EA046736B1 - PHARMACEUTICAL COMBINATIONS - Google Patents

Description

Introduction

The present invention generally relates to the fields of immunology and molecular biology. More specifically, provided herein are pharmaceutical combinations comprising antibodies (A) or antigen binding portions thereof directed against LY75 and (B) venetoclax; methods for preparing pharmaceutical combinations and methods for treating diseases, such as cancers, mediated by the expression or activity of LY75.

BACKGROUND OF THE INVENTION

Leukemia and lymphoma types belong to a broad 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 enlarged 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 lymphomas (HL) and non-Hodgkin lymphomas (NHL). The World Health Organization (WHO) includes two other groups as types of lymphoma: multiple myeloma and immunoproliferative diseases. Approximately 90% of lymphoma types are non-Hodgkin lymphoma.

Leukemia is a group of cancers that usually start in the bone marrow and result in large numbers of abnormal white blood cells. Symptoms may include problems with bleeding and bruising, feeling tired, fever and an increased risk of infections. These symptoms occur 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 for types of leukemia and lymphoma may include one or more of chemotherapy, radiation therapy, targeted therapy, and surgery (and bone marrow transplant 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 lymphoma treatment depends on the subtype, with some being curable and in most cases treatment increases 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. Chemotherapy 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 that it minimizes the chance of developing resistance to any one drug. In addition, drugs can often be administered in smaller doses, reducing toxicity. Combination therapies for treating Hodgkin's disease include MOPP (mustargen, vincristine, procarbazine, prednisone) and ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine). Combination therapies for non-Hodgkin's lymphoma include CHOP (cyclophosphamide, doxorubicin, vincristine, prednisolone). Considering the number of drugs that are known for use in the treatment of types of leukemia and lymphoma, the number of permutations and combinations of possible drug treatments is certainly large. In addition, the above combination therapies do not provide antibodies.

There remains, however, a need for new treatments for types of leukemia and lymphoma, and in particular for effective combination therapies. Lymphocyte antigen 75 acts as an endocytic receptor that directs 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, colorectal, esophageal, skin, thyroid and lung (non-small cell) cancers, as well as in multiple myeloma and many different subtypes of lymphoma and leukemia. WO2009/061996 discloses isolated monoclonal antibodies that bind to human DEC-205 (LY75) and corresponding antibody compositions and molecules. Also disclosed are pharmaceutical compositions containing antibodies, as well as therapeutic and diagnostic methods for using antibodies. WO2008/104806 discloses affinity reagents capable of binding to LY75 for use in the treatment or prevention of cancer. WO2015/052537 discloses specific isolated antibodies capable of binding to LY75 and their use in the treatment of various forms of cancer.

Venetoclax is an oral small molecule drug that blocks the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2), leading to programmed cell death. It is indicated for chronic lymphocytic leukemia (CLL) in patients

- 1,046,736 ents with a specific chromosomal abnormality (17p deletion). In 2015, the US Food and Drug Administration (FDA) granted breakthrough therapy designation to venetoclax for patients with CLL who had relapsed or were refractory to previous treatment and had a 17p deletion genetic mutation.

Combinations of certain anti-LY75 antibodies with venetoclax have now been found to demonstrate synergistic results in the treatment of lymphomas.

Brief description of the invention

In one aspect, the present invention provides a pharmaceutical combination comprising:

(A) an anti-LY75 antibody or antigen-binding portion thereof that competes for binding to LY75 with an antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; or an antibody to LY75 or an antigen-binding portion thereof, wherein said antibody contains:

a) a heavy chain variable region, which contains:

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

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

b) a light chain variable region, which contains:

i) the 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 optionally any one or more of the above SEQ ID NOs independently comprise one, two, three, four or five amino acid substitutions, additions or deletions; and (B) venetoclax or a pharmaceutically acceptable salt thereof;

where the pharmaceutical combination is presented in the form of a combination preparation for simultaneous, separate or sequential use.

In one embodiment, an anti-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 NO: 8, 9 and 10.

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

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

Antibodies containing variable regions of heavy and light chains 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, for example, heavy and light chain variable regions characterized by at least 80-85%, 85-90%, 90-95% or 95-100% sequence identity with any of the above sequences, are also intended to be covered herein invention.

In one embodiment, the anti-LY75 antibody comprises a heavy chain variable region comprising SEQ ID NO: 1 or a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1. In another embodiment, anti

- 2 046736 the body to LY75 contains a light chain variable region containing SEQ ID NO: 2 or a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 2 In another embodiment, the anti-LY75 antibody comprises a heavy chain framework region comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92% of the amino acid sequence. 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 variable heavy chain region of SEQ ID NO: 1 shown under SEQ ID NOs: 16, 17, 18 and 19. In another embodiment, the anti-LY75 antibody comprises a light chain framework region comprising an amino acid sequence of at least 80%, at least 85%, at least 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 of SEQ ID NO: 2 shown under SEQ ID NO: 20, 21, 22 and 23.

In a further embodiment, the anti-LY75 antibody comprises a heavy chain comprising SEQ ID NO: 38 or a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 38. In another embodiment, the anti-LY75 antibody comprises a light chain comprising SEQ ID NO: 39 or a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 39. The heavy chain may contain sequences of SEQ ID NO: 5-7 or 1. The light chain may contain sequences of SEQ ID NO: 8-10 or 2.

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

Other antibodies of the present invention bind to the same epitope or to the 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 the amino acid sequences respectively represented by SEQ ID NOs: 1 and 2, or amino acid sequences, 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 set forth in 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 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 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 NO: 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 antibodies to LY75 contains one or more peptides, for example, two or three peptides selected from the group consisting of SEQ ID NOs: 30, 36 and 37, or fragments thereof, wherein said fragments contain at least 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, anti-LY75 antibodies contain different CDRs compared to the parent antibodies described herein. Thus, the present invention provides antibody variants containing variants of the variable regions of the original

- 3 046736 antibodies, where the parent antibody contains a first vhCDR containing SEQ ID NO: 5, a second vhCDR containing SEQ ID NO: 6, a third vhCDR containing SEQ ID NO: 7, a first vlCDR containing SEQ ID NO: 8, a second a vlCDR comprising SEQ iD NO: 9 and a third vlCDR comprising SEQ ID NO: 10, and wherein the antibody variant collectively has 1, 2, 3, 4, 5 or 6 amino acid substitutions in the set of a first vhCDR, a second vhCDR, a third vhCDR , a first vlCDR, a second vlCDR, and a third vlCDR, wherein 1-4, 1-3, or 1-2 substitutions are particularly useful, and wherein the antibody retains specific binding to LY75.

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

In other embodiments, anti-LY75 antibodies are in the form of an immunoconjugate (ie, they further include a covalently attached functional moiety). In a specific embodiment, the functional moiety is a drug such as a maytansinoid, dolastatin, auristatin, trichothecin, calicheamicin, CC1065, or derivatives thereof. In a preferred embodiment, the drug moiety is DM1 or DM4.

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 different from SEQ ID NO: 3 and 4 due to degeneracy of the genetic code.

In a further aspect, there is provided a method of treating cancer in a patient, comprising simultaneously, sequentially or separately administering to a patient in need thereof therapeutically effective amounts of components (A) and (B) of a pharmaceutical combination of the present invention.

In a further aspect of the present invention, a pharmaceutical combination of the present invention is provided for use in the treatment of cancer.

Also provided is the use of components (A) and (B), as defined herein, in the preparation of a pharmaceutical combination for simultaneous, separate or sequential use for the treatment of cancer. In one embodiment, the cancer is preferably leukemia or lymphoma.

In some embodiments, the cancer is selected from the group consisting of non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, mucosal lymphoid tissue (MALT) lymphoma, B-cell lymphoma, T-cell/histiocyte-rich lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, small cell lymphocytic lymphoma, marginal zone lymphoma, T-cell lymphoma, peripheral T-cell lymphoma, anaplastic large cell lymphoma and angioimmunoblastic T-cell lymphoma, acute myeloid leukemia for and chronic lymphocytic leukemia. More preferably, the cancer is DLBCL or non-Hodgkin's lymphoma.

Also within the scope of the present invention are kits containing the pharmaceutical combination of the present invention and optionally instructions for use. The kit may further contain at least one additional reagent or one or more additional antibodies.

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

Brief description of graphic materials

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

In fig. 2 shows a light chain alignment of LY75_A1 (SEQ ID NO: 2), human germline VK O12 (SEQ ID NO: 13), and human germline JK4 (SEQ ID NO: 14). The LY75_A1 light chain CDR regions are underlined.

In fig. 3a shows the cytotoxic activity of anti-LY75 monoclonal antibodies conjugated to DM1 against HT-29 and shows that while most antibodies bind to LY75, only a few are effective.

In fig. 3b shows the cytotoxic activity of antibodies to LY75 conjugated to DM1

- 4 046736 bo DM4, in relation to HT-29.

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

In fig. Figure 3d shows the cytotoxic activity of antibodies to LY75 conjugated to DM1 or DM4 against Namalwa cells.

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

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

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

In fig. 3h shows the cytotoxic activity of antibodies to LY75 conjugated to DM1 or DM4 against HPAFFII cells.

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

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

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

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

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

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

In fig. Figure 3o shows the cytotoxic activity of antibodies to LY75 conjugated to DM1 or DM4 against RT4 cells.

In fig. 3p shows the cytotoxic activity of antibodies to LY75 conjugated to DM1 or DM4 against 5637 cells.

In fig. 3q shows the cytotoxic activity of antibodies to LY75 conjugated to DM1 or DM4 against SW780 cells.

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

In fig. Figure 3 shows the cytotoxic activity of antibodies to LY75 conjugated to DM1 or DM4 against OE 19 cells.

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

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

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

In fig. Figure 3w shows the cytotoxic activity of antibodies to LY75 conjugated to DM1 or DM4 against RPMI8226 cells.

In fig. Figure 4a shows the effectiveness of anti-LY75 antibodies conjugated to DM1 or DM4 against Raji Burkitt lymphoma cells in a xenograft SCID mouse model.

In fig. 4b shows the effectiveness of anti-LY75 antibodies conjugated to DM1 or DM4 against Namalwa Burkitt lymphoma cells in a xenograft SCID mouse model.

In fig. Figure 4c shows the effectiveness of anti-LY75 antibodies conjugated to DM1 or DM4 against HPAFII pancreatic adenocarcinoma cells in a xenograft model in athymic nude mice.

In fig. Figure 4d shows the effectiveness of anti-LY75 antibodies conjugated to DM1 or DM4 against human bladder carcinoma SW780 cells in a xenograft model in SCID mice.

In fig. Figure 4e shows the effectiveness of anti-LY75 antibodies conjugated to DM1 or DM4 against MDA-MB-468 cells in a xenograft model in athymic nude mice.

In fig. Figure 4f shows the effectiveness of anti-LY75 antibodies conjugated to DM1 or DM4 against COLO205 colorectal adenocarcinoma cells in a xenograft model in athymic nude mice.

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

In fig. 5b shows non-competitive binding of LY75_A1 and anti-LY75 mAb conjugated to MCC-DM1.

- 5 046736

Figures 6a-6j show graphical representations of the binding of the LY75_A1 antibody to the LY75 peptides in the peptide microarray.

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

In fig. Figure 8 shows the algebraic evaluation graph: CI. versus the fractional effect of different doses of LY75_DM4 in combination with venetoclax against the U2932 ABC-DLBCL cell line.

In fig. 9 shows a plot of algebraic evaluation: CI versus fractional effect of different doses of LY75_DM4 in combination with venetoclax against the HBL-1 ABC-DLBCL cell line.

In fig. 10 shows a plot of algebraic evaluation: CI versus fractional effect of different doses of LY75_DM4 in combination with venetoclax against the HBL-1 ABC-DLBCL cell line.

In fig. 11 shows a plot of algebraic evaluation: CI versus fractional effect of different doses of LY75_DM4 in combination with venetoclax against the TMD8 ABC-DLBCL cell line.

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 presented in the form of a combination preparation for simultaneous, separate or sequential use. Component (A) refers to an anti-LY75 antibody as defined herein. Component (B) refers to venetoclax or a pharmaceutically acceptable salt thereof.

One example of the LY75 protein is shown at 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 lymphoma, mucosal lymphoid tissue (MALT) lymphoma, T-cell/histiocyte-rich B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, small cell lymphocytic lymphoma, marginal zone cell lymphoma, T-cell lymphoma, peripheral T-cell lymphoma , anaplastic large cell lymphoma and angioimmunoblastic T-cell lymphoma.

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

Antibodies to LY75 have specific structural features, such as CDR regions with specific amino acid sequences. Disclosed herein is a set of CDRs that can form an affinity reagent, for example an antibody, that is characterized by binding to LY75.

Any of the anti-LY75 antibodies of the present invention can be an isolated antibody.

Thus, the present invention provides antibodies, preferably isolated antibodies (which, as summarized below, include a wide variety of well-known antibody structures, derivatives, mimetics and conjugates), nucleic acids encoding antibody combinations, host cells used to produce the 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 that directs 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, designated herein as OGTA076b and OGTA076c, are expressed in malignant Hodgkin lymphoma cells called Hodgkin and Reed Sternberg (HRS) cells. LY75 acts as an endocytic receptor that directs 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 pancreatic, bladder, ovarian, breast (including triple negative), colorectal, esophageal, skin, thyroid and lung (non-small cell) cancers, as well as in multiple myeloma and many different subtypes of lymphoma (including DLBCL) and leukemia.

Anti-LY75 antibody may in some cases cross-react with LY75 from a different species

- 6 046736 from a person. For example, to facilitate clinical research, antibodies to LY75 may cross-react with mouse or primate LY75 molecules. Alternatively, in some embodiments, the antibodies may have full specificity for human LY75 and may not exhibit species or other types of cross-reactivity with non-human molecules.

Antibodies useful in the present invention can take a number of formats described herein, including traditional antibodies, as well as derivatives, fragments and mimetics of antibodies described below. In one embodiment, the present invention provides antibody structures comprising the set of 6 CDRs defined herein (containing a small number of amino acid changes described below).

Antibody, as used herein, includes a wide variety of structures understood by those skilled in the art, which in some embodiments contain at least the set of 6 CDRs defined herein; including, but not limited to, conventional antibodies (including both monoclonal and polyclonal antibodies), humanized and/or chimeric antibodies, antibody fragments, engineered antibodies (e.g., having amino acid modifications summarized below), multispecific antibodies (including bispecific antibodies ) and other analogues known from the prior art.

The structural units of traditional 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 approximately 25 kDa) and one heavy chain (typically having a molecular weight of approximately 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 accordingly define the antibody isotype as IgM, IgD, IgG, IgA and IgE. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3 and IgG4. IgM has subclasses including, but not limited to, IgM1 and IgM2. Thus, an isotype, as used herein, means any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. The 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 particularly useful 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 assembled together to form an antigen binding region. Each of the loops is referred to as a complementarity determining region (hereinafter referred to as a CDR) in which amino acid sequence variation is greatest. Variable refers to the fact that certain segments of the variable region vary significantly in sequence among antibodies. Variability within a variable region is not evenly distributed. In fact, V regions are composed of relatively invariant regions, called framework regions (FR), 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, located from the amino terminus to the carboxyl terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The hypervariable region generally covers amino acid residues from amino acid residues about 24-34 (LCDR1; L is light chain), 50-56 (LCDR2) and 89-97 (LCDR3) in the light chain variable region and about about 31-35B (HCDR1; H is heavy chain), 5065 (HCDR2) and 95-102 (HCDR3) in the heavy chain variable region; 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 (for example, 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. 196:901-917). Specific CDRs of the present invention are described below.

Throughout the present specification, when referring to a residue in the variable domain (approximately light chain variable region residues 1107 and heavy chain variable region residues 1-113), the Kabat numbering system is generally used (eg, Kabat et al., supra (1991)).

CDRs contribute to the formation of the antigen-binding or, more specifically, epitope-binding region of antibodies. The term epitope or antigenic determinant refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed either by contiguous amino acids or by non-contiguous amino acids located next to each other due to protein folding into a tertiary structure. Epitopes formed by adjacent amino acids are usually

- 7 046736 but are retained when exposed to denaturing solvents, whereas epitopes formed by folding into a tertiary structure are usually lost when exposed 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. 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 adjacent LY75 peptides are examined for reactivity with the indicated anti-LY75 antibody. Methods for determining the spatial conformation of epitopes include techniques known in the art and techniques described herein, for example, X-ray crystallography and 2-dimensional 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 of identifying the molecular determinants of antigen recognition by an antibody.

The carboxy-terminal part of each chain defines constant regions that are primarily responsible for effector function. Kabat et al. collected data on multiple primary sequences of heavy chain and light chain variable regions. 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.).

In the IgG subclass of immunoglobulins, the heavy chain contains several immunoglobulin domains. By immunoglobulin (Ig) domain, as used herein, is meant a region of an immunoglobulin having a distinct tertiary structure. Of interest in the present invention are heavy chain domains including constant (CH) and hinge heavy chain domains. In relation to IgG antibodies, each of the IgG isotypes has three CH regions. Accordingly, the CH'' domains in relation to IgG are as follows: CH1 refers to positions 118-220 according to the Kabat EU index. CH2 refers to positions 237-340 according to the Kabat EU index, and CH3 refers to positions 341-447 according to the Kabat EU index.

Another type of Ig heavy chain domain is the hinge region. By hinge region or hinge region or antibody hinge region or immunoglobulin hinge region, as used herein, is meant a flexible polypeptide comprising amino acids between the first and second constant domains of an antibody. Structurally, the CH1 domain of IgG ends at position 220 according to EU, and the CH2 domain of IgG begins at position position 237 according to EU. Thus, for an IgG antibody, the hinge region is defined herein to include positions 221 (D221 in IgG1) to 236 (G236 in IgG1), where the numbering corresponds to the Kabat EU index. In some embodiments, such as the Fc region, a lower hinge portion is included, with the lower hinge portion typically being at positions 226 or 230.

Of particular interest in the present invention are the Fc regions. By Fc, or Fc region, or Fc domain, as used herein, is meant a polypeptide containing the constant region of an antibody, excluding the first domain of the immunoglobulin constant region and, in some cases, part of the hinge region. Thus, Fc refers to the last two constant region domains of the immunoglobulins IgA, IgD and IgG, the last three constant region domains of the immunoglobulins IgE and IgM, and the flexible hinge region located N-terminal to 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 the human IgG heavy chain is generally defined to include residues C226 or P230 to its carboxyl terminus, where numbering corresponds to the Kabat EU index. In some embodiments, as more fully described below, amino acid modifications are made to the Fc region, eg, altering binding to one or more FcYR receptors or an FcRn receptor.

In some embodiments, the antibodies are full length antibodies. By full-length antibody, as used herein, is meant a structure that is the natural biological form of an antibody, comprising variable and constant regions containing one or more of the modifications summarized herein.

Alternatively, antibodies may be a variety 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, antibody fusion products (sometimes referred to herein as antibody conjugates) and, respectively, fragments of each. Structures based on the use of the CDR set are included in the definition of an antibody.

In one embodiment, the antibody is an antibody fragment. Specific antibody fragments include, but are not limited to (i) a Fab fragment consisting of VL-, VH-, CL- and CH1

- 8,046,736 domains, (ii) Fd fragment, consisting of VH and CHI domains, (iii) Fv fragment, consisting of VL and VH domains of a separate antibody; (iv) a dAb fragment (Ward et al., 1989, Nature 341:544-546, incorporated by reference in its entirety) consisting of a separate variable region, (v) isolated CDR regions, (vi) P(ab ')2-fragments, bivalent fragments containing two linked Fab fragments, (vii) single-chain Fv (scFv) molecules, where the VH domain and VL domain are linked by a peptide linker, allowing the two domains to join to form the antigen-binding site (Bird et al., 1988, Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883, (viii) bispecific single chain Fv (WO 03/ 11161, incorporated by reference in its entirety) and (ix) diabodies or tribodies, polyvalent or polyspecific fragments constructed by gene fusion (Tomlinson et. al., 2000, Methods Enzymol. 326:461-479; WO94/13804; Holliger et al., 1993, Proc. Natl. Sci. 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, for example, a chimeric antibody and/or a humanized antibody. That is, in the present invention, CDR sets can be used with framework and constant regions other than those specifically described in sequence herein.

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 mouse (or, in some cases, rat) variable region(s) and human constant region(s). 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 CDRs, 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 transplanted into a beta sheet framework in the variable region of a human antibody to produce an antibody whose specificity is determined by the transplanted CDRs. The preparation 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. A reverse mutation by replacing selected residues of the acceptor framework regions with the corresponding donor residues is often necessary to restore the affinity lost by the construct after the initial transplantation (US 5530101; US 5585089; US 5693761; US 5693762; US 6180370; US 5859205; US 5821337; US 60 54297; US 6,407,213, all of which are incorporated by reference in their entirety). The humanized antibody optimally also contains at least a portion of an immunoglobulin constant region, typically human immunoglobulin, and accordingly typically contains a human Fc region. Humanized antibodies can also be produced 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 for humanizing and reconstituting non-human antibodies are well known in the art (see Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA), and literature referenced therein, all of which are incorporated by reference in their entirety). Humanization methods include, but are not limited to, 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 described, for example, in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973, incorporated by reference in its entirety. In one embodiment, the parent antibody has been subjected to affinity maturation known in the art. Structural methods, such as those described in USSN 11/004590, can be used for humanization and affinity maturation. Selection-based methods can be used to humanize and/or affinity mature the variable regions of antibodies, 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 transplanting only parts of the CDR, including, but not limited to, 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.

Antibodies disclosed herein may be isolated or recombinant. You

- 9 046736 divided, as 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 is expressed. Thus, an isolated antibody is intended to mean an antibody that is substantially free from other antibodies with different antigen specificities (eg, an isolated antibody that specifically binds to LY75 is substantially free from antibodies that specifically bind to antigens other than LY75). Thus, the isolated antibody is presented in a form not typically 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 with which it is normally associated in its natural state, for example, comprising at least about 5% or at least about 50% by weight of the total protein in the sample. It is understood that the isolated protein may range from 5 to 99.9% by weight of the total protein content depending on the circumstances. For example, the protein can be produced at a significantly higher concentration by using an inducible promoter or a promoter that allows for high expression, thereby producing the protein at increased concentration levels. In the case of recombinant proteins, the definition includes production of the antibody in a wide variety of organisms and/or host cells known in the art in which it is not produced naturally. Typically, the isolated polypeptide is obtained through at least one purification step. An isolated antibody refers to an antibody that is substantially free from other antibodies with different antigen specificities. For example, an isolated antibody that specifically binds to LY75 contains virtually no antibodies that specifically bind antigens other than LY75. Of course, the isolated anti-LY75 antibody may be related to venetoclax.

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

The anti-LY75 antibodies of the present invention specifically bind to LY75 (eg, SEQ ID NO: 15). Specific binding, or specifically binds to, or specific to, with respect to a particular antigen or epitope means binding that is measurably different from a nonspecific interaction. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is typically 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 exhibited, for example, by 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 approximately 10 ^-12 M or greater, where KD refers to the dissociation rate constant for a particular antibody-antigen interaction. An antibody that specifically binds to an antigen will typically have a K _D 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 antibody ADC of the present invention, it is important that the KD is sufficient to ensure internalization and hence cell death without significant side effects.

Specific binding to a particular antigen or epitope may also be exhibited, for example, by an antibody having a K _A or K _a for the antigen or epitope at least 20, 50, 100, 500, 1000, 5000, 10,000 or more times that of the epitope. compared to control, where K _A or K _a refers to the rate constant of association for a particular antibody-antigen interaction.

Standard assays for assessing the ability of antibodies to bind to LY75 can be performed at the protein or cellular level and are known in the art, including, for example, ELISA, Western blot assays, RIA, BIAcore® assays, and flow cytometry assays. Suitable assays are described in detail 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 the Biacore® system assay. To assess binding to Raji or Daudi cells of a B-cell tumor, Raji cells (ATCC deposit number CCL-86) or Daudi cells (ATCC deposit number CCL-213) can be obtained from publicly available sources such as the American

- 10 046736 collection of type cultures, and used in standard assays such as flow cytometry 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 LY75-binding antibodies or, for ease of description, anti-LY75 antibodies. Both terms are used interchangeably throughout this document.

Antibodies to LY75 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 briefly described herein, using standard assays, such as using MAbZap. It will be apparent to one skilled 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, thereby bringing the drug into the cell. A toxic drug will have the ability to destroy cells, i.e. to destroy 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 in vitro assays, anti-LY75 antibodies are added together with an anti-LY75 antibody containing the toxin; for example, the anti-LY75 antibody may be murine or humanized, and the anti-LY75 antibody may be anti-mouse or anti-humanized 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, leading to 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 limited by any 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 comprises complementarity determining regions (CDRs) or variable regions (VRs) of the heavy and light chains of a particular antibody described herein (eg, referred to herein as LY75_A1). Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2 and CDR3 domains of the variable heavy chain (VH) region of the antibody LY75_A1 having the sequence shown in SEQ ID NO: 1, and the CDR1, CDR2 and CDR3 domains of the variable region of the light chain (VL) of the antibody LY75_A1 having the sequence shown under SEQ ID NO: 2.

In another embodiment, an anti-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 comprising SEQ ID NO: 7; and a light chain variable region comprising a first vlCDR comprising 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 the amino acid sequence comprising SEQ ID NO: 1 and conservative sequence modifications thereof. The antibody may further comprise a light chain variable region comprising the amino acid sequence comprising SEQ ID NO: 2 and 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 set forth in SEQ ID NO: 1 and/or 2, respectively, and conservative sequence modifications thereof.

In a further embodiment, anti-LY75 antibodies bind to human LY75 and comprise a heavy chain and light chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 38 and/or 39, respectively, and modifications of conserved sequences thereof.

As used herein, the term conservative sequence modification refers, for example, to the replacement of an amino acid with an amino acid having similar characteristics. It is generally common knowledge to one skilled 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 NO: 5-10 independently provide one, two, three, or

- 11 046736 one or five conservative amino acid substitutions; optionally, one or more of SEQ ID NO: 510 independently provide one or two conservative amino acid substitutions.

Preferably, the term conservative sequence modifications is intended to include amino acid modifications that do not affect or significantly alter the binding characteristics of the antibody comprising 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 identified in the prior 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 (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. 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 variable regions of heavy and light chains 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, for example, heavy and light chain variable regions characterized by at least 80-85%, 85-90%, 90-95% or 95-100% sequence identity with any of the above sequences, are also intended to be covered herein invention. In one embodiment, the anti-LY75 antibody comprises a heavy chain variable region comprising SEQ ID NO: 1 or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In another embodiment, the anti-LY75 antibody comprises a variable region light chain containing SEQ ID NO: 2 or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2. In another embodiment, an anti-LY75 antibody comprises a heavy chain framework region comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the heavy chain variable region framework of SEQ ID NO: 1 comprising SEQ ID NOs: 16, 17 and 18. In another embodiment, an anti-LY75 antibody comprises a light chain framework comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least is at least 98% or at least 99% identical to the light chain variable region framework of SEQ ID NO: 2 containing SEQ ID NOs: 19, 20 and 21.

In a further embodiment, the anti-LY75 antibody comprises a heavy chain comprising SEQ ID NO: 38 or a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 38. In another embodiment, the anti-LY75 antibody comprises a light chain comprising SEQ ID NO: 39 or a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 39. The heavy chain may contain sequences of SEQ ID NO: 5-7 or 1. The light chain may contain sequences of SEQ ID NO: 8-10 or 2.

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

- 12 046736

A1 SEQ Ш NO Heavy chain variable region CDR1 5 CDR2 variable heavy chain region 6 CDR3 variable heavy chain region 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 contain the sets of CDRs of the present invention, as well as full-length heavy and light chains (eg, also containing constant regions). Those skilled in the art will appreciate that anti-LY75 antibody CDR sets may be included in murine, humanized, or human constant regions (including framework regions). Accordingly, provided herein are variable heavy and light chains, as well as full-length heavy and light chains, that are at least 90-99% identical to the SEQ IDs disclosed herein, all with values of 90, 91, 92, 93 94, 95, 96, 97, 98 and 99% are applicable 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 set forth in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence set forth in 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.e. Competitive binding assay. Competitive binding is determined in an assay in which the immunoglobulin of interest inhibits the specific binding of a reference antibody to a common antigen, such as LY75. Many types of competitive binding assays are known, for example, solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme-linked immunosorbent assay (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)); solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct RIA labeled using the I-125 tag (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid-phase direct EIA using a biotin-avidin complex (Cheung et al., Virology 176:546 (1990)) and direct labeling RIA (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either, an unlabeled test immunoglobulin, and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of label bound to a solid surface or to cells in the presence of the immunoglobulin of interest. The immunoglobulin being tested is usually present in excess. If the competing antibody is present in excess, it will generally 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%, 8590%, 90-95%, 95-99% or more.

Monoclonal antibodies can be characterized for binding to LY75 using many known methods. Typically, antibodies are first characterized using ELISA. Briefly, microtiter plates can be coated with purified LY75 in PBS and then blocked with irrelevant proteins such as bovine serum albumin (BSA) diluted in PBS. Dilutions of plasma from mice immunized with LY75 are added to each well 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, plates are developed with ABTS substrate and assayed at OD 405. Preferably, mice that exhibit the highest titers will be used for fusion procedures.

As described above, the ELISA assay can be used to screen for antibodies and thus hybridomas that produce antibodies that exhibit positive reactivity with the LY75 immunogen. Hybridomas that bind preferentially with high affinity to LY75 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 banking and antibody purification.

To purify anti-LY75 antibodies, selected hybridomas can be grown in roller bottles, two-liter spinner flasks, or other culture systems. Supernatant samples can be filtered and concentrated prior to protein A affinity chromatography on Sepharose

- 13 046736 (Pharmacia, Piscataway, NJ, USA) for protein purification. After buffer exchange 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 bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL, USA). 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, microtiter plate wells can be coated with 10 µg/ml anti-Ig 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 two hours. The wells can then be reacted with conjugated probes specific for IgG1 or other isotypes. The plates were developed and analyzed as described above.

Flow cytometry can be used to test the binding of monoclonal antibodies to living cells expressing LY75. Briefly, cell lines and/or human PBMCs expressing membrane-bound LY75 (grown under standard growth conditions) were mixed with various concentrations of monoclonal antibodies in PBS containing 0.1% BSA at 4°C for 1 h. After washing, the cells were reacted with fluorescein-labeled anti-IgG antibody under the same conditions as for primary antibody staining. Samples can be analyzed using a FACScan instrument using light scattering and side scattering properties to impose a logic cut-off on individual cells and determine the binding of labeled antibodies. An alternative analysis using fluorescence microscopy can be used in addition to or instead of flow cytometry analysis. Cells can be stained exactly as described above and examined using fluorescence microscopy. This method provides visualization of individual cells, but may have reduced sensitivity depending on the density of the antigen.

Anti-LY75 IgG antibodies can be further tested for reactivity to the LY75 antigen by Western blotting. Briefly, cell extracts from cells expressing LY75 can be prepared and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked with 20% mouse serum and probed with monoclonal antibodies to be studied. IgG binding can be detected with alkaline phosphatase-conjugated anti-IgG antibody and demonstrated 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, for example, 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 containing 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 _KD of 5x10 ^-8 M or less, binds to the LY75 protein with a KD of 2x10 ^-8 M or less, binds to the LY75 protein with a KD of 5x10 ^-9 M or less, binds to the LY75 protein with a KD of 4x10 ^-9 M or less, binds to the LY75 protein with a KD of 3x10 ^-9 M or less, binds to the LY75 protein with a KD of 2x10 ^-9 M or less, binds to the LY75 protein with a KD of 1x10 ^-9 M or less, binds to LY75 protein with a KD of 5 x 10 ^-10 M or less, or binds to a LY75 protein with a KD of 1 x 10 ^-10 M or less.

In one embodiment, anti-LY75 antibodies compete (eg, cross-compete) for binding to LY75 with specific anti-LY75 antibodies described herein (eg, LY75_A1). Such competitive 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 unlabeled antibodies to eliminate the labeled antibody from competition for binding is assessed. Additionally or alternatively, the BIAcore assay can be used to assess the ability of antibodies to cross-compete. The ability of the test antibody to inhibit 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 antibody.

- 14 046736 described in this document (eg LY75_A1). Standard epitope mapping techniques, such as X-ray crystallography and 2-dimensional nuclear magnetic resonance spectroscopy, can be used to determine whether 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.

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 has been isolated having the desired properties described herein, other mAbs with similar properties, eg, having the same epitope, can be generated. For example, mice can be immunized with LY75 as described herein, hybridomas can be generated, and the resulting mAbs can be screened for the ability to compete with the parent mAb for binding to LY75. Mice can also be immunized with a smaller fragment of LY75 containing the epitope to which the parent mAb binds. The localization of the epitope can be determined by, for example, screening for binding to a series 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 original antibody's heavy chain is first paired with a pool of (preferably human) light chains to select mAbs that bind to LY75, and then the new light chain is paired with a pool of (preferably human) heavy chains to select (preferably human) mAbs that bind LY75, having the same epitope as the original mAb. Alternatively, variants of the original mAb can be obtained 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, the LY75 antigen can be incubated with a saturating amount of a first anti-LY75 antibody or antigen-binding fragment thereof conjugated to a tag compound (eg, H, 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 unlabeled blocking antibody compared to the absence of blocking antibody. Thus, if there is 50% inhibition of binding of a labeled antibody 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, reference to competition between the first and second antibodies being 50% or greater, 60% or greater, 70% or greater, 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 the binding of the second antibody to the antigen in the absence of the first antibody). Thus, inhibition of binding of a first antibody to an antigen by a second antibody of 50, 60, 70, 71, 72, 73, 74, 75, 80, 85, 90, 95, 96, 97, 98, 99% or greater means that two antibodies bind to the same epitope.

The present invention covers antibody variants, sometimes also referred to as antibody derivatives or antibody analogs. That is, 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 (eg , for the addition of conjugated drugs, etc.).

By variant, as used herein, is meant a polypeptide sequence that differs from that of the parent polypeptide due to at least one amino acid modification. In this case, the parent polypeptide is a full length heavy or light chain variable region, such as those set forth in SEQ ID NOs: 1 or 2, respectively, or the CDR regions or framework regions of the heavy and light chains set forth in SEQ ID NOs: 5-10 and 16- 21 for LY75. Amino acid modifications can include substitutions, insertions and deletions, with the former being preferred in many cases. It will be appreciated 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 with a naturally occurring or non-naturally occurring amino acid.

In general, the variants may contain any number of modifications, provided that the antibody function will still be present, as described herein. In other words, in the case of LY75_A1, for example, the antibody should still specifically bind to human LY75. If amino acid variants are produced with an Fc region, for example, then the antibody variants should retain the receptor binding functions required for the particular antibody application or indication

- 15 046736 to him.

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

However, in general, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions are typically used since the goal is often to change function with a minimum number of modifications. In some cases, there are from 1 to 5 modifications (eg, individual 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 area being modified; for example, CDR areas generally require fewer modifications. 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 CDR1, CDR2 or CDR3 of the heavy and/or light chains. In a further embodiment, modifications are made to any of the CDR1 or CDR2 of the heavy and/or light chains. In yet another further 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 present in functional domains: for example, it may be necessary to have 1-5 modifications in the Fc region of wild-type or engineered proteins, as well as 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 parent sequences (e.g., variable regions, constant regions, and/or heavy and light chain sequences, and/or CDR LY75_A1). It should be noted that, depending on the size of the sequence, the percentage identity will depend on the number of amino acids.

By amino acid substitution or substitution as used herein is meant the replacement of an amino acid at a specific position in the original polypeptide sequence with another amino acid, which may be a naturally occurring or non-naturally occurring amino acid. For example, the S100A substitution refers to a polypeptide variant in which the 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 form a variant. In general, the starting polypeptide herein is LY75_A1. Accordingly, by parent antibody as used herein is meant an antibody that is 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, etc. WT have an amino acid sequence or nucleotide sequence that has not been intentionally modified.

By Fc region variant, as used herein, is meant an Fc sequence that differs from the wild-type Fc sequence due to at least one amino acid modification. An Fc variant may refer to the Fc polypeptide itself, 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. Typically, only 1, or 2, or 3 amino acids are changed in any single CDR, and in a set of 6 CDRs, typically no more than 4, 5, 6, 7, 8, 9, or 10 changes are made. 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 obvious that substitutions can be made in any of the 6 CDRs. In one embodiment, substitutions are made in CDR1 of the heavy and/or light chains.

In some cases, amino acid modifications in CDRs are called affinity maturation. An antibody subjected to affinity maturation has one or more changes in one or more CDRs that result in improved affinity of the antibody for an antigen compared to a parent antibody lacking 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 not usually 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%, about 90 %, approximately 100%, approximately 110%, approximately 120%, approx.

- 16 046736 significantly 130%, approximately 140%, approximately 150% or more or 1, 2, 3, 4-5 times compared to the original antibody. Preferred antibodies subjected to affinity maturation will have nanomolar or even picomolar affinity values 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 variable light chain (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. 226:889896, for example.

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

Thus, the definition of CDRs and antibodies disclosed herein includes variants of CDRs and antibodies; those. antibodies may include amino acid modifications in one or more of the LY75_A1 CDRs. In addition, as summarized below, amino acid modifications can also be independently and optionally made to any region outside the CDR, including the framework and constant regions described herein.

In some embodiments, anti-LY75 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, conferring a number of important functional abilities called 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 isoforms FcYRIIIa (including allotypes V158 and F158 associated with antibody-dependent cellular cytotoxicity (ADCC)) and FcYRIIIb (including allotypes FcYRIIIb-NAl and FcYRIIIb-NA2), FcRn (neonatal receptor) , C1q (a complement protein involved in complement-dependent cytotoxicity (CDC)) and FcRn (a neonatal receptor involved in the regulation of serum half-life). Suitable modifications can be made to one or more provisions, as generally set forth in, for example, US patent application 11/841654 and the literature cited therein, 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, particularly 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 incorporating disulfide bridges linking the 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 antibody-drug conjugate (ADC) pathways, further described below. In some embodiments, the antibody constant region may be designed to contain one or more particularly thiol-reactive cysteine residues to provide more specific and controlled placement of the drug moiety. See, for example, US Pat. No. 7,521,541, which is 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, some 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 often reacted with α-haloacetates (and 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, etc.

Histidyl residues are derivatized by reaction with diethyl pyrocarbonate at pH 5.5

- 17 046736

7.0 because the agent is relatively specific to the histidyl side chain. Also useful is p-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 succinic anhydrides 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; 2,4pentanedione and glyoxylate in a transaminase-catalyzed reaction.

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 functionality. In addition, these reagents can react with lysine groups as well as the epsilon amino group of arginine.

Specific modifications of tyrosyl residues can be made, with particular interest being the introduction of spectral tags into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. To form 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 radioimmunoassays, 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' optionally represent 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 methods in addition to the methods described below. Commonly used cross-linking agents include, for example, 1,1bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, such as 4-azidosalicylic acid esters, homobifunctional imido esters, including disuccinimidyl esters such as 3 ,3'-dithiobis(succinimidyl propionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizers such as methyl 3-[(p-azidophenyl)dithio]propioimidate provide photoactivatable intermediates capable of cross-linking in the presence of light. Alternatively, reactive water-insoluble matrices, such as cyanogen bromide activated carbohydrates and reactive substrates described in US Pat. Nos. 3,969,287, are used to immobilize proteins; 3691016; 4195128; 4247642; 4,229,537 and 4,330,440, 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 slightly 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 the α-amino groups of lysine, arginine and histidine side chains (T. E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, pp. 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 appreciated by those skilled in the art, any labels (including fluorescent, enzymatic, magnetic, radioactive, etc.) can be added to the 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 linking 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 in, for example, PEG 2005-2006 from Nektar Therapeutics (available on the website Nektar), US patents 4640835; 4496689; 4301144; 4670417; 4791192 or 4179337, all of which are incorporated by reference in their entirety. In addition, as is known in the art, amino acid substitutions can be made at various positions in the antibody to facilitate the addition of polymers such as PEG. See, for example, US Patent Application Publication No. 2005/0114037A1, incorporated by reference in its entirety.

- 18 046736 completeness.

In additional embodiments, the antibodies may contain a label. By labeled as used herein, it is meant that a compound has at least one element, isotope, or chemical compound attached to provide detection of the compound. Typically, tracers are divided into three classes: a) isotopic tracers, which may be radioactive or heavy isotopes; b) magnetic, electrical, temperature marks and c) colored or luminescent dyes; although tags 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, erythrosine, 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 to drugs to form antibody-drug conjugates (ADCs). Typically, ADCs are used in oncology pathways where the use of antibody-drug conjugates for local delivery of cytotoxic or cytostatic agents provides targeted delivery of the drug moiety to tumors, which can provide higher efficacy, lower toxicity, etc. P. This technology is reviewed in 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, among other things, anti-LY75 antibodies conjugated to drugs. Typically, conjugation is accomplished by covalent attachment to an antibody, as detailed below, and typically relies on the use of a linker, often a peptide bond (which, as described below, may or may not be sensitive to protease cleavage at the target site). In addition, as described above, linker-drug unit (LU-D) binding can be accomplished by attachment to cysteine residues in the antibody. As will be appreciated by those skilled in the art, the number of drug functional moieties per antibody can vary depending on the reaction conditions, and the drug:antibody ratio can vary from 1:1 to 10:1. As will be appreciated by those skilled in the art, the actual amount is an average.

Thus, antibodies to LY75 can be conjugated to drugs. As described below, the ADC drug can be any number of agents, including, but not limited to, cytotoxic agents such as chemotherapeutic agents, growth inhibitors, toxins (eg, 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 of using ADCs.

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

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

Toxins can be used in antibody-toxin conjugates and include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, low molecular weight toxins such as geldanamycin (Mandler et al (2000) J. Nat. Cancer Inst. 92(19) : 1573-1581; Mandler et al.

- 19 046736 (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 (WO2004/026293; 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 low 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 DM4. Maytansine compounds suitable for use as maytansinoid functional moieties representing drugs are well known in the art and can be isolated from natural sources according to known methods, obtained using genetic engineering techniques (see Yu et al ( 2002) PNAS 99:7968-7973) or maytansinol and maytansinol analogues obtained synthetically according to known methods. As described below, drugs can be modified by including a functional group, such as a thiol group or an amino group, for conjugation with an antibody.

Exemplary maytansinoid drug functional moieties include components that have a modified aromatic ring, such as C19 dechloro derivatives (US Pat. No. 4,256,746) (obtained by reducing ansamitocin P2 with lithium aluminum hydride); C-20-hydroxy- (or C-20-demethyl-) +/-C-19-dechloro derivatives (US Pat. Nos. 4,361,650 and 4,307,016) (obtained by demethylation with Streptomyces or Actinomyces or dechlorination with LAH) and C- 20-demethoxy, C-20-acyloxy (--OCOR), +/dechloro derivatives (US Patent No. 4,294,757) (obtained by acylation with acid chlorides), as well as components that have modifications at other positions.

Exemplary maytansinoid drug functional moieties also include components that have modifications such as C-9-SH (US Pat. No. 4,424,219) (produced by reacting maytansinol with H2S or P2S5); C-14alkoxymethyl(demethoxy/CH ₂ OR) (US patent No. 4331598); C-14-hydroxymethyl or acyloxymethyl (CH2OH or _CH2OAc ) (US Patent No. 4450254) (derived from Nocardia); C-15-hydroxy/acyloxy (US Patent No. 4364866) (obtained by the conversion of maytansinol by Streptomyces); C15-methoxy (US Pat. Nos. 4,313,946 and 4,315,929) (isolated from Trewia nudlflora); C-18-N-demethyl (US Patent Nos. 4362663 and 4322348) (obtained by demethylation of maytansinol by Streptomyces) and 4,5-deoxy (US Patent No. 4371533) (obtained by reduction of maytansinol with titanium trichloride/LAH).

Particularly applicable 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, WO02/098883, 6441163, 7368565, WO02/16368 and WO04/1033272, all of which are explicitly included through links in their entirety.

ADCs containing maytansinoids, methods for their preparation and their therapeutic uses are disclosed, for example, in US patent No. 5208020; 5416064; 6441163 and European patent EP 0425235 B1, the disclosures of which are expressly incorporated herein by reference. In Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996), describes ADCs containing a maytansinoid designated DM1 linked to the monoclonal antibody C242 directed against human colorectal 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 the maytansinoid was conjugated via a disulfide linker to mouse A7 antibody, which binds antigen in human colon cancer cell lines, or to another mouse monoclonal antibody. 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 3x10 ⁵ HER-2 surface antigens per cell. The conjugate 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.

For compositions containing multiple antibodies, drug loading is represented as p, the average number of drug molecules per antibody. The drug load can vary from 1 to 20 drug molecules (D) per antibody. Average number of drug molecules per antibody in a product produced by

- 20 046736 conjugation reactions can be characterized using traditional 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, the separation, purification and characterization of homogeneous antibody-drug conjugates, where p is a certain value, from antibody-drug conjugates with a different drug load can be accomplished using techniques such as reverse phase HPLC or electrophoresis. In illustrative embodiments, p is equal to or a fraction of 2, 3, 4, 5, 6, 7, or 8.

The preparation of antibody-drug conjugate compounds can be accomplished according to any technique known to one skilled in the art. Briefly, antibody-drug conjugate compounds may comprise an anti-LY75 antibody as the antibody building block, a drug, and optionally a linker that connects the drug and the binding agent.

Many different reactions are available for covalently attaching drugs and/or linkers to binding agents. This can be accomplished by reaction with amino acid residues of the binding agent, for example, antibody molecules, including amino groups of lysine, free carboxylic acid groups of glutamic and aspartic acids, sulfhydryl groups of cysteine and various functional parts of aromatic amino acids. A widely used non-specific method of covalent addition is the carbodiimide reaction of coupling 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 addition of drugs to binders. This method involves the oxidation with periodate of a drug containing glycol groups or hydroxy groups, thereby forming an aldehyde, which is then reacted with a binding agent. The attachment occurs through the formation of a Schiff base with the amino groups of the coupling agent. Isothiocyanates can also be used as coupling agents to covalently attach drugs to binding agents. Other techniques are known to one 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 in the drug and/or intermediate are used. The reaction product between the drug and the intermediate, or derivatized drug, is then reacted with the 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 the compound more suitable for the purpose 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 minimal or tolerable effect on the activity or other properties of the drug.

Typically, antibody-drug conjugates contain a linker unit between the drug unit and the antibody unit. In some embodiments, the linker is cleaved under intracellular or extracellular conditions such that upon cleavage of the linker, the drug entity is released from the antibody into the appropriate environment. For example, solid tumors that secrete certain proteases can serve as targets for a cleavable linker; in other embodiments, intracellular proteases are used. In still several other embodiments, the linker unit is not cleavable and the drug is released, for example, by degradation of antibodies in lysosomes.

In some embodiments, the linker is cleaved by a cleavage agent present in the intracellular environment (eg, a lysosome or endosome or a caveola). The linker may be, for example, a peptidyl linker that is 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 in length, or is at least three amino acids in length or more.

Degrading agents may include, but are not limited to, cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide derivatives of drugs, resulting in the release of the active drug into target cells (see, e.g., Dubowchik and Walker, 1999, Pharm Therapeutics 83:67-123). Peptidyl linkers can be cleaved by enzymes that are present in cells expressing LY75. For example, you can use pepti

- 21 046736 dil linker, cleaved by the thiol-dependent protease cathepsin B, characterized by high expression in cancer tissue (for example, Phe-Leu or Gly-Phe-Leu-Gly linker (SEQ ID NO: 46)). Other examples of such linkers are described, for example, in US Pat. No. 6,214,345, which is 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 cleavable linker is pH sensitive, that is, sensitive to hydrolysis at certain pH values. Typically, the pH-sensitive linker is hydrolyzable under acidic conditions. For example, an acid-labile linker that is hydrolyzable in the lysosome (eg, hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic acid amide, orthoester, acetal, ketal, etc.) can be used. (See, for example, US Patent Nos. 5122368; 5824805; 5622929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable below pH 5.5 or 5.0, the approximate pH in the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (such as, for example, a thioester attached to a therapeutic agent via an acylhydrazone linkage (see, for example, US Pat. No. 5,622,929)).

In still several other embodiments, the linker is cleaved under reducing conditions (eg, a disulfide linker). A number of disulfide linkers are known from the prior art, including, for example, those that can be formed using SATA N-succinimidyl-5acetylthioacetate), SPDP N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB N-succinimidyl-3 (2-pyridyldithio)butyrate) and SMPT N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2pyridyldithio)toluene)-, SPDB and SMPT. (See, for example, 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 US Patent No. 4880935).

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

In still several other embodiments, the linker unit is not cleavable and the drug is released by degradation of the antibodies. (See US 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 linking two spaced apart chemical moieties into a stable three-part molecule. It will spontaneously separate from the second chemical functional moiety upon cleavage of its bond with the first component. See for example WO 2007/059404A2, WO06/110476A2, WO05/112919A2, WO2010/062171, WO09/017394, WO07/089149, WO07/018431, WO04/043493 and WO02/083180 , which refer to drug-cleavable substrate conjugates, wherein the drug and the cleavable substrate are optionally linked by a self-cleavable linker, all of which are expressly incorporated by reference.

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

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

In other non-mutually exclusive embodiments, the linker facilitates cellular internalization. In certain embodiments, the linker promotes cellular internalization when conjugated to a therapeutic agent (that is, within the functional linker-therapeutic portion of an antibody-drug conjugate compound described herein). In still several embodiments, the linker promotes cellular internalization when conjugated to both the auristatin compound and the anti-LY75 antibodies of the present invention.

A number of illustrative linkers that can be used in the compositions and methods of the present

- 22046736 invention, 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 (Nsuccinimidyl-3-(2pyridyldithio)butyrate).

Drug loading is expressed as p and is the average number of functional drug moieties per antibody in a molecule. The drug load (p) may 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 amount is a fraction or decimal. Generally, a drug load of 1 to 4 is frequently used, and 1 to 2 is also used. ADCs of the present invention include groups of antibodies conjugated to a number of drug components in an amount from 1 to 20, for example, 1-15, 1-10, 2-9, 3-8, 4-7, 5-6. The average number of drug functional moieties per antibody in ADC preparations obtained from conjugation reactions can be characterized using traditional methods such as mass spectroscopy and ELISA analysis.

It is also possible to determine the quantitative distribution of ADC by 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 using 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 occurs 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 more sufficiently reactive thiol groups through which the linker can be attached. In certain embodiments, higher drug loading, eg, p > 5, may cause aggregation, insolubility, toxicity, or loss of cellular permeability of certain antibody-drug conjugates. In certain embodiments, the drug loading for the ADCs of the present invention ranges from 1 to about 8; from about 2 to about 6; from about 3 to about 5; from about 3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8; from about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3 to about 3.8 or from about 3.3 to about 3.7. 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 some embodiments, less than the theoretical maximum of drug moieties are coupled to the antibody during the conjugation reaction. The antibody may contain, for example, lysine residues that are not reactive with the drug linker intermediate or linker reagent, as discussed below. Antibodies typically contain few free and reactive cysteine thiol groups that may be associated with the drug component; in fact, most thiol groups on 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 on lysine or cysteine.

The loading (drug/antibody ratio) of the ADC can be adjusted 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 the cysteine thiol groups, (iv) designing, 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 (as , for example, in the case of thio-Mab or thio-Fab prepared as disclosed herein and in WO2006/034488 (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 followed by a drug moiety reagent, the resulting product is a mixture of compounds

- 23 046736

An ADC with the distribution of one or more drug moieties attached to an antibody. In a mixture, the average amount of drug per antibody can be calculated using a dual antibody ELISA, antibody-specific and drug-specific. 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 ADCs.

Methods are known for determining whether a drug or antibody-drug conjugate causes 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-based assays can be used to measure viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) by the antibody-drug conjugate.

A thymidine incorporation assay can be used to determine whether 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 I-thymidine for the last 8 hours of the 72 hour period. Incorporation of ^3I -thymidine into culture cells is measured in the presence and absence of an antibody-drug conjugate.

Necrosis or apoptosis (programmed cell death) can be measured to determine cytotoxicity. Necrosis is usually accompanied by increased permeability of the plasma membrane; swelling of the cell and rupture of the plasma membrane. Apoptosis is typically characterized by membrane blebbing, cytoplasmic condensation, and activation of endogenous endonucleases. 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 the cellular 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 this assay, cells are incubated in a medium containing a dye, the cells are washed, and the remaining dye, which reflects the cells' uptake of the dye, is measured spectrophotometrically. The protein-binding dye sulforhodamine 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 for the survival and proliferation of mammalian cells by detecting living, but not dead, cells (see, for example, 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 the incorporation of labeled nucleotides into fragmented DNA) and ELISA-based assays, are described in Biochemica, 1999, p. 2, pp. 34-37 (Roche Molecular Biochemicals).

Apoptosis can also be determined by measuring morphological changes in a cell. For example, as with necrosis, loss of plasma membrane integrity can be determined by measuring the uptake of certain dyes (eg, a fluorescent dye such as acridine orange or ethidium bromide). A method for measuring the number of apoptotic cells was described in Duke and Cohen, Current Protocols in Immunology (Coligan et al. eds., 1992, pp. 3.17.1-3.17.16). It is also possible to label cells with a DNA dye (eg, acridine orange, ethidium bromide, or propidium iodide) and observe chromatin condensation and marginalization 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 blebbing, and cell shrinkage.

The presence of apoptotic cells can be measured in both fixed and floating compartments of the cultures. For example, both compartments can be collected by removing the supernatant, trypsinizing the adherent cells, pooling the slides after a washing step by centrifugation (eg, for 10 min at 2000 rpm), and detecting apoptosis (eg, 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 assessed in a suitable animal model. For example, xenogeneic cancer models can be used where cancer 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 using assays that measure inhibition of

- 24 046736 detection of tumor formation, tumor regression or metastasis, etc.

Therapeutic compositions used in the practice of the above methods can be formulated into pharmaceutical compositions containing a carrier suitable for the desired mode of delivery. Suitable carriers include any material that, when combined with a therapeutic composition, retains the antitumor function of the therapeutic composition and to which the patient's immune system does not typically 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 produced by any suitable method. Such methods involve culturing a host cell containing the isolated nucleic acid(s) encoding the antibodies. As will be appreciated by those skilled in the art, this can be accomplished 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 formed and can be isolated.

The heavy and light chain variable regions of LY75_A1 are disclosed herein (both protein and nucleic acid sequences); as will be appreciated in the art, they can be easily scaled up to produce full length heavy and light chains. In other words, in the presence of DNA fragments encoding the VH and VK segments briefly described 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 genes of Fab fragments or into the scFv gene. During these manipulations, a DNA fragment encoding _VK or VH is functionally linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term operably linked as used herein is intended to mean that two DNA fragments are joined in such a way 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 with another DNA molecule encoding the heavy chain constant regions ( _CH1 , CH2 and CH3). Mouse heavy chain constant region gene sequences are known in the art [see, for example, Kabat, EA, 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 PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is most preferably an IgG1 or IgG4 constant region. In the case of a Fab fragment heavy chain gene, it is possible to form a functional link between the DNA encoding the V _H and another DNA molecule encoding only the heavy chain constant region _CH 1 .

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 DNA encoding the VL with another DNA molecule encoding the light chain constant region CL. Mouse light chain constant region gene sequences 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 PCR amplification. In preferred embodiments, the light chain constant region may be a kappa or lambda chain constant region.

To obtain the scFv gene, a functional link is formed between DNA fragments encoding V _H - and V _L /V _K with another fragment encoding a flexible linker, for example, encoding the amino acid sequence (Gly ₄ -Ser) ₃ , so that the sequences VH and V _L / _VK can be expressed as a continuous single-chain protein, with the _VL / _VK and VH regions 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 85:58795883; McCafferty et al., (1990) Nature 348:552-554].

Provided are nucleic acids encoding the antibodies disclosed herein. Such polynucleotides encode both the variable and constant regions of each of the heavy and light chains, although other combinations are also contemplated in accordance with the compositions described herein.

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

In some embodiments, the nucleic acid(s) encoding antibodies disclosed herein are included in expression vectors, which may be extrachromosomal or intended to be integrated into the genome of a host cell, in which it was entered. Expression vectors may contain any number of appropriate regulatory sequences (including, but not limited to, transcriptional and translational control sequences, promoters, ribosome binding sites, enhancers, origins of replication, etc.) or other components (selectable genes, etc. ), all of which are functionally 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 a first expression vector, light chain in a 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 the choice of host cell, the 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 appropriate to the host cell of choice (e.g. transformation, transfection, electroporation, infection) such that the molecule(s) ) nucleic acid operably linked(s) to one or more expression control elements (eg, in a vector, in a construct produced by cellular processes integrated into the genome of a host cell). 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 formation of the encoded 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, USA, 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 many 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, antibodies can be produced 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 Patent Nos. 4816567, 4816397, 6331415 and 7923221, and Antibody Engineering edited by Kontermann & Dubel, Springer, Heidelberg, 2001 and 2010 Hayhurst & Georgiou, 2001, Curr Opin Chem Biol 5:683-689; Maynard & Georgiou, 2000, Annu Rev Biomed Eng 2:339-76; and Morrison, S. (1985) Science 229:1202.

Component (B) of the pharmaceutical combination is venetoclax or a pharmaceutically acceptable salt thereof. Venetoclax is an oral small molecule drug that blocks the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2), leading to programmed cell death. It is indicated for chronic lymphocytic leukemia (CLL) in patients with a specific chromosomal abnormality (17p deletion). In 2015, the US Food and Drug Administration (FDA) granted breakthrough therapy designation to venetoclax for patients with CLL who had relapsed or were refractory to previous treatment and had a 17p deletion genetic mutation.

The structural formula of venetoclax is given below

C.I.

Its IUPAC name is 4-(4-{[2-(4-chlorophenyl)-4,4-dimethyl-1-cyclohexene-1

- 26 046736 yl]methyl}-1-piperazinyl)-No({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2(1H-pyrrolo[2,3 -b]pyridin-5-yloxy)benzamide. Venetoclax is sold under the trade names Venclexta® in the United States and Venclyxto® in Europe.

Pharmaceutical compositions.

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

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

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

The term "unfixed combination" means that the active ingredients (eg, components (A) and (B)) are present in different units or dosages (eg, as separate compositions or formulations), for example, as a set of parts. Independent components (A) and (B) (in their desired compositions or compositions) can then be administered separately or sequentially, at the same time point or at different time points.

In the case where administration is carried out sequentially, the delay in administration of the second component should not be such that the beneficial effect obtained from the combination is lost. Therefore, in one embodiment, sequential treatment includes administration of 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 first component (B) and then component (A).

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

Components (A) and (B), whether present in the same composition or in separate compositions, may independently be 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 routes of use of the antibodies disclosed herein.

As used herein, a pharmaceutically acceptable carrier includes any possible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents, and the like that 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.e. 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 necessary 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. Sci. 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, 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. 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'-dibenzylethylene diamine. N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylene

- 27 046736 diamine, procaine, etc.

The pharmaceutical combination of the present invention or part 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 glycerin, 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 auxiliary agents such as preservatives, wetting agents, emulsifiers and dispersants. Prevention of microorganisms can be achieved either through the sterilization procedures described above or through the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, etc. 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 including absorption retarding 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 means for pharmaceutically active substances is known from the prior art. Unless any conventional media or agent is incompatible with the active compound, their use is contemplated in the pharmaceutical compositions of the present invention. Additional active compounds may also be included in the compositions.

Therapeutic compositions generally must be sterile and stable under conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentrations. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (eg, glycerin, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants. In many cases it will be preferable to include isotonic agents in the composition, for example, sugars, polyhydric alcohols such as mannitol, sorbitol, or sodium chloride. Prolonged absorption of injectable compositions can be achieved by including in the composition an absorption delaying agent, for example, monostearate salts and gelatin.

Sterile injection solutions can be prepared 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 typically prepared by incorporating the active compound into a sterile vehicle that contains the basic dispersion medium and other necessary ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze drying (lyophilization), which produce a powder form of the active ingredient, as well as any additional required ingredient, from a solution thereof previously subjected to sterilization 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. The amount of active ingredient that can be combined with the carrier material to form a unit dosage form will generally be that amount of the composition that produces 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 a pharmaceutically acceptable carrier.

Dosing regimens are adjusted to achieve the optimal desired response (eg, si

- 28 046736 ergic combination, therapeutic response). For example, a single bolus dose may be administered, multiple divided doses may be administered over time, or the dose may be proportionally decreased 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 dosage. Unit dosage form, as used herein, refers to physically discrete units suitable as unitary dosages for subjects to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect, together with the required pharmaceutical carrier. The specifications for unit dosage forms of the present invention are due to, and are directly affected by (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the inherent limitations in the preparation of such active compound in relation to the treatment of susceptible individuals. .

For administration of anti-LY75 antibody, the dose ranges from about 0.0001 to 100 mg/kg, such as 0.001 to 50 mg/kg, 0.005 to 20 mg/kg, 0.01 to 10 mg/kg, and more typically from 0.01 to 5 mg/kg based on the host’s body weight. 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 body weight or be in the range of 0.1-20 mg/kg, 0.5-15 mg/kg, 1-10 mg/kg, 2-8 mg/kg, 3-7 mg/kg, 4-6 mg/kg. An exemplary treatment regimen includes administration once daily, once every 2 days, once per week, once every two weeks, once every three weeks, once every four weeks, once every month, once every 6 weeks, once every 3 months or once every 3-6 months. Preferred dosage regimens for the 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 schedules: (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 10 to 1500 nM or 18 to 1200 nM (e.g., about 18.75, 37.5, 75, 150, 300, 600 or 1200 nM). Preferably, the anti-LY75 antibody (eg, LY75_DM4) is adjusted to achieve a blood concentration of the antibody of 15 to 50 nM, 50 to 100 nM, 100 to 500 nM, or 500 to 1200 nM.

In some embodiments, the dose of venetoclax (or a pharmaceutically acceptable salt thereof) is adjusted to achieve a plasma concentration of 0.5 to 500 nM or 0.64 to 400 nM (e.g., about 0.64, 3.2, 16 , 80, 400 or 2000 nM). Preferably, venetoclax (or a pharmaceutically acceptable salt thereof) is adjusted to achieve a plasma concentration of 0.5 to 20 nM, 20 to 50 nM, 50 to 100 nM, or 100 to 500 nM. It can be administered orally. In some embodiments, the dose of venetoclax (or a pharmaceutically acceptable salt thereof) is about 100 mg, 200 mg, 300 mg, 400 mg, or 500 mg. Venetoclax or a pharmaceutically acceptable salt thereof can be administered with one or more of cyclophosphamide, hydroxydaunorubicin, Oncovin and prednisone or prednisolone (ie, CHOP therapy).

Preferably, the combination of components (A) and (B) is a synergistic combination. One skilled in the art will appreciate 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. Synergy can be quantified using the ChouTalalay Additivity Index (CI) (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 TC, Motzer RJ, Tong Y, Bosl GJ ., 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. This provides a quantification for strong synergy (CI<0.3), synergism (CI=0.3-0.9), additive effect (CI=0.9-1.1) or antagonism/no benefit (CI>1 ,1), and this provides an algorithm for computer software to automatically model drug combinations. Both the effectiveness (D(m) value) and the shape of the dose-effect curve (m value) of each drug individually and their combinations are taken into account. The Chou-Talalay index of additivity (CI) can be estimated using the Synergy R package (see Preclinical versus Clinical Drugs Combination Studies, Chou TC. Leuk. Lymphoma. (2008); 49(11):2059-2080, and references therein, all of which are specifically incorporated herein by reference). The CI combination can be tested in a suitable cell line, for example, the ABC-DLBLC cell line (such as TMD8 or HBL1), for example, under the conditions used

- 29 046736 in example 26.

Preferably, the pharmaceutical combination of the present invention is a synergistic combination in which the Chow-Talalay 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 of treating cancer in a patient is provided, comprising 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, amounts of components (A) and (B) are administered to a patient to achieve the plasma concentrations disclosed above.

Also provided is the use of synergistic amounts of components (A) and (B) of the pharmaceutical combination of the present invention in the preparation of a pharmaceutical combination for simultaneous, separate or sequential use for 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 anti-LY75 monoclonal antibodies with different types of binding specificities are administered simultaneously, and in this case, the dose of each antibody administered is within specified ranges. The antibody is usually administered several times. The intervals between individual doses may correspond, for example, to once daily, twice weekly, once weekly, once monthly, every three months, every six months or once a year. The intervals may also be irregular, as indicated by measuring antibody levels to the target antigen in the patient's blood. In some methods, the dose is adjusted to achieve plasma antibody concentrations of approximately 1-1000 μg/ml, 5-750 μg/ml, 10-600 μg/ml, 15500 μg/ml, 20-400 μg/ml, and in some methods methods approximately 25-300 µg/ml.

Alternatively, anti-LY75 antibodies can be administered as sustained release formulations, which require less frequent administration. The dose and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dosage 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. Therapeutic routes of administration sometimes require the administration of relatively high doses at relatively short intervals until disease progression is reduced or stopped, and preferably until the patient demonstrates partial or complete relief of disease symptoms. After this, a prophylactic regimen can be applied to the patient.

The actual dosage levels of the active ingredients in the pharmaceutical combinations of the present invention can be varied in order to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition and route of administration without toxicity to the patient. The dose level selected will depend on a number of pharmacokinetic factors, including the potency of the particular 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 drugs, compounds and /or materials used in combination with the particular compositions used, age, sex, body weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical field.

A therapeutically effective dose of an anti-LY75 antibody preferably results in a reduction in the severity of symptoms of the disease, an increase in the frequency and duration of periods without symptoms of the disease, or the prevention of impairment or disability due to the disease. For example, for treating LY75-mediated tumors, a therapeutically effective dose preferably inhibits cell growth or tumor growth by at least about 20%, at least about 30%, more preferably by at least about 40%, at least about 50% , even more preferably at least about 60%, at least about 70%, and even more preferably at least about 80% or at least about 90% compared to subjects who were not treated. The ability of a compound to inhibit tumor growth can be assessed 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. Therapeutically effective amount

- 30 046736 therapeutic compound can reduce the size of a tumor or otherwise reduce the severity of symptoms in a subject. One of ordinary skill in the art will be able to determine such amounts based on factors such 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 through 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 through the same route or through different routes. As will be appreciated by one skilled in the art, the route and/or mode of administration will vary depending on the results desired. Preferred routes of administration of the antibodies of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes, such as by injection or infusion. The phrase parenteral administration as used herein means methods of administration other than enteral and topical administration, typically by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrathoracic injection and infusion.

Alternatively, the anti-LY75 antibody may be administered via a route other than the parenteral route, such as the topical, epidermal, or mucosal route, such as intranasally, orally, vaginally, rectally, sublingually, or topically.

Preferably, venetoclax or a pharmaceutically acceptable salt thereof is administered orally, for example in the form of a tablet.

The 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 copolymer, 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 needle-free hypodermic injection device, such as the devices 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: the implantable microinfusion pump for controlled rate drug dosing, disclosed in US Pat. No. 4,487,603; a therapeutic device for administering drugs through the skin, disclosed in US Pat. No. 4,486,194; a drug infusion pump for delivering a drug at a precise infusion rate, disclosed in US Pat. No. 4,447,233; an implantable variable volume infusion device for continuous drug delivery, 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 an 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, an anti-LY75 antibody may be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) does not allow many highly hydrophilic compounds to pass through. To ensure that therapeutic compounds cross the BBB (if required), they can be formulated, for example, in liposomes. For methods of preparing liposomes, see, for example, US Pat. No. 4,522,811; 5374548 and 5399331. Liposomes can contain one or functional parts that are selectively transported to specific cells or organs, thereby improving the quality of targeted drug delivery [see, for example, 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.

Applications and methods.

As used herein, the term subject is intended to include human and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammalian animals, such as non-humans

- 31 046736 primates, sheep, dogs, cats, cows, horses, chickens, amphibians and reptiles. Preferred subjects include human patients having disorders mediated by impaired LY75 activity.

In some preferred embodiments, the subject is a human subject who has a 17p deletion, for example a person who exhibits features of chronic lymphocytic leukemia.

The methods are particularly suitable for treating human patients having a disorder associated with aberrant LY75 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 medicament for the treatment of such a disorder, including, for example, leukemia , including chronic lymphocytic leukemia and acute myeloid leukemia, non-Hodgkin 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 cell lymphocytic lymphoma, marginal zone lymphoma, T-cell lymphoma, peripheral T-cell lymphoma, anaplastic large cell lymphoma and angioimmunoblastic T-cell lymphoma. LY75 has been demonstrated to be internalized upon binding to an antibody, as illustrated in Examples 5 and 7 below, thus allowing the use of anti-LY75 antibodies in any loading mechanism, such as an ADC approach, a radioimmunoconjugate approach, or an ADEPT approach. .

Antibodies to LY75, usually administered as an ADC, can be used to inhibit or block the function of LY75, which in turn may be associated with the prevention or amelioration of certain disease symptoms, thereby implicating LY75 as a disease mediator . This can be achieved by bringing the sample and control into contact with an anti-LY75 antibody under conditions that allow the formation of a complex between the antibody and LY75. In the sample and control samples, any complexes formed between the antibody and LY75 are detected and compared.

Suitable routes for administering 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 dosages of 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 described above, anti-LY75 antibodies can be administered in conjunction 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 anticancer therapy, such as radiation therapy. Such therapeutic agents include, among others, antineoplastic agents such as doxorubicin (Adriamycin), cisplatin, bleomycin sulfate, carmustine, chlorambucil, as well as cyclophosphamide and hydroxyurea, which themselves are 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, for example gastric cancer, colorectal cancer, prostate cancer, breast cancer, ovarian cancer or lung cancer, such as Avastin®, 5FU and gemcitabine. Co-administration of the anti-LY75 antibodies or antigen-binding fragments thereof of the present invention with chemotherapeutic agents provides two anticancer agents acting through different mechanisms that achieve a cytotoxic effect on human tumor cells. Such co-administration may address problems caused by the development of drug resistance or changes in the antigenicity of tumor cells that may render them unresponsive to the antibody.

The pharmaceutical combinations of the present invention can also be administered together with blood 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 complement or serum may be administered separately.

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

- 32 046736 for the first antibody).

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

In other embodiments, the subject may receive additional treatment with an agent that modulates, for example enhances or inhibits, the expression or activity of Fcy or FcY receptors, for example by treating the subject with a cytokine. Preferred cytokines to administer 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 literary sources referred to herein, including, without limitation, all documents, publications, patents, patent applications, presentations, texts, reports, manuscripts, brochures, books, Internet publications, journal articles, periodicals, newsletters about products, etc., are hereby incorporated by reference in their entirety. The discussion of references in this document is intended only to summarize the statements made by their authors and does not make any acknowledgment that any reference constitutes part of the prior art, and applicants reserve the right to challenge the accuracy and significance of references cited.

Although the foregoing invention has been described in some detail by way of illustration and example for the purpose of clarity of understanding, those of ordinary skill in the art will readily appreciate, in light of the teachings of the present invention, that certain changes and modifications can be made thereto 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.

Following standard procedures, mice (XenoMouse IgG1) were immunized with CHO cells transfected with full-length LY75.

The specificity of antibodies raised against LY75 was examined by flow cytometry using HEK293 cells transfected with LY75 and subsequently using HT29 cells expressing LY75. To examine the ability of the 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 min and then washed twice with the FACS buffer described above. The cell-antibody 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 FACS buffer. Samples were loaded onto a BD FACScanto II flow cytometer and data were analyzed using BD FACSdiva software (results not shown).

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

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

Antibody sequences can be mutagenized to return to germline residues at one or more residue positions.

The nucleotide and amino acid sequences of the heavy chain variable region of LY75_A1 are shown under SEQ ID NO: 3 and 1, respectively. The nucleotide and amino acid sequences of the light chain variable region of LY75_A1 are shown under SEQ ID NO: 4 and 2, respectively.

The amino acid and nucleotide sequences of the heavy chain of LY75_A1 are shown in SEQ ID NO: 38 and 202, respectively. The amino acid and nucleotide sequences of the LY75_A1 light chain are shown in SEQ ID NO: 39 and 203, 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 utilizes a VH segment from the human germline VH 3-15 and a JH segment from the human germline _JH JH4. Further analysis of the LY75_A1 VH sequence using the Kabat system to determine the CDR region resulted in the identification of the heavy chain CDR1, CDR2 and CDR3 regions shown at SEQ ID NO: 5, 6 and 7, respectively. Alignments of the CDR1, CDR2 and CDR3 sequences of VH LY75_A1 with the germline VH 3-15 and germline JH JH4 sequences are shown in FIG. 1.

- 33 046736

Comparison of the LY75_A1 immunoglobulin light chain sequence with known human immunoglobulin light chain germline sequences demonstrated that the LY75_A1 light chain utilizes a VK segment from the human germline V _K O12 and a JK segment from the human germline J _K JK4. Further analysis of the _VK LY75_A1 sequence using the Kabat system to determine the CDR region led to the identification of the light chain CDR1, CDR2 and CDR3 regions shown at SEQ ID NO: 8, 9 and 10, respectively. Alignments of V _K LY75_A1 CDR1, CDR2 and CDR3 sequences with V _K O12 germline and J _K JK4 germline sequences are shown in FIG. 2.

Example 3. Immunohistochemical study using monoclonal antibody to LY75.

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

Materials and methods.

Materials.

Xylenes (X5P-lgal) 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, MA, USA.

Thermo Scientific* Pierce* Peroxidase Inhibitor (35000) from Thermo Scientific, Massachusetts, USA.

Serum-free protein blocking solution (X0909) from Dako, California, USA.

Secondary antibody: Fab-specific goat anti-human IgG antibody conjugated to FITC (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, MA, 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, California, USA.

DAB 2 Solution Set (882014) from Invitrogen, New York, USA.

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

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

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

Preparation of FFPE microslides - paraffin removal and rehydration.

FFPE slides were dewaxed in xylene (2x3 min) and then rehydrated using a 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).

Preparation of FFPE microslides - antigen unmasking (microwave radiation).

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

Obtaining FF microslides.

Microspecimens were removed from storage at -80°C and dried at room temperature in a fume hood for 20-30 minutes. Microspecimens were fixed for 10 minutes in ice-cold acetone at -20°C, then dried for 20 minutes in a fume hood at room temperature. Microspecimens were washed and rehydrated in PBS with 3 washes, each for 3 min. The sections were outlined using 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 provide a solution at 20 times the desired final concentration (20 μg/ml for a final concentration of 1 μg/ml). Goat antigen-binding secondary antibody

- 34 046736 human immunoglobulin G (IgG) fragment (Fab) was prepared in a similar manner in SFPB to create a solution of equal concentration.

Equal volumes of primary and secondary antibody were combined 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 min at room temperature, resulting in a primary antibody concentration twice the required final concentration (2 μg/mL for a final concentration of 1 μg/mL). .

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

Immunostaining.

Meanwhile, endogenous tissue peroxidase activity was blocked by incubating tissues with a peroxidase inhibitor for 5–10 min at room temperature in a humidification chamber. The slides were then washed in PBS 3 times for 3 min for each wash. Tissues were incubated in SFPB for 30 min at room temperature in a humidification chamber. The final staining complexes were applied to each tissue section and/or microarray, and the microslides were incubated for 30 min at room temperature in a humidification chamber. Slides were then washed once in PBS and once in PBST (PBS+0.125% Tween-20) for 3 min for each wash. Mouse anti-FITC tertiary antibody was applied at a concentration of 2 μg/ml for 30 min at room temperature in a humidification chamber. Sections were then washed once in PBS and once in PBST for 3 min for each wash. The HRP-conjugated polymer Dual Link EnVision+ for mouse and rabbit antibodies was then applied to the tissues, and the slides were incubated for 30 min at room temperature in a humidification 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. The slides were then washed once in running tap water for 2 min and once in PBS for 3 min. Microslides 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 the slides were then mounted under coverslips using Faramount mounting medium.

Results.

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

LY75 staining in normal FF tissues was generally absent to low. The ductal epithelium of the mammary gland, salivary gland, and pancreas showed markedly low to moderate levels of staining, and low levels of positive staining were observed in the spleen. Thus, antibodies directed to LY75 could be used as therapeutics and diagnostics in some of the cancers under investigation and possibly in other types of cancer characterized by LY75 expression.

Example 4. Efficacy of anti-LY75 monoclonal antibodies conjugated to DM1 against HT-29 cells.

Materials.

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

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

RPMI 1640 media (MT-10-041-CM) from Fisher Scientific, Pennsylvania, USA.

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

Way.

Cells were dissociated using CellStripper and their number was counted. 5e3 cells/well were centrifuged to obtain a pellet (in the case of cells in suspension, a larger number could 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.

Add 50 μl/well of cell suspension to the wells of a 96-well plate with white walls and a transparent bottom. Antibodies were diluted and titrated at 8 points (titration with 3-fold dilutions), corresponding to concentrations of 0-20 nM (twice the test concentrations). Diluted antibodies or medium (for untreated samples) (50 μl/well) were added accordingly.

- 35 046736 howling holes.

Excess medium (200 μl/well) was added to the outer rows and columns of the plate to prevent evaporation. The plate was incubated for 72 hours at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 minutes. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed once with 100 µl/well PBS (for cells in suspension, the plate was first centrifuged to pellet the cells). 100 μl/well of PBS and 100 μl of 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 efficient cell lysis had occurred. The plate was then read on a Glomax luminometer.

Results.

The results are presented in Fig. 3a, which shows a subpopulation of antibodies known to bind to LY75, which 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. Antibodies were then 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 colorectal cancer cells.

Materials.

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

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

RPMI 1640 media (MT-10-041-CM) from Fisher Scientific, Pennsylvania, USA.

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

Way.

Cells were dissociated using Cell Stripper and their number was counted. 5e3 cells/well were centrifuged to obtain a pellet (in the case of cells in suspension, a larger number could 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.

Add 50 μl/well of cell suspension to the wells of a 96-well plate with white walls and a transparent bottom. Antibodies were diluted and titrated at 8 points (titration with 3-fold dilutions), corresponding to concentrations of 0-20 nM (twice the test concentrations). Diluted antibodies or medium (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 plate was incubated for 72 hours at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 minutes. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed once with 100 µl/well PBS (for cells in suspension, the plate was first centrifuged to pellet the cells). 100 μl/well of PBS and 100 μl of 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 efficient 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 against 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).

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

Materials.

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

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

RPMI 1640 media (MT-10-041-CM) from Fisher Scientific, Pennsylvania, USA.

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

Way.

Cells were dissociated using CellStripper and their number was counted. 5e3 cells/well were centrifuged to obtain a pellet (in the case of cells in suspension, a larger number could 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.

Add 50 μl/well of cell suspension to the wells of a 96-well plate with white walls and a transparent bottom. Antibodies were diluted and titrated at 8 points (titration with 3-fold dilutions

- 36 046736 mi), corresponding to concentrations of 0-20 nM (twice the concentrations studied). Diluted antibodies or medium (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 plate was incubated for 72 hours at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 minutes. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed once with 100 µl/well PBS (for cells in suspension, the plate was first centrifuged to pellet the cells). 100 μl/well of PBS and 100 μl of 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 efficient cell lysis had occurred. The plate was then read on a Glomax luminometer.

Results.

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

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

Materials.

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

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

RPMI 1640 media (MT-10-041-CM) from Fisher Scientific, Pennsylvania, USA.

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

Way.

Cells were dissociated using CellStripper and their number was counted. 5e3 cells/well were centrifuged to obtain a pellet (in the case of cells in suspension, a larger number could 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.

Add 50 μl/well of cell suspension to the wells of a 96-well plate with white walls and a transparent bottom. Antibodies were diluted and titrated at 8 points (titration with 3-fold dilutions), corresponding to concentrations of 0-20 nM (twice the test concentrations). Diluted antibodies or medium (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 plate was incubated for 72 hours at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 minutes. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed once with 100 µl/well PBS (for cells in suspension, the plate was first centrifuged to pellet the cells). 100 μl/well of PBS and 100 μl of 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 efficient 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 against BxPC3 cells. In fig. 3g shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 against HupT4 cells. In fig. 3h shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 against HPAFFII cells. These results demonstrate an increase in cytotoxic activity proportional to antibody concentration compared to other anti-LY75 antibodies conjugated to DM1 and DM4 (selected from Example 1).

Example 8. Efficacy of monoclonal antibodies to LY75, conjugated to DM1 and conjugated to DM4, against cell lines that are substrates of chronic lymphocytic leukemia.

Materials.

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

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

RPMI 1640 media (MT-10-041-CM) from Fisher Scientific, Pennsylvania, USA.

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

Way.

- 37 046736

Cells were dissociated using CellStripper and their number was counted. 5e3 cells/well were centrifuged to obtain a pellet (in the case of cells in suspension, a larger number could 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.

Add 50 μl/well of cell suspension to the wells of a 96-well plate with white walls and a transparent bottom. Antibodies were diluted and titrated at 8 points (titration with 3-fold dilutions), corresponding to concentrations of 0-20 nM (twice the test concentrations). Diluted antibodies or medium (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 plate was incubated for 72 hours at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 minutes. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed once with 100 µl/well PBS (for cells in suspension, the plate was first centrifuged to pellet the cells). 100 μl/well of PBS and 100 μl of 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 efficient 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 against EHEB cells. In fig. Figure 3j shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 against Mec-1 cells. These results demonstrate an increase in cytotoxic activity proportional to antibody concentration compared to other anti-LY75 antibodies conjugated to DM1 and DM4 (selected from Example 1).

Example 9. Efficacy of monoclonal antibodies to LY75, conjugated to DM1 and conjugated to DM4, against cell lines that are substrates of acute monocytic leukemia.

Materials.

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

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

RPMI 1640 media (MT-10-041-CM) from Fisher Scientific, Pennsylvania, USA.

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

Way.

Cells were dissociated using CellStripper and their number was counted. 5e3 cells/well were centrifuged to obtain a pellet (in the case of cells in suspension, a larger number could 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.

Add 50 μl/well of cell suspension to the wells of a 96-well plate with white walls and a transparent bottom. Antibodies were diluted and titrated at 8 points (titration with 3-fold dilutions), corresponding to concentrations of 0-20 nM (twice the test concentrations). Diluted antibodies or medium (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 plate was incubated for 72 hours at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 minutes. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed once with 100 µl/well PBS (for cells in suspension, the plate was first centrifuged to pellet the cells). 100 μl/well of PBS and 100 μl of 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 efficient cell lysis had occurred. The plate was then read on a Glomax luminometer.

Results.

In fig. 3k shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 against AML-193 cells. These results demonstrate an increase in cytotoxic activity proportional to antibody concentration compared to other anti-LY75 antibodies conjugated to DM1 and DM4 (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 dissociation agent) (MT-25-056CI) from Fisher Scientific, Pennsylvania, USA.

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

RPMI 1640 media (MT-10-041-CM) from Fisher Scientific, Pennsylvania, USA.

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

- 38 046736

Way.

Cells were dissociated using Cell Stripper and their number was counted. 5e3 cells/well were centrifuged to obtain a pellet (in the case of cells in suspension, a larger number could 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.

Add 50 μl/well of cell suspension to the wells of a 96-well plate with white walls and a transparent bottom. Antibodies were diluted and titrated at 8 points (titration with 3-fold dilutions), corresponding to concentrations of 0-20 nM (twice the test concentrations). Diluted antibodies or medium (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 plate was incubated for 72 hours at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 minutes. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed once with 100 µl/well PBS (for cells in suspension, the plate was first centrifuged to pellet the cells). 100 μl/well of PBS and 100 μl of 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 efficient 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 against HCC 70 cells (ER-negative, PR-negative and Her2-negative). In fig. 3m shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 against HCC 1806 cells (ER-negative, PR-negative and Her2-negative). In fig. 3n shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 against MDA-MB-468 cells. These results demonstrate an increase in cytotoxic activity proportional to antibody concentration.

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

Materials.

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

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

RPMI 1640 media (MT-10-041-CM) from Fisher Scientific, Pennsylvania, USA.

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

Way.

Cells were dissociated using CellStripper and their number was counted. 5e3 cells/well were centrifuged to obtain a pellet (in the case of cells in suspension, a larger number could 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.

Add 50 μl/well of cell suspension to the wells of a 96-well plate with white walls and a transparent bottom. Antibodies were diluted and titrated at 8 points (titration with 3-fold dilutions), corresponding to concentrations of 0-20 nM (twice the test concentrations). Diluted antibodies or medium (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 plate was incubated for 72 hours at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 minutes. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed once with 100 µl/well PBS (for cells in suspension, the plate was first centrifuged to pellet the cells). 100 μl/well of PBS and 100 μl of 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 efficient cell lysis had occurred. The plate was then read on a Glomax luminometer.

Results.

In fig. Figure 3o shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 against RT4 cells. In fig. 3p shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 against 5637 cells. FIG. 3q shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 against 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

- 39 046736

Scientific, Pennsylvania, USA.

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

RPMI 1640 media (MT-10-041-CM) from Fisher Scientific, Pennsylvania, USA.

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

Way.

Cells were dissociated using CellStripper and their number was counted. 5e3 cells/well were centrifuged to obtain a pellet (in the case of cells in suspension, a larger number could 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.

Add 50 μl/well of cell suspension to the wells of a 96-well plate with white walls and a transparent bottom. Antibodies were diluted and titrated at 8 points (titration with 3-fold dilutions), corresponding to concentrations of 0-20 nM (twice the test concentrations). Diluted antibodies or medium (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 plate was incubated for 72 hours at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 minutes. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed once with 100 µl/well PBS (for cells in suspension, the plate was first centrifuged to pellet the cells). 100 μl/well of PBS and 100 μl of 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 efficient 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 against 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 dissociation agent) (MT-25-056CI) from Fisher Scientific, Pennsylvania, USA.

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

RPMI 1640 media (MT-10-041-CM) from Fisher Scientific, Pennsylvania, USA.

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

Way.

Cells were dissociated using Cell Stripper and their number was counted. 5e3 cells/well were centrifuged to obtain a pellet (in the case of cells in suspension, a larger number could 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.

Add 50 μl/well of cell suspension to the wells of a 96-well plate with white walls and a transparent bottom. Antibodies were diluted and titrated at 8 points (titration with 3-fold dilutions), corresponding to concentrations of 0-20 nM (twice the test concentrations). Diluted antibodies or medium (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 plate was incubated for 72 hours at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 minutes. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed once with 100 µl/well PBS (for cells in suspension, the plate was first centrifuged to pellet the cells). 100 μl/well of PBS and 100 μl of 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 efficient 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 against OE 19 cells. These results demonstrate an increase in cytotoxic activity proportional to the antibody concentration.

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

Materials.

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

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

- 40 046736

RPMI 1640 media (MT-10-041-CM) from Fisher Scientific, Pennsylvania, USA.

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

Way.

Cells were dissociated using CellStripper and their number was counted. 5e3 cells/well were centrifuged to obtain a pellet (in the case of cells in suspension, a larger number could 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.

Add 50 μl/well of cell suspension to the wells of a 96-well plate with white sides and a clear bottom. Antibodies were diluted and titrated at 8 points (titration with 3-fold dilutions), corresponding to concentrations of 0-20 nM (twice the test concentrations). Diluted antibodies or medium (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 plate was incubated for 72 hours at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 minutes. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed once with 100 µl/well PBS (for cells in suspension, the plate was first centrifuged to pellet the cells). 100 μl/well of PBS and 100 μl of 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 efficient cell lysis had occurred. The plate was then read on a Glomax luminometer.

Results.

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

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

Materials.

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

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

RPMI 1640 media (MT-10-041-CM) from Fisher Scientific, Pennsylvania, USA.

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

Way.

Cells were dissociated using CellStripper and their number was counted. 5e3 cells/well were centrifuged to obtain a pellet (in the case of cells in suspension, a larger number could 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.

Add 50 μl/well of cell suspension to the wells of a 96-well plate with white sides and a clear bottom. Antibodies were diluted and titrated at 8 points (titration with 3-fold dilutions), corresponding to concentrations of 0-20 nM (twice the test concentrations). Diluted antibodies or medium (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 plate was incubated for 72 hours at 37°C.

The plate was removed from the incubator and incubated at room temperature for 30 minutes. Meanwhile, the CellTiter Glo solution was thawed. The plate was shaken and washed 1x with 100 µl/well PBS (for suspended cells, the plate was first centrifuged to pellet the cells). Add 100 μl/well PBS and 100 μl Glo cell titer to each well and triturate until mixed. The plate was incubated in the dark at room temperature for 15 min and observed by microscopy to ensure that efficient cell lysis had 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 against MOLP-8 cells. In fig. Figure 3w shows the cytotoxic activity of anti-LY75 antibodies conjugated to DM1 and DM4 against RPMI8226 cells. These results demonstrate an increase in cytotoxic activity proportional to antibody concentration.

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

The efficacy of LY75_DM1 and LY75_DM4 was studied in a xenograft model using subcutaneous injection of Raji Burkitt lymphoma cells into SCID mice.

Immunodeficient SCID mice were inoculated subcutaneously with Raji tumor cells (lymphoma

- 41 046736

Burkitt man). Tumors were allowed to form and mice were assigned to five treatment groups of 3-6 mice per group. When the mean tumor volume reached a mean 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 to determine 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 occurred first. Efficacy was determined based on tumor growth delay (TGD), increase in median time to endpoint (TTE), and log-rank analysis of differences in Kaplan-Meier survival curves for ADC-treated versus mice treated with ADCs. treated with PBS. Tumor samples were collected from the first five control vehicle-treated mice that reached the endpoint and 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 a Raji cell 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 complete but transient tumor regression in 5 of 6 mice. All treatments were well tolerated and no clinical signs of toxicity were observed. These data suggest that ADCs targeting LY75, such as LY75_DM1 and LY75_DM4, may provide clinical benefit 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 studied in a xenograft model using subcutaneous injection of Namalwa Burkitt lymphoma cells into SCID mice.

Immunodeficient SCID mice were inoculated subcutaneously with Namalwa (human Burkitt's lymphoma) tumor cells. Tumors were allowed to form and mice were assigned to five treatment groups of 6 mice per group. When the mean tumor volume reached a mean 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 to determine 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 occurred first. Efficacy was determined based on tumor growth delay (TGD), increase in median time to endpoint (TTE), and log-rank analysis of differences in Kaplan-Meier survival curves for ADC-treated versus mice treated with ADCs. treated with PBS. Tumor samples were collected from the first five control vehicle-treated mice that reached the endpoint and processed by formalin fixation and paraffin embedding.

Results.

In fig. 4b shows that LY75_DM1 and LY75_DM4 each demonstrated significant antitumor activity and prolongation of survival in a Namalwa cell xenograft model of Burkitt's lymphoma in SCID mice compared to controls; however, the 5 mg/kg dose of 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 targeting LY75, such as LY75_DM1 and LY75_DM4, may provide clinical benefit 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 xenograft models of pancreatic cancer.

The effectiveness of LY75_DM1 and LY75_DM4 was studied in a xenograft model with subcutaneous injection of HPAFII pancreatic adenocarcinoma cells into athymic nude mice.

Immunodeficient nude mice were inoculated subcutaneously with HPAFII (human pancreatic adenocarcinoma) tumor cells. Tumors were allowed to form and mice were assigned to five treatment groups of 6 mice per group. When the average

- 42 046736 tumor volume reached a mean 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 to determine health status and unwanted side effects, and tumors were measured three times per week. Mice were euthanized when their tumors reached a tumor volume endpoint of 2000 mm ³ or after 90 days, whichever occurred first. Efficacy was determined based on the effect of treatment on tumor volume and based on log-rank analysis of differences in Kaplan-Meier survival curves for ADC-treated mice compared with PBS-treated mice. Tumors were collected from control vehicle-treated mice and processed by formalin fixation and paraffin embedding.

Results.

In fig. Figure 4c shows that LY75_DM1 and LY75_DM4 exhibited significant and similar antitumor activity and prolonged survival in the HPAFII xenograft nude mouse model compared to controls. All treatments were well tolerated and no clinical signs of toxicity were observed. These data suggest that ADCs targeting LY75, such as LY75_DM1 and LY75_DM4, may provide clinical benefit 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 studied in a xenograft model by subcutaneously injecting SCID mice with SW780 human bladder carcinoma cells.

Immunodeficient nude mice were inoculated subcutaneously with HPAFII (human pancreatic adenocarcinoma) tumor cells. Tumors were allowed to form and mice were assigned to five treatment groups of 6 mice per group. When the mean tumor volume reached a mean 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 to determine health status and adverse side effects, and tumors were measured three times per week. Mice were euthanized when their tumors reached a tumor volume endpoint of 2000 mm ³ or after 90 days, whichever occurred first. Efficacy was determined based on the effect of treatment on tumor volume and based on log-rank analysis of differences in Kaplan-Meier survival curves for ADC-treated mice compared with PBS-treated mice. Tumors were collected from control vehicle-treated 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 and prolonged survival in the SW780 xenograft nude mouse model compared to controls. All treatments were well tolerated and no clinical signs of toxicity were observed. These data suggest that ADCs targeting LY75, such as LY75_DM1 and LY75_DM4, may provide clinical benefit 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 effectiveness of LY75_DM1 and LY75_DM4 was studied in a xenograft model with subcutaneous injection of MDA-MB-468 cells into athymic nude mice.

Immunodeficient nude mice were inoculated subcutaneously with MDA-MB-468 (triple-negative human mammary adenocarcinoma) tumor cells. Tumors were allowed to form and mice were assigned to seven treatment groups of 10 mice per group. When the mean tumor volume reached a mean 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-DM4; 5 mg/kg). Body weight (BW) values were monitored, mice were examined frequently to determine health status and unwanted side effects, and tumors were measured twice a week. Mice were euthanized 82 days after tumor inoculation. Efficacy was determined based on antitumor activity (mean tumor size in treatment group/mean tumor size in control

- 43 046736 group x 100) and an increase in mean time to endpoint (TTE) in ADC-treated mice compared to PBS-treated mice. The five largest tumors from vehicle control mice were sampled at day 71 post-inoculation and processed by formalin fixation and paraffin embedding.

Results.

In fig. 4e shows that LY75_DM1 and LY75_DM4 each exhibited significant antitumor activity in the MDA-MB-468 xenograft nude mouse model compared to controls. Dose-dependent activity was observed with LY75_DM4, with 2.5 and 5 mg/kg having much greater effects than 1 mg/kg. At 5 mg/kg, LY75_DM1 and LY75_DM4 had similar efficacy. Long-term regressions of mean tumor volume were observed in the case of 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 targeting 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 DM1-conjugated and DM4-conjugated anti-LY75 monoclonal antibodies in xenograft models of colorectal cancer.

The efficacy of LY75_DM1 and LY75_DM4 was studied in a xenograft model with subcutaneous injection of COLO205 colorectal adenocarcinoma cells into athymic nude mice.

Immunodeficient nude mice were inoculated subcutaneously with COLO205 tumor cells (human colorectal adenocarcinoma). Tumors were allowed to form and mice were assigned to five treatment groups of 6 mice per group. When the mean tumor volume reached a mean 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-EM4; 5 mg/kg). The second dose was administered twelve days after the first. Body weight (BW) values were monitored, mice were examined frequently to determine 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 occurred first. Efficacy was determined based on tumor growth delay (TGD), increase in median time to endpoint (TTE), and log-rank analysis of differences in Kaplan-Meier survival curves for ADC-treated versus mice treated with ADCs. treated with PBS. Tumor samples were collected from the first five control mice treated with vehicle to reach the endpoint and were treated with formalin fixation and paraffin embedding.

Results.

In fig. Figure 4f shows that LY75_DM1 and LY75_DM4 exhibited similar modest antitumor activity and prolonged survival in a COLO205 cell xenograft model of colorectal adenocarcinoma in nude mice compared with controls. All treatments were well tolerated and no clinical signs of toxicity were observed. These data suggest that ADCs targeting 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-conjugated and DM4-conjugated anti-LY75 monoclonal antibodies to cynomolgus monkeys.

Six male monkeys were assigned to the study at 2 monkeys/group. Vehicle (PBS), LY75_DM4 (cleavable) or LY75_DM1 (non-cleavable) was administered twice (day 1 and day 29) by intravenous infusion over 15 min 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 collected for toxicokinetic studies before dosing (day 1) and 1, 2, 3, 7, 14, 21, and 28 days after each dose. Blood samples were collected for clinicopathological analyzes before dosing (day 1) and 1, 3, 7, 14, 21, and 28 days after each dose (the 28-day post-dose time point was also used as the pre-dose time point). dose administration for the 2nd dose). All study animals were euthanized and necropsied after the final blood draw on day 57. Blood plasma separated from each blood sample was isolated, frozen, and transported to Oxford BioTherapeutics, Inc. for analysis regarding ADC concentration using ELISA.

Treatment-related clinicopathological findings observed included mild regenerative anemia and transient decreases in leukocyte cell counts, most significant in neutrophil counts. Anemia was observed in both animals treated with 5 mg/kg LY75_DM4 and in one of the two animals treated with 10 mg/kg LY75_DM1. Severe neutropenia with maximum decrease in neutrophil count one week after injection

- 44 046736 dose changes and rapid restoration of their quantity were observed in all animals; however, the maximum decrease in the absolute number of neutrophils was smaller in animals treated with LY75_DM4. No effects were observed on the coagulation parameters APTT and PT associated with the 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 LY75_DM4-treated animals. The short duration and/or magnitude of the increases in serum chemistry parameters suggested that they were not related to adverse events. There were no observable findings related to the study drug on urinalysis. When examined at necropsy after a 4-week recovery period, there were no observable macropathological findings associated with processing or changes in absolute and relative organ weights. Observed histopathological findings only for the thyroid gland (changes in the morphological characteristics of the colloidal contents in the follicles) and the kidney (dilated tubules in the outer layer of the cortex) were classified as characterized by minimal severity; not related to changes in other studied parameters; and also not related to adverse events and having minimal toxicological significance. Conclusion. Repeated dosing treatments at two doses of 5 mg/kg LY75_DM4 or 10 mg/kg LY75_DM1 were well tolerated by cynomolgus macaques. All observed treatment-related toxicities were reversible after a 4-week recovery period.

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

Way.

COLO205 cells (ATCC, catalog no. CCL-222) were separated from tissue culture pads using a CellStripper (Cellgro, catalog 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 per well in a 96-well plate with a V-shaped well bottom. Cells were washed once with FAcS buffer (PBS (Fisher, Cat# SH30028-03) +2% FBS). mAb to LY75 (selected from Example 1) or LY75_A1 was added to the wells at an initial concentration of 250 nM, and 3-fold serial dilutions were made and added to the appropriate wells for 45 minutes on ice. Test well contents that required one or more staining steps were maintained in FACS buffer as necessary to ensure that the final staining was completed simultaneously for all test conditions. The contents of two wells containing FACS buffer 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, resuspended in FACS buffer plus 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 antibody conjugated to RPE. Cells were incubated on ice for 45 min and 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 cytometer (96-well plate formats) and the raw data was analyzed using Guava Cytosoft.

Results.

In fig. 5a shows that blocking using anti-LY75 mAb conjugated to MCCDM1 resulted in decreased binding of anti-LY75 mAb. LY75_A1 binding assay to COLO205 cells demonstrated that LY75_A1 was unable to block the binding of anti-LY75 mAb conjugated to MCC-DM1 (see Fig. 5b). Thus, it can be determined that the anti-LY75 mAb and LY75_A1 are not competing antibodies, and yet LY75_A1 recognizes a distinct and unique epitope of LY75 compared to other anti-LY75 antibodies.

Example 24 Characterization of the LY75_A1 epitope by peptide microarray analysis.

Way.

Peptide microarray analysis was performed at LC Sciences, Houston, Texas, and, briefly, the method involved the following steps: contiguous 8-mer LY75 protein peptides, overlapping at one amino acid, were synthesized, 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 probed using LY75_A1 to identify the peptides to which the antibody bound. The binding assay was performed under the following conditions: the microarray containing adjacent 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

- 45 046736 for 2 hours. The matrix was washed again with 0.5 ml of wash buffer at 4°C for 30 minutes, then incubated with 25 ng/ml anti-human IgG conjugate with Alexa 647 in binding buffer (pH 7, 0) at 4°C for 1 hour. The matrix was washed again with 0.5 ml of 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 valid duplicate samples. The average signal intensity for replicates was reported as the final signal intensity.

Results.

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

Example 25: LY75_A1 peptide precipitation assay.

Way.

1.1. Co-precipitation analysis.

Recombinant LY75 protein was cleaved by tryptic proteolysis of bead-associated protein (Promega, USA). The resulting digestion peptides were recovered using a C18 capture column (Thermo Fisher Scientific). The purified peptides were then incubated with 200 μl of protein A beads cross-linked to the LY75_A1 antibody overnight at 4°C. The next day, unbound peptides were collected and the beads were washed twice with 1 ml PBS. 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-mass spectrometry using a Waters nanoACQUITY UPLC system equipped with a nanoACQUITY UPLC BEH 130 C18, 75 μm x 250 mm column (186003545) and an LTQ Orbitrap Velos column (Thermo Fisher Scientific). Peptides were eluted using a gradient of increasing acetonitrile from 3 to 35% at 300 nL/min for 120 min. Mass spectra were obtained in full scan mode at a resolution of 60,000 in the mass range of 400-2000 wt/charge using an Orbitrap. In each run, the twenty highest intensity peptides 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 were processed using Mascot software (Matrix Science), which uses the Mowse algorithm (Curr Biol. 1993 Jun 1;3(6):327-3) to infer amino acid sequences based on peak set 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. Peptide identification criteria included trypsin cleavage, up to 2 missing cleavage sites, and various biological and chemical modifications (oxidized methionine, modification of cysteine by 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 and a spectral match degree of 28 or more were uploaded to the OGAP database.

1.4. Recognition of peptides associated with LY75.

The LY75 identification method used peptide sequences obtained experimentally 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 WO2009/087462.

Results.

The results of the peptide coprecipitation assay using the LY75_A1 antibody are shown in Table 1. 1 below and in FIG. 7. Peptides that were identified in both peptide 1a and 1b elutions in the coprecipitation assay and in the microarray analysis were considered the most likely candidates for epitope formation.

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Table 1

Comparison of peptide microarray and peptide coprecipitation experiments

SEQ ID NO: Peptide identified by microarray analysis Peptide identified by precipitation assay Region 1 (amino acids 609-618) - Region 2 (amino acids 651-662) - 40 Region 3 (amino acids 761-780) GWHFYDDR (765-772) 41 42 Region 4 (amino acids 883-901) ISEWPIDDHFTYSR(877-890) FP VTFGEECLYMS AK(896-910) 43 Region 5 (amino acids 1029-1040) ELTYSNFHPLLVSGR (10301044) 44 Region 6 (amino acids 1077-1093) HFVSLCQK (1084-1091) 45 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) -

Table 1 shows that a number of overlapping peptide regions of LY75 were identified in both the peptide microarray analysis and in both elutions 1a and 1b in the peptide precipitation assay. These regions were considered most likely to contain the epitope recognized by the LY75_A1 antibody because they bind to LY75_A1 when examined using both techniques used.

Example 26: Synergistic combinations of LY75_DM4 and venetoclax.

A series of activated diffuse B-cell lymphoma (ABC-DLBCL) B-cell lines (i.e., U2932, HBL1, OCI-Ly10, TMD8 cell lines) were exposed for 72 hours to increasing doses of LY75_DM4 (i.e., 18.75 - 37.5 - 75 - 150 - 300 - 600 - 1200 nM) either alone or in combination with increasing doses of venetoclax (0.64 - 3.2 - 16 - 80 - 400 - 2000 - 10000 nM). This was followed by analysis using MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazol bromide].

The Chou-Talalay additivity index (C.I.) could be assessed using the Synergy R package (Preclinical versus Clinical Drugs Combination Studies. Chou T.C. Leuk. Lymphoma. 2008;49(11):2059-2080). It provides quantification of strong synergism (<0.3), synergism (0.3-0.9), additive effect (0.9-1.1) or antagonism/lack of beneficial effect (>1.1).

Plots of algebraic CI scores versus fractional effect of different doses of LY75_DM4 in combination with venetoclax against different ABC-DLBCL cell lines are shown in FIG. 8-11.

The supporting data are given in table. 2-5 below. (Fractional effect is the effect of a drug dose, or in this case a combination of two different drug doses, on cell viability. See Drug combination studies and their synergy quantification using the Chou-Talalay method, Chou TC Cancer Res. 2010 Jan 15 ; 70(2):440-6. doi: 10.1158/0008-5472.CAN-091947. Epub 2010 Jan 12; Evaluation of combination chemotherapy: integration of nonlinear regression, curve shift, and combination index analyses, Zhao L1, Wientjes MG, Au JL. Clin Cancer Res. 2004 Dec 1; 10(23):7994-8004 and Computerized quantitation of synergism and antagonism of taxol, topotecan, and cisplatinagainst human teratocarcinoma cell growth: a rational approach to clinical protocol design. TC1, Motzer RJ, Tong Y, Bosl GJ. J Natl Cancer Inst. 1994 Oct 19; 86(20):1517-24.

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table 2

Chou-Talalay index of additivity (CI) of different doses of LY75_DM4 in combination with venetoclax against the U2932 ABC-DLBCL cell line

No. Venetoclax (nM) LY75DM4 (nM) C.I. 1 0.64 18.75 0.228628 2 3.2 18.75 48390.23 3 16 18.75 0.832871 4 80 18.75 48571.11 5 400 18.75 1.283932 6 2000 18.75 4.300007 7 0.64 37.5 0.20187 8 3.2 37.5 0.158138 9 16 37.5 0.162302 10 80 37.5 0.359423 AND 400 37.5 1.063934 12 2000 37.5 4.016023 13 0.64 75 0.240453 14 3.2 75 0.318658 15 16 75 0.269793 16 80 75 0.449203 17 400 75 1.319344 18 2000 75 4.535702 19 0.64 150 0.668392 20 3.2 150 0.503223 21 16 150 0.368141 22 80 150 0.78697 23 400 150 1.229211 24 2000 150 4.080756 25 0.64 300 0.674222 26 3.2 300 0.614852 27 16 300 0.471318 28 80 300 0.612392 29 400 300 1.0508 thirty 2000 300 2.887408

31 0.64 600 0.398468 32 3.2 600 0.269578 33 16 600 0.215265 34 80 600 0.262392 35 400 600 0.482253 36 2000 600 1.556613 37 0.64 1200 0.311401 38 3.2 1200 0.201658 39 16 1200 0.131998 40 80 1200 0.143223 41 400 1200 0.264552 42 2000 1200 0.889985

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Table 3

Chou-Talalay index of additivity (CI) of different doses of LY75_DM4 in combination with venetoclax against the HBL-1 ABC-DLBCL cell line

No. Venetoclax (nM) LY75 DM4 (nM) C.I. 1 0.64 18.75 0.060475 2 3.2 18.75 0.066231 3 16 18.75 0.072936 4 80 18.75 0.103875 5 400 18.75 0.2431 6 2000 18.75 0.723887 7 10000 18.75 2.513073 8 0.64 37.5 0.2091 9 3.2 37.5 0.126913 10 16 37.5 0.130206 AND 80 37.5 0.181304 12 400 37.5 0.257498 13 2000 37.5 0.723106 14 10000 37.5 2.455379 15 0.64 75 0.307332 16 3.2 75 0.251612 17 16 75 0.235462 18 80 75 0.185182 19 400 75 0.304787 20 2000 75 0.717305 21 10000 75 2.213852 22 0.64 150 0.429262 23 3.2 150 0.324116 24 16 150 0.379524 25 80 150 0.276266 26 400 150 0.341981 27 2000 150 0.641475 28 10000 150 1.688396 29 0.64 300 0.530256 thirty 3.2 300 0.558872 31 16 300 0.48397 32 80 300 0.427943 33 400 300 0.411042 34 2000 300 0.581318 35 0.64 600 0.81368 36 3.2 600 0.7166 37 16 600 0.688208 38 80 600 0.539914 39 400 600 0.447228 40 2000 600 0.576687 41 10000 600 0.927968 42 0.64 1200 1.462271 43 3.2 1200 1.463631 44 16 1200 1.240995 45 80 1200 0.962969 46 400 1200 0.699419 47 2000 1200 0.834858 48 10000 1200 1.20449

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Table 4

Chou-Talalay index of additivity (CI) of different doses of LY75_DM4 in combination with venetoclax against the OCI-LY10 ABC-DLBCL cell line

No. Venetoclax (nM) LY75 DM4 (nM) C.I. 1 0.64 18.75 0.358713 2 3.2 18.75 0.390664 3 16 18.75 0.528223 4 80 18.75 0.519535 5 400 18.75 0.48347 6 2000 18.75 0.449909 7 0.64 37.5 0.76338 8 3.2 37.5 0.792829 9 16 37.5 0.863246 10 80 37.5 0.849432 AND 400 37.5 0.842251 12 2000 37.5 0.83696 13 0.64 75 1.620332 14 3.2 75 1.497024 15 16 75 1.52695 16 80 75 1.247205 17 400 75 1.03465 18 2000 75 0.679064 19 0.64 150 2.591237 20 3.2 150 2.543499 21 16 150 2.319816 22 80 150 2.028179 23 400 150 1.529779 24 2000 150 1.245879

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Table 5

Chou-Talalay additivity index (CI) of different doses of LY75_DM4 in combination with venetoclax against the TMD8 ABC-DLBCL cell line

No. Venetoclax (nM) LY75 DM4 (nM) C.I. 1 0.64 18.75 3.857478 2 3.2 18.75 0.280543 3 16 18.75 0.384029 4 80 18.75 0.317572 5 400 18.75 0.746108 6 2000 18.75 1.517033 7 10000 18.75 5.015455 8 0.64 37.5 0.563791 9 3.2 37.5 0.501728 10 16 37.5 0.518688 AND 80 37.5 0.54784 12 400 37.5 0.769738 13 2000 37.5 1.419712 14 10000 37.5 5.250996 15 0.64 75 1.002662 16 3.2 75 0.925877 17 16 75 0.941806 18 80 75 0.952971 19 400 75 1.073273 20 2000 75 1.592762 21 10000 75 4.559974 22 0.64 150 1.564785 23 3.2 150 1.540005 24 16 150 1.522806 25 80 150 1.396608 26 400 150 1.273743 27 2000 150 1.376175 28 10000 150 0.217978

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Sequences

SEQ ID NO Description Subsequence 1 A.K. A1VH EVQLVESGGGLVKPGGSLRLSCAASGFTYSNAWMSWVRQ APGKELEWVGRIKSKTDGGTTDYAAPVQGRFTISRDDSKN TLYLQMNSLKTEDTAVYYCTIFGVVSFDYWGQGTLVTVSS 2 A.K. A1VL DVQMTQSPSSLSASVGDRVTITCRASQSISDYLSWYQQRPG KAPNLLIYAASNLKTGVPSRFSGSGSGTDFTLTISTLQPEDF ATYYCQQSYRSPWTFGQGTKVEIKR 3 Nucleotides A1VH gaggtgcagctggtggagtctgggggaggcttggtaaagccggggggtcccttagactct cctgtgcagcctctggcttcacttacagtaacgcctggatgagctgggtccgccaggctccag ggaaggggctggagtgggttggccgtattaaaagcaaaactgatggtgggacaacagacta cgctgcacccgtgcaagg cagattcaccatctcaagagatgattcaaaaaacacgctgtatct gcaaatgaacagcctgaaaaccgaggacacagccgtgtattactgtacgatttttggagtggtt agctttgactactggggccagggaaccctggtcaccgtctcctca 4 Nucleotides A1VL gacgtccagatgacccagtctccatcctccctgtctgcatctgttggagacagagtcaccatca cttgccgggcaagtcagagcattagcgactatttaagttggtatcagcagagaccagggaaa gcccctaacctcctgatctatgctgcatccaatttaaagactggggtcccatcaaggttcagtg gcagtggatctgggacagattt cactctcaccatcagcactctgcaacctgaagattttgcaac gtactactgtcaacagagttacaggtccccgtggacgttcggccaagggaccaaggtggaa atcaaacga 5 A.K. A1VHCDR1 NAWMS 6 A.K. A1VHCDR2 RIKSKTDGGTTDYAAPVQG 7 A.K. A1VHCDR3 FGVVSFDY 8 A.K. A1VLCDR1 RASQSISDYLS 9 A.K. A1VLCDR2 AASNLKT 10 A.K. A1VLCDR3 QQSYRSPWT eleven VH3|315/D4|411 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQ APGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKN TLYLQMNSLKTEDTAVYYCTTTTVT 12 JH4 YFD YWGQGTL VT VS S 13 012 DIQMTQ SP S SLS AS VGDRVTITCRASQ SIS S YLNW YQQKPG KAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDF ATYYCQQSYS 14 JK1 WTFGQGTKVEIKR

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15 LY75 (DEC205) MRTGWATPRRPAGLLMLLFWFFDLAEPSGRAANDPFTIVH GNTGKCIKPVYGWIVADDCDETEDKLWKWVSQHRLFHLH SQKCLGLDITKSVNELRMF SCDS S AMLWWKCEHHSL YGA ARYRLALKDGHGTAISNASDVWKKGGSEESLCDQPYHEIY TRDGNSYGRPCEFPFLIDGTWHHDCILDEDHSGPWCATTL NYEYDRKWGICLKPENGCEDNWEKNEQFGSCYQFNTQTA LSWKEAYVSCQNQGADLLSINSAAELTYLKEKEGIAKIFWI GLNQLYSARGWEWSDHKPLNFLNWDPDRPSAPTIGGSSCA RMDAESGLWQSFSCEAQLPYVCRKPLNNTVELTDVWTYS DTRCD AGWLPNNGFCYLLVNESNSWDI<AHAI<CI<AF S SDL ISIHSLADVEVVVTKLHNEDIKEEVWIGLKNINIPTLFQWSD GTEVTLTYWDENEPNVPYNKTPNCVSYLGELGQWKVQSC EEKLKYVCKRKGEKLNDASSDKMCPPDEGWKRHGETCY I<IYEDEVPFGTNCNLTITSRFEQEYLNDLMI<I<YDI<SLRI<YF WTGLRDVD SCGEYNWATVGGRRRAVTF SNWNFLEP ASPG GCVAMSTGKSVGKWEVKDCRSFKALSICKKMSGPLGPEE ASPKPDDPCPEGWQSFPASLSCYKVFHAERIVRKRNWEEA ERFCQ ALGAHLS SF SHVDEIKEFLHFLTDQF SGQHWLWIGL NKRSPDLQGSWQWSDRTPVSTIIMPNEFQQDYDIRDCAAV I <VFHRPWRRGWHFYDDREFIYLRPFACDTI<LEWVCQIPI<G RTPKTPDWYNPDRAGIHGPPLIIEGSEYWFVADLHLNYEEA VLYCASNHSFLATITSFVGLKAIKNKIANISGDGQKWWIRIS EWPIDDHFTYSRYPWHRFPVTFGEECLYMSAKTWLIDLGK PTDCSTKLPFICEKYNVSSLEKYSPDSAAKV QCSEQWIPFQ NKCFLKIKPVSLTFSQASDTCHSYGGTLPSVLSQIEQDFITSL LPDMEATLWIGLRWTAYEKINKWTDNRELTYSNFHPLLVS GRLRIPENFFEEESRYHCALILNLQKSPFTGTWNFTSCSERH FVSLCQKYSEVKSRQTLQNASETVKYLNNLYKIIPKTLTW HSAKRECLKSNMQLVSITDPYQ QAFLSVQALLHNSSLWIG LF SQDDELNFGWSDGKRLHF SRWAETNGQLEDC VVLDTD GFWKTVDCNDNQPGAICYYSGNETEKEVKPVDSVKCPSPV LNTPWIPFQNCCYNFIITKNRHMATTQDEVHTKCQKLNPK SHILSIRDEKENNFVLEQLLYFNYMASWVMLGITYRNKSL MWFDKTPLSYTH WRAGRPTIKNEKFLAGLSTDGFWDIQTF KVIEEAVYFHQHSILACKIEMVDYKEEYNTTLPQFMPYED GIYSVIQKKVTWYEALNMCSQSGGHLASVHNQNGQLFLE DIVKRDGFPLWVGL S SHDGSES SFEWSDGSTFD YIPWKGQ TSPGNCVLLDPKGTWKHEKCNSVKDGAICYKPTKSKKLSR LT YS SRCP AAKENGSRWIQYKGHC YKSDQ ALHSF SEAKKL CSKHDHSATIVSIKDEDENKFVSRLMRENNNITMRVWLGL SQHSVDQSWSWLDGSEVTFVKWENKSKSGVGRCSMLIAS NETWKKVECEHGFGRVVCKVPLGPDYTAIAIIVATLSILVL MGGLIWFLFQRHRLHL AGF S VRYAQGV NEDEIMLPSFHD 16 A1VHFR1 EVQLVESGGGLVKPGGSLRLSCAASGFTYS 17 A1VHFR2 WVRQAPGKGLEWVG 18 A1VHFR3 RFTISRDDSKNTLYLQMNSLKTEDTAVYYCTI 19 A1VHFR4 WGQGTLVTVSS

- 53 046736

20 A1VLFR1 DVQMTQSPSSLSSASVGDRVTITTC 21 A1VLFR2 WYQQRPGKAPNLLIY 22 A1VLFR3 GVPSRFSGSGSGTDFTLTISTLQPEDFATYYC 23 A1VLFR4 FGQGTKVEIKR 24 LY75, 609-618 WEVKDCRSSFK 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, 1107- 1118 TVKYLNNLYKII 31 LY75, 1368- 1378 EAVYFHQHSIL 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 39 40 41 42 A1_H (amino acid) AIL (amino acid) (765-772) (877-890) (896-910) MEWSWVFLFFLSVTTGVHSEVQLVESGGGLVKPGGSLRLS CAASGFTYSNAWMSWVRQAPGKELEWVGRIKSKT DGGTTDYAAPVQGRFTISRDDSKNTLYLQMNSLKTEDTAV YYCTIFGVVSFDYWGQGTLVTVSSASTKFPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKFQPREPQVYTLPPSREEMTKNQV SLTCLVKFFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK MSVPTQVLGLLLLWLTDARCDVQMTQSPSSLSASVGDRV TITCRASQSISDYLSWYQQRPGKAPNLLIYAASN LKTGVPSRFSGSGSGTDFTLTISTLQPEDFATYYCQQSYRSP WTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC GWHFYDDR ISEWPIDDHFTYSR FPVTFGEECLYMSAK 43 (1030-1044) ELTYSNFHPLLVSGR 44 (1084-1091) HFVSLCQK 45 (1099-1109) QTLQNASETVK 46 Linker Gly-Phe-Leu-Gly

- 54 046736

Sequence listing in free text format SEQ ID NO: 38 <223> Heavy chain A1

SEQ ID NO: 39 <223> Light chain A1

SEQ ID NO: 46 <223> Linker

SEQ ID NO: 47-157 <223> Peptide

SEQ ID NO: 159-201 <223> Peptide

List of sequences <110> Berlin-Chemie AG <120> PHARMACEUTICAL COMBINATIONS <130> 489.138735/02 <160> 203 <170> PatentIn version 3.5 <210> 1 <211> 119 <212> PROTEIN <213> Human <400> 1

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr Ser Asn Ala 20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60

Pro Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95

Tyr Cys Thr Ile Phe Gly Val Val Ser Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115 <210> 2

- 55 046736 <211> 108 <212> PROTEIN <213> Human <400> 2

Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr 20 25 30

Leu Ser Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45

Tyr Ala Ala Ser Asn Leu Lys Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Thr Leu Gln Pro 65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Arg Ser Pro Trp 85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105 <210> 3 <211> 357 <212> DNA <213> Human <400> 3 gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc cggggggggtc ccttagactc 60 tcctgtgcag cctctggctt cacttacagt aacgcctgga tgagctgggt ccg ccaggct 120 ccagggaagg ggctggagtg ggttggccgt attaaaagca aaactgatgg tgggacaaca 180 gactacgctg cacccgtgca aggcagattc accatctcaa gagatgattc aaaaaacacg 240 ctgtatctgc aaatgaacag cctgaaaacc gaggacacag ccgtgtatta ctgtacgatt 300 tttggagtgg ttagctttga ctactggggc cagggaaccc tggtcaccgt ctcctca 357 <210> 4 <211> 324 <212> DNA <213> Human <400> 4 tccaga tgacccagtc tccatcctcc ctgtctgcat ctgttggaga cagagtcacc 60 atcacttgcc gggcaagtca gagcattagc gactatttaa gttggtatca gcagagacca 120

- 56 046736 gggaaagccc ctaacctcct gatctatgct gcatccaatt taaagactgg ggtcccatca 180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcac tctgcaacct 240 gaagattttg caacgtacta ctgtcaacag agttacaggt cccc gtggac gttcggccaa 300 gggaccaagg tggaaatcaa acga 324 <210>5 <211>5 <212> PROTEIN <213> Human <400> 5

Asn Ala Trp Met Ser 1 5 <210>6 <211> 19 <212> PROTEIN <213> Human <400> 6

Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro 1 5 10 15

Val Gln Gly <210>7 <211> 8 <212> PROTEIN <213> Human <400> 7

Phe Gly Val Val Ser Phe Asp Tyr 1 5 <210> 8 <211> 11 <212> PROTEIN <213> Human <400> 8

Arg Ala Ser Gln Ser Ile Ser Asp Tyr Leu Ser

5 10 <210> 9 <211> 7 <212> PROTEIN <213> Human

- 57 046736 <400> 9

Ala Ala Ser Asn Leu Lys Thr 1 5 <210> 10 <211> 9 <212> PROTEIN <213> Human <400> 10

Gln Gln Ser Tyr Arg Ser Pro Trp Thr 1 5 <210> 11 <211> 104 <212> PROTEIN <213> Human <400> 11

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60

Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95

Tyr Cys Thr Thr Thr Thr Val Thr 100 <210> 12 <211> 15 <212> PROTEIN <213> Human <400> 12

Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 15

- 58 046736 <210> 13 <211> 93 <212> PROTEIN <213> Human <400> 13

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser 85 90 <210> 14 <211> 13 <212> PROTEIN <213> Human <400> 14

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 1 5 10 <210> 15 <211> 1722 <212> PROTEIN <213> Human <400> 15

Met Arg Thr Gly Trp Ala Thr Pro Arg Arg Pro Ala Gly Leu Leu Met 1 5 10 15

Leu Leu Phe Trp Phe Phe Asp Leu Ala Glu Pro Ser Gly Arg Ala Ala 20 25 30

Asn Asp Pro Phe Thr Ile Val His Gly Asn Thr Gly Lys Cys Ile Lys 35 40 45

- 59 046736

Pro Val Tyr Gly Trp Ile Val Ala Asp Asp Cys Asp Glu Thr Glu Asp 50 55 60

Lys Leu Trp Lys Trp Val Ser Gln His Arg Leu Phe His Leu His Ser 65 70 75 80

Gln Lys Cys Leu Gly Leu Asp Ile Thr Lys Ser Val Asn Glu Leu Arg 85 90 95

Met Phe Ser Cys Asp Ser Ser Ala Met Leu Trp Trp Lys Cys Glu His

100 105 110

His Ser Leu Tyr Gly Ala Ala Arg Tyr Arg Leu Ala Leu Lys Asp Gly

115 120 125

His Gly Thr Ala Ile Ser Asn Ala Ser Asp Val Trp Lys Lys Gly Gly

130 135 140

Ser Glu Glu Ser Leu Cys Asp Gln Pro Tyr His Glu Ile Tyr Thr Arg

145 150 155 160

Asp Gly Asn Ser Tyr Gly Arg Pro Cys Glu Phe Pro Phe Leu Ile Asp

165 170 175

Gly Thr Trp His His Asp Cys Ile Leu Asp Glu Asp His Ser Gly Pro

180 185 190

Trp Cys Ala Thr Thr Leu Asn Tyr Glu Tyr Asp Arg Lys Trp Gly Ile

195 200 205

Cys Leu Lys Pro Glu Asn Gly Cys Glu Asp Asn Trp Glu Lys Asn Glu

210 215 220

Gln Phe Gly Ser Cys Tyr Gln Phe Asn Thr Gln Thr Ala Leu Ser Trp

225 230 235 240

Lys Glu Ala Tyr Val Ser Cys Gln Asn Gln Gly Ala Asp Leu Leu Ser

245 250 255

Ile Asn Ser Ala Ala Glu Leu Thr Tyr Leu Lys Glu Lys Glu Gly Ile

260 265 270

Ala Lys Ile Phe Trp Ile Gly Leu Asn Gln Leu Tyr Ser Ala Arg Gly

275 280 285

Trp Glu Trp Ser Asp His Lys Pro Leu Asn Phe Leu Asn Trp Asp Pro

- 60 046736

290 295 300

Asp Arg Pro Ser Ala Pro Thr Ile Gly Gly Ser Ser Cys Ala Arg Met 305 310 315 320

Asp Ala Glu Ser Gly Leu Trp Gln Ser Phe Ser Cys Glu Ala Gln Leu

325 330 335

Pro Tyr Val Cys Arg Lys Pro Leu Asn Asn Thr Val Glu Leu Thr Asp

340 345 350

Val Trp Thr Tyr Ser Asp Thr Arg Cys Asp Ala Gly Trp Leu Pro Asn

355 360 365

Asn Gly Phe Cys Tyr Leu Leu Val Asn Glu Ser Asn Ser Trp Asp Lys

370 375 380

Ala His Ala Lys Cys Lys Ala Phe Ser Ser Asp Leu Ile Ser Ile His 385 390 395 400

Ser Leu Ala Asp Val Glu Val Val Val Thr Lys Leu His Asn Glu Asp 405 410 415

Ile Lys Glu Glu Val Trp Ile Gly Leu Lys Asn Ile Asn Ile Pro Thr

420 425 430

Leu Phe Gln Trp Ser Asp Gly Thr Glu Val Thr Leu Thr Tyr Trp Asp

435 440 445

Glu Asn Glu Pro Asn Val Pro Tyr Asn Lys Thr Pro Asn Cys Val Ser

450 455 460

Tyr Leu Gly Glu Leu Gly Gln Trp Lys Val Gln Ser Cys Glu Glu Lys

465 470 475 480

Leu Lys Tyr Val Cys Lys Arg Lys Gly Glu Lys Leu Asn Asp Ala Ser

485 490 495

Ser Asp Lys Met Cys Pro Pro Asp Glu Gly Trp Lys Arg His Gly Glu

500 505 510

Thr Cys Tyr Lys Ile Tyr Glu Asp Glu Val Pro Phe Gly Thr Asn Cys

515 520 525

Asn Leu Thr Ile Thr Ser Arg Phe Glu Gln Glu Tyr Leu Asn Asp Leu

530 535 540

- 61 046736

Met Lys Lys Tyr Asp Lys Ser Leu Arg Lys Tyr Phe Trp Thr Gly Leu 545 550 555 560

Arg Asp Val Asp Ser Cys Gly Glu Tyr Asn Trp Ala Thr Val Gly Gly

565 570 575

Arg Arg Arg Ala Val Thr Phe Ser Asn Trp Asn Phe Leu Glu Pro Ala

580 585 590

Ser Pro Gly Gly Cys Val Ala Met Ser Thr Gly Lys Ser Val Gly Lys

595 600 605

Trp Glu Val Lys Asp Cys Arg Ser Phe Lys Ala Leu Ser Ile Cys Lys

610 615 620

Lys Met Ser Gly Pro Leu Gly Pro Glu Glu Ala Ser Pro Lys Pro Asp 625 630 635 640

Asp Pro Cys Pro Glu Gly Trp Gln Ser Phe Pro Ala Ser Leu Ser Cys

645 650 655

Tyr Lys Val Phe His Ala Glu Arg Ile Val Arg Lys Arg Asn Trp Glu

660 665 670

Glu Ala Glu Arg Phe Cys Gln Ala Leu Gly Ala His Leu Ser Ser Phe

675 680 685

Ser His Val Asp Glu Ile Lys Glu Phe Leu His Phe Leu Thr Asp Gln 690 695 700

Phe Ser Gly Gln His Trp Leu Trp Ile Gly Leu Asn Lys Arg Ser Pro 705 710 715 720

Asp Leu Gln Gly Ser Trp Gln Trp Ser Asp Arg Thr Pro Val Ser Thr

725 730 735

Ile Ile Met Pro Asn Glu Phe Gln Gln Asp Tyr Asp Ile Arg Asp Cys

740 745 750

Ala Ala Val Lys Val Phe His Arg Pro Trp Arg Arg Gly Trp His Phe

755 760 765

Tyr Asp Asp Arg Glu Phe Ile Tyr Leu Arg Pro Phe Ala Cys Asp Thr

770 775 780

Lys Leu Glu Trp Val Cys Gln Ile Pro Lys Gly Arg Thr Pro Lys Thr

- 62 046736

785 790 795 800

Pro Asp Trp Tyr Asn Pro Asp Arg Ala Gly Ile His Gly Pro Pro Leu

805 810 815

Ile Ile Glu Gly Ser Glu Tyr Trp Phe Val Ala Asp Leu His Leu Asn

820 825 830

Tyr Glu Glu Ala Val Leu Tyr Cys Ala Ser Asn His Ser Phe Leu Ala

835 840 845

Thr Ile Thr Ser Phe Val Gly Leu Lys Ala Ile Lys Asn Lys Ile Ala

850 855 860

Asn Ile Ser Gly Asp Gly Gln Lys Trp Trp Ile Arg Ile Ser Glu Trp 865 870 875 880

Pro Ile Asp Asp His Phe Thr Tyr Ser Arg Tyr Pro Trp His Arg Phe

885 890 895

Pro Val Thr Phe Gly Glu Glu Cys Leu Tyr Met Ser Ala Lys Thr Trp

900 905 910

Leu Ile Asp Leu Gly Lys Pro Thr Asp Cys Ser Thr Lys Leu Pro Phe

915 920 925

Ile Cys Glu Lys Tyr Asn Val Ser Ser Leu Glu Lys Tyr Ser Pro Asp

930 935 940

Ser Ala Ala Lys Val Gln Cys Ser Glu Gln Trp Ile Pro Phe Gln Asn

945 950 955 960

Lys Cys Phe Leu Lys Ile Lys Pro Val Ser Leu Thr Phe Ser Gln Ala

965 970 975

Ser Asp Thr Cys His Ser Tyr Gly Gly Thr Leu Pro Ser Val Leu Ser

980 985 990

Gln Ile Glu Gln Asp Phe Ile Thr Ser Leu Leu Pro Asp Met Glu Ala 995 1000 1005

Thr Leu Trp Ile Gly Leu Arg Trp Thr Ala Tyr Glu Lys Ile Asn 1010 1015 1020

Lys Trp Thr Asp Asn Arg Glu Leu Thr Tyr Ser Asn Phe His Pro 1025 1030 1035

- 63 046736

Leu Leu Val Ser Gly Arg Leu Arg Ile Pro Glu Asn Phe Phe Glu 1040 1045 1050

Glu Glu Ser Arg Tyr His Cys Ala Leu Ile Leu Asn Leu Gln Lys 1055 1060 1065

Ser Pro Phe Thr Gly Thr Trp Asn Phe Thr Ser Cys Ser Glu Arg 1070 1075 1080

His Phe Val Ser Leu Cys Gln Lys Tyr Ser Glu Val Lys Ser Arg 1085 1090 1095

Gln Thr Leu Gln Asn Ala Ser Glu Thr Val Lys Tyr Leu Asn Asn 1100 1105 1110

Leu Tyr Lys Ile Ile Pro Lys Thr Leu Thr Trp His Ser Ala Lys 1115 1120 1125

Arg Glu Cys Leu Lys Ser Asn Met Gln Leu Val Ser Ile Thr Asp 1130 1135 1140

Pro Tyr Gln Gln Ala Phe Leu Ser Val Gln Ala Leu Leu His Asn 1145 1150 1155

Ser Ser Leu Trp Ile Gly Leu Phe Ser Gln Asp Asp Glu Leu Asn 1160 1165 1170

Phe Gly Trp Ser Asp Gly Lys Arg Leu His Phe Ser Arg Trp Ala 1175 1180 1185

Glu Thr Asn Gly Gln Leu Glu Asp Cys Val Val Leu Asp Thr Asp 1190 1195 1200

Gly Phe Trp Lys Thr Val Asp Cys Asn Asp Asn Gln Pro Gly Ala 1205 1210 1215

Ile Cys Tyr Tyr Ser Gly Asn Glu Thr Glu Lys Glu Val Lys Pro 1220 1225 1230

Val Asp Ser Val Lys Cys Pro Ser Pro Val Leu Asn Thr Pro Trp 1235 1240 1245

Ile Pro Phe Gln Asn Cys Cys Tyr Asn Phe Ile Ile Thr Lys Asn 1250 1255 1260

Arg His Met Ala Thr Thr Gln Asp Glu Val His Thr Lys Cys Gln

- 64 046736

1265 1270 1275

Lys Leu Asn Pro Lys Ser His Ile Leu Ser Ile Arg Asp Glu Lys 1280 1285 1290

Glu Asn Asn Phe Val Leu Glu Gln Leu Leu Tyr Phe Asn Tyr Met 1295 1300 1305

Ala Ser Trp Val Met Leu Gly Ile Thr Tyr Arg Asn Lys Ser Leu 1310 1315 1320

Met Trp Phe Asp Lys Thr Pro Leu Ser Tyr Thr His Trp Arg Ala 1325 1330 1335

Gly Arg Pro Thr Ile Lys Asn Glu Lys Phe Leu Ala Gly Leu Ser 1340 1345 1350

Thr Asp Gly Phe Trp Asp Ile Gln Thr Phe Lys Val Ile Glu Glu 1355 1360 1365

Ala Val Tyr Phe His Gln His Ser Ile Leu Ala Cys Lys Ile Glu 1370 1375 1380

Met Val Asp Tyr Lys Glu Glu Tyr Asn Thr Thr Leu Pro Gln Phe 1385 1390 1395

Met Pro Tyr Glu Asp Gly Ile Tyr Ser Val Ile Gln Lys Lys Val 1400 1405 1410

Thr Trp Tyr Glu Ala Leu Asn Met Cys Ser Gln Ser Gly Gly His 1415 1420 1425

Leu Ala Ser Val His Asn Gln Asn Gly Gln Leu Phe Leu Glu Asp 1430 1435 1440

Ile Val Lys Arg Asp Gly Phe Pro Leu Trp Val Gly Leu Ser Ser 1445 1450 1455

His Asp Gly Ser Glu Ser Ser Phe Glu Trp Ser Asp Gly Ser Thr 1460 1465 1470

Phe Asp Tyr Ile Pro Trp Lys Gly Gln Thr Ser Pro Gly Asn Cys 1475 1480 1485

Val Leu Leu Asp Pro Lys Gly Thr Trp Lys His Glu Lys Cys Asn 1490 1495 1500

- 65 046736

Ser Val Lys Asp Gly Ala Ile Cys Tyr Lys Pro Thr Lys Ser Lys 1505 1510 1515

Lys Leu Ser Arg Leu Thr Tyr Ser Ser Arg Cys Pro Ala Ala Lys 1520 1525 1530

Glu Asn Gly Ser Arg Trp Ile Gln Tyr Lys Gly His Cys Tyr Lys 1535 1540 1545

Ser Asp Gln Ala Leu His Ser Phe Ser Glu Ala Lys Lys Leu Cys 1550 1555 1560

Ser Lys His Asp His Ser Ala Thr Ile Val Ser Ile Lys Asp Glu 1565 1570 1575

Asp Glu Asn Lys Phe Val Ser Arg Leu Met Arg Glu Asn Asn Asn 1580 1585 1590

Ile Thr Met Arg Val Trp Leu Gly Leu Ser Gln His Ser Val Asp 1595 1600 1605

Gln Ser Trp Ser Trp Leu Asp Gly Ser Glu Val Thr Phe Val Lys 1610 1615 1620

Trp Glu Asn Lys Ser Lys Ser Gly Val Gly Arg Cys Ser Met Leu 1625 1630 1635

Ile Ala Ser Asn Glu Thr Trp Lys Lys Val Glu Cys Glu His Gly 1640 1645 1650

Phe Gly Arg Val Val Cys Lys Val Pro Leu Gly Pro Asp Tyr Thr 1655 1660 1665

Ala Ile Ala Ile Ile Val Ala Thr Leu Ser Ile Leu Val Leu Met 1670 1675 1680

Gly Gly Leu Ile Trp Phe Leu Phe Gln Arg His Arg Leu His Leu 1685 1690 1695

Ala Gly Phe Ser Ser Val Arg Tyr Ala Gln Gly Val Asn Glu Asp 1700 1705 1710

Glu Ile Met Leu Pro Ser Phe His Asp 1715 1720 <210> 16

- 66 046736 <211> 30 <212> PROTEIN <213> Human <400> 16

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr Ser 20 25 30 <210> 17 <211> 14 <212> PROTEIN <213> Human <400> 17

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 1 5 10 <210> 18 <211> 32 <212> PROTEIN <213> Human <400> 18

Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15

Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile 20 25 30 <210> 19 <211> 11 <212> PROTEIN <213> Human <400> 19

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

5 10 <210> 20 <211> 23 <212> PROTEIN <213> Human <400> 20

Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

- 67 046736

Asp Arg Val Thr Ile Thr Cys 20 <210> 21 <211> 15 <212> PROTEIN <213> Human <400> 21

Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Asn Leu Leu Ile Tyr 1 5 10 15 <210> 22 <211> 32 <212> PROTEIN <213> Human <400> 22

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15

Leu Thr Ile Ser Thr Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 <210> 23 <211> 11 <212> PROTEIN <213> Human <400> 23

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

5 10 <210> 24 <211> 10 <212> PROTEIN <213> Human <400> 24

Trp Glu Val Lys Asp Cys Arg Ser Phe Lys

5 10 <210> 25 <211> 12 <212> PROTEIN <213> Human <400> 25

Pro Ala Ser Leu Ser Cys Tyr Lys Val Phe His Ala 1 5 10

- 68 046736 <210> 26 <211> 20 <212> PROTEIN <213> Human <400> 26

Pro Trp Arg Arg Gly Trp His Phe Tyr Asp Asp Arg Glu Phe Ile Tyr 1 5 10 15

Leu Arg Pro Phe 20 <210> 27 <211> 19 <212> PROTEIN <213> Human <400> 27

Asp Asp His Phe Thr Tyr Ser Arg Tyr Pro Trp His Arg Phe Pro Val 1 5 10 15

Thr Phe Gly <210> 28 <211> 12 <212> PROTEIN <213> Human <400> 28

Arg Glu Leu Thr Tyr Ser Asn Phe His Pro Leu Leu 1 5 10 <210> 29 <211> 17 <212> PROTEIN <213> Human <400> 29

Phe Thr Ser Cys Ser Glu Arg His Phe Val Ser Leu Cys Gln Lys Tyr 1 5 10 15

Ser <210> 30 <211> 12 <212> PROTEIN <213> Human

- 69 046736 <400> 30

Thr Val Lys Tyr Leu Asn Asn Leu Tyr Lys Ile Ile 1 5 10 <210> 31 <211> 11 <212> PROTEIN <213> Human <400> 31

Glu Ala Val Tyr Phe His Gln His Ser Ile Leu

5 10 <210> 32 <211> 11 <212> PROTEIN <213> Human <400> 32

Lys Lys Leu Ser Arg Leu Thr Tyr Ser Ser Cys

5 10 <210> 33 <211> 20 <212> PROTEIN <213> Human <400> 33

Asn Gly Ser Arg Trp Ile Gln Tyr Lys Gly His Cys Tyr Lys Ser Asp 1 5 10 15

Gln Ala Leu His 20 <210> 34 <211> 25 <212> PROTEIN <213> Human <400> 34

Ile Ser Glu Trp Pro Ile Asp Asp His Phe Thr Tyr Ser Arg Tyr Pro 1 5 10 15

Trp His Arg Phe Pro Val Thr Phe Gly 20 25 <210> 35 <211> 20 <212> PROTEIN <213> Human

- 70 046736 <400> 35

Gln Thr Leu Gln Asn Ala Ser Glu Thr Val Lys Tyr Leu Asn Asn Leu 1 5 10 15

Tyr Lys Ile Ile 20 <210> 36 <211> 10 <212> PROTEIN <213> Human <400> 36

Asp Asp His Phe Thr Tyr Ser Arg Tyr Pro

5 10 <210> 37 <211> 15 <212> PROTEIN <213> Human <400> 37

Phe Thr Ser Cys Ser Glu Arg His Phe Val Ser Leu Cys Gln Lys 1 5 10 15 <210> 38 <211> 468 <212> PROTEIN <213> Artificial sequence <220>

<223> Heavy chain A1 <400> 38

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15

Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr 35 40 45

Ser Asn Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60

Glu Trp Val Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp 65 70 75 80

- 71 046736

Tyr Ala Ala Pro Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser 85 90 95

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Thr Ile Phe Gly Val Val Ser Phe Asp Tyr Trp

115 120 125

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

130 135 140

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

145 150 155 160

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

165 170 175

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

180 185 190

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

195 200 205

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

210 215 220

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 225 230 235 240

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

245 250 255

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

260 265 270

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

275 280 285

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

290 295 300

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

305 310 315 320

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

- 72 046736

325 330 335

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

340 345 350

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

355 360 365

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

370 375 380

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

385 390 395 400

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

405 410 415

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

420 425 430

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

435 440 445

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

450 455 460

Ser Pro Gly Lys 465 <210> 39 <211> 234 <212> PROTEIN <213> Artificial Sequence <220>

<223> Light chain A1 <400> 39

Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15

Asp Ala Arg Cys Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser 35 40 45

Ile Ser Asp Tyr Leu Ser Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro

- 73 046736

55 60

Asn Leu Leu Ile Tyr Ala Ala Ser Asn Leu Lys Thr Gly Val Pro Ser 65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95

Thr Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr

100 105 110

Arg Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

115 120 125

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

130 135 140

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

165 170 175

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

180 185 190

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

195 200 205

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

210 215 220

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 <210> 40 <211> 8 <212> PROTEIN <213> Homo sapiens <400> 40

Gly Trp His Phe Tyr Asp Asp Arg 1 5 <210> 41 <211> 14 <212> PROTEIN <213> Homo sapiens

- 74 046736 <400> 41

Ile Ser Glu Trp Pro Ile Asp Asp His Phe Thr Tyr Ser Arg 1 5 10 <210> 42 <211> 15 <212> PROTEIN <213> Homo sapiens <400> 42

Phe Pro Val Thr Phe Gly Glu Glu Cys Leu Tyr Met Ser Ala Lys 1 5 10 15 <210> 43 <211> 15 <212> PROTEIN <213> Homo sapiens <400> 43

Glu Leu Thr Tyr Ser Asn Phe His Pro Leu Leu Val Ser Gly Arg 1 5 10 15 <210> 44 <211>8 <212> PROTEIN <213> Homo sapiens <400> 44

His Phe Val Ser Leu Cys Gln Lys 1 5 <210> 45 <211> 11 <212> PROTEIN <213> Homo sapiens <400> 45

Gln Thr Leu Gln Asn Ala Ser Glu Thr Val Lys

5 10 <210> 46 <211> 4 <212> PROTEIN <213> Artificial sequence <220>

<223> Linker <400> 46

Gly Phe Leu Gly

- 75 046736 <210> 47 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 47

Trp Glu Val Lys Asp Cys Arg Ser

5 <210> 48 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 48

Glu Val Lys Asp Cys Arg Ser Phe

5 <210> 49 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 49

Val Lys Asp Cys Arg Ser Phe Lys

5 <210> 50 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 50

Lys Asp Cys Arg Ser Phe Lys Ala

5 <210> 51 <211> 8 <212> PROTEIN

- 76 046736 <213> Artificial sequence <220>

<223> Peptide <400> 51

Asp Cys Arg Ser Phe Lys Ala Leu

5 <210> 52 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 52

Cys Arg Ser Phe Lys Ala Leu Ser

5 <210> 53 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 53

Arg Ser Phe Lys Ala Leu Ser Ile

5 <210> 54 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 54

Pro Ala Ser Leu Ser Cys Tyr Lys

5 <210> 55 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 55

- 77 046736

Ala Ser Leu Ser Cys Tyr Lys Val 1 5 <210> 56 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 56

Ser Leu Ser Cys Tyr Lys Val Phe

5 <210> 57 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 57

Leu Ser Cys Tyr Lys Val Phe His

5 <210> 58 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 58

Ser Cys Tyr Lys Val Phe His Ala

5 <210> 59 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 59

Cys Tyr Lys Val Phe His Ala Glu

5 <210> 60

- 78 046736 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 60

Tyr Lys Val Phe His Ala Glu Arg

5 <210> 61 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 61

Lys Val Phe His Ala Glu Arg Ile

5 <210> 62 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 62

Val Phe His Ala Glu Arg Ile Val

5 <210> 63 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 63

Phe His Ala Glu Arg Ile Val Arg

5 <210> 64 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide

- 79 046736 <400> 64

Asp Cys Ala Ala Val Lys Val Phe 1 5 <210> 65 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 65

Ala Ala Val Lys Val Phe His Arg

5 <210> 66 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 66

Val Lys Val Phe His Arg Pro Trp

5 <210> 67 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 67

Val Phe His Arg Pro Trp Arg Arg

5 <210> 68 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 68

His Arg Pro Trp Arg Arg Gly Trp

5

- 80 046736 <210> 69 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 69

Pro Trp Arg Arg Gly Trp His Phe

5 <210> 70 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 70

Arg Arg Gly Trp His Phe Tyr Asp

5 <210> 71 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 71

Gly Trp His Phe Tyr Asp Asp Arg

5 <210> 72 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 72

His Phe Tyr Asp Asp Arg Glu Phe

5 <210> 73 <211> 8 <212> PROTEIN <213> Artificial sequence

- 81 046736 <220>

<223> Peptide <400> 73

Tyr Asp Asp Arg Glu Phe Ile Tyr 1 5 <210> 74 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 74

Asp Arg Glu Phe Ile Tyr Leu Arg

5 <210> 75 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 75

Glu Phe Ile Tyr Leu Arg Pro Phe

5 <210> 76 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 76

Pro Ile Asp Asp His Phe Thr Tyr

5 <210> 77 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 77

Ile Asp Asp His Phe Thr Tyr Ser

- 82 046736 <210> 78 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 78

Asp Asp His Phe Thr Tyr Ser Arg

5 <210> 79 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 79

Asp His Phe Thr Tyr Ser Arg Tyr

5 <210> 80 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 80

His Phe Thr Tyr Ser Arg Tyr Pro

5 <210> 81 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 81

Phe Thr Tyr Ser Arg Tyr Pro Trp

5 <210> 82 <211> 8 <212> PROTEIN

- 83 046736 <213> Artificial sequence <220>

<223> Peptide <400> 82

Thr Tyr Ser Arg Tyr Pro Trp His

5 <210> 83 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 83

Tyr Ser Arg Tyr Pro Trp His Arg

5 <210> 84 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 84

Ser Arg Tyr Pro Trp His Arg Phe

5 <210> 85 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 85

Arg Tyr Pro Trp His Arg Phe Pro

5 <210> 86 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 86

- 84 046736

Tyr Pro Trp His Arg Phe Pro Val 1 5 <210> 87 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 87

Pro Trp His Arg Phe Pro Val Thr

5 <210> 88 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 88

Trp His Arg Phe Pro Val Thr Phe

5 <210> 89 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 89

His Arg Phe Pro Val Thr Phe Gly

5 <210> 90 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 90

Arg Glu Leu Thr Tyr Ser Asn Phe

5 <210> 91

- 85 046736 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 91

Glu Leu Thr Tyr Ser Asn Phe His

5 <210> 92 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 92

Leu Thr Tyr Ser Asn Phe His Pro

5 <210> 93 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 93

Thr Tyr Ser Asn Phe His Pro Leu

5 <210> 94 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 94

Tyr Ser Asn Phe His Pro Leu Leu

5 <210> 95 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide

- 86 046736 <400> 95

Ser Asn Phe His Pro Leu Leu Val 1 5 <210> 96 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 96

Asn Phe His Pro Leu Leu Val Ser

5 <210> 97 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 97

Phe His Pro Leu Leu Val Ser Gly

5 <210> 98 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 98

His Pro Leu Leu Val Ser Gly Arg

5 <210> 99 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 99

Pro Leu Leu Val Ser Gly Arg Leu

5

- 87 046736 <210> 100 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 100

Leu Leu Val Ser Gly Arg Leu Arg

5 <210> 101 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 101

Leu Val Ser Gly Arg Leu Arg Ile

5 <210> 102 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 102

Val Ser Gly Arg Leu Arg Ile Pro

5 <210> 103 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 103

Phe Thr Ser Cys Ser Glu Arg His

5 <210> 104 <211> 8 <212> PROTEIN <213> Artificial sequence

- 88 046736 <220>

<223> Peptide <400> 104

Thr Ser Cys Ser Glu Arg His Phe 1 5 <210> 105 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 105

Ser Cys Ser Glu Arg His Phe Val

5 <210> 106 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 106

Cys Ser Glu Arg His Phe Val Ser

5 <210> 107 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 107

Ser Glu Arg His Phe Val Ser Leu

5 <210> 108 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 108

Glu Arg His Phe Val Ser Leu Cys

- 89 046736 <210> 109 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 109

Arg His Phe Val Ser Leu Cys Gln

5 <210> 110 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400>110

His Phe Val Ser Leu Cys Gln Lys

5 <210> 111 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400>111

Phe Val Ser Leu Cys Gln Lys Tyr

5 <210> 112 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400>112

Val Ser Leu Cys Gln Lys Tyr Ser

5 <210> 113 <211> 8 <212> PROTEIN

- 90 046736 <213> Artificial sequence <220>

<223> Peptide <400>113

Ser Leu Cys Gln Lys Tyr Ser Glu

5 <210> 114 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400>114

Leu Cys Gln Lys Tyr Ser Glu Val

5 <210> 115 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400>115

Cys Gln Lys Tyr Ser Glu Val Lys

5 <210>116 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400>116

Gln Lys Tyr Ser Glu Val Lys Ser

5 <210>117 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400>117

- 91 046736

Thr Val Lys Tyr Leu Asn Asn Leu 1 5 <210>118 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400>118

Val Lys Tyr Leu Asn Asn Leu Tyr

5 <210>119 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400>119

Lys Tyr Leu Asn Asn Leu Tyr Lys

5 <210> 120 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 120

Tyr Leu Asn Asn Leu Tyr Lys Ile

5 <210> 121 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 121

Leu Asn Asn Leu Tyr Lys Ile Ile

5 <210> 122

- 92 046736 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 122

Asn Asn Leu Tyr Lys Ile Ile Pro

5 <210> 123 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 123

Asn Leu Tyr Lys Ile Ile Pro Lys

5 <210> 124 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 124

Leu Tyr Lys Ile Ile Pro Lys Thr

5 <210> 125 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 125

Tyr Lys Ile Ile Pro Lys Thr Leu

5 <210> 126 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide

- 93 046736 <400> 126

Lys Ile Ile Pro Lys Thr Leu Thr 1 5 <210> 127 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 127

Ile Ile Pro Lys Thr Leu Thr Trp

5 <210> 128 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 128

Ile Pro Lys Thr Leu Thr Trp His

5 <210> 129 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 129

Pro Lys Thr Leu Thr Trp His Ser

5 <210> 130 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400>130

Lys Thr Leu Thr Trp His Ser Ala

5

- 94 046736 <210> 131 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 131

Thr Leu Thr Trp His Ser Ala Lys

5 <210> 132 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400>132

Glu Ala Val Tyr Phe His Gln His

5 <210> 133 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400>133

Ala Val Tyr Phe His Gln His Ser

5 <210> 134 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400>134

Val Tyr Phe His Gln His Ser Ile

5 <210> 135 <211> 8 <212> PROTEIN <213> Artificial sequence

- 95 046736 <220>

<223> Peptide <400>135

Tyr Phe His Gln His Ser Ile Leu 1 5 <210>136 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400>136

Phe His Gln His Ser Ile Leu Ala

5 <210>137 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400>137

Lys Lys Leu Ser Arg Leu Thr Tyr

5 <210> 138 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 138

Lys Leu Ser Arg Leu Thr Tyr Ser

5 <210> 139 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 139

Leu Ser Arg Leu Thr Tyr Ser Ser

- 96 046736 <210> 140 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 140

Ser Arg Leu Thr Tyr Ser Ser Arg

5 <210> 141 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 141

Arg Leu Thr Tyr Ser Ser Arg Cys

5 <210> 142 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 142

Asn Gly Ser Arg Trp Ile Gln Tyr

5 <210> 143 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 143

Gly Ser Arg Trp Ile Gln Tyr Lys

5 <210> 144 <211> 8 <212> PROTEIN

- 97 046736 <213> Artificial sequence <220>

<223> Peptide <400> 144

Ser Arg Trp Ile Gln Tyr Lys Gly

5 <210> 145 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 145

Arg Trp Ile Gln Tyr Lys Gly His

5 <210> 146 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 146

Trp Ile Gln Tyr Lys Gly His Cys

5 <210> 147 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 147

Ile Gln Tyr Lys Gly His Cys Tyr

5 <210> 148 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 148

- 98 046736

Gln Tyr Lys Gly His Cys Tyr Lys 1 5 <210> 149 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 149

Tyr Lys Gly His Cys Tyr Lys Ser

5 <210> 150 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400>150

Lys Gly His Cys Tyr Lys Ser Asp

5 <210> 151 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 151

Gly His Cys Tyr Lys Ser Asp Gln

5 <210> 152 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400>152

His Cys Tyr Lys Ser Asp Gln Ala

5 <210> 153

- 99 046736 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 153

Cys Tyr Lys Ser Asp Gln Ala Leu

5 <210> 154 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 154

Tyr Lys Ser Asp Gln Ala Leu His

5 <210> 155 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 155

Lys Ser Asp Gln Ala Leu His Ser

5 <210> 156 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 156

Ser Asp Gln Ala Leu His Ser Phe

5 <210> 157 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide

- 100 046736 <400> 157

Asp Gln Ala Leu His Ser Phe Ser 1 5 <210> 158 <211> 1722 <212> PROTEIN <213> Homo sapiens <400> 158

Met Arg Thr Gly Trp Ala Thr Pro Arg Arg Pro Ala Gly Leu Leu Met 1 5 10 15

Leu Leu Phe Trp Phe Phe Asp Leu Ala Glu Pro Ser Gly Arg Ala Ala 20 25 30

Asn Asp Pro Phe Thr Ile Val His Gly Asn Thr Gly Lys Cys Ile Lys 35 40 45

Pro Val Tyr Gly Trp Ile Val Ala Asp Asp Cys Asp Glu Thr Glu Asp 50 55 60

Lys Leu Trp Lys Trp Val Ser Gln His Arg Leu Phe His Leu His Ser 65 70 75 80

Gln Lys Cys Leu Gly Leu Asp Ile Thr Lys Ser Val Asn Glu Leu Arg 85 90 95

Met Phe Ser Cys Asp Ser Ser Ala Met Leu Trp Trp Lys Cys Glu His

100 105 110

His Ser Leu Tyr Gly Ala Ala Arg Tyr Arg Leu Ala Leu Lys Asp Gly

115 120 125

His Gly Thr Ala Ile Ser Asn Ala Ser Asp Val Trp Lys Lys Gly Gly

130 135 140

Ser Glu Glu Ser Leu Cys Asp Gln Pro Tyr His Glu Ile Tyr Thr Arg

145 150 155 160

Asp Gly Asn Ser Tyr Gly Arg Pro Cys Glu Phe Pro Phe Leu Ile Asp

165 170 175

Gly Thr Trp His His Asp Cys Ile Leu Asp Glu Asp His Ser Gly Pro

180 185 190

- 101 046736

Trp Cys Ala Thr Thr Leu Asn Tyr Glu Tyr Asp Arg Lys Trp Gly Ile

195 200 205

Cys Leu Lys Pro Glu Asn Gly Cys Glu Asp Asn Trp Glu Lys Asn Glu

210 215 220

Gln Phe Gly Ser Cys Tyr Gln Phe Asn Thr Gln Thr Ala Leu Ser Trp

225 230 235 240

Lys Glu Ala Tyr Val Ser Cys Gln Asn Gln Gly Ala Asp Leu Leu Ser

245 250 255

Ile Asn Ser Ala Ala Glu Leu Thr Tyr Leu Lys Glu Lys Glu Gly Ile

260 265 270

Ala Lys Ile Phe Trp Ile Gly Leu Asn Gln Leu Tyr Ser Ala Arg Gly

275 280 285

Trp Glu Trp Ser Asp His Lys Pro Leu Asn Phe Leu Asn Trp Asp Pro

290 295 300

Asp Arg Pro Ser Ala Pro Thr Ile Gly Gly Ser Ser Cys Ala Arg Met 305 310 315 320

Asp Ala Glu Ser Gly Leu Trp Gln Ser Phe Ser Cys Glu Ala Gln Leu

325 330 335

Pro Tyr Val Cys Arg Lys Pro Leu Asn Asn Thr Val Glu Leu Thr Asp

340 345 350

Val Trp Thr Tyr Ser Asp Thr Arg Cys Asp Ala Gly Trp Leu Pro Asn

355 360 365

Asn Gly Phe Cys Tyr Leu Leu Val Asn Glu Ser Asn Ser Trp Asp Lys

370 375 380

Ala His Ala Lys Cys Lys Ala Phe Ser Ser Asp Leu Ile Ser Ile His 385 390 395 400

Ser Leu Ala Asp Val Glu Val Val Val Thr Lys Leu His Asn Glu Asp 405 410 415

Ile Lys Glu Glu Val Trp Ile Gly Leu Lys Asn Ile Asn Ile Pro Thr

420 425 430

Leu Phe Gln Trp Ser Asp Gly Thr Glu Val Thr Leu Thr Tyr Trp Asp

435 440 445

- 102 046736

Glu Asn Glu Pro Asn Val Pro Tyr Asn Lys Thr Pro Asn Cys Val Ser

450 455 460

Tyr Leu Gly Glu Leu Gly Gln Trp Lys Val Gln Ser Cys Glu Glu Lys

465 470 475 480

Leu Lys Tyr Val Cys Lys Arg Lys Gly Glu Lys Leu Asn Asp Ala Ser

485 490 495

Ser Asp Lys Met Cys Pro Pro Asp Glu Gly Trp Lys Arg His Gly Glu

500 505 510

Thr Cys Tyr Lys Ile Tyr Glu Asp Glu Val Pro Phe Gly Thr Asn Cys

515 520 525

Asn Leu Thr Ile Thr Ser Arg Phe Glu Gln Glu Tyr Leu Asn Asp Leu

530 535 540

Met Lys Lys Tyr Asp Lys Ser Leu Arg Lys Tyr Phe Trp Thr Gly Leu 545 550 555 560

Arg Asp Val Asp Ser Cys Gly Glu Tyr Asn Trp Ala Thr Val Gly Gly

565 570 575

Arg Arg Arg Ala Val Thr Phe Ser Asn Trp Asn Phe Leu Glu Pro Ala

580 585 590

Ser Pro Gly Gly Cys Val Ala Met Ser Thr Gly Lys Ser Val Gly Lys

595 600 605

Trp Glu Val Lys Asp Cys Arg Ser Phe Lys Ala Leu Ser Ile Cys Lys

610 615 620

Lys Met Ser Gly Pro Leu Gly Pro Glu Glu Ala Ser Pro Lys Pro Asp 625 630 635 640

Asp Pro Cys Pro Glu Gly Trp Gln Ser Phe Pro Ala Ser Leu Ser Cys

645 650 655

Tyr Lys Val Phe His Ala Glu Arg Ile Val Arg Lys Arg Asn Trp Glu

660 665 670

Glu Ala Glu Arg Phe Cys Gln Ala Leu Gly Ala His Leu Ser Ser Phe

675 680 685

- 103 046736

Ser His Val Asp Glu Ile Lys Glu Phe Leu His Phe Leu Thr Asp Gln 690 695 700

Phe Ser Gly Gln His Trp Leu Trp Ile Gly Leu Asn Lys Arg Ser Pro 705 710 715 720

Asp Leu Gln Gly Ser Trp Gln Trp Ser Asp Arg Thr Pro Val Ser Thr

725 730 735

Ile Ile Met Pro Asn Glu Phe Gln Gln Asp Tyr Asp Ile Arg Asp Cys

740 745 750

Ala Ala Val Lys Val Phe His Arg Pro Trp Arg Arg Gly Trp His Phe

755 760 765

Tyr Asp Asp Arg Glu Phe Ile Tyr Leu Arg Pro Phe Ala Cys Asp Thr

770 775 780

Lys Leu Glu Trp Val Cys Gln Ile Pro Lys Gly Arg Thr Pro Lys Thr 785 790 795 800

Pro Asp Trp Tyr Asn Pro Asp Arg Ala Gly Ile His Gly Pro Pro Leu

805 810 815

Ile Ile Glu Gly Ser Glu Tyr Trp Phe Val Ala Asp Leu His Leu Asn

820 825 830

Tyr Glu Glu Ala Val Leu Tyr Cys Ala Ser Asn His Ser Phe Leu Ala

835 840 845

Thr Ile Thr Ser Phe Val Gly Leu Lys Ala Ile Lys Asn Lys Ile Ala

850 855 860

Asn Ile Ser Gly Asp Gly Gln Lys Trp Trp Ile Arg Ile Ser Glu Trp 865 870 875 880

Pro Ile Asp Asp His Phe Thr Tyr Ser Arg Tyr Pro Trp His Arg Phe

885 890 895

Pro Val Thr Phe Gly Glu Glu Cys Leu Tyr Met Ser Ala Lys Thr Trp

900 905 910

Leu Ile Asp Leu Gly Lys Pro Thr Asp Cys Ser Thr Lys Leu Pro Phe

915 920 925

Ile Cys Glu Lys Tyr Asn Val Ser Ser Leu Glu Lys Tyr Ser Pro Asp

930 935 940

- 104 046736

Ser Ala Ala Lys Val Gln Cys Ser Glu Gln Trp Ile Pro Phe Gln Asn

945 950 955 960

Lys Cys Phe Leu Lys Ile Lys Pro Val Ser Leu Thr Phe Ser Gln Ala

965 970 975

Ser Asp Thr Cys His Ser Tyr Gly Gly Thr Leu Pro Ser Val Leu Ser

980 985 990

Gln Ile Glu Gln Asp Phe Ile Thr Ser Leu Leu Pro Asp Met Glu Ala 995 1000 1005

Thr Leu Trp Ile Gly Leu Arg Trp Thr Ala Tyr Glu Lys Ile Asn 1010 1015 1020

Lys Trp Thr Asp Asn Arg Glu Leu Thr Tyr Ser Asn Phe His Pro 1025 1030 1035

Leu Leu Val Ser Gly Arg Leu Arg Ile Pro Glu Asn Phe Phe Glu 1040 1045 1050

Glu Glu Ser Arg Tyr His Cys Ala Leu Ile Leu Asn Leu Gln Lys 1055 1060 1065

Ser Pro Phe Thr Gly Thr Trp Asn Phe Thr Ser Cys Ser Glu Arg 1070 1075 1080

His Phe Val Ser Leu Cys Gln Lys Tyr Ser Glu Val Lys Ser Arg 1085 1090 1095

Gln Thr Leu Gln Asn Ala Ser Glu Thr Val Lys Tyr Leu Asn Asn 1100 1105 1110

Leu Tyr Lys Ile Ile Pro Lys Thr Leu Thr Trp His Ser Ala Lys 1115 1120 1125

Arg Glu Cys Leu Lys Ser Asn Met Gln Leu Val Ser Ile Thr Asp 1130 1135 1140

Pro Tyr Gln Gln Ala Phe Leu Ser Val Gln Ala Leu Leu His Asn 1145 1150 1155

Ser Ser Leu Trp Ile Gly Leu Phe Ser Gln Asp Asp Glu Leu Asn 1160 1165 1170

- 105 046736

Phe Gly Trp Ser Asp Gly Lys Arg Leu His Phe Ser Arg Trp Ala 1175 1180 1185

Glu Thr Asn Gly Gln Leu Glu Asp Cys Val Val Leu Asp Thr Asp 1190 1195 1200

Gly Phe Trp Lys Thr Val Asp Cys Asn Asp Asn Gln Pro Gly Ala 1205 1210 1215

Ile Cys Tyr Tyr Ser Gly Asn Glu Thr Glu Lys Glu Val Lys Pro 1220 1225 1230

Val Asp Ser Val Lys Cys Pro Ser Pro Val Leu Asn Thr Pro Trp 1235 1240 1245

Ile Pro Phe Gln Asn Cys Cys Tyr Asn Phe Ile Ile Thr Lys Asn 1250 1255 1260

Arg His Met Ala Thr Thr Gln Asp Glu Val His Thr Lys Cys Gln 1265 1270 1275

Lys Leu Asn Pro Lys Ser His Ile Leu Ser Ile Arg Asp Glu Lys 1280 1285 1290

Glu Asn Asn Phe Val Leu Glu Gln Leu Leu Tyr Phe Asn Tyr Met 1295 1300 1305

Ala Ser Trp Val Met Leu Gly Ile Thr Tyr Arg Asn Lys Ser Leu 1310 1315 1320

Met Trp Phe Asp Lys Thr Pro Leu Ser Tyr Thr His Trp Arg Ala 1325 1330 1335

Gly Arg Pro Thr Ile Lys Asn Glu Lys Phe Leu Ala Gly Leu Ser 1340 1345 1350

Thr Asp Gly Phe Trp Asp Ile Gln Thr Phe Lys Val Ile Glu Glu 1355 1360 1365

Ala Val Tyr Phe His Gln His Ser Ile Leu Ala Cys Lys Ile Glu 1370 1375 1380

Met Val Asp Tyr Lys Glu Glu Tyr Asn Thr Thr Leu Pro Gln Phe 1385 1390 1395

Met Pro Tyr Glu Asp Gly Ile Tyr Ser Val Ile Gln Lys Lys Val 1400 1405 1410

- 106 046736

Thr Trp Tyr Glu Ala Leu Asn Met Cys Ser Gln Ser Gly Gly His 1415 1420 1425

Leu Ala Ser Val His Asn Gln Asn Gly Gln Leu Phe Leu Glu Asp 1430 1435 1440

Ile Val Lys Arg Asp Gly Phe Pro Leu Trp Val Gly Leu Ser Ser 1445 1450 1455

His Asp Gly Ser Glu Ser Ser Phe Glu Trp Ser Asp Gly Ser Thr 1460 1465 1470

Phe Asp Tyr Ile Pro Trp Lys Gly Gln Thr Ser Pro Gly Asn Cys 1475 1480 1485

Val Leu Leu Asp Pro Lys Gly Thr Trp Lys His Glu Lys Cys Asn 1490 1495 1500

Ser Val Lys Asp Gly Ala Ile Cys Tyr Lys Pro Thr Lys Ser Lys 1505 1510 1515

Lys Leu Ser Arg Leu Thr Tyr Ser Ser Arg Cys Pro Ala Ala Lys 1520 1525 1530

Glu Asn Gly Ser Arg Trp Ile Gln Tyr Lys Gly His Cys Tyr Lys 1535 1540 1545

Ser Asp Gln Ala Leu His Ser Phe Ser Glu Ala Lys Lys Leu Cys 1550 1555 1560

Ser Lys His Asp His Ser Ala Thr Ile Val Ser Ile Lys Asp Glu 1565 1570 1575

Asp Glu Asn Lys Phe Val Ser Arg Leu Met Arg Glu Asn Asn Asn 1580 1585 1590

Ile Thr Met Arg Val Trp Leu Gly Leu Ser Gln His Ser Val Asp 1595 1600 1605

Gln Ser Trp Ser Trp Leu Asp Gly Ser Glu Val Thr Phe Val Lys 1610 1615 1620

Trp Glu Asn Lys Ser Lys Ser Gly Val Gly Arg Cys Ser Met Leu 1625 1630 1635

- 107 046736

Ile Ala Ser Asn Glu Thr Trp Lys Lys Val Glu Cys Glu His Gly 1640 1645 1650

Phe Gly Arg Val Val Cys Lys Val Pro Leu Gly Pro Asp Tyr Thr 1655 1660 1665

Ala Ile Ala Ile Ile Val Ala Thr Leu Ser Ile Leu Val Leu Met 1670 1675 1680

Gly Gly Leu Ile Trp Phe Leu Phe Gln Arg His Arg Leu His Leu 1685 1690 1695

Ala Gly Phe Ser Ser Val Arg Tyr Ala Gln Gly Val Asn Glu Asp 1700 1705 1710

Glu Ile Met Leu Pro Ser Phe His Asp 1715 1720 <210> 159 <211> 23 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 159

Cys Ile Lys Pro Val Tyr Gly Trp Ile Val Ala Asp Asp Cys Asp Glu 1 5 10 15

Thr Glu Asp Lys Leu Trp Lys 20 <210> 160 <211> 11 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 160

Cys Glu His His Ser Leu Tyr Gly Ala Ala Arg

5 10 <210> 161 <211> 34 <212> PROTEIN <213> Artificial sequence <220>

- 108 046736 <223> Peptide <400> 161

Asp Gly His Gly Thr Ala Ile Ser Asn Ala Ser Asp Val Trp Lys Lys 1 5 10 15

Gly Gly Ser Glu Glu Ser Leu Cys Asp Gln Pro Tyr His Glu Ile Tyr 20 25 30

Thr Arg <210> 162 <211> 19 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 162

Lys Gly Gly Ser Glu Glu Ser Leu Cys Asp Gln Pro Tyr His Glu Ile 1 5 10 15

Tyr Thr Arg <210> 163 <211> 18 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 163

Gly Gly Ser Glu Glu Ser Leu Cys Asp Gln Pro Tyr His Glu Ile Tyr 1 5 10 15

Thr Arg <210> 164 <211> 15 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 164

- 109 046736

Asp Gly His Gly Thr Ala Ile Ser Asn Ala Ser Asp Val Trp Lys 1 5 10 15 <210> 165 <211> 17 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 165

Trp Gly Ile Cys Leu Lys Pro Glu Asn Gly Cys Glu Asp Asn Trp Glu 1 5 10 15

Lys <210> 166 <211> 13 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 166

Ile Phe Trp Ile Gly Leu Asn Gln Leu Tyr Ser Ala Arg 1 5 10 <210> 167 <211> 15 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 167

Leu His Asn Glu Asp Ile Lys Glu Glu Val Trp Ile Gly Leu Lys 1 5 10 15 <210> 168 <211> 15 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 168

Thr Pro Asn Cys Val Ser Tyr Leu Gly Glu Leu Gly Gln Trp Lys 1 5 10 15

- 110 046736 <210> 169 <211> 23 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 169

Tyr Phe Trp Thr Gly Leu Arg Asp Val Asp Ser Cys Gly Glu Tyr Asn 1 5 10 15

Trp Ala Thr Val Gly Gly Arg 20 <210> 170 <211> 11 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 170

Ser Val Gly Lys Trp Glu Val Lys Asp Cys Arg

5 10 <210> 171 <211> 10 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 171

Trp Glu Val Lys Asp Cys Arg Ser Phe Lys

5 10 <210> 172 <211> 7 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 172

Ala Leu Ser Ile Cys Lys Lys

5

- 111 046736 <210> 173 <211> 12 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 173

Pro Ala Ser Leu Ser Cys Tyr Lys Val Phe His Ala 1 5 10 <210> 174 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 174

Arg Asn Trp Glu Glu Ala Glu Arg

5 <210> 175 <211> 33 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 175

Arg Ser Pro Asp Leu Gln Gly Ser Trp Gln Trp Ser Asp Arg Thr Pro 1 5 10 15

Val Ser Thr Ile Ile Met Pro Asn Glu Phe Gln Gln Asp Tyr Asp Ile 20 25 30

Arg <210> 176 <211> 19 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 176

Thr Pro Val Ser Thr Ile Ile Met Pro Asn Glu Phe Gln Gln Asp Tyr 1 5 10 15

- 112 046736

Asp Ile Arg <210> 177 <211> 20 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 177

Pro Trp Arg Arg Gly Trp His Phe Tyr Asp Asp Arg Glu Phe Ile Tyr 1 5 10 15

Leu Arg Pro Phe 20 <210> 178 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 178

Gly Trp His Phe Tyr Asp Asp Arg

5 <210> 179 <211> 14 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 179

Ile Ser Glu Trp Pro Ile Asp Asp His Phe Thr Tyr Ser Arg 1 5 10 <210> 180 <211> 19 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 180

- 113 046736

Asp Asp His Phe Thr Tyr Ser Arg Tyr Pro Trp His Arg Phe Pro Val 1 5 10 15

Thr Phe Gly <210> 181 <211> 30 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 181

Phe Pro Val Thr Phe Gly Glu Glu Cys Leu Tyr Met Ser Ala Lys Thr 1 5 10 15

Trp Leu Ile Asp Leu Gly Lys Pro Thr Asp Cys Ser Thr Lys 20 25 30 <210> 182 <211> 15 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 182

Thr Trp Leu Ile Asp Leu Gly Lys Pro Thr Asp Cys Ser Thr Lys 1 5 10 15 <210> 183 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 183

Tyr Asn Val Ser Ser Leu Glu Lys

5 <210> 184 <211> 12 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide

- 114 046736 <400> 184

Val Gln Cys Ser Glu Gln Trp Ile Pro Phe Gln Asn 1 5 10 <210> 185 <211> 12 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 185

Arg Glu Leu Thr Tyr Ser Asn Phe His Pro Leu Leu 1 5 10 <210> 186 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 186

Glu Leu Thr Tyr Ser Asn Phe His Pro Leu Leu Val Ser Gly Arg Leu 1 5 10 15

Arg Ile Pro Glu Asn Phe Phe Glu Glu Glu Ser Arg Tyr His Cys Ala 20 25 30

Leu Ile Leu Asn Leu Gln Lys 35 <210> 187 <211> 11 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 187

Tyr His Cys Ala Leu Ile Leu Asn Leu Gln Lys

5 10 <210> 188 <211> 17 <212> PROTEIN <213> Artificial sequence <220>

- 115 046736 <223> Peptide <400> 188

Phe Thr Ser Cys Ser Glu Arg His Phe Val Ser Leu Cys Gln Lys Tyr 1 5 10 15

Ser <210> 189 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 189

His Phe Val Ser Leu Cys Gln Lys

5 <210> 190 <211> 11 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 190

Gln Thr Leu Gln Asn Ala Ser Glu Thr Val Lys

5 10 <210> 191 <211> 12 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 191

Thr Val Lys Tyr Leu Asn Asn Leu Tyr Lys Ile Ile

5 10 <210> 192 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide

- 116 046736 <400> 192

Thr Leu Thr Trp His Ser Ala Lys 1 5 <210> 193 <211> 14 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 193

Asn Arg His Met Ala Thr Thr Gln Asp Glu Val His Thr Lys 1 5 10 <210> 194 <211>7 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 194

Ser His Ile Leu Ser Ile Arg

5 <210> 195 <211> 16 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 195

Ser Leu Met Trp Phe Asp Lys Thr Pro Leu Ser Tyr Thr His Trp Arg 1 5 10 15 <210> 196 <211> 7 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 196

Ser Leu Met Trp Phe Asp Lys

5

- 117 046736 <210> 197 <211> 11 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 197

Glu Ala Val Tyr Phe His Gln His Ser Ile Leu

5 10 <210> 198 <211> 10 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 198

Lys Lys Leu Ser Arg Leu Thr Tyr Ser Ser

5 10 <210> 199 <211> 20 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 199

Asn Gly Ser Arg Trp Ile Gln Tyr Lys Gly His Cys Tyr Lys Ser Asp 1 5 10 15

Gln Ala Leu His 20 <210> 200 <211> 21 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 200

His Asp His Ser Ala Thr Ile Val Ser Ile Lys Asp Glu Asp Glu Asn 1 5 10 15

Lys Phe Val Ser Arg

- 118 046736 <210> 201 <211>9 <212> PROTEIN <213> Artificial sequence <220>

<223> Peptide <400> 201

Val Glu Cys Glu His Gly Phe Gly Arg

5 <210> 202 <211> 1404 <212> DNA <213> Homo sapiens <400> 202 atggaatgga gctgggtgtt cctgttcttt ctgtccgtga ccacaggcgt gcattctgaa 60 gttcagctgg tcgaaagcgg aggaggtctg gtgaaacccg gtgg ctccct gaggctgagc 120 tgcgccgcct ccggctttac ttacagtaat gcctggatgt cctgggtcag acaggcccca 180 ggtaagggtc tggagtgggt gggtaggatt aagtctaaaa ctgatggcgg gacaacagac 240 tatgccgccc cagtgcaagg acggttcacc atttctaggg acgactctaa gaatacactg 300 tatctgcaga tgaacagcct caaaacagaa gacactgccg tttactactg taccatcttt 360 ggcgttgtct cctttgatta ttggggacag ggtacactcg tgaccgtttc caagt 420 acaaaggggc catcagtgtt tccactggcc ccatcctcta agagcactag tggcggcaca 480 gccgccctgg gatgtctggt gaaggactat ttcccagagc ctgtgaccgt cagctggaac 540 agtggtgctc tcacctcagg tgtgcacaca ttccccgctg tgctcca atc cagtggcctc 600 tacagtctga gcagcgttgt gactgttccc agtagctcac tgggcaccca aacctacata 660 tgcaatgtga accataaacc tagcaatacc aaagtggaca agaaagtgga acctaagtcc 720 tgtgacaaga ctcatacctg tcctccttgt cctgccccag agctgctcgg aggcccttcc 780 gtctttctct tcccaccaaa gccaaaggat accctgatga tcagccggac acctgaggtt 840 acctgc gttg tggtcgacgt ttcacacgag gatcctgaag tcaaattcaa ctggtacgtt 900 gatggagtcg aggtccacaa cgccaaaacc aagcctcgcg aagaacaata caatagcaca 960 tatagggtgg tgtctgtgct cactgtcctg caccaggact ggctgaacgg caaggtac 10 20 aaatgcaagg ttagtaacaa ggccctgccc gcacccattg agaagactat cagtaaagct 1080 aagggccagc ctcgcgagcc tcaggtttac accctgcctc cctctagaga ggaaatgaca 1140 aagaaccagg tgtctctcac ctgcctggtt aaaggattct atccatccga cattgctgtg 1200

- 119 -

Claims (1)

gaatgggaat ccaacggaca gcccgaaaac aactataaga caacaccacc tgttctggat 1260 tccgatggtt ccttctttct gtattccaaa ctcacagtgg acaagagtcg ctggcagcaa 1320 ggtaacgtgt tttcttgctc cgtgatgcac gaagcactcc acaatcacta cact cagaag 1380 agtctcagcc tctctccagg caaa 1404 <210> 203 <211> 702 <212> DNA <213> homo sapiens <400> 203 atgtctgtgc ctacccaggt gctgggactg ctgctgctgt ggctgacaga cgcccgctgt 60 gatgttcaga tgacacagtc tccaagtagt ctcagcgcaa gcgttggcga cagagtgact 120 atcacatgca gagcctctca gtctatctct gactatctgt cttggtacca gcagaggcca 180 ggcaaagctc caaacctcct gatctatgct gccagta atc tgaagacagg cgtgcctagt 240 agattctccg ggtccggtag tgggactgat ttcaccctga caatctccac actgcaacct 300 gaggattttg ctacctacta ttgtcagcaa tcttatcgca gcccttggac cttcggacag 360 gggactaagg ttgagattaa acgcaccgtg caccca gcgtctttat ctttcctccc 420 tccgacgagc agctcaagtc cggaacagca tcagtcgttt gcctcctgaa taacttttat 480 ccaagggagg ccaaggtcca gtggaaagtc gacaatgccc tccaatctgg taactcccag 540 gagtctgtga ctgaacaaga ttctaaggac agtacctatt cactcagctc caccctgacc 600 ctcagcaaag cagactacga aaagcataaa gtttacgctt gc gaagtgac ccaccaaggc 660 ctgtcttctc ctgtcacaaa gagttttaat agaggggagt gt 702 CLAIM 1. A pharmaceutical combination containing (A) an anti-LY75 antibody or an antigen-binding portion thereof, wherein said antibody or portion comprises: a) a heavy chain variable region, which contains: i) the first vhCDR containing SEQ ID NO: 5; ii) a second vhCDR comprising SEQ ID NO: 6; and iii) a third vhCDR comprising SEQ ID NO: 7; And b) a light chain variable region, which contains: i) the 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; or an anti-LY75 antibody or antigen-binding portion thereof, wherein said antibody or portion comprises: a) a heavy chain variable region, which contains: i) the first vhCDR containing SEQ ID NO: 5; ii) a second vhCDR comprising SEQ ID NO: 6; and iii) a third vhCDR comprising SEQ ID NO: 7; And b) a light chain variable region, which contains: i) the first vlCDR containing SEQ ID NO: 8; ii) a second vlCDR containing SEQ ID NO: 9; And - 120 -