate is reacted with a drug under appropriate conditions. In other embodiments, reactive groups are used on the drug and/or intermediate. The reaction product between drug and intermediate, or derivatized drug, is then reacted with an anti-LY75 antibody of the present invention under appropriate conditions.

It will be understood that chemical modifications can also be made to the desired compound to make the reactions of that compound more suitable for purposes of preparing the conjugates of the present invention. For example, a functional group, such as an amino group, a hydroxyl group, or a sulfhydryl group, can be attached to a drug at a position that has a minimal or acceptable effect on the activity or other properties of the drug.

Typically, antibody-drug conjugate compounds contain a linker entity between the drug entity and the antibody entity. In some embodiments, the linker is cleaved under intracellular or extracellular conditions such that cleavage of the linker from the antibody releases the drug entity into the appropriate environment. For example, solid tumors secreting certain proteases can serve as targets for a cleavable linker; in other embodiments, it is intracellular proteases that are used. In a few other embodiments, the linker entity is not cleavable and the drug is released, for example, by destroying antibodies in lysosomes.

In some embodiments, the linker is cleaved by a cleaving agent present in the intracellular environment (eg, in the lysosome or endosome, or in the caveolus). The linker may be, for example, a peptidyl linker cleavable 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 greater.

Cleaving agents may include, without limitation, cathepsins B and D and plasmin, all of

- 22 042216 which are known to hydrolyze dipeptide derivatives of drugs, resulting in the release of the active drug into target cells (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics, 83:67-123). Peptidyl linkers are cleaved by enzymes present in cells expressing LY75. For example, a peptidyl linker cleavable by the thiol-dependent protease cathepsin B, which is highly expressed in cancer tissue, can be used (eg Phe-Leu or Gly-Phe-Leu-Gly linker (SEQ ID NO: 52)). Other examples of such linkers are described, for example, in US patent No. 6214345, incorporated herein by reference in its entirety and in all respects.

In some embodiments, the intracellular protease-cleavable peptidyl linker is a Val-Cit linker or a Phe-Lys linker (see, for example, US Pat. No. 6,214,345, which describes the synthesis of doxorubicin using a Val-Cit linker).

In other embodiments, the implementation of the cleavable linker is pH-sensitive, in other words, sensitive to hydrolysis at certain pH values. As a rule, the pH-sensitive linker is hydrolyzed under acidic conditions. For example, an acid-labile lysosome-hydrolysable linker (eg, hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic acid amide, orthoester, acetal, ketal, etc.) can be used. (See, for example, US Pat. are relatively stable under neutral pH conditions, such as in blood, but are unstable below pH 5.5 or 5.0, the approximate pH in the lysosome. In certain embodiments, the hydrolysable linker is a thioether linker (such as, for example, a thioether attached to a therapeutic agent via an acylhydrazone bond (see, for example, US Pat. No. 5,622,929).

In a few other embodiments, the implementation of the linker is cleaved under reducing conditions (for example, a disulfide linker). A number of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB -3(2-pyridyldithio)butyrate) and SMPT (N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2pyridyldithio)toluene)-, SPDB and SMPT (see e.g. Thorpe et al., 1987, Cancer Res., 47 :5924-5931; Wawrzynczak et al., in Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987. See also U.S. Patent No. 4,880,935).

In other embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res., 15:1387-93), maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299 -1304) or 3'-N-α-sweet analogue (Lau et al., 1995, Bioorg-Med-Chem., 3(10):1305-12).

In still a few other embodiments, the linker entity is not cleavable and the drug is released by disruption of the antibodies (See U.S. Patent Application Publication No. 2005/0238649, incorporated herein by reference in its entirety and in all respects).

In many embodiments, the linker is self-cleaving. As used herein, the term self-cleaving spacer refers to a bifunctional chemical moiety capable of covalently bonding two separated chemical moieties into a stable three-part molecule. It will spontaneously separate from the second chemical functional part when its bond with the first component is cleaved. See, for example, WO 2007/059404A2, WO 06/110476A2, WO 05/112919A2, WO 2010/062171, WO 09/017394, WO 07/089149, WO 07/018431, WO 04/043493 and WO 04/043493 and WO 04/043493 and WO 080.08. which refer to drug-cleavable substrate conjugates wherein the drug and cleavable substrate are optionally linked via a self-cleaving linker, all of which are expressly incorporated by reference.

The linker is often substantially insensitive to the action of the extracellular environment. As used herein, substantially insensitive to extracellular media in relation to a linker means that no more than about 20%, 15%, 10%, 5%, 3%, or no more than about 1% of the linkers are cleaved in a sample of an antibody-drug conjugate compound. if the antibody-drug conjugate compound is present in the extracellular environment (eg, in blood plasma).

Whether the linker is substantially insensitive to the action of the extracellular environment can be determined, for example, by incubating the antibody drug conjugate compound with blood plasma for a predetermined period of time (eg, 2, 4, 8, 16, or 24 hours) and subsequent quantification of the amount of free drug present in plasma.

In other non-mutually exclusive embodiments, the implementation of the linker promotes cellular internalization. In certain embodiments, the linker promotes cellular internalization by being conjugated to a therapeutic agent (in other words, in the environment of the linker-therapeutic agent functional portion of the antibody-drug conjugate compound described in

- 23 042216 of this document). In several other embodiments, the linker promotes cellular internalization by being conjugated to both the auristatin compound and the anti-LY75 antibodies of the present invention.

A number of exemplary linkers that can be used in the compositions and methods of the present invention are described in WO 2004/010957, US Patent Application Publication No. 2006/0074008, US Patent Application Publication No. 20050238649, and US Patent Application Publication No. 2006/0024317 ( each of which is incorporated herein by reference in its entirety and in all respects). Preferably the linker is SPDB (K-succinimidyl-3-(2-pyridyldithio)butyrate).

Drug loading is represented as p and is the average number of functional drug moieties per antibody per molecule. The drug load (p) can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more functional parts (D) per antibody, although often the average is a fraction or decimal. Typically, a drug load of 1 to 4 is commonly used and 1 to 2 is also used. The ADCs of the present invention comprise groups of antibodies conjugated to a range of drug components from 1 to 20, eg 1-15, 1-10, 2-9, 3-8, 4-7, 5-6. The average number of drug functional moieties per antibody in ADC preparations resulting from conjugation reactions can be characterized using conventional methods such as mass spectroscopy and ELISA analysis.

It is also possible to determine the quantitative distribution of ADC over p. In some cases, separating, purifying homogeneous ADCs, where p is a certain value, from ADCs with a different drug load, and characterizing them can be accomplished by methods such as electrophoresis.

For some antibody-drug conjugates, p may be limited by the number of attachment sites in the antibody. For example, if the attachment is at a cysteine thiol group, as in the exemplary embodiments described above, the antibody may have only one or more cysteine thiol groups, or may have only one or a few sufficiently reactive thiol groups through which a linker can be attached. In certain embodiments, higher drug loading, eg p>5, may cause aggregation, insolubility, toxicity or loss of cell permeability of certain antibody drug conjugates. In certain embodiments, drug loading for an ADC of the present invention ranges from 1 to about 8; from about 2 to about 6; about 3 to about 5; about 3 to about 4; from about 3.1 to about 3.9; 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. In fact, it has been shown that for certain ADCs, the optimal number of drug functional moieties per antibody may be less than 8 and may be from about 2 to about 5. See US 2005/0238649 A1 (incorporated herein by reference). in its entirety).

In certain embodiments, the conjugation reaction conjugates less than the theoretical maximum drug functional moieties to the antibody. The antibody may contain, for example, lysine residues that are not reactive with a drug-linker intermediate or linker reagent, as discussed below. Antibodies usually contain few free and reactive cysteine thiol groups that can be associated with the drug component; in fact, most of the thiol groups of cysteine residues in antibodies exist as disulfide bridges. In certain embodiments, the antibody can be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) under partial or complete reduction conditions to form reactive cysteine thiol groups. In certain embodiments, the antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups, such as in lysine or cysteine.

The loading (drug/antibody ratio) in the ADC can be controlled in various ways, for example, by (i) limiting the molar excess of the drug-linker intermediate or linker reagent relative to the antibody;

(ii) limiting the duration or temperature of the conjugation reaction;

(iii) applying partial or limited reduction conditions to modify cysteine thiol groups;

(iv) constructing, by recombinant techniques, the amino acid sequence of the antibody so as to modify the number and position of cysteine residues to control the number and/or position of the linker-drug components to be attached.

- 24 042216 (as, for example, in the case of thio-Mab or thio-Fab, obtained as disclosed in this document and in

WO 2006/034488 (incorporated herein by reference in its entirety)).

It should be understood that if more than one nucleophilic group reacts with a drug-linker intermediate or linker reagent and then with a drug functional moiety, the resulting product is a mixture of ADC compounds with a distribution of one or more functional parts that are drugs attached to the antibody. In the mixture, the average number of drugs per antibody can be calculated using a dual antibody-specific and drug-specific ELISA antibody assay. Individual ADC molecules can be identified in a mixture using mass spectroscopy and separated using HPLC, such as hydrophobic interaction chromatography.

In some embodiments, a homogeneous ADC with a single loading value can be isolated from a mixture of conjugates using electrophoresis or chromatography.

Methods for determining the cytotoxic effect of ADC.

Methods are known for determining whether a drug or antibody drug conjugate produces a cytostatic and/or cytotoxic effect on a cell. Typically, the cytotoxic or cytostatic activity of an antibody-drug conjugate can be measured by exposing mammalian cells expressing the target protein to the antibody-drug conjugate in cell culture medium; culturing the cells for a period of about 6 hours to about 5 days; and measuring cell viability. In vitro cell assays can be used to measure the viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) by an antibody-drug conjugate.

Thymidine incorporation assay can be used to determine if an antibody-drug conjugate produces a cytostatic effect. For example, cancer cells expressing the target antigen at a density of 5000 cells/well in 96-well plates can be cultured for a period of 72 hours and exposed to 0.5 μCi ³ H-thymidine during the last 8 hours of a 72 hour period. Incorporation of 3H-thymidine into culture cells is measured in the presence and absence of the antibody-drug conjugate.

Necrosis or apoptosis (programmed cell death) can be measured to determine cytotoxicity. Necrosis is usually accompanied by an increase in the permeability of the plasma membrane; cell edema and plasma membrane rupture. Apoptosis is typically characterized by membrane blistering, cytoplasmic condensation, and activation of endogenous endonucleases. The determination of any of these effects on cancer cells indicates that the antibody-drug conjugate is useful in the treatment of forms of cancer.

Cell viability can be measured by determining cell uptake of a dye such as neutral red, trypan blue, or ALAMAR™ blue (see, for example, Page et al., 1993, Intl. J. Oncology, 3:473-476). In such an assay, cells are incubated in a medium containing a dye, the cells are washed, and the remaining dye, reflecting the uptake of the dye by the cells, is measured spectrophotometrically. The protein-binding dye sulforodamine B (SRB) can also be used to measure cytotoxicity (Skehan et al., 1990, J. Natl. Cancer Inst., 82:1107-12).

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

Apoptosis can be quantified by measuring, for example, DNA fragmentation. Commercial photometric methods are available to quantify DNA fragmentation in vitro. Examples of such assays, including TUNEL (which detects incorporation of labeled nucleotides into fragmented DNA) and ELISA-based assays, are described in Biochemica, 1999, no. 2, p. 34-37 (Roche Molecular Biochemicals).

Apoptosis can also be determined by measuring morphological changes in the cell. For example, as with necrosis, loss of plasma membrane integrity can be determined by measuring the uptake of certain dyes (eg, a fluorescent dye such as, for example, acridine orange or ethidium bromide). A method for measuring the number of apoptotic cells has been described in Duke and Cohen, Current Protocols in Immunology (Coligan et al., eds., 1992, p. 3.17.1-3.17.16). It is also possible to label cells with a DNA stain (eg, acridine orange, ethidium bromide, or propidium iodide) and observe chromatin condensation and its marginal arrangement along the inner nuclear membrane in the cells. Other morphological changes that can be measured to determine apoptosis include, for example, cytoplasmic condensation, increased membrane bubbling, and cell contraction.

The presence of apoptotic cells can be measured in both fixed and floating culture compartments. For example, both compartments can be harvested by removing the supernatant, trypsinizing the anchored cells, pooling the preparations after a washing step by centrifugation (e.g., for 10 min at 2000 rpm), and detecting apoptosis (e.g., by measuring DNA fragmentation) (See, for example, Piazza et al., 1995, Cancer Research, 55:3110-16).

The in vivo effect of the anti-LY75 antibody therapeutic composition of the present invention can be evaluated in a suitable animal model. For example, xenogeneic cancer models can be used where tumor explants or passaged xenograft tissues are administered to immunocompromised animals such as nude or SCID mice (Klein et al., 1997, Nature Medicine, 3:402-408). Efficacy can be measured by assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.

Therapeutic compositions used in the practice of the methods described above can be formulated into pharmaceutical compositions containing a carrier suitable for the desired mode of delivery. Suitable carriers include any material which, when combined with the therapeutic composition, retains the antitumor function of the therapeutic composition and to which the patient's immune system does not normally respond. Examples include, without limitation, any of a variety of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like. (See generally Remington's Pharmaceutical Sciences 16th edition, A. Osal., ed., 1980).

Methods for obtaining antibodies.

The antibodies disclosed herein can be obtained using any suitable method. Such methods include culturing a host cell containing isolated nucleic acid(s) encoding antibodies. As will be appreciated by those skilled in the art, this can be done in a number of ways depending on the nature of the antibody. In the case where the antibodies are conventional full length antibodies, for example, the heavy chain variable region and the light chain variable region are under such conditions that the antibody is generated and can be isolated.

The variable regions of the LY75_A1 heavy and light chains are disclosed herein (both protein and nucleic acid sequences); as will be appreciated in the art, they can easily be extended to give full length heavy and light chains. In other words, given DNA fragments encoding the VH and VK segments summarized herein, these DNA fragments can be further manipulated using standard recombinant DNA techniques, for example, to convert variable region genes into full length antibody chain genes, into the Fab fragment genes or into the scFv gene. These manipulations form a functional linkage of a DNA fragment encoding a VK or VH with another DNA fragment encoding a different protein, such as an antibody constant region or a flexible linker. The term operably linked as used herein is meant to mean that two DNA fragments are linked 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 into a full length heavy chain gene by operably linking the DNA encoding the VH to another DNA molecule encoding the heavy chain constant regions (CH1, CH2 and CH3). The gene sequences for mouse heavy chain constant regions 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. 91-3242], and DNA fragments covering these regions can be obtained by standard SPR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably is an IgG1 or IgG4 constant region. In the case of a Fab fragment heavy chain gene, a DNA encoding VH can be operably linked to another DNA molecule encoding only the CH1 constant region of the heavy chain.

The isolated DNA encoding the VL/VK region can be converted into a full length light chain gene (as well as a Fab light chain gene) by operably linking the VL encoding DNA to another DNA molecule encoding the CL light chain constant region. The gene sequences for mouse light chain constant regions are known in the art [see, for example, Kabat, E.A. et al. (1991), Sequences of Proteins of Immunological Interest, fifth edition, US Department of Health and Human Services, NIH Publication, No. 91-3242], and DNA fragments covering these regions can be obtained by standard SPR amplification. In preferred embodiments, the light chain constant region may be a kappa or lambda chain constant region.

To obtain the scFv gene, the DNA fragments encoding VH- and VL/VK are functionally linked to another fragment encoding a flexible linker, for example encoding the amino acid sequence (Gly ₄ -Ser) ₃ , so that the V _H and VL/VK sequences can be expressed in the form of a continuous single-stranded protein, while the VL/VK and VH regions are connected by a flexible linker [see, for example, Bird et al. (1988), Science, 242:423-426; Huston et al. (1988), Proc. Natl. Acad. Sci., USA, 85:5879-5883; McCafferty et al. (1990), Nature, 348:552-554].

Nucleic acids encoding the antibodies disclosed herein are provided. Such polynucleotides encode both the variable and constant regions of each of the severe and mild

- 26 042216 chains, although other combinations are also provided in accordance with the compositions described in this document.

Polynucleotides may be in the form of RNA or DNA. Polynucleotides in the form of DNA, cDNA, genomic DNA, nucleic acid analogs, and synthetic DNA are also useful. The DNA may be double-stranded or single-stranded, and in the case of single-stranded, it may be a coding (sense) strand or a non-coding (antisense) strand. The coding sequence that encodes a polypeptide may be identical to the coding sequence provided herein, or may be a different coding sequence, and this sequence, due to redundancy or degeneracy of the genetic code, encodes the same polypeptides as the DNA presented herein.

In some embodiments, the nucleic acid(s) encoding the antibodies disclosed herein are included in expression vectors, which may be extrachromosomal or designed to be integrated into the genome of the host cell into which they are introduced. Expression vectors may contain any number of appropriate regulatory sequences (including, but not limited to, transcription and translation control sequences, promoters, ribosome binding sites, enhancers, origins of replication, and the like) or other components (selectable genes, and the like). ), all of which are operatively related, as is well known in the art. In some cases, two nucleic acids are used and each is placed in a separate expression vector (eg, heavy chain in the first expression vector, light chain in the second expression vector), or alternatively they can be placed in the same expression vector. Those skilled in the art will appreciate that the design of the expression vector(s), including the choice of regulatory sequences, may depend on factors such as choice of host cell, desired level of protein expression, and the like.

Nucleic acids and/or expression systems can generally be introduced into a suitable host cell to produce a recombinant host cell by any method suitable for the selected host cell (e.g., transformation, transfection, electroporation, infection), such that the molecule(s) nucleic acids are operably linked to one or more expression control elements (eg, in a vector, in a construct produced by in-cell processes integrated into the host cell's genome). The resulting recombinant host cell can be maintained under conditions suitable for expression (e.g., in the presence of an inducer, in a suitable non-human animal, in a suitable culture medium supplemented with appropriate salts, growth factors, antibiotics, nutritional supplements, and the like. ), resulting in the encoded(s) polypeptide(s). In some cases, heavy chains are formed in one cell and light chains in another.

Mammalian cell lines available as hosts for expression are known in the art and include a large number of immortalized cell lines available from the American Type Culture Collection (ATCC), Manassas, Virginia, including, but not limited to, Chinese Hamster Ovary (CHO) cells, HEK 293 cells, NSO cells, HeLa cells, newborn hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (eg, Hep G2), and a variety of other cell lines. Cells other than mammalian cells, including but not limited to bacterial, yeast, insect, and plant cells, can also be used to express recombinant antibodies. In some embodiments, the implementation of antibodies can be obtained in transgenic animals, such as cows or chickens.

General methods for molecular biology, expression, purification and screening of antibodies are well known, see for example US Pat. , 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.

In a further embodiment of the present invention, component (B) of the pharmaceutical combination is ibrutinib or a pharmaceutically acceptable salt thereof. Ibrutinib (Imbravica®) is a small molecule drug that permanently binds to Bruton's tyrosine kinase (BTK). It has previously been used to treat B-cell cancers such as mantle cell lymphoma, chronic lymphocytic leukemia, and Waldenström's macroglobulinemia (a form of non-Hodgkin's lymphoma). The structural and chemical formulas of ibrutinib are shown below:

(R)-1 -(3-(4-Amino-3-(4-phenoxyphenyl)-1 H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2en-1 -He.

- 27 042216

Ibrutinib is marketed under the trade name Imbravica®. It is available from Pharmacyclics, Inc. (USA).

pharmaceutical compositions.

The pharmaceutical combination of the present invention is in the form of a combined preparation for simultaneous, separate or sequential use. Similarly, within the methods of the present invention, the components (A) and (B) of the pharmaceutical combination can be administered to the patient simultaneously, separately or sequentially.

The term combination preparation includes both fixed combinations and non-fixed combinations.

The term fixed combination means that the active ingredients (eg components (A) and (B)) are in the form of a single agent or dose. In other words, the active ingredients are present in a single composition or composition.

The term non-fixed combination means that the active ingredients (eg, components (A) and (B)), are present in different means or doses (eg, as separate compositions or formulations), for example, as a set of parts. The independent components (A) and (B) (in their desired compositions or formulations) can then be administered separately or sequentially at the same time or at different times.

In the case where the introduction is carried out sequentially, the delay in the introduction of the second component should not be such that the beneficial effect obtained due to the use of the combination is lost. Therefore, in one embodiment, sequential treatment comprises administering each component of the combination over a period of 11 days. In another embodiment, this period is 10 days. In another embodiment, this period is 9 days. In another embodiment, this period is 8 days. In another embodiment, this period is 7 days. In another embodiment, this period is 6 days. In another embodiment, this period is 5 days. In another embodiment, this period is 4 days. In another embodiment, this period is 3 days. In another embodiment, this period is 2 days. In another embodiment, this period is 24 hours. In another embodiment, this period is 12 hours.

Components (A) and (B) can be entered in any order, for example, component (A) first, and then component (B); or component (B) first, and then component (A).

The ratio of the total amounts of component (A) and component (B) to be administered in a combined preparation may vary, for example, to meet the needs of a subpopulation of patients to be treated, or the needs of one patient, while the different needs of patients may be due to their age, gender, body weight, etc.

Components (A) and (B) present in the same composition or in separate compositions may be independently formulated with one or more pharmaceutically acceptable carriers. The pharmaceutical combinations of the present invention may also contain at least one other antitumor agent or anti-inflammatory or immunosuppressive agent. Examples of therapeutic agents that can be used in combination therapy are described in more detail below in the section on the ways of using the antibodies disclosed in this document.

As used herein, a pharmaceutically acceptable carrier includes any possible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. The carrier is preferably suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg by injection or infusion). Depending on the route of administration, the active compound, i. the antibody, immunoconjugate, or bispecific molecule may be coated with a material to protect the compound from acids and other natural conditions that may inactivate the compound.

Components (A) and/or (B) may be in the form of 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 cause any undesirable toxic effects [see, for example, Berge, S.M. et al. (1977), J. Pharm. Sc., 66:1-19]. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include salts derived from non-toxic inorganic acids such as hydrochloric, nitric, phosphoric,

- 28 042216 sulfuric, hydrobromic, hydroiodic, phosphorous, etc., as well as from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. P. Base addition salts include salts derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, as well as from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

The pharmaceutical combination of the present invention, or a portion thereof, may also contain a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like;

(2) fat-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical combinations of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) 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 maintaining the required particle size in the case of dispersions, and by the use of surfactants.

These combinations or parts thereof may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersants. Microorganism control can be achieved both by the sterilization procedures described above and by the inclusion of various antibacterial and antifungal agents, eg 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 in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be achieved by the inclusion of absorption delaying agents such as aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for preparing sterile injectable solutions or dispersions for immediate administration. The use of such media and agents for pharmaceutically active substances is known from the prior art. Unless any conventional media or agent is incompatible with the active compound, their use in the pharmaceutical compositions of the present invention is contemplated. Additional active compounds may also be included in the compositions.

Therapeutic compositions generally 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 structure suitable for high drug concentration. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be maintained, for example, by applying a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using surfactants. In many cases it will be preferable to include isotonic agents, for example sugars, polyhydric alcohols such as mannitol, sorbitol, or sodium chloride, in the composition. Prolonged absorption of injectable compositions can be achieved by the inclusion in the composition of an agent that slows absorption, for example, monostearate salts and gelatin.

Sterile injectable solutions can be obtained by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of the ingredients listed above, if necessary, followed by sterilizing microfiltration. Dispersions are generally prepared by incorporating the active compound into a sterile vehicle which contains the basic dispersion medium and the other necessary ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization), by which the powder form of the active ingredient, as well as any additional desired ingredient, is obtained from their solution, previously subjected to sterilizing filtration.

The amount of active ingredient that can be combined with the carrier material to form a unit dosage form will vary depending on the subject being treated and the particular route of administration. Amount of active ingredient that can be combined

- 29 042216 a thread with a carrier material to obtain a unit dosage form, as a rule, will be such an amount of the composition that causes a therapeutic effect. From 100%, this amount will typically range from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30% of the active ingredient in combination with pharmaceutically acceptable carrier.

Dosing regimens are adjusted to obtain the optimal desired response (eg, synergistic combination, therapeutic response). For example, a single bolus dose may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the needs of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosing. Dosage unit form, as used herein, refers to physically discrete units suitable as single doses for subjects to be treated; each unit contains a predetermined amount of active compound, calculated to obtain the desired therapeutic effect, together with the necessary pharmaceutical carrier. The technical requirements for unit dosage forms of the present invention are due to (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations in the preparation of such an active compound in relation to the treatment of individuals with sensitivity, and depend directly on them. .

For administration of an anti-LY75 antibody or an anti-CD20 antibody, the dose ranges from about 0.0001 to 100 mg/kg, e.g., 0.001 to 50 mg/kg, 0.005 to 20 mg/kg, 0.01 to 10 mg/kg and more often from 0.01 to 5 mg/kg body weight of the patient receiving it. For example, doses may 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 weight, 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 weight, 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 of body weight or be in the range of 0.1-20, 0.5-15, 1-10, 2-8, 3-7, 4-6 mg/kg. An exemplary treatment regimen includes administration once a day, once every two days, once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every six weeks, once every three months or once every three six months. Preferred dosing regimens for an anti-LY75 antibody of the present invention include 1 mg/kg body weight or 3 mg/kg body weight via intravenous administration, with the antibody administered using one of the following dosing regimens:

(i) every four weeks for six doses, 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 embodiments, the dose of the anti-LY75 antibody (e.g., LY75-DM4) is adjusted to achieve a plasma antibody concentration of 0.01 to 1.5 nM, or 0.018 to 1.2 nM (e.g., about 0.018, 0.037, 0.075, 0.15, 0.3, 0.6 or 1.2 nM). Preferably, the dose of anti-LY75 antibody is adjusted to achieve a plasma antibody concentration of 0.03 to 0.30 nM.

In some embodiments, the anti-CD20 antibody is formulated in a solution at a concentration of 10 mg/ml. In some embodiments, the anti-CD20 antibody is administered intravenously at a dose of 350-400 mg/m ² , preferably once a week. In some embodiments, the dose of an anti-CD20 antibody (e.g., rituximab) is adjusted to achieve a plasma antibody concentration of between about 2.34 and 150 nM (e.g., about 2.34, 4.68, 9.37, 18.75 , 37.5, 75, or 150 nM). An anti-CD20 antibody (eg, rituximab) can be administered subcutaneously at a dose of approximately 1400 mg/23400 units. In some embodiments, the dose is 200, 400, 600, 800, 1000, or 1200 mg. An anti-CD20 antibody (eg, rituximab) can be administered with one or more of cyclophosphamide, hydroxydaunorubicin, oncovin, and prednisone or prednisolone (ie, as part of CHOP therapy).

In some embodiments, the dose of ibrutinib is adjusted to achieve a plasma concentration of between about 15.6 and 1000 nM (e.g., about 15.6, 31.2, 62.5, 125, 250, 500, or 1000 nM). In other embodiments, the dose of ibrutinib is adjusted to achieve a plasma concentration of about 1.56 to 100 nM (e.g., about 1.5, 3.1, 6.2, 12.5, 25, 50, or 100 nM) . The dose of ibrutinib may be approximately 560 mg (eg, four 140 mg capsules). It can be administered orally. In some embodiments, the dose of ibrutinib is about 100, 200, 300, 400, or 500 mg.

Preferably the combination of components (A) and (B) is a synergistic combination. A person skilled in the art will understand that a synergistic combination is a combination in which the effect of the combination is greater than the sum of the effects of its individual components. The synergistic effect can be quantified using the addi

- 30 042216 Chow-Talalai (CI) activity (see Evaluation of combination chemotherapy: integration of nonlinear regression, curve shift, isobologram, and combination index analyses, Zhao L. et al., Clin Cancer Res. (2004), Dec 1 ; 10(23):7994-8004; and Computerized quantitation of synergism and antagonism of taxol, topotecan, and cisplatin against human teratocarcinoma cell growth: a rational approach to clinical protocol design, Chou T.C., Motzer R.J., Tong Y., Bosl G.J. , J Natl Cancer Inst (1994), Oct 19, 86(20):1517-24). This Additivity Index (CI) method is based on the multiple drug effect equation derived from the median effect principle of the law of mass action. It quantifies strong synergistic effect (CI<0.3), synergistic effect (CI=0.3-0.9), additive effect (CI=0.9-1.1), or antagonism/no beneficial effect (CI >1.1) and this provides an algorithm for computer software for automated simulation of drug combinations. Both the efficacy (D(m) value) and the shape of the dose-response curve (m value) of each drug individually and in combination are taken into account. The Chow-Talalai Additivity Index (CI) can be estimated using the Synergy R package (see Preclinical versus Clinical Drugs Combination Studies, Chou T.C., Leuk. Lymphoma. (2008), 49(11):2059-2080, and references therein, all of which are specifically incorporated herein by reference). The CI of the combination can be tested in a suitable cell line, such as the ABC-DLBLC cell line (such as TMD8 or HBL1), such as under the conditions used in Example 26.

Preferably the pharmaceutical combination of the present invention is a synergistic combination wherein the Chow-Talalai Additivity Index (CI) is less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 or 0.2. Preferably the CI is 0.1-0.5, 0.1-0.3 or 0.1-0.2.

In particular, a method is provided for treating cancer in a patient, comprising the simultaneous, sequential or separate administration to a patient in need thereof of therapeutically effective synergistic amounts of components (A) and (B) of a pharmaceutical combination of the present invention. Also provided is a pharmaceutical combination of the present invention for use in the treatment of cancer, wherein synergistic amounts of components (A) and (B) are administered to a patient simultaneously, separately or sequentially for the treatment of cancer. Preferably, the amounts of components (A) and (B) are administered to the patient in order to provide the plasma concentrations disclosed above.

Also presented is the use of synergistic amounts of components (A) and (B) of the pharmaceutical combination of the present invention in the manufacture of a pharmaceutical combination for simultaneous, separate or sequential use in the treatment of cancer. Also provided is a synergistic pharmaceutical combination of the present invention for use in therapy or for use as a drug.

In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dose of each antibody administered falls within the indicated ranges. The antibody is usually administered several times. Dosing intervals may correspond to, for example, administration once a day, twice a week, once a week, once a month, every three months, every six months or once a year. The intervals may also be irregular, as indicated by measuring levels of antibody to the target antigen in the patient's blood. In some methods, the dose is adjusted to achieve a plasma antibody concentration of about 1-1000, 5-750, 10-600, 15-500, 20-400 µg/ml, and in some methods about 25-300 µg/ml.

Alternatively, anti-LY75 antibodies and or anti-CD20 antibodies can be administered as sustained release formulations, in which case less frequent administration is required. The dose and frequency vary depending on the half-life of the antibody from the patient's body. Typically, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dose and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic routes of administration, a relatively low dose is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic routes of administration, administration of relatively high doses at relatively short intervals is sometimes required until the progression of the disease is reduced or stopped, and preferably until the patient demonstrates a partial or complete reduction in the intensity of the symptoms of the disease. After that, a prophylactic regimen can be applied to the patient.

Actual dosage levels of active ingredients in the pharmaceutical combinations of the present invention may be varied in order to obtain an amount of active ingredient effective to achieve the desired therapeutic response for a particular patient, composition and route of administration without toxicity to the patient. The selected dose level will depend on a number of pharmacokinetic factors, including the potency of the specific compositions of the present invention or their ester, salt or amide used, route of administration, time of administration, rate of excretion of the particular compound used, duration of treatment, other medicinal products.

- 31 042216 means, compounds and/or materials used in combination with specific compositions used, age, sex, body weight, condition, general health and history of the patient being treated, and similar factors well known in the field of medicine.

A therapeutically effective dose of an anti-LY75 antibody or an anti-CD20 antibody preferably results in a reduction in the severity of the symptoms of the disease, an increase in the frequency and duration of asymptomatic periods of the disease, or the prevention of deterioration or disability due to the involvement of the disease. For example, for the treatment of tumors mediated by LY75 or CD20, a therapeutically effective dose preferably inhibits cell growth or tumor growth by at least about 20%, at least about 30%, more preferably at least about 40%, at least at least about 50%, even more preferably at least about 60%, at least about 70%, and even more preferably at least about 80% or at least about 90% compared to subjects not receiving treatment. The ability of a compound to inhibit tumor growth can be evaluated in an animal model system predictive of efficacy against human tumors. Alternatively, this property of the composition can be assessed by examining the ability of the compound to inhibit cell growth, which inhibition can be measured in vitro using assays known to the practitioner. A therapeutically effective amount of a therapeutic compound may reduce the size of a tumor or otherwise reduce the intensity of symptoms in a subject. One of ordinary skill in the art will be able to determine such amounts based on such factors as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration chosen.

The pharmaceutical combination of the present invention can be administered via one or more routes of administration using one or more of a number of methods known in the art. Components (A) and (B) may be administered via the same route or via different routes. As will be appreciated by one of skill in the art, the route and/or mode of administration will vary depending on the desired results. Preferred routes of administration of the antibodies of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, such as by injection or infusion. The phrase parenteral administration as used herein means administration methods other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular , intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

Alternatively, an anti-LY75 antibody or an anti-CD20 antibody can be administered via a non-parenteral route, such as a topical, epidermal, or transmucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually, or topically.

Active compounds can be formulated with carriers that will protect the compound from rapid release, such as in controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Many methods for preparing such formulations are patented or well known to those skilled in the art [see, for example, Sustained and Controlled Release Drug Delivery Systems (1978), J.R. Robinson, ed., Marcel Dekker, Inc., N.Y].

Therapeutic compositions can be administered using medical devices known in the art. For example, in a preferred embodiment, component (A) and/or (B) can be administered using a needleless hypodermic injection device such as those disclosed in US Pat. Nos. 5,399,163; 5383851; 5312335; 5064413; 4941880; 4,790,824 or 4,596,556. Examples of well known implants and modules useful in the present invention include an implantable microinfusion pump for controlled rate drug dispensing as disclosed in US Pat. No. 4,487,603; therapeutic device for the introduction of drugs through the skin, disclosed in US patent No. 4486194; a drug infusion pump for delivering a drug at a precise infusion rate as disclosed in US Pat. No. 4,447,233; an implantable variable volume infusion device for continuous drug delivery as disclosed in US Pat. No. 4,447,224; an osmotic drug delivery system having multi-chamber compartments disclosed in US Pat. No. 4,439,196; and the osmotic drug delivery system disclosed in US Pat. No. 4,475,196. These patents are incorporated herein by reference. Many other such implants, delivery systems and modules are known to those skilled in the art.

In some embodiments, anti-LY75 antibodies and/or anti-CD20 antibodies can be formulated

- 32 042216 to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) blocks many highly hydrophilic compounds. To ensure that the BBB is crossed by therapeutic compounds (if necessary), they can be formulated, for example, in liposomes. For methods of preparing liposomes, see, for example, US Pat. Nos. 4,522,811; 5,374,548 and 5,399,331. Liposomes may contain one or more functional moieties that are selectively transported to specific cells or organs, thereby improving targeted drug delivery [see, e.g., V.V. Ranade (1989), J. Clin. Pharmacol., 29:685]. Exemplary targeting functional moieties include folate or biotin (see, for example, US Pat. No. 5,416,016); 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. Keinanen; M.L. Laukkanen (1994), FEBS Lett. 346:123; J.J. killion; I.J. Fidler (1994) Immunomethods 4:273.

Ways of application and methods.

As used herein, the term subject is intended to include humans and non-human animals. Non-human animals include all vertebrate animals, such as 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 and/or CD20 activity.

The methods are particularly suitable for the treatment of human patients having a disorder associated with abnormal LY75 and/or CD20 expression. In view of the expression of LY75 on tumor cells, the combinations and methods of the present invention can be used to treat a subject with an oncogenic disorder, for example, a disorder characterized by the presence of tumor cells expressing LY75, or in the manufacture of a drug for the treatment of such a disorder, including, for example, leukemia, including chronic lymphocytic leukemia and acute myeloid leukemia, non-Hodgkin's lymphoma, including DLBCL, B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, mucosal lymphoid tissue (MALT) lymphoma, T-cell-rich B-cell lymphoma/ histiocytes, Burkitt's lymphoma, lymphoplasmacytic lymphoma, small lymphocytic lymphoma, marginal zone cell lymphoma, T-cell lymphoma, peripheral T-cell lymphoma, anaplastic large cell lymphoma, and angioimmunoblastic T-cell lymphoma. LY75 has been shown to be internalized when bound to an antibody, as illustrated in Examples 5 and 7 below, thus allowing the use of anti-LY75 antibodies in any loading mechanism, e.g., in an ADC approach, in a radioimmunoconjugate, or in an ADEPT approach. .

Anti-LY75 antibodies, usually administered as ADC, can be used to inhibit or block the function of LY75, which in turn can be associated with the prevention or reduction of certain symptoms of the disease, thus implying LY75 as a mediator of the disease. . This can be achieved by bringing the sample and the control sample into contact with the antibody to LY75 under conditions that ensure the formation of a complex between the antibody and LY75. In the sample and the control sample, any complexes formed between the antibody and LY75 are identified and compared.

Suitable routes of administration of antibody compositions (eg, monoclonal antibodies and immunoconjugates) in vivo and in vitro are well known in the art and can be selected by those of ordinary skill in the art. For example, antibody compositions can be administered by injection (eg, intravenous or subcutaneous). Suitable doses of the molecules used will depend on the age and body weight of the subject, as well as the concentration and/or composition of the antibody composition.

As previously described, anti-LY75 antibodies and/or anti-CD20 antibodies can be co-administered with one or more other therapeutic agents, such as a cytotoxic agent, a radiotoxic agent, or an immunosuppressive agent. The antibody may be associated with the agent (as an immune complex) or may be administered separately from the agent. In the latter case (separate administration), the antibody may be administered before, after, or simultaneously with the administration of the agent, or may be administered in conjunction with other known therapies, such as anti-cancer therapy, such as radiation therapy. Such therapeutic agents include, among others, antitumor agents such as doxorubicin (adriamycin), cisplatin, bleomycin sulfate, carmustine, chlorambucil, as well as cyclophosphamide and hydroxyurea, which are themselves effective only at levels that are toxic or subtoxic to the patient. Cisplatin is administered intravenously at a dose of 100 mg/kg once every four weeks, and adriamycin is administered intravenously at a dose of 60-75 mg/ml once every 21 days. Other agents suitable for co-administration with the antibodies of the present invention include other agents used to treat forms of cancer such as gastric cancer, colon cancer, prostate cancer, breast cancer, ovarian cancer, or lung cancer, such as Avastin ®, 5FU and gemcitabine. Co-administration of antibodies to

- 33 042216

LY75 or their antigen-binding fragments of the present invention with chemotherapeutic agents provides two anti-cancer agents acting through different mechanisms that provide a cytotoxic effect on human tumor cells. Such co-administration may solve problems associated with the development of drug resistance or alteration in the antigenicity of tumor cells which may render them unreactive with the antibody.

The pharmaceutical combinations of the present invention can also be administered with serum and/or the complement system. These compositions may be advantageous if the complement system is in close proximity to the antibodies. Alternatively, antibodies and the complement system or blood serum can be administered separately.

Also within the scope of the present invention are kits containing components (A) and (B) together with instructions for use. The kit may further comprise one or more additional reagents, such as an immunosuppressive reagent, a cytotoxic agent, or a radiotoxic agent, or one or more additional antibodies (e.g., an antibody having complementary activity that binds to a different epitope on the LY75 antigen than that of the first antibody). ).

Accordingly, patients receiving treatment with the pharmaceutical combinations of the present invention may additionally be administered (before, simultaneously with, or after administration of the antibody disclosed herein) another therapeutic agent, such as a cytotoxic or radiotoxic agent, that enhances or enhances the therapeutic effect. antibodies.

In other embodiments, the subject may receive additional treatment with an agent that modulates, eg enhances or inhibits, the expression or activity of Fcy or Fcy receptors, through, for example, treating the subject with a cytokine. Preferred cytokines for administration during multispecific molecule treatment include granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-γ (IFN-γ) and tumor necrosis factor (TNF).

All literature referenced in this specification, including without limitation all documents, publications, patents, patent applications, presentations, texts, reports, manuscripts, brochures, books, online publications, journal articles, periodicals, newsletters about products, and the like, are hereby incorporated into this specification by reference in their entirety. The discussion of literature sources in this document is intended only to summarize the statements made by their authors and does not acknowledge the fact that any literature source forms part of the prior art, and applicants reserve the right to challenge the reliability and significance of the referenced literature sources.

Although the above invention has been described in some detail by way of illustration and example for the sake of clarity of understanding, those of ordinary skill in the art, in light of the teachings of the present invention, will readily appreciate that certain changes and modifications may be made to it without departing from the spirit or scope of the dependent claims. inventions.

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

Example 1 Preparation of human monoclonal antibodies against the LY75 antigen.

Mice (XenoMouse IgG1) were immunized with CHO cells transfected with full length LY75 according to standard procedures.

The specificity of antibodies raised against LY75 was examined by flow cytometry using LY75-transfected HEK293 cells and subsequently using LY75-expressing HT29 cells. In order to examine the ability of antibodies to bind to the LY75 protein on the cell surface, the antibodies were incubated with cells expressing LY75. Cells were washed with FACS buffer (DPBS, 2% FBS), centrifuged and resuspended in 100 μl of diluted anti-LY75 primary antibody (also diluted in FACS buffer). The antibody-cell line complex was incubated on ice for 60 minutes and then washed twice with the FACS buffer described above. The antibody cell pellet was resuspended in 100 μl of diluted secondary antibody (also diluted in FACS buffer) and incubated on ice for 60 min on ice. The pellet was washed as described above and resuspended in 200 µl of FACS buffer. Samples were loaded into a BD FACScanto II flow pitometer and data was analyzed using the BD FACSdiva software (results not shown).

Example 2 Establishment of structural characteristics of monoclonal antibodies to LY75.

The cDNA sequences encoding the heavy and light chain variable regions of the LY75_A1 monoclonal antibody were obtained using standard PCR techniques and sequenced using standard DNA sequencing techniques.

Antibody sequences can be mutated to revert to germline residues at one or more residue positions.

Nucleotide and amino acid sequences of the heavy chain variable region

LY75_A1 are shown under SEQ ID NO: 3 and 1, respectively.

The nucleotide and amino acid sequences of the LY75_A1 light chain variable region are shown under SEQ ID NOs: 4 and 2, respectively.

Comparison of the LY75_A1 immunoglobulin heavy chain sequence with known human immunoglobulin heavy chain germline sequences demonstrated that the LY75_A1 heavy chain uses the VH segment from human germline VH 3-15 and the JH segment from human germline JH JH4. Further analysis of the LY75_A1 VH sequence using the Kabat CDR region detection system resulted in the identification of the heavy chain CDR1, CDR2 and CDR3 regions shown under SEQ ID NOS: 5, 6 and 7, respectively. Sequence alignments of CDR1, CDR2, and CDR3 of LY75_A1 VH with the VH 3-15 germline and JH JH4 germline sequences are shown in FIG. 1.

Comparison of the LY75_A1 immunoglobulin light chain sequence with known human immunoglobulin light chain germline sequences demonstrated that the LY75_A1 light chain uses the VK segment from human germline VK 012 and the JK segment from human JK4 human germline JK. Further analysis of the LY75_A1 VK sequence using the Kabat CDR region detection system resulted in the light chain CDR1, CDR2 and CDR3 regions shown under SEQ ID NOS: 8, 9 and 10, respectively. Sequence alignments of CDR1, CDR2, and CDR3 VK LY75_A1 with the VK O12 germline and JH JK4 germline sequences are shown in FIG. 2.

Example 3 Immunohistochemical study using a monoclonal antibody to LY75.

Using human monoclonal antibodies specific for LY75, an immunohistochemical study was performed using FFPE HT-29 and A549 cell pellets, non-Hodgkin's lymphoma and pancreatic cancer FFPE specimens and fresh frozen lymphoma/leukemia tumor specimens, sections of ovarian cancer, pancreatic cancer pancreatic tumors and breast cancer and matrix with normal tissues.

Materials and methods.

Materials.

Xylenes (X5P-1gal) from Fisher Scientific, Pennsylvania, USA.

100% Ethanol HistoPrep (HC-800-1GAL) from Fisher Scientific, Pennsylvania, USA.

10x citrate buffer for thermal epitope unmasking (AP9003125) from Thermo Scientific, Massachusetts, USA.

Peroxidase Inhibitor Thermo Scientific* Pierce* (35000) from Thermo Scientific, Massachusetts, USA. Serum Free Protein Blocking Solution (X0909) from Dako, CA, USA.

Secondary antibody: FITC-conjugated goat anti-human IgG Fab-specific antibody (109-097-003) from Jackson ImmunoResearch, Pennsylvania, USA.

ChromPure human IgG, whole molecule (09-000-003), from Jackson ImmunoResearch, Pennsylvania, USA.

Tertiary antibody: mouse anti-FITC antibody (ab10257) from Abcam, Massachusetts, USA.

Purified human IgG for isotype control (1-001A) from R&D Systems, Minnesota, USA.

Tween-20 (BP337-100) from Fisher Scientific, Pennsylvania, USA.

Acetone (BP2403-4) from Fisher Scientific, Pennsylvania, USA.

HRP-conjugated polymer Dual Link EnVision+ for mouse and rabbit antibodies (K4063) from Dako, CA, USA.

DAB kit with 2 solutions (882014) from Invitrogen, New York, USA.

Harris Hematoxylin (23-245-677) from Fisher Scientific, Pennsylvania, USA.

Faramount embedding medium (S302580) from Dako, California, USA.

Tissue sections and matrices were purchased from US Biomax Inc., MD, USA or Origene, MD, USA.

Obtaining micropreparations FFPE - dewaxing and rehydration.

Dewaxing of FFPE slides in xylene (2x3 min) followed by rehydration using 1:1 mixture of xylene and 100% ethanol (1x3 min), 100% ethanol (2x3 min), 95% ethanol (1x3 min), 70 % ethanol (1x3 min), 50% ethanol (1x3 min) and tap water (1x3 min).

Obtaining micropreparations FFPE - antigen unmasking (microwave radiation).

The LY75 antigen was unmasked using high power microwave heating to boiling then low power for 10 minutes in 50 ml 1x citrate buffer in a Coplin flask. Slides were then allowed to cool to room temperature for an additional 15 min, then washed with tap water for 3 min. Each tissue section/TMA was circled with a pencil to create a hydrophobic barrier, and then the slides were washed 3 times in PBS for 3 minutes for each wash.

Preparation of micropreparations FF.

Micropreparations were removed from storage at -80°C and dried at room temperature in a fume hood for 20-30 min. Micropreparations were fixed for 10 min in ice-cold acetone at -20°C, then they were dried for 20 min in a fume hood at room temperature. Micropreparations were washed and rehydrated in PBS with 3 washes, each for 3 min. Sections were outlined with a pencil to create a hydrophobic barrier.

Preparation of antibody complexes.

Primary anti-LY75 antibody was diluted in serum free protein blocking solution (SFPB) to give a solution at 20 times the desired final concentration (20 μg/mL for a final concentration of 1 μg/mL). A goat secondary antibody to human immunoglobulin G (IgG) antigen-binding fragment (Fab) was similarly prepared in SFPB to create an equal concentration solution.

Equal volumes of primary and secondary antibody were pooled in a labeled tube, mixed gently, and incubated for 3 min at room temperature, resulting in a primary antibody concentration 10 times the required final concentration (10 μg/mL for a final concentration of 1 μg/mL). ml). This mixture was diluted 1:5 with SFPB, mixed gently, and incubated for 30 minutes at room temperature, resulting in a primary antibody concentration twice the required final concentration (2 µg/mL for a 1 µg/mL final concentration) .

To obtain the final staining complexes, a 1% (10 μg/μl) solution of human IgG was prepared in SFPB with an equal volume added to the primary/secondary antibody mixture. This combination was gently mixed and incubated at room temperature for 30 min diluting the primary/secondary antibody mixture to half the primary antibody concentration, resulting in the desired final primary antibody concentration (1 μg/mL).

Immunostaining.

Meanwhile, endogenous tissue peroxidase activity was blocked by incubating the tissues with a peroxidase inhibitor for 5-10 minutes at room temperature in a humidification chamber. The slides were then washed in PBS 3 times for 3 min for each wash. The tissues were incubated in SFPB for 30 min at room temperature in a humidifying chamber. The final staining complexes were applied to each tissue section and/or microarray, while the micropreparations were incubated for 30 min at room temperature in a humidification chamber. The slides were then washed once in PBS and once in PBST (PBS+0.125% Tween-20) for 3 minutes for each wash. Mouse tertiary antibody to FITC was applied at a concentration of 2 μg/ml for 30 minutes at room temperature in a humidification chamber. Sections were then washed once in PBS and once in PBST for 3 min for each wash. Then, HRP-conjugated polymer Dual Link EnVision+ for mouse and rabbit antibodies was applied to the tissues, and the slides were incubated for 30 min at room temperature in a humidifying chamber. The slides were then washed once in PBS, once in PBST for 3 min for each wash. Tissues were incubated in DAB solution prepared according to the manufacturer's instructions at room temperature for 10 min. Then the micropreparations were washed once with running tap water for 2 min and once in PBS for 3 min. Micropreparations were counterstained with hematoxylin for 30 s at room temperature and washed with running tap water. The slides were dried at room temperature for 30 min and then the slides were placed under coverslips using Faramount embedding medium.

Results.

LY75_A1 was positive with triple negative breast cancer FFPE specimens, with 77% of the sections showing positive staining and 55% showing intense (+++) staining.

Staining for LY75 of normal FF tissues generally ranged from absent to low. The epithelium of the ducts of the breast, salivary gland and pancreas showed a level of staining from marked low to moderate, while there was a low level of positive staining of the spleen. Thus, antibodies directed to LY75 can be used as therapeutics and diagnostics in some of the investigated cancers and possibly other cancers characterized by LY75 expression.

Example 4 Efficacy of DM1 Conjugated Anti-LY75 Monoclonal Antibodies on HT-29 Cells.

Materials.

CellStripper (non-enzymatic cell dissociator) (MT-25-056CI) from Fisher

- 36 042216

Scientific, Pennsylvania, USA.

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

RPMI 1640 medium (MT-10-041-CM) from Fisher Scientific, PA, USA.

CellTiter Glo (G7572) from Promega, Wisconsin, USA.

Way.

The cells were dissociated using a CellStripper and their number was counted. 5e3 cells/well were centrifuged to pellet (in the case of cells in suspension, more can be used depending on the cell doubling time, such as 10e3 cells/well). The pellet was resuspended in culture medium to obtain a concentration of 1e5 cells/ml.

μl/well of the cell suspension was added to the wells of a 96-well plate with white walls and a transparent bottom. Antibodies were diluted and titrated at 8 points (titrations with 3-fold dilutions) corresponding to concentrations of 0-20 nM (twice the test concentrations). Diluted antibodies or media (for untreated samples) (50 µl/well) were added to the appropriate wells. Excess medium (200 μl/well) was added to the outer rows and columns of the plate to prevent evaporation. The tablet was incubated for 72 h at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 min. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed 1 time using 100 μl/well PBS (in the case of cells in suspension, the plate was first centrifuged to pellet the cells). 100 μl/well PBS and 100 μl CellTiter Glo were added to each well and mixed thoroughly to obtain a mixture. The plate was incubated in the dark at room temperature for 15 minutes and observed by microscopy to ensure that effective cell lysis had occurred. The plate was then read on a Glomax luminometer.

Results.

The results are shown in FIG. 3a, which shows a subpopulation of antibodies known to bind to LY75 that can induce killing of HT-29 cells. This suggests that while these antibodies can bind to LY75, only some of them are effective when conjugated to DM1. Then, antibodies were selected from the subpopulation for further analysis of cytotoxic activity.

Example 5 Efficacy of anti-LY75 monoclonal antibodies conjugated to DM1 and conjugated to DM4 against colon and rectal cancer cells.

Materials.

CellStripper (non-enzymatic cell dissociator) (MT-25-056CI) from Fisher Scientific, Pennsylvania, USA.

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

RPMI 1640 medium (MT-10-041-CM) from Fisher Scientific, PA, USA.

CellTiter Glo (G7572) from Promega, Wisconsin, USA.

Way.

The cells were dissociated using a CellStripper and their number was counted. 5e3 cells/well were centrifuged to pellet (in the case of cells in suspension, more can be used depending on the cell doubling time, such as 10e3 cells/well). The pellet was resuspended in culture medium to obtain a concentration of 1e5 cells/ml.

μl/well of the cell suspension was added to the wells of a 96-well plate with white walls and a transparent bottom. Antibodies were diluted and titrated at 8 points (titrations with 3-fold dilutions) corresponding to concentrations of 0-20 nM (twice the test concentrations). Diluted antibodies or media (for untreated samples) (50 µl/well) were added to the appropriate wells. Excess medium (200 μl/well) was added to the outer rows and columns of the plate to prevent evaporation. The tablet was incubated for 72 h at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 min. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed 1 time using 100 μl/well PBS (in the case of cells in suspension, the plate was first centrifuged to pellet the cells). 100 μl/well PBS and 100 μl CellTiter Glo were added to each well and mixed thoroughly to obtain a mixture. The plate was incubated in the dark at room temperature for 15 minutes and observed by microscopy to ensure that effective cell lysis had occurred. The plate was then read on a Glomax luminometer.

Results.

In FIG. 3b shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 on HT-29 cells. These results demonstrate an increase in cytotoxic activity proportional to antibody concentration compared to other toxin-conjugated anti-LY75 antibodies (selected from Example 1).

- 37 042216

Example 6 Efficacy of anti-LY75 monoclonal antibodies conjugated to DM1 and conjugated to DM4 on lymphoma cell lines.

Materials.

CellStripper (non-enzymatic cell dissociator) (MT-25-056CI) from Fisher

Scientific, Pennsylvania, USA.

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

RPMI 1640 medium (MT-10-041-CM) from Fisher Scientific, PA, USA.

CellTiter Glo (G7572) from Promega, Wisconsin, USA.

Way.

The cells were dissociated using a CellStripper and their number was counted. 5e3 cells/well were centrifuged to pellet (in the case of cells in suspension, more can be used depending on the cell doubling time, such as 10e3 cells/well). The pellet was resuspended in culture medium to obtain a concentration of 1e5 cells/ml.

μl/well of the cell suspension was added to the wells of a 96-well plate with white walls and a transparent bottom. Antibodies were diluted and titrated at 8 points (titrations with 3-fold dilutions) corresponding to concentrations of 0-20 nM (twice the test concentrations). Diluted antibodies or media (for untreated samples) (50 µl/well) were added to the appropriate wells. Excess medium (200 μl/well) was added to the outer rows and columns of the plate to prevent evaporation. The tablet was incubated for 72 h at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 min. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed 1 time using 100 μl/well PBS (in the case of cells in suspension, the plate was first centrifuged to pellet the cells). 100 μl/well PBS and 100 μl CellTiter Glo were added to each well and mixed thoroughly to obtain a mixture. The plate was incubated in the dark at room temperature for 15 minutes and observed by microscopy to ensure that effective cell lysis had occurred. The plate was then read on a Glomax luminometer.

Results.

In FIG. 3c shows the cytotoxic activity of DM1 and DM4 conjugated anti-LY75 antibodies against RAJI cells. In FIG. 3d shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 on Namalwa cells. In FIG. 3e shows the cytotoxic activity of DM1 and DM4 conjugated anti-LY75 antibodies against Karpas 299 cells. These results demonstrate an increase in cytotoxic activity proportional to antibody concentration compared to other DM1 and DM4 conjugated anti-LY75 antibodies (selected from Example 1) .

Example 7 Efficacy of DM1- and DM4-conjugated anti-LY75 monoclonal antibodies against pancreatic cancer cell lines.

Materials.

CellStripper (non-enzymatic cell dissociator) (MT-25-056CI) from Fisher Scientific, Pennsylvania, USA.

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

RPMI 1640 medium (MT-10-041-CM) from Fisher Scientific, PA, USA.

CellTiter Glo (G7572) from Promega, Wisconsin, USA.

Way.

The cells were dissociated using a CellStripper and their number was counted. 5e3 cells/well were centrifuged to pellet (in the case of cells in suspension, more can be used depending on the cell doubling time, such as 10e3 cells/well). The pellet was resuspended in culture medium to obtain a concentration of 1e5 cells/ml.

μl/well of the cell suspension was added to the wells of a 96-well plate with white walls and a transparent bottom. Antibodies were diluted and titrated at 8 points (titrations with 3-fold dilutions) corresponding to concentrations of 0-20 nM (twice the test concentrations). Diluted antibodies or media (for untreated samples) (50 µl/well) were added to the appropriate wells. Excess medium (200 μl/well) was added to the outer rows and columns of the plate to prevent evaporation. The tablet was incubated for 72 h at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 min. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed 1 time using 100 μl/well PBS (in the case of cells in suspension, the plate was first centrifuged to pellet the cells). 100 μl/well PBS and 100 μl CellTiter Glo were added to each well and mixed thoroughly to obtain a mixture. The plate was incubated in the dark at room temperature for 15 minutes and observed by microscopy to ensure that effective cell lysis had occurred. The plate was then read on a Glomax luminometer.

Results.

In FIG. 3f shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 on BxPC3 cells. In FIG. 3g shows the cytotoxic activity of anti-LY75 antibodies conjugated with DM1 and DM4 on HupT4 cells. In FIG. 3h shows the cytotoxic activity of anti-LY75 antibodies conjugated with DM1 and DM4 against HPAFFII cells. These results demonstrate an increase in cytotoxic activity proportional to antibody concentration compared to other DM1 and DM4 conjugated anti-LY75 antibodies (selected from Example 1).

Example 8 Efficacy of DM1-conjugated and DM4-conjugated anti-LY75 monoclonal antibodies against cell lines that are a substrate of chronic lymphocytic leukemia.

Materials.

CellStripper (non-enzymatic cell dissociator) (MT-25-056CI) from Fisher Scientific, Pennsylvania, USA.

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

RPMI 1640 medium (MT-10-041-CM) from Fisher Scientific, PA, USA.

CellTiter Glo (G7572) from Promega, Wisconsin, USA.

Way.

The cells were dissociated using a CellStripper and their number was counted. 5e3 cells/well were centrifuged to pellet (in the case of cells in suspension, more can be used depending on the cell doubling time, such as 10e3 cells/well). The pellet was resuspended in culture medium to obtain a concentration of 1e5 cells/ml.

μl/well of the cell suspension was added to the wells of a 96-well plate with white walls and a transparent bottom. Antibodies were diluted and titrated at 8 points (titrations with 3-fold dilutions) corresponding to concentrations of 0-20 nM (twice the test concentrations). Diluted antibodies or media (for untreated samples) (50 µl/well) were added to the appropriate wells. Excess medium (200 μl/well) was added to the outer rows and columns of the plate to prevent evaporation. The tablet was incubated for 72 h at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 min. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed 1 time using 100 μl/well PBS (in the case of cells in suspension, the plate was first centrifuged to pellet the cells). 100 μl/well PBS and 100 μl CellTiter Glo were added to each well and mixed thoroughly to obtain a mixture. The plate was incubated in the dark at room temperature for 15 minutes and observed by microscopy to ensure that effective cell lysis had occurred. The plate was then read on a Glomax luminometer.

Results.

In FIG. 3i shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 on EHEB cells. In FIG. 3j shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 on Mec-1 cells. These results demonstrate an increase in cytotoxic activity proportional to antibody concentration compared to other DM1 and DM4 conjugated anti-LY75 antibodies (selected from Example 1).

Example 9 Efficacy of DM1-conjugated and DM4-conjugated anti-LY75 monoclonal antibodies against cell lines that are a substrate of acute monocytic leukemia.

Materials.

CellStripper (non-enzymatic cell dissociator) (MT-25-056CI) from Fisher Scientific, Pennsylvania, USA.

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

RPMI 1640 medium (MT-10-041-CM) from Fisher Scientific, PA, USA.

CellTiter Glo (G7572) from Promega, Wisconsin, USA.

Way.

The cells were dissociated using a CellStripper and their number was counted. 5e3 cells/well were centrifuged to pellet (in the case of cells in suspension, more can be used depending on the cell doubling time, such as 10e3 cells/well). The pellet was resuspended in culture medium to obtain a concentration of 1e5 cells/ml.

μl/well of the cell suspension was added to the wells of a 96-well plate with white walls and a transparent bottom. Antibodies were diluted and titrated at 8 points (titrations with 3-fold dilutions) corresponding to concentrations of 0-20 nM (twice the test concentrations). Diluted antibodies or media (for untreated samples) (50 µl/well) were added to the appropriate wells. Excess medium (200 μl/well) was added to the outer rows and columns of the plate to prevent evaporation. The tablet was incubated for 72 h at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 min. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed 1 time using 100 μl/well PBS (in the case of cells in suspension, the plate was first centrifuged to pellet the cells). 100 μl/well PBS and 100 μl CellTiter Glo were added to each well and mixed thoroughly to obtain a mixture. The plate was incubated in the dark at room temperature for 15 min and observed by microscopy to ensure that effective

- 39 042216 cell lysis. The plate was then read on a Glomax luminometer.

Results.

In FIG. 3k shows the cytotoxic activity of DM1 and DM4 conjugated anti-LY75 antibodies against AML-193 cells. These results demonstrate an increase in cytotoxic activity proportional to antibody concentration compared to other DM1 and DM4 conjugated anti-LY75 antibodies (selected from Example 1).

Example 10 Efficacy of anti-LY75 monoclonal antibodies conjugated to DM1 and conjugated to DM4 against breast cancer cell lines.

Materials.

CellStripper (non-enzymatic cell dissociator) (MT-25-056CI) from Fisher Scientific, Pennsylvania, USA.

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

RPMI 1640 medium (MT-10-041-CM) from Fisher Scientific, PA, USA.

CellTiter Glo (G7572) from Promega, Wisconsin, USA.

Way.

The cells were dissociated using a CellStripper and their number was counted. 5e3 cells/well were centrifuged to pellet (in the case of cells in suspension, more can be used depending on the cell doubling time, such as 10e3 cells/well). The pellet was resuspended in culture medium to obtain a concentration of 1e5 cells/ml.

μl/well of the cell suspension was added to the wells of a 96-well plate with white walls and a transparent bottom. Antibodies were diluted and titrated at 8 points (titrations with 3-fold dilutions) corresponding to concentrations of 0-20 nM (twice the test concentrations). Diluted antibodies or media (for untreated samples) (50 µl/well) were added to the appropriate wells. Excess medium (200 μl/well) was added to the outer rows and columns of the plate to prevent evaporation. The tablet was incubated for 72 h at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 min. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed 1 time using 100 μl/well PBS (in the case of cells in suspension, the plate was first centrifuged to pellet the cells). 100 μl/well PBS and 100 μl CellTiter Glo were added to each well and mixed thoroughly to obtain a mixture. The plate was incubated in the dark at room temperature for 15 minutes and observed by microscopy to ensure that effective cell lysis had occurred. The plate was then read on a Glomax luminometer.

Results.

In FIG. 3l shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 on HCC 70 cells (ER-negative, PR-negative and Her2-negative). In FIG. 3m shows the cytotoxic activity of DM1 and DM4 conjugated anti-LY75 antibodies against HCC 1806 cells (ER-negative, PR-negative and Her2-negative). In FIG. 3n shows the cytotoxic activity of DM1 and DM4 conjugated anti-LY75 antibodies against MDA-MB-468 cells. These results demonstrate an increase in cytotoxic activity proportional to antibody concentration.

Example 11 Efficacy of DM1- and DM4-conjugated anti-LY75 monoclonal antibodies against bladder cancer cell lines.

Materials.

CellStripper (non-enzymatic cell dissociator) (MT-25-056CI) from Fisher Scientific, Pennsylvania, USA.

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

RPMI 1640 medium (MT-10-041-CM) from Fisher Scientific, PA, USA.

CellTiter Glo (G7572) from Promega, Wisconsin, USA.

Way.

The cells were dissociated using a CellStripper and their number was counted. 5e3 cells/well were centrifuged to pellet (in the case of cells in suspension, more can be used depending on the cell doubling time, such as 10e3 cells/well). The pellet was resuspended in culture medium to obtain a concentration of 1e5 cells/ml.

μl/well of the cell suspension was added to the wells of a 96-well plate with white walls and a transparent bottom. Antibodies were diluted and titrated at 8 points (titrations with 3-fold dilutions) corresponding to concentrations of 0-20 nM (twice the test concentrations). Diluted antibodies or media (for untreated samples) (50 µl/well) were added to the appropriate wells. Excess medium (200 μl/well) was added to the outer rows and columns of the plate to prevent evaporation. The tablet was incubated for 72 h at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 min. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed once with 100 μl/well PBS (in the case of cells in suspension, the plate was first centrifuged to pellet the cells). 100 μl/well PBS and 100 μl CellTiter Glo were added to each well and mixed thoroughly to obtain a mixture. The plate was incubated in the dark at room temperature for minutes and observed by microscopy to ensure that effective cell lysis had occurred. The plate was then read on a Glomax luminometer.

Results.

In FIG. 3o shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 on RT4 cells. In FIG. 3p shows the cytotoxic activity of DM1 and DM4 conjugated anti-LY75 antibodies on 5637 cells. FIG. 3q shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 on SW780 cells. These results demonstrate an increase in cytotoxic activity proportional to antibody concentration.

Example 12 Efficacy of anti-LY75 monoclonal antibodies conjugated to DM1 and conjugated to DM4 against head and neck cancer cell lines.

Materials.

CellStripper (non-enzymatic cell dissociator) (MT-25-056CI) from Fisher Scientific, Pennsylvania, USA.

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

RPMI 1640 medium (MT-10-041-CM) from Fisher Scientific, PA, USA.

CellTiter Glo (G7572) from Promega, Wisconsin, USA.

Way.

The cells were dissociated using a CellStripper and their number was counted. 5e3 cells/well were centrifuged to pellet (in the case of cells in suspension, more can be used depending on the cell doubling time, such as 10e3 cells/well). The pellet was resuspended in culture medium to obtain a concentration of 1e5 cells/ml.

μl/well of the cell suspension was added to the wells of a 96-well plate with white walls and a transparent bottom. Antibodies were diluted and titrated at 8 points (titrations with 3-fold dilutions) corresponding to concentrations of 0-20 nM (twice the test concentrations). Diluted antibodies or media (for untreated samples) (50 µl/well) were added to the appropriate wells. Excess medium (200 μl/well) was added to the outer rows and columns of the plate to prevent evaporation. The tablet was incubated for 72 h at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 min. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed 1 time using 100 μl/well PBS (in the case of cells in suspension, the plate was first centrifuged to pellet the cells). 100 μl/well PBS and 100 μl CellTiter Glo were added to each well and mixed thoroughly to obtain a mixture. The plate was incubated in the dark at room temperature for 15 minutes and observed by microscopy to ensure that effective cell lysis had occurred. The plate was then read on a Glomax luminometer.

Results.

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

Example 13 Efficacy of anti-LY75 monoclonal antibodies conjugated to DM1 and conjugated to DM4 against esophageal cancer cell lines.

Materials.

CellStripper (non-enzymatic cell dissociator) (MT-25-056CI) from Fisher Scientific, Pennsylvania, USA.

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

RPMI 1640 medium (MT-10-041-CM) from Fisher Scientific, PA, USA.

CellTiter Glo (G7572) from Promega, Wisconsin, USA.

Way.

The cells were dissociated using a CellStripper and their number was counted. 5e3 cells/well were centrifuged to pellet (in the case of cells in suspension, more can be used depending on the cell doubling time, such as 10e3 cells/well). The pellet was resuspended in culture medium to obtain a concentration of 1e5 cells/ml.

μl/well of the cell suspension was added to the wells of a 96-well plate with white walls and a transparent bottom. Antibodies were diluted and titrated at 8 points (titrations with 3-fold dilutions) corresponding to concentrations of 0-20 nM (twice the test concentrations). Diluted antibodies or media (for untreated samples) (50 µl/well) were added to the appropriate wells. Excess medium (200 μl/well) was added to the outer rows and columns of the plate to prevent evaporation. The tablet was incubated for 72 h at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 min. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed once with 100 μl/well PBS (in the case of cells in suspension, the plate was first centrifuged to pellet the cells). 100 μl/well PBS and 100 μl CellTiter Glo were added to each well and mixed thoroughly to obtain a mixture. The plate was incubated in the dark at room temperature for minutes and observed by microscopy to ensure that effective cell lysis had occurred. The plate was then read on a Glomax luminometer.

Results.

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

Example 14 Efficacy of DM1- and DM4-conjugated anti-LY75 monoclonal antibodies against ovarian cancer cell lines.

Materials.

CellStripper (non-enzymatic cell dissociator) (MT-25-056CI) from Fisher Scientific, Pennsylvania, USA.

PBS, pH 7.4 (1X) (SH30028LS) from Fisher Scientific, PA, USA. RPMI 1640 medium (MT-10041-CM) from Fisher Scientific, PA, USA. CellTiter Glo (G7572) from Promega, Wisconsin, USA.

Way.

The cells were dissociated using a CellStripper and their number was counted. 5e3 cells/well were centrifuged to pellet (in the case of cells in suspension, more can be used depending on the cell doubling time, such as 10e3 cells/well). The pellet was resuspended in culture medium to obtain a concentration of 1e5 cells/ml.

μl/well of the cell suspension was added to the wells of a 96-well plate with white walls and a transparent bottom. Antibodies were diluted and titrated at 8 points (titrations with 3-fold dilutions) corresponding to concentrations of 0-20 nM (twice the test concentrations). Diluted antibodies or media (for untreated samples) (50 µl/well) were added to the appropriate wells. Excess medium (200 μl/well) was added to the outer rows and columns of the plate to prevent evaporation. The tablet was incubated for 72 h at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 min. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed 1 time using 100 μl/well PBS (in the case of cells in suspension, the plate was first centrifuged to pellet the cells). 100 μl/well PBS and 100 μl CellTiter Glo were added to each well and mixed thoroughly to obtain a mixture. The plate was incubated in the dark at room temperature for 15 minutes and observed by microscopy to ensure that effective cell lysis had occurred. The plate was then read on a Glomax luminometer.

Results.

In FIG. 3t shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 on OVCAR-3 cells. In FIG. 3u shows the cytotoxic activity of DM1 and DM4-conjugated anti-LY75 antibodies against SK-OV-3 cells. These results demonstrate an increase in cytotoxic activity proportional to antibody concentration.

Example 15 Efficacy of DM1- and DM4-conjugated anti-LY75 monoclonal antibodies against multiple myeloma cell lines.

Materials.

CellStripper (non-enzymatic cell dissociator) (MT-25-056CI) from Fisher Scientific, Pennsylvania, USA.

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

RPMI 1640 medium (MT-10-041-CM) from Fisher Scientific, PA, USA.

CellTiter Glo (G7572) from Promega, Wisconsin, USA.

Way.

The cells were dissociated using a CellStripper and their number was counted. 5e3 cells/well were centrifuged to pellet (in the case of cells in suspension, more can be used depending on the cell doubling time, such as 10e3 cells/well). The pellet was resuspended in culture medium to obtain a concentration of 1e5 cells/ml.

μl/well of the cell suspension was added to the wells of a 96-well plate with white walls and a transparent bottom. Antibodies were diluted and titrated at 8 points (titrations with 3-fold dilutions) corresponding to concentrations of 0-20 nM (twice the test concentrations). Diluted antibodies or media (for untreated samples) (50 µl/well) were added to the appropriate wells. Excess medium (200 μl/well) was added to the outer rows and columns of the plate to prevent evaporation. The tablet was incubated for 72 h at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 min. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed 1 time using 100 μl/well PBS (in the case of cells in suspension, the plate was first centrifuged to pellet the cells). 100 μl/well PBS and 100 μl CellTiter Glo were added to each well and mixed thoroughly to obtain a mixture. The plate was incubated in the dark at room temperature for

- 42 042216 min and observed by microscopy to ensure that effective cell lysis has occurred. The plate was then read on a Glomax luminometer.

Results.

In FIG. 3v shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 on MOLP-8 cells. In FIG. 3w shows the cytotoxic activity of DM1 and DM4 conjugated anti-LY75 antibodies against RPMI8226 cells. These results demonstrate an increase in cytotoxic activity proportional to antibody concentration.

Example 16 Efficacy of DM1- and DM4-conjugated anti-LY75 monoclonal antibodies in Raji xenograft models.

The efficacy of LY75_DM1 and LY75_DM4 was investigated in a subcutaneous xenograft model in SCID mice with Burkitt's Raji lymphoma cells.

Immunodeficient SCID mice were subcutaneously inoculated with Raji tumor cells (human Burkitt's lymphoma). Tumors were allowed to form and mice were divided into five treatment groups of 3-6 mice per group. When the mean tumor volume reached an average of 129-132 mm ³ per group, each group was treated with one of the following compounds administered intravenously at the indicated doses: group 1 (vehicle; phosphate buffered saline (PBS)), group 2 (LY75_DM1; 10 mg/kg), group 3 (isotype control-DMI; 10 mg/kg), group 4 (LY75_DM4; 5 mg/kg), group 5 (isotype control-SPBDDM4; 5 mg/kg). The second dose was administered one week later. Body weight (BW) values were monitored, mice were examined frequently for health status and unwanted side effects, and tumors were measured twice a week. Mice were euthanized when their tumors reached a tumor volume endpoint of 2000 mm ³ or after 60 days, whichever came first. Efficacy was determined from tumor growth delay (TGD), increase in median time to end point (TTE), and from log-rank analysis of differences in Kaplan-Meier survival curves for mice treated with ADC compared with mice treated with PBS. The first five vehicle-treated control mice that reached the endpoint were sampled tumors, which were processed by formalin fixation and paraffin embedding.

Results.

In FIG. 4a shows that LY75_DM1 and LY75_DM4 each demonstrated significant antitumor activity and significantly prolonged survival in the Raji xenograft model of Burkitt's lymphoma in SCID mice compared to controls; however, doses of 5 mg/kg LY75_DM4 were significantly more effective than doses of 10 mg/kg LY75_DM1, resulting in 5 out of 6 mice with complete but temporary tumor regression. All treatments were well tolerated and no clinical signs of toxicity were observed. These data suggest that ADCs directed to LY75, such as LY75_DM1 and LY75_DM4, may provide a clinically beneficial effect in the treatment of human cancer patients with non-Hodgkin's lymphoma.

Example 17 Efficacy of DM1-conjugated and DM4-conjugated anti-LY75 monoclonal antibodies in Namalwa xenograft models.

The efficacy of LY75_DM1 and LY75_DM4 was investigated in a subcutaneous xenograft model in mice with SCID Burkitt's Namalwa cells.

Immunodeficient SCID mice were subcutaneously inoculated with Namalwa tumor cells (human Burkitt's lymphoma). Tumors were allowed to form and mice were divided into five treatment groups of six mice per group. When the mean tumor volume reached an average of 114 mm ³ per group, each group was treated with one of the following compounds administered intravenously at the indicated doses: group 1 (vehicle; phosphate buffered saline (PBS)); group 2 (LY75_DM1; 10 mg/kg), group 3 (isotype control-DMI; 10 mg/kg), group 4 (LY75_DM4; 5 mg/kg), group 5 (isotype control-SPBDDM4; 5 mg/kg). Body weight (BW) values were monitored, mice were examined frequently for health status and unwanted side effects, and tumors were measured twice a week. Mice were euthanized when their tumors reached a tumor volume endpoint of 2000 mm ³ or after 60 days, whichever came first. Efficacy was determined from tumor growth delay (TGD), increase in median time to end point (TTE), and from log-rank analysis of differences in Kaplan-Meier survival curves for mice treated with ADC compared with mice treated with PBS. The first five vehicle-treated control mice that reached the endpoint were sampled tumors, which were processed by formalin fixation and paraffin embedding.

Results.

In FIG. 4b shows that LY75_DM1 and LY75_DM4 each showed significant antitumor activity and prolongation of survival in the Namalwa xenograft model of Burkitt's lymphoma in SCID mice compared to controls; however, a dose of 5 mg/kg

- 43 042216

LY75_DM4 was significantly more effective than the 10 mg/kg dose of LY75_DM1 in causing a transient reduction in tumor volume. All treatments were well tolerated and no clinical signs of toxicity were observed. These data suggest that ADCs directed to LY75, such as LY75_DM1 and LY75_DM4, may provide a clinically beneficial effect in the treatment of human cancer patients with non-Hodgkin's lymphoma.

Example 18 Efficacy of anti-LY75 monoclonal antibodies conjugated to DM1 and conjugated to DM4 in pancreatic cancer xenograft models.

The efficacy of LY75_DM1 and LY75_DM4 was studied in a xenograft model with subcutaneous administration of HPAFII pancreatic adenocarcinoma cells to athymic nude mice.

Immunodeficient athymic nude mice were subcutaneously inoculated with HPAFII (human pancreatic adenocarcinoma) tumor cells. Tumors were allowed to form and mice were divided into five treatment groups of 6 mice per group. When the mean tumor volume reached an average of 114 mm ³ per group, each group was treated with one of the following compounds administered intravenously at the indicated doses: group 1 (vehicle; phosphate buffered saline (PBS)), group 2 (LY75_DM1; 10 mg/kg), group 3 (isotype control-DMI; 10 mg/kg), group 4 (LY75_DM4; 5 mg/kg), group 5 (isotype control-SPBDDM4; 5 mg/kg). Body weight (BW) values were monitored, mice were examined frequently for health status and unwanted side effects, and tumors were measured three times a week. Mice were euthanized when their tumors reached a tumor volume endpoint of 2000 mm ³ 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 for ADC-treated mice compared to PBS-treated mice. Tumor samples were taken from vehicle-treated control mice and processed by formalin fixation and paraffin embedding.

Results.

In FIG. 4c shows that LY75_DM1 and LY75_DM4 exhibited significant and similar antitumor activity, with prolongation of survival observed in the HPAFII xenograft model in nude mice compared to controls. All treatments were well tolerated and no clinical signs of toxicity were observed. These data suggest that ADCs directed to LY75, such as LY75_DM1 and LY75_DM4, may provide a clinically beneficial effect in the treatment of human patients with pancreatic cancer.

Example 19 Efficacy of anti-LY75 monoclonal antibodies conjugated to DM1 and conjugated to DM4 in xenograft models of bladder cancer.

The efficacy of LY75_DM1 and LY75_DM4 was investigated in a subcutaneous xenograft model in mice with SCID human bladder carcinoma SW780 cells.

Immunodeficient athymic nude mice were subcutaneously inoculated with HPAFII (human pancreatic adenocarcinoma) tumor cells. Tumors were allowed to form and mice were divided into five treatment groups of six mice per group. When the mean tumor volume reached an average of 114 mm ³ per group, each group was treated with one of the following compounds administered intravenously at the indicated doses: group 1 (vehicle; phosphate buffered saline (PBS)), 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 weight (BW) values were monitored, mice were examined frequently for health status and unwanted side effects, and tumors were measured three times a week. Mice were euthanized when their tumors reached a tumor volume endpoint of 2000 mm ³ 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 for ADC-treated mice compared to PBS-treated mice. Tumor samples were taken from vehicle-treated control mice and processed by formalin fixation and paraffin embedding.

Results.

In FIG. 4d shows that LY75_DM1 and LY75_DM4 exhibited significant and similar antitumor activity, with prolongation of survival observed in the SW780 xenograft model in nude mice compared to controls. All treatments were well tolerated and no clinical signs of toxicity were observed. These data suggest that ADCs directed to LY75, such as LY75_DM1 and LY75_DM4, may provide a clinically beneficial effect in the treatment of human patients with bladder cancer.

Example 20 Efficacy of anti-LY75 monoclonal antibodies conjugated to DM1 and conjugated to DM4 in xenograft models of breast cancer.

The efficacy of LY75_DM1 and LY75_DM4 was studied in a xenograft model with subcutaneous injection of MDA-MB-468 cells into athymic nude mice.

- 44 042216

Immunodeficient athymic nude mice were subcutaneously inoculated with MDA-MB-468 tumor cells (triple negative human mammary adenocarcinoma). Tumors were allowed to form and mice were divided into seven treatment groups of 10 mice per group. When the mean tumor volume reached an average of 167 mm ³ per group, each group was treated with one of the following compounds administered intravenously at the indicated doses: group 1 (vehicle; 20 mM sodium succinate, pH 5.0, 6% trehalose, 0.04 % polysorbate); group 2 (LY75_DM1; 5 mg/kg), group 3 (LY75_DM1; 10 mg/kg), group 4 (LY75_DM4; 5 mg/kg), group 5 (LY75_DM4; 2.5 mg/kg), group 6 (LY75_DM4 ; 1 mg/kg), group 7 (isotype control-OM4; 5 mg/kg). Body weight (BW) values were monitored, mice were examined frequently for health status and unwanted side effects, and tumors were measured twice a week. Mice were euthanized 82 days after tumor inoculation. Efficacy was determined in terms of antitumor activity (mean treatment group tumor size/mean control group tumor size100) and the increase in mean time to endpoint (TTE) in ADC-treated mice compared to PBS-treated mice. The five largest tumors were sampled from vehicle-treated control mice on day 71 post inoculation and processed by formalin fixation and paraffin embedding.

Results.

In FIG. 4e shows that LY75_DM1 and LY75_DM4 each showed significant antitumor activity in the MDA-MB-468 xenograft model in nude mice compared to controls. Dose dependent activity was observed with LY75_DM4, with 2.5 and 5 mg/kg having a much stronger effect than 1 mg/kg. At 5 mg/kg, LY75_DM1 and LY75_DM4 showed similar efficacy. Long-term regressions in mean tumor 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 that ADCs directed to LY75, such as LY75_DM1 and LY75_DM4, may provide clinical benefit in the treatment of human patients with triple negative breast cancer.

Example 21 Efficacy of anti-LY75 monoclonal antibodies conjugated to DM1 and conjugated to DM4 in colon and rectal cancer xenograft models.

The efficacy of LY75_DM1 and LY75_DM4 was studied in a xenograft model with subcutaneous administration of COLO205 colon and rectal adenocarcinoma cells to athymic nude mice.

Immunodeficient athymic nude mice were subcutaneously inoculated with tumor cells COLO205 (adenocarcinomas of the human colon and rectum). Tumors were allowed to form and mice were divided into five treatment groups of six mice per group. When the mean tumor volume reached an average of 117 mm ³ per group, each group was treated with one of the following compounds administered intravenously at the indicated doses: group 1 (vehicle; phosphate buffered saline (PBS)), group 2 (LY75_DM1; 10 mg /kg), group 3 (isotype control-DMI; 10 mg/kg), group 4 (LY75_DM4; 5 mg/kg), group 5 (isotype control-DM4; 5 mg/kg). The second dose was administered 12 days after the first. Body weight (BW) values were monitored, mice were examined frequently for health status and unwanted side effects, and tumors were measured twice a week. Mice were euthanized when their tumors reached a tumor volume endpoint of 1000 mm ³ or after 60 days, whichever came first. Efficacy was determined from tumor growth delay (TGD), increase in median time to end point (TTE), and from log-rank analysis of differences in Kaplan-Meier survival curves for ADC-treated mice compared to mice treated with ADC. treated with PBS. The first five vehicle-treated control mice that reached the endpoint were sampled tumors, which were processed by formalin fixation and paraffin embedding.

Results.

In FIG. 4f shows that LY75_DM1 and LY75_DM4 showed similar modest antitumor activity and prolongation of survival in a nude mouse COLO205 xenograft model of colorectal adenocarcinoma from COLO205 cells compared to controls. All treatments were well tolerated and no clinical signs of toxicity were observed. These data suggest that ADCs directed to LY75, such as LY75_DM1 and LY75_DM4, may provide clinical benefit in the treatment of human patients with colorectal cancer.

Example 22 Toxicity of DM1- and DM4-conjugated monoclonal antibodies to LY75 against cynomolgus monkeys.

Six male monkeys were distributed in the study at 2 monkeys/group. Vehicle (PBS), LY75_DM4 (cleavable) or LY75_DM1 (non-cleavable) was administered twice (on days 1 and 29) via 15 min intravenous infusion at 0 mg/kg/dose (PBS, vehicle), 5 mg/kg/dose ( LY75_DM4, cleavable) or 10 mg/kg/dose (LY75_DM1, non-cleavable). Blood samples were taken for toxicokinetic studies before the start of dosing (day 1) and after 1, 2,

- 45 042216

3, 7, 14, 21 and 28 days after each dose. Blood samples were taken for clinicopathological analyzes before dosing (Day 1) and 1, 3, 7, 14, 21 and 28 days after each dose (28 days after 1st dose was also used as the time before dose administration for the 2nd dose). All study animals were euthanized and necropsied after the last blood draw on day 57. Blood plasma separated from each blood sample was isolated, frozen and transported to Oxford BioTherapeutics, Inc. for analysis in relation to the concentration of ADC using ELISA.

Clinical and pathological manifestations associated with treatment have been observed to include mild regenerative anemia and transient decreases in white blood cell counts, most notable for neutrophil counts. Anemia was observed in both animals treated with 5 mg/kg LY75_DM4 and one of the two animals treated with 10 mg/kg LY75_DM1. Severe neutropenia with a maximum decrease in the number of neutrophils one week after dosing and a rapid recovery of their number was observed in all animals; while the maximum decrease in the absolute number of neutrophils was less in animals treated with LY75_DM4. No effects were observed on APTT and PT coagulation parameters associated with study drug. Changes in serum chemistry included transient increases in AST, CK, LDH (in 1 of 2 animals in each treatment group) and globulin following administration of 5 mg/kg LY75_DM4 and 10 mg/kg LY75_DM1. In addition, a transient increase in liver-specific ALT enzyme levels was observed only in animals treated with LY75_DM4. The short duration and/or magnitude of increases in serum chemistry parameters suggested that they were not adverse events. Urinalysis showed no observed manifestations associated with the study drug. During the post-mortem examination after a 4-week recovery period, there were no observed macropathological manifestations associated with processing, or changes in absolute and relative organ mass values. The observed histopathological manifestations only for the thyroid gland (change in the morphological characteristics of the colloidal content in the follicles) and kidney (dilated tubules in the outer layer of the cortical substance) were classified as characterized by minimal severity; not related to changes in other studied parameters; as well as those not related to adverse events and having minimal toxicological significance.

Conclusion.

Repeated dose treatment at two doses of 5 mg/kg LY75_DM4 or 10 mg/kg LY75_DM1 was well tolerated by cynomolgus monkeys. All observed treatment-related toxicity was reversible after a 4-week recovery period.

Example 23 Epitope characterization for LY75 A1 by competition binding assay using fluorescence activated cell sorting (FACS).

Way.

COLO205 cells (ATCC, cat. no. CCL-222) were separated from tissue culture mats using a CellStripper (Cellgro, cat. no. MT-25-056CI). Cells were washed and resuspended in FACS buffer (PBS+2% FBS), neutralized with growth medium and counted. Cells were seeded at 50,000 cells/well in a 96-well V-bottom plate. Cells were washed once with FACS buffer (PBS (Fisher, cat. no. SH30028-03) + 2% FBS). mAb to LY75 (selected from Example 1) or LY75_A1 was added to wells at an initial concentration of 250 nM, 3-fold serial dilutions were made and added to the respective wells for 45 minutes on ice. Test well contents that required one or more staining steps were maintained in FACS buffer as needed to ensure that final staining was completed simultaneously for all test conditions. The contents of two FACS buffer wells were left unstained as controls.

After incubation with blocking antibody, cells were washed twice with FACS buffer. Cells were resuspended in FACS buffer containing anti-LY75 mAb conjugated to MCC-DM1 (1 nM) and incubated on ice for 45 min. Cells were washed as described above and resuspended in FACS buffer supplemented with 1 μg/ml mouse anti-maytansine antibody and incubated on ice for 45 min. Cells were washed as described above and resuspended in FACS buffer containing 2 μg/ml goat anti-mouse Ig kappa chain RPE conjugated. Cells were incubated on ice for 45 min, then washed as described above. Cells were resuspended in FACS buffer at 200 µl per well. The mean fluorescence intensity for each sample was determined using a Guava EasyCyte Plus HT flow meter (96-well plate formats) and raw data was analyzed using Guava Cytosoft.

Results.

In FIG. 5a shows that blocking with an anti-LY75 mAb conjugated to MCC-DM1 resulted in reduced binding of the LY75 mAb. Binding analysis of LY75_A1 to COLO205 cells demonstrated that LY75_A1 was unable to block the binding of MCC-DM1 conjugated anti-LY75 mAb (see Fig. 5b). Thus, it can be determined that mAb to LY75 and LY75_A1

- 46 042216 are non-competing antibodies, while LY75_A1 recognizes a distinct and unique LY75 epitope compared to other anti-LY75 antibodies.

Example 24 Epitope characterization for LY75 A1 by peptide microarray analysis.

Way.

Microarray analysis of peptides was performed at LC Sciences, Houston, Texas, in brief, the method involved the following steps: synthesized contiguous 8-mer peptides of the LY75 protein, overlapping at one amino acid, spanning residues 216-1666 of the full-length LY75 protein, and immobilized on a microarray chip. The chip contained three panels, so the experiment was performed in triplicate. The microarray was examined using LY75_A1 to identify peptides to which the antibody bound. The binding assay was performed under the following conditions.

The microarray containing contiguous peptides in triplicate was washed with 1 ml of binding buffer at 4°C for 20 min. It was then incubated with 1 μg/ml LY75_A1 in binding buffer (pH 7.0) at 4°C for 2 h. 25 ng/ml anti-human IgG Alexa 647 conjugate in binding buffer (pH 7.0) at 4°C for 1 hour. The matrix was washed again with 0.5 ml wash buffer at 4°C for 30 minutes.

The matrix was then scanned at 635 nm and PMT 500 and the signal intensity was recorded. A peptide was classified as detectable if it was present in at least 2/3 of the allowed duplicate samples. The mean signal intensity for replicates was presented as the final signal intensity.

Results.

As shown in FIG. 6, the LY75_A1 antibody was characterized by specific binding to a number of peptides located on the matrix. The maximum signal level observed for binding to LY75_A1 was 25,000 (1-65535 scale), with the average signal level for all spots on the matrix being approximately 885. A signal intensity of 3,000 was set as the background cut-off point due to non-specific binding. Based on the observed level of antibody binding signal intensity, potential epitope sequences for LY75_A1 were identified. These areas are shown in Fig. 6a-6j and under SEQ ID NO: 22-31.

Example 25 Peptide analysis by co-precipitation using LY75 A1.

Way.

1.1. Analysis by co-precipitation method.

The recombinant LY75 protein was cleaved by tryptic proteolysis of the granule-associated protein (Promega, USA). The resulting digested peptides were recovered using a C18 capture column (Thermo Fisher Scientific). The purified peptides were then incubated with 200 μl of protein A beads crosslinked with LY75_A1 antibody overnight at 4°C. The next day, unbound peptides were collected, while the beads were washed twice with 1 ml of PBS. The antibody bound peptides were eluted from the beads by heating them at 90° C. in 100 μl PBS for 5 min. This elution step was repeated.

1.2. Mass spectrometry.

Samples were analyzed by liquid chromatography with mass spectrometry using a Waters nanoACQUITY UPLC system equipped with a nanoACQUITY UPLC BEH 130 C18, 75 µm x 250 mm column (186003545) and LTQ Orbitrap Velos (Thermo Fisher Scientific). The peptides were eluted using a gradient of 3 to 35% acetonitrile at 300 nl/min for 120 minutes. Received mass spectra in full scan mode at a resolution of 60,000 in the mass range 400-2000 mass/charge using Orbitrap. In each cycle, twenty peptides with the highest intensity were selected for CID MS/MS scans in a linear ion trap with a nanospray ion source mounted on the instrument.

1.3. Analysis of the amino acid sequence of the peptide.

Raw data obtained using the LTQ Orbitrap Velos was processed using Mascot software (Matrix Science), which uses the Mowse algorithm (Curr. Biol., 1993 Jun 1, 3(6):327-3) to derive amino acid sequences from from a set of peaks by searching the sequence database consisting of Ensembl (http://www.ensembl.org/index.html), IPI (www.ebi.ac.uk/IPI/IPIhuman.html) and SwissProt (http ://www.uniprot.org) along with sequences of contaminant proteins. Criteria for peptide identification included trypsin cleavage to 2 missing cleavage sites and various biological and chemical modifications (oxidized methionine, modification of cysteine with MMTS or iodoacetamide, and phosphorylation of serine, threonine and tyrosine). Peptides that were assigned a rank of 1 with an expected value of 0.05% or less, a degree of coincidence of the spectra of 28 or more, were loaded into the OGAP database.

1.4. Recognition of peptides associated with LY75.

The method for identifying LY75 used peptide sequences experimentally obtained by mass spectrometry, as described above, of naturally occurring human proteins to identify and sequence coding exons in the published human genomic sequence. These experimentally determined sequences were compared to the OGAP® database, which was compiled by processing and integrating peptide masses, peptide signatures, ESTs and publicly available genomic sequence data as described in international patent application WO 2009/087462.

Results.

The results of the analysis of peptides by the method of codeposition using antibodies LY75_A1 are shown in table. 1 below and in FIG. 7. The peptides that were identified in both elutions of peptides 1a and 1b in the coprecipitation and microarray analysis were considered the most likely candidates for epitope formation.

Table 1

Comparison of Experiments with Peptide Microarray Analysis and Peptide Coprecipitation

SEQ GO NO: Peptide identified by microarray analysis Peptide identified by codeposition analysis Region 1 (amino acids 609-618) - Region 2 (amino acids 651-662) - 46 Region 3 (amino acids 761-780) GWHFYDDR (765-772) 47 Region 4 (amino acids 883-901) ISEWPIDDHFTYSR(877-890) 48 FPVTFGEECLYMSAK(896-910) 49 Region 5 (amino acids 1029-1040) ELTYSNFHPLLVSGR (1030-1044) 50 Region 6 (amino acids 1077-1093) HFVSLCQK (1084-1091) 51 Region 7 (amino acids 1107-1118) QTLQNASETVK (1099-1109) Region 8 (amino acids 1368-1378) - Region 9 (amino acids 1518-1528) - Region 10 (amino acids 1535-1554) -

In table. 1 shows that a number of overlapping regions of LY75 peptides were identified both in microarray peptide analysis and in both elutions 1a and 1b in peptide coprecipitation analysis. These regions were considered to be the most likely to contain an epitope recognized by the LY75_A1 antibody, since they bind to LY75_A1 when examined by both methods used.

Example 26.

A number of activated B cells from diffuse large B cell lymphoma (ABC-DLBCL) cell lines (i.e., TMD8 and HBL1 cell lines) were exposed to increasing doses of LY75_DM4 (i.e., 0.018 - 0.037 - 0.075 - 0.15 - 0.3 - 0.6 - 1.2 nM) either alone or in combination with increasing doses of rituximab (2.34 - 4.68 - 9.37 - 18.75 - 37.5 - 75 - 150 nM) or ibrutinib (15.6 - 31.2 - 62.5 - 125 - 250 - 500 - 1000 nM) for 72 h. This was followed by analysis using MTT [3-(4,5-dimethylthiazolyl-2)-2 ,5-diphenyltetrazol bromide].

The Chow-Talalai additivity index (CI) was evaluated using the Synergy R package (Preclinical versus Clinical Drugs Combination Studies, Chou T.C., Leuk., Lymphoma., 2008;49(11):2059-2080). It quantifies strong synergistic effect (<0.3), synergistic effect (0.3-0.9), additive effect (0.9-1.1), or antagonism/no beneficial effect (>1.1).

In FIG. 8a, 8b show synergistic effects observed between LY75_DM4 and rituximab. In FIG. 9 shows synergistic effects observed between LY75_DM4 and ibrutinib. Additional data are shown in table. 2, 3 and 4 below.

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Table 2 Chow-Talalai additivity index (CI) for various doses of LY75_DM4 in combination with rituximab against three TMD8 ABC-DLBCL cell lines

No. Rituximab (nM) LY75_DM4 (nM) CI 1 2.3438 0.01875 17.124 2 2.3438 0.075 0.225 3 2.3438 0.15 0.346 4 2.3438 0.3 0.583 5 2.3438 0.6 1.165 6 2.3438 1.2 2.514 7 2.3438 0.0375 0.145 8 4.6875 0.01875 1.499 9 4.6875 0.0375 0.147 10 4.6875 0.075 0.216 eleven 4.6875 0.15 0.346 12 4.6875 0.3 0.628 13 4.6875 0.6 1.165 14 4.6875 1.2 2.514 15 9.375 0.01875 2.918 16 9.375 0.0375 0.144 17 9.375 0.075 0.219 18 9.375 0.15 0.332 19 9.375 0.3 0.583 20 9.375 0.6 1.025 21 9.375 1.2 2.514 22 18.75 0.01875 0.8

- 49 042216

23 18.75 0.0375 0.145 24 18.75 0.075 0.213 25 18.75 0.15 0.346 26 18.75 0.3 0.628 27 18.75 0.6 1.165 28 18.75 1.2 2.514 29 37.5 0.01875 11.427 thirty 37.5 0.075 0.216 31 37.5 0.15 0.332 32 37.5 0.3 0.583 33 37.5 0.6 1.165 34 37.5 1.2 2.655 35 37.5 0.0375 0.151 36 75 0.01875 0.216 37 75 0.0375 0.147 38 75 0.075 0.209 39 75 0.15 0.332 40 75 0.3 0.628 41 75 0.6 1.165 42 75 1.2 2.514 43 150 0.01875 45.466 44 150 0.0375 0.152 45 150 0.075 0.209 46 150 0.15 0.332 47 150 0.3 0.628 48 150 0.6 1.257 49 150 1.2 2,770

- 50 042216

Table 3 Chow-Talalai additivity score (C1) for various doses of LY75_DM4 in combination with rituximab against three HBL-1 ABC-DLBCL cell lines

No. Rituximab (nM) LY75 DM4 (nM) CI 1 2.3438 0.0188 0.534 2 2.3438 0.0750 0.958 3 2.3438 0.1500 0.603 4 2.3438 0.3000 0.509 5 2.3438 0.6000 0.454 6 2.3438 1.2000 0.817 7 2.3438 0.0375 0.413 8 4.6875 0.0188 0.568 9 4.6875 0.0375 0.837 10 4.6875 0.0750 0.919 eleven 4.6875 0.1500 0.635 12 4.6875 0.3000 0.580 13 4.6875 0.6000 0.501 14 4.6875 1.2000 0.908 15 9.375 0.0188 0.249 16 9.375 0.0375 1.576 17 9.375 0.0750 0.717 18 9.375 0.1500 0.439 19 9.375 0.3000 0.618 20 9.375 0.6000 0.501 21 9.375 1.2000 0.908 22 18.75 0.0188 0.787 23 18.75 0.0375 0.497

- 51 042216

24 18.75 0.0750 0.562 25 18.75 0.1500 0.545 26 18.75 0.3000 0.510 27 18.75 0.6000 0.366 28 18.75 1.2000 0.817 29 37.50 0.0375 0.734 thirty 37.50 0.0750 0.548 31 37.50 0.0188 0.543 32 37.50 0.1500 0.465 33 37.50 0.3000 0.476 34 37.50 0.6000 0.324 35 37.50 1.2000 0.648 36 75 0.0188 0.442 37 75 0.0375 1.578 38 75 0.0750 0.432 39 75 0.1500 0.493 40 75 0.3000 0.383 41 75 0.6000 0.409 42 75 1.2000 0.908 43 150 0.0188 0.680 44 150 0.0375 0.640 45 150 0.0750 0.911 46 150 0.1500 0.498 47 150 0.3000 0.355 48 150 0.6000 0.366 49 150 1.2000 0.817

- 52 042216

Table 4 Chow-Talalai additivity index (CI) for various doses of LY75 DM4 in combination with ibrutinib against _______ three HBL-1 ABC-DLBCL cell lines_______

No. Ibrutinib (nM) LY75_DM4 (nM) CI 1 1.5625 0.01875 0.481 2 1.5625 0.0375 0.627 3 1.5625 0.075 0.862 4 1.5625 0.15 0.360 5 1.5625 0.3 0.287 6 1.5625 0.6 0.270 7 1.5625 1.2 0.343 8 3.125 0.01875 0.411 9 3.125 0.0375 0.358 10 3.125 0.075 0.461 eleven 3.125 0.15 0.287 12 3.125 0.3 0.251 13 3.125 0.6 0.246 14 3.125 1.2 0.394 15 6.25 0.01875 0.204 16 6.25 0.0375 0.219 17 6.25 0.075 0.275 18 6.25 0.15 0.226 19 6.25 0.3 0.216 20 6.25 0.6 0.222 21 6.25 1.2 0.343 22 12.5 0.01875 0.205 23 12.5 0.0375 0.152 24 12.5 0.075 0.287

- 53 042216

25 12.5 0.15 0.217 26 12.5 0.3 0.228 27 12.5 0.6 0.222 28 12.5 1.2 0.343 29 25 0.01875 0.152 thirty 25 0.0375 0.183 31 25 0.075 0.202 32 25 0.15 0.199 33 25 0.3 0.193 34 25 0.6 0.222 35 25 1.2 0.343 36 50 0.01875 0.374 37 50 0.0375 0.473 38 50 0.075 0.287 39 50 0.15 0.192 40 50 0.3 0.194 41 50 0.6 0.246 42 50 1.2 0.394 43 100 0.01875 0.715 44 100 0.0375 0.748 45 100 0.075 0.457 46 100 0.15 0.187 47 100 0.3 0.195 48 100 0.6 0.222 49 100 1.2 0.394

- 54 042216

Sequences

SEQID NO Description Subsequence 1 A. to. A1VH EVQLVESGGGLVKPGGSLRLSCAASGFTYSNAWMSWVRQAPGKTLE WVGRIKSKTDGGTTDYAAPVQGRFTISRDDSKNTLYLQMNSLKTEDT AVYYCTIFGWSFDYWGQGTLVTVSS 2 A. to. A1VL DVQMTQSPSSLSASVGDRVTITCRASQSISDYLSWYQQRPGKAPNLLI YAASNLKTGVPSRFSGSGSGTDFTLTISTLQPEDFATYYCQQSYRSPW TFGQGTKVEIKR 3 Nucleotides A1VH gaggtgcagctggtggagtctgggggaggcttggtaaagccgggggggtcccttagactctcctgtgcagcc tctggcttcacttacagtaacgcctggatgagctgggtccgccaggctccagggaaggggctggagtgggttg gccgtattaaaagcaaaactgatggtgggacaacagactacgctgcacccgtgcaaggcagattcaccatctc aagagatgattcaaaaaacacgctgtatctgcaaatgaacagcctgaaaaccgaggacacagccgtgtattac tgtacgatttttggagtggttagctttgactactggggccagggaaccctggtcaccgtctcctca 4 Nucleotides A1VL gacgtccagatgacccagtctccatcctccctgtctgcatctgttggagacagagtcaccatcacttgccgggc aagtcagagcattagcgactatttaagttggtatcagcagagaccagggaaagcccctaacctcctgatctatg ctgcatccaatttaaagactggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcacca tcagcactctgcaacctgaagattttgcaacgtactactgtcaacagagttacaggtccccgtggacgttcggcc aagggaccaaggtggaaatcaaacga 5 A. to. A1VHCDR1 NAWMS 6 A. to. A1VHCDR2 RIKSKTDGGTTDYAAPVQG 7 A. to. A1VHCDR3 FGWSFDY 8 A. to. A1VLCDR1 RASQSISDYLS 9 A. to. A1VLCDR2 AASNLKT 10 A. to. QQSYRSPWT

- 55 042216

A1VLCDR3 eleven VH3|315/D4|411 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE WVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDT AVYYCTTTTVT 12 JH4 YFDYWGQGTLVTVSS 13 012 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYS 14 JK1 WTFGQGTKVEIKR 15 LY75 (DEC205) MRTGWATPRRPAGLLMLLFWFFDLAEPSGRAANDPFTIVHGNTGKCI KPVYGWIVADDCDETEDKLWKWVSQHRLFHLHSQKCLGLDITKSVN ELRMFSCDSSAMLWWKCEHHSLYGAARYRLALKDGHGTAISNASDV WKKGGSEESLCDQPYHEIYTRDGNSYGRPCEFPFLIDGTWHHDCILDE DHSGPWCATTLNYEYDRKWGICLKPENGCEDNWEKNEQFGSCYQFN TQTALSWKEAYVSCQNQGADLLSINSAAELTYLKEKEGIAKIFWIGLN QLYSARGWEWSDHKPLNFLNWDPDRPSAPTIGGSSCARMDAESGLW QSFSCEAQLPYVCRKPLNNTVELTDVWTYSDTRCDAGWLPNNGFCY LLVNESNSWDKAHAKCKAFSSDLISIHSLADVEVWTKLHNEDIKEEV WIGLKNINIPTLFQWSDGTEVTLTYWDENEPNVPYNKTPNCVSYLGE LGQWKVQSCEEKLKYVCKRKGEKLNDASSDKMCPPDEGWKRHGET CYKIYEDEVPFGTNCNLTITSRFEQEYLNDLMKKYDKSLRKYFWTGL RDVDSCGEYNWATVGGRRRAVTFSNWNFLEPASPGGCVAMSTGKSV GKWEVKDCRSFKALSICKKMSGPLGPEEASPKPDDPCPEGWQSFPAS LSCYKVFHAERIVRKRNWEEAERFCQALGAHLSSFSHVDEIKEFLHFL TDQFSGQHWLWIGLNKRSPDLQGSWQWSDRTPVSTIIMPNEFQQDYD IRDCAAVKVFHRPWRRGWHFYDDREFIYLRPFACDTKLEWVCQIPKG RTPKTPDWYNPDRAGIHGPPLIIEGSEYWFVADLHLNYEEAVLYCASN HSFLATITSFVGLKAIKNKIANISGDGQKWWIRISEWPIDDHFTYSRYP WHRFPVTFGEECLYMSAKTWLIDLGKPTDCSTKLPFICEKYNVSSLEK YSPDSAAKVQCSEQWIPFQNKCFLKIKPVSLTFSQASDTCH SYGGTLP SVLSQIEQDFITSLLPDMEATLWIGLRWTAYEKINKWTDNRELTYSNF HPLLVSGRLRIPENFFEEESRYHCALILNLQKSPFTGTWNFTSCSERHF VSLCQKYSEVKSRQTLQNASETVKYLNNLYKIIPKTLTWHSAKRECL KSNMQLVSITDPYQQAFLSVQALLHNSSLWIGLFSQDDELNFGWSDG KRLHFSRWAETNGQLEDCWLDTDGFWKTVDCNDNQPGAICYYSGN ETEKEVKPVDSVKCPSPVLNTPWIPFQNCCYNFIITKNRHMATTQDEV HTKCQKLNPKSHILSIRDEKENNFVLEQLLYFNYMASWVMLGITYRN

- 56 042216

KSLMWFDKTPLSYTHWRAGRPTIKNEKFLAGLSTDGFWDIQTFKVIE EAVYFHQHSILACKIEMVDYKEEYNTTLPQFMPYEDGIYSVIQKKVT WYEALNMCSQSGGHLASVHNQNGQLFLEDIVKRDGFPLWVGLSSHD GSESSFEWSDGSTFDYIPWKGQTSPGNCVLLDPKGTWKHEKCNSVKD GAICYKPTKSKKLSRLIYSSRCPAAKENGSRWIQYKGHCYKSDQALH SFSEAKKLCSKHDHSATIVSIKDEDENKFVSRLMRENNNITMRVWLG LSQHSVDQSWSWLDGSEVTFVKWENKSKSGVGRCSMLIASNETWKK VECEHGFGRWCKVPLGPDYTAIAIIVATLSILVLMGGLIWFLFQRHRL HLAGFSSVRYAQGVNEDEIMLPSFHD 16 A1VHFR1 EVQLVESGGGLVKPGGSLRLSCAASGFTYS 17 A1VHFR2 WVRQAPGKGLEWVG 18 A1VHFR3 RFTISRDDSKNTLYLQMNSLKTEDTAVYYCTI 19 A1VHFR4 WGQGTLVTVSS 20 A1VLFR1 DVQMTQSPSSLSASVGDRVTITC 21 A1VLFR2 WYQQRPGKAPNLLIY 22 A1VLFR3 GVPSRFSGSGSGTDFTLTISTLQPEDFATYYC 23 A1VLFR4 FGQGTKVEIKR 24 LY75, 609-618 WEVKDCRSFK 25 LY75, 651-662 PASLSCYKVFHA 26 LY75, 761-780 PWRRGWHFYDDREFIYLRPF 27 LY75, 883-901 DDHFTYSRYPWHRFPVTFG 28 LY75, 1029- 1040 RELTYSNFHPLL 29 LY75, 1077- 1093 FTSCSERHFVSLCQKYS thirty LY75, HOT- HIS TVKYLNNLYKII 31 LY75, 1368- EAVYFHQHSIL

- 57 042216

1378 32 LY75, 1518- 1528 KKLSRLTYSSC 33 LY75, 1535- 1554 NGSRWIQYKGHCYKSDQALH 34 LY75, 877-901 ISEWPIDDHFTYSRYPWHRFPVTFG 35 LY75, 1099- 1118 QTLQNASETVKYLNNLYKII 36 LY75, 883-892 DDHFTYSRYP 37 LY75, 1077- 1091 FTSCSERHFVSLCQK 38 VH of rituximab QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGL EWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSA VYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 39 VL of rituximab QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYA TSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTF GGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTKTLSKADYEKHKVY ACEVNSFQS 40 rituximab, VHCDR1 SYNMH 41 rituximab, AIYPGNGDTSYNQKFKG

- 58 042216

VH_CDR2 42 rituximab, VH_CDR3 STYYGGDWYFNV 43 rituximab, VLCDR1 RASSSVSYIH 44 rituximab, VL_CDR2 ATSNLAS 45 rituximab, VLCDR3 QQWTSNPPTF 46 (765-772) GWHFYDDR 47 (877-890) ISEWPIDDHFTYSR 48 (896-910) FPVTFGEECLYMSAK 49 (1030-1044) ELTYSNFHPLLVSGR 50 (1084-1091) HFVSLCQK 51 (1099-1109) QTLQNASETVK 52 Linker Gly-Phe-Leu-Gly

Sequence listing in free text format.

SEQ YU NO: 38 <223> VH sequence of rituximab

SEQ ID NO: 39 <223> VL sequence of rituximab

SEQ ID NO: 40 <223> Rituximab VH CDR1

SEQ ID NO: 41 <223> Rituximab VH CDR2

SEQ ID NO: 42 <223> Rituximab VH CDR3

SEQ ID NO: 43 <223> VL CDR1 of rituximab

SEQ ID NO: 44 <223> VL CDR2 of rituximab

SEQ ID NO: 45 <223> VL CDR3 of rituximab

SEQ ID NO: 52 <223> Linker

SEQ ID NO: 53-163 <223> Peptide

SEQ ID NO: 165-207 <223> Peptide

Claims (9)

CLAIM 1. A pharmaceutical combination containing (A) (i) an anti-LY75 antibody or an antigen-binding portion thereof, said antibody or portion containing a) a heavy chain variable region containing i) a first vhCDR containing SEQ ID NO: 5; ii) a second vhCDR containing SEQ ID NO: 6; and iii) a third vhCDR containing SEQ ID NO: 7; And b) a light chain variable region containing i) a first vlCDR containing SEQ ID NO: 8; ii) a second vlCDR containing SEQ ID NO: 9; and iii) a third vlCDR containing SEQ ID NO: 10; or (ii) an anti-LY75 antibody or antigen-binding portion thereof, wherein said antibody or portion contains a) a heavy chain variable region containing i) a first vhCDR containing SEQ ID NO: 5; ii) a second vhCDR containing SEQ ID NO: 6; and iii) a third vhCDR containing SEQ ID NO: 7; And b) a light chain variable region containing i) a first vlCDR containing SEQ ID NO: 8; ii) a second vlCDR containing SEQ ID NO: 9; and iii) a third vlCDR comprising SEQ ID NO: 10, wherein any one or more of the above SEQ ID NOs independently comprise one or two amino acid substitutions, wherein the anti-LY75 antibody or antigen-binding portion thereof is covalently attached to a drug, wherein said drug is is maytansinoid drug 1 (DM1) or maytansinoid drug 4 (DM4); and (B) rituximab, wherein the pharmaceutical combination is in the form of a combined preparation suitable for simultaneous, separate or sequential use in the treatment of diffuse large B-cell lymphoma (DLBCL). 2. The pharmaceutical combination of claim 1, wherein any one or more of SEQ ID NOs: 5-10 independently contain one or two conservative amino acid substitutions. 3. A pharmaceutical combination according to claim 1 or 2, wherein the antibody or antigen-binding portion thereof is internalizable. 4. A pharmaceutical combination according to any one of claims 1 to 3, wherein the anti-LY75 antibody or antigen-binding portion thereof comprises a heavy chain variable region characterized by at least 80%, 85%, 90%, 95%, 99% or 100% identity with the amino acid sequence under SEQ ID NO: 1, and a light chain variable region having at least 80%, 85%, 90%, 95%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:2. 5. Pharmaceutical combination according to any one of claims 1 to 4, wherein the anti-LY75 antibody is a human IgG1 monoclonal antibody. 6. Pharmaceutical combination according to any one of claims 1 to 5, where (A) and/or (B) additionally contain one or more pharmaceutically acceptable diluents, excipients or carriers. 7. A method of treating diffuse large B-cell lymphoma (DLBCL) in a patient, comprising simultaneous, sequential or separate administration to a patient in need thereof, therapeutically effective amounts of components (A) and (B) of the pharmaceutical combination according to any one of claims 1-6 . 8. The method of claim 7, wherein the anti-LY75 antibody or antigen-binding portion thereof is internalized by a cell expressing LY75. 9. The use of a pharmaceutical combination according to any one of claims 1 to 6 in therapy or as a drug.