AU2018200685A1 - Monoclonal antibodies against claudin-18 for treatment of cancer - Google Patents

Abstract

The present invention provides antibodies useful as therapeutics for treating and/or preventing diseases associated with cells expressing CLDI 8, including tumor-related diseases such as 5 gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, hepatic cancer, head-neck cancer, and cancer of the gallbladder.

Description

The present invention provides antibodies useful as therapeutics for treating and/or preventing diseases associated with cells expressing CLDI 8, including tumor-related diseases such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, hepatic cancer, head-neck cancer, and cancer of the gallbladder.

MONOCLONAL ANTIBODIES AGAINST CLAUDIN-18 FOR TREATMENT OF

CANCER

2018200685 30 Jan 2018

The present application is a divisional application from Australian patent application number

2016202322,

2014208256, 2012202225, which in turn is a which in turn is a which in turn is a divisional from divisional from divisional from

Australian patent Australian patent Australian patent

2006316767, the entire disclosures of which are incorporated herein by reference.

application number application number application number

Antibody-based therapies for cancer have the potential of higher specificity and lower side effect profile as compared to conventional drugs. The reason is a precise distinction between normal and neoplastic cells by antibodies and the fact, that their mode of action relies on less toxic immunological anti-tumor mechanisms, such as complement activation and recruitment of cytotoxic immune cells.

L5

Targets for antibody-based therapies need to have particular qualities, which form the basis for proper discrimination between normal and neoplastic cells. Obviously, a target with either exclusive restriction to tumor cells and entirely undetectable on normal tissues is ideal for the development of efficient and safe antibody therapeutics. In another aspect, a high-level

0 overexpression may be the basis for the therapeutic window and low side effects exemplified by the human epidermal growth factor receptor type 2 (HER-2), which as a result of gene amplification Is a good target for the antibody trastuzumab (Herceptin).

Other targets for antibodies which are either already approved or m clinical development for

5 tumor therapy have distinct qualities, which are not based on a numeric overexpression of target molecules on tumor cells. In the case of antibodies to the proteoglycan MUC-1, a peptide repeat epitope in the backbone of the target is underglycosylated in tumor cells and thus altered to its normal counterpart. In the case of antibodies to CD20 (rituximab), CD52 (Campath-IH) and CD22 ( epratuzumab ), antibody targets have comparable expression levels

0 on tumor cells and normal lymphocytes. Here, the ablation of normal cells by the antibody is tolerable since target-negative stem cells restore the normal lymphocyte repertoire. Other examples of differential accessibility of antibody targets are carcinoembryonal antigen (CEA) and carboanhydrase IX (CA9). Both antigens are expressed on normal epithelia of colon and kidney, respectively. However, radioactively labeled imaging antibodies do distinguish well between tumor and normal tissue, and cytotoxic antibodies are well tolerated. This is most likely due to a restricted expression of CA9 and CEA on the luminal side of

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2018200685 30 Jan 2018 normal epithelial tissue where IgG antibodies do not have access. Also antigen epithelial cell adhesion molecule (Ep-CAM) belongs to this category. As a homotypic cell adhesion molecule for epithelial cells it is localized in the intercellular space. Intriguingly, whereas high-affinity anti-Ep-CAM antibodies are very toxic, intermediate-affinity antibodies are well tolerated. This suggests accessibility of the Ep-CAM target on normal cells but also indicates that kinetics of antibody binding may open a therapeutic window.

One possibility is that other epithelial cell-specific proteins involved in cell/cell adhesion may be also attractive for antibody approaches, since they may be barely accessible in well-structured epithelia to antibodies but become exposed on tumor cells. We therefore analyzed proteins involved in organizing epithelial tissue architecture for their suitability as targets for therapeutic antibodies. A protein, which particularly attracted our attention is claudin 18.

The claudin 18 (CLD18) molecule (Genbank accession number: splice variant 1 (CLD18A1): NP_057453, NM_016369, and splice variant 2 (CLD18A2):

NM_001002026, NP_001002026) is an integral transmembrane protein with a molecular weight of approximately 27,9 / 27,72 kD. Claudins are integral membrane proteins located within the tight junctions of epithelia and endothelia. Tight junctions organize a network of interconnected strands of intramembranous

0 particles between adjacent cells. In tight junctions, occludin and claudins are the most prominent transmembrane protein components. Due to their strong intercellular adhesion properties they create a primary barrier to prevent and control the paracellular transport of solutes and restrict the lateral diffusion of membrane lipids and proteins to maintain cellular polarity. Tight junction forming

5 proteins are critically involved in organizing epithelial tissue architecture. We assumed that such proteins may be barely accessible to antibodies in wellstructured epithelia but become exposed on tumor cells.

CLD18 is a tetraspanin and has as such 4 hydrophobic regions. We have generated data indicating that CLD18 displays several different conformations, which may be

0 selectively addressed by antibodies. One conformation (CLD18-Conformation-1) implies, that all four hydrophobic regions serve as regular transmembrane domains (TM) and two extracellular loops (loopl embraced by hydrophobic region 1 and

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WO 2007/059997 PCT/EP2006/011302 hydrophobic region 2; loop2 embraced by hydrophobic regions 3 and 4) are formed, as described for the vast majority of claudin family members. A second conformation (CLD18-Conformation-2) implies that, as described for PMP22, another member of the tetraspanin family (Taylor et al., J. Neurosc. Res. 62:15-27,

2000), that the second and third hydrophobic domains do not fully cross the plasma membrane so that portion (loopD3) in between the first and fourth transmembrane domain is extracellular. A third conformation (CLD18Conformation-3) implies, a large extracelluar domain with two internal hydrophobic regions embraced by the first and fourth hydrophobic region, which serve as regular transmembrane domains. Due to the presence of classical Nglycosylation site in loopD3 the Claudin-18 topology variants CLD18 topology-2and CLD18 topology-3 harbour an additional extracellular N-glycosylation site.

Another level of complexity is added to CLD18 molecule by the presence of two different splice variants, which are described in mouse and in human (Niimi, Mol.

Cell. Biol. 21:7380-90, 2001). The splice variants CLD18A1 and CLD18A2 differ in the first 21 N-terminal amino acids, which comprise the first TM and loopl, whereas the primary protein sequence of the C-terminus is identical.

CLD18A1 is selectively expressed on normal lung and stomach epithelia, whereas

0 CLD18A2 is expressed only on gastric cells (Niimi, Mol. Cell. Biol. 21:7380-90, 2001). Most importantly, CLD18A2 is restricted to the differentiated short-lived cells of stomach epithelium but is devoid from the gastric stem cell region. Using sensitive RT-PCR, we have shown that both variants are not detectable at all in any other normal human organ, but are robustly expressed in several cancer types

5 including stomach, esophageal, pancreatic and lung tumors as well as human cancer cell lines. Expression is most prominent in the adenocarcinoma subtypes of these indications.

The molecular weight of the protein differs in some cancers and adjacent normal

0 tissue. The higher molecular weight protein observed in healthy tissue can be transferred into the same molecular weight as observed in cancer by treating tissue lysates with the deglycosylating compound PNGase F. This suggests, that CLD18 is less N-glycosylated in cancer as compared to its normal tissue counterpart. This structural difference is likely to give rise to an altered epitope. A classical N3

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2018200685 30 Jan 2018 glycosylation motif is in position aa 116 within the loopD3 domain of the molecule.

The terms “CLD18” and “CLD18-variant” according to the invention shall 5 encompass (i) CLD18-splice variants, (ii) CLD18-N-glycosylation variants, (iii)

CLD18-conformation variants, (iv) CLD18-ffee and homotypically/heterotypically associated variants localized at intercellular tight junctions and (v) CLD18-cancer related and CLD18-non-cancer cell related variants.

The molecular and functional characteristics of CLD 18 make this molecule a highly interesting target for antibody based cancer therapy. These are in particular (i) the absence of CLD 18 from the vast majority of toxicity relevant normal tissues, (ii) the restriction of CLD18A2 variant expression to a dispensible cell population as differentiated gastric cells, which can be replenished by target15 negative stem cells of the stomach, (iii) hints to potential differential glycosylation between normal and neoplastic cells, and (iv) the presence of different conformational topologies. Moreover, the role of CLD 18 as tight junction protein may further contribute to a good therapeutic window. Because tumor cells express claudins but often do not form classical tight junctions by homotypic and

0 heterotypic association of claudins as found in normal epithelial tissue, tumor cells may have a considerable pool of free claudin that is amenable to extracellular antibody binding and immunotherapy. It is possible that binding epitopes of claudins in healthy epithelium are shielded within tight junctions from the access by such antibodies.

The object of the invention is to provide antibodies useful for therapy of diseases wherein CLD 18 is expressed, such as tumor diseases. The antibodies described herein have also utility in diagnosing such diseases.

0 SUMMARY OF THE INVENTION

The present invention generally provides antibodies useful as therapeutics for treating and/or preventing diseases associated with cells expressing CLD 18, including tumor-related diseases such as gastric cancer, esophageal cancer,

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2018200685 30 Jan 2018 pancreatic cancer, lung cancer, ovarian cancer, colon cancer, hepatic cancer, headneck cancer, and cancer of the gallbladder.

In one aspect the invention relates to an antibody having the ability of binding to 5 CLD18 and mediating killing of cells expressing CLD18. Preferably, the antibody binds to CLD18A1 and CLD18A2 and more preferably binds to CLD18A2 but not to CLD18A1. Preferably, antibodies of the invention bind to and are specific for loopl or loop2 of CLD-conformation-1. In further preferred embodiments, the antibody of the invention binds to and is specific for loopD3 of CLD10 conformation-2 and, in particular, binds at or around a potential N-glycosylation site at position 116 within loopD3. In further embodiments, the antibody of the invention is specific for the unglycosylated form of the potential N-glycosylation site at position 116 within loopD3.

Killing of cells by the antibody of the invention is preferably induced by binding of the antibody to CLD18 expressed by said cells, more preferably by binding of the antibody to CLD18A2 expressed by said cells. In one embodiment, binding of the antibody of the invention to CLD18A1 expressed by said cells does not induce killing of said cells.

The cells expressing CLD18 are preferably cancer cells and are, in particular, selected from the group consisting of tumorigenic gastric, esophageal, pancreatic, lung, ovarian, colon, hepatic, head-neck, and gallbladder cancer cells.

5 Preferably the antibody of the invention mediates killing of cells by inducing complement dependent cytotoxicity (CDC) mediated lysis, antibody dependent cellular cytotoxicity (ADCC) mediated lysis, apoptosis, homotypic adhesion, and/or phagocytosis, preferably by inducing CDC mediated lysis and/or ADCC mediated lysis.

In one embodiment the antibody of the invention does not induce CDC mediated lysis of cells.

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Preferably, ADCC mediated lysis of cells takes place in the presence of effector cells, which in particular embodiments are selected from the group consisting of monocytes, mononuclear cells, NK cells and PMNs, and phagocytosis is by macrophages.

The antibody of the invention may be a monoclonal, chimeric, human, or humanized antibody, or a fragment of an antibody and may be selected from the group consisting of an IgGl, an IgG2, preferably IgG2a and IgG2b, an IgG3, an IgG4, an IgM, an IgAl, an IgA2, a secretory IgA, an IgD, and an IgE antibody.

According to all aspects of the invention, CLD18 is preferably human CLD18, preferably human CLD18A2, and CLD18A2 preferably has the amino acid sequence according to SEQ ID NO:2 and CLD18A1 preferably has the amino acid sequence according to SEQ ID NO:8.

In particular preferred embodiments, the antibody of the invention binds to native epitopes of CLD18 present on the surface of living cells. In further preferred embodiments, the antibody of the invention is specific for cancer cells, preferably stomach cancer cells.

In certain embodiments of the invention CLD18 is expressed on the surface of cells.

Antibodies of the invention may be obtained by a method comprising the step of 2 5 immunizing an animal with a protein or peptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2, 4, 6, 16, 18, 20, 21-23, and

26-31, or an immunogenic fragment thereof, or a nucleic acid or host cell expressing said protein or peptide, or immunogenic fragment thereof. Preferably, an antibody of the invention is specific for the afore mentioned proteins, peptides or immunogenic fragments thereof.

In a particularly preferred embodiment, the antibody of the invention is produced by a clone having the accession no. DSM ACC2737 (182-D1106-055), DSM ACC2738 (182-D1106-056), DSM ACC2739 (182-D1106-057), DSM ACC2740

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WO 2007/059997 PCT/EP2006/011302 (182-D1106-058), DSM ACC2741 (182-D1106-059), DSM ACC2742 (182D1106-062), DSM ACC2743 (182-D1106-067), DSM ACC2745 (182-D758-035),

DSM ACC2746 (182-D758-036), DSM ACC2747 (182-D758-040), DSM

ACC2748 (182-D1106-061), DSM ACC2808 (182-D1106-279), DSM ACC2809 (182-D1106-294), or DSM ACC2810 (182-D 1106-362).

In one embodiment the antibody of the invention is coupled to a therapeutic agent such as a toxin, a radioisotope, a drug or a cytotoxic agent.

In a further aspect the invention relates to a hybridoma capable of producing the antibody of the invention. Preferred hybridomas are those having the accession no. DSM ACC2737 (182-D1106-055), DSM ACC2738 (182-D 1106-056), DSM ACC2739 (182-D 1106-057), DSM ACC2740 (182-D1106-058), DSM ACC2741 (182-D 1106-059), DSM ACC2742 (182-D1106-062), DSM ACC2743 (18215 DI 106-067), DSM ACC2745 (182-D758-035), DSM ACC2746 (182-D758-036), DSM ACC2747 (182-D758-040), DSM ACC2748 (182-D1106-061), DSM ACC2808 (182-D1106-279), DSM ACC2809 (182-D 1106-294), or DSM ACC2810 (182-D1106-362).

0 Antibodies of the invention are designated herein by referring to the designation of the antibody , e.g. 182-D758-035, and/or by referring to the clone producing the antibody, e.g. 26D12.

The invention also relates to a pharmaceutical composition comprising an antibody

5 of the invention and/or a conjugate thereof with a therapeutic agent, and a pharmaceutically acceptable carrier.

In a further aspect the invention relates to a method of inhibiting growth and/or killing of a cell expressing CLD18, preferably CLD18A2, comprising contacting

0 the cell with an effective amount of an antibody of the invention and/or a conjugate thereof with a therapeutic agent. CLD18 is preferably expressed on the surface of said cell.

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In a further aspect the invention relates to a method of treating or preventing a disease or disorder involving cells expressing CLD18, preferably CLD18A2, comprising administering to a subject an antibody of the invention, a conjugate thereof with a therapeutic agent, or a pharmaceutical composition comprising the antibody of the invention or the conjugate thereof with a therapeutic agent. Preferably the disease or disorder is a tumor-related disease and in particular embodiments is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, hepatic cancer, head-neck cancer, and cancer of the gallbladder. CLD18 is preferably expressed on the surface of said cells.

Preferably, the antibodies of the invention have the ability to discriminate CLD18variants expressed by different cell types including cancer cells and non-malignant cells. In a particularly preferred embodiment, the antibodies of the invention have the ability to bind to CLD18A2 while they do not bind to CLD18A1, or bind to CLD18A1 with a lower specificity compared to the binding specificity to CLD18A2.

The term binding according to the invention preferably relates to a specific

0 binding. “Specific binding” means that an agent such as an antibody binds stronger to a target such as an epitope for which it is specific compared to the binding to another target. An agent binds stronger to a first target compared to a second target if it binds to the first target with a dissociation constant (Kd) which is lower than the dissociation constant for the second target. Preferably the dissociation constant

5 (K_D) for the target to which the agent binds specifically is more than 10-fold, preferably more than 20-fold, more preferably more than 50-fold, even more preferably more than 100-fold, 200-fold, 500-fold or 1000-fold lower than the dissociation constant (Kd) for the target to which the agent does not bind specifically.

The antibodies of the invention mediate killing of cells expressing CLD18, preferably CLD18A2, by binding to CLD18, preferably expressed on the surface of said cells. In one embodiment, antibodies of the invention induce complement dependent cytotoxicity (CDC), e.g. at least about 20-40% CDC mediated lysis,

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2018200685 30 Jan 2018 preferably about 40-50% CDC mediated lysis, and more preferably more than 50%

CDC mediated lysis of cells expressing CLD18. Such antibodies are exemplified herein by the following antibodies: 37H8, 38G5, 38H3, 39F11, 61C2, 26B5,

26D12, 28D10, 163E12, 175D10, 45C1, 125E1, ch-163E12, and ch-175D10.

Alternatively or in addition to inducing CDC, antibodies of the invention may induce antibody dependent cellular cytotoxicity (ADCC) of cells expressing CLD18 in the presence of effector cells (e.g., monocytes, mononuclear cells, NK cells and PMNs). Such antibodies are exemplified herein by the following antibodies: 37G11, 37H8, 38G5, 38H3, 39F11, 43A11, 61C2, 26B5, 26D12,

28D10, 42E12, 163E12, 175D10, 45C1, and 125E1. Antibodies of the invention may have the ability to induce apoptosis of cells expressing CLD18, induce homotypic adhesion of cells expressing CLD18 and/or induce phagocytosis of cells expressing CLD18 in the presence of macrophages. The antibodies of the invention may have one or more of the above described functional properties.

Preferably, antibodies of the invention induce CDC mediated lysis and ADCC mediated lysis of cells expressing CLD18 and more preferably induce ADCC mediated lysis of cells expressing CLD18 while they do not induce CDC mediated lysis of said cells. Exemplary target cells for antibodies of the present invention include, but are not limited to, cancer cells expressing CLD18, preferably

0 CLD18A2, such as tumorigenic gastric, pancreatic, esophageal and lung cancer cells. In a particular preferred embodiment, killing of cells mediated by antibodies of the invention is CLD18A2 specific, i.e. antibodies of the invention mediate killing of cells, preferably CDC and/or ADCC mediated lysis of cells, expressing CLD18A2 but do not mediate killing of cells expressing CLD18A1 but not

5 expressing CLD18A2. The antibodies described above may be used to mediate killing of tumor cells in the treatment or prevention of cancer such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, hepatic cancer, head-neck cancer, and cancer of the gallbladder.

0 Antibodies of the invention may be categorized into distinct classes according to their binding properties and their ability to mediate effector function on cells expressing CLD18. The antibodies of the invention may be categorized according to their

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2018200685 30 Jan 2018 • binding properties to and/or effector functions mediated on cells expressing either CLD18A1 or CLD18A2 (discrimination of CLD18 splice variants), • binding properties to and/or effector functions mediated on cells expressing 5 either glycosylated or non-glycosylated CLD18 variants (discrimination betweeen CLD18-variants with and without N-glycosylation), • binding properties to and/or effector functions mediated on either cancer cells or normal cell types (discrimination between CLD18-variants expressed by tumor cells or normal nonmalignant cells), • binding properties to CLD18-epitopes masked by the formation of tight junctions, · abilities to induce aggregate formation of CLD18 on living cells, and • abilities to bind a non-human CLD18 variant, particularly CLD18 variants from mice, rats, rabbits and primates.

0 Antibodies of the invention may have one or more of the following properties whereby reference is given to specific examples of antibodies of the invention described herein (24H5, 26B5, 26D12, 28D10, 37G11, 37H8, 38G5, 38H3, 39F11, 41C6, 42E12, 43A11, 44E10, 47D12, 61C2, 75B8, 85A3, 9E8, 19B9, 45C1, 125E1, 163E12, 166E2, 175D10, ch-43All, ch-45Cl, ch-125El, ch-163E12, ch2 5 166E2, ch-175D10):

a) binding to CLD18A2 as well as to CLD18A1 (e.g. 26D12, 28D10, 37H8, 38H3, 39F11, 61C2, and 41C6)

b) binding to CLD18A2 but not to CLD18A1 (e.g. 26B5, 37G11, 38G5, 42E12, and 43A11, 45C1, 125E1, 163E12, 166E2, 175D10, ch-43All, ch-45Cl, ch30 125E1, ch-163E12, ch-166E2, ch-175D10)

c) binding to CLD18 naturally expressed by tumor cells but not to CLD18 naturally expressed by non-cancer cells or tissues such as cells of stomach and lung (e.g 26B5, 75B8, 24H5, 39F11,45C1, 125E1, 163E12, 166E2, 175D10).

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d) mediating CDC induced killing of cells, which express CLD18A2 but not of cells which express CLD18A1 (e.g. 26D12, 28D10, 37H8, and 39F11, 163E12, ch-125El, ch-163E12, ch-175D10)

e) mediating ADCC induced killing of cells expressing CLD18 (e.g. 26B5,

37G11, 37H8, 38G5, 38H3, 39F11, 43A11, 47D12, and 61C2, ch-163E12, ch175D10)

f) mediating ADCC induced killing but not CDC mediated killing of cells expressing CLD18 (e.g. 37G11, 42E12, and 43A11)

g) mediating ADCC induced killing and CDC induced killing of cells expressing 10 CFD18A2 (e.g. 37H8, 38H3, 39F11, ch-163E12, ch-175D10).

As exemplified herein, antibodies of the invention further encompasses molecules, which

a) bind to differentiated cells of normal stomach, but not to stem cells of stomach (e.g. 39F11)

b) do not bind to normal gastric tissue as well as other normal organs but exclusively to cancer cells (e.g. 26B5)

c) bind to an epitope encompassing a non-glycosylated Asn at position 116 of 20 CLD18

d) which bind to human as well as to mouse CLD18 allowing to thoroughly perform preclinical toxicity studies in mice.

Antibodies of the invention may be derived from different species, including but 25 not limited to mouse, rat, rabbit, guinea pig and human. Antibodies of the invention also include chimeric molecules in which an antibody constant region derived from one species, preferably human, is combined with the antigen binding site derived from another species. Moreover antibodies of the invention include humanized molecules in which the antigen binding sites of an antibody derived

0 from a non-human species are combined with constant and framework regions of human origin.

Antibodies of the invention include polyclonal and monoclonal antibodies and include IgG2a (e.g. IgG2a, κ, λ), IgG2b (e.g. IgG2b, κ, λ), IgG3 (e.g. IgG3, κ, λ)

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2018200685 30 Jan 2018 and IgM antibodies. However, other antibody isotypes are also encompassed by the invention, including IgGl, IgAl, IgA2, secretory IgA, IgD, and IgE antibodies.

The antibodies can be whole antibodies or antigen-binding fragments thereof including, for example, Fab, F(ab')2, Fv, single chain Fv fragments or bispecific antibodies. Furthermore, the antigen-binding fragments include binding-domain immunoglobulin fusion proteins comprising (i) a binding domain polypeptide (such as a heavy chain variable region or a light chain variable region) that is fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. Such bindingdomain immunoglobulin fusion proteins are further disclosed in US2003/0118592 and US 2003/0133939.

Antibodies of the present invention preferably dissociate from CLD18 with a dissociation equilibrium constant (KD) of approximately l-100nM or less. Preferably, antibodies of the invention do not cross-react with related cell-surface antigens and thus do not inhibit their function.

In preferred embodiments, antibodies of the present invention can be characterized by one or more of the following properties:

a) specificity for CLD18, in particular specificity for CLD18A2;

b) a binding affinity to CLD18, in particular CLD18A2, of about 100 nM or less, preferably, about 5-10 nM or less and, more preferably, about 1-3 nM or less,

5 c) the ability to mediate a high level of CDC on either CD55/59 negative or

CD55/59 positive cells;

d) the ability to inhibit the growth of cells which express CLD18;

e) the ability to induce apoptosis of cells which express CLD18;

f) the ability to induce homotypic adhesion of cells which express CLD18;

0 g) the ability to induce ADCC of cells which express CLD18 in the presence of effector cells;

h) the ability to prolong survival of a subject having tumor cells which express CLD18;

i) the ability to deplete cells which express CLD18;

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j) the ability to deplete cells which express low levels of CLD18 and/or

k) the ability to aggregate CLD18 on the surface of living cells

The anti-CLD18 antibodies of the present invention can be derivatized, linked to or 5 co-expressed to other binding specificities. In a particular embodiment, the invention provides a bispecific or multispecific molecule comprising at least one first binding specificity for CLD18 (e.g., an anti-CLD18 antibody or mimetic thereof), and a second binding specificity for a effector cell, such as a binding specificity for an Fc receptor (e.g., a Fc-gamma receptor, such as Fc-gamma RI, or any other Fc receptor) or a T cell receptor, e.g., CD3.

Accordingly, the present invention includes bispecific and multispecific molecules that bind to both CLD18 and to an Fc receptor or a T cell receptor, e.g. CD3. Examples of Fc receptors are IgG receptor, Fc-gamma receptor (FcyR), such as

FcyRI (CD64), FcyRII (CD32), and FcyRIII (CD 16). Other Fc receptors, such as IgA receptors (e.g., FcaRI), also can be targeted. The Fc receptor is preferably located on the surface of an effector cell, e.g., a monocyte, macrophage or an activated mononuclear cell. In a preferred embodiment, the bispecific and multispecific molecules bind to an Fc receptor at a site which is distinct from the

0 immunoglobulin Fc (e.g., IgG or IgA) binding site of the receptor. Therefore, the binding of the bispecific and multispecific molecules is not blocked by physiological levels of immunoglobulins.

In yet another aspect, anti-CLD18 antibodies of the invention are derivatized,

5 linked to or co-expressed with another functional molecule, e.g., another peptide or protein (e.g., a Fab' fragment). For example, an antibody of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g. to produce a bispecific or a multispecific antibody), a cytotoxin, cellular ligand or antigen (e.g. to produce an immunoconjugate, such as an immunotoxin). An antibody of the present invention can be linked to other therapeutic moieties, e.g., a radioisotope, a small molecule anti-cancer drug, a recombinant cytokine or chemokine. Accordingly, the present invention encompasses a large variety of antibody conjugates, bispecific and multispecific

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2018200685 30 Jan 2018 molecules, and fusion proteins, all of which bind to CLD18 expressing cells and which can be used to target other molecules to such cells.

In still another aspect, the invention provides compositions, e.g., pharmaceutical 5 and diagnostic compositions/kits, comprising a pharmaceutically acceptable carrier formulated along with one or a combination of antibodies of the invention. In a particular embodiment, the composition includes a combination of antibodies which bind to distinct epitopes or which possess distinct functional characteristics, such as inducing CDC and/or ADCC and inducing apoptosis. In this embodiment of the invention, antibodies may be used in combination, e. g., as a pharmaceutical composition comprising two or more anti-CLD18 monoclonal antibodies. For example, anti-CLD18 antibodies having different but complementary activities can be combined in a single therapy to achieve a desired therapeutic effect. In a preferred embodiment, the composition includes an anti-CLD18 antibody that mediates CDC combined with another anti-CLD18 antibody that induces apoptosis. In another embodiment, the composition includes an anti-CLD18 antibody that mediates highly effective killing of target cells in the presence of effector cells, combined with another anti-CLD18 antibody that inhibits the growth of cells expressing CLD18.

The present invention also includes the simultaneous or sequential administration of two or more anti-CLD18 antibodies of the invention, wherein at least one of said antibodies is a chimeric anti-CLD18 antibody and at least one further antibody is a human anti-CLD18 antibody, the antibodies binding to the same or different

5 epitopes of CLD18. Preferably, a chimeric CLD18 antibody of the invention is administered first followed by the administration of a human anti-CLD18 antibody of the invention, wherein the human anti-CLD18 antibody is preferably administered for an extended period of time, i.e. as maintenance therapy.

0 Antibodies, immunoconjugates, bispecific and multispecific molecules and compositions of the present invention can be used in a variety of methods for inhibiting growth of cells expressing CLD18, in particular CLD18A2 and/or selectively killing cells expressing CLD18, in particular CLD18A2 by contacting the cells with an effective amount of the antibody, immunconjugate,

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2018200685 30 Jan 2018 bispecific/multispecific molecule or composition, such that the growth of the cell is inhibited and/or the cell is killed. In one embodiment, the method includes killing of the cell expressing CLDI8, optionally in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis, or by a combination of two or more of these mechanisms. Cells expressing CLDI8 which can be inhibited or killed using the antibodies of the invention include cancer cells such as tumorigenic stomach, pancreatic, esophageal, lung, ovarian, colon, hepatic, headneck, and gallbladder cells.

Accordingly, antibodies of the present invention can be used to treat and/or prevent a variety of diseases involving cells expressing CLDI8 by administering the antibodies to patients suffering from such diseases. Exemplary diseases that can be treated (e.g., ameliorated) or prevented include, but are not limited to, tumorigenic diseases. Examples of tumorigenic diseases, which can be treated and/or prevented include gastric cancer, pancreatic cancer, esophageal cancer, lung cancer, ovarian cancer, colorectal cancer, hepatic cancer, head-neck cancer, and cancer of the gallbladder.

In a particular embodiment of the invention, the subject being administered the antibody is additionally treated with a chemotherapeutic agent, radiation, or an agent that modulates, e.g., enhances or inhibits, the expression or activity of an Fc receptor, e.g. an Fc-gamma receptor, such as a cytokine. Typical cytokines for administration during treatment include granulocyte colony-stimulating factor (GCSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ

5 (IFN-γ), and tumor necrosis factor (TNF). Typical therapeutic agents include, among others, anti-neoplastic agents such as doxorubicin, cisplatin, taxotere, 5fluoruracil, methotrexat, gemzitabin and cyclophosphamide.

In yet another aspect, the invention relates to an immunization strategy to

0 immunize non-human animals such as mice with human CLDI8 or a peptide fragment thereof, preferably CLD18A2 or a peptid fragment thereof to obtain antibodies. Preferred peptides for immunization are those selected from the group consisting of SEQ ID NO:2, 4, 6, 16, 18, 20-23, and 26-31. Accordingly, in preferred embodiments, the antibodies of the invention are those obtained by

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2018200685 30 Jan 2018 immunization using peptides selected from the group consisting of SEQ ID NO:2,

4, 6, 16, 18, 20-23, and 26-31. Analogously, antibodies to CLD18 can be generated in a transgenic non-human animal, such as a transgenic mouse. The transgenic non-human animal may be a transgenic mouse having a genome comprising a heavy chain transgene and a light chain transgene encoding all or a portion of an antibody.

Wildtype as well as transgenic non-human animals can be immunized with a purified or enriched preparation of CLD18 antigen and/or nucleic acids and/or cells expressing CLD18 or a peptide fragment thereof. Preferably, the non-human animal, is capable of producing multiple isotypes of human monoclonal antibodies to CLD18 (e.g., IgG, IgA and/or IgM) by undergoing V-D-J recombination and isotype switching. Isotype switching may occur by e.g., classical or non-classical isotype switching.

Accordingly, in yet another aspect, the invention provides isolated B cells from a non-human animal as described above. The isolated B cells can then be immortalized by fusion to an immortalized cell to provide a source (e.g., a hybridoma) of antibodies of the invention. Such hybridomas (i.e., which produce

0 antibodies of the invention) are also included within the scope of the invention.

As exemplified herein, antibodies of the invention can be obtained directly from hybridomas which express the antibody, or can be cloned and recombinantly expressed in a host cell (e.g., a CHO cell, or a lymphocytic cell). Further examples of host cells are microorganisms, such as E. coli, and fungi, such as yeast. Alternatively, they can be produced recombinantly in a transgenic non-human animal or plant.

Preferred hybridoma cells for producing antibodies of the invention are those

0 sequenced or deposited at the DSMZ (Mascheroder Weg lb, 31824 Braunschweig,

Germany; new address: Inhoffenstr. 7B, 31824 Braunschweig, Germany) having the following designations and accession numbers:

a. 182-D1106-055, accesssion no. DSM ACC2737, deposited on October 19, 2005

b. 182-D1106-056, accesssion no. DSM ACC2738, deposited on October 19, 2005

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c. 182-D1106-057, accesssion no. DSM ACC2739, deposited on October 19, 2005

d. 182-D1106-058, accesssion no. DSM ACC2740, deposited on October 19, 2005

e. 182-D1106-059, accesssion no. DSM ACC2741, deposited on October 19, 2005

f. 182-D1106-062, accesssion no. DSM ACC2742, deposited on October 19, 2005, 5 g. 182-D1106-067, accesssion no. DSM ACC2743, deposited on October 19, 2005

h. 182-D758-035, accesssion no. DSM ACC2745, deposited on Nov. 17, 2005

i. 182-D758-036, accesssion no. DSM ACC2746, deposited on Nov. 17, 2005

j. 182-D758-040, accesssion no. DSM ACC2747, deposited on Nov. 17, 2005

k. 182-D1106-061, accesssion no. DSM ACC2748, deposited on Nov. 17, 2005

1. 182-D1106-279, accesssion no. DSM ACC2808, deposited on Oct. 26, 2006

m. 182-D1106-294, accesssion no. DSM ACC2809, deposited on Oct. 26, 2006,

n. 182-D1106-362, accesssion no. DSM ACC2810, deposited on Oct. 26, 2006.

Preferred antibodies of the invention are those produced by and obtainable from 15 the above-described hybridomas; i.e. 37G11 in the case of 182-D1106-055, 37H8 in the case of 182-D1106-056, 38G5 in the case of 182-D1106-057, 38H3 in the case of 182-D1106-058, 39F11 in the case of 182-D1106-059, 43A11 in the case of 182-D1106-062, 61C2 in the case of 182-D1106-067, 26B5 in the case of 182D758-035, 26D12 in the case of 182-D758-036, 28D10 in the case of 182-D7582 0 040, 42E12 in the case of 182-D 1106-061, 125E1 in the case of 182-D 1106-279,

163E12 in the case of 182-D1106-294, and 175D10 in the case of 182-D 1106-362; and the chimerized and humanized forms thereof.

In preferred embodiments, antibodies, in particular chimerised forms of antibodies 25 according to the invention include antibodies comprising a heavy chain constant region (CH) comprising an amino acid sequence derived from a human heavy chain constant region such as the amino acid sequence represented by SEQ ID NO:

or 150 or a fragment thereof. In further preferred embodiments, antibodies, in particular chimerised forms of antibodies according to the invention include

0 antibodies comprising a light chain constant region (CL) comprising an amino acid sequence derived from a human light chain constant region such as the amino acid sequence represented by SEQ ID NO: 41 or 148 or a fragment thereof. In a particular preferred embodiment, antibodies, in particular chimerised forms of antibodies according to the invention include antibodies which comprise a CH

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2018200685 30 Jan 2018 comprising an amino acid sequence derived from a human CH such as the amino acid sequence represented by SEQ ID NO: 46 or 150 or a fragment thereof and which comprise a CL comprising an amino acid sequence derived from a human

CL such as the amino acid sequence represented by SEQ ID NO: 41 or 148 or a fragment thereof.

A CH comprising the amino acid sequence represented by SEQ ID NO: 46 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 45. A CH comprising the amino acid sequence represented by SEQ

ID NO: 150 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 149. A CL comprising the amino acid sequence represented by SEQ ID NO: 41 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 40. A CL comprising the amino acid sequence represented by SEQ ID NO: 148 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 147.

In certain preferred embodiments, chimerised forms of antibodies include antibodies comprising a heavy chain comprising an amino acid sequence selected

0 from the group consisting of SEQ ID NO: 115, 116, 117, 118, 119, 120, and a fragment thereof and/or comprising a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 121, 122, 123, 124, 125, 126, 127, 128, 129, and a fragment thereof.

5 In certain preferred embodiments, chimerised forms of antibodies include antibodies comprising a combination of heavy chains and light chains selected from the following possibilities (i) to (ix):

(i) the heavy chain comprises an amino acid sequence represented by SEQ ID NO:

115 or a fragment thereof and the light chain comprises an amino acid sequence

0 represented by SEQ ID NO: 122 or a fragment thereof, (ii) the heavy chain comprises an amino acid sequence represented by SEQ ID NO:

116 or a fragment thereof and the light chain comprises an amino acid sequence represented by SEQ ID NO: 121 or a fragment thereof,

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2018200685 30 Jan 2018 (iii) the heavy chain comprises an amino acid sequence represented by SEQ ID NO: 117 or a fragment thereof and the light chain comprises an amino acid sequence represented by SEQ ID NO: 123 or a fragment thereof, (iv) the heavy chain comprises an amino acid sequence represented by SEQ ID 5 NO: 119 or a fragment thereof and the light chain comprises an amino acid sequence represented by SEQ ID NO: 126 or a fragment thereof, (v) the heavy chain comprises an amino acid sequence represented by SEQ ID NO: 118 or a fragment thereof and the light chain comprises an amino acid sequence represented by SEQ ID NO: 125 or a fragment thereof, (vi) the heavy chain comprises an amino acid sequence represented by SEQ ID NO: 120 or a fragment thereof and the light chain comprises an amino acid sequence represented by SEQ ID NO: 124 or a fragment thereof, (vii) the heavy chain comprises an amino acid sequence represented by SEQ ID NO: 120 or a fragment thereof and the light chain comprises an amino acid sequence represented by SEQ ID NO: 127 or a fragment thereof, (viii) the heavy chain comprises an amino acid sequence represented by SEQ ID NO: 120 or a fragment thereof and the light chain comprises an amino acid sequence represented by SEQ ID NO: 128 or a fragment thereof, and (ix) the heavy chain comprises an amino acid sequence represented by SEQ ID

0 NO: 120 or a fragment thereof and the light chain comprises an amino acid sequence represented by SEQ ID NO: 129 or a fragment thereof.

“Fragment” or “fragment of an amino acid sequence” as used above relates to a part of an antibody sequence, i.e. a sequence which represents the antibody

5 sequence shortened at the N- and/or C-terminus, which when it replaces said antibody sequence in an antibody retains binding of said antibody to CLD18 and preferably functions of said antibody as described herein, e.g. CDC mediated lysis or ADCC mediated lysis. Preferably, a fragment of an amino acid sequence comprises at least 80%, preferably at least 90%, 95%, 96%, 97%, 98%, or 99% of

0 the amino acid residues from said amino acid sequence. A fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, and 129 preferably relates to said sequence wherein 17, 18, 19, 20, 21, 22 or 23 amino acids at the Nterminus are removed. Fragments of amino acid sequences described herein may

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2018200685 30 Jan 2018 be encoded by respective fragments of nucleic acid sequences encoding said amino acid sequences.

A heavy chain comprising an amino acid sequence represented by SEQ ID NO: 5 115 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 100. A heavy chain comprising an amino acid sequence represented by SEQ ID NO: 116 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 101. A heavy chain comprising an amino acid sequence represented by SEQ ID NO: 117 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 102. A heavy chain comprising an amino acid sequence represented by SEQ ID NO: 119 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 104. A heavy chain comprising an amino acid sequence represented by SEQ ID NO: 118 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 103. A heavy chain comprising an amino acid sequence represented by SEQ ID NO: 120 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 105.

0 A light chain comprising an amino acid sequence represented by SEQ ID NO: 122 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 107. A light chain comprising an amino acid sequence represented by SEQ ID NO: 121 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 106. A light chain comprising an amino acid sequence represented by SEQ ID NO: 123 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO:

108. A light chain comprising an amino acid sequence represented by SEQ ID NO: 126 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 111. A light chain comprising an amino acid sequence

0 represented by SEQ ID NO: 125 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 110. A light chain comprising an amino acid sequence represented by SEQ ID NO: 124 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO:

109. A light chain comprising an amino acid sequence represented by SEQ ID NO:

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127 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 112. A light chain comprising an amino acid sequence represented by SEQ ID NO: 128 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 113. A light chain comprising an amino acid sequence represented by SEQ ID NO: 129 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 114.

In a preferred embodiment, an antibody of the invention comprises a heavy chain 10 variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 132, 133, 134, 135, 136, 137, and a fragment thereof.

In a preferred embodiment, an antibody of the invention comprises a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 138, 139, 140, 141, 142, 143, 144, 145, 146, and a fragment thereof.

In certain preferred embodiments, an antibody of the invention comprises a combination of heavy chain variable region (VH) and light chain variable region

0 (VL) selected from the following possibilities (i) to (ix):

(i) the VH comprises an amino acid sequence represented by SEQ ID NO: 132 or a fragment thereof and the VL comprises an amino acid sequence represented by SEQ ID NO: 139 or a fragment thereof, (ii) the VH comprises an amino acid sequence represented by SEQ ID NO: 133 or

5 a fragment thereof and the VL comprises an amino acid sequence represented by

SEQ ID NO: 138 or a fragment thereof, (iii) the VH comprises an amino acid sequence represented by SEQ ID NO: 134 or a fragment thereof and the VL comprises an amino acid sequence represented by SEQ ID NO: 140 or a fragment thereof,

0 (iv) the VH comprises an amino acid sequence represented by SEQ ID NO: 136 or a fragment thereof and the VL comprises an amino acid sequence represented by

SEQ ID NO: 143 or a fragment thereof,

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2018200685 30 Jan 2018 (v) the VH comprises an amino acid sequence represented by SEQ ID NO: 135 or a fragment thereof and the VL comprises an amino acid sequence represented by SEQ ID NO: 142 or a fragment thereof, (vi) the VH comprises an amino acid sequence represented by SEQ ID NO: 137 or 5 a fragment thereof and the VL comprises an amino acid sequence represented by

SEQ ID NO: 141 or a fragment thereof, (vii) the VH comprises an amino acid sequence represented by SEQ ID NO: 137 or a fragment thereof and the VL comprises an amino acid sequence represented by SEQ ID NO: 144 or a fragment thereof, (viii) the VH comprises an amino acid sequence represented by SEQ ID NO: 137 or a fragment thereof and the VL comprises an amino acid sequence represented by SEQ ID NO: 145 or a fragment thereof, (ix) the VH comprises an amino acid sequence represented by SEQ ID NO: 137 or a fragment thereof and the VL comprises an amino acid sequence represented by

SEQ ID NO: 146 or a fragment thereof.

A VH comprising an amino acid sequence represented by SEQ ID NO: 132 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 55. A VH comprising an amino acid sequence represented by SEQ

0 ID NO: 133 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 56. A VH comprising an amino acid sequence represented by SEQ ID NO: 134 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 57. A VH comprising an amino acid sequence represented by SEQ ID NO: 136 may be

5 encoded by a nucleic acid comprising the nucleic acid sequence represented by

SEQ ID NO: 59. A VH comprising an amino acid sequence represented by SEQ ID NO: 135 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 58. A VH comprising an amino acid sequence represented by SEQ ID NO: 137 may be encoded by a nucleic acid

0 comprising the nucleic acid sequence represented by SEQ ID NO: 60.

A VL comprising an amino acid sequence represented by SEQ ID NO: 139 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 62. A VL comprising an amino acid sequence represented by SEQ ID

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NO: 138 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 61. A VL comprising an amino acid sequence represented by SEQ ID NO: 140 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 63. A VL comprising an amino acid sequence represented by SEQ ID NO: 143 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 66. A VL comprising an amino acid sequence represented by SEQ ID NO: 142 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 65. A VL comprising an amino acid sequence represented by SEQ ID

NO: 141 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 64. A VL comprising an amino acid sequence represented by SEQ ID NO: 144 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 67. A VL comprising an amino acid sequence represented by SEQ ID NO: 145 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 68. A VL comprising an amino acid sequence represented by SEQ ID NO: 146 may be encoded by a nucleic acid comprising the nucleic acid sequence represented by SEQ ID NO: 69.

0 In a preferred embodiment, an antibody of the invention comprises a VH comprising a set of complementarity-determining regions CDR1, CDR2 and CDR3 selected from the following embodiments (i) to (vi):

(i) CDR1: positions 45-52 of SEQ ID NO: 115, CDR2: positions 70-77 of SEQ ID NO: 115, CDR3: positions 116-125 of SEQ ID NO: 115,

5 (ii) CDR1: positions 45-52 of SEQ ID NO: 116, CDR2: positions 70-77 of SEQ ID NO: 116, CDR3: positions 116-126 of SEQ ID NO: 116, (iii) CDR1: positions 45-52 of SEQ ID NO: 117, CDR2: positions 70-77 of SEQ ID NO: 117, CDR3: positions 116-124 of SEQ ID NO: 117, (iv) CDR1: positions 45-52 of SEQ ID NO: 118, CDR2: positions 70-77 of SEQ

ID NO: 118, CDR3: positions 116-126 of SEQ ID NO: 118, (v) CDR1: positions 44-51 of SEQ ID NO: 119, CDR2: positions 69-76 of SEQ ID NO: 119, CDR3: positions 115-125 of SEQ ID NO: 119, and (vi) CDR1: positions 45-53 of SEQ ID NO: 120, CDR2: positions 71-78 of SEQ ID NO: 120, CDR3: positions 117-128 of SEQ ID NO: 120.

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In a preferred embodiment, an antibody of the invention comprises a VL comprising a set of complementarity-determining regions CDR1, CDR2 and

CDR3 selected from the following embodiments (i) to (ix):

(i) CDR1: positions 47-58 of SEQ ID NO: 121, CDR2: positions 76-78 of SEQ ID

NO: 121, CDR3: positions 115-123 of SEQ ID NO: 121, (ii) CDR1: positions 49-53 of SEQ ID NO: 122, CDR2: positions 71-73 of SEQ ID NO: 122, CDR3: positions 110-118 of SEQ ID NO: 122, (iii) CDR1: positions 47-52 of SEQ ID NO: 123, CDR2: positions 70-72 of SEQ ID NO: 123, CDR3: positions 109-117 of SEQ ID NO: 123, (iv) CDR1: positions 47-58 of SEQ ID NO: 124, CDR2: positions 76-78 of SEQ ID NO: 124, CDR3: positions 115-123 of SEQ ID NO: 124, (v) CDR1: positions 47-58 of SEQ ID NO: 125, CDR2: positions 76-78 of SEQ ID NO: 125, CDR3: positions 115-123 of SEQ ID NO: 125, (vi) CDR1: positions 47-58 of SEQ ID NO: 126, CDR2: positions 76-78 of SEQ 15 ID NO: 126, CDR3: positions 115-122 of SEQ ID NO: 126, (vii) CDR1: positions 47-58 of SEQ ID NO: 127, CDR2: positions 76-78 of SEQ ID NO: 127, CDR3: positions 115-123 of SEQ ID NO: 127, (viii) CDR1: positions 47-58 of SEQ ID NO: 128, CDR2: positions 76-78 of SEQ ID NO: 128, CDR3: positions 115-123 of SEQ ID NO: 128, and

0 (ix) CDR1: positions 47-52 of SEQ ID NO: 129, CDR2: positions 70-72 of SEQ ID NO: 129, CDR3: positions 109-117 of SEQ ID NO: 129.

In a preferred embodiment, an antibody of the invention comprises a combination of VH and VL each comprising a set of complementarity-determining regions

5 CDR1, CDR2 and CDR3 selected from the following embodiments (i) to (ix):

(i) VH: CDR1: positions 45-52 of SEQ ID NO: 115, CDR2: positions 70-77 of SEQ ID NO: 115, CDR3: positions 116-125 of SEQ ID NO: 115, VL: CDR1: positions 49-53 of SEQ ID NO: 122, CDR2: positions 71-73 of SEQ ID NO: 122, CDR3: positions 110-118 of SEQ ID NO: 122,

0 (ii) VH: CDR1: positions 45-52 of SEQ ID NO: 116, CDR2: positions 70-77 of

SEQ ID NO: 116, CDR3: positions 116-126 of SEQ ID NO: 116, VL: CDR1: positions 47-58 of SEQ ID NO: 121, CDR2: positions 76-78 of SEQ ID NO: 121, CDR3: positions 115-123 of SEQ ID NO: 121,

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2018200685 30 Jan 2018 (iii) VH: CDR1: positions 45-52 of SEQ ID NO: 117, CDR2: positions 70-77 of

SEQ ID NO: 117, CDR3: positions 116-124 of SEQ ID NO: 117, VL: CDR1:

positions 47-52 of SEQ ID NO: 123, CDR2: positions 70-72 of SEQ ID NO: 123,

CDR3: positions 109-117 of SEQ ID NO: 123, (iv) VH: CDR1: positions 44-51 of SEQ ID NO: 119, CDR2: positions 69-76 of SEQ ID NO: 119, CDR3: positions 115-125 of SEQ ID NO: 119, VL: CDR1: positions 47-58 of SEQ ID NO: 126, CDR2: positions 76-78 of SEQ ID NO: 126, CDR3: positions 115-122 of SEQ ID NO: 126, (v) VH: CDR1: positions 45-52 of SEQ ID NO: 118, CDR2: positions 70-77 of 10 SEQ ID NO: 118, CDR3: positions 116-126 of SEQ ID NO: 118, VL: CDR1:

positions 47-58 of SEQ ID NO: 125, CDR2: positions 76-78 of SEQ ID NO: 125, CDR3: positions 115-123 of SEQ ID NO: 125, (vi) VH: CDR1: positions 45-53 of SEQ ID NO: 120, CDR2: positions 71-78 of SEQ ID NO: 120, CDR3: positions 117-128 of SEQ ID NO: 120, VL: CDR1:

positions 47-58 of SEQ ID NO: 124, CDR2: positions 76-78 of SEQ ID NO: 124, CDR3: positions 115-123 of SEQ ID NO: 124, (vii) VH: CDR1: positions 45-53 of SEQ ID NO: 120, CDR2: positions 71-78 of SEQ ID NO: 120, CDR3: positions 117-128 of SEQ ID NO: 120, VL: CDR1: positions 47-58 of SEQ ID NO: 127, CDR2: positions 76-78 of SEQ ID NO: 127,

CDR3: positions 115-123 of SEQ ID NO: 127, (viii) VH: CDR1: positions 45-53 of SEQ ID NO: 120, CDR2: positions 71-78 of SEQ ID NO: 120, CDR3: positions 117-128 of SEQ ID NO: 120, VL: CDR1: positions 47-58 of SEQ ID NO: 128, CDR2: positions 76-78 of SEQ ID NO: 128, CDR3: positions 115-123 of SEQ ID NO: 128, and

5 (ix) VH: CDR1: positions 45-53 of SEQ ID NO: 120, CDR2: positions 71-78 of

SEQ ID NO: 120, CDR3: positions 117-128 of SEQ ID NO: 120, VL: CDR1: positions 47-52 of SEQ ID NO: 129, CDR2: positions 70-72 of SEQ ID NO: 129, CDR3: positions 109-117 of SEQ ID NO: 129.

0 In further preferred embodiments, an antibody of the invention preferably comprises one or more of the complementarity-determining regions (CDRs), preferably at least the CDR3 variable region, of the heavy chain variable region (VH) and/or of the light chain variable region (VL) of a monoclonal antibody against CLD18, preferably of a monoclonal antibody against CLD18 described

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2018200685 30 Jan 2018 herein, and preferably comprises one or more of the complementarity-determining regions (CDRs), preferably at least the CDR3 variable region, of the heavy chain variable regions (VH) and/or light chain variable regions (VL) described herein. In one embodiment said one or more of the complementarity-determining regions (CDRs) are selected from a set of complementarity-determining regions CDR1, CDR2 and CDR3 described herein. In a particularly preferred embodiment, an antibody of the invention preferably comprises the complementarity-determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region (VH) and/or of the light chain variable region (VL) of a monoclonal antibody against CLDI8, preferably of a monoclonal antibody against CLDI8 described herein, and preferably comprises the complementarity-determining regions CDR1, CDR2 and CDR3 of the heavy chain variable regions (VH) and/or light chain variable regions (VL) described herein.

In one embodiment an antibody of the invention comprising one or more CDRs, a set of CDRs or a combination of sets of CDRs as described herein comprises said CDRs together with their intervening framework regions. Preferably, the portion will also include at least about 50% of either or both of the first and fourth framework regions, the 50% being the C-terminal 50% of the first framework

0 region and the N-terminal 50% of the fourth framework region. Construction of antibodies of the present invention made by recombinant DNA techniques may result in the introduction of residues N- or C-terminal to the variable regions encoded by linkers introduced to facilitate cloning or other manipulation steps, including the introduction of linkers to join variable regions of the invention to

5 further protein sequences including immunoglobulin heavy chains, other variable domains (for example in the production of diabodies) or protein labels.

In one embodiment an antibody of the invention comprising one or more CDRs, a set of CDRs or a combination of sets of CDRs as described herein comprises said

0 CDRs in a human antibody framework.

Reference herein to an antibody comprising with respect to the heavy chain thereof a particular chain, or a particular region or sequence preferably relates to the

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2018200685 30 Jan 2018 situation wherein all heavy chains of said antibody comprise said particular chain, region or sequence. This applies correspondingly to the light chain of an antibody.

The present invention also relates to nucleic acids comprising genes or nucleic acid 5 sequences encoding antibodies or parts thereof, e.g. an antibody chain, as described herein. The nucleic acids may be comprised in a vector, e.g., a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering. The vector may comprise further genes such as marker genes which allow for the selection of the vector in a suitable host cell and under suitable conditions. Furthermore, the vector may comprise expression control elements allowing proper expression of the coding regions in suitable hosts. Such control elements are known to the artisan and may include a promoter, a splice cassette, and a translation initiation codon.

Preferably, the nucleic acid of the invention is operatively attached to the above expression control sequences allowing expression in eukaryotic or prokaryotic cells. Control elements ensuring expression in eukaryotic or prokaryotic cells are well known to those skilled in the art.

0 Methods for construction of nucleic acid molecules according to the present invention, for construction of vectors comprising the above nucleic acid molecules, for introduction of the vectors into appropriately chosen host cells, for causing or achieving the expression are well-known in the art.

5 A further aspect of the present invention relates to a host cell comprising a nucleic acid or vector as disclosed herein.

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

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BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows an immunfluorescence analysis of HEK293 cells transfected with CLD18A2 coupled to a green fluorochrome and reacted with mouse serum after

DNA immunisation with SEQ ID NO: 15 fused to a helper epitope.

Fig. 2 shows a Western blot analysis of HEK293 cells transfected with CLD18A2myc (SEQ ID NO: 3) and untransfected HEK293 cells with the monoclonal mouse-anti-c-myc antibody 9E11 (Serotec, CRL MCA2200).

Fig. 3 shows an immunfluorescence analysis using CHO cells transfected with CLD18A2 and a polyclonal rabbit-anti-CLD18 antibody (Zymed, CRL 38-8000).

Fig. 4A and B show the binding of hybridoma supernatants 24H5 and 85A3 to

HEK293 cells transiently transfected with human CLD18A2 and a fluorescent marker as determined by flow cytometry. Figure 4C shows the binding of hybridoma supernatants 45C1, 125E1, 163E12, 166E2 and 175D10 to HEK293 cells stably transfected with human CLD18A2 and counterstained with propidium iodide.

Fig. 5 shows binding of hybridoma supernatants 24H5 (A), 9E8 (Β), 26B5 (C) and 19B9 (D) to HEK293 cells transiently transfected with a fluorescent marker and either human CLD18A2 or CLD18A2-Myc or CLD18A2-HA as analyzed by flow cytometry.

Fig. 6A and B show binding of hybridoma supernatants 37H8, 43A11, 45C1 and 163E12 to HEK293 cells stably transfected with either human CLD18A2 or CLD18A1 as determined by flow cytometry.

0 Fig. 7 shows an immunofluorescence analysis of the CLD18A2 isoform specific monoclonal antibody 37G11 by staining HEK293 cells transfected with CLD18A2 (A, C) and CLD18A1 (B, D), respectively, under native (A, B) and paraformaldehyde fixation (C, D) conditions.

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Fig. 8 shows an immunfluorescence analysis of the CLD18 monoclonal antibody

26B5 by staining HEK293 cells transfected with CLD18A2 (A, C) and CLD18A1 (B, D), respectively, under native (A, B) and paraformaldehyde fixation (C, D) conditions.

Fig. 9. Cell line RT-PCR

RT-PCR analyis with CLD18A2-specific primers showed clear expression in 4/5 tested cell lines.

Fig. 10 shows an immunfluorescence analysis of DAN-G cells (subclone F2) and a polyclonal rabbit-anti-CLD18 antibody (Zymed, CRL 38-8000).

Fig. 11 shows an immunfluorescence analysis of KATO-III cells (subclone 3B9 4D5) and a polyclonal rabbit-anti-CLD18 antibody (Zymed, CRL 38-8000).

Fig. 12 A shows an immunfluorescence analysis of SNU-16 cells (subclone G5) with a polyclonal rabbit-anti-CLD18 antibody (Zymed, CRL 38-8000). Fig. 12 B shows an immunfluorescence analysis of KATO-III cells with monoclonal antibodies of the invention.

Fig. 13 shows surface expression of CLD18 on KATO-III and NUGC-4 cells as analyzed by staining of cells with monoclonal antibodies 61C2 and 163E12 followed by flow cytometrical analysis.

Fig. 14. Protein-alignment of human CLD18A1 (NP_057453), human CLD18A2 (NP_001002026), mouse CLD18A1 (NP_062789) and mouse CLD18A2 (AAL15636).

Fig. 15 A and B show binding of hybridoma supernatants 38G5, 38H3, 37G11,

0 45C1, and 163E12, respectively, to HEK293 cells transiently transfected with a fluorescent marker and either murine CLD18A1 or murine CLD18A2 as analyzed by flow cytometry.

Fig. 16. Immunhistochemical analyses with polyclonal AB pi 05.

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Immunhistochemical stainings on a subset of normal tissues (stomach, lung, bone marrow and prostate) confirm gastric tissue specificity (A). Expression was also detected in stomach carcinomas (upper row) and lung carcinomas (B). Only differentiated cells but not stem cells do express CLD18A2 (C).

Fig. 17. Immunhistochemical analyses with monoclonal AB 39F11D7 (A) Specific protein expression was detected in normal stomach mucosa, whereas all other tested normal tissue were negative.

(B) Strong CLD18A2 expression was found in stomach and lung carcinomas.

Fig. 18. Immunhistochemical analyses with monoclonal AB 26B5 (A), 175D10 (B), 43 A11 (C), 163E12 (D), and 45C1 (E). All antibodies show strong staining of HEK293-CLD18A2 xenograft tumors and gastric cancer specimens, but not HEK293-Mock control-transfected tumors.

Fig. 19 is a graph comparing the percentage of dead cells after induction of CDC by 85A3, 28D10, 24H5, or 26D12 against HEK293 cells stably transfected with human CLD18A2 using flow cytometry.

0 Fig. 20 is a graph comparing the percentage of specific cell lysis after induction of

CDC by 24H5, 26D12, 28D10, 37G11, 37H8, 38G5, 38H3, 39F11, 41C6, 42E12, 43A11, 44E10, 47D12, or 61C2 against adherent CHO cells stably transfected with either human CLD18A2 or human CLD18A1 as determined by fluorescence measurement.

Fig. 21 shows concentration-dependent induction of CDC against CHO cells stably transfected with human CLD18A2 by 75B8 (A), 28D10 (B), or 37H8 (C) as determined by fluorescence measurement.

0 Fig. 22 shows lysis of HEK293-CLD18A2 cells by 26B5, 37H8, 38G5, 47D12, and 61C2, respectively, in the presence of MNCs.

Fig. 23 shows lysis of HEK293-CLD18A1 cells by 26B5, 37H8, 38G5, 47D12, and 61C2, respectively, in the presence of MNCs.

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Fig. 24 shows tumor growth inhibition by antibodies of the invention in an early treatment xenograft model with HEK293-CLD18A2 cells.

Fig. 25A and B show prolonged survival by treatment with antibodies of the invention in two early treatment xenograft models with HEK293-CLD18A2 cells.

Fig. 26 shows prolongation of survival by antibodies of the invention in an advanced treatment xenograft model with HEK293-CLD18A2 cells.

Fig. 27A shows tumor growth inhibition by antibodies of the invention in an early treatment xenograft model. Fig. 27B shows prolongation of survival by antibodies of the invention in an early treatment xenograft model. Endogenously CLD18A2 expressing DAN-G cells were used.

Fig. 28 shows CLD18A2 mRNA expression in mouse tissues. RT-PCR investigations with CLD18A2-specfic primers showed no significant expression within all tested normal tissues except stomach. The following normal tissues were analysed: 1: small intestine, 2: spleen, 3: skin, 4: stomach, 5: lung, 6: pancreas, 7:

0 lymph node, 8: thymus, 9: negative control

Fig. 29 shows CLD18 expression in normal stomach. Immunohistochemical analysis with CLD18 specific antibody of mouse stomach reveals conserved expression pattern. While the surface epithelia and deeper crypts express CLD18 in their cell surface, the central neck region is CLD18 negative.

Fig. 30 shows haematoxylin and eosin staining of mice stomach tissues. Shown is in overview (A) and in detail (B) the stomach of a 37G11 -treated mouse in comparison to a control mouse (C and D), which was treated with PBS only.

Fig 31A and B show flowcytometric staining of HEK293 cells stably transfected with human CLD18A1 and A2, respectively, as well as endogenously expressing KATO-III cells with antibodies of the invention (43A11, 125E1, 163E12, 166E2, and 175D10).

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Fig. 32 shows CDC on CLD18A2 expressing cells mediated by chimeric antibodies of the invention.

Fig. 33 shows ADCC on KATO-III cells mediated by chimeric antibodies of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The antibodies described herein may be isolated monoclonal antibodies which specifically bind to an epitope present on CLD18. Isolated monoclonal antibodies encompassed by the present invention include IgA, IgG 1-4, IgE, IgM, and IgD antibodies. In one embodiment the antibody is an IgGi antibody, more particularly an IgGi, kappa or IgGi, lambda isotype. In another embodiment the antibody is an

IgG3 antibody, more particularly an IgG3, kappa or IgG3, lambda isotype. In yet another embodiment the antibody is an IgG4 antibody, more particularly an IgG4, kappa or IgG4, lambda isotype. In still another embodiment the antibody is an IgAl or IgA2 antibody. In still another embodiment the antibody is an IgM antibody.

In one embodiment the invention relates to antibodies which specifically bind to cells expressing CLD18, and preferably (i) bind to cells expressing CLD18A2, and (ii) do not bind to cells not expressing CLD18A2 but expressing CLD18A1. The antibodies of the invention preferably (i) mediate killing of cells expressing

5 CLD18A2, and (ii) do not mediate killing of cells not expressing CLD18A2 but expressing CLD18A1.

In another embodiment, the invention relates to antibodies which (i) bind to tumor cells expressing CLD18, (ii) do not bind to CLD18 expressing cells of normal

0 stomach mucosa, and/or (iii) do not bind to CLD18 expressing cells of non-cancer lung tissue.

The invention also includes antibodies which (i) mediate killing of tumor cells expressing CLD18, (ii) do not mediate killing of CLD18 expressing cells of

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2018200685 30 Jan 2018 normal stomach mucosa, and/or (iii) do not mediate killing of CLD18 expressing cells of non-cancer lung tissue.

In particular embodiments, the antibodies of the invention (i) bind to an epitope on 5 CLD18A2 which is not present on CLD18A1, preferably SEQ ID NO: 21, 22, and

23, (ii) bind to an epitope localized on the CLD18A2-loopl, preferably SEQ ID NO: 28, (iii) bind to an epitope localized on the CLD18A2-loop2, preferably SEQ ID NO: 30, (iv) bind to an epitope localized on the CLD18A2-loopD3, preferably SEQ ID NO: 31, (v) bind to an epitope, which encompass CLD18A2-loopl and

CLD18A2-loopD3, (vi) bind to a non-glycosylated epitope localized on the CLD18A2-loopD3, preferably SEQ ID NO: 29, or (vii) bind to an epitope present in human and mouse CLD18 (SEQ ID NO: 2, SEQ ID NO: 8 and SEQ ID NO: 35, SEQ ID NO: 37, respectively).

In particularly preferred embodiments, the antibodies of the invention bind to an epitope on CLD18A2 which is not present on CLD18A1.

Antibodies of the invention include fully human antibodies. Such antibodies may be produced in a non-human transgenic animal, e.g., a transgenic mouse, capable

0 of producing multiple isotypes of human monoclonal antibodies to CLD18 by undergoing V-D-J recombination and isotype switching. Such transgenic animal can also be a transgenic rabbit for producing polyclonal antibodies such as disclosed in US 2003/0017534.

Binding of an antibody of the invention to the CLD18 antigen may mediate the killing of cells expressing CLD18 (e.g. a tumor cell), e.g. by activation of the complement system. The killing of cells expressing CLD18 may occur by one or more of the following mechanisms: complement dependent cytotoxity (CDC) of cells expressing CLD18; apoptosis of cells expressing CLD18; effector cell

0 phagocytosis of cells expressing CLD18; or effector cell antibody dependent cellular cytotoxicity (ADCC) of cells expressing CLD18.

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In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

DEFINITION OF TERMS

The term CLD18 relates to claudin-18 and includes any variants, including CLD18A1 and CLD18A2, conformations, isoforms and species homologs of CLD18 which are naturally expressed by cells or are expressed by cells transfected with the CLD18 gene. Preferably, CLD18 relates to human CLD18, in particular CLD18A2 (SEQ ID NOs: 1, 2) and/or CLD18A1 (SEQ ID NOs: 7, 8), more preferably CLD18A2.

The term CLD18A1 includes posttranslationally modified variants, isoforms and species homologs of human CLD18A1 which are naturally expressed by cells or are expressed on cells transfected with the CLD18A1 gene.

The term CLD18A2 includes posttranslationally modified variants, isoforms and species homologs of human CLD18A2 which are naturally expressed by cells or

0 are expressed on cells transfected with the CLD18A2 gene.

The term CLD18 variant shall encompass (i) CLD18 splice variants, (ii) CLD18posttranslationally modified variants, particularly including variants with different N-glycosylation status, (iii) CLD18 conformation variants, particularly including

5 CLD18-conformation-l, CLD18-conformation-2 and CLD18-conformation-3, (iv)

CLD18 free and homotypically/heterotypically associated variants localized at intercellular tight junctions, (v) CLD18 cancer related and CLD18 non-cancer related variants.

0 The term raft refers to the sphingolipid- and cholesterol-rich membrane microdomains located in the outer leaflet area of the plasma membrane of a cell. The ability of certain proteins to associate within such domains and their abbility of forming “aggregates” or “focal aggregates” can effect the protein's function. For example, the translocation of CLD18 molecules into such structures, after being

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2018200685 30 Jan 2018 bound by antibodies of the present invention, creates a high density of CLD18 antigen-antibody complexes in the plasma membranes. Such a high density of

CLD18 antigen-antibody complexes can enable efficient activation of the complement system during CDC.

The terms “conformation” and “topology” describe how an integrale membrane molecule is positioned in the cell surface membrane, and, in particular, which of its regions are extracellular and thus eligible for antibodies. CLD18 for example can exist in three different conformations, which most likely depend on whether it is prevalent as homomers or heteromers and whether it is integrated in supramolecular tight junction structures or “free”. These different states result in different epitopes eligible to antibodies.

According to the invention, the term “disease” refers to any pathological state, including cancer, in particular those forms of cancer described herein.

By tumor is meant an abnormal group of cells or tissue that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease. Tumors show partial or complete lack of structural

0 organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue, which may be either benign or malignant.

By metastasis is meant the spread of cancer cells from its original site to another part of the body. The formation of metastasis is a very complex process and

5 depends on detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membranes to enter the body cavity and vessels, and then, after being transported by the blood, infiltration of target organs. Finally, the growth of a new tumor at the target site depends on angiogenesis. Tumor metastasis often occurs even after the removal of

0 the primary tumor because tumor cells or components may remain and develop metastatic potential. In one embodiment, the term “metastasis” according to the invention relates to “distant metastasis” which relates to a metastasis which is remote from the primary tumor and the regional lymph node system.

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The term “treatment of a disease” includes curing, shortening the duration, ameliorating, preventing, slowing down or inhibiting progression or worsening, or preventing or delaying the onset of a disease or the symptoms thereof.

According to the invention, a sample may be any sample useful according to the present invention, in particular a biological sample such a tissue sample, including bodily fluids, and/or a cellular sample and may be obtained in the conventional manner such as by tissue biopsy, including punch biopsy, and by taking blood, bronchial aspirate, sputum, urine, feces or other body fluids. According to the invention, the term “biological sample” also includes fractions of biological samples.

The term antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. The term antibody also includes all recombinant forms of antibodies, in particular of the antibodies described herein, e.g., antibodies expressed in prokaryotes, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives as described below. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a

0 heavy chain constant region. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune

0 system (e.g., effector cells) and the first component (Clq) of the classical complement system.

The term “humanized antibody” refers to a molecule having an antigen binding site that is substantially derived from an immunoglobulin from a non-human

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2018200685 30 Jan 2018 species, wherein the remaining immunoglobulin structure of the molecule is based upon the structure and/or sequence of a human immunoglobulin. The antigen binding site may either comprise complete variable domains fused onto constant domains or only the complementarity determining regions (CDR) grafted onto appropriate framework regions in the variable domains. Antigen binding sites may be wild-type or modified by one or more amino acid substitutions, e.g. modified to resemble human immunoglobulins more closely. Some forms of humanized antibodies preserve all CDR sequences (for example a humanized mouse antibody which contains all six CDRs from the mouse antibody). Other forms have one or more CDRs which are altered with respect to the original antibody.

The term “chimeric antibody” refers to those antibodies wherein one portion of each of the amino acid sequences of heavy and light chains is homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class, while the remaining segment of the chain is homologous to corresponding sequences in another. Typically the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals, while the constant portions are homologous to sequences of antibodies derived from another. One clear advantage to such

0 chimeric forms is that the variable region can conveniently be derived from presently known sources using readily available B-cells or hybridomas from nonhuman host organisms in combination with constant regions derived from, for example, human cell preparations. While the variable region has the advantage of ease of preparation and the specificity is not affected by the source, the constant region being human, is less likely to elicit an immune response from a human subject when the antibodies are injected than would the constant region from a non human source. However the definition is not limited to this particular example.

The term antigen-binding portion of an antibody (or simply binding portion),

0 as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigenbinding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term antigenbinding portion of an antibody include (i) Fab fragments, monovalent fragments

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2018200685 30 Jan 2018 consisting of the VL, VH, CL and CH domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting of the VH and CH domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody, (v) dAb fragments (Ward et al., (1989) Nature 341: 544-546), which consist of a VH domain; (vi) isolated complementarity determining regions (CDR), and (vii) combinations of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also intended to be encompassed within the term antigen-binding portion of an antibody. A further example is binding-domain immunoglobulin fusion proteins comprising (i) a binding domain polypeptide that is fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The binding domain

0 polypeptide can be a heavy chain variable region or a light chain variable region.

The binding-domain immunoglobulin fusion proteins are further disclosed in US 2003/0118592 and US 2003/0133939. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

The term epitope means a protein determinant capable of binding to an antibody, wherein the term binding herein preferably relates to a specific binding. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural

0 characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

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The term discontinuous epitope as used herein, means a conformational epitope on a protein antigen which is formed from at least two separate regions in the primary sequence of the protein.

The term bispecific molecule is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has two different binding specificities. For example, the molecule may bind to, or interact with (a) a cell surface antigen, and (b) an Fc receptor on the surface of an effector cell. The term multispecific molecule or heterospecific molecule is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has more than two different binding specificities. For example, the molecule may bind to, or interact with (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, and (c) at least one other component. Accordingly, the invention includes, but is not limited to, bispecific, trispecific, tetraspecific, and other multispecific molecules which are directed to CLD18, and to other targets, such as Fc receptors on effector cells. The term bispecific antibodies also includes diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby

0 forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g. , Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, R. J., et al. (1994) Structure 2: 11211123).

5 The invention also includes derivatives of the antibodies described herein. The term antibody derivatives refers to any modified form of an antibody, e.g., a conjugate of the antibody and another agent or antibody. As used herein, an antibody is derived from a particular germline sequence if the antibody is obtained from a system by immunizing an animal or by screening an

0 immunoglobulin gene library, and wherein the selected antibody is at least 90%, more preferably at least 95%, even more preferably at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, an antibody derived from a particular germline sequence will display no more than 10 amino acid differences, more

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2018200685 30 Jan 2018 preferably, no more than 5, or even more preferably, no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

As used herein, the term heteroantibodies refers to two or more antibodies, derivatives thereof, or antigen binding regions linked together, at least two of which have different specificities. These different specificities include a binding specificity for an Fc receptor on an effector cell, and a binding specificity for an antigen or epitope on a target cell, e.g., a tumor cell.

The antibodies described herein may be human antibodies. The term human antibody, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

The term monoclonal antibody as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a

0 single binding specificity and affinity for a particular epitope. In one embodiment, the monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a non-human animal, e.g., mouse, fused to an immortalized cell.

The term recombinant antibody, as used herein, includes all antibodies that are

5 prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal with respect to the immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant,

0 combinatorial antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of immunoglobulin gene sequences to other DNA sequences.

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The term transfectoma, as used herein, includes recombinant eukaryotic host cells expressing an antibody, such as CHO cells, NS/0 cells, HEK293 cells,

HEK293T cells, plant cells, or fungi, including yeast cells.

As used herein, a heterologous antibody is defined in relation to a transgenic organism producing such an antibody. This term refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism not consisting of the transgenic organism, and being generally derived from a species other than the transgenic organism.

As used herein, a heterohybrid antibody refers to an antibody having light and heavy chains of different organismal origins. For example, an antibody having a human heavy chain associated with a murine light chain is a heterohybrid antibody.

The antibodies described herein are preferably isolated. An isolated antibody as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to CLD18 is substantially free of antibodies that specifically

0 bind antigens other than CLD18). An isolated antibody that specifically binds to an epitope, isoform or variant of human CLD18 may, however, have cross-reactivity to other related antigens, e.g., from other species (e.g., CLD18 species homologs). Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals. In one embodiment of the invention, a combination of isolated

5 monoclonal antibodies relates to antibodies having different specificities and being combined in a well defined composition.

According to the invention, the term “binding” preferably relates to “specific binding”. As used herein, specific binding refers to antibody binding to a

0 predetermined antigen. Typically, the antibody binds with an affinity corresponding to a KD of about 1 x 1 O'⁷ M or less, and binds to the predetermined antigen with an affinity corresponding to a KD that is at least two orders of magnitude lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen.

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The term KD (M), as used herein, is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction.

As used herein, isotype refers to the antibody class (e.g., IgM or IgGl) that is encoded by heavy chain constant region genes.

As used herein, isotype switching refers to the phenomenon by which the class, or isotype, of an antibody changes from one Ig class to one of the other Ig classes.

The term naturally occurring as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.

The term rearranged as used herein refers to a configuration of a heavy chain or light chain immunoglobulin locus wherein a V segment is positioned immediately adjacent to a D-J or J segment in a conformation encoding essentially a complete

0 VH or VL domain, respectively. A rearranged immunoglobulin (antibody) gene locus can be identified by comparison to germline DNA; a rearranged locus will have at least one recombined heptamer/nonamer homology element.

The term unrearranged or germline configuration as used herein in reference to 25 a V segment refers to the configuration wherein the V segment is not recombined so as to be immediately adjacent to a D or J segment.

The term nucleic acid molecule, as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or

0 double-stranded, but preferably is double-stranded DNA.

The nucleic acids described according to the invention have preferably been isolated. The term “isolated nucleic acid” means according to the invention that the nucleic acid was (i) amplified in vitro, for example by polymerase chain reaction

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2018200685 30 Jan 2018 (PCR), (ii) recombinantly produced by cloning, (iii) purified, for example by cleavage and gel-electrophoretic fractionation, or (iv) synthesized, for example by chemical synthesis. An isolated nucleic acid is a nucleic acid which is available for manipulation by recombinant DNA techniques.

Nucleic acids may, according to the invention, be present alone or in combination with other nucleic acids, which may be homologous or heterologous. In preferred embodiments, a nucleic acid is functionally linked to expression control sequences which may be homologous or heterologous with respect to said nucleic acid. The term “homologous” means that a nucleic acid is also functionally linked to the expression control sequence naturally and the term “heterologous” means that a nucleic acid is not functionally linked to the expression control sequence naturally.

A nucleic acid, such as a nucleic acid expressing RNA and/or protein or peptide, and an expression control sequence are “functionally” linked to one another, if they are covalently linked to one another in such a way that expression or transcription of said nucleic acid is under the control or under the influence of said expression control sequence. If the nucleic acid is to be translated into a functional protein, then, with an expression control sequence functionally linked to a coding

0 sequence, induction of said expression control sequence results in transcription of said nucleic acid, without causing a frame shift in the coding sequence or said coding sequence not being capable of being translated into the desired protein or peptide.

5 The term “expression control sequence” comprises according to the invention promoters, ribosome binding sites, enhancers and other control elements which regulate transcription of a gene or translation of a mRNA. In particular embodiments of the invention, the expression control sequences can be regulated. The exact structure of expression control sequences may vary as a function of the

0 species or cell type, but generally comprises 5’-untranscribed and 5’- and 3’untranslated sequences which are involved in initiation of transcription and translation, respectively, such as TATA box, capping sequence, CAAT sequence, and the like. More specifically, 5’-untranscribed expression control sequences comprise a promoter region which includes a promoter sequence for transcriptional

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2018200685 30 Jan 2018 control of the functionally linked nucleic acid. Expression control sequences may also comprise enhancer sequences or upstream activator sequences.

According to the invention the term “promoter” or “promoter region” relates to a 5 nucleic acid sequence which is located upstream (5’) to the nucleic acid sequence being expressed and controls expression of the sequence by providing a recognition and binding site for RNA-polymerase. The “promoter region” may include further recognition and binding sites for further factors which are involved in the regulation of transcription of a gene. A promoter may control the transcription of a prokaryotic or eukaryotic gene. Furthermore, a promoter may be “inducible” and may initiate transcription in response to an inducing agent or may be “constitutive” if transcription is not controlled by an inducing agent. A gene which is under the control of an inducible promoter is not expressed or only expressed to a small extent if an inducing agent is absent. In the presence of the inducing agent the gene is switched on or the level of transcription is increased. This is mediated, in general, by binding of a specific transcription factor.

Promoters which are preferred according to the invention include promoters for SP6, T3 and T7 polymerase, human U6 RNA promoter, CMV promoter, and

0 artificial hybrid promoters thereof (e.g. CMV) where a part or parts are fused to a part or parts of promoters of genes of other cellular proteins such as e.g. human GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and including or not including (an) additional intron(s).

5 According to the invention, the term “expression” is used in its most general meaning and comprises the production of RNA or of RNA and protein/peptide. It also comprises partial expression of nucleic acids. Furthermore, expression may be carried out transiently or stably.

0 In a preferred embodiment, a nucleic acid molecule is according to the invention present in a vector, where appropriate with a promoter, which controls expression of the nucleic acid. The term “vector” is used here in its most general meaning and comprises any intermediary vehicle for a nucleic acid which enables said nucleic acid, for example, to be introduced into prokaryotic and/or eukaryotic cells and,

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2018200685 30 Jan 2018 where appropriate, to be integrated into a genome. Vectors of this kind are preferably replicated and/or expressed in the cells. Vectors comprise plasmids, phagemids, bacteriophages or viral genomes. The term “plasmid” as used herein generally relates to a construct of extrachromosomal genetic material, usually a circular DNA duplex, which can replicate independently of chromosomal DNA.

As the vector for expression of an antibody, either of a vector type in which the antibody heavy chain and light chain are present in different vectors or a vector type in which the heavy chain and light chain are present in the same vector can be used.

The teaching given herein with respect to specific nucleic acid and amino acid sequences, e.g. those shown in the sequence listing, is to be construed so as to also relate to modifications of said specific sequences resulting in sequences which are functionally equivalent to said specific sequences, e.g. amino acid sequences exhibiting properties identical or similar to those of the specific amino acid sequences and nucleic acid sequences encoding amino acid sequences exhibiting properties identical or similar to those of the amino acid sequences encoded by the specific nucleic acid sequences. One important property is to retain binding of an

0 antibody to its target or to sustain effector functions of an antibody. Preferably, a sequence modified with respect to a specific sequence, when it replaces the specific sequence in an antibody retains binding of said antibody to CLD18 and preferably functions of said antibody as described herein, e.g. CDC mediated lysis or ADCC mediated lysis.

It will be appreciated by those skilled in the art that in particular the sequences of the CDR, hypervariable and variable regions can be modified without losing the ability to bind CLD18. For example, CDR regions will be either identical or highly homologous to the regions specified herein. By highly homologous it is

0 contemplated that from 1 to 5, preferably from 1 to 4, such as 1 to 3 or 1 or 2 substitutions may be made in the CDRs. In addition, the hypervariable and variable regions may be modified so that they show substantial homology with the regions specifically disclosed herein.

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It is to be understood that the specific nucleic acids described herein also include nucleic acids modified for the sake of optimizing the codon usage in a particular host cell or organism. Differences in codon usage among organisms can lead to a variety of problems concerning heterologous gene expression. Codon optimization by changing one or more nucleotides of the original sequence can result in an optimization of the expression of a nucleic acid, in particular in optimization of translation efficacy, in a homologous or heterologous host in which said nucleic acid is to be expressed. For example if nucleic acids derived from human and encoding constant regions and/or framework regions of antibodies are to be used according to the present invention, e.g. for preparing chimeric or humanised antibodies, it may be preferred to modify said nucleic acids for the sake of optimization of codon usage, in particular if said nucleic acids, optionally fused to heterologous nucleic acids such as nucleic acids derived from other organisms as described herein, are to be expressed in cells from an organism different from human such as mouse or hamster. For example, the nucleic acid sequences encoding human light and heavy chain constant regions such as those according to SEQ ID NOs: 40 and 45, respectively, can be modified to include one or more, preferably, at least 1, 2, 3, 4, 5, 10, 15, 20 and preferably up to 10, 15, 20, 25, 30, 50, 70 or 100 or more nucleotide replacements resulting in an optimized codon

0 usage but not resulting in a change of the amino acid sequence. Such nucleotide replacements preferably relate to replacements of nucleotides in SEQ ID Nos: 40 and 45, respectively, selected from the replacements shown in the following alignment of SEQ ID Nos: 40 and 45, respectively, with their modified counterparts and not resulting in a change in the encoded amino acid sequence or

5 relate to corresponding replacements at corresponding positions in other nucleic acid sequences encoding human light and heavy chain constant regions, respectively. Preferably, all of the replacements shown in the following alignments of SEQ ID Nos: 40 and 45, respectively, with their modified counterparts not resulting in a change in the encoded amino acid sequence are effected in nucleic

0 acid sequences encoding human light and heavy chain constant regions, respectively.

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Alignment of SEQ ID NO: 40 and SEQ ID NO: 147:

CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT lllllllllll II II II lllllllllll II II mill nil II

CGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGTCC

GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAG ii ii iii ii iiiiiiiiiiiiii ilium hi i mum 11 hi

GGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAG

TGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGAC iiiiiiiim mum 11 11 in mmm iiiii mmm

TGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGAC

AGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAG is mmm mum mi iiiiiiiiiiiiii iiiiiiii 11 mmm

AGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAG

AAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAG ii mil ii mmmii 11 iiiii mmm i iiiii u iii

0 AAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAG

AGCTTCAACAGGGGAGAGTGTTAG 324

AGCTTCAACAGGGGCGAGTGCTAG 324 25

Alignment of SEQ ID NO: 45 and SEQ ID NO: 149:

GGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC mm ii mmmii ιιι i mmmi 11 iiiiiiii hi

GGCCCAAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCC

CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGC iiiiiiiiiiiiii mmmii mm u iiiii hi ihih n

CTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGA

GCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCC mmm mimmimim π u n hi i ii ii hi i

GCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGC

CTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAAC η 111111111111111111111 mi iii mmimmmmimm

CTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAAC

GTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGAC

IIIII 1111111111111111111111111111111 III IIIIIIII II III

5 GTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGAC

AAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTC

II II IIIII IIIII II IIIIIIII II II II IIIII IIIII II III

AAGACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTGGGCGGACCCAGCGTGTTC

CTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC ii iiiiiiii ii 11111111111111 mm i mini iiiii ii iii

CTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGC

5 GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGC

11111111111111111111111 iiiii iiiii mmmmmmmm

GTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGC

120

120

180

180

240

240

300

300

120

120

180

180

240

240

300

300

360

360

420

420

480

480

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GTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGT 540 lllllllllll II llllllll Hill I 11111111111111111111 III I

GTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGG 540

GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC 600

Hill III II Hill llllllllllllllllllll llllllll lllllllll

GTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGC 600

AAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGG 660

AAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGCCAAGGGC 660

CAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAAC 720

CAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCCGGGAGGAGATGACCAAGAAC 720

CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGG 780

Hill lllllllll IIIII II llllllll III111111111111111111111

0 CAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGG 780

GAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC 840 iiiiiiii π mu mmiimuuum u n iimiiii mi

GAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGAC 840 25

GGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAAC 900 iii iimiiii ii iiiiiiii imimiui mumiimu hi

GGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAAC 900

0 GTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC 960 π hi hi iimiiii iiiii πιπιιιιιιιπιπ mm hi

GTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACC ACTACACCCAGAAGTCCCTG 960

TCCCTGTCTCCGGGTAAATGA 981 mi || II II I

AGCCTGAGCCCCGGCAAGTAG 981

Furthermore, it may be desired according to the present invention to modify the amino acid sequences described herein, in particular those of human heavy chain

0 constant regions to adapt the sequence to a desired allotype, e.g. an allotype found in the Caucasian population. Such modifications are preferably selected from the group consisting of the following amino acid replacments within SEQ ID NO: 46 or at corresponding positions within other human heavy chain constant regions: K93R, D235E, and L237M. Preferably, all of these modifications are included in

5 amino acid sequences of human heavy chain constant regions.

According the invention, the term “corresponding positions” relates to nucleotides or amino acid residues which in a sequence alignment of two nucleic acid or protein sequences are aligned to each other.

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Preferably the degree of identity between a specific nucleic acid sequence described herein and a nucleic acid sequence which is modified with respect to said specific nucleic acid sequence will be at least 70%, preferably at least 75%, more preferably at least 80%, even more preferably at least 90% or most preferably at least 95%, 96%, 97%, 98% or 99%. Preferably, the two sequences are capable of hybridizing and forming a stable duplex with one another, with hybridization preferably being carried out under conditions which allow specific hybridization between polynucleotides (stringent conditions). Stringent conditions are described, for example, in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Editors, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989 or Current Protocols in Molecular Biology, F.M. Ausubel et al., Editors, John Wiley & Sons, Inc., New York and refer, for example, to hybridization at 65°C in hybridization buffer (3.5 x SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH2PO4 (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride/0.15 M sodium citrate, pH 7. After hybridization, the membrane to which the DNA has been transferred is washed, for example, in 2 χ SSC at room temperature and then in 0.1-0.5 χ SSC/0.1 χ SDS at temperatures of up to 68°C.

Preferably the degree of similarity, preferably identity between a specific amino acid sequence described herein and an amino acid sequence which is modified with respect to said specific amino acid sequence such as between amino acid sequences showing substantial homology will be at least 70%, preferably at least

80%, even more preferably at least 90% or most preferably at least 95%, 96%,

97%, 98% or 99%.

All of the above described modified sequences are within the scope of the present invention.

Sequence similarity indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions. Sequence identity between two polypeptide or nucleic acid sequences indicates the percentage of amino acids or nucleotides that are identical between the sequences.

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The “percentage identity” is obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length. Sequence comparisons between two nucleotide or amino acid sequences are conventionally carried out by comparing these sequences after having aligned them optimally, said comparison being carried out by segment or by “window of comparison” in order to identify and compare local regions of sequence similarity. The optimal alignment of the sequences for comparison may be produced, besides manually, by means of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by means of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by means of the similarity search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444, or by means of computer programs which use these algorithms (GAP, BESTFIT, FASTA, BLAST P,

BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

The percentage identity is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the

0 number of positions compared and multiplying the result obtained by 100 so as to obtain the percentage identity between these two sequences.

Conservative substitutions, may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example: (a) nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; (b) polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positively charged (basic) amino acids include arginine, lysine, and histidine; and

0 (d) negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitutions typically may be made within groups (a)-(d). In addition, glycine and proline may be substituted for one another based on their ability to disrupt [alpha]-helices. Some preferred substitutions may be made among the following groups: (i) S and T; (ii) P and G; and (iii) A, V, L and I. Given the

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PCT/EP2006/011302 known genetic code, and recombinant and synthetic DNA techniques, the skilled scientist readily can construct DNAs encoding the conservative amino acid variants.

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The present invention comprises antibodies in which alterations have been made in the Fc region in order to change the functional or pharmacokinetic properties of the antibodies. Such alterations may result in a decrease or increase of Clq binding and CDC or of FcyR binding and ADCC. Substitutions can, for example, be made in one or more of the amino acid residues of the heavy chain constant region, thereby causing an alteration in an effector function while retaining the ability to bind to the antigen as compared with the modified antibody, cf. US 5,624,821 and US 5,648,260.

The in vivo half-life of antibodies can be improved by modifying the salvage receptor epitope of the Ig constant domain or an Ig-like constant domain such that the molecule does not comprise an intact CH2 domain or an intact Ig Fc region, cf. US 6,121,022 and US 6,194,551. The in vivo half-life can furthermore be increased by making mutations in the Fc region, e.g., by substituting threonine for leucine at position 252, by substituting threonine for serine at position 254, or by

0 substituting threonine for phenylalanine at position 256, cf. US 6,277,375.

Furthermore, the glycosylation pattern of antibodies can be modified in order to change the effector function of the antibodies. For example, the antibodies can be expressed in a transfectoma which does not add the fucose unit normally attached

5 to Asn at position 297 of the Fc region in order to enhance the affinity of the Fc region for Fc-Receptors which, in turn, will result in an increased ADCC of the antibodies in the presence of NK cells, cf. Shield et al. (2002) JBC, 277: 26733. Furthermore, modification of galactosylation can be made in order to modify CDC.

0 Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a anti-CLD18 antibody coding sequence, such as by saturation mutagenesis, and the resulting modified anti-CLD18 antibodies can be screened for binding activity.

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The term recombinant host cell (or simply host cell), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term host cell as used herein. Recombinant host cells include, for example, transfectomas, such as CHO cells, NS/0 cells, and lymphocytic cells.

As used herein, the term subject includes any human or non-human animal. The term non-human animal includes all vertebrates, e.g., mammals and nonmammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.

The terms transgenic animal refers to an animal having a genome comprising one or more transgenes, preferably heavy and/or light chain transgenes, or transchromosomes (either integrated or non-integrated into the animal's natural genomic DNA) and which is preferably capable of expressing the transgenes. For

0 example, a transgenic mouse can have a human light chain transgene and either a human heavy chain transgene or human heavy chain transchromosome, such that the mouse produces human anti-CLD18 antibodies when immunized with CLD18 antigen and/or cells expressing CLD18. The human heavy chain transgene can be integrated into the chromosomal DNA of the mouse, as is the case for transgenic mice, e.g., HuMAb mice, such as HCo7 or HCol2 mice, or the human heavy chain transgene can be maintained extrachromosomally, as is the case for transchromosomal (e.g., KM) mice as described in WO 02/43478. Such transgenic and transchromosomal mice may be capable of producing multiple isotypes of human monoclonal antibodies to CLD18 (e.g., IgG, IgA and/or IgE) by

0 undergoing V-D-J recombination and isotype switching.

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Mechanisms of mAb action

Although the following provides considerations regarding the mechanism underlying the therapeutic efficacy of antibodies of the invention it is not to be considered as limiting to the invention in any way.

The antibodies described herein preferably interact with components of the immune system, preferably through ADCC or CDC. Antibodies of the invention can also be used to target payloads (e.g., radioisotopes, drugs or toxins) to directly kill tumor cells or can be used synergistically with traditional chemotherapeutic agents, attacking tumors through complementary mechanisms of action that may include anti-tumor immune responses that may have been compromised owing to a chemotherapeutic's cytotoxic side effects on T lymphocytes.

Antibody-dependent cell-mediated cytotoxicity. ADCC describes the cell-killing ability of effector cells as described herein, in particular lymphocytes, which preferably requires the target cell being marked by an antibody.

ADCC preferably occurs when antibodies bind to antigens on tumor cells and the antibody Fc domains engage Fc receptors (FcR) on the surface of immune effector

0 cells. Several families of Fc receptors have been identified, and specific cell populations characteristically express defined Fc receptors. ADCC can be viewed as a mechanism to directly induce a variable degree of immediate tumor destruction that leads to antigen presentation and the induction of tumor-directed T-cell responses. Preferably, in vivo induction of ADCC will lead to tumor2 5 directed T-cell responses and host-derived antibody responses.

Complement-dependent cytotoxicity. CDC is another cell-killing method that can be directed by antibodies. IgM is the most effective isotype for complement activation. IgGl and IgG3 are also both very effective at directing CDC via the

0 classical complement-activation pathway. Preferably, in this cascade, the formation of antigen-antibody complexes results in the uncloaking of multiple Clq binding sites in close proximity on the Ch2 domains of participating antibody molecules such as IgG molecules (Clq is one of three subcomponents of complement Cl). Preferably these uncloaked Clq binding sites convert the

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2018200685 30 Jan 2018 previously low-affinity Clq-IgG interaction to one of high avidity, which triggers a cascade of events involving a series of other complement proteins and leads to the proteolytic release of the effector-cell chemotactic/activating agents C3a and

C5a. Preferably, the complement cascade ends in the formation of a membrane attack complex, which creates pores in the cell membrane that facilitate free passage of water and solutes into and out of the cell.

Production of antibodies

Antibodies of the invention can be produced by a variety of techniques, including 10 conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibodies can be employed, e.g., viral or oncogenic transformation of B-lymphocytes or phage display techniques using libraries of antibody genes.

The preferred animal system for preparing hybridomas that secrete monoclonal antibodies is the murine system. Hybridoma production in the mouse is a very well established procedure. Immunization protocols and techniques for isolation of

0 immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

Other preferred animal systems for preparing hybridomas that secrete monoclonal antibodies are the rat and the rabbit system (e.g. described in Spieker-Polet et al.,

5 Proc. Natl. Acad. Sci. U.S.A. 92:9348 (1995), see also Rossi et al., Am. J. Clin.

Pathol. 124: 295 (2005)).

In yet another preferred embodiment, human monoclonal antibodies directed against CLD18 can be generated using transgenic or transchromosomal mice

0 carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomic mice include mice known as HuMAb mice and KM mice, respectively, and are collectively referred to herein as transgenic mice. The production of human antibodies in such transgenic mice can be performed as described in detail for CD20 in W02004 035607

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Yet another strategy for generating monoclonal antibodies is to directly isolate genes encoding antibodies from lymphocytes producing antibodies of defined strategy e.g. see Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined strategy. For details of recombinant antibody engineering see also Welschof and Kraus, Recombinant antibodes for cancer therapy ISBN-0-89603-918-8 and Benny K.C. Lo Antibody Engineering ISBN 1-58829-092-1.

Immunizations

To generate antibodies to CLD18, mice can be immunized with carrier-conjugated peptides derived from the CLD18 sequence, an enriched preparation of recombinantly expressed CLD18 antigen or fragments thereof and/or cells expressing CLD18, as described. Alternatively, mice can be immunized with DNA encoding full length human CLD18 (e.g. SEQ ID NO: 1) or fragments thereof, in particular those of SEQ ID Nos: 15, 17, and 19. In the event that immunizations using a purified or enriched preparation of the CLD18 antigen do not result in antibodies, mice can also be immunized with cells expressing CLD18, e.g., a cell line, to promote immune responses.

The immune response can be monitored over the course of the immunization protocol with plasma and serum samples being obtained by tail vein or retroorbital bleeds. Mice with sufficient titers of anti-CLD18 immunoglobulin can be used for fusions. Mice can be boosted intraperitonealy or intravenously with CLD18

5 expressing cells 3 days before sacrifice and removal of the spleen to increase the rate of specific antibody secreting hybridomas.

Generation of Hybridomas Producing Monoclonal Antibodies

To generate hybridomas producing monoclonal antibodies to CLD18, splenocytes

0 and lymph node cells from immunized mice can be isolated and fused to an appropriate immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can then be screened for the production of antigen-specific antibodies. Individual wells can then be screened by ELISA for antibody secreting hybridomas. By Immunofluorescence and FACS analysis using CLD18 expressing

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2018200685 30 Jan 2018 cells, antibodies with specificity for CLD18 can be identified. The antibody secreting hybridomas can be replated, screened again, and if still positive for antiCLD18 monoclonal antibodies can be subcloned by limiting dilution. The stable subclones can then be cultured in vitro to generate antibody in tissue culture medium for characterization.

Generation of Transfectomas Producing Monoclonal Antibodies

Antibodies of the invention also can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as are well known in the art (Morrison, S. (1985) Science 229: 1202).

For example, in one embodiment, the gene(s) of interest, e.g., antibody genes, can be ligated into an expression vector such as a eukaryotic expression plasmid such as used by the GS gene expression system disclosed in WO 87/04462, WO

89/01036 and EP 338 841 or other expression systems well known in the art. The purified plasmid with the cloned antibody genes can be introduced in eukaryotic host cells such as CHO cells, NS/0 cells, HEK293T cells or HEK293 cells or alternatively other eukaryotic cells like plant derived cells, fungal or yeast cells. The method used to introduce these genes can be methods described in the art such as electroporation, lipofectine, lipofectamine or others. After introduction of these antibody genes in the host cells, cells expressing the antibody can be identified and selected. These cells represent the transfectomas which can then be amplified for their expression level and upscaled to produce antibodies. Recombinant antibodies can be isolated and purified from these culture supernatants and/or cells.

5 Alternatively, the cloned antibody genes can be expressed in other expression systems, including prokaryotic cells, such as microorganisms, e.g. E. coli. Furthermore, the antibodies can be produced in transgenic non-human animals, such as in milk from sheep and rabbits or in eggs from hens, or in transgenic plants; see e.g. Verma, R., et al. (1998) J. Immunol. Meth. 216: 165-181; Pollock, et al. (1999) J. Immunol. Meth. 231: 147-157; and Fischer, R., et al. (1999) Biol.

Chem. 380: 825-839.

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Use of Partial Antibody Sequences to Express Intact Antibodies (i.e.

humanization and chimerisation).

a) Chimerization

Murine monoclonal antibodies can be used as therapeutic antibodies in humans 5 when labeled with toxins or radioactive isotopes. Nonlabeled murine antibodies are highly immunogenic in man when repetitively applied leading to reduction of the therapeutic effect. The main immunogenicity is mediated by the heavy chain constant regions. The immunogenicity of murine antibodies in man can be reduced or completely avoided if respective antibodies are chimerized or humanized.

Chimeric antibodies are antibodies, the different portions of which are derived from different animal species, such as those having a variable region derived from a murine antibody and a human immunoglobulin constant region. Chimerisation of antibodies is achieved by joining of the variable regions of the murine antibody heavy and light chain with the constant region of human heavy and light chain (e.g. as described by Kraus et al., in Methods in Molecular Biology series,

Recombinant antibodies for cancer therapy ISBN-0-89603-918-8). In a preferred embodiment chimeric antibodies are generated by joining human kappa-light chain constant region to murine light chain variable region. In an also preferred embodiment chimeric antibodies can be generated by joining human lambda-light

0 chain constant region to murine light chain variable region. The preferred heavy chain constant regions for generation of chimeric antibodies are IgGl, IgG3 and IgGA Other preferred heavy chain constant regions for generation of chimeric antibodies are IgG2, IgA, IgD and IgM.

5 b) Humanization

Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of

0 CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different

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2018200685 30 Jan 2018 properties (see, e.g., Riechmann, L. et al. (1998) Nature 332: 323-327; Jones, P. et al. (1986) Nature 321: 522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci.

U. S. A. 86: 10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they will not include completely assembled variable genes, which are formed by V (D) J joining during B cell maturation. Germline gene sequences will also differ from the sequences of a high affinity secondary repertoire antibody at individual evenly across the variable region. For example, somatic mutations are relatively infrequent in the amino terminal portion of framework region 1 and in the carboxyterminal portion of framework region 4. Furthermore, many somatic mutations do not significantly alter the binding properties of the antibody. For this reason, it is not necessary to obtain the entire DNA sequence of a particular antibody in order to recreate an intact recombinant antibody having binding properties similar to those of the original antibody (see WO 99/45962). Partial heavy and light chain sequences spanning the CDR regions are typically sufficient for this purpose. The partial sequence is used to determine which germline variable and joining gene segments contributed to the recombined antibody variable genes. The germline sequence is then used to fill in missing portions of the variable regions. Heavy and

0 light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody. To add missing sequences, cloned cDNA sequences can be combined with synthetic oligonucleotides by ligation or PCR amplification. Alternatively, the entire variable region can be synthesized as a set of short, overlapping, oligonucleotides and combined by PCR amplification to create an entirely synthetic variable region clone. This process has certain advantages such as elimination or inclusion or particular restriction sites, or optimization of particular codons.

The nucleotide sequences of heavy and light chain transcripts from hybridomas are

0 used to design an overlapping set of synthetic oligonucleotides to create synthetic

V sequences with identical amino acid coding capacities as the natural sequences. The synthetic heavy and kappa chain sequences can differ from the natural sequences in three ways: strings of repeated nucleotide bases are interrupted to facilitate oligonucleotide synthesis and PCR amplification; optimal translation

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2018200685 30 Jan 2018 initiation sites are incorporated according to Kozak's rules (Kozak, 1991, J. Biol.

Chem. 266: 19867-19870); and Hindlll sites are engineered upstream of the translation initiation sites.

For both the heavy and light chain variable regions, the optimized coding and corresponding non-coding, strand sequences are broken down into 30-50 nucleotides approximately at the midpoint of the corresponding non-coding oligonucleotide. Thus, for each chain, the oligonucleotides can be assembled into overlapping double stranded sets that span segments of 150-400 nucleotides. The pools are then used as templates to produce PCR amplification products of 150400 nucleotides. Typically, a single variable region oligonucleotide set will be broken down into two pools which are separately amplified to generate two overlapping PCR products. These overlapping products are then combined by PCR amplification to form the complete variable region. It may also be desirable to include an overlapping fragment of the heavy or light chain constant region in the PCR amplification to generate fragments that can easily be cloned into the expression vector constructs.

The reconstructed chimerized or humanized heavy and light chain variable regions

0 are then combined with cloned promoter, leader, translation initiation, constant region, 3' untranslated, polyadenylation, and transcription termination sequences to form expression vector constructs. The heavy and light chain expression constructs can be combined into a single vector, co-transfected, serially transfected, or separately transfected into host cells which are then fused to form a host cell

5 expressing both chains. Plasmids for use in construction of expression vectors for human IgGK are described below. The plasmids were constructed so that PCR amplified V heavy and V kappa light chain cDNA sequences could be used to reconstruct complete heavy and light chain minigenes. These plasmids can be used to express completely human, or chimeric IgGl, Kappa or IgG4, Kappa antibodies.

0 Similar plasmids can be constructed for expression of other heavy chain isotypes, or for expression of antibodies comprising lambda light chains.

Thus, in another aspect of the invention, the structural features of the anti-CLD18 antibodies of the invention, are used to create structurally related humanized anti59

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CLD18 antibodies that retain at least one functional property of the antibodies of the invention, such as binding to CLD18. More specifically, one or more CDR regions of mouse monoclonal antibodies can be combined recombinantly with known human framework regions and CDRs to create additional, recombinantly5 engineered, humanized anti-CLD 18 antibodies of the invention.

Binding to antigen expressing cells

The ability of the antibody to bind CLD18 can be determined using standard binding assays, such as those set forth in the examples (e.g., ELISA, Western Blot,

Immunofluorescence and flow cytometric analysis)

Characterization of binding of antibodies

To purify anti-CLD 18 antibodies, selected hybridomas can be grown in two-liter spinner-flasks for monoclonal antibody purification. Alternatively, anti-CLD 18 antibodies can be produced in dialysis based bioreactors. Supernatants can be filtered and, if necessary, concentrated before affinity chromatography with protein G-sepharose or protein A-sepharose. Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be

0 determined by OD280 using 1.43 extinction coefficient. The monoclonal antibodies can be aliquoted and stored at -80°C.

To determine if the selected anti-CLD 18 monoclonal antibodies bind to unique epitopes, site-directed or multi-site directed mutagenesis can be used.

5 Isotype determination

To determine the isotype of purified antibodies, isotype ELIS As with various commercial kits (e.g. Zymed, Roche Diagnostics) can be performed. Wells of microtiter plates can be coated with anti-mouse Ig. After blocking, the plates are reacted with monoclonal antibodies or purified isotype controls, at ambient

0 temperature for two hours. The wells can then be reacted with either mouse IgGl,

IgG2a, IgG2b or IgG3, IgA or mouse IgM-specific peroxidase-conjugated probes. After washing, the plates can be developed with ABTS substrate (1 mg/ml) and analyzed at OD of 405-650. Alternatively, the IsoStrip Mouse Monoclonal

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Antibody Isotyping Kit (Roche, Cat. No. 1493027) may be used as described by the manufacturer.

Flow cytometric analysis

In order to demonstrate presence of anti-CLD18 antibodies in sera of immunized mice or binding of monoclonal antibodies to living cells expressing CLD18, flow cytometry can be used. Cell lines expressing naturally or after transfection CLD18 and negative controls lacking CLD18 expression (grown under standard growth conditions) can be mixed with various concentrations of monoclonal antibodies in hybridoma supernatants or in PBS containing 1% FBS, and can be incubated at 4°C for 30 min. After washing, the APC- or Alexa647-labeled anti IgG antibody can bind to CLD18-bound monoclonal antibody under the same conditions as the primary antibody staining. The samples can be analyzed by flow cytometry with a FACS instrument using light and side scatter properties to gate on single, living cells. In order to distinguish CLD18-specific monoclonal antibodies from nonspecific binders in a single measurement, the method of co-transfection can be employed. Cells transiently transfected with plasmids encoding CLD18 and a fluorescent marker can be stained as described above. Transfected cells can be detected in a different fluorescence channel than antibody-stained cells. As the

0 majority of transfected cells express both transgenes, CLD18-specific monoclonal antbodies bind preferentially to fluorescence marker expressing cells, whereas non-specific antibodies bind in a comparable ratio to non-transfected cells. An alternative assay using fluorescence microscopy may be used in addition to or instead of the flow cytometry assay. Cells can be stained exactly as described

5 above and examined by fluorescence microscopy.

Tight junction proteins tend to be internalized, if cell cell contact to neighbouring cells of particularly adherent cells is lost by e.g. detachment of cells. Cell surface expression of CLD18 can be optimized by a) adjusting culture conditions, e.g.

0 culturing in higher cell densitiy in a standardized manner, using mild detachment (e.g. 2mM EDTA/PBS or accutase), processing at room temperature, and adding inhibitors of endocytosis (e.g. sodium azide) or activators of CLD18 transcription or translation, and by b) selecting and cloning of cells maintaining CLD18 in high

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2018200685 30 Jan 2018 levels at the cell surface, e.g. by selection with antibiotics in terms of transfected cells, by immunomagnetic or FACS cell sorting, and by limited dilution cloning.

Immunofluorescence microscopy

In order to demonstrate presence of anti-CLD18 antibodies in sera of immunized mice or binding of monoclonal antibodies to living cells expressing CLD18, immunofluorescence microscopy analysis can be used. For example, cell lines expressing either spontaneously or after transfection CLD18 and negative controls lacking CLD18 expression are grown in chamber slides under standard growth conditions in DMEM/F12 medium, supplemented with 10 % fetal calf serum (FCS), 2 mM L-glutamine, 100 IU/ml penicillin and 100 pg/ml streptomycin. Cells can then be fixed with methanol or paraformaldehyde or left untreated. Cells can then be reacted with monoclonal antibodies against CLD18 for 30 min. at 25°C. After washing, cells can be reacted with an Alexa555-labelled anti-mouse IgG secondary antibody (Molecular Probes) under the same conditions. Cells can then be examined by fluorescence microscopy.

Total CLD18 levels in cells can be observed when cells are methanol fixed or paraformaldehyde fixed and permeabilized with Triton X-100. In living cells and

0 non-permeabilized, paraformaldehyde fixed cells surface localization of CLD18 can be examined. Additionally targeting of CLD18 to tight junctions can be analyzed by co-staining with tight junction markers such as ZO-1. Furthermore, effects of antibody binding and CLD18 localization within the cell membrane can be examined.

Western Blot

Anti-CLD18 IgG can be further tested for reactivity with CLD18 antigen by Western Blotting. Briefly, cell extracts from cells expressing CLD18 and appropriate negative controls can be prepared and subjected to sodium dodecyl

0 sulfate (SDS) polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens will be transferred to nitrocellulose membranes, blocked, and probed with the monoclonal antibodies to be tested. IgG binding can be detected using anti-mouse IgG peroxidase and developed with ECL substrate.

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Immunohistochemistry

Anti-CLD18 mouse IgGs can be further tested for reactivity with CLDI8 antigen by Immunohistochemistry in a manner well known to the skilled person, e.g. using paraformaldehyde or acetone fixed cryosections or paraffin embedded tissue sections fixed with paraformaldehyde from non-cancer tissue or cancer tissue samples obtained from patients during routine surgical procedures or from mice carrying xenografited tumors inoculated with cell lines expressing spontaneously (e.g. DAN-G, SNU-16, or KATO-III) or after transfection (e.g. HEK293) CLD18. For immunostaining antibodies reactive to CLDI8 can be incubated followed by horseradish-peroxidase conjugated goat anti-mouse or goat anti-rabbit antibodies (DAKO) according to the vendors instructions.

Phagocytic and Cell Killing Activities of Antibodies in vitro

In addition to binding specifically to CLDI8, anti-CLD18 antibodies can be tested 15 for their ability to mediate phagocytosis and killing of cells expressing CLDI8.

The testing of monoclonal antibody activity in vitro will provide an initial screening prior to testing in vivo models.

Antibody dependent cell-mediated cytotoxicity (ADCC):

0 Briefly, polymorphonuclear cells (PMNs), NK cells, monocytes, mononuclear cells or other effector cells, from healthy donors can be purified by Ficoll Hypaque density centrifugation, followed by lysis of contaminating erythrocytes. Washed effector cells can be suspended in RPMI supplemented with 10% heat-inactivated fetal calf serum or, alternatively with 5% heat-inactivated human serum and mixed

5 with ⁵¹Cr labeled target cells expressing CLDI 8, at various ratios of effector cells to target cells. Alternatively, the target cells may be labeled with a fluorescence enhancing ligand (BATDA). A highly fluorescent chelate of Europium with the enhancing ligand which is released from dead cells can be measured by a fluorometer. Another alternative technique may utilize the transfection of target

0 cells with luciferase. Added lucifer yellow may then be oxidated by viable cells only. Purified anti-CLD18 IgGs can then be added at various concentrations. Irrelevant human IgG can be used as negative control. Assays can be carried out for 4 to 20 hours at 37°C depending on the effector cell type used. Samples can be assayed for cytolysis by measuring ⁵¹Cr release or the presence of the EuTDA

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2018200685 30 Jan 2018 chelate in the culture supernatant. Alternatively, luminescence resulting from the oxidation of lucifer yellow can be a measure of viable cells.

Anti-CLD18 monoclonal antibodies can also be tested in various combinations to determine whether cytolysis is enhanced with multiple monoclonal antibodies.

Complement dependent cytotoxicity (CDC):

Monoclonal anti-CLD18 antibodies can be tested for their ability to mediate CDC using a variety of known techniques. For example, serum for complement can be obtained from blood in a manner known to the skilled person. To determine the

CDC activity of mAbs, different methods can be used. ⁵¹Cr release can for example be measured or elevated membrane permeability can be assessed using a propidium iodide (PI) exclusion assay. Briefly, target cells can be washed and 5 x 10⁵/ml can be incubated with various concentrations of mAb for 10-30 min. at room temperature or at 37°C. Serum or plasma can then be added to a final concentration of 20% (v/v) and the cells incubated at 37°C for 20-30 min. All cells from each sample can be added to the PI solution in a FACS tube. The mixture can then be analyzed immediately by flow cytometry analysis using FACSArray.

In an alternative assay, induction of CDC can be determined on adherent cells. In one embodiment of this assay, cells are seeded 24 h before the assay with a density

0 of 3 x 10⁴/well in tissue-culture flat-bottom microtiter plates. The next day growth medium is removed and the cells are incubated in triplicates with antibodies. Control cells are incubated with growth medium or growth medium containing 0.2% saponin for the determination of background lysis and maximal lysis, respectively. After incubation for 20 min. at room temperature supernatant is removed and 20% (v/v) human plasma or serum in DMEM (prewarmed to 37°C) is added to the cells and incubated for another 20 min. at 37°C. All cells from each sample are added to propidium iodide solution (10 pg/ml). Then, supernatants are replaced by PBS containing 2.5 pg/ml ethidium bromide and fluorescence emission upon excitation at 520 nm is measured at 600 nm using a Tecan Safire.

0 The percentage specific lysis is calculated as follows: % specific lysis (fluorescence sample-fluorescence background)/ (fluorescence maximal lysisfluorescence background) x 100.

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Inhibition of cell proliferation by monoclonal antibodies:

To test for the ability to initiate apoptosis, monoclonal anti-CLD18 antibodies can, for example, be incubated with CLD18 positive tumor cells, e.g., SNU-16, DANG, KATO-III or CLD18 transfected tumor cells at 37°C for about 20 hours. The cells can be harvested, washed in Annexin-V binding buffer (BD biosciences), and incubated with Annexin V conjugated withFITC or APC (BD biosciences) for 15 min. in the dark. All cells from each sample can be added to PI solution (10 pg/ml in PBS) in a FACS tube and assessed immediately by flow cytometry (as above). Alternatively, a general inhibition of cell-proliferation by monoclonal antibodies can be detected with commercially available kits. The DELFIA Cell Proliferation Kit (Perkin-Elmer, Cat. No. AD0200) is a non-isotopic immunoassay based on the measurement of 5-bromo-2’-deoxyuridine (BrdU) incorporation during DNA synthesis of proliferating cells in microplates. Incorporated BrdU is detected using europium labelled monoclonal antibody. To allow antibody detection, cells are fixed and DNA denatured using Fix solution. Unbound antibody is washed away and DELFIA inducer is added to dissociate europium ions from the labelled antibody into solution, where they form highly fluorescent chelates with components of the DELFIA Inducer. The fluorescence measured - utilizing timeresolved fluorometry in the detection - is proportional to the DNA synthesis in the

0 cell of each well.

Preclinical studies

Monoclonal antibodies which bind to CLD18 also can be tested in an in vivo model (e.g. in immune deficient mice carrying xenografted tumors inoculated with

5 cell lines expressing CLD18, e.g. DAN-G, SNU-16, or KATO-III, or after transfection, e.g. HEK293) to determine their efficacy in controlling growth of CLD18-expressing tumor cells.

In vivo studies after xenografting CLD18 expressing tumor cells into

0 immunocompromised mice or other animals can be performed using antibodies of the invention. Antibodies can be adminstered to tumor free mice followed by injection of tumor cells to measure the effects of the antibodies to prevent formation of tumors or tumor-related symptoms. Antibodies can be adminstered to tumor-bearing mice to determine the therapeutic efficacy of respective antibodies

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2018200685 30 Jan 2018 to reduce tumor growth, metastasis or tumor related symptoms. Antibody application can be combined with application of other substances as cystostatic drugs, growth factor inhibitors, cell cycle blockers, angiogenesis inhibitors or other antibodies to determine synergistic efficacy and potential toxicity of combinations.

To analyze toxic side effects mediated by antibodies of the invention animals can be inoculated with antibodies or control reagents and thoroughly investigated for symptoms possibly related to CLD18-antibody therapy. Possible side effects of in vivo application of CLD18 antibodies particularly include toxicity at CLD18 expressing tissues including stomach and lung. Antibodies recognizing CLD18 in human and in other species, e.g. mice, are particularly useful to predict potential side effects mediated by application of monoclonal CLD18-antibodies in humans.

Epitope mapping

Mapping of epitopes recognized by antibodies of invention can be performed as 15 described in detail in “Epitope Mapping Protocols (Methods in Molecular Biology) by Glenn E. Morris ISBN-089603-375-9 and in „Epitope Mapping: A Practical

Approach¹¹ Practical Approach Series, 248 by Olwyn M. R. Westwood, Frank C. Hay.

0 I. Bispecific/Multispeciflc Molecules Which Bind to CLD18

In yet another embodiment of the invention, antibodies to CLD18 can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., an Fab' fragment) to generate a bispecific or multispecific molecule which binds to multiple binding sites or target epitopes. For example, an antibody of the invention can be functionally linked (e.g. by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, peptide or binding mimetic.

Accordingly, the present invention includes bispecific and multispecific molecules

0 comprising at least one first binding specificity for CLD18 and a second binding specificity for a second target epitope. In a particular embodiment of the invention, the second target epitope is an Fc receptor, e.g. human Fc-gammaRI (CD64) or a human Fc-alpha receptor (CD89), or a T cell receptor, e.g. CD3. Therefore, the invention includes bispecific and multispecific molecules capable of binding both

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2018200685 30 Jan 2018 to Fc-gammaR, Fc-alphaR or Fc-epsilonR expressing effector cells (e.g.

monocytes, macrophagesor polymorphonuclear cells (PMNs)), and to target cells expressing CLD18. These bispecific and multispecific molecules may target

CLD18 expressing cells to effector cell and may trigger Fc receptor-mediated effector cell activities, such as phagocytosis of CLD18 expressing cells, antibody dependent cellular cytotoxicity (ADCC), cytokine release, or generation of superoxide anion.

Bispecific and multispecific molecules of the invention can further include a third 10 binding specificity, in addition to an anti-Fc binding specificity and an anti-CLD18 binding specificity. In one embodiment, the third binding specificity is an antienhancement factor (EF) portion, e.g. a molecule which binds to a surface protein involved in cytotoxic activity and thereby increases the immune response against the target cell. The anti-enhancement factor portion can be an antibody, functional antibody fragment or a ligand that binds to a given molecule, e.g., an antigen or a receptor, and thereby results in an enhancement of the effect of the binding determinants for the Fc receptor or target cell antigen. The antienhancement factor portion can bind an Fc receptor or a target cell antigen. Alternatively, the anti-enhancement factor portion can bind to an entity that is

0 different from the entity to which the first and second binding specificities bind.

For example, the anti-enhancement factor portion can bind a cytotoxic T cell (e.g., via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell that results in an increased immune response against the target cell).

In one embodiment, the bispecific and multispecific molecules of the invention comprise as a binding specificity at least one antibody, including, e.g., an Fab, Fab', F(ab')2, Fv, or a single chain Fv. The antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof such as a Fv or a single chain construct as described in Ladner et al., US 4,946,778. The antibody may also be a

0 binding-domain immunoglobulin fusion protein as disclosed in US2003/0118592 and US 2003/0133939.

In one embodiment bispecific and multispecific molecules of the invention comprise a binding specificity for an Fc-gammaR or an Fc-alphaR present on the

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2018200685 30 Jan 2018 surface of an effector cell, and a second binding specificity for a target cell antigen, e.g., CLD18.

In one embodiment, the binding specificity for an Fc receptor is provided by a 5 monoclonal antibody, the binding of which is not blocked by human immunoglobulin G (IgG). As used herein, the term IgG receptor refers to any of the eight gamma-chain genes located on chromosome 1. These genes encode a total of twelve transmembrane or soluble receptor isoforms which are grouped into three Fc-gamma receptor classes: Fc-gammaRI (CD64), Fc-gammaRII (CD32), and Fc-gammaRIII (CD 16). In one preferred embodiment, the Fc-gamma receptor is a human high affinity Fc-gammaRI.

The production and characterization of these preferred monoclonal antibodies are described by Fanger et al. in WO 88/00052 and in US 4,954,617. These antibodies bind to an epitope of Fc-gammaRI, Fc-gammaRII or Fc-gammayRIII at a site which is distinct from the Fey binding site of the receptor and, thus, their binding is not blocked substantially by physiological levels of IgG. Specific anti-FcgammaRI antibodies useful in this invention are mAb 22, mAb 32, mAb 44, mAb 62 and mAb 197. In other embodiments, the anti-Fcy receptor antibody is a

0 humanized form of monoclonal antibody 22 (H22). The production and characterization of the H22 antibody is described in Graziano, R. F. et al. (1995) J. Immunol. 155 (10): 4996-5002 and WO 94/10332. The H22 antibody producing cell line was deposited at the American Type Culture Collection on November 4, 1992 under the designation HA022CL1 and has the accession No. CRL 11177.

In still other preferred embodiments, the binding specificity for an Fc receptor is provided by an antibody that binds to a human IgA receptor, e.g., an Fc-alpha receptor (Fc-alphaRI (CD89)), the binding of which is preferably not blocked by human immunoglobulin A (IgA). The term IgA receptor is intended to include

0 the gene product of one alpha-gene (Fc-alphaRI) located on chromosome 19. This gene is known to encode several alternatively spliced transmembrane isoforms of 55 to 110 kDa. Fc-alphaRI (CD89) is constitutively expressed on monocytes/macrophages, eosinophilic and neutrophilic granulocytes, but not on non-effector cell populations. Fc-alphaRI has medium affinity for both IgAl and

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IgA2, which is increased upon exposure to cytokines such as G-CSF or GM-CSF (Morton, H. C. et al. (1996) Critical Reviews in Immunology 16: 423-440). Four

Fc-alphaRI-specific monoclonal antibodies, identified as A3, A59, A62 and A77, which bind Fc-alphaRI outside the IgA ligand binding domain, have been described (Monteiro, R. C. et al. (1992) J.Immunol. 148: 1764).

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

In another embodiment the bispecific molecule is comprised of two monoclonal antibodies according to the invention which have complementary functional activities, such as one antibody predominately working by inducing CDC and the other antibody predominately working by inducing apoptosis.

An effector cell specific antibody as used herein refers to an antibody or

0 functional antibody fragment that binds the Fc receptor of effector cells. Preferred antibodies for use in the subject invention bind the Fc receptor of effector cells at a site which is not bound by endogenous immunoglobulin.

As used herein, the term effector cell refers to an immune cell which is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, e.g, lymphocytes (e.g., B cells and T cells including cytolytic T cells (CTLs), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils. Some effector cells express specific Fc receptors and carry out specific immune functions. In preferred embodiments, an effector cell is capable of inducing antibody-dependent cellular cytotoxicity (ADCC), e.g., a neutrophil capable of inducing ADCC. For example, monocytes, macrophages, which express FcR are involved in specific killing of target cells and presenting antigens to other

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2018200685 30 Jan 2018 components of the immune system, or binding to cells that present antigens. In other embodiments, an effector cell can phagocytose a target antigen, target cell, or microorganism. The expression of a particular FcR on an effector cell can be regulated by humoral factors such as cytokines. For example, expression of Fc5 gammaRI has been found to be up-regulated by interferon gamma (IFN-γ). This enhanced expression increases the cytotoxic activity of Fc-gammaRI-bearing cells against targets. An effector cell can phagocytose or lyse a target antigen or a target cell.

Target cell shall mean any undesirable cell in a subject (e.g., a human or animal) that can be targeted by an antibody of the invention. In preferred embodiments, the target cell is a cell expressing or overexpressing CLD18. Cells expressing CLD18 typically include tumor cells.

Bispecific and multispecific molecules of the present invention can be made using chemical techniques (see e.g., D. M. Kranz et al. (1981) Proc. Natl. Acad. Sci. USA 78:5807), polydoma techniques (See US 4,474,893, to Reading), or recombinant DNA techniques.

0 In particular, bispecific and multispecific molecules of the present invention can be prepared by conjugating the constituent binding specificities, e.g., the anti-FcR and anti-CLD18 binding specificities, using methods known in the art. For example, each binding specificity of the bispecific and multispecific molecule can be generated separately and then conjugated to one another. When the binding

5 specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), Nsuccinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl-4-(N3 0 maleimidomethyl)cyclohexane-l -carboxylate (sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160: 1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82: 8648). Other methods include those described by Paulus (Behring Ins. Mitt. (1985) No. 78,118-132); Brennan et al. (Science (1985) 229: 81-83), and Glennie et al. (J. Immunol. (1987) 139: 2367-2375). Preferred conjugating agents

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2018200685 30 Jan 2018 are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford,

IL).

When the binding specificities are antibodies, they can be conjugated via 5 sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.

Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the bispecific and multispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab')2 or ligand x Fab fusion protein. A bispecific and multispecific molecule of the invention, e.g., a bispecific molecule, can be a single chain molecule, such as a single chain bispecific antibody, a single chain bispecific molecule comprising one single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific and multispecific molecules can also be single chain molecules or may comprise at least two single chain molecules. Methods for preparing bi-and multispecific molecules are described for example in US 5,260,203; US 5,455,030; US 4,881,175; US

5,132,405; US 5,091,513; US 5,476,786; US 5,013,653; US 5,258,498; and US

5,482,858.

Binding of the bispecific and multispecific molecules to their specific targets can be confirmed by enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), FACS analysis, a bioassay (e.g., growth inhibition), or a Western Blot Assay. Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, the FcRantibody complexes can be detected using e.g., an enzyme-linked antibody or

0 antibody fragment which recognizes and specifically binds to the antibody-FcR complexes. Alternatively, the complexes can be detected using any of a variety of other immunoassays. For example, the antibody can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques,

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The Endocrine Society, March, 1986). The radioactive isotope can be detected by such means as the use of a γ-counter or a scintillation counter or by autoradiography.

II. Immunoconjugates

In another aspect, the present invention features an anti-CLD18 antibody conjugated to a therapeutic moiety or agent, such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radioisotope. Such conjugates are referred to herein as immunoconjugates. Immunoconjugates which include one or more cytotoxins are referred to as immunotoxins. A cytotoxin or cytotoxic agent includes any agent that is detrimental to and, in particular, kills cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 115 dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.

Suitable therapeutic agents for forming immunoconjugates of the invention include, but are not limited to, antimetabolites (e.g., methotrexate, 62 0 mercaptopurine, 6-thioguanine, cytarabine, fludarabin, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin)

5 and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC), and anti-mitotic agents (e.g., vincristine and vinblastine). In a preferred embodiment, the therapeutic agent is a cytotoxic agent or a radiotoxic agent. In another embodiment, the therapeutic agent is an immunosuppressant. In yet another embodiment, the therapeutic agent is GM3 0 CSF. In a preferred embodiment, the therapeutic agent is doxorubicin, cisplatin, bleomycin, sulfate, carmustine, chlorambucil, cyclophosphamide or ricin A.

Antibodies of the present invention also can be conjugated to a radioisotope, e.g., iodine-131, yttrium-90 or indium-111, to generate cytotoxic radiopharmaceuticals

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2018200685 30 Jan 2018 for treating a CLD18-related disorder, such as a cancer. The antibody conjugates of the invention can be used to modify a given biological response, and the drug moiety is not to be construed as limited to classical chemical therapeutic agents.

For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, an enzymatically active toxin, or active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or interferonγ; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Amon et al., Monoclonal Antibodies For Immunotargeting Of Drugs In

Cancer Therapy, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds. ), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., Antibodies For Drug Delivery, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review, in Monoclonal Antibodies '84: Biological

0 And Clinical Applications, Pincheraet al. (eds. ), pp. 475-506 (1985); Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., The Preparation And Cytotoxic Properties Of Antibody-Toxin

5 Conjugates, Immunol. Rev., 62: 119-58 (1982).

In a further embodiment, the antibodies according to the invention are attached to a linker-chelator, e.g., tiuxetan, which allows for the antibody to be conjugated to a radioisotope.

III. Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., a pharmaceutical composition, containing one or a combination of antibodies of the present invention. The pharmaceutical compositions may be formulated with

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2018200685 30 Jan 2018 pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition,

Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. In one embodiment, the compositions include a combination of multiple (e.g., two or more) isolated antibodies of the invention which act by different mechanisms, e.g., one antibody which predominately acts by inducing CDC in combination with another antibody which predominately acts by inducing apoptosis.

Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e., combined with other agents. For example, the combination therapy can include a composition of the present invention with at least one anti-inflammatory agent or at least one immunosuppressive agent. In one embodiment such therapeutic agents include one or more anti-inflammatory agents, such as a steroidal drug or a NSAID (nonsteroidal anti-inflammatory drug). Preferred agents include, for example, aspirin and other salicylates, Cox-2 inhibitors, such as rofecoxib (Vioxx) and celecoxib (Celebrex), NSAIDs such as ibuprofen (Motrin, Advil), fenoprofen (Nalfon), naproxen (Naprosyn), sulindac (Clinoril), diclofenac (Voltaren), piroxicam (Feldene), ketoprofen (Orudis), diflunisal (Dolobid), nabumetone (Relafen), etodolac (Lodine), oxaprozin (Daypro), and indomethacin (Indocin).

In another embodiment, such therapeutic agents include agents leading to the depletion or functional inactivation of regulatory T cells like low dose

5 cyclophosphamid, anti-CTLA4 antibodies, anti-IL2 or anti-IL2-receptor antibodies.

In yet another embodiment, such therapeutic agents include one or more chemotherapeutics, such as Taxol derivatives, taxotere, gemcitabin, 5-Fluoruracil,

0 doxorubicin (Adriamycin), cisplatin (Platinol), cyclophosphamide (Cytoxan,

Procytox, Neosar). In another embodiment, antibodies of the present invention may be administered in combination with chemotherapeutic agents, which preferably show therapeutic efficacy in patients suffering from stomach, esophageal, pancreatic and lung cancer.

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In yet another embodiment, the antibodies of the invention may be administered in conjunction with radiotherapy and/or autologous peripheral stem cell or bone marrow transplantation.

In still another embodiment, the antibodies of the invention may be administered in combination with one or more antibodies selected from anti-CD25 antibodies, antiEPCAM antibodies, anti-EGFR, anti-Her2/neu, and anti-CD40 antibodies.

In yet a further embodiment, the antibodies of the invention may be administered in combination with an anti-C3b(i) antibody in order to enhance complement activation.

As used herein, pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody,

0 bispecific and multispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

A pharmaceutically acceptable salt refers to a salt that retains the desired

5 biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66: 1-19).

Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as

0 hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium,

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2018200685 30 Jan 2018 magnesium, calcium and the like, as well as from nontoxic organic amines, such as

Ν,Ν'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

A composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for the preparation of such formulations are generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery

Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

To administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. For example, the compound may be

0 administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7: 27).

5 Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof

0 in the pharmaceutical compositions of the invention is contemplated.

Supplementary active compounds can also be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution,

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2018200685 30 Jan 2018 microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.

Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other

0 ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It

0 is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with

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2018200685 30 Jan 2018 the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

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) oil-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, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

For the therapeutic compositions, formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods known in the art of pharmacy. The amount of active ingredient which can be combined with a

0 carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect.

Generally, out of one hundred per cent, this amount will range from about 0.01 per cent to about ninety-nine percent of active ingredient, preferably from about 0.1 percent to about 70 percent, most preferably from about 1 percent to about 30 percent.

Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate. Dosage forms for the topical or transdermal administration of compositions of this invention

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2018200685 30 Jan 2018 include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

The phrases parenteral administration and administered parenterally as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size

0 in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such

0 as aluminum monostearate and gelatin.

In one embodiment the monoclonal antibodies of the invention are administered in crystalline form by subcutaneous injection, cf. Yang et al. (2003) PNAS, 100 (12): 6934-6939. When the compounds of the present invention are administered as

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2018200685 30 Jan 2018 pharmaceuticals, to humans and animals, they can be given alone or as a pharmaceutical composition containing, for example, 0.01 to 99.5% (more preferably, 0.1 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of

0 the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase

0 the dosage until the desired effect is achieved. In general, a suitable daily dose of a composition of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous,

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2018200685 30 Jan 2018 preferably administered proximal to the site of the target. If desired, the effective daily dose of a therapeutic composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).

In one embodiment, the antibodies of the invention may be administered by infusion, preferably slow continuous infusion over a long period, such as more than 24 hours, in order to reduce toxic side effects. The administration may also be performed by continuous infusion over a period of from 2 to 24 hours, such as of from 2 to 12 hours. Such regimen may be repeated one or more times as necessary, for example, after 6 months or 12 months. The dosage can be determined or adjusted by measuring the amount of circulating monoclonal anti-CLD18 antibodies upon administration in a biological sample by using anti-idiotypic antibodies which target the anti-CLD18 antibodies.

In yet another embodiment, the antibodies are administered by maintenance therapy, such as, e.g., once a week for a period of 6 months or more.

In still another embodiment, the antibodies according to the invention may be administered by a regimen including one infusion of an antibody against CLD18 followed by an infusion of an antibody against CLD18 conjugated to a radioisotope. The regimen may be repeated, e.g., 7 to 9 days later.

Therapeutic compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in US 5,399,163; US 5,383,851; US 5,312,335; US

0 5,064,413; US 4,941,880; US 4,790,824; or US 4,596,556. Examples of wellknown implants and modules useful in the present invention include those described in: US 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; US 4,486,194, which discloses a therapeutic device for administering medicants through the skin; US 4,447,233,

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2018200685 30 Jan 2018 which discloses a medication infusion pump for delivering medication at a precise infusion rate; US 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; US 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and US 4,475,196, which discloses an osmotic drug delivery system.

Many other such implants, delivery systems, and modules are known to those skilled in the art. In certain embodiments, the antibodies of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., US 4,522,811; US 5,374,548; and US 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, and thus enhance targeted drug delivery (see, e.g., V.V. Ranade (1989) J. Clin. Pharmacol. 29: 685). Exemplary targeting moieties include folate or biotin (see, e.g., US 5,416,016 to Low et al.); 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); and surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134).

In one embodiment of the invention, the therapeutic compounds of the invention are formulated in liposomes. In a more preferred embodiment, the liposomes include a targeting moiety. In a most preferred embodiment, the therapeutic compounds in the liposomes are delivered by bolus injection to a site proximal to the desired area, e.g., the site of a tumor. The composition must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of

0 microorganisms such as bacteria and fungi.

In a further embodiment, antibodies of the invention can be formulated to prevent or reduce their transport across the placenta. This can be done by methods known in the art, e.g., by PEGylation of the antibodies or by use of F(ab)2' fragments.

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Further references can be made to Cunningham-Rundles C, Zhuo Z, Griffith B,

Keenan J. (1992) Biological activities of polyethylene-glycol immunoglobulin conjugates. Resistance to enzymatic degradation. J. Immunol. Methods, 152: 177190; and to Landor M. (1995) Maternal-fetal transfer of immunoglobulins, Ann.

Allergy Asthma Immunol. 74: 279-283.

A therapeutically effective dosage for tumor therapy can be measured by objective tumor responses which can either be complete or partial. A complete response (CR) is defined as no clinical, radiological or other evidence of disease. A partial response (PR) results from a reduction in aggregate tumor size of greater than 50%. Median time to progression is a measure that characterizes the durability of the objective tumor response.

A therapeutically effective dosage for tumor therapy can also be measured by its ability to stabilize the progression of disease. The ability of a compound to inhibit cancer can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit cell growth or apoptosis by in vitro assays known to the skilled practitioner. A therapeutically effective amount

0 of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.

The composition must be sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, the carrier can be an isotonic buffered saline solution, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Proper fluidity

0 can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by

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2018200685 30 Jan 2018 including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

When the active compound is suitably protected, as described above, the 5 compound may be orally administered, for example, with an inert diluent or an assimilable edible carrier.

IV. Uses and Methods of the Invention

The antibodies (including immunoconjugates, bispecifics/multispecifics, compositions and other derivatives described herein) of the present invention have numerous therapeutic utilities involving the treatment of disorders involving cells expressing CLD18. For example, the antibodies can be administered to cells in culture, e.g., in vitro or ex vivo, or to human subjects, e.g., in vivo, to treat or prevent a variety of disorders such as those described herein. As used herein, the term subject is intended to include human and non-human animals which respond to the antibodies against CLD18. Preferred subjects include human patients having disorders that can be corrected or ameliorated by killing diseased cells, in particular cells characterized by an altered expression pattern of CLD18 compared to normal cells.

A therapeutic effect in the treatments discussed herein is preferably achieved through the functional properties of the antibodies of the invention to mediate killing of cells e.g. by inducing complement dependent cytotoxicity (CDC) mediated lysis, antibody dependent cellular cytotoxicity (ADCC) mediated lysis,

5 apoptosis, homotypic adhesion, and/or phagocytosis, preferably by inducing CDC mediated lysis and/or ADCC mediated lysis.

For example, in one embodiment, antibodies of the present invention can be used to treat a subject with a tumorigenic disorder, e.g., a disorder characterized by the

0 presence of tumor cells expressing CLD18 including, for example, gastric cancer.

Examples of tumorigenic diseases which can be treated and/or prevented encompass all CLD18 expressing cancers and tumor entities including stomach cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, breast cancer, colorectal cancer, hepatic cancer, cancer of the gallbladder and head-neck

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metastasis.

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The pharmaceutical compositions and methods of treatment described according to 5 the invention may also be used for immunization or vaccination to prevent a disease described herein.

In another embodiment, antibodies of the invention can be used to detect levels of CLD18 or particular forms of CLD18, or levels of cells which contain CLD18 on their membrane surface, which levels can then be linked to certain diseases or disease symptoms such as described above. Alternatively, the antibodies can be used to deplete or interact with the ftmction of CLD18 expressing cells, thereby implicating these cells as important mediators of the disease. This can be achieved by contacting a sample and a control sample with the anti-CLD18 antibody under conditions that allow for the formation of a complex between the antibody and CLD18. Any complexes formed between the antibody and CLD18 are detected and compared in the sample and a control sample, i.e. a reference sample.

Antibodies of the invention can be initially tested for their binding activity

0 associated with therapeutic or diagnostic uses in vitro. For example, the antibodies can be tested using flow cytometric assays as described herein.

Moreover, activity of the antibodies in triggering at least one effector-mediated effector cell activity, including inhibiting the growth of and/or killing of cells expressing CLD18, can be assayed. For example, the ability of the antibodies to trigger CDC and/or apoptosis can be assayed. Protocols for assaying for CDC, homotypic adhesion, molecular clustering or apoptosis are described herein.

The antibodies of the invention can be used to elicit in vivo or in vitro one or more

0 of the following biological activities: to inhibit the growth of and/or differentiation of a cell expressing CLD18; to kill a cell expressing CLD18; to mediate phagocytosis or ADCC of a cell expressing CLD18 in the presence of effector cells; to mediate CDC of a cell expressing CLD18 in the presence of complement;

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2018200685 30 Jan 2018 to mediate apoptosis of a cell expressing CLD18; to induce homotypic adhesion;

and/or to induce translocation into lipid rafts upon binding CLD18.

In a particular embodiment, the antibodies are used in vivo or in vitro to treat, 5 prevent or diagnose a variety of CLD18-related diseases. Examples of CLD18related diseases include, among others, cancers such as gastric cancer, pancreatic cancer, esophageal cancer, lung cancer and cancers as those listed above.

CLD18A2 is also expressed in differentiated normal stomach cells. Possible antibody induced clinical side effects by killing of these cells may be reduced or avoided by parallel administration of stomach protective drugs such as antacida, or inhibitors of the gastric proton pump such as omeprazol or related drugs.

Suitable routes of administering the antibody compositions of the invention in vivo and in vitro are well known in the art and can be selected by those of ordinary skill.

As described above, anti-CLD18 antibodies of the invention can be coadministered with one or other more therapeutic agents, e.g., a cytotoxic agent, a

0 radiotoxic agent, antiangiogeneic agent or and immunosuppressive agent to reduce the induction of immune responses against the antibodies of invention. The antibody can be linked to the agent (as an immunocomplex) or can be administered separate from the agent. In the latter case (separate administration), the antibody can be administered before, after or concurrently with the agent or can be co2 5 administered with other known therapies, e.g., an anti-cancer therapy, e.g., radiation. Such therapeutic agents include, among others, anti-neoplastic agents such as listed above. Co-administration of the anti-CLD18 antibodies of the present invention with chemotherapeutic agents provides two anti-cancer agents which operate via different mechanisms yielding a cytotoxic effect to tumor cells.

0 Such co-administration can solve problems due to development of resistance to drugs or a change in the antigenicity of the tumor cells which would render them unreactive with the antibody.

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In another particular embodiment of the invention, the subject being administered the antibody is additionally treated with an antiagionic agent including antibodies targeting VEGF or VEGFR and one or more chemical compounds inhibiting angiogenesis. Pretreatment with or parallel applicatition of these drugs may improve the penetration of antibodies in bulk tumors.

In another particular embodiment of the invention, the subject being administered the antibody is additionally treated with a compound inhibiting growth factor receptor signaling including monoclonal antibodies binding to the EGFR receptor as well as chemical compounds inhibiting signaling initiated by the EGFR, Herl or Her2/neu receptor.

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

0 solution. The number of cells administered can be in the order of 10 to 10 but will vary depending on the therapeutic purpose. In general, the amount will be sufficient to obtain localization at the target cell, e.g., a tumor cell expressing CLD18, and to effect cell killing by, e.g., phagocytosis. Routes of administration can also vary.

Therapy with target-specific effector cells can be performed in conjunction with other techniques for removal of targeted cells. For example, anti-tumor therapy using the compositions of the invention and/or effector cells armed with these compositions can be used in conjunction with chemotherapy. Additionally,

0 combination immunotherapy may be used to direct two distinct cytotoxic effector populations toward tumor cell rejection. For example, anti-CLD18 antibodies linked to anti-Fc-RI or anti-CD3 may be used in conjunction with IgG- or IgAreceptor specific binding agents.

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Bispecific and multispecific molecules of the invention can also be used to modulate Fc-gammaR or Fc-alphaR levels on effector cells, such as by capping and eliminating receptors on the cell surface. Mixtures of anti-Fc receptors can also be used for this purpose.

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

The compositions of the invention can also be administered together with complement. Accordingly, within the scope of the invention are compositions

0 comprising antibodies, multispecific or bispecific molecules and serum or complement. These compositions are advantageous in that the complement is located in close proximity to the antibodies, multispecific or bispecific molecules.

Alternatively, the antibodies, multispecific or bispecific molecules of the invention

5 and the complement or serum can be administered separately. Binding of the compositions of the present invention to target cells causes translocation of the CLD18 antigen-antibody complex into lipid rafts of the cell membrane. Such translocation creates a high density of antigen-antibody complexes which may efficiently activate and/or enhance CDC.

Also within the scope of the present invention are kits comprising the antibody compositions of the invention (e.g., antibodies and immunoconjugates) and instructions for use. The kit can further contain one or more additional reagents, such as an immunosuppressive reagent, a cytotoxic agent or a radiotoxic agent, or

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2018200685 30 Jan 2018 one or more additional antibodies of the invention (e.g., an antibody having a complementary activity).

Accordingly, patients treated with antibody compositions of the invention can be 5 additionally administered (prior to, simultaneously with, or following administration of a antibody of the invention) with another therapeutic agent, such as a cytotoxic or radiotoxic agent, which enhances or augments the therapeutic effect of the antibodies of the invention.

In other embodiments, the subject can be additionally treated with an agent that modulates, e.g., enhances or inhibits, the expression or activity of Fc-gamma or Fc-alpha receptors by, for example, treating the subject with a cytokine. Preferred cytokines include granulocyte colony-stimulating factor (G-CSF), granulocytemacrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), and tumor necrosis factor (TNF). Other important agents for increasing the therapeutic efficacy of the antibodies and pharmaceutical compositions described herein are βglucans which are homopolysaccharides of branched glucose residues and are produced by a variety of plants and microorganisms, for example, bacteria, algae, fungi, yeast and grains. Fragments of β-glucans produced by organisms may be

0 also be used. Preferably, the β-glucan is a polymer of β(1,3) glucose wherein at least some of the backbone glucose units, e.g. 3-6 % of the backbone glucose units, possess branches such as β(1,6) branches.

In a particular embodiment, the invention provides methods for detecting the presence of CLD 18 antigen in a sample, or measuring the amount of CLD 18 antigen, comprising contacting the sample, and a control sample, with a antibody which specifically binds to CLD 18, under conditions that allow for formation of a complex between the antibody or portion thereof and CLD 18. The formation of a complex is then detected, wherein a difference complex formation between the

0 sample compared to the control sample is indicative for the presence of CLD 18 antigen in the sample.

In still another embodiment, the invention provides a method for detecting the presence or quantifying the amount of CLD18-expressing cells in vivo or in vitro.

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The method comprises (i) administering to a subject a composition of the invention conjugated to a detectable marker; (ii) exposing the subject to a means for detecting said detectable marker to identify areas containing CLD18-expressing cells.

Methods as described above are useful, in particular, for diagnosing CLD18related diseases and/or the localization of CLD18-related diseases such as cancer diseases. Preferably an amount of CLD18, preferably CLD18-A2 in a sample which is higher than the amount of CLD18, preferably CLD18-A2, in a control sample is indicative for the presence of a CLD18-related disease in a subject, in particular a human, from which the sample is derived.

In yet another embodiment immunoconjugates of the invention can be used to target compounds (e.g., therapeutic agents, labels, cytotoxins, radiotoxins immunosuppressants, etc.) to cells which have CLD18 expressed on their surface by linking such compounds to the antibody. Thus, the invention also provides methods for localizing ex vivo or in vitro cells expressing CLD18, such as circulating tumor cells.

0 The present invention is further illustrated by the following examples which are not be construed as limiting the scope of the invention.

EXAMPLES

5 1. Generation of murine antibodies against CLD18

a. Immunizations:

Balb/c or C57/BL6 mice were immunized with eucaryotic expression vectors, encoding human CLD18 fragments (SEQ ID NO: 15, 16; 17, 18). 50 pg or 25 pg of plasmid DNA was injected into the quadriceps (intramuscular, i.m.) on days 1

0 and 10 for generation of monoclonal antibodies of Setl or alternatively on days 1 and 9, 1 and 11, or 1, 16 and 36 for generation of monoclonal antibodies of Set2 in the presence of adjuvants, for example CpG (for details see Tab. lb). CpG as well as cells transfected with CLD18A2 (SEQ ID NO: 1) alone or co-transfected additionally with murine soluble CD40L encoding RNA were injected

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2018200685 30 Jan 2018 intramuscularly, PEI-Man was injected intramuscularly or intraperitonally. The presence of antibodies directed against human CLD18 in sera of mice was monitored by immune fluorescence microscopy between day 16 and 43 depending on the specific immunization protocol used. The immune fluorescence was determined using HEK293 cells transiently transfected with a nucleic acid encoding a fusion construct comprising human CLD18A2 (SEQ ID NOs: 1, 2) and a fluorescent reporter protein. Mice with detectable immune responses (Fig. 1) were boosted three days prior to splenectomy for generation of monoclonal antibodies of Setl, or mice were boosted three days, three and two days, or mice were boosted four, three and two days prior to splenectomy for generation of monoclonal antibodies of Set2 by intraperitonal injection of 5 x 10⁷ or alternatively 1 x 10⁸ HEK293 cells transiently transfected with a nucleic acid encoding human CLD18A2 (SEQ ID NOs: 1, 2) (for details see Tab. lb). In Tab. la the immunization protocols used are dedicated to the respective monoclonal antibodies.

Tab. la: Immunisation protocols used for generation of monoclonal antibodies

mAB	Immunisation protocol*	mAB	Immunisation protocol
Setl
24H5	40	42E12	45
26B5	40	43A11	45
26D12	40	44E10	45
28D10	40	47D12	45
37G11	45	61C2	45
37H8	45	75B8	6
38G5	45	85A3	6
38H3	45	9E8	40
39F11	45	19B9	40
41C6	45
Set2
45C1	53	166E2	51
125E1	45	175D10	51
163E12	51

0 * For specific immunization protocols see Tab. lb

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Boosts with transfected cells	days prior to splenectomy	CG	CG	CG	3 and 2	4, 3 and 2
Cells co-transfected with CLD18A2 (SEQ ID NO: 1) and with murine soluble CD40L encoding RNA	none	5 χ 107 HEK293 cells; lOOpg CPG as adjuvant	1 χ 108 HEK293 cells	none	none
Cells transfected with CLD18A2 (SEQ ID NO: 1) alone	5x10' transfected MC3T3 cells			5x10' transfected HEK293 cells	5 x 107 transfected HEK293 cells
Serum -monitoring	on day	00	00		22, 30 and 43	20
Immunisation (prime and boosts with DNA)	o -o	go *— cd —	C © — CO	— § ©s	1, 16 and 36	1 and 11
with adjuvant 1	o Q. u on =i_ © in	50pg CpG	a a. O on © in	2,5 μΐ PEIMan*(150 mM) in H2O with 5% Glucose	50pg CpG in in H2O with 5% Glucose
with DNA vectors encoding CLD18 fragments	SEQ ID NO: 15: 50pg	SEQ ID NO: 17: 50pg	SEQ ID NO: 15: 50pg	on m CM <n O z g σ ω OO	Priming: SEQ ID NO: 15:25pg, and SEQ ID NO: 17:25pg; Boosting: SEQ ID NO: 17: 50pg
Immu- nisation protocol			40	45	in	CG •n

in vivo-jetPEI™-Man from PolyPlus Transfection

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b. Generation of hybridomas producing human monoclonal antibodies to CLD18: Mouse spienocytes were isolated and fused with PEG to a mouse myeloma cell line based on standard protocols. The resulting hybridomas were then screened for production of immunoglobulines with CLD18 specificity using HEK293 cells transfected with a nucleic acid encoding human CLD18 by FACS analysis.

Single cell suspensions of splenic lymphocytes from immunized mice were fused with P3X63Ag8U.l nonsecreting mouse myeloma cells (ATCC, CRL 1597) in a 2:1 ratio using 50% PEG (Roche Diagnostics, CRL 738641). Cells were plated at approximately 3 x 10⁴/well in flat bottom microtiter plates, followed by about two week incubation in selective medium containing 10% fetal bovine serum, 2% hybridoma fusion and cloning supplement (HFCS, Roche Diagnostics, CRL 1 363 735) plus 10 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 pg/ml gentamycin and lx HAT (Sigma, CRL H0262). After 10 to 14 days individual wells were screened by flow cytometry for anti-CLD18 monoclonal antibodies. The antibody secreting hybridomas were replated, screened again and, if still positive for antiCLD18 monoclonal antibodies, were subcloned by limiting dilution. The stable subclones were then cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization. At least one clone from each hybridoma, which retained the reactivity of parent cells (by FACS), was chosen. 9

0 vial cell banks were generated for each clone and stored in liquid nitrogen.

c. Selection of monoclonal antibodies binding to CLD18:

To determine the isotype of antibodies, an isotype ELISA was performed. The mouse monoAB ID Kit (Zymed, CRL 90-6550) or alternatively the IsoStrip Mouse

5 Monoclonal Antibody Isotyping Kit (Roche, Cat. No. 1493027) was used to determine Ig subclasses of the identified CLD18 reactive monoclonal antibodies. Defined as Setl, nineteen hybridoma cell lines were generated, six from a fusion of cells from a C57/BL6 mouse immunized with CLD18A2-LoopD3 (SEQ ID NOs: 17, 18), thirteen from a fusion of cells from a Balb/c mouse immunized with

0 CLD18A2-Loopl (SEQ ID NOs: 15, 16), expressing the following antibodies:

24H5, 26B5, 26D12, 28D10, 37G11, 37H8, 38G5, 38H3, 39F11, 41C6, 42E12, 43A11,44E10, 47D12, 61C2, 75B8, 85A3, 9E8, 19B9

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24H5: mouse monoclonal IgG2b, κ antibody, 182-D758-034

26B5: mouse monoclonal IgG2a, κ antibody, 182-D758-035, DSM ACC2745

26D12: mouse monoclonal IgG3, κ antibody, 182-D758-036, DSM ACC2746

28D10: mouse monoclonal IgG3, κ antibody, 182-D758-040, DSM ACC2747

37G11: mouse monoclonal IgG2a, κ antibody, 182-D1106-055, DSM ACC2737

37H8: mouse monoclonal IgG3, κ antibody, 182-D1106-056, DSM ACC2738 38G5: mouse monoclonal IgG3, κ antibody, 182-D1106-057, DSM ACC2739 38H3: mouse monoclonal IgG3, κ antibody, 182-D1106-058, DSM ACC2740 39F11: mouse monoclonal IgG3, κ antibody, 182-D1106-059, DSM ACC2741

41C6: mouse monoclonal IgG2a, κ antibody, 182-D1106-060

42E12: mouse monoclonal IgG2a, κ antibody, 182-D1106-061, DSM ACC2748 43A11: mouse monoclonal IgG2a, κ antibody, 182-D 1106-062, DSM ACC2742 44E10: mouse monoclonal IgG3, κ antibody, 182-D 1106-063

47D12: mouse monoclonal IgG3, κ antibody, 182-D1106-064

61C2: mouse monoclonal IgG2b, κ antibody, 182-D1106-067, DSM ACC2743

75B8: mouse monoclonal IgM, κ antibody, 182-D756-001 85A3: mouse monoclonal IgM, κ antibody, 182-D756-002 9E8: mouse monoclonal IgM, κ antibody, 182-D758-011 19B9: mouse monoclonal IgM, κ antibody, 182-D758-024

Defined as Set2, five hybridoma cell lines were generated, one from a fusion of cells from a Balb/c mouse immunized with CLD18A2-LoopD3 (SEQ ID NOs: 17, 18) and CLD18A2-LoopDl (SEQ ID NOs: 15, 16), four from a fusion of cells from a Balb/c mouse immunized with CLD18A2-LoopDl (SEQ ID NOs: 15, 16),

5 expressing the following antibodies:

45C1, 125E1, 163E12, 166E2, 175D10

45C1: mouse monoclonal IgG2a, κ antibody, 182-D758-187

0 125E1: mouse monoclonal IgG2a, κ antibody, 182-D 1106-279, DSM ACC2808

163E12: mouse monoclonal IgG3, κ antibody, 182-D1106-294, DSM ACC2809

166E2: mouse monoclonal IgG3, κ antibody, 182-D1106-308

175D10: mouse monoclonal IgGl, κ antibody, 182-D1106-362, DSM ACC2810

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2. Production of Monoclonal Antibodies

Production and purification of monoclonal antibodies reactive to CLD18:

To produce mg amounts of antibody for functional characterization, hybridoma cells were seeded in dialysis based bioreactors (CELLine CL 1000, Integra, Chur,

CH) at 2 x 10⁶ cells / ml. Antibody containing supernatant was harvested once weekly. Mouse monoclonal antibody was purified using Melon Gel (Pierce, Rockford, USA) and concentrated by ammonium sulphate precipitation or alternatively purified by ProteinA using FPLC. Antibody concentration and purity was determined by BCA-Assay and purity checked by sodium dodecylsulphate gel electrophoresis and coomassie staining.

3. Binding Characteristics of Monoclonal Antibodies

a. Quality control of transfectants in WB, IF:

To generate CLD18A2 expressing cells, HEK293 or CHO cells were transfected 15 with nucleic acids encoding CLD18A2 (SEQ ID NOs: 1, 2) or CLD18A2-myc (SEQ ID NOs: 3, 4).

HEK293 cells were transfected with CLDN18A2-myc (SEQ ID NOs: 3, 4) or left untransfected. 24 hours post transfection, cells were harvested, lysed and subjected to sodium dodecylsulphate gel electrophoresis. The gel was blotted and stained

0 with a mouse anti-myc antibody. After incubation with a peroxidase labelled anti mouse antibody, the blot was developed with ECL reagent and visualized using a LAS-3000 imager (Fuji). Only in the transfected cells but not in the negative control, a band with the expected molecular weight of CLD18-myc was observed (Fig- 2).

5 CHO cells were transfected with CLD18A2 (SEQ ID NOs: 1, 2) and grown on chamber slides for 24 h. Cells were fixed with methanol and stained with a rabbit polyclonal antibody against CLD18 at 1 pg/ml for 60 min. at 25°C. After washing, cells were stained with an Alexa488 labelled goat anti-rabbit IgG (Molecular Probes) and evaluated by fluorescence microscopy. Fig. 3 shows transfected CHO

0 cells, expressing CLD18 on the cell membrane as well as untransfected cells.

These heterologously CLD18 expressing cells were used for the following assays to test the specificity of antibody binding.

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b. Selection of Monoclonal Antibodies Binding to CLD18/Primary Screens by Flow Cytometry:

HEK293 cells were co-transfected with expression vectors encoding human CLD18A2 (SEQ ID NOs: 1, 2) and a fluorescing reporter protein 40 h prior to the assay or alternatively HEK293 cells stably expressing human CLD18A2 (HEK293-CLD18A2) were used and counterstained with propidium iodide (PI). After cell detachment using 2mM EDTA/PBS cells were washed with complete growth medium and plated at approximately 1-5 x 10⁵ cells/well in U-bottom microtiter plates. Cells were incubated for 30 min. at 4° C with hybridoma supernatant followed by two washing steps with 1% heatinactivated FBS/PBS and finally incubation with APC or Alexa647-conjugated anti-mouse IgG specific secondary antibody. After two washing steps, co-transfected cells were fixed with CellFIX (BD Biosciences). Binding was assessed by flow cytometry using a BD FACSArray. Fluorescence marker expression is plotted on the horizontal axis against antibody binding on the vertical axis. All mouse antibodies 24H5, 26B5, 26D12, 28D10, 37G11, 37H8, 38G5, 38H3, 39F11, 41C6, 42E12, 43A11, 44E10, 47D12, 61C2, 75B8, 85A3, 9E8, 19B9, 45C1, 125E1, 163E12, 166E2, and 175D10 were dectected to bind specifically to the surface of fluorescence marker expressing cells (Fig. 4, cells in Q2) as exemplified for hybridoma supernatants

0 containing monoclonal antibodies 24H5 (Fig. 4A, cells in Q2), 85A3 (Fig. 4B), 175D10, 125E1, 163E12, 166E2 and 45C1 (Fig. 4C, cells in Ql).

c. Comparison of antibody binding to Myc- or HA-tagged CLD18A2:

The binding characteristics of the identified CLD18-specific monoclonal

5 antibodies were further specified. Therefore, monoclonal antibody binding was analyzed to CLD18A2 mutants, created by insertion of epitope tags. CLD18A2HA (SEQ ID NO: 6) contains a HA-epitope tag in CLD18A2-loopl, whereas CLD18A2-Myc (SEQ ID NO: 4) contains a Myc-epitope tag inserted into CLD18A2-loop2. As insertion of these tags causes destruction of epitopes, the

0 identified monoclonal antibodies, can be grouped according to the loss of binding to any of the mutants. HEK293 cells transiently co-transfected with a fluorescence marker and human CLD18A2, or with a fluorescence marker and CLD18A2-HA, or with a fluorescence marker and CLD18A2-Myc were incubated with hybridoma supernatants containing CLD18-specific monoclonal antibodies for 30 min. at 4°C,

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WO 2007/059997 PCT/EP2006/011302 followed by incubation with Alexa647-conjugated anti-mouse IgG secondary antibody. Before analysis on a BD FACSArray, cells were fixed using CellFIX. As exemplified for 24H5, 9E8, 26B5 and 19B9 in Fig. 5, monoclonal antibodies could be separated based on their binding characteristics into four different groups: (i) antibodies that bind to unmodified CLD18A2 as well as to CLD18A2-HA and CLD18A2-Myc, e.g. 24H5, (Fig. 5A), or (ii) antibodies that do not bind to CLD18A2-HA, e.g. 9E8, (Fig. 5B), or (iii) antibodies that do not bind to CLD18A2-Myc, e.g. 26B5, (Fig. 5C), or (iv) antibodies that do not bind to CLD18A2-HA nor to CLD18A2-Myc, e.g. 19B9, (Fig. 5D).

d. Comparison of antibody binding to human CLD18A1 versus CLD18A2 transfectants by flow cytometry:

Binding specificity of the identified monoclonal antibodies to CLD18A2 isoforms was analyzed by flow cytometry. HEK293 cells stably expressing human

CLD18A2 (HEK293-CLD18A2) and HEK293 cells stably expressing human CLD18A1 (SEQ ID NOs: 7, 8) (HEK293-CLD18A1) were incubated for 30 min. at 4°C with hybridoma supernatants containing monoclonal antibodies, followed by incubation with Alexa647-conjugated anti-mouse IgG secondary antibody and fixation of cells or alternatively without fixation but with PI counterstaining.

0 Binding was assessed by flow cytometry using a BD FACSArray. Fig. 6 shows examples for the two groups of monoclonal antibodies that were identified in the panel comprised of 24H5, 26B5, 26D12, 28D10, 37G11, 37H8, 38G5, 38H3, 39F11, 41C6, 42E12, 43A11, 44E10, 47D12, 61C2, 75B8, 85A3, 9E8, 19B9, 45C1, 125E1, 163E12, 166E2, 175D10: (i) monoclonal antibodies 43A11, 45C1, and 163E12 bind specifically to human CLD18A2 but not to human CLD18A1 (Fig 6A,B), and (ii) monoclonal antibody 37H8 binds to both human isoforms (Fig 6A).

e. Comparison of antibody binding to human CLD18A1 versus CLD18A2

0 transfectants by immunofluorescence microscopy:

HEK293 cells were transiently transfected with an expression vector encoding a fusion protein of CLD18A1 (SEQ ID NO: 8) or CLD18A2 (SEQ ID NO: 2) with a fluorescence reporter and grown on chamber slides. Cells were either stained unfixed or after paraformaldehyde fixation with monoclonal antibody containing

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WO 2007/059997 PCT/EP2006/011302 tissue culture supernatant for 30 min. at 37°C. After washing, cells were stained with an Alexa555-labelled anti-mouse Ig antibody (Molecular Probes). Binding of antibodies was evaluated by fluorescence microscopy. As shown in Fig. 7, antibody 37G11 specifically reacted with CLD18A2 (Fig. 7A) but not with

CLD18A1 (Fig. 7B). In contrast, antibody 26B5 was reactive with both, CLD18A2 andCLD18Al (Fig. 8).

For antibodies 24H5, 26B5, 26D12, 28D10, 37G11, 37H8, 38G5, 38H3, 39F11, 41C6, 42E12, 43A11, 44E10, 47D12, 61C2, 75B8, 85A3, 9E8, 19B9, a clear difference between staining of living cells and paraformaldehyde fixed cells was observed. The antibodies formed an uniform membrane staining when cells were fixed (Fig. 7C, 8C, 8D). In contrast, incubation of living cells with these antibodies leads to the generation of protein clusters, visible as a speckle like staining pattern (Fig. 7A, 8A, 8B). This shows that all antibodies bind to native epitopes as found on the surface of living cells.

f. Determination of endogenously expressing cell lines:

A CLD18A2 gene-specific primer pair (SEQ ID NO: 11, 12) was used in RT-PCR analyses to screen cell lines for expression of CLD18A2. Human gastric carcinoma cell lines NCI-SNU-16 (ATCC CRL-5974), NUGC-4 (JCRB0834) and KATO-III

0 (ATCC HTB-103) and human pancreas adenocarcinoma cell line DAN-G (DSMZ

ACC249) were found to display robust endogenous expression of CLD18 (Fig. 9). Expression was confirmed on protein level by staining with a rabbit polyclonal serum against CLD18.

g. Staining of endogenously expressing cell lines with CLD18 specific antibodies and immunofluorescence analysis:

DAN-G, SNU-16, NUGC-4 and KATO-III cells were grown on chamber slides under standard conditions. Cells were unfixed or alternatively fixed with methanol and stained with the respective antibodies. For antibodies 24H5, 26B5, 26D12,

0 28D10, 37G11, 37H8, 38G5, 38H3, 39F11, 41C6, 42E12, 43A11, 44E10, 47D12,

61C2, 75B8, 85A3, 9E8, 19B9 staining of the cell surface was observed as exemplified in Fig. 10, 11 and 12A. For antibodies 45C1, 125E1, 163E12, 166E2, and 175D10 native epitope recognition was assayed and cell surface staining was observed on unfixed cells as shown in Fig 12B. Subgroups of antibodies showed

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2018200685 30 Jan 2018 homogenous staining of the cell membrane either preponderantly at cell-cell interfaces or at free parts of the membrane not adjacent to other cells. Other antibodies stained discrete foci and aggregates on the cell membrane altogether demonstrating that the respective antibodies bind to different epitopes including epitopes which are masked by homotypic or heterotypic association of CLD18 as well as CLD18 epitopes accessible in preformed tight junctions.

h. Staining of endogenously expressing cell lines by flow cytometry:

Surface expression of constitutively expressed CLD18A2 on KATO-III and 10 NUGC-4 living cells was analyzed by flow cytometry. This is exemplified by

KATO-III and NUGC-4 cells stained with monoclonal antibody 61C2 or 163E12, followed by incubation with Alexa647-conjugated anti-mouse IgG secondary antibody and fixation of cells or alternatively without fixation. Binding was assessed by flow cytometry using a BD FACS Array. Fig. 13 shows a strong binding of 61C2 to at least 70.3% of KATO-III cells and of 163E12 to CLD18A2 on KATO-III and NUGC-4 cells.

i. Sequence alignment of mouse and human CLD18A1 and CLD18A2:

Human CLD18A2 (NP_ 001002026) and human CLD18A1 (NP_057453) in a

0 sequence comparison differ in the N-terminus and mouse CLD18 variants (NP_062789 and AAL15636) demonstrate high homology and sequence variation sites between the molecules (see Fig. 14).

j. Reactivity of antibodies with murine CLD18A1 and murine CLD18A2 analyzed 25 by flow cytometry:

Binding of the identified monoclonal antibodies to murine CLD18A2 and CLD18A1 was analyzed by flow cytometry. HEK293 cells transiently cotransfected with a fluorescence marker and murine CLD18A2 (SEQ ID NOs: 33, 35) or with a fluorescence marker and murine CLD18A1 (SEQ ID NOs: 36, 37)

0 were incubated with hybridoma supernatants containing the human CLD18specific monoclonal antibodies 38G5, 38H3, 37G11, 45C1 and 163E12, respectively, for 30 min. at 4°C, followed by incubation with Alexa647-conjugated anti-mouse IgG secondary antibody and fixation of cells. Binding was assessed by flow cytometry using a BD FACSArray. Fig. 15 shows three different binding

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WO 2007/059997 PCT/EP2006/011302 profiles: 38G5, and 45C1 do not bind to any of the murine CLD18 isoforms,

37G11, and 163E12 bind to murine CLD18A2 but not to murine CLD18A1, and

38H3 binds to murine CLD18A1 and CLD18A2. These antibodies are valuable tools to determine a potential toxicity of CLD18 monoclonal antibodies in preclinical studies.

Altogether these data show, that monoclonal antibodies of the invention 24H5, 26B5, 26D12, 28D10, 37G11, 37H8, 38G5, 38H3, 39F11, 41C6, 42E12, 43A11, 44E10, 47D12, 61C2, 75B8, 85A3, 9E8, 19B9, 45C1, 125E1, 163E12, 166E2, and 175D10 generated against CLD18 represents a diversity of binding characteristics to different epitopes and topologies of human CLD18.

A combination of different properties described in examples 3b, c, d, e, g, h, and j can be used to categorize monoclonal antibodies into such different classes.

4. Immunohistochemistry (IHC)

A CLD18A2 epitope specific antibody generated by immunization with the peptide of SEQ ID NO: 21 was used for immunohistochemical characterisation of CLD18A2 expression. Paraffin embedded tissue sections derived from a comprehensive panel of normal and tumor tissues were used for protein expression and localisation analyses. No significant expression was detected in any other

0 normal organ tissue except stomach (see Tab. 2, Fig. 16A). In contrast, CLD18A2 expression was verified by immunohistochemistry in different cancers including stomach cancer and lung cancer (Fig. 16B).

Interestingly, expression of CLD18A2 protein in gastric mucosa was restricted to terminally differentiated cells of the gastric epithelium in the base and pit regions.

5 In contrast, cells in the neck region of gastric mucosa, in particular gastric stem cells in the isthmus part, which replenish the entire mucosa, do not express CLD18A2 (Fig. 16C).

too

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Tab. 2: CLD18A2 expression in normal and tumor tissues as analysed by IHC

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Tissue type Result

Adrenal

Bladder

Blood cells

Bone Marrow

Breast

Colon

Endothelium

Esophagus

Fallopian tube

Heart

Kidney (glomerulus, tubule)

Liver

Lung

Lymph node

Ovary

Pancreas

Parathyroid

Pituitary

Placenta

Prostate

Skin

Spleen

Stomach +

Striated muscle

Testis

Thymus

Thyroid

Ureter

Uterus (cervix, endometrium)

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The monoclonal antibody 39F11 was used for immunohistochemical CLD18A2 specific studies. As shown in Fig. 17A, no significant reactivity was detectable on all tested normal tissues except stomach (Fig. 17A), whereas stomach carcinomas and lung carcinomas remain 5 strongly positive (Fig. 17B).

Another group of antibodies of the invention shows a specific cancer staining pattern with binding to stomach cancer but no reactivity with normal stomach tissue. Such a staining pattern is shown in Fig. 18A with monoclonal antibody 26B5.

Immunohistochemistry was used for specificity analysis of 175D10 (Fig. 18B), 43A11 (Fig. 0 18C), 163E12 (Fig.l8D) and 45C1 (Fig. 18E) on sections derived from HEK293 tumor cell lines: HEK293 tumor cell lines stably expressing human CLD18A2 (HEK293-CLD18A2) or

CLD18A1 (HEK293-CLD18A1) or being transfected with an expression control plasmid containing only the antibiotic resistence gene for selection (HEK293-mock) were xenografted into mice to form solid tumors. No expression was detectable in mock-transfected HEK293 5 xenograft tumors. In contrast, strong and homogeneous membran-staining was observed in HEK293-CLD18A2 xenograft tumors and in stomach carcinoma specimens.

5. Complement Dependent Cytotoxicity (CDC)

a. CDC of monoclonal antibodies of Setl as measured by flow cytometry:

D Plasma for complement lysis was prepared by drawing blood from healthy volunteers into SMonovette-EDTA vacutainer tubes (Sarstedt, Niirmbrecht, Germany) which were then centrifuged at 600 g for 20 min. Plasma was harvested and stored at -20°C.

In a first set of experiments hybridoma supernatants were analyzed for their capability to induce complement dependent cytotoxicity (CDC) against HEK293 cells stably expressing human CLD18A2 (HEK293-CLD18A2). Cells were incubated with hybridoma supernatants containing monoclonal antibodies 85A3, 28D10, 24H5 or 26D12, respectively for 20 min. at room temperature. Following centrifugation (5 min. at 450 g) the supernatant was removed and 20% human plasma in DMEM (prewarmed to 37°C) was added to the cells and incubated

0 for another 20 min. at 37°C. Thereafter, cell lysis was determined on FACS by using the propidium iodide (PI) staining method. PI was added to a final concentration of 2.5 pg/ml. For flow cytometry, a BD FACSArray flow cytometer was used (BD Biosciences, Mountain View, CA). At least 10000 events were collected for analysis with cell debris excluded by adjustment of the forward sideward scatter (FCS) threshold. The percentage of lysed cells (PI102

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2018200685 30 Jan 2018 positive cells) is shown in Figure 19. Monoclonal antibodies 85A3, 28D10 and 26D12 induced lysis of 33.5%, 38.2% and 39.2%, respectively of HEK293-CLD18A2 cells, whereas

CDC mediated by 24H5 was only 19.3%.

b. CDC of monoclonal antibodies of Setl:

In a second set of experiments the specificity of monoclonal antibodies to induce CDC on CLD18A2 expressing cells was analyzed. Therefore, a set of antibodies binding either specific to human CLD18A2 or also binding to human CLD18A1 was tested for CDCinduction against CHO cells stably transfected with human CLD18A2 (CHO-CLD18A2) or 0 human CLD18A1 (CHO-CLD18A1). CHO-CLD18A2 and CHO-CLD18A1 cells were seeded 24 h before the assay with a density of 3 x 10⁴/well in tissue-culture flat-bottom microtiter plates. The next day growth medium was removed and the cells were incubated in triplicates with hybridoma supernatants adjusted to a concentration of 10 pg/ml containing monoclonal antibodies 24H5, 26D12, 28D10, 37G11, 37H8, 38G5, 38H3, 39F11, 41C6, 5 42E12, 43A11, 44E10, 47D12, and 61C2, respectively. Control cells were incubated with growth medium or growth medium containing 0.2% saponin for the determination of background lysis and maximal lysis, respectively. After incubation for 20 min. at room temperature supernatant was removed and 20% human plasma in DMEM (prewarmed to 37°C) was added to the cells and incubated for another 20 min. at 37°C. Then, supernatants 0 were replaced by PBS containing 2.5 pg/ml ethidium bromide and fluorescence emission after excitation at 520 nm was measured using a Tecan Safire. The percentage specific lysis was calculated as follows: % specific lysis = (fluorescence sample - fluorescence background) / (fluorescence maximal lysis - fluorescence background) x 100. Fig. 20 shows that monoclonal antibodies 26D12, 28D10, 37H8, 38H3 and 39F11 mediate high, monoclonal antibody 38G5 25 mediates medium, monoclonal antibodies 41C6 and 61C2 mediate low, and monoclonal antibodies 24H5, 37G11, 42E12, 43A11, 44E10 and 47D12 mediate no CDC against CHOCLD18A2 cells. In contrast, none of the antibodies is capable of inducing CDC against CHOCLDA1 cells, although 26D12, 28D10, 37H8, 38H3, 39F11, 41C6, 47D12 and 61C2 also bind to CLD18A1 as determined by flow cytometry and immunofluorescence.

c. Monoclonal antibody titration and CDC using monoclonal antibodies of Setl:

To measure the ability of the anti-CLD18 antibodies to induce CDC at low concentrations, an experiment was performed where three different antibodies were titrated. CHO-CLD18A2 cells growing in microtiter plates were incubated with a concentration range of 75B8 (100, 30,

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10, 3 and 1 pg/ml), 37H8 (10, 3.3 and 1 pg/ml) and 28D10 (10, 1 and 0.1 pg/ml), respectively, for 20 min. at room temperature. Supernatant was removed and 20% human plasma in DMEM (prewarmed to 37°C) was added to the cells and incubated for another 20 min. at 37°C. Before analysis using a Tecan Safire, supernatants were replaced by PBS 5 containing 2.5 μg/ml ethidium bromide. Figures 21A-C show the percentage of specific lysis as a function of antibody concentration. Monoclonal antibody 75B8 induces lysis of 31.0% CHO-CLD18A2 cells at 10 pg/ml, and drops to 6.2% at 1 pg/ml (Fig. 21 A), whereas monoclonal antibodies 28D10 and 37H8 still induce 39% and 26.5% specific lysis at 1 pg/ml (Fig. 2IB, C), respectively.

d. CDC of monoclonal antibodies of Set2 as measured by flow cytometry:

Serum for complement lysis was prepared by drawing blood from healthy volunteers into Serum-Monovette vacutainer tubes (Sarstedt, Nurmbrecht, Germany) which were then centrifuged at 600 g for 20 min. Serum was harvested and stored at -20°C. Control serum 5 was heat inactivated at 56°C for 30 min before storage.

Hybridoma supernatants were analyzed for their capability to induce complement dependent cytotoxicity (CDC) against KATO-III cells endogenously expressing human CLD18A2. Cells were incubated with crude or purified hybridoma supernatants containing monoclonal antibodies 45C1, 125E1, 163E12, 166E2, and 175D10, respectively for 30 min. at 37°C. 20% 0 human serum in RPMI was added to the cells and incubated for another 30 min. at 37°C. Thereafter, cell lysis was determined on FACS by using the propidium iodide (PI) staining method. PI was added to a final concentration of 2,5 pg/ml. For flow cytometry a BD FACSArray flow cytometer was used (BD Biosciences, Mountain View, CA). At least 10000 events were collected for analysis with cell debris excluded by adjustment of the forward 25 sideward scatter (FSC/SSC) threshold. Specific lysis was calculated by the following formula: specific lysis = (% Pi-positive cells in sample - % Pi-positive cells in sample with heat inactivated serum). Robust CDC mediated lysis was observed in particular for 163E12.

6. Antibody-Dependent Cellular Cytotoxicity (ADCC)

0 Hybridoma supernatants were analyzed for their capability to induce antibody-dependent cellular cytotoxicity (ADCC) against HEK293 cells stably expressing human CLD18A2 (HEK293-CLD18A2) or human CLD18A1 (HEK293-CLD18A1).

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a. Enrichment of human peripheral blood mononuclear cells: Human blood from healthy donors was diluted twice in phosphate buffer (PBS) and blood cells were layered on Ficoll (Lymphocyte Separation Medium 1077 g/ml, PAA Laboratories, cat. no. J15-004). Peripheral blood mononuclear cells (MNCs) were collected from the interphase, washed and resuspended in RPMI 1640 culture medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L- glutamine.

b. ADCC set up: Target cells were labeled with with a fluorescence enhancing ligand (BADTA, Perkin Elmer cytotoxicity assay kit DELFIA EuTDA Cytotoxicity Reagents, cat.

no. AD0116) for 30 minutes. After extensive washing in RPMI-10 supplemented with 10 mM probenecid (Sigma, cat. no. P8761), 10-20 mM HEPES, and 10% heat-inactivated fetal calf serum, the cells were adjusted to 1 x 10⁵ cells/ml. Labeled target cells, effector cells (MNCs), and supernatants containing monoclonal antibodies adjusted to a concentration of 10 pg/ml were added to round-bottom microtiter plates. For isolated effector cells, an effector to target (E:T) ratio of 100:1 (data not shown for 50:1 and 25:1) was used. After incubation (2 hours,

37°C), assays were stopped by centrifugation, and fluorescence ligand release from duplicates was measured in europium counts in a time-resolved fluorometer. Percentage of cellular cytotoxicity was calculated using the following formula: % specific lysis = (experimental release counts - spontaneous release counts) / (maximal release counts - spontaneous release counts) x 100, with maximal fluorescence ligand release determined by adding Triton X-100 (0,25% final concentration) to target cells, and spontaneous release measured in the absence of antibodies and effector cells. Figure 22 shows that monoclonal antibodies 26B5, 37H8, 38G5, 47D12, and 61C2 mediate ADCC against HEK293-CLD18A2 cells. In contrast, these antibodies induce no significant or only low level cytotoxicity on CLD18A1 targets 2 5 demonstrating a CLD18A2 specific ADCC (Figure 23).

7. Proliferation Inhibition

Purified murine monoclonal antibodies were analyzed for their capability to inhibit cell growth of KATO-III cells endogenously expressing human CLD18A2.

lxl0⁴ target cells endogenously expressing CLD18A2 (KATO-III) were cultured in the presence of approximatly 10pg monoclonal antibodies.

DELFIA Cell Proliferation Kit (Perkin-Elmer, Cat. No. AD0200) is a non-isotopic immunoassay based on the measurement of 5-bromo-2’-deoxyuridine (BrdU) incorporation during DNA synthesis of proliferating cells in microplates. Incorporated BrdU is detected

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2018200685 30 Jan 2018 using europium labelled monoclonal antibody. To allow antibody detection cells are fixed and DNA denatured using Fix solution. Unbound antibody is washed away and DELFIA inducer is added to dissociate europium ions from the labelled antibody into solution, where they form highly fluorescent chelates with components of the DELFIA Inducer. The fluorescence 5 measured - utilizing time-resolved fluorometry in the detection - is proportional to the DNA synthesis in the cell of each well.

Strong inhibition of proliferation was observed with antibodies 125E1, 163E12, 45C1, 37G11, 37H8, 28D10 and 166E2, respectively. . Moderate inhibition of proliferation was observed with murine antibodies 43A11, 175D10, 42E12, 26D12, 61C2 and 38H3, 0 respectively.

8. Performance in therapeutic mouse xenograft models

Therapeutic potential of the identified monoclonal antibodies binding specifically to CLD18A2 was studied in therapeutic xenograft models.

a. Early treatment of highly CLD18A2 expressing tumors in mice

SCID mice were subcutaneously inoculated with 1 x 10⁷ HEK293 cells stably expressing high levels of human CLD18A2 (HEK293-CLD18A2). Expression levels of human CLD18A2 in HEK293-CLD18A2 cells were comparable with expression levels in primary gastric cancers from patients. Each experimental treatment group comprised 10 mice (number of mice per group n=10). Therapy of mice started 3 days after tumor inoculation. 200 pg of purified hybridoma supernatants representing murine monoclonal antibodies 26B5, 26D12, 28D10, 37G11, 37H8, 38G5, 39F11, 42E12, 43A11, 38H3, or 61C2 were injected once per week for 4 weeks intravenously. Alternatively 200 pg of purified hybridoma supernatants containing murine monoclonal antibodies 45C1, 125E1, 163E12, 166E2, or 175D10 were administered twice per week for 6 weeks by alternating intravenous and intraperitoneal injection. Tumor growth of treated mice was monitored twice per week (Tumor Volume = Length x Width x Width divided by 2 in mm ). The mice were killed if the tumor reached a volume of 500 mm or in case of severe morbidity. Fig. 24 exemplifies robust inhibition of HEK293-CLD18A2

0 tumor cell growth by antibodies of the invention. Fig. 25A and 25B show prolongation of survival by treatment with antibodies of the invention in an early treatment xenograft model using HEK293-CLD18A2 cells.

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b. Late onset treatment of advanced highly CLD18A2 expressing tumors in mice

The same tumor xenograft model based on HEK293-CLD18A2 cells was designed as a late therapy onset protocol as opposed to the early treatment described above. On day 27 after tumor cell inoculation mice were randomized in test groups each comprising 5-6 mice and 5 therapy was initiated with 200 pg of purified hybridoma supernatants containing murine monoclonal antibodies 43A11, 163E12, and 175D10, respectively. Antibodies were administered twice per week for 6 weeks by alternating intravenous and intraperitoneal injection. Also in this model antibodies of the invention were shown to inhibit tumor growth. For several antibodies this resulted in prolongation of survival (Fig. 26).

c. Early treatment of tumors expressing low levels of CLD18A2

SCID mice were subcutaneously inoculated with 2 x 10⁵ cells of the DAN-G tumor cell line, an infiltrating human pancreatic adenocarcinoma cell line that constitutively expresses CLD18A2 protein at low level. Treatment of mice (10 per group) was initiated 3 days after tumor grafting: 200 pg of purified hybridoma supernatants containing murine monoclonal antibodies 45C1, 125E1, 163E12, 166E2, or 175D10 were administered twice per week for 6 weeks by alternating intravenous and intraperitoneal injection. Owing to the aggressive and fast tumor growth of the pancreatic DAN-G tumor cell line in vivo mice developed tumor cachexia and died within a few days. Even though, as a consequence, the window for measuring therapeutic effects was narrow, tumor growth inhibition and prolonged survival mediated by antibodies of the invention was also observed in this model (Fig. 27A and 27B).

d. Antibodies of the invention do not elicit side effects in mice

A murine CLD18A2-specific primer pair (s: CTA CCA AGG GCT ATG GCG TTC, as: GCA 25 CCG AAG GTG TAC CTG GTC) was used in RT-PCR analyses to amplify cDNA derived from a comprehensive panel of normal mouse tissues (see Fig. 28).

Expression of murine CLD18A2 was not detectable in any tested normal tissues, except stomach (see Fig. 28). Furthermore, an CLD18A2 specific antibody, which crossreacts with human and mouse CLD18A2, was used for immunohistochemical analysis of CLD18A2

0 expression in a large panel of normal mouse tissues (see Tab. 3). Except for normal gastric tissue all tested normal tissues show no CLD18A2 expression. As we observed for the human CLD18A2, we also found for the mouse counterpart that while the surface epithelia- and deeper crypt cells express CLD18A2 at their cell surface, the central neck region is CLD18A2 negative (see Fig. 29 A-C). In summary, tissue distribution of CLD18A2 appears to be

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Tab. 3: CLD18 expression within murine normal tissues as analysed by immunhistochemistry tissue CLD18 expression cerebellum cerebrum colon esophagus heart kidney liver lung lymph node ovary pancreas skeletal muscle spleen stomach + thymus bladder 5 We further investigated potential side effects mediated by antibodies 125E1, 163E12, 166E2 and 175D10 in mice. All of these antibodies had been previously shown by FACS analysis to react with the murine CLD18A2 as well as with the human protein.

Neither were any visible side effects observed in mice during and after treatment with these 10 antibodies, nor were any histomorphological correlates of toxicity observed in the gastric mucosa of antibody treated mice as compared to untreated (PBS- treated) mice (see Figure

30).

9. Chimerization of antibodies

a. Generation of mouse/human chimeric monoclonal antibodies

Total RNA and subsequently single stranded cDNA was prepared from human peripheral blood mononuclear cells (PBMC) and from human spleen tissue by standard methods known to those skilled in the art, for example by using RNeasy Midi Kit (Qiagen) and Superscript II

0 reverse transcriptase (Invitrogen).

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The constant region of the human kappa light chain was amplified from PBMC cDNA by PCR. The sense oligomer (SEQ ID NO:38) added a BamHI restriction site at the 5’ end of the constant region and changed the original nucleic acid sequence 5’-CGAACT-3’ coding for the 5 first two amino acids (Arg-Thr) of the constant region into 5’-CGTACG-3’, generating a BsiWI restriction site without changing the amino acid sequence. The antisense oligomer (SEQ ID NO:39) included a stop codon and added a Notl restriction site at the 3’ end of the amplified constant region. The PCR product as well as a standard expression vector (for example pcDNA3.1(+), Invitrogen) were sequentially incubated with BamHI and Notl 0 restriction enzymes. The vector was additionally treated with calf intestinal alkaline phosphatase to prevent recirculation. The constant region was finally ligated into the vector, so that any forthcoming fusion of a variable region in front of the constant region is now possible via a Hindlll restriction site (5’-AAGCTT-3’) from the residual vector multiple cloning site and via the BsiWI restriction site (5’-CGTACG-3’) generated with the PCR 5 product. The sequence of the human kappa light chain constant region inserted into the vector is listed as SEQ ID NO:40, the amino acid sequence of the human kappa constant region is listed as SEQ ID NO:41.

The constant region of the human gamma-1 heavy chain was amplified from spleen cDNA by 3 PCR. The 5’ phosphorylated sense oligomer (SEQ ID NO:42) was placed over the naturally occurring Apal restriction site, located 11 nucleotides downstream of the beginning of the constant region, and added a Hindlll restriction site at the 5’ end of the amplified part of the constant region. The 5’ phosphorylated antisense oligomer (SEQ ID NO: 43) included a stop codon and added a Notl restriction site at the 3 ’ end of the thus amplified constant region. The

5 thus generated PCR product was blunt ended and 5’ phosphorylated. The amplified gamma constant region was verified to be of the IgGl subclass by PCR with a discriminating antisense oligomer (SEQ ID NO: 44) and by sequencing. A standard expression vector (for example pcDNA3.1(+)/Hygro, Invitrogen) with a different antibiotic resistance (for example hygromycin) than that of the vector used for expression of the light chain (for example

0 neomycin) was incubated with Pmel restriction enzyme to completely remove the multiple cloning site leaving blunt ends. The vector was additionally treated with calf intestinal alkaline phosphatase to prevent recirculation. The constant region was finally ligated into the vector, so that any forthcoming fusion of a variable region in front of the constant region is now possible via the Hindlll restriction site (5’-AAGCTT-3’) and via the Apal restriction site

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2018200685 30 Jan 2018 (5’-GGGCCC-3’), both generated with the PCR product. The correct orientation of the constant region in the vector, i.e. suitable for the preceeding promoter of the vector, was verified by sequencing. Due to the position of the Apal restriction site, any amplification of a variable region for this purpose has to include the first 11 nucleotides of the sequence of the human gamma-1 constant region in addition to the sequence of the Apal site. The sequence of the thus amplified human gamma-1 heavy chain constant region inserted into the vector is listed as SEQ ID NO:45, the amino acid sequence of the thus expressed human gamma-1 constant region is listed as SEQ ID NO: 46.

Tab. 4: mouse hybridoma cell lines used for antibody cloning

	clone	mAb	Isotype	variable region	oligomer pair in PCR	chimerized antibody
heavy
chain	43A11	182-D1106-062	IgG2a	SEQIDNO:55, 132	SEQ ID NO:70, 71	SEQ ID NO: 100, 115
	163E12	182-D1106-294	IgG3	SEQIDNO:56, 133	SEQ ID NO:72, 73	SEQ IDNO:101, 116
	125E1	182-D 1106-279	IgG2a	SEQ IDNO:57, 134	SEQ ID NO:74, 75	SEQ ID NO: 102, 117
	166E2	182-D1106-308	IgG3	SEQ IDNO:59, 136	SEQ ID NO:78, 79	SEQ ID NO: 104, 119
	175D10	182-D 1106-362	IgGI	SEQ IDNO:58, 135	SEQ ID NO:76, 77	SEQ ID NO: 103, 118
	45C1	182-D758-187	IgG2a	SEQ IDNO:60, 137	SEQ ID NO:80,81	SEQ ID NO: 105, 120
light
chain	43A11	182-D 1106-062	IgK	SEQ IDNO:62, 139	SEQ ID NO:84, 85	SEQ ID NO: 107, 122
	163E12	182-D 1106-294	IgK	SEQ IDNO:61, 138	SEQ ID NO:82, 83	SEQ ID NO: 106, 121
	125E1	182-D 1106-279	IgK	SEQ ID NO:63, 140	SEQ ID NO: 86, 87	SEQ ID NO: 108, 123
	166E2	182-D1106-308	IgK	SEQ ID NO:66, 143	SEQ ID NO:92, 93	SEQ IDNO:111, 126
	175D10	182-D 1106-362	IgK	SEQ ID NO:65, 142	SEQ ID NO:90, 91	SEQ IDNO:110, 125
	45C1	182-D758-187	IgK	SEQ IDNO:64, 141	SEQ ID NO:88, 89	SEQ ID NO: 109, 124
	45C1	182-D758-187	IgK	SEQ ID NO:67, 144	SEQ ID NO:94, 95	SEQ ID NO: 112, 127
	45C1	182-D758-187	IgK	SEQ IDNO:68, 145	SEQ ID NO:96, 97	SEQ ID NO: 113, 128
	45C1	182-D758-187	IgK	SEQ IDNO:69, 146	SEQ ID NO:98, 99	SEQ ID NO: 114, 129

Corresponding to their murine counterparts the chimeric monoclonal antibodies were named adding the prefix “ch-“ e.g. ch-43All, ch-163E12, ch-125El, ch-166E2, ch-175D10, ch45C1.

Amplification of the murine variable regions of light and heavy chains was carried out according to the “step-out PCR” method described in Matz et al. (Nucleic Acids Research, 1999, Vol. 27, No. 6). For this, total RNA was prepared from monoclonal hybridoma cell no

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2018200685 30 Jan 2018 lines (see Tab. 4) by standard methods known to those skilled in the art, for example with the use of RNeasy Mini Kit (Qiagen). Single stranded cDNA was prepared according to the “template-switch” method also described in Matz et al. (Nucleic Acids Research, 1999, Vol. 27, No. 6, 1558). In addition to an (dT)30 oligomer (SEQ ID NO: 47), it included a 5 DNA/RNA hybrid oligomer (SEQ ID NO: 48) serving as an 5’ adaptor for template switching during polymerization of the cDNA strand. In this adaptor oligomer the last three nucleotides were ribo- instead of deoxyribonucleotides. The subsequent “step-out PCR” used an antisense oligomer targeted to the constant region of the mouse kappa chain or to the constant region of the subclasses 1, 2a or 3 of the gamma chains (SEQ ID NO: 49 to 52, respectively). The IgG D subclass of the murine monoclonal antibody produced by the hybridoma cell lines was afore immunologically analyzed with IsoStrip (see Example 1), and the appropriate antisense oligomer was chosen accordingly (see Tab. 4). A primer mix served as the sense oligomer in the “step-out PCR”, comprising the two oligomers listed in SEQ ID NO: 53 and 54. Some hybridoma cell lines expressed more than one heavy or light chain (in addition to the chains 5 expressed by the myeloma cell line used for the generation of hybridomas). Table 4 summarizes the SEQ ID NOs of the cloned and sequenced variable regions of the murine antibody chains (SEQ ID NO: 55 to 69 and SEQ ID NO: 132 to 146) and of the cloned and sequenced full-length chimieric antibody chains (SEQ ID NO: 100 to 129).

The identified murine variable regions were then amplified by PCR omitting the 5’ UTR and the 3’ mouse constant region, adding restriction sites to the ends which allowed subcloning into the prepared expression vectors carrying the human constant regions. In addition, the sense oligomers provided a consensus Kozak sequence (5’-GCCGCCACC-3’ or 5’AGCCACC-3’) and the antisense oligomers for heavy chain variable regions included the

5 first 11 nucleotides of the human gamma-1 constant region in addition to the Apal restriction site (see Tab. 4, SEQ ID NO: 70 to 99). Kappa light chain variable regions were cloned using Hindlll and BsiWI restriction enzymes, gamma heavy chain variable regions demanded Hindlll and Apal restriction enzymes. The heavy gamma chain variable region of monoclonal antibody 45C1 contained an internal Hindlll restriction site - here, the compatible Bsal

0 enzyme was used instead (see SEQ ID NO: 80). SEQ ID NO: 100 to 114 show the nucleic acid sequences of the resulting chimerized antibodies (see Tab. 4). SEQ ID NO: 115 to 129 show the amino acid sequences of the accordingly expressed chimerized antibodies (see Tab. 4)·

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b. Generation and production of chimeric antibodies against CLD18

Mammalian cell lines producing chimeric antibodies with CLDI8 specificity were generated. The cell lines derived from HEK293T cells (ATCC CRL-11268). One day before transfection, 2.5 x 10 cells were plated in a 14.5 cm tissue culture dish and cultured in 20 ml 5 of complete medium, or alternatively 1 x 10⁷ cells were plated in a 10 cm tissue culture dish and cultured in 10 ml of complete medium, or alternatively 0.6 x 10⁶ cells were plated in a well of a 12-well tissue plate and cultured in 2-3 ml of complete medium (complete medium: DMEM:F12 medium supplemented with 10% FBS without antibiotics). The recommended cell density at the time of transfection should be 90% confluence. Immediately before D transfection, medium was replaced by fresh medium. HEK293T cells were transfected with transfection reagents, e.g. Lipofectamine 2000 (Invitrogen, 11668-019) or alternatively Polyethylenimine (Sigma-Aldrich, 408727). Exemplified for transfection of HEK293T cells a total DNA amount of 110 pg or 296 pg was used for a 14.5 cm tissue dish, and the ratio of transfection agent to DNA was 1:2.5 and 1:12 for Lipofectamine 2000 and PEI, respectively. 5 24 h after transfection medium was replaced with a GMP suitable medium, e.g. X-Vivo 15 (Cambrex) or a chemical defined medium like Pro293a (Cambrex) without serum. Transfected HEK293T cells producing chimeric monoclonal antibodies against CLDI8 were cultured for further 96 h. Crude supernatants were harvested, sterile filtered and purified by protein A-sepharose. Antibody concentration was determined by BCA Assay and purity 0 checked by sodium dodecylsulphate gel electrophoresis and coomassie staining.

c. Binding Characteristics of Chimeric Monoclonal Antibodies

Binding specificity of the cloned and generated chimeric monoclonal antibodies to CLD18A2 was analyzed by flow cytometry as described in Example 3. HEK293 living cells stably expressing human CLD18A2 (HEK293-CLD18A2) and HEK293 cells stably expressing human CLD18A1 (SEQ ID NOs: 7, 8) (HEK293-CLD18A1) were incubated for 30 min. at 4°C with purified HEK293T cell culture supernatants containing chimeric monoclonal antibodies, followed by incubation with APC-conjugated F(ab’)2 fragment goat anti-human IgG Fey secondary antibody and counterstained with PI. Binding was assessed by flow

0 cytometry using a BD FACSArray.

Similarly, endogenously CLD18A2 expressing human tumor cell lines, for example ΚΑΤΟΠΙ and NUGC-4 cells, were analyzed by flow cytometry.

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Fig. 31A and B show flowcytometric analyses of chimeric antibodies ch-43All, ch-125El, ch-163E12, ch-166E2, and ch-175D10. All of them show native epitope recognition and exhibit specific and strong binding to CLD18A2 but not CLD18A1 expressing cells.

d. Complement Dependent Cytotoxicity (CDC)

Serum for complement lysis was prepared by drawing blood from healthy volunteers into Serum-Monovette vacutainer tubes (Sarstedt, Nurmbrecht, Germany) which were then centrifuged at 600 g for 20 min. Serum was harvested and stored at -20°C. Control serum was heat inactivated at 56°C for 30 min before storage.

Protein A-sepharose-purified chimeric antibodies of this invention were analyzed for their capability to induce complement dependent cytotoxicity (CDC) against KATO-III cells endogenously expressing human CLD18A2, as well as stably transfected CHO-CLD18A2 cells. Cells were incubated with monoclonal antibodies ch-163E12, ch-166E2, and ch175D10, respectively, in a final concentration of 2.5 pg/ml to 35 pg/ml for 30 min. at 37°C.

20% human serum in RPMI was added to the cells and incubated for another 30 min. at 37°C.

Thereafter, dead and living cells were discriminated by PI staining in a final concentration of 2.5 pg/ml and percentage of antibody-mediated cell lysis was determined by flow cytometry. For flow cytometric analysis a BD FACSArray flow cytometer was used (BD Biosciences, Mountain View, CA). At least 10000 events were collected for analysis with cell debris excluded by adjustment of the forward sideward scatter (FSC/SSC) threshold. Specific lysis was calculated by the following formula: specific lysis = (% Pi-positive cells in sample - % Pi-positive cells in sample with heat inactivated serum). Specific lysis mediated by CDC was shown for several antibodies. All three antibodies mediated robust CDC on CHO-CLD18A2 cells (Figure 32). On KATO-III cells antibodies ch-163E12 and ch-175D10 were inducers of 25 robust CDC.

e. Antibody-Dependent Cellular Cytotoxicity (ADCC)

FPLC-purified, chimeric antibodies of the invention were analyzed for their capability to induce antibody-dependent cellular cytotoxicity (ADCC) against KATO-III cells

0 endogenously expressing human CLD18A2.

Human blood from healthy donors was diluted twice in phosphate buffer (PBS) and blood cells were layered on Ficoll (1077 g/ml, Pharmacia). After centrifugation, peripheral blood mononuclear cells (PBMC) were collected from the interphase, washed and resuspended in

5 X-Vivo-15 culture medium supplemented with 5% heat-inactivated human serum.

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15h before the assay, KATO-III cells were transfected with luciferase and plated at 5 x 10⁴ cells/well in a white microplate.

For the assay, effector cells (PBMC, prepared as described above) at an effector to target (E:T) ratio of 20:1 and FPLC-purified chimeric antibodies were added and incubated for 2 5 3h at 37°C, 5% CO2. Final concentration of the antibody in the well was 50μg/ml. After 2-3h of pre-incubation, lucifer yellow (BD Biosciences, San Jose USA) was added at lmg/ml. Luminescence resulting from the oxidation of lucifer yellow by the luciferase of viable cells was measured continually for up to 6h using a microplate-reader (Infinite200, Tecan, Switzerland). Percentage of cellular cytotoxicity was calculated using the following formula: D % specific lysis = 100-((sample luminescence counts - spontaneous luminescence counts) / (maximal luminescence counts - spontaneous luminescence counts) x 100), with the spontaneous luminescence determined by adding Triton X-100 (0,2% final concentration), and the maximal signal measured in the absence of antibodies.

Using this assay it was shown that monoclonal antibodies ch-163E12 and ch-175D10 mediate 5 strong ADCC on KATO-III cells (Fig. 33).

f. Proliferation Inhibition

FPLC-purified chimeric antibodies of the invention were analyzed for their capability to D inhibit cell growth of KATO-III cells endogenously expressing human CLD18A2.

Target cells (KATO-III) were cultured in the presence of chimeric respective antibodies (see proliferation inhibition of murine antibodies, Example 7). FPLC purified chimeric antibodies ch-163E12 and ch-166E2 were shown to inhibit cell proliferation.

5 10. Selection of antibodies as clinical lead candidates

Ideal clinical leads may cover a wide range of therapeutic and diagnostic applications (see also section IV - Uses and Methods of the Invention). According to the invention antibodies directed to CLD18-A2 are provided. It is shown that the antibodies provided according to the

0 invention offer a broad spectrum of properties regarding specificity, ability to induce CDC and ADCC and inhibit proliferation of cells expressing CLD18, in particular tumor cells. Furthermore, it has been demonstrated that chimerisation of antibodies may lead to the aquisition of additional Fc-dependent effector functions not present in the parental murine molecule. For example, it is shown herein that antibody 175D10 with murine IgGI does not

5 induce complement dependent cytotoxicity (see Example 5), while ch-175D10 with human

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IgGl induces specific lysis of constitutively CLD18 expressing tumor cells (see Tab. 5 and

Tab. 6).

Antibodies provided according to the present invention may be categorized into distinct classes according to their binding properties and their ability to mediate effector functions on 5 cells expressing CLD18. From the antibodies provided according to the present invention, clinical lead candidates may be selected based on their functional characteristics. An overview of properties for selected murine and chimeric antibodies of the invention is given in Tab. 5 and Tab. 6, respectively.

Clinical lead candidates of the invention may have one or more of the following properties:

a) binding to human CLD18A2 but not to human CLD18A1 (e.g. 43A11, 45C1, 125E1, 163E12, 166E2 and 175D10, and ch-43All, ch-45Cl, ch-125El, ch-163E12, ch-166E2 and ch-175D10). For examples, see figures 6A and 6B.

b) binding to mouse CLD18A2 but not to mouse CLD18A1 (e.g. 125E1, 163E12, 166E2 and 175D10). For examples, see figures 15A and 15B.

c) binding to CLD18 naturally expressed by tumor cells (e.g. 45C1, 43A11, 125E1, 163E12, 166E2 and 175D10, and ch-45Cl, ch-43All, ch-125El, ch-163E12, ch-166E2 and ch175D10). For examples, see figure 13

d) binding to CLD18 in intercellular contact zones (e.g. 45C1, 43A11, 125E1, 163E12, 166E2 and 175D10). For examples, see figures 12A and 12B.

e) mediating CDC induced killing of cells, which express CLD18 (e.g. 45C1, 125E1, 163E12, 166E2 and 175D10, and ch-163E12 and ch-175D10). For examples, see figure 32.

f) mediate ADCC induced killing of cells expressing CLD18 (e.g. ch-163E12 and ch175D10). For examples, see figure 33.

g) inhibiting proliferation of cells expressing CLD18 (e.g. 45C1, 125E1, 163E12, 166E2 and 175D10, and ch-163E12 and ch-166E2).

h) inhibiting tumor growth in xenograft models with cells expressing CLD18 (e.g. 43All, 125E1, 163E12, 166E2, and 175D10). For examples, see figure 24.

i) prolonging survival in xenograft models with cells expressing CLD18 (e.g. 43All,

0 125E1, 163E12, 166E2 and 175D10). For examples, see figure 25B.

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Exemplary overview of properties for lead candidate selection

Table 5: murine antibodies

antibody	binding of human CLD18A2 but not Al	binding of mouse CLD18A2 but not Al	binding of CLD18 on naturally expressing tumor cells	binding to CLD18 in contact zones	mediating CDC on CLD18 expressing cells	inhibiting proliferation of cells expressing CLD18	inhibiting tumor growth in xenograft expressing CLD18	prolonging survival in xenograft expressing CLD18
45C1	4-	-	+	+	(+)	+	(+)	(+)
125E1	+	+	+	+	(+)	+	+	+
163E12	+	+	+	+	+	+	+	+
175D10	+	+	+	+	(+)	(+)	+	+

legend: + excellent performance, (+) performance in different setups.

Table 6: chimeric antibodies

antibody	binding of human CLD18A2 but not Al	binding of CLD18 on naturally expressing tumor cells	mediating CDC on CLD18 expressing cells	mediating ADCC on CLD18 expressing cells	inhibiting proliferation of cells expressing CLD18
ch-45Cl	+	+	n.d.	n.d.	n.d.
ch-125El	+	+	n.d.	n.d.	n.d.
ch-163E12	+	+	+	+	+
ch-175D10	+	+	+	+	n.d.

legend: + excellent performance, (+) performance in different setups, n.d. not done.

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New International Patent Application

Ganymed Pharmaceuticals AG, et al.

„Monoclonal Antibodies Against Claudin-18 For Treatment Of Cancer”

Our Ref.: 342-31 PCT

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Additional Sheet for Biological Material

Identification of further deposits:

1) The Name and Address of depositary institution for the deposits (DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM ACC2741, DSM ACC2742, DSM ACC2743, DSM ACC-2745, DSM ACC2746, DSM ACC2747, DSM ACC2748) are:

DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH Mascheroder Weg lb 38124 Braunschweig DE

2) The Name and Address of depositary institution for the deposits (DSM ACC2808, DSM ACC2809, DSM ACC2810) are:

DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH Inhoffenstr. 7 B 38124 Braunschweig DE

Date of desposits	Accession Numbers	The indications made below relate to the deposited microorganism in the description on the following page(s)
October 19, 2005	DSM ACC2738	page 16, line 34
October 19, 2005	DSM ACC2739	page 17, line 1
October 19, 2005	DSM ACC2740	page 17, line 2
October 19, 2005	DSM ACC2741	page 17, line 3
October 19, 2005	DSM ACC2742	page 17, line 4
October 19, 2005	DSM ACC2743	page 17, line 5
November 17, 2005	DSM ACC2745	page 17, line 6
November 17, 2005	DSM ACC2746	page 17, line 7
November 17, 2005	DSM ACC2747	page 17, line 8
November 17, 2005	DSM ACC2748	page 17, line 9
October 26, 2006	DSM ACC2808	page 17, line 10
October 26, 2006	DSM ACC2809	page 17, line 11
October 26, 2006	DSM ACC2810	page 17, line 12

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Additional Indications for all above mentioned deposits:

- Mouse (Mus musculus) myeloma P3X63 Ag8U. 1 fused with mouse (Mus musculus) splenocytes

- Hybridoma secreting antibody against human claudin-18 A2

3) Depositor:

All above mentioned depositions were made by:

Ganymed Pharmaceuticals AG FreiligrathstraBe 12 55131 Mainz DE

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Applicant's or agent's	International applicationNo.
file reference 342-31 PCT	PCT/EP2006/011302

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INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL (PCT Rule 13Z>w)

A. The indications made below relate to the deposited microorganism or other biological material referred to in the description on page 16 , line ?3 .

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet |X|

Name of depositary institution

DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH

Address of depositary institution (including postal code and country)

Mascheroder Weg 1b 38124 Braunschweig DE

Date of deposit	Accession Number
October 19, 2005	DSM ACC2737

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet □

- Mouse (Mus musculus) myeloma P3X63Ag8U.1 fused with mouse (Mus musculus) splenocytes

- Hybridoma secreting antibody against human claudin-18A2

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e.g., Accession Number of Deposit)

For receiving Office use only

For International Bureau use only □ This sheet was received with the international application □ This sheet was received by the International Bureau on:

Authorized officer

Authorized officer

Form PCT/RO/134 (July 1998; reprint January 2004)

119

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WO 2007/059997

2018200685 30 Jan 2018 inymed Pharmaceuticals AG eiligraihstr. 12 >131 Mainz

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

DSMZ

Devbch'

SammlunQ A

Mikroorgon’isrnon W und Zallkullvren GmbH ·**

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at die bottom of ibis page

IDENTIFICATION OF THE MICROORGANISM

dentiEcation reference givoi by the DEPOSITOR: 182-D 1106-055	Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY:
	DSM ACC2737

it. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

Tbe microorganism identified under I. above was accompanied by;

( χ ) a scientific description ( ) a proposed taxonomic designation (Mark with a cross where ipplicablc).

HI. RECEIPT AND ACCEPTANCE

ThiB fntcmotiontii Deposlsiy Authority accepts the rnicrtrargHniim ifienilikd under I. ahev—, -Much web received by it On 2005-10-19 (Date ofthe original deposit)'.

IV. RECEIPT OF REQUEST FOR CONVERSION

The microorganism identified under! above was received by this International Depositary Authority on and a request to eoovottlbe original deposit to a deposit under the Budapest Treaty was received by it on for convention).

(date of original deposit) (date of receipt nf request

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: DSMZ-DEtirSCHE SAMMLUNG VON	SigaatnrefB) of pnson(s) having fhe power to represent flte
MIKROORGANISMEN UND ZELLKULTUREN GmbH	International Depository Authority or Of authorised oJtiCial(s):
Address; MnscherodcrWeg lb D-38124 Enunacbweig
	Date 2005-11-01

¹ Where Rule C.4 (d) applies, sack date is the date on which tbe status of intcmadousl depositary authority wns Required,

Foito DSMZ-BP/4 (sole page) 11/2001

120

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2018200685 30 Jan 2018 ianymed Pharmaceuticals AG Teiligrathstr. 12

55131 Mainz

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL form νΐΑΒΠ-ΠΎ STATEMENT issued pursuant to Rule 10.2 by the

INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page

Dfubchfi Sommiuoq won Mikraorgnnisman und folliishiiftn GmbH *

I, DEPOSITOR	n. IDENTIFICATION OF THE MICROORGANISM
Name:	Ganymed Pharmaceuticals AG Freiligratbslr, 12	Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY:
Address:	55131 Mainz	DSM ACC2737
		Date of the deposit nr the transfer’:
		2005-10-19

ΙΠ. VIABILITY STATEMENT

The viobllity of the microorganism identified under Π above was tested on 2005-.10-.19

On that date, the said microorganism won (χ)⁴ viable ( )' no longer viable

IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED

V. INTERNATIONAL DEPOSITARY AUTHORITY

Nunte: DSMZ-DEUTSCHE SAMMLUNG VON MfKROORGANISMEN UND ZELLKULTUREN GmbH	5ignaturc(a) of person[s) having the power to represent the International Depositary Authority or of authorized qfTieialfs):
Address: MascheroderWcg 1¼ £>-38124 Braunschweig	Dote: 2005-11-01

¹ Indicate the date of original deposit or, where a new deposit or a transfer has been made, the boost recent relevant date (date of the flew deposit or date of the transfer).

² In the eases refereed to in Rulo 10.2(a) (ii) and (iii), refer to the most recent viability test, ’ Mark with a cross the applicable box.

⁴ Fill in if the information hns been requested nnd if the results of the test were negative.

Form DSMZ-BP/9 (solcpage) 12/2001

121

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2018200685 30 Jan 2018 mypied Pharmaceuticals AG nligrathstr. 12 131 Mainz

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

DSMZ

SofnrftluriS *°ⁿ MikroorgowiMn irn<l ZftBkuitumn GmbH *

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page

IDENTIFICATION OF THE MICROORGANISM

rotificatiem reference fiivcn by the DEPOSITOR:	Accession number given by the
1.82-D 1106-056	INTERNATIONAL DEPOSITAR Y AUTHORITY:
DSM ACC2738

SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION ie microorganism identified under! above was accompanied by:

( X ) ^a scientific description ( ) a proposed taxonomic designation lark with, a cross where applicable).

. RECEIPT AND ACCEPTANCE

This International Depositaty Authority accepts the microorganism identified under I. above, which was received by it on 2005-10-19 (Date of the original deposit)¹.

IV. RECEIPT OF REQUEST FOR CONVERSION
The microorganism identified under I above was received by this International Dcpositaiy Authority on and n. request to convert the original deposit to n deposit under the Budapest Treaty was received by it on for conversion).	(date of original depoaiL) (date of receipt of request'
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON MIKROORGANISMENUND ZELLKULTUREN GmbH	Signature^) of peiKin(o) having the power to represent the International Depositary Authority or of authorised officials,:
Address: Mascheroder Wee lb D-38124 Braunschweig	. (// Date: 2005-11-01

¹ Where Role 6.4 (d) applies, such date ie the date on which the smtvs of international depositary authority was acquired.

Form DSM2-BP/4 (sole pop) 12/2001

122

WO 2007/059997

PCT/EP2006/011302

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BUDAPEST TREATY ON THE INTERNATIONAL · RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR. THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM ’

Ganymed Pharmaceuticals AG

Freiligrathslr. 12

55131 Mainz

VIABILITY STATEMENT issued pursue nt to Rule 10.2 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page

I. DEPOSITOR

Name;

Address; S5I31 _Majnz

Ganymed Pharmaceuticals ΛΟ Freiligrathslr. 12

Deutsche Sammlunp von MifcrOOVQOnismnn ' und Zollkuiturcn GmbH '

Π. IDENTIFICATION OF THE MICROORGANISM

Accession number given by the

INTERNATIONAL DEPOSITA RY AUTHORITY:

DSM ACC2738

Date of the deposit or the transfer';

2005-10-19

ΠΤ. VIABILITY STATEMENT

The viability of the microorganism identified under Π above was tested on 2005-10-19 On that date, the said microorganism was (x)^J viable ( )^Λ no longot viable

IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED⁴

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: DSMZ-DEUTSCHE SAMMLUNG VON MK.ROORGAN1SMEN UND ZELLKULTUREN GmbH	Signature^) of peison(s) having the power to represent the International Depositary Authority or of authorized o(Iieial(s):
Address: ManehcoodcrWeg lb D-38I24 Braunschweig	// C/-'/
•	Date: 2005-11-01

¹ Indicate the date of original deposit or, where a new deposit or a transfer has been made, the most recent relevant date (date of the new deposit or date of the transfer).

’ In the eases referred to in Rule 10.2(a) (ii) and (iii). refer to the most recent viability test ’ Mark with a crass the applicable box.

* Fill in if the infonnation has been requested and if the results of the test were negative.

Form DSMZ-BP/9 (sole page) 12/2001

123

PCT/EP2006/011302

WO 2007/059997

2018200685 30 Jan 2018 lanymed Pharmaceuticals AG rciligrathstr. 12 5131 Mainz

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION ΟΓ THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OP PATENT PROCEDURE

INTERNATIONAL FORM

DSMZ.

Dauhcta 9

Sommlunjj von / imd Zcllkvllwron GmbH «*

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page

L IDENTIFICATION OF IHE MICROORGANISM

identification reference given by the DEPOSITOR: 182-D 1106-057	Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY:
	DSM ACC2739

Π. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism Identified tinder I. above was accompanied by.

( X ) a scientific description ( ) n proposed taxonomic designation (Mr.rk with a cross where Applicable).

IB. RECEIPT .AND ACCEPTANCE

Tns” International Depositary Authority accepts die microorganism identified nndor I. above, which was received by If on 2005-10-19 (Dote of the original deposit)¹.

IV. RECEIPT OF REQUEST FOR CONVERSION

The nticTOTTgitnism identified under I above was received by thia International Depositary Authority on and a request to convert die original deposit to a deposit under the Budapest Treaty was received by it on for conversion), (date of original deposit) (date of receipt of request

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name; DSMZ-DEUTSCHE SAMMLUNG VON	Signature^) of pcraon(s) having the power to represent (he
MKR.OORGANKMEN UND 2ELLKULTUREN GmbH	International Depositary Authority or of authorized officinlfs):
Address; Mnschcrodcr Weg lb D-38 J 24 Braunschweig
	Date: 2005-11-01

¹ Where Rule 6.4 (d) applies. such date is the date on which the status of international depositary authority was acquired.

Form DSMZ-BP/4 (soltpnge) 12/2001

124

WO 2007/059997

2018200685 30 Jan 2018

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR. THE PURPOSES OF PATENT PROCEDURE

PCT/EP2006/0U302

DcuUcho Snmmlting von ΜίΙσ©θΓηοπί?Γη·>η und Zollkuliuron GmbH

INTERNATIONAL FORM

Sanymcd Pharmaceuticals AG

Yeiligrathstr. 12 >5131 Mainz

VIABILITY STATEMENT ipeucd pursuant to Rule 10.2 by the • INTERNATIONAL DEPOSITARY AUTHORITY identified nt the bottom of th is page

t. DEPOSITOR	Π. .IDENTIFICATION OF THE MICROORGANISM
Name.·	Ganymed Pharmaceuticals AG Freiligrathstr. 12	Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY:
Address:	55131 Mainz	DSM ACC2739
		Dele ofthe deposit or the transfer* 1:
		2005-10-19

ITT. VIABILITY STATEMENT

The viability of the microorganism identified under Π above was tested on 2005-10-19 On that date, the said microorganism was (_x)’ viable ( )’ no longer viable rv. CONDITIONS UNDER WHICH THE VIABILITY ΊΈ5Τ HAS BEEN PERFORMED'

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: D5M2-DJEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH	Signaturc(s) of pcrcon(a) having the power to represent the International Depositary Authority or of authorized offi eiaJ ($):
Address: MasehorodorWeg 1b D-38124 Braunschweig
	owe: 2005-11-01

¹ Indicate the date of original deposit or. where a new deposit or a transfer has been made; the most recent relevant date (date of the new deposit or date of the transfer).

¹ In the eases rctoted to in Rule 10.2(a) (ii) and (iii), refer to the most recent vinbility test, ¹ Mark with a craa the applicable box.

' Fill in if die information has been requested and i.f the results of the test were negntive.

Form DSMZ-BP/9 (sole page) 12/2001

125 f

WO 2007/059997

2018200685 30 Jan 2018 mymed Pharmaceuticals AG eiligrathstr. 12 i 131 Mainz

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

PCT/EP2006/011302

DSMZ tfcunche 4

SaRimivnfl *σπ Mikroorgonisman wnrf Zvllkwhvr^ft GmbH ***

INTERNATIONAL FORM

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified ot the bottom of this page

IDENTIFICATION OF 7HE MICROORGANISM

dcntification reference given by die DEPOSITOR: 182-D 1106-058	Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY:
	DSM ACC2740

I. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism Identified under I. above was oteompnoied by;

C X ) * scientific description ( ) Λ prepnsed taxonomic designation fMark with a cross where explicable).

ΓΠ. RECEIPT AND ACCEPTANCE

This International Depositary Authority accepts the microorganism identified under I. above, which was received by it on 2005-10-19 (Date of the original deposit)¹.

IV. RECEIPT OF REQUEST FOR CONVERSION

The microorganism identified under I above was received by this International Depositary Authority on and a request to convert die original deposit to a deposit under flic Budapest Treaty was received by it on for conversion).

(data of original deposit) (date of receipt of request

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: DSMZ-DEUTSCHE SAMMLUNG VON MKROOKGANISMEN UND ZELLKULTUREN GmbH	Signttorcfs) ofpttson(s) having the power to represent the International Depositary Authority or of authorized ofScialfs):
Address: Mascherochr Weg lb
D-38124 Braunschweig
	Dm 2005-11-01

¹ Where Rule 6.4 (d.) applies, such date is the date on which the status of international depositary authority was acquired.

Form DSMZ-BP/4 (sole j»gt>) 12/2001

126

WO 2007/059997

PCT/EP2006/0U302

2018200685 30 Jan 2018

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

DSMZ

Dadlacha Somrnlyrg von Mikrocrganiiman vnd ZellkwUvrif) GmbH [NTERNATIONAL FORM

Ganymed Pharmaceuticals AG

Freiligrathstr. 12

55131 Mainz

VIABILITY STATEMENT issued pursuant to Rule 10.2 by the INTERNATIONAL DEPOSITARY AUTHORITY identified nt the bottom of this page

I. DEPOSITOR	Π. IDENTIFICATION OF THE MICROORGANISM
Name- Ganymed Pharmaceuticals AG Freiligrathstr. 12 Address: 55137 Mainz	Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY·. DSM ACC2740 Dote of the deposit or the transfer': 2005-10-19
HI. VIABILITY STATEMENT -
The viability of the microorganism identified under II above was tested on 2005-1.0-J 9 3 · On dial dare, the said microorganism was (χ)- viable ( )3 no longer viable
IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED’
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE S AMMLUNO VON MKROORG ANJSMEN UND ZELLKULTUREN GmbH Addtess: Moscbcrodcr Wcg lb D-38124 Braunschweig	Signature!*) of person(s) having the power to represent die Incarnntiottal Depository Authority or of authorized ofiicialfs): Date: 2005-11-01

¹ Indicate the date of original deposit or, where a new deposit or a transfer has been made, the most recent relevant date (date of the new deposit or date Of the transfcrl.

² In the eaxcs refined to in Rule 10.2(a) (il) at)d (Hi). refer to the most recent viability test.

⁵ Mark with a cross die applicable boa.

* Fill in if the information bos been requested and if the results of the test were negative.

Form DSMZ-BP/9 (aolepagc) 12/2001

127

WO 2007/059997

PCT/EP2006/011302

2018200685 30 Jan 2018

Sanymed Pharmaceuticals AG ‘reiligrathstr. 12 >5131 Mainz

BUDAPEST TREATY ON THE INTERNATIONAL ·

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

DSMZ

Dnjloch* Λ

Sorr.mlunp von V Mikreoifjnnlim-n unH WlkuHurcn GmbH ·

RECEIPT IN THE CASE OF AN ORIGINAL DEPOST issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page

L IDENTIFICATION OF IHE MICROORGANISM

Identification reference given by the DEPOSITOR: 182-D 1.106-059	Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY;
	DSM ACC2741

U. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified under I. above was accompanied by;

( X ) # scientific description ( ) a proposed taxonomic designation (Mark with a cross whcrtipplicablo).

ΠΙ. RECEIPT AND ACCEPTANCE

This International Depositary .Authority accepts the ntieraorgx-iiim identified under I. above, which was received fcv it on 2005-10-19 (Date of the original deposit)'.

IV. RECEIPT OF REQUEST FOR CONVERSION

The microorganism identified under I above was received by this International Depositary Authority on and a request to convert fc original deposit to a deposit under the Budapest Treaty was received by it on for coovcraion).

(date of original deposit) (date of taeojpt of request

V. INTERNATIONALDEPOSITARY AUTHORITY

Name¹

Address;

DSMZ-DEUTSCHE SAMMUING VON

MKROORGANISMEN UNIT ZEU.KULTUREN GmbH

MajcheroderWcg lb

D-38I24 Braunschweig

Signature^) of penon(s) having the power to represent the International Depository Authority or of authorized official(s):

if

Date: 2005-11-01 ¹ Where Rule 0.4 (d) applies, aneh date is the date on which the status of international depositary authority was acquired.

Form DSMZ-BP/4 (aofcpagc) 12/2001

128

WO 2007/059997

PCT/EP2006/0U302

2018200685 30 Jan 2018

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

DAuheh·

Sonunlvno von ΜϋσοοΓθαηΙιηΊΑή • •ndZillktfllvron GmbH «

INTERNATIONAL FORM

Gaoymed Pharmaceuticals AG

Freiligrathstr. 12

55131 Mainz

VIABILITY STATEMENT issued puisiiant to Rule 10.2 by the • INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom Dfthis page

I. DEPOSITOR	Π. IDEN riFICATION OF ΤΙΙΕ MICROORGANISM
Name·:	Ganymed Pharmaceuticals AG	Ai cession number given by the
	Freiligrathstr. 12	IN TERN ATONAL DEPOSITARY AUTHORITY:
Address:	55131 Mainz	DSM ACC2741
		D itc of the deposit or the transfer’: 2005-10-19

HI. VIABILITY STATEMENT

The suability ofthe microorganism identified under II above was tested on 2005-10- 19 On that date, the raid microorganism was (χ)’ viable ( )’ no longer viable

IV. CONDITIONS UNDER WHICH THE VIABtLITY TEST HAS BEEN PERFORMED*

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: DSMZ-DEUT5CHE SAMMLUNG VON MKROORGANISMEN UNO ZELLKULTUREN GmbH	Sigiiaturofs) of person(s) having the power to represent the International Depositary Authority or of authorized offieial(s):
Address: Maschcrodet Weg 1 b D-38124 Braunschweig	Daco: 2005-11-01

Indicate the date of original deposit or, where a new deposit or a transfer has been made, the most recent relevant date (date of the new deposit or date of the transfer).

In the ease* referred to in Rvlc 10.2(a) (ii) and (iii), refer tn the most recent viability test.

Mark with a cross the applicable box.

Fill in if the iofonnntion tins been requested and if the results of the test were negative.

Form DSMZ-0P/9 (sole page) 12/2001

129

WO 2007/059997

PCT/EP2006/011302 ο

<Ν

BUDAPEST TREATY ON THE INTERN ATONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE cd

Η—5 ο

DSMZ.

Peur.che ®

Sotnffduftgvan MlIireerQtinismen untfZdikvflurcn GmbH «

INTERNATIONAL FORM

ΓΟ lymed Pharmaceuticals AG iligrathstr. 12 in oo

O o

CM

Mainz

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7.] by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page

O ---

CM

PENTITICATION OP TEE MICROORGANISM

ntifscation reference given by the DEPOSITOR: 182-Dl 106-062	Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY:
	DSM ACC2742

SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION e microorganism identified under I. above was accompanied bit ( χ I a scientist description . { ) a proposedtasonomic designation lurk with a croaa where applicable).

. RECEIPT AND ACCEPTANCE

ι nif mtcreMkmal Dtporit'tfy Authority accepts ihe njicrocrgsnism identified under f. above, which wps received, by it on 2005-10-19 (Dote of the original dcpoail)\
IV. RECEIPT OF REQUEST FOR CONVERSION
The microorganism idcnti&d under I above was received by thia International Depositary Authority on (date of original deposit) and b request to convert the original deposit to u dtpoeit under the Budapest Treaty was received by it on (date of receipt of request fnr conversion).
V. INTERNATIONAL DEPOSITARY AUTHORITY
Nome: DSMZ-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH Address: MaiCbcrodctWcg lb D-38I24 Braunschweig	Signatories) of pcreon(a) having the power to represent file International Depositary Authnrity or of authorized officials): . Date: 2005-11-01

• Where Rule 6.4 (d) applet, such date is tie diw on which the status of international depositary authority was acquired.

Form DSMZ-BP/4 (sole page) 12/2001

130

WO 2007/059997

PCT/EP2006/011302

2018200685 30 Jan 2018

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR. THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

Ganymed Pharmaceuticals AG

Freiligrathstr. 12

55131 Mainz

VIABILITY STATEMENT issued pursuaoi Io Rule 10.2 by the

·. INTERNATIONAL DEPOSITARY AUTHORITY ideal! (led ot the bottom of this page

Samnilupp'Ofl Mlkroorganinrivsn vftdZellkuHurenGmfcH «

I. DEPOSITOR	Π. IDENTIFICATION OF THE MICROORGANISM.
Name: Ganymed Pharmaceuticals AO Freiligrathstr. 12 Address: 55131 Mains;	AcccsBJDn number given by the INTERNATIONAL DEPOSITARY AUTHORITY: DSM ACC2742 Date of the deposit or the transfer': 2005-10-19
m. VIABILITY STATEMENT
The viability of the rtticnwrgnnisni identified under Π above was tested on 2005-10-19 1 · On that date, the said microorganism was (χ)3 viable ( )’ no longer viable
TV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED*
V. INTERN ATONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON MIKROOROAN1SMEN UND ZELLKULTUREN GmbH Address: Mosehcruffcr Wcg lb D-38124 Braunschweig	Signaturefa) of pcrson(s) having the power to represent the International Depositary Authority or of authorized official^): Date: 2005-11-01

' Indicate the date of origrnnl deposit or, where a new deposit or a transfer has ban nude, the most recent relevant date (date nf die new deposit or date Ofthe transfer).

* In the eases rofared to in Rule 10.2(a) (ii) and (Hi), refer to the most recent viability test.

³ Mark with a crass the applicable box.

⁴ Fit] in if the information has boen requested and if the results ofthe teat wore negative.

Form DSMZ-BP/9 (sole page) 12/2001

131

I

WO 2007/059997

PCT/EP2006/011302

2018200685 30 Jan 2018

Ganymed Pharmaceuticals AG Freiligrathstr. 12 55131 Mainz

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

DSMZ.

D^vDckt @

Soffiffift/ng won

MlkroOQonitmen “ und ZcDkullurnn GmbH «

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page

L rDENTmCATlON OFTHE MICROORGANISM

Identification reference given by the DEPOSITOR: 182-DI 106-067	Accession number given by die INTERNATIONAL DEPOSITARY AUTHORITY:
	DSM ACC2743

Π. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The miproorgnnisni idennfied under I. above was accompanied by:

( X ) ^a scientific description f ) a proposed taxonomic designation (Mark with a cross where applicable).

ΠΙ. RECEIPT AND ACCEPTANCE

This International Depositary Authority accepts 'die microorganism identified under I. above, which wits received by it on 2005-10-19 (Date of the original deposit)¹.

IV. RECEIPT OF REQUESTTOR CONVERSION

The microorganism idaitified under I above ’.vss received by ibis International Depositary Authority on and a request to con ven (be original deposit lo a deposit under the Budapest Treaty was received by it on for conversion).

(date of original deposit) (date of receipt of request

V. INTERNATIONALDEPOSrTARY AUTHORITY

• Name; DSMZ-DcUTSCHE SAMMLUNG VON	Signaturefs) of pason(s) having the power to represent the
MKROORGANISMEN UND ZELLKULTUREN GmbH	International Depositary Authority or of authorized official(s):
Address: MasehcredcrWcg Jb D-3 8124 Braunschweig
	Date: 2005-11-01

Where Rule <5.4 (d) eppllci, such date is the date on which the status of international depositary authority was acquired

Fftnn DSMZ-BP/4 (sofcpage) 12/7001

132

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PCT/EP2006/011302

2018200685 30 Jan 2018

Ganymed Pharmaceuticals AG PreiUgrathstr. 12 55131 Mainz

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

VIABILITY STATEMENT issued pursuant to Rule 10.2 by the

INTERNA TONAL DEPOSITARY AUTHORITY identified at the bottom of this page

Douiiche Sommluna won Mikronr0onl»m*o unci Zdlkvliuron GmbH «

I. DEPOSITOR	Π. IDENTIFICATION OF THE MICROORC ANISM
Name- Ganymed Phaimaceuticala AG Frciligralhstr. 12 Addre?s: 5513, Mainz	Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY: DSM ACC2743 Date of the deposit or the transfer': 2005-10-19
HI. VIABILITY STATEMENT
The viability ofthe microorganism identified under Π above wrs tested on 2005-10-19 2 · On that date, the said microorganism was (x)J viable ( / no longer viable
IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED*
v. international depositary authority
Name: DSMZ-DEUTSCHE SAMMLUNG VON MKROORGANISMEN UNO ZELLKULTUREN GmbH Address: MaseberoderWeg lb D-3SI24 Braunschweig	Signatures) of perscm(s) having the power to represent the International Depositary Authority or nf authorized offi cial(s): Date: 2005-11-01

¹ Indicate the date of original deposit or. where a new deposit or a transfer has been made, the most recent relevant date (date of the newdeposit or date of the transfer), ¹ In the eases referred to in Rule I 0.2(e) (ii) and (lii), refer to the most recent viability rest.

¹ Mark with a crouthe applicable box.

⁴ Fill in if the information has heen requested and if the results of the test were negative.

Porm DSMZ-BP/9 (sole page) 12/2001

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Ganyracd Pharmaceuticals AG Freiligrathstr. 12 55131 Mainz

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL form

Dnobcho 4Bik

Somm'ung von Sgy Mikrocrgor»iiH«n und7,*llkulfuron GrhH

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued nW6uant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identilied at the bottom of thie page

I. IDENTIFICATION OFTHE MICROORGANISM
Identification reference given by the DEPOSITOR: 182-D758-O35	Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY: DSM ACC2745
H. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The microorganism identified under J, above was accompanied by: ( ) a scientific description ( ) λ proposed taxonomic designation {M vk with a cross where applicable).
HI. RECEIPT AND ACCEPTANCE
This International Ocpositaiy Authority accepts the microorganism identified undor 1. above, which was received by it on 2005-11-17 (Date of tbe original deposit)1.
TV. RECEIPT OF REQUEST FOR CONVERSION
The microorganism identified under I above was received by this International Depositary Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion).
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON MIKROORGANISMENUND ZELLKULTUREN GmbH Addresfc MoseberoderWeg Ib D-3S124 Braunschweig	Signatures) of pcrson(s) having the power to represent the International Depositary Authority or of authorized offirial(s): (/, Date: 2005-12-05

¹ Where Rule fi.4 (d) applies, such date is the dote on which the fltttua of international depositary authority was acquired.

Form DSMZ-BP/4 (sole page) 12/2001

134

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2018200685 30 Jan 2018

BUDAPEST TREATY ON THE INTERNATIONAL · RECOGNITION OF THB DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

Ganymed Pharmaceuticals AG

Freiligrathstr. 12

55131 Mainz

VIABILITY STATEMENT issued pursuant to Rule 10.2 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page

I. DEPOSITOR	IT. IDENTIFICATION OF THE MICROOROANISM
Name· Gsaymed Pharmaceuticals AG Freiligrathstr. 12 Address: 55,3, Μώ)ζ	Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY: DSM ACC2745 Date nf the deposit or the transfer1: 2005-11-17
ΠΤ. VIABILITY STATEMENT
The viability of the microorganism identified under B above was tested on 2005-11-21 1. On that date, the said microorganism was (χ)’ viable ( )’ no longer viable
IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED·1
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON MTKROORGAN1SMEN UND ZELLKULTUREN GmbH Address: Maschertxta Weg lb D-38124 Braunschweig	Signature^) of person(s) having the power to represent the International Depositary Authority or of authorized official(s): Date: 2005-12-05

Indicate the dote of original depositor, where a new depositor a transfer has been made, the most recent relevant date (date of the new deposit or date of the transfer).

⁴ In the cases referred to in Rule 10.2(a) (ii) Mid (iii). refer to the most recent viability test.

¹ Mark with a cross the applicable box.

⁴ Fill in if the information naa been requested and i f the rcsultn of the teat were negative.

Form DSMZ-BP/9 (sole page) 12/2001

135

WO 2007/059997

PCT/EP2006/011302

2018200685 30 Jan 2018

BUDAPEST TREATV ON THE INTERNA TONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

DMrcfie Somffitvnp ’on MftroefjjoniEmtn (mrfXpJAdWinnGnibH «

INTERNATIONAL FORM

Ganyrned Pbajroacduticals AG Freiligrathstr. 12 55131 Mainz	RECEIPT IN TOE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Ruin 7,1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page
LIDENTIFICA WN OP THE MICROORGANISM
Haiti ficutioft reference givea by the OEPOSITOR; 1S2-D758-036	Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY: DSM ACC2746
Π. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The microorganism tdcntifcd under !. above was aecestipauied by; ( ) a seiretiEe description { ) a profosedasonotpic designation IMruit with a cross where «pplicab ie).

Iff. RECEIPT ANO ACCEPTANCE

This InumatiMi! Depositny Authority accepts fa mrewoigf-nisra identified under I. above, which was received by it on 2005-11-17 (Date of the original deposit)¹,

IV. RECEIPT OF REQUEST FOR CONVERSION

The ndcroorgnnisrrt identified unfa I above was received by das International Depositary Authority on and a request to convert the original deposit to a deposit under the Budapest Treat}' was received by it on for conversion).

(date of original deposit) (date of receipt of request

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name;

DSMZ-DEUTSCHE SAMMLUNG VON MKROORCANBMEN UND ZELLKULTUREN GmbH

Signature//;) of peaon(s) having the power ro represent the tolemationsl Dcpoaiuuy Authority or of authorized offiriai(s).·

Address: MaseberoderWeg lb

Mascheroder Weg i D-38l24Bnans«n

IWCIg

Date 2005-12-05 ' Where Rule SA (d) applia, saeh date «the date on which the atoms of intereaSonaJ depositary authority w acquired.

Form DSMZ-BP/4 ftole page) 1MOO1

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V TREATY ON THE INTERNATIONAL

OF THE DEPOSIT OF MICROORGANISMS VRPOSES OF PATENT PROCEDURE \rnationalform

VIABILITY STATEMENT issued pursuant io Rule 10.2 by the

INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page

-USfTOR	Π. IDENTIFICATION OF THE MICROORGANISM
e Ganymed Pharmaceuticals AG	Accession number given by the
Freiligftttlistr. 12	INTERNATIONAL DEPOSITARY AUTHORITY:
€5S; 55131 Mainz	DSM ACC2746
	Date of the deposit or the traiuffcr1;
	2005-11-17
1ABILITY STATEMENT
'lability of the microorganism identified under Π above wos tested on 2005-11-21. 2· jat date, the said microorganism wss (χ)5 viable ( / no longer viable

ONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED*

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: DSMZ-DEUTSCHE SAMMLUNC VON

MIKROORGANISMEN UND ZELLKULTUREN GmbH

Address· Maschcrodcr Weg lb D-38124 Btatmsclttvoig

Signature^) of person(s) having the power to represent the International Depositary Authority or of authorized official(s):

1/.

Date: 2005-12-05

Indicate the date of original deposit or. where a new deposit or a transfer has been made, the most recent relevant date (dato of the new deposit or date of the transfer).

In the cases refened to in Rule I 0.2(b) (ii) and (iii). refer to the most recent viability test.

MatR with a cross the applicable box.

Fill in if Che infottnation has been requested and if the results of the test were negative.

Form DSMZ-BP/9 (solcpage) 12/2001

137

WO 2007/059997

2018200685 30 Jan 2018

Ganymed Pharmaceuticals AG Freiligrathstr. 12 55131 Mainz

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issuod pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY idctiti Tied nt the bottom of this page

PCT/EP2006/011302 bomm'urg von Mltcroorganijmen und ZaHkulluron GmbH

I. IDENTIFICATION OF THE MICROORGANISM
Identification reference given by tbc DEPOSITOR! 182-D758-040	Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY! DSM ACC2747
Π. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The microorganism identified under I. above was accompanied by: ( ) q scientific description ( ) nprapoF.cd uxonomicdesignation (Mark with a cross where applicable).
in. RECEIPT AND ACCEPTANCE
Tltis International Depositary Authority accepts the microorganism identified under I. above, which was received by it on 2005-11-17 (Date of the original deposit)1.
IV. RECEIPT OF REQUEST FOR CONVERSION
The microorganism identified under I above was received by this International Depositary Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion).
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON MIKROOR.GANTSMEN UND ZELLKULTUREN GmbH Address: MaschcrederWeg lb 1X38124 Braunschweig	Signature(s) of personfj) having the power to represent the International Depositary Authority or of authorized official^); IS, Date: 2005-12-05

¹ Where Rulo 6.4(d) applies, such date is the date on which the status of international depositary authority was acquired.

Form DSMZ-BP/4 (sole page) 12/2001

138

WO 2007/059997

PCT/EP2006/011302

2018200685 30 Jan 2018

Ganymed Pharmaceuticals AG Freiligrathstr. 12 55131 Mainz

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

VIABILITY STATEMENT issued pursuant to Rule 10.2 by the

INTERNATIONAL DEPOSITARY AUTHORITY identified nt the bottom of this page

Douhdin Sommlvfla von MikroDrganismen und Z*IIknlu·»<jn GmbH

I. DEPOSITOR	IT, IDENTIFICATION OF THE MICROORGANISM
Name- Ganymed Pharmaceuticals ΛΟ Freiligrathslr. 12 Address; 55 J3 χ	Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY; DSM ACC2747 Date of the deposit or the transfer1: 2005-11-17
IB. VIABILITY STATEMENT
The viability of the microorganism identified under JI above was tcsicden 2005-11-21 3 · On thnt date, the snid microorgiinism was (χ)3 viable ( }’ no longer viable
IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED*
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH Address: Maschcrodcr Weg lb D-38124 Braunschweig	Signaturc(s) of persnn(s) having the power to represent the International Depositary Authority or of authorised officials); (/ edz-'Ey Date: 2005-12-05

¹ Indicate the dnteof original deposit or. where a new deposit or a bun? fcr has been made, the most recent relevant date (date of the new deposit or date of the transfer).

¹ In the eases refotred to in Rule 10.2(a) (ii) and fiii). refer to the most recent viability test.

’ Marie with a cross the applicable box.

⁴ Fill in if the infonnation has been requested and if the results of the test were negative.

Form DSMZ-BP/9 (boIc page) 12/2001

139

WO 2007/059997

PCT/EP2006/011302

2018200685 30 Jan 2018

Ganymed Pharmaceuticals AG

Freiligrathstr. 12

55131 Mainz

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THB DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

L IDENTIFICATION OFTHE MICROORGANISM

DSMZ

p-jubchs Sommlung won ΜίΙσοο'&δηίίΓη'ϊπ und Zollkulluron GmbH ·

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued puraoant [O Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified et the bottom of this page

Identification reference given by the DEPOSITOR;

182-D1106-061

Accession number given by the

INTERNATIONAL DEPOSITARY AUTHORITY·.

DSM ACC2748

Π. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified under I. above was accompanied by:

( ) a sciestlfic description { ) a proposed taxonomic designation (Mark with a cross where applicable).

ΠΙ. RECEIPT AND ACCEPTANCE

This International Depositary Authority accepts the microorganism identified under I. above, which was received by it On 2005-11-17 (Date of the original deposit)'.

IV. RECEIPT OF REQUEST FOR CONVERSION

The microorganism identified under I above was received by this International Depository Authority on

Hilda request to convert theoriginol deposit to a deposit under the Budapest Treaty was received by it on for convetsian).

(date of original deposit) (date of receipt of request

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name; DSMZ-DEUTSCHE SAMMLUNG VON	Signatures) of pcrson(s) having the power to represent the
MIKROORGANISMEN UND ZELLKULTUREN GmbH	International Depositary Authority et of authorized official^);
Address; MasehcnodcrVcg lb	/ Date: 2005-12-05
0*38124 Braunschweig

¹ Where Rule 6.4 (d) applies, such date is the date on which the status of international depositary authority was acquired,

Form DSMZ-BP/4 (sole pap) 12/2001 140

WO 2007/059997

PCT/EP2006/011302

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BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

Douischn SarnmlurtB *°n Mikroargoni>mflft unri JeUMfuren GmbH ·

Ganymed Pharmaceuticals AG

Freiligrathstr. 12

55131 Mainz

VIABILITY STATEMENT issued pursuant to Rule 10.2 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page

i. depositor	R. IDENTIFICATION OF THE MICROORGANISM
Name· Ganymed Pharmaceuticals AG .Freiligrathstr. 12 Address: 5513] Mainz	Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY: DSM ACC2748 Date ofthe deposit or the transfer': 2005-11-17
HL VIABILITY STATEMENT
The viability of the microorganism identified under Π above was tested on 2005-1.1-21 1 - On that datei the said microorganism was (x)3 viable ( / no longcrviablc
IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED'
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON MKROORGANISMEN UND ZELLKULTUREN GmbH Address; MaschcroderWcg lb D-3S124 Braunschweig	Signnturc(s) of pentoti(s) having the power to represent the International Depositary Authority orpf authorized afiicial(a): (// A/efEn Date: 2005-12-05

Indicate the dele of original deposit or, where a now deposit or a transfer has been made, the most recent relevant date (date of the new depoait or date of the transfer).

' · In the cases reffetted to in Rule 10.2(a) (ii) and (iii), refer to the most recent viability test.

_t Mark with a cron the applicable box.

‘ Fill in iftheinfotmntion has been requested and ifthercsults ofthe teatwere negative.

Form DSMZ-BP/9 (aolepagc) 12/2001

141

WO 2007/059997

PCT/EP2006/011302

2018200685 30 Jan 2018

Ganymed Pharmaceuticals AG Freiligrathstr. 12 55131 Mainz

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

Deutsche Sammlung von Mikroorgonismen und Zellkulturen GmbH <

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified nt the bottom of this page

1. IDENTIFICATION OF THE MICROORGANISM
Identification reference given by (he DEPOSITOR: 182-D1106-279	Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY: DSM ACC2808
Π. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The microorganism identified under I. above was accompanied by: ( X ) a scientific description ( ) a proposed taxonomic designation (Mark with a cross where applicable).
ΙΠ. RECEIPT AND ACCEPTANCE
This International Depositary Aulhnriiy accepts the microorganism identified under 1. above, which was received by it on 2006-10-26 (Date of the original deposit)’.
IV. RECEIPT OF REQUEST FOR CONVERSION
The microorganism identified under I above was received by this International Depositary Authority on (date of original deposit) and a request to convert (he original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion).
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH Address: Inliofiensfr. 7 B D-38124 Braunschweig	Signature^) of pcrson(s) having the power to represent the International Depositary Authority or of authorized official(s): (/ Date: 2006-11-08

¹ Where Rule 6.4 (d) applies, such date is the date on which the status of international depositary authority was acquired.

Form DSMZ-BP/4 (sole page) 08/2006

142

WO 2007/059997

PCT/EP2006/011302

2018200685 30 Jan 2018

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

Ganymed Pharmaceuticals AG

Freiligrathstr. 12

55131 Mainz

VIABILITY STATEMENT issued pursuant to Rule i 0.2 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page

Deutsche Sofnrrdung von Milcroorgonismen und Zellkulluien GmbH <

I. DEPOSITOR	II. IDENTIFICATION OF THE MICROORGANISM
Name· Ganymed Pharmaceuticals AG Freiligrathstr. 12 Address: 55131 Mainz	Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY: DSM ACC2808 Date of the deposit or the transfer1: 2006-10-26
IB. VIABILITY STATEMENT
The viability of themicroorganism identified under 11 above was tested on 2006-10-30 2 · On that date, the said microorganism was (^)3 viable ( )3 no longer viable
IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED4
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAM MLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH Address: Inhoflenslr. 7 B D-38124 Braunschweig	Signature(s) of person(s) having the power to represent the International Depositary Authority or of authorized officials): Dale: 2006-11-08

¹ Indicate the date of original depositor, where a new deposit or a transfer has been made, the most recent relevant dale (date of the new deposit or date of the transfer).

¹ In the cases referred to in Rule 10.2(a) (ii)and (iii), refer to the most recent viability test.

³ Mark with a cross the applicable box.

^J Fill in if the information has been requested and if the results of the test were negative.

Form DSMZ-BP/9 (sole page) 08/2006

143

WO 2007/059997

PCT/EP2006/011302

2018200685 30 Jan 2018

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

Deulsche Sommlung von Mikroorgonismen und Zellkulluren GmbH <

Ganymed Pharmaceuticals AG

Freiligrathstr. 12

55131 Mainz receipt in the case of an original deposit issued pursuant to Rule 7.1 fay the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page

I. IDENTIFICATION OF THE MICROORGANISM
Identification reference given by the DEPOSITOR: 182-D 1106-294	Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY: DSM ACC2809
If. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The microorganism identified under I. above was accompanied by: ( X ) n scientific description ( ) a proposed taxonomic designation (Mark with a cross where applicable).
III. RECEIPT AND ACCEPTANCE
This international Depositary Authority accepts the microorganism identified under I. above, which was received by il on 2006-10-26 (Date of the original deposit)*.
IV. RECEIPT OF REQUEST FOR CONVERSION
The microorganism identified under f above was received by this international Depositary Authority on (dale of original deposit) and a request lo convert the original deposit to a deposit under the Budapest Treaty was received by il on (dale of receipt of request for conversion).
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHESAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH Address: Inlioflenstr. 7 B D-38124 Braunschweig	Signature(s) ofpcrson(s) having the power to represent the International Depositary Authority or of authorized officiaf(s): (/ Dale: 2006-11-08

¹ Where Rule 6.4 (d) applies, such date is tile dote on which the status of international depositary authority was acquired,

Fonn DSMZ-BP/4 (sole page) 08/2006

144

WO 2007/059997

PCT/EP2006/011302

2018200685 30 Jan 2018

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

Ganymed Pharmaceuticals AG

Freiligrathstr. 12

55131 Mainz

VIABILITY STATEMENT issued pursuant to Rule 10.2 by the INTERNATIONAL DEPOSITARY AUTHORITY identified al lliebollom of Ibis page

I. DEPOSITOR	Π. IDENTIFICATION OF THE MICROORGANISM
Nanlc. Ganymed Pharmaceuticals AG Freiligrathstr. 12 Address: 55131 Majnz	Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY: DSM ACC2809 Date of the deposit or the transfer1: 2006-10-26
ΠΙ. VIABILITY STATEMENT
Tlie viability of the microorganism identified under II above was tested on 2006-10-30 1 On that dale, ihe said microorganism was (χ)’1 viable ( )3 no longer viable
rv. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED'
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH Address: lnhofienslr. 7 B D-38124 Braunschweig	Signalurc(s) of pcrson(s) having the power to represent the International Depository Authority or of authorized officials): Date: 2006-11-08

’ Indicate thedate of original depositor, where a new deposit or a transfer lias been made, the most recent relevant date (date of the new deposit or date of the transfer).

⁵ In Ihe cases referred to in Rule 3 0.2(a) (ii)and (iii), refer to the most recent viability lest ³ Mark with a cross the applicable box.

^M Fill in if the information has been requested and if the results of the test were negative.

Form DSMZ-BP/9(solc page) 08/2006

145

WO 2007/059997

PCT/EP2006/011302

2018200685 30 Jan 2018

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

Deutsche Sornmlung von Mikroorgonismen und Zellkuiluren GmbH *

Ganymed Pharmaceuticals AG

Freiligrathstr. 12

55131 Mainz RECEIPT IN THE CASE OFAN ORIGINAL DEPOSIT issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at (lie bottom of this page

I. IDENTIFICATION OF THE MICROORGANISM
Identification reference given by the DEPOSITOR: 182-Dl 106-362	Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY: DSM ACC2810
II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The microorganism identified under I. above was accompanied by: ( X ) a scientific description ( ) a proposed taxonomic designation (Mark with a cross where applicable).
in. RECEIPT AND ACCEPTANCE
This international Depositary Authority accepts the mic-iwrganism identified under I. above, which was received by it on 2006-10-26 (Dale of the original deposit)'.
IV. RECEIPT OF REQUEST FOR CONVERSION
The microorganism identified under I above was received by this International Depositary Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion).
V. INTERNATIONALDEPOS1TARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON MIKR00RGANISMEN UND ZELLKULTUREN GmbH Address: Inhoffenstr. 7 B D-38124 Braunschweig	Signaturc(s) of pcrson(s) having the power to represent the International Depositary Authority or of authorized officials): (/. // Date: 2006-11-08

¹ Where Rule 6.4 (d) applies, such date is the date on which the status of international depository authority was acquired.

Form DSMZ-BP/4 (sole page) 08/2005

146

WO 2007/059997

PCT/EP2006/011302

2018200685 30 Jan 2018

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

Ganymed Pharmaceuticals AG

Freiligrathstr. 12

55131 Mainz

VIABILITY STATEMENT issued pursuant to Rule 10.2 by the INTERNATIONAL DEPOSITARY AUTHORITY identified nt the bottom of this page

I. DEPOSITOR	II. IDENTIFICATION OF THE MICROORGANISM
Name· Ganymed Pharmaceuticals AG Freiligrathstr. 12 Address: 55131 Mainz	Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY: DSM ACC2810 Dale ofthe deposit or the transfer': 2006-10-26
ΠΙ. VIABILITY STATEMENT
The viability of the microorganism identified under II above was tested on 2006-10-30 2 · On that date, the said microorganism was (^)J viable ( )3 no longer viable
IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED'
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH Address: Inbofienslr. 7 B D-3S124 Braunschweig	Signatures) of pcrson(s) having the power to represent the International Depositary Authority or of authorized official(s): Date: 2006-11-08

¹ Indicate the date of original deposit or, where a new deposit or a transfer has been made, the most recent relevant dale (duteof the new deposit or date ofthe transfer).

¹ In the cases referred to in Rule 10.2(a) (ii) and (iii), refer to the most recent viability test.

³ Mark with a cross the applicable box.

⁴ Fill in if the information has been requested and if the results of the test were negative.

Form DSMZ-BP/S (sole page) 08/2006

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2018200685 30 Jan 2018

Claims (51)

1. Claims:

   1. A method of expressing an antibody, or a polypeptide comprising an antigen binding fragment thereof, that binds to CLD18A2 but not to CLD18A1, and mediates killing of cells

   5 expressing CLDN18A2, the method comprising the steps of:

   (a) transforming a human host cell with one or more expression vectors; and (b) culturing the transformed human host cell under conditions in which the host cell expresses the polypeptide encoded by said expression vectors;

   wherein the one or more expression vectors comprise:

   LO (i) 125E1 all 6 CDRs a nucleic acid sequence encoding a polypeptide comprising the antibody heavy chain CDR1, CDR2, and CDR3 regions having the amino acid sequences of positions 45-52, positions 70-77, and positions 116-124 of SEQ ID NO: 117, respectively; and a nucleic acid sequence encoding a polypeptide comprising the antibody light chain CDR1, CDR2, and

   L5 CDR3 regions having the amino acid sequences of positions 47-52, positions

   70-72, and positions 109-117 of SEQ ID NO: 123, respectively; or (ii) 163E12 all 6 CDRs a nucleic acid sequence encoding a polypeptide comprising the antibody heavy chain CDR1, CDR2, and CDR3 regions having the amino acid sequences of positions 45-52, positions 70-77, and positions
2. 2 0 116-126 of SEQ ID NO: 116, respectively; and a nucleic acid sequence encoding a polypeptide comprising the antibody light chain CDR1, CDR2, and CDR3 regions having the amino acid sequences of positions 47-58, positions 76-78, and positions 115-123 of SEQ ID NO: 121, respectively; or (iii) 175D1 all 6 CDRs a nucleic acid sequence encoding a polypeptide

   2 5 comprising the antibody heavy chain CDR1, CDR2, and CDR3 regions having the amino acid sequences of positions 45-52, positions 70-77, and positions 116-126 of SEQ ID NO: 118, respectively; and a nucleic acid sequence encoding a polypeptide comprising the antibody light chain CDR1, CDR2, and CDR3 regions having the amino acid sequences of positions 47-58, positions
3. 3 0 76-78, and positions 115-123 of SEQ ID NO: 125, respectively.

   2. The method of claim 1, wherein the human host cell is a HEK293 cell, HEK293T cell, dendritic cell, B cell, K562 cell, HELA cell, or a lymphocytic cell.

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   2018200685 30 Jan 2018

   3. The method of claim 1, wherein the human host cell is a B cell.
4. 4. The method of any one of claims 1 to 3, wherein the expression vector comprises a promoter sequence, a leader sequence, a translation initiation sequence, a light chain constant
5. 5 region, a heavy chain constant region, 3’ untranslated sequence, a polyadenylation sequence, or a transcription termination sequence.

   5. The method of any one of claims 1 to 4, wherein the antigen binding fragment is a Fab, F(ab)2, Fv, or single chain Fv.

   LO
6. 6. A recombinant nucleic acid comprising a nucleic acid sequence selected from the group consisting of:

   (i) 125E1 HC CDRs a nucleic acid sequence encoding a polypeptide comprising heavy chain CDR1, CDR2, and CDR3 regions having the amino acid sequences of

   L5 positions 45-52, positions 70-77, and positions 116-124 of SEQ ID NO: 117, respectively;

   (ii) 163E12 HC CDRs a nucleic acid sequence encoding a polypeptide comprising heavy chain CDR1, CDR2, and CDR3 regions having the amino acid sequences of positions 45-52, positions 70-77, and positions 116-126 of SEQ ID NO: 116,

   2 0 respectively;

   (iii) 175D1 HC CDRs a nucleic acid sequence encoding a polypeptide comprising heavy chain CDR1, CDR2, and CDR3 regions having the amino acid sequences of positions 45-52, positions 70-77, and positions 116-126 of SEQ ID NO: 118, respectively;

   2 5 (iv) 125E1 LC CDRs a nucleic acid sequence encoding a polypeptide comprising light chain CDR1, CDR2, and CDR3 regions having the amino acid sequences of positions 47-52, positions 70-72, and positions 109-117 of SEQ ID NO: 123, respectively;

   (ν) 163E12 LC CDRs a nucleic acid sequence encoding a polypeptide comprising

   3 0 light chain CDR1, CDR2, and CDR3 regions having the amino acid sequences of positions 47-58, positions 76-78, and positions 115-123 of SEQ ID NO: 121, respectively; and (vi) 175D1 LC CDRs a nucleic acid sequence encoding a polypeptide comprising light chain CDR1, CDR2, and CDR3 regions having the amino acid sequences of

   149

   2018200685 30 Jan 2018 positions 47-58, positions 76-78, and positions 115-123 of SEQ ID NO: 125, respectively.
7. 7. The recombinant nucleic acid of claim 6, wherein the nucleic acid sequence encodes a 5 heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 133, 134, and 135.
8. 8. The recombinant nucleic acid of claim 6, wherein the nucleic acid sequence encodes a light chain variable region comprising an amino acid sequence selected from the group

   10 consisting of SEQ ID NO: 138, 140, and 142.
9. 9. The recombinant nucleic acid of any one of claims 6 to 8, wherein the nucleic acid sequence is operatively linked to expression control sequences.

   15 10. A transformed human cell comprising the recombinant nucleic acid of any one of claims 6 to 9.

   11. The transformed cell of claim 10, wherein the cell is a HEK293 cell, HEK293T cell, dendritic cell, B cell, K562 cell, HELA cell, or a lymphocytic cell.

   12. A polypeptide encoded by the recombinant nucleic acid of any one of claims 6 to 9.

   13. The polypeptide of claim 12, further comprising an immunoglobulin hinge region.

   2 5 14. A recombinant nucleic acid comprising a nucleic acid sequence selected from the group consisting of:

   (i) 125E1 V_H & V_L a nucleic acid sequence encoding a polypeptide comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 134 and a light chain variable region having the amino acid sequence of SEQ ID NO: 140;

   3 0 (ii) 163E12 Vh & Vl a nucleic acid sequence encoding a polypeptide comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 133 and a light chain variable region having the amino acid sequence of SEQ ID NO: 138; and

   150

   2018200685 30 Jan 2018 (iii) 175D1 V_H & V_L a nucleic acid sequence encoding a polypeptide comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 135 and a light chain variable region having the amino acid sequence of SEQ ID NO: 142.

   5 15. The recombinant nucleic acid of claim 14, wherein the nucleic acid sequence is operatively linked to expression control sequences.

   16. A transformed human cell comprising the recombinant nucleic acid of claim 14 or 15.

   LO 17. The transformed cell of claim 16, wherein the cell is a HEK293 cell, HEK293T cell, dendritic cell, B cell, K562 cell, HELA cell, or a lymphocytic cell.

   18. A polypeptide encoded by the recombinant nucleic acid of claim 14 or 15.

   15 19. The polypeptide of claim 18, further comprising an immunoglobulin hinge region.

   20. An anti-CLD18A2 antibody expressed by the transformed human cell of claim 10, 11, 16 or 17, wherein the antibody binds to CLD18A2 but not to CLD18A1.

   2 0 21. An antigen binding fragment expressed by the transformed human cell of claim 10, 11,

   16 or 17, wherein the antigen binding fragment binds to CLD18A2 but not to CLD18A1.

   22. The antigen binding fragment of claim 21, wherein the antigen binding fragment is a Fab, F(ab)2, Fv, or single chain Fv.

   23. The antigen binding fragment of claim 21 or 22, wherein the antigen binding fragment is fused to an immunoglobulin hinge region.

   24. A method of producing a recombinant eukaryotic host cell, comprising the steps of:

   3 0 a. transforming a eukaryotic cell with an expression vector comprising the recombinant nucleic acid of claim 6 to 9 or 14 or 15; and b. obtaining the transformed cell, wherein the transformed cell comprises the recombinant nucleic acid.

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   2018200685 30 Jan 2018

   25. The method of claim 24, wherein the recombinant eukaryotic host cell is a human cell.

   26. The method of claim 24 or 25, wherein the recombinant eukaryotic host cell is a HEK293 cell, HEK293T cell, dendritic cell, B cell, K562 cell, HELA cell, or a lymphocytic

   5 cell.

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    2018200685 30 Jan 2018

    Sequences_342-31PCT. txt SEQUENCE LISTING <110> Ganymed Pharmaceuticals AG <120> Monoclonal antibodies against claudin-18 for treatment of cancer <130> 342-31PCT <150> EP 05 025 657.7 <151> 2005-11-24 <160> 150 <170> Patentln version 3.3 <210> 1 <211> 786 <212> DNA <213> Homo sapiens <400> 1

    atggccgtga ctgcctgtca gggcttgggg ttcgtggttt cactgattgg gattgcgggc 60 atcattgctg ccacctgcat ggaccagtgg agcacccaag acttgtacaa caaccccgta 120 acagctgttt tcaactacca ggggctgtgg cgctcctgtg tccgagagag ctctggcttc 180 accgagtgcc ggggctactt caccctgctg gggctgccag ccatgctgca ggcagtgcga 240 gccctgatga tcgtaggcat cgtcctgggt gccattggcc tcctggtatc catctttgcc 300 ctgaaatgca tccgcattgg cagcatggag gactctgcca aagccaacat gacactgacc 360 tccgggatca tgttcattgt ctcaggtctt tgtgcaattg ctggagtgtc tgtgtttgcc 420 aacatgctgg tgactaactt ctggatgtcc acagctaaca tgtacaccgg catgggtggg 480 atggtgcaga ctgttcagac caggtacaca tttggtgcgg ctctgttcgt gggctgggtc 540 gctggaggcc tcacactaat tgggggtgtg atgatgtgca tcgcctgccg gggcctggca 600 ccagaagaaa ccaactacaa agccgtttct tatcatgcct cgggccacag tgttgcctac 660 aagcctggag gcttcaaggc cagcactggc tttgggtcca acaccaaaaa caagaagata 720 tacgatggag gtgcccgcac agaggacgag gtacaatctt atccttccaa gcacgactat 780

    gtgtaa 786 <210> 2 <211> 261 <212> PRT <213> Homo sapiens <400> 2

    Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile 15 10 15

    Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr 20 25 30

    Page 1

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    Sequences_342-31PCT.txt

    Gin Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gin Gly 35 40 45

    Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60

    Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gin Ala Val Arg 65 70 75 80

    Ala Leu Met Xie Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95

    Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110

    Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125

    Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140

    Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160

    Met Val Gin Thr Val Gin Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175

    Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190

    Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205

    Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220

    Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240

    Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gin Ser Tyr Pro Ser 245 250 255

    Lys His Asp Tyr Val 260 <210> 3 <211> 816 <212> DNA

    Page 2

    2018200685 30 Jan 2018

    Sequences_3 42-31PCT. txt <213> Homo sapiens <400> 3

    atggccgtga ctgcctgtca gggcttgggg ttcgtggttt cactgattgg gattgcgggc 60 atcattgctg ccacctgcat ggaccagtgg agcacccaag acttgtacaa caaccccgta 120 acagctgttt tcaactacca ggggctgtgg cgctcctgtg tccgagagag ctctggcttc 180 accgagtgcc ggggctactt caccctgctg gggctgccag ccatgctgca ggcagtgcga 240 gccctgatga tcgtaggcat cgtcctgggt gccattggcc tcctggtatc catctttgcc 300 ctgaaatgca tccgcattgg cagcatggag gactctgcca aagccaacat gacactgacc 360 tccgggatca tgttcattgt ctcaggtctt tgtgcaattg ctggagtgtc tgtgtttgcc 420 aacatgctgg tgactaactt ctggatgtcc acagctaaca tgtacaccgg catgggtgaa 480 caaaaactca tctcagaaga ggatctgggg atggtgcaga ctgttcagac caggtacaca 540 tttggtgcgg ctctgttcgt gggctgggtc gctggaggcc tcacactaat tgggggtgtg 600 atgatgtgca tcgcctgccg gggcctggca ccagaagaaa ccaactacaa agccgtttct 660 tatcatgcct cgggccacag tgttgcctac aagcctggag gcttcaaggc cagcactggc 720 tttgggtcca acaccaaaaa caagaagata tacgatggag gtgcccgcac agaggacgag 780 gtacaatctt atccttccaa gcacgactat gtgtaa 816

    <210> 4 <211> 271 <212> PRT

    <213> Homo <400> 4 sapiens Met 1 Ala Val Thr Ala 5 Cys Gln Gly Leu Gly 10 Phe Val Val Ser Leu 15 Ile Gly Ile Ala Gly 20 Ile Ile Ala Ala Thr 25 Cys Met Asp Gln Trp 30 Ser Thr Gln Asp Leu 35 Tyr Asn Asn Pro Val 40 Thr Ala Val Phe Asn 45 Tyr Gln Gly Leu Trp 50 Arg Ser Cys Val Arg 55 Glu Ser Ser Gly Phe 60 Thr Glu Cys Arg Gly 65 Tyr Phe Thr Leu Leu 70 Gly Leu Pro Ala Met 75 Leu Gln Ala Val Arg 80 Ala Leu Met Ile Val 85 Gly Ile Val Leu Gly 90 Ala Ile Gly Leu Leu 95 Val Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Page 3 Ser Met Glu Asp Ser

    2018200685 30 Jan 2018

    Sequences_342-31PCT. txt 100 105 110

    Ala Lys Ala Asn Met Thr Leu Thr Ser 120 Gly Ile Met Phe 125 Ile Val Ser 115 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Glu 145 150 155 160 Gin Lys Leu Ile Ser Glu Glu Asp Leu Gly Met Val Gin Thr Val Gin 165 170 175 Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe Val Gly Trp Val Ala Gly 180 185 190 Gly Leu Thr Leu Ile Gly Gly Val Met Met Cys Ile Ala Cys Arg Gly 195 200 205 Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala Val Ser Tyr His Ala Ser 210 215 220 Gly His Ser Val Ala Tyr Lys Pro Gly Gly Phe Lys Ala Ser Thr Gly 225 230 235 240 Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile Tyr Asp Gly Gly Ala Arg 245 250 255 Thr Glu Asp Glu Val Gin Ser Tyr Pro Ser Lys His Asp Tyr Val 260 265 270

    <210> 5 <211> 813 <212> DNA <213> Homo sapiens <400> 5

    atggccgtga ctgcctgtca gggcttgggg ttcgtggttt cactgattgg gattgcgggc 60 atcattgctg ccacctgcat ggaccagtgg agcacccaag acttgtacaa caaccccgta 120 acagctgttt tcaactacca ggggctgtgg cgctcctgtg tccgagagag ctctggcttc 180 accgagtgcc ggggetactt caccctgtac ccatacgacg tgccagacta cgcactgggg 240 ctgccagcca tgctgcaggc agtgcgagcc ctgatgatcg taggcatcgt cctgggtgcc 300 attggcctcc tggtatccat ctttgccctg aaatgcatcc gcattggcag catggaggac 360 tctgccaaag ccaacatgac actgacctcc gggatcatgt tcattgtctc aggtctttgt 420 gcaattgctg gagtgtctgt gtttgccaac atgctggtga Page 4 ctaacttctg gatgtccaca 480

    Sequences_342-31PCT.txt

    2018200685 30 Jan 2018

    gctaacatgt acaccggcat gggtgggatg gtgcagactg ttcagaccag gtacacattt 540 ggtgcggctc tgttcgtggg ctgggtcgct ggaggcctca cactaattgg gggtgtgatg 600 atgtgcatcg cctgccgggg cctggcacca gaagaaacca actacaaagc cgtttcttat 660 catgcctcgg gccacagtgt tgcctacaag cctggaggct tcaaggccag cactggcttt 720 gggtccaaca ccaaaaacaa gaagatatac gatggaggtg cccgcacaga ggacgaggta 780 caatcttatc cttccaagca cgactatgtg taa 813

    <210> 6 <211> 270 <212> PRT <213> Homo sapiens <400> 6

    Met 1 Ala Val Thr Ala 5 Cys Gln Gly Leu Gly 10 Phe Val Val Ser Leu 15 He Gly lie Ala Gly 20 lie He Ala Ala Thr 25 Cys Met Asp Gln Trp 30 Ser Thr Gln Asp Leu 35 Tyr Asn Asn Pro Val 40 Thr Ala Val Phe Asn 45 Tyr Gln Gly Leu Trp 50 Arg Ser Cys Val Arg 55 Glu Ser Ser Gly Phe 60 Thr Glu Cys Arg Gly 65 Tyr Phe Thr Leu Tyr 70 Pro Tyr Asp Val Pro 75 Asp Tyr Ala Leu Gly 80 Leu Pro Ala Met Leu 85 Gln Ala Val Arg Ala 90 Leu Met He Val Gly 95 He Val Leu Gly Ala 100 lie Gly Leu Leu Val 105 Ser He Phe Ala Leu 110 Lys Cys He Arg He 115 Gly Ser Met Glu Asp 120 Ser Ala Lys Ala Asn 125 Met Thr Leu Thr Ser 130 Gly He Met Phe He 135 Val Ser Gly Leu Cys 140 Ala He Ala Gly Val 145 Ser Val Phe Ala Asn 150 Met Leu Val Thr Asn 155 Phe Trp Met Ser Thr 160 Ala Asn Met Tyr Thr Gly Met Gly Gly Met Val Gln Thr Val Gln Thr

    165 170 175

    Page 5

    Sequences_342-31PCT.txt

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    Arg Tyr Thr Phe 180 Gly Ala Ala Leu Phe Val Gly Trp Val Ala Gly Gly 185 190 Leu Thr Leu Ile Gly Gly Val Met Met Cys Ile Ala Cys Arg Gly Leu 195 200 205 Ala Pro Glu Glu Thr Asn. Tyr Lys Ala Val Ser Tyr His Ala Ser Gly 210 215 220 His Ser Val Ala Tyr Lys Pro Gly Gly Phe Lys Ala Ser Thr Gly Phe 225 230 235 240 Gly Ser Asn Thr Lys Asn Lys Lys Ile Tyr Asp Gly Gly Ala Arg Thr 245 250 255 Glu Asp Glu Val Gln Ser Tyr Pro Ser Lys His Asp Tyr Val 260 265 270

    <210> 7 <211> 786 <212> DNA <213> Homo sapiens <400> 7 atgtccacca ccacatgcca agtggtggcg ttcctcctgt ccatcctggg gctggccggc 60 tgcatcgcgg ccaccgggat ggacatgtgg agcacccagg acctgtacga caaccccgtc 120 acctccgtgt tccagtacga agggctctgg aggagctgcg tgaggcagag ttcaggcttc 180 accgaatgca ggccctattt caccatcctg ggacttccag ccatgctgca ggcagtgcga 240 gccctgatga tcgtaggcat cgtcctgggt gccattggcc tcctggtatc catctttgcc 300 ctgaaatgca tccgcattgg cagcatggag gactctgcca aagccaacat gacactgacc 360 tccgggatca tgttcattgt ctcaggtctt tgtgcaattg ctggagtgtc tgtgtttgcc 420 aacatgctgg tgactaactt ctggatgtcc acagctaaca tgtacaccgg catgggtggg 480 atggtgcaga ctgttcagac caggtacaca tttggtgcgg ctctgttcgt gggctgggtc 540 gctggaggcc tcacactaat tgggggtgtg atgatgtgca tcgcctgccg gggcctggca 600 ccagaagaaa ccaactacaa agccgtttct tatcatgcct caggccacag tgttgcctac 660 aagcctggag gcttcaaggc cagcactggc tttgggtcca acaccaaaaa caagaagata 720 tacgatggag gtgcccgcac agaggacgag gtacaatctt atccttccaa gcacgactat 780 gtgtaa <210> 8 <211> 261 <212> PRT <213> Homo sapiens 786

    Page 6

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    Sequences_342-31PCT. txt <400> 8

    Met Ser Thr Thr Thr Cys Gln Val Val Ala Phe Leu Leu Ser Ile Leu 15 10 15

    Gly Leu Ala Gly Cys Ile Ala Ala Thr Gly Met Asp Met Trp Ser Thr 20 25 30

    Gln Asp Leu Tyr Asp Asn Pro Val Thr Ser Val Phe Gln Tyr Glu Gly 35 40 45

    Leu Trp Arg Ser Cys Val Arg Gln Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60

    Pro Tyr Phe Thr Ile Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80

    Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95

    Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110

    Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125

    Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140

    Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160

    Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175

    Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190

    Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205

    Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220

    Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240

    Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser Page 7

    2018200685 30 Jan 2018

    Sequences_3 42-31PCT. txt 245 250 255

    Lys His Asp Tyr Val 260 <210> 9 <211> 795 <212> DNA <213> Mus musculus <400> 9

    atggccacca ccacgtgcca ggtggtaggg cttctcctgt ccctcctggg tctggccggc 60 tgcatagccg ccactgggat ggacatgtgg agcactcaag acctgtatga caacccagtc 120 accgccgtgt tccagtatga agggctctgg aggagttgcg tgcaacagag ctcggggttc 180 accgagtgcc ggccatactt caccatcctg ggccttccag ccatgctgca agctgtacga 240 gccctgatga tcgtgggcat tgttctgggg gtcatcggta tcctcgtgtc catcttcgcc 300 ctgaagtgca ttcgcattgg tagcatggat gactctgcca aggccaagat gactctgact 360 tctgggatct tgttcatcat ctccggcatc tgtgcaatca ttggtgtgtc tgtgtttgcc 420 aacatgctgg tgaccaactt ctggatgtcc acagctaaca tgtacagcgg catgggcggc 480 atgggtggca tggtgcagac cgttcagacc aggtacacct ttggtgcagc tctgttcgtg 540 ggctgggttg ctggaggcct caccctgatt gggggagtga tgatgtgcat cgcctgccgt 600 ggcctgacac cagatgacag caacttcaaa gctgtgtctt accatgcctc tggccaaaat 660 gttgcctaca ggcctggagg ctttaaggcc agcactggct ttgggtccaa caccagaaac 720 aagaagatct acgatggggg tgcccgcaca gaagacgatg aacagtctca tcctaccaag 780 tatgactatg tgtag 795 <210> 10 <211> 795 <212> DNA <213> Mus musculus <400> 10 atgtcggtga ccgcctgcca gggcttgggg tttgtggtgt cactgatcgg gtttgcgggc 60 atcattgcag ccacttgtat ggaccagtgg agcacccagg atttatacaa caacccggtg 120 accgctgtat tcaactacca agggctatgg cgttcatgcg tccgagagag ctctggcttc 180 accgagtgcc gaggctactt caccctgttg gggttgccag ccatgctgca agctgtacga 240 gccctgatga tcgtgggcat tgttctgggg gtcatcggta tcctcgtgtc catcttcgcc 300 ctgaagtgca ttcgcattgg tagcatggat gactctgcca aggccaagat gactctgact 360 tctgggatct tgttcatcat ctccggcatc tgtgcaatca ttggtgtgtc tgtgtttgcc 420 aacatgctgg tgaccaactt ctggatgtcc acagctaaca tgtacagcgg catgggcggc 480

    Page 8

    Sequences_342-31PCT.txt

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    atgggtggca tggtgcagac cgttcagacc aggtacacct ttggtgcagc tctgttcgtg 540 ggctgggttg ctggaggcct caccctgatt gggggagtga tgatgtgcat cgcctgccgt 600 ggcctgacac cagatgacag caacttcaaa gctgtgtctt accatgcctc tggccaaaat 660 gttgcctaca ggcctggagg ctttaaggcc agcactggct ttgggtccaa caccagaaac 720 aagaagatct acgatggggg tgcccgcaca gaagacgatg aacagtctca tcctaccaag 780 tatgactatg tgtag 795

    <210> 11 <211> 21 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 11 tggctctgtg tcgacactgt g 21 <210> 12 <211> 21 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 12 gtgtacatgt tagctgtgga c 21 <210> 13 <211> 55 <212> PRT <213> Homo sapiens <400> 13

    Met 1 Asp Met Trp Ser 5 Thr Gin Asp Leu Tyr 10 Asp Asn Pro Val Thr 15 Ser Val Phe Gin Tyr 20 Glu Gly Leu Trp Arg 25 Ser Cys Val Arg Gin 30 Ser Ser Gly Phe Thr 35 Glu Cys Arg Pro Tyr 40 Phe Thr Ile Leu Gly 45 Leu Pro Ala Met Leu Gin Ala Val Arg Ala

    50 55 <210> 14 <211> 153 <212> PRT <213> Homo sapiens

    Page 9

    Sequences_342-31PCT. txt <400> 14

    2018200685 30 Jan 2018

    Met Asp Met Trp 1 Ser 5 Thr Gln Asp Leu Tyr Asp Asn Pro Val Thr Ser 10 15 val Phe Gln Tyr Glu Gly Leu Trp Arg Ser Cys Val Arg Gln Ser Ser 20 25 30 Gly Phe Thr Glu Cys Arg Pro Tyr Phe Thr Ile Leu Gly Leu Pro Ala 35 40 45 Met Leu Gln Ala Val Arg Ala Leu Met Ile Val Gly Ile Val Leu Gly 50 55 60 Ala Ile Gly Leu Leu Val Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile 65 70 75 80 Gly Ser Met Glu Asp Ser Ala Lys Ala Asn Met Thr Leu Thr Ser Gly 85 90 95 Ile Met Phe Ile Val Ser Gly Leu Cys Ala Ile Ala Gly Val Ser Val 100 105 110 Phe Ala Asn Met Leu Val Thr Asn Phe Trp Met Ser Thr Ala Asn Met 115 120 125 Tyr Thr Gly Met Gly Gly Met Val Gln Thr Val Gln Thr Arg Tyr Thr 130 135 140 Phe Gly Ala Ala Leu Phe Val Gly Trp

    145 150 <210> 15 <211> 390 <212> DNA <213> Homo sapiens <400> 15

    atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60 gacgcggccc agccggccag gcgcgcgcgc cgtacgaagc ttggtaccga gctcggatcc 120 actccagtgt ggtggaattc tgcagatggc cgcatggacc agtggagcac ccaagacttg 180 tacaacaacc ccgtaacagc tgttttcaac taccaggggc tgtggcgctc ctgtgtccga 240 gagagctctg gcttcaccga gtgccggggc tacttcaccc tgctggggct gccagccatg 300 ctgcaggcag tgcgagcggc catccagcac agtggcggcc gctcgaggag ggcccgaaca 360 aaaactcatc tcagaagagg atctgaatag 390

    Page 10

    Sequences_342-31PCT. txt

    2018200685 30 Jan 2018

    <210> <211> <212> <213> : <400> 16 129 PRT Homo 16 sapiens Met 1 Glu Thr Asp Thr 5 Leu Leu Leu Trp Val 10 Gly Ser Thr Gly 20 Asp Ala Ala Gln Pro 25 Ala Lys Leu Gly 35 Thr Glu Leu Gly Ser 40 Thr Pro Asp Gly 50 Arg Met Asp Gln Trp 55 Ser Thr Gln Val 65 Thr Ala Val Phe Asn 70 Tyr Gln Gly Leu Glu Ser Ser Gly Phe 85 Thr Glu Cys Arg Gly 90 Leu Pro Ala Met 100 Leu Gln Ala Val Arg 105 Ala Gly Arg Ser 115 Arg Arg Ala Arg Thr 120 Lys Thr

    Glu

    110

    125

    Val Pro 15

    Arg Thr

    Ser Ala

    Asn Pro

    Val Arg 80

    Leu Gly 95

    Ser Gly

    Gly Ser <210> 17 <211> 411 <212> DNA <213> Homo sapiens <400> 17

    atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60 gacgcggccc agccggccag gcgcgcgcgc cgtacgaagc ttggtaccga gctcggatcc 120 actccagtgt ggtggaattc tgcagatggc cgcgccctga tgatcgtagg catcgtcctg 180 ggtgccattg gcctcctggt atccatcttt gccctgaaat gcatccgcat tggcagcatg 240 gaggactctg ccaaagccaa catgacactg acatccggga tcatgttcat tgtctcaggt 300 ctttgtgcaa ttgctggagt gtctgtgttt gccaacgcgg ccatccagca cagtggcggc 360 cgctcgagga gggcccgaac aaaaactcat ctcagaagag gatctgaata g 411

    Page 11

    Sequences_342-31PCT.txt

    2018200685 30 Jan 2018

    <210> 18 <211> 136 <212> PRT <213> Homo <400> 18 Met Glu Thr

    Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Arg Arg Ala Arg Arg Thr 20 25 30

    Lys Leu Gly Thr Glu Leu Gly Ser Thr Pro Val Trp Trp Asn Ser Ala 35 40 45

    Asp Gly Arg Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly 50 55 60

    Leu Leu Val Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met 65 70 75 80

    Glu Asp Ser Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe 85 90 95

    Ile Val Ser Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn 100 105 110

    Ala Ala Ile Gln His Ser Gly Gly Arg Ser Arg Arg Ala Arg Thr Lys 115 120 125

    Thr His Leu Arg Arg Gly Ser Glu 130 135 <210> 19 <211> 531 <212> DNA <213> Homo sapiens <400> 19

    atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60 gacgcggccc agccggccag gcgcgccatg gaccagtgga gcacccaaga cttgtacaac 120 aaccccgtaa cagctgtttt caactaccag gggctgtggc gctcctgtgt ccgagagagc 180 tctggcttca ccgagtgccg gggctacttc accctgctgg ggctgccagc catgctgcag 240 gcagtgcgag ccctgatgat cgtaggcato gtcctgggtg ccattggcct cctggtatcc 300 atctttgccc tgaaatgcat ccgcattggc agcatggagg actctgccaa agccaacatg 360 acactgacct ccgggatcat gttcattgtc tcaggtcttt gtgcaattgc tggagtgtct 420 gtgtttgcca acatgctggt gactaacttc tggatgtcca cagctaacat gtacaccggc 480

    Page 12

    Sequences_342-31PCT.txt atgggtggga tggtgcagac tgttcagacc aggtacacat ttggtgcgta g

    531

    2018200685 30 Jan 2018 <210> 20 <211> 176 <212> PRT <213> Homo sapiens <400> 20

    Met 1 Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val 15 Pro 5 10 Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Arg Arg Ala Met Asp Gln 20 25 30 Trp Ser Thr Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn 35 40 45 Tyr Gln Gly Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr 50 55 60 Glu Cys Arg Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln 65 70 75 80 Ala Val Arg Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly 85 90 95 Leu Leu Val Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met 100 105 110 Glu Asp Ser Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe 115 120 125 Ile Val Ser Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn 130 135 140 Met Leu Val Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly 145 150 155 160 Met Gly Gly Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala

    165 170 175 <210> 21

    <211> <212> <213> 10 PRT Homo sapiens <400> 21 Asp Gln Trp Ser Thr Gln Asp Leu Tyr Asn 1 5 10

    Page 13

    Sequences_342-31PCT.txt

    2018200685 30 Jan 2018

    <210> 22 <211> 11 <212> PRT <213> Homo <400> 22 Asn Asn Pro 1 <210> 23 <211> 14 <212> PRT <213> Homo <400> 23 Ser Thr Gln 1 <210> 24 <211> 12 <212> PRT <213> Homo <400> 24 Asp Met Trp 1 <210> 25 <211> 12 <212> PRT <213> Homo <400> 25 Cys Arg Pro 1 <210> 26 <211> 13 <212> PRT <213> Homo <400> 26 Thr Asn Phe 1 <210> 27 <211> 13 <212> PRT <213> Homo <400> 27

    sapiens

    Val Thr Ala 5 sapiens

    Asp Leu Tyr 5 sapiens

    Ser Thr Gln 5 sapiens

    Tyr Phe Thr 5 sapiens

    Trp Met Ser 5 sapiens

    Val Phe Asn Tyr Gln 10

    Asn Asn Pro Val Thr Ala Val Phe 10

    Asp Leu Tyr Asp Asn Pro 10

    Ile Leu Gly Leu Pro Ala 10

    Thr Ala Asn Met Tyr Thr Gly 10

    Page 14

    Sequences_342-31PCT.txt

    2018200685 30 Jan 2018

    Asp Ser Ala 1 Lys Ala 5 Asn Met Thr Leu Thr 10 Ser Gly Ile <210> 28 <211> 55 <212> PRT <213> Homo sapiens <400> 28 Met Asp Gln Trp Ser Thr Gln Asp Leu Tyr Asn Asn Pro Val Thr 1 5 10 15 Val Phe Asn Tyr Gln Gly Leu Trp Arg Ser Cys Val Arg Glu Ser 20 25 30 Gly Phe Thr Glu Cys Arg Gly Tyr Phe Thr Leu Leu Gly Leu Pro 35 40 45 Met Leu Gln Ala Val Arg Ala 50 55 <210> 29 <211> 24 <212> PRT <213> Homo sapiens <400> 29 Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser Ala 1 5 10 15 Ala Asn Met Thr Leu Thr Ser Gly 20 <210> 30 <211> 40 <212> PRT <213> Homo sapiens <400> 30 Ala Asn Met Leu Val Thr Asn Phe Trp Met Ser Thr Ala Asn Met 1 5 10 15 Thr Gly Met Gly Gly Met Val Gln Thr Val Gln Thr Arg Tyr Thr 20 25 30 Gly Ala Ala Leu Phe Val Gly Trp

    35 <210> 31 <211> 153 <212> PRT

    Page 15

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    Sequences_342-31PCT.txt <213> Homo sapiens <400> 31

    Met 1 Asp Gln Trp Ser 5 Thr Gln Asp Leu Tyr Asn Asn Pro Val 10 Thr 15 Ala Val Phe Asn Tyr Gln Gly Leu Trp Arg Ser Cys Val Arg Glu Ser Ser 20 25 30 Gly Phe Thr Glu Cys Arg Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala 35 40 45 Met Leu Gln Ala Val Arg Ala Leu Met Ile Val Gly Ile Val Leu Gly 50 55 60 Ala Ile Gly Leu Leu Val Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile 65 70 75 80 Gly Ser Met Glu Asp Ser Ala Lys Ala Asn Met Thr Leu Thr Ser Gly 85 90 95 Ile Met Phe Ile Val Ser Gly Leu Cys Ala Ile Ala Gly Val Ser Val 100 105 110 Phe Ala Asn Met Leu Val Thr Asn Phe Trp Met Ser Thr Ala Asn Met 115 120 125 Tyr Thr Gly Met Gly Gly Met Val Gln Thr Val Gln Thr Arg Tyr Thr 130 135 140 Phe Gly Ala Ala Leu Phe Val Gly Trp

    145 150 <210> 32 <211> 3359 <212> DNA <213> Homo sapiens <400> 32

    cacaccttcg gcagcaggag ggcggoagct tctcgcaggc ggcagggcgg gcggccagga 60 tcatgtccac caccacatgc caagtggtgg cgttcctcct gtccatcctg gggctggccg 120 gctgcatcgc ggccaccggg atggacatgt ggagcaccca ggacctgtac gacaaccccg 180 tcacctccgt gttccagtac gaagggctct ggaggagctg cgtgaggcag agttcaggct 240 tcaccgaatg caggccctat ttcaccatcc tgggacttcc agccatgctg caggcagtgc 300 gagccctgat gatcgtaggc atcgtcctgg gtgccattgg cctcctggta tccatctttg 360 ccctgaaatg catccgcatt ggcagcatgg aggactctgc caaagccaac atgacactga 420

    Page 16

    Sequences_342-31PCT.txt

    2018200685 30 Jan 2018

    cctccgggat catgttcatt gtctcaggtc tttgtgcaat tgctggagtg tctgtgtttg 480 ccaacatgct ggtgactaac ttctggatgt ccacagctaa catgtacacc ggcatgggtg 540 ggatggtgca gactgttcag accaggtaca catttggtgc ggctctgttc gtgggctggg 600 tcgctggagg cctcacacta attgggggtg tgatgatgtg catcgcctgc cggggcctgg 660 caccagaaga aaccaactac aaagccgttt cttatcatgc ctcaggccac agtgttgcct 720 acaagcctgg aggcttcaag gccagcactg gctttgggtc caacaccaaa aacaagaaga 780 tatacgatgg aggtgcccgc acagaggacg aggtacaatc ttatccttcc aagcacgact 840 atgtgtaatg ctctaagacc tctcagcacg ggcggaagaa actcccggag agctcaccca 900 aaaaacaagg agatcccatc tagatttctt cttgcttttg actcacagct ggaagttaga 960 aaagcctcga tttcatcttt ggagaggcca aatggtctta gcctcagtct ctgtctctaa 1020 atattccacc ataaaacagc tgagttattt atgaattaga ggctatagct cacattttca 1080 atcctctatt tcttttttta aatataactt tctactctga tgagagaatg tggttttaat 1140 ctctctctca cattttgatg atttagacag actccccctc ttcctcctag tcaataaacc 1200 cattgatgat ctatttccca gcttatcccc aagaaaactt ttgaaaggaa agagtagacc 1260 caaagatgtt attttctgct gtttgaattt tgtctcccca cccccaactt ggctagtaat 1320 aaacacttac tgaagaagaa gcaataagag aaagatattt gtaatctctc cagcccatga 1380 tctcggtttt cttacactgt gatcttaaaa gttaccaaac caaagtcatt ttcagtttga 1440 ggcaaccaaa cctttctact gctgttgaca tcttcttatt acagcaacac cattctagga 1500 gtttcctgag ctctccactg gagtcctctt tctgtcgcgg gtcagaaatt gtccctagat 1560 gaatgagaaa attatttttt ttaatttaag tcctaaatat agttaaaata aataatgttt 1620 tagtaaaatg atacactatc tctgtgaaat agcctcaccc ctacatgtgg atagaaggaa 1680 atgaaaaaat aattgctttg acattgtcta tatggtactt tgtaaagtca tgcttaagta 1740 caaattccat gaaaagctca ctgatcctaa ttctttccct ttgaggtctc tatggctctg 1800 attgtacatg atagtaagtg taagccatgt aaaaagtaaa taatgtctgg gcacagtggc 1860 tcacgcctgt aatcctagca ctttgggagg ctgaggagga aggatcactt gagcccagaa 1920 gttcgagact agcctgggca acatggagaa gccctgtctc tacaaaatac agagagaaaa 1980 aatcagccag tcatggtggc ctacacctgt agtcccagca ttccgggagg ctgaggtggg 2040 aggatcactt gagcccaggg aggttggggc tgcagtgagc catgatcaca ccactgcact 2100 ccagccaggt gacatagcga gatcctgtct aaaaaaataa aaaataaata atggaacaca 2160 gcaagtccta ggaagtaggt taaaactaat tctttaaaaa aaaaaaaaag ttgagcctga 2220 attaaatgta atgtttccaa gtgacaggta tccacatttg catggttaca agccactgcc 2280 agttagcagt agcactttcc tggcactgtg gtcggttttg ttttgttttg ctttgtttag 2340

    Page 17

    Sequences_342-31PCT. txt

    2018200685 30 Jan 2018

    agacggggtc tcactttcca ggctggcctc aaactcctgc actcaagcaa ttcttctacc 2400 ctggcctccc aagtagctgg aattacaggt gtgcgccatc acaactagct ggtggtcagt 2460 tttgttactc tgagagctgt tcacttctct gaattcacct agagtggttg gaccatcaga 2520 tgtttgggca aaactgaaag ctctttgcaa ccacacacct tccctgagct tacatcactg 2580 cccttttgag cagaaagtct aaattccttc caagacagta gaattccatc ccagtaccaa 2640 agccagatag gccccctagg aaactgaggt aagagcagtc tctaaaaact acccacagca 2700 gcattggtgc aggggaactt ggccattagg ttattatttg agaggaaagt cctcacatca 2760 atagtacata tgaaagtgac ctccaagggg attggtgaat actcataagg atcttcaggc 2820 tgaacagact atgtctgggg aaagaacgga ttatgcccca ttaaataaca agttgtgttc 2880 aagagtcaga gcagtgagct cagaggccct tctcactgag acagcaacat ttaaaccaaa 2940 ccagaggaag tatttgtgga actcactgcc tcagtttggg taaaggatga gcagacaagt 3000 caactaaaga aaaaagaaaa gcaaggagga gggttgagca atctagagca tggagtttgt 3060 taagtgctct ctggatttga gttgaagagc atccatttga gttgaaggcc acagggcaca 3120 atgagctctc ccttctacca ccagaaagtc cctggtcagg tctcaggtag tgcggtgtgg 3180 ctcagctggg tttttaatta gcgcattctc tatccaacat ttaattgttt gaaagcctcc 3240 atatagttag attgtgcttt gtaattttgt tgttgttgct ctatcttatt gtatatgcat 3300 tgagtattaa cctgaatgtt ttgttactta aatattaaaa acactgttat cctacagtt 3359 <210> 33 <211> 849 <212> DNA <213> Mus musculus <400> 33 gagaacctgc ctgtctcttg tcctctccat ttgtgtggac tctgtgctcc atcatgtcgg 60 tgaccgcctg ccagggcttg gggtttgtgg tgtcactgat cgggtttgcg ggcatcattg 120 cagccacttg tatggaccag tggagcaccc aggatttata caacaacccg gtgaccgctg 180 tattcaacta ccaagggcta tggcgttcat gcgtccgaga gagctctggc ttcaccgagt 240 gccgaggcta cttcaccctg ttggggttgc cagccatgct gcaagctgta cgagccctga 300 tgatcgtggg cattgttctg ggggtcatcg gtatcctcgt gtccatcttc gccctgaagt 360 gcattcgcat tggtagcatg gatgactctg ccaaggccaa gatgactctg acttctggga 420 tcttgttcat catctccggc atctgtgcaa tcattggtgt gtctgtgttt gccaacatgc 480 tggtgaccaa cttctggatg tccacagcta acatgtacag cggcatgggc ggcatgggtg 540 gcatggtgca gaccgttcag accaggtaca ccttcggtgc agctctgttc gtgggctggg 600 ttgctggagg cctcaccctg attgggggag tgatgatgtg catcgcctgc cgtggcctga 660 caccagatga cagcaacttc aaagctgtgt cttaccatgc ctctggccaa aatgttgcct 720

    Page 18

    Sequences_342-31PCT. txt

    2018200685 30 Jan 2018

    acaggcctgg aggctttaag gccagcactg gctttgggtc caacaccaga aacaagaaga 780 tctacgatgg gggtgcccgc acagaagacg atgaacagtc tcatcctacc aagtatgact 840 atgtgtagt 849

    <210> 34 <211> 3350 <212> DNA <213> Homo sapiens <400> 34

    agaattgcgc tgtccacttg tcgtgtggct ctgtgtcgac actgtgcgcc accatggccg 60 tgactgcctg tcagggcttg gggttcgtgg tttcactgat tgggattgcg ggcatcattg 120 ctgccacctg catggaccag tggagcaccc aagacttgta caacaacccc gtaacagctg 180 ttttcaacta ccaggggctg tggcgctcct gtgtccgaga gagctctggc ttcaccgagt 240 gccggggcta cttcaccctg ctggggctgc cagccatgct gcaggcagtg cgagccctga 300 tgatcgtagg catcgtcctg ggtgccattg gcctcctggt atccatcttt gccctgaaat 360 gcatccgcat tggcagcatg gaggactctg ccaaagccaa catgacactg acctccggga 420 tcatgttcat tgtctcaggt ctttgtgcaa ttgctggagt gtctgtgttt gccaacatgc 480 tggtgactaa cttctggatg tccacagcta acatgtacac cggcatgggt gggatggtgc 540 agactgttca gaccaggtac acatttggtg cggctctgtt cgtgggctgg gtcgctggag 600 gcctcacact aattgggggt gtgatgatgt gcatcgcctg ccggggcctg gcaccagaag 660 aaaccaacta caaagccgtt tcttatcatg cctcaggcca cagtgttgcc tacaagcctg 720 gaggcttcaa ggccagcact ggctttgggt ccaacaccaa aaacaagaag atatacgatg 780 gaggtgcccg cacagaggac gaggtacaat cttatccttc caagcacgac tatgtgtaat 840 gctctaagac ctctcagcac gggcggaaga aactcccgga gagctcaccc aaaaaacaag 900 gagatcccat ctagatttct tcttgctttt gactcacagc tggaagttag aaaagcctcg 960 atttcatctt tggagaggcc aaatggtctt agcctcagtc tctgtctcta aatattccac 1020 cataaaacag ctgagttatt tatgaattag aggctatagc tcacattttc aatcctctat 1080 ttcttttttt aaatataact ttctactctg atgagagaat gtggttttaa tctctctctc 1140 acattttgat gatttagaca gactccccct cttcctccta gtcaataaac ccattgatga 1200 tctatttccc agcttatccc caagaaaact tttgaaagga aagagtagac ccaaagatgt 1260 tattttctgc tgtttgaatt ttgtctcccc acccccaact tggctagtaa taaacactta 1320 ctgaagaaga agcaataaga gaaagatatt tgtaatctct ccagcccatg atctcggttt 1380 tcttacactg tgatcttaaa agttaccaaa ccaaagtcat tttcagtttg aggcaaccaa 1440 acctttctac tgctgttgac atcttcttat tacagcaaca ccattctagg agtttcctga 1500

    Page 19

    Sequences_342-31PCT.txt

    2018200685 30 Jan 2018

    gctctccact ggagtcctct ttctgtcgcg ggtcagaaat tgtccctaga tgaatgagaa 1560 aattattttt tttaatttaa gtcctaaata tagttaaaat aaataatgtt ttagtaaaat 1620 gatacactat ctctgtgaaa tagcctcacc cctacatgtg gatagaagga aatgaaaaaa 1680 taattgcttt gacattgtct atatggtact ttgtaaagtc atgcttaagt acaaattcca 1740 tgaaaagctc actgatccta attctttccc tttgaggtct ctatggctct gattgtacat 1800 gatagtaagt gtaagccatg taaaaagtaa ataatgtctg ggcacagtgg ctcacgcctg 1860 taatcctagc actttgggag gctgaggagg aaggatcact tgagcccaga agttcgagac 1920 tagcctgggc aacatggaga agccctgtct ctacaaaata cagagagaaa aaatcagcca 1980 gtcatggtgg cctacacctg tagtcccagc attccgggag gctgaggtgg gaggatcact 2040 tgagcccagg gaggttgggg ctgcagtgag ccatgatcac accactgcac tccagccagg 2100 tgacatagcg agatcctgtc taaaaaaata aaaaataaat aatggaacac agcaagtcct 2160 aggaagtagg ttaaaactaa ttctttaaaa aaaaaaaaaa gttgagcctg aattaaatgt 2220 aatgtttcca agtgacaggt atccacattt gcatggttac aagccactgc cagttagcag 2280 tagcactttc ctggcactgt ggtcggtttt gttttgtttt gctttgttta gagacggggt 2340 ctcactttcc aggctggcct caaactcctg cactcaagca attcttctac cctggcctcc 2400 caagtagctg gaattacagg tgtgcgccat cacaactagc tggtggtcag ttttgttact 2460 ctgagagctg ttcacttctc tgaattcacc tagagtggtt ggaccatcag atgtttgggc 2520 aaaactgaaa gctctttgca accacacacc ttccctgagc ttacatcact gcccttttga 2580 gcagaaagtc taaattcctt ccaagacagt agaattccat cccagtacca aagccagata 2640 ggccccctag gaaactgagg taagagcagt ctctaaaaac tacccacagc agcattggtg 2700 caggggaact tggccattag gttattattt gagaggaaag tcctcacatc aatagtacat 2760 atgaaagtga cctccaaggg gattggtgaa tactcataag gatcttcagg ctgaacagac 2820 tatgtctggg gaaagaacgg attatgcccc attaaataac aagttgtgtt caagagtcag 2880 agcagtgagc tcagaggccc ttctcactga gacagcaaca tttaaaccaa accagaggaa 2940 gtatttgtgg aactcactgc ctcagtttgg gtaaaggatg agcagacaag tcaactaaag 3000 aaaaaagaaa agcaaggagg agggttgagc aatctagagc atggagtttg ttaagtgctc 3060 tctggatttg agttgaagag catccatttg agttgaaggc cacagggcac aatgagctct 3120 cccttctacc accagaaagt ccctggtcag gtctcaggta gtgcggtgtg gctcagctgg 3180 gtttttaatt agcgcattct ctatccaaca tttaattgtt tgaaagcctc catatagtta 3240 gattgtgctt tgtaattttg ttgttgttgc tctatcttat tgtatatgca ttgagtatta 3300 acctgaatgt tttgttactt aaatattaaa aacactgtta tcctacagtt 3350

    <210> 35

    Page 20

    2018200685 30 Jan 2018

    Sequences_342-31PCT.txt <211> 264 <212> PRT <213> Mus musculus <400> 35

    Met Ser Val Thr Ala Cys Gin Gly Leu Gly Phe Val Val Ser Leu Ile 15 10 15

    Gly Phe Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gin Trp Ser Thr 20 25 30

    Gin Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gin Gly 35 40 45

    Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60

    Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gin Ala Val Arg 65 70 75 80

    Ala Leu Met Ile Val Gly Ile Val Leu Gly Val Ile Gly Ile Leu Val 85 90 95

    Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Asp Asp Ser 100 105 110

    Ala Lys Ala Lys Met Thr Leu Thr Ser Gly Ile Leu Phe Ile Ile Ser 115 120 125

    Gly Ile Cys Ala Ile Ile Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140

    Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Ser Gly Met Gly Gly 145 150 155 160

    Met Gly Gly Met Val Gin Thr Val Gin Thr Arg Tyr Thr Phe Gly Ala 165 170 175

    Ala Leu Phe Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly 180 185 190

    Val Met Met Cys Ile Ala Cys Arg Gly Leu Thr Pro Asp Asp Ser Asn 195 200 205

    Phe Lys Ala Val Ser Tyr His Ala Ser Gly Gin Asn Val Ala Tyr Arg 210 215 220

    Pro Gly Gly Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Arg Asn 225 230 235 240

    Page 21

    2018200685 30 Jan 2018

    Sequences_342-31PCT. txt

    Lys Lys Ile Tyr Asp Gly Gly Ala Arg Thr Glu Asp Asp Glu Gln Ser 245 250 255

    His Pro Thr Lys Tyr Asp Tyr Val 260 <210> 36 <211> 2786 <212> DNA <213> Mus musculus <400> 36

    ggccgggaac cttcccagca agagggtggt ggttgctcct ggaagcctgc gcccagcagc 60 tgaagccatg gccaccacca cgtgccaggt ggtagggctt ctcctgtccc tcctgggtct 120 ggccggctgc atagccgcca ctgggatgga catgtggagc actcaagacc tgtatgacaa 180 cccagtcacc gccgtgttcc agtatgaagg gctctggagg agttgcgtgc aacagagctc 240 ggggttcacc gagtgccggc catacttcac catcctgggc cttccagcca tgctgcaagc 300 tgtacgagcc ctgatgatcg tgggcattgt tctgggggtc atcggtatcc tcgtgtccat 360 cttcgccctg aagtgcattc gcattggtag catggatgac tctgccaagg ccaagatgac 420 tctgacttct gggatcttgt tcatcatctc cggcatctgt gcaatcattg gtgtgtctgt 480 gtttgccaac atgctggtga ccaacttctg gatgtccaca gctaacatgt acagcggcat 540 gggcggcatg ggtggcatgg tgcagaccgt tcagaccagg tacaccttcg gtgcagctct 600 gttcgtgggc tgggttgctg gaggcctcac cctgattggg ggagtgatga tgtgcatcgc 660 ctgccgtggc ctgacaccag atgacagcaa cttcaaagct gtgtcttacc atgcctctgg 720 ccaaaatgtt gcctacaggc ctggaggctt taaggccagc actggctttg ggtccaacac 780 cagaaacaag aagatctacg atgggggtgc ccgcacagaa gacgatgaac agtctcatcc 840 taccaagtat gactatgtgt agtgctctaa gacccgccaa cctgtgtgca ggaggaaccc 900 ttccccaaga agagctcacc ccaaagcaac gggagtctac cttgttccct tgttgatttc 960 aactgacatc tgaaagttgg taaagcctga ttttcatcca tagggaggct agacagtctt 1020 ggccacatgt gtctgcctct aaatatccca tcacaaaaca gctgagttat cgtttatgag 1080 ttagaggcca taacactcac tttagcccaa ccctctgctt tttaccgtag actttctttt 1140 catctggtga tggaatggaa tttgactcac agactaatac tttaatggtt tagagaaact 1200 ttccttcctc gtacttaata agcctgctga tggtcgattt tccagcttga ccaccaaggg 1260 aaattttaaa aggaaaaaaa aatacattaa aaggcattat ttcctactca attgtgcctt 1320 acccaccccc aacttgactg ataataataa tgaacaccac ttaaagaaag aatgccagag 1380 gaaagatagt tgtgtttccc cccagccagt catctgagtc cccctatgtg gtgatctaga 1440

    Page 22

    Sequences_342-31PCT.txt

    2018200685 30 Jan 2018

    acattactcg ccacagtgat tttcaaagaa ggcaagcgag cctgttcgct ctgctcagca 1500 tctgctgatt ccagcaaggc ccttccagag ctttccacta gaagtcctcc ttctctcgga 1560 agtcagaaat tccccctaga agagtaagaa atagattctt ttgggtaacc tgagtcctag 1620 gtatagttat aataaatagt atattagcaa aacggtttgg tatctcagtg aattagtttc 1680 agccttacat atagaaaaag ctggggaaaa aaaaagcatc ccttgacatt gtctatagcg 1740 taagatccta tataaatcca agcttcaaca aaagctcact gagtctaata gttttctttt 1800 gaggtctcca cggccttagt actcatagat gcagcccctg tttaaaagta aaaaaattaa 1860 agtagcttaa aacgggttct tttttttttt ttttttttca aaaaatccaa tagagacctg 1920 tgtgtctggc atagctacag ttactgccaa tcgacagggc cacttctttg gtcctgtagg 1980 cagttttgca gttctgacag ctgcgccggg catcaatatg cagaccacac ccttctctgt 2040 gcttgtagga cgacccgttc aaggagaaag catgaactcc atctccatgt gagcctgaat 2100 gctcccagga aatggagata gggtgctctc caaaacccac ctgaacctga aacagctgta 2160 gcgctatgct gtaagagcct ggccatcaag ttcctatgga gaaaaagggc agtccttgca 2220 ttaatagtgc atatataagt ggcctctggg gggcagggat gaatattcag tggtggctcc 2280 gagtatgtac agaccgtcta aggagctgtg ttgaccaaga gccaggttaa tacgcagagt 2340 ttttcccact gggactacag tgattttaga ctatactgaa gaaggccctc tggaaaatca 2400 ttatctgaaa tggcataaag aatgaacaga ccaaacaatt taaggggagg gggcaggtgg 2460 aaggaggggg aaggaggtag aaataagaat ctagggcatg aagattgtta aggttcttgg 2520 ggtccaaatg gaaggtcacc cctttgaggc catggacaca atgcacccca cccctacccc 2580 cacctgccca cccaccagaa agtccctggt cggactggag gcagtgagaa tcagctgttt 2640 tcagttagtg ggtctcggtg tagcacctgg ctgtttcaaa gcttcccctt gctttgccgt 2700 tttttccgcc attgctgtct tgttttctgt gttattaacc tccatgtttt gtacgttaaa 2760 tattaaaaca ctgttaacat ccattc 2786

    <210> 37 <211> 264 <212> PRT <213> Mus musculus <400> 37

    Met 1 Ala Thr Thr Thr Cys 5 Gin Val Val Gly Leu Leu Leu Ser 10 Leu 15 Leu Gly Leu Ala Gly Cys Ile Ala Ala Thr Gly Met Asp Met Trp Ser Thr 20 25 30 Gin Asp Leu Tyr Asp Asn Pro Val Thr Ala Val Phe Gin Tyr Glu Gly

    35 40 45

    Page 23

    Sequences_342-31PCT.txt

    2018200685 30 Jan 2018

    Leu Trp Arg Ser Cys Val Gin Gin Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60

    Pro Tyr Phe Thr Ile Leu Gly Leu Pro Ala Met Leu Gin Ala Val Arg 65 70 75 80

    Ala Leu Met Ile Val Gly Ile Val Leu Gly Val Ile Gly Ile Leu Val 85 90 95

    Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Asp Asp Ser 100 105 110

    Ala Lys Ala Lys Met Thr Leu Thr Ser Gly Ile Leu Phe Ile Ile Ser 115 120 125

    Gly Ile Cys Ala Ile Ile Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140

    Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Ser Gly Met Gly Gly 145 150 155 160

    Met Gly Gly Met Val Gin Thr Val Gin Thr Arg Tyr Thr Phe Gly Ala 165 170 175

    Ala Leu Phe Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly 180 185 190

    Val Met Met Cys Ile Ala Cys Arg Gly Leu Thr Pro Asp Asp Ser Asn 195 200 205

    Phe Lys Ala Val Ser Tyr His Ala Ser Gly Gin Asn Val Ala Tyr Arg 210 215 220

    Pro Gly Gly Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Arg Asn 225 230 235 240

    Lys Lys Ile Tyr Asp Gly Gly Ala Arg Thr Glu Asp Asp Glu Gin Ser 245 250 255

    His Pro Thr Lys Tyr Asp Tyr Val

    260 <210> 38 <211> 40 <212> DNA <213> Artificial <220>

    Page 24

    2018200685 30 Jan 2018

    Sequences_342-31PCT. txt <223> Description of artificial sequence: Oligonucleotide <400> 38 gagaggatcc cgtacggtgg ctgcaccatc tgtcttcatc 40 <210> 39 <211> 37 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 39 gagagcggcc gcctaacact ctcccctgtt gaagctc 37 <210> 40 <211> 324 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: : PCR product <400> 40 cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300 agcttcaaca ggggagagtg ttag 324

    <210> 41 <211> 107 <212> PRT <213> Artificial <220>

    <223> Description of artificial sequence: Translation of PCR product <400> 41

    Arg 1 Thr Val Ala Ala 5 Pro Ser Val Phe Ile 10 Phe Pro Pro Ser Asp 15 Glu Gln Leu Lys Ser 20 Gly Thr Ala Ser Val 25 Val Cys Leu Leu Asn 30 Asn Phe Tyr Pro Arg 35 Glu Ala Lys Val Gln 40 Trp Lys Val Asp Asn 45 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

    50 55 60

    Page 25

    Sequences_342- 31PCT. txt

    2018200685 30 Jan 2018

    Thr Tyr Ser Leu 65

    Lys His Lys Val

    Ser Ser 70

    Thr

    Tyr Ala Cys 85

    Leu Thr Leu

    Glu Val

    Ser Lys Ala Asp 75

    Tyr Glu 80

    Thr His Gln Gly Leu Ser Ser 90 95

    Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 42 <211> 34 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 42 gagaaagctt tccaccaagg gcccatcggt cttc <210> 43 <211> 36 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 43 gagagcggcc gctcatttac ccggagacag ggagag <210> 44 <211> 21 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 44 taccagttga acttgacctc a <210> 45 <211> 981 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: PCR product <400> 45 ggcccatcgg tcttccccct ggcaccctcc tccaagagca cctctggggg cacagcggcc 60 ctgggctgcc tggtcaagga ctacttcccc gaaccggtga cggtgtcgtg gaactcaggc 120 gccctgacca gcggcgtgca caccttcccg gctgtcctac agtcctcagg actctactcc 180

    Page 2 6

    Sequences_342-31PCT. txt

    2018200685 30 Jan 2018

    ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 240 gtgaatcaca agcccagcaa caccaaggtg gacaagaaag ttgagcccaa atcttgtgac 300 aaaactcaca catgcccacc gtgcccagca cctgaactcc tggggggacc gtcagtcttc 360 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 420 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 480 gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt 540 gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc 600 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg 660 cagccccgag aaccacaggt gtacaccctg cccccatccc gggatgagct gaccaagaac 720 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 780 gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 840 ggctccttct tcctctatag caagctcacc gtggacaaga gcaggtggca gcaggggaac 900 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 960 tccctgtctc cgggtaaatg a 981

    <210> 46 <211> 326 <212> PRT <213> Artificial <220>

    <223> Description of artificial sequence: Translation of PCR product <400> 46

    Gly 1 Pro Ser Val Phe 5 Pro Leu Ala Pro Ser 10 Ser Lys Ser Thr Ser 15 Gly Gly Thr Ala Ala 20 Leu Gly Cys Leu Val 25 Lys Asp Tyr Phe Pro 30 Glu Pro Val Thr Val 35 Ser Trp Asn Ser Gly 40 Ala Leu Thr Ser Gly 45 Val His Thr Phe Pro 50 Ala Val Leu Gln Ser 55 Ser Gly Leu Tyr Ser 60 Leu Ser Ser Val Val 65 Thr Val Pro Ser Ser 70 Ser Leu Gly Thr Gln 75 Thr Tyr Ile Cys Asn 80 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

    85 90 95

    Page 27

    2018200685 30 Jan 2018

    Sequences_342-31PCT.txt

    Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 100 105 110

    Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125

    Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140

    Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 145 150 155 160

    Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 165 170 175

    Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 180 185 190

    Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 195 200 205

    Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 210 215 220

    Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 225 230 235 240

    Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255

    Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270

    Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285

    Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300

    Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320

    Ser Leu Ser Pro Gly Lys 325

    <210> 47 <211> 32 <212> DNA <213> Artificial

    Page 28

    2018200685 30 Jan 2018

    S equences_3 42-31PCT. txt <220>

    <223> Description of artificial sequence: Oligonucleotide <220>

    <221> misc_feature <222> (31) . . (32) <223> n is a, c, g, or t <400> 47 tttttttttt tttttttttt tttttttttt nn 32 <210> 48 <211> 30 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 48 aagcagtggt atcaacgcag agtacgcggg <210> 49 <211> 26 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 49 ctgctcactg gatggtggga agatgg 26 <210> 50 <211> 25 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 50 gggacagtca ctgagctgct cagag <210> 51 <211> 25 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 51 acaggggcca gtggatagac cgatg 25 <210> 52 <211> 27

    Page 2 9

    2018200685 30 Jan 2018

    Sequences_342-31PCT. txt <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 52 agccagggac caagggatag acagatg 27 <210> 53 <211> 45 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 53 gtaatacgac tcactatagg gcaagcagtg gtatcaacgc agagt 45 <210> 54 <211> 22 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 54 gtaatacgac tcactatagg gc 22 <210> 55 <211> 351 <212> DNA <213> Artificial <220>

    <223 > Description of artificial sequence: PCR product <400> 55 caggttcagc tgcagcagtc tggagctgag ctgatgaagc ctggggcctc agtgaagata 60 tcctgcaagg ctactggcta cacattcagt agctactgga tagagtgggt aaagcagagg 120 cctggacatg gccttgagtg gattggagag attttacctg gaagtggtag tactaactac 180 aatgagaagt tcaagggcaa ggccacattc actgcagata catcctccaa cacagcctac 240 atgcaactca gcagcctgac atctgaggac tctgccgtct attactgtgc aagatatgat 300 tacccctggt ttgcttactg gggccaaggg actctggtca ctgtctctgc a 351 <210> 56 <211> 354 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: PCR product

    Page 30

    2018200685 30 Jan 2018

    Sequences_342-31PCT.txt <400> 56 cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60 tcctgcaagg cttctgggta taccttcaca aactatggaa tgaactgggt gaagcaggct 120 ccaggaaagg gtttaaagtg gatgggctgg ataaacacca acactggaga gccaacatat 180 gctgaagagt tcaagggacg gtttgccttc tctttggaaa cctctgccag cactgcctat 240 ttgcagatca acaacctcaa aaatgaggac acggctacat atttctgtgc aagactgggt 300 tttggtaatg ctatggacta ctggggtcaa ggaacctcag tcaccgtctc ctca 354 <210> 57 <211> 348 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: PCR product <400> 57 caggttcagc tgcagcagtc tggagctgag ctggcgaggc ccggggcttc agtgaagctg 60 tcctgcaagg cttctggcta caccttcact gactactata taaactgggt gaagcagagg 120 actggacagg gccttgagtg gattggagag atttatcctg gaagtggtaa tacttactac 180 aatgagaagt tcaagggcaa ggccacactg actgcagaca aatcctccag cacagcctac 240 atgcagctca gcagcctgac atctgaggac tctgcagtct atttctgtgc aagatcgtat 300 ggtgcctttg actactgggg ccaaggcacc actctcacag tctcctca 348 <210> 58 <211> 354 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: PCR product <400> 58 caggtccaac tgcagcagcc tggggctgag ctggtgaggc ctggggcttc agtgaagctg 60 tcctgcaagg cttctggcta caccttcacc agctactgga taaactgggt gaagcagagg 120 cctggacaag gccttgagtg gatcggaaat atttatcctt ctgatagtta tactaactac 180 aatcaaaagt tcaaggacaa ggccacattg actgtagaca aatcctccag cacagcctac 240 atgcagctca gcagcccgac atctgaggac tctgcggtct attactgtac aagatcgtgg 300 aggggtaact cctttgacta ctggggccaa ggcaccactc tcacagtctc ctca 354 <210> 59 <211> 354 <212> DNA <213> Artificial <220>

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    2018200685 30 Jan 2018

    Sequences_342-31PCT. txt <223> Description of artificial sequence: PCR product <400> 59 caggttcagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaagatg 60 tcctgcaagg cttctggata cacattcact gactatgtta taagctgggt gaagcagaga 120 actggacagg gccttgagtg gattggagag atttatcctg gaagtggtag tacttactac 180 aatgagaagt tcaagggcaa ggccacactg actgcagaca aatcctccaa cacagcctac 240 atgcagctca gcagcctgac atctgaggac tctgcggtct atttctgtgc aagaggggta 300 ttactacggg ctatggacta ctggggtcaa ggaacctcag tcaccgtctc ctca 354 <210> 60 <211> 360 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: PCR product <400> 60 caggttcacc tacaacagtc tggttctgaa ctgaggagtc ctgggtcttc agtaaagctt 60 tcatgcaagg attttgattc agaagtcttc ccttttgctt atatgagttg gattaggcag 120 aagcctgggc atggatttga atggattgga gacatactcc caagtattgg tagaacaatc 180 tatggagaga agtttgagga caaagccaca ctggatgcag acacagtgtc caacacagcc 240 tacttggagc tcaacagtct gacatctgag gactctgcta tctactactg tgcaaggggg 300 gagggctacg gtgcctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca 360 <210> 61 <211> 339 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: PCR product <400> 61 gacattgtga tgacacagtc tccatcctcc ctgactgtga cagcaggaga gaaggtcact 60 atgagctgca agtccagtca gagtctgtta aacagtggaa atcaaaagaa ctacttgacc 120 tggtaccagc agaaaccagg gcagcctcct aaactgttga tctactgggc atccactagg 180 gaatctgggg tccctgatcg cttcacaggc agtggatctg gaacagattt cactctcacc 240 atcagcagtg tgcaggctga agacctggca gtttattact gtcagaatga ttatagttat 300 ccgctcacgt tcggtgctgg gaccaagctg gagctgaaa 339 <210> 62 <211> 318 <212> DNA <213> Artificial

    Page 3 2

    2018200685 30 Jan 2018

    Sequences_342-31PCT. txt <220>

    <223> Description of artificial sequence: PCR product <400> 62 caaattgttc tcacccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60 ataacctgca gtgccagctc aagtgtaagt tacatgcact ggttccagca gaagccaggc 120 acttctccca aactctggat ttatagcaca tccaacctgg cttctggagt ccctgctcgc 180 ttcagtggca gtggatctgg gacctcttac tctctcacaa tcagccgaat ggaggctgaa 240 gatgctgcca cttattactg ccagcaaagg agtagttacc cacccacgtt cggagggggg 300 accaagctgg aaataaaa 318 <210> 63 <211> 321 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: PCR product <400> 63 gacattgtga tgacccagtc tcaaaaattc atgtccacat cagtaggaga cagggtcagc 60 atcacctgca aggccagtca gaatgttcgt actgctgtag cctggtatca acagaaacca 120 gggcagtctc ctaaagcact gatttacttg gcatccaacc ggcacactgg agtccctgat 180 cgcttcacag gcagtggatc tgggacagat ttcactctca ccattagcaa tgtgcaatct 240 gaagacctgg cagattattt ctgtctgcaa cattggaatt atcctctgac gttcggtgga 300 ggcaccaagc tggaaatcaa a 321 <210> 64 <211> 339 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: PCR product <400> 64 gacattgtga tgtcacagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact 60 atgagctgca agtccagtca gagcctttta tatagtagca atcaaaagaa ctacttggcc 120 tggtaccagc agaaaccagg gcagtctcct aaactgctga tttactgggc atccactagg 180 gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240 atcagcagtg tgaaggctga agacctggca gtttattact gtcagcaata ttatagctat 300 ccgctcacgt tcggtgctgg gaccaagctg gagctgaaa 339 <210> 65 <211> 339

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    Sequences_342-31PCT. txt <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: PCR product <400> 65 gacattgtga tgacacagtc tccatcctcc ctgactgtga cagcaggaga gaaggtcact 60 atgagctgca agtccagtca gagtctgtta aacagtggaa atcaaaagaa ctacttgacc 120 tggtaccagc agaaaccagg gcagcctcct aaactgttga tctactgggc atccactagg 180 gaatctgggg tccctgatcg cttcacaggc agtggatctg gaacagattt cactctcacc 240 atcagcagtg tgcaggctga agacctggca gtttattact gtcagaatga ttatagttat 300 ccattcacgt tcggctcggg gacaaagttg gaaataaaa 339 <210> 66 <211> 336 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: PCR product <400> 66 gacattgtga tgtcacagtc tccatcctcc ctggctgtgt cagcaggaga gaaggtcact 60 atgagctgca aatccagtca gagtctgctc aacagtagaa cccgaaagaa ctacttggct 120 tggtaccagc agaaaccagg gcagtctcct aaactgctga tctactgggc atccactagg 180 gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240 atcagcagtg tgcaggctga agacctggca gtttattact gcaagcaatc ttataatctg 300 tacacgttcg gaggggggac caagctggaa ataaaa 336 <210> 67 <211> 339 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: PCR product <400> 67 gacatcgtga tgtcacagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact 60 atgagctgca agtccagtca gagcctttta tatagtagca atcaaaagaa ctacttggcc 120 tggtaccagc agaaaccagg gcagtctcct aaactgctga tttactgggc atccactagg 180 gaatctgggg tccctgatcg cttcacaggc agtggatctg caacagattt cactctgacc 240 atcagcagtg tgcaggctga agaccttgca gattatcact gtggacaggg ttacagctat 300 ccgtacacgt tcggaggggg gaccaagctg gaaataaaa 339

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    Sequences_342-31PCT.txt <210> 68 <211> 339 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: PCR product <400> 68 gacattgtga tgtcacagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact 60 atgagctgca agtccagtca gagcctttta tatagtagca atcaaaagaa ctacttggcc 120 tggtaccagc agaaaccagg gcagtctcct aaactgctga tttactgggc atccactagg 180 gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240 atcagcagtg tgaaggctga agacctggca gtttattact gtcagcaata ttatagctat 300 ccgctcacgt tcggtgctgg gaccaagctg gagctgaaa 339 <210> 69 <211> 321 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: PCR product <400> 69 aacattgtaa tgacccaatc tcccaaatcc atgtccatgt cagtaggaga gagggtcacc 60 ttgacctgca aggccagtga gaatgtggtt acttatgttt cctggtatca acagaaacca 120 gagcagtctc ctaaactgct gatatacggg gcatccaacc ggtacactgg ggtccccgat 180 cgcttcacag gcagtggatc tgcaacagat ttcactctca ccatcagcag tgtgaaggct 240 gaagacctgg cagtttatta ctgtcagcaa tattatagct atccgctcac gttcggtgct 300 gggaccaagc tggagctgaa a 321 <210> 70 <211> 43 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 70 gagaaagctt gccgccacca tggaatggac ctgggtcttt etc 43 <210> 71 <211> 43 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide

    Page 35

    Sequences_342-31PCT.txt <400> 71

    2018200685 30 Jan 2018

    gagagggccc ttggtggagg ctgcagagac agtgaccaga gtc 43 <210> 72 <211> 47 <212> DNA <213> Artificial <220> <223> Description of artificial sequence: Oligonucleotide <400> 72 gagaaagctt gccgccacca tggattggct gtggaacttg ctattcc 47 <210> 73 <211> 44 <212> DNA <213> Artificial <220> <223> Description of artificial sequence: Oligonucleotide <400> 73 gagagggccc ttggtggagg ctgaggagac ggtgactgag gttc 44 <21O> 74 <211> 46 <212> DNA <213> Artificial <220> <223> Description of artificial sequence : Oligonucleotide <400> 74 gagaaagctt gccgccacca tggaatggat ctggatcttt ctcttc 46 <210> 75 <211> 44 <212> DNA <213> Artificial <220> <223> Description of artificial sequence: Oligonucleotide <400> 75 gagagggccc ttggtggagg ctgaggagac tgtgagagtg gtgc 44 <210> 76 <211> 46 <212> DNA <213> Artificial <220> <223> Description of artificial sequence : Oligonucleotide <400> 76 gagaaagctt gccgccacca tgggatggag ctgtatcatc ctcttc 46

    Page 3 6

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    Sequences_342-31PCT. txt <210> 77 <211> 43 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 77 gagagggccc ttggtggagg ctgaggagac tgtgagagtg gtg 43 <210> 78 <211> 47 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 78 gagaaagctt gccgccacca tggaatggag gatctttctc ttcatcc 47 <210> 79 <211> 44 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 79 gagagggccc ttggtggagg ctgaggagac ggtgactgag gttc 44 <210> 80 <211> 51 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 80 gagaggtctc aagcttagcc accatggact ggatttggat catgctccat c 51 <210> 81 <211> 44 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 81 gagagggccc ttggtggagg ctgcagagac agtgaccaga gtcc 44 <210> 82 <211> 43 <212> DNA <213> Artificial

    Page 3 7

    Sequences_342-31PCT.txt

    2018200685 30 Jan 2018 <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 82 gagaaagctt gccgccacca tggaatcaca gactcaggtc etc 43 <210> 83 <211> 34 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 83 cacacgtacg tttcagctcc agcttggtcc cage 34 <210> 84 <211> 46 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 84 gagaaagctt gccgccacca tgcattttca agtgcagatt ttcagc 46 <210> 85 <211> 30 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 85 cacacgtacg ttttatttcc agcttggtcc 30 <210> 86 <211> 44 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 86 gagaaagctt gccgccacca tggagtttca gacccaggtc tttg 44 <210> 87 <211> 33 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide

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    Sequences_342-31PCT. txt

    2018200685 30 Jan 2018

    <400> 87 cacacgtacg tttgatttcc agcttggtgc etc <210> 88 <211> 46 <212> DNA <213> Artificial <220> <223> Description of artificial sequence : Oligonucleotide <400> 88 gagaaagctt gccgccacca tggattcaca ggcccaggtt ettatg

    <210> 89 <211> 30 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 89 cacacgtacg tttcagctcc agcttggtcc <210> 90 <211> 46 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 90 gagaaagctt gccgccacca tggaatcaca gactcaggtc ctcatg 46 <210> 91 <211> 30 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 91 cacacgtacg ttttatttcc aactttgtcc 30 <210> 92 <211> 49 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 92 gagaaagctt gccgccacca tggattcaca ggcccaggtt cttatattg 49

    Page 39

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    Sequences_342-31PCT.txt <210> 93 <211> 30 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 93 cacacgtacg ttttatttcc agcttggtcc 30 <210> 94 <211> 46 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 94 gagaaagctt gccgccacca tggattcaca ggcccaggtt cttatg 46 <210> 95 <211> 30 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 95 cacacgtacg ttttatttcc agcttggtcc 30 <210> 96 <211> 46 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 96 gagaaagctt gccgccacca tggattcaca ggctcaggtt cttatg 46 <210> 97 <211> 33 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 97 cacacgtacg tttcagctcc agcttggtcc cag 33 <210> 98 <211> 41 <212> DNA <213> Artificial

    Page 40

    Sequences_342-31PCT. txt <220>

    2018200685 30 Jan 2018

    <223> Description of <400> 98 gagaaagctt agccaccatg artificial sequence: Oligonucleotide gaatcacaga ctctggtctt c 41 <210> 99 <211> 30 <212> DNA <213> Artificial <220> <223> Description of artificial sequence: Oligonucleotide <400> 99 cacacgtacg tttcagctcc agcttggtcc 30 <210> 100 <211> 1401 <212> DNA <213> Artificial <220> <223> Description of artificial sequence : chimeric monoclonal antibody

    <400> 100

    atggaatgga cctgggtctt tctcttcctc ctgtcagtaa ctgcaggtgt ccactcccag 60 gttcagctgc agcagtctgg agctgagctg atgaagcctg gggcctcagt gaagatatcc 120 tgcaaggcta ctggctacac attcagtagc tactggatag agtgggtaaa gcagaggcct 180 ggacatggcc ttgagtggat tggagagatt ttacctggaa gtggtagtac taactacaat 240 gagaagttca agggcaaggc cacattcact gcagatacat cctccaacac agcctacatg 300 caactcagca gcctgacatc tgaggactct gccgtctatt actgtgcaag atatgattac 360 ccctggtttg cttactgggg ccaagggact ctggtcactg tctctgcagc ctccaccaag 420 ggcccatcgg tcttccccct ggcaccctcc tccaagagca cctctggggg cacagcggcc 480 ctgggctgcc tggtcaagga ctacttcccc gaaccggtga cggtgtcgtg gaactcaggc 540 gccctgacca gcggcgtgca caccttcccg gctgtcctac agtcctcagg actctactcc 600 ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 660 gtgaatcaca agcccagcaa caccaaggtg gacaagaaag ttgagcccaa atcttgtgac 720 aaaactcaca catgcccacc gtgcccagca cctgaactcc tggggggacc gtcagtcttc 780 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 840 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 900 gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt 960 gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc 1020 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg 1080

    Page 41

    Sequences_342-31PCT.txt

    2018200685 30 Jan 2018

    cagccccgag aaccacaggt gtacaccctg cccccatccc gggatgagct gaccaagaac 1140 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1200 gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1260 ggctccttct tcctctatag caagctcacc gtggacaaga gcaggtggca gcaggggaac 1320 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 1380 tccctgtctc cgggtaaatg a 1401

    <210> 101 <211> 1404

    <212> DNA <213> Artificial <220> <223> Description of artificial sequence: chimeric monoclonal antibody <400> 101 atggattggc tgtggaactt gctattcctg atggcagctg cccaaagtat ccaagcacag 60 atccagttgg tgcagtctgg acctgagctg aagaagcctg gagagacagt caagatctcc 120 tgcaaggctt ctgggtatac cttcacaaac tatggaatga actgggtgaa gcaggctcca 180 ggaaagggtt taaagtggat gggctggata aacaccaaca ctggagagcc aacatatgct 240 gaagagttca agggacggtt tgccttctct ttggaaacct ctgccagcac tgcctatttg 300 cagatcaaca acctcaaaaa tgaggacacg gctacatatt tctgtgcaag actgggtttt 360 ggtaatgcta tggactactg gggtcaagga acctcagtca ccgtctcctc agcctccacc 420 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 660 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 720 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 780 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 840 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 900 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 960 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 1020 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1080 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 1140 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1200 tgggagagca atgggcagcc ggagaacaac tacaagacca Page 42 cgcctcccgt gctggactcc 1260

    Sequences_342-31PCT.txt

    2018200685 30 Jan 2018

    gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg 1320 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1380 ctctccctgt ctccgggtaa atga 1404 <210> 102 <211> 1398 <212> DNA <213> Artificial <220> <223> Description of artificial sequence: chimeric monoclonal antibody <400> 102 atggaatgga tctggatctt tctcttcatc ctctcaggaa ctgcaggtgt ccactcccag 60 gttcagctgc agcagtctgg agctgagctg gcgaggcccg gggcttcagt gaagctgtcc 120 tgcaaggctt ctggctacac cttcactgac tactatataa actgggtgaa gcagaggact 180 ggacagggcc ttgagtggat tggagagatt tatcctggaa gtggtaatac ttactacaat 240 gagaagttca agggcaaggc cacactgact gcagacaaat cctccagcac agcctacatg 300 cagctcagca gcctgacatc tgaggactct gcagtctatt tctgtgcaag atcgtatggt 360 gcctttgact actggggcca aggcaccact ctcacagtct cctcagcctc caccaagggc 420 ccatcggtct tccccctggc accctcctcc aagagcacct ctgggggcac agcggccctg 480 ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 540 ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc 600 agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc agacctacat ctgcaacgtg 660 aatcacaagc ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa 720 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 780 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 840 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 900 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 960 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 1020 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 1080 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 1140 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1200 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 1260 tccttcttcc tctatagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1320 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1380 ctgtctccgg gtaaatga 1398

    Page 43

    Sequences_342-31PCT.txt

    2018200685 30 Jan 2018 <210> 103 <211> 1404 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: chimeric monoclonal antibody <400> 103

    atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt ccactcccag 60 gtccaactgc agcagcctgg ggctgagctg gtgaggcctg gggcttcagt gaagctgtcc 120 tgcaaggctt ctggctacac cttcaccagc tactggataa actgggtgaa gcagaggcct 180 ggacaaggcc ttgagtggat cggaaatatt tatccttctg atagttatac taactacaat 240 caaaagttca aggacaaggc cacattgact gtagacaaat cctccagcac agcctacatg 300 cagctcagca gcccgacatc tgaggactct gcggtctatt actgtacaag atcgtggagg 360 ggtaactcct ttgactactg gggccaaggc accactctca cagtctcctc agcctccacc 420 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 660 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 720 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 780 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 840 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 900 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 960 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 1020 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1080 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 1140 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1200 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1260 gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg 1320 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1380 ctctccctgt ctccgggtaa atga 1404

    <210> 104 <211> 1401 <212> DNA <213> Artificial

    Page 44

    Sequences_342-31PCT.txt

    2018200685 30 Jan 2018 <220>

    <223> Description of artificial sequence: chimeric monoclonal antibody <400> 104

    atggaatgga ggatctttct cttcatcctg tcaggaactg caggtgtcca ctcccaggtt 60 cagctgcagc agtctggacc tgagctggtg aagcctgggg cttcagtgaa gatgtcctgc 120 aaggcttctg gatacacatt cactgactat gttataagct gggtgaagca gagaactgga 180 cagggccttg agtggattgg agagatttat cctggaagtg gtagtactta ctacaatgag 240 aagttcaagg gcaaggccac actgactgca gacaaatcct ccaacacagc ctacatgcag 300 ctcagcagcc tgacatctga ggactctgcg gtctatttct gtgcaagagg ggtattacta 360 cgggctatgg actactgggg tcaaggaacc tcagtcaccg tctcctcagc ctccaccaag 420 ggcccatcgg tcttccccct ggcaccctcc tccaagagca cctctggggg cacagcggcc 480 ctgggctgcc tggtcaagga ctacttcccc gaaccggtga cggtgtcgtg gaactcaggc 540 gccctgacca gcggcgtgca caccttcccg gctgtcctac agtcctcagg actctactcc 600 ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 660 gtgaatcaca agcccagcaa caccaaggtg gacaagaaag ttgagcccaa atcttgtgac 720 aaaactcaca catgcccacc gtgcccagca cctgaactcc tggggggacc gtcagtcttc 780 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 840 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 900 gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt 960 gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc 1020 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg 1080 cagccccgag aaccacaggt gtacaccctg cccccatccc gggatgagct gaccaagaac 1140 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1200 gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1260 ggctccttct tcctctatag caagctcacc gtggacaaga gcaggtggca gcaggggaac 1320 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 1380 tccctgtctc cgggtaaatg a 1401

    <210> 105 <211> 1410 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: chimeric monoclonal antibody <400> 105 atggactgga tttggatcat gctccatctg ctggcagcag ctacaggtat ccaatcccag Page 45

    Sequences_342-31PCT. txt

    2018200685 30 Jan 2018

    gttcacctac aacagtctgg ttctgaactg aggagtcctg ggtcttcagt aaagctttca 120 tgcaaggatt ttgattcaga agtcttccct tttgcttata tgagttggat taggcagaag 180 cctgggcatg gatttgaatg gattggagac atactcccaa gtattggtag aacaatctat 240 ggagagaagt ttgaggacaa agccacactg gatgcagaca cagtgtccaa cacagcctac 300 ttggagctca acagtctgac atctgaggac tctgctatct actactgtgc aaggggggag 360 ggctacggtg cctggtttgc ttactggggc caagggactc tggtcactgt ctctgcagcc 420 tccaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 480 acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540 aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 600 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 660 atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa 720 tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 780 tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 840 gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 900 gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 960 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 1020 tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1080 gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg 1140 accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1200 gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1260 gactccgacg gctccttctt cctctatagc aagctcaccg tggacaagag caggtggcag 1320 caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1380 aagagcctct ccctgtctcc gggtaaatga 1410

    <210> 106 <211> 723 <212> DNA <213> Artificial

    <220> <223> Description of artificial sequence: chimeric monoclonal antibody <400> 106 atggaatcac agactcaggt cctcatgtcc ctgctgttct gggtatctgg tacctgtggg 60 gacattgtga tgacacagtc tccatcctcc ctgactgtga cagcaggaga gaaggtcact 120 atgagctgca agtccagtca gagtctgtta aacagtggaa atcaaaagaa ctacttgacc 180 tggtaccagc agaaaccagg gcagcctcct aaactgttga Page 46 tctactgggc atccactagg 240

    Sequences_342-31PCT.txt

    2018200685 30 Jan 2018

    gaatctgggg tccctgatcg cttcacaggc agtggatctg gaacagattt cactctcacc 300 atcagcagtg tgcaggctga agacctggca gtttattact gtcagaatga ttatagttat 360 ccgctcacgt tcggtgctgg gaccaagctg gagctgaaac gtacggtggc tgcaccatct 420 gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 480 ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 540 caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 600 ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 660 gaagtcaccc atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 720

    tag 723 <210> 107 <211> 708 <212> DNA <213> Artificial

    <220> <223> Description of artificial sequence : < chimeric monoclonal antibody <400> 107 atgcattttc aagtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtcc 60 agaggacaaa ttgttctcac ccagtctcca gcaatcatgt ctgcatctcc aggggagaag 120 gtcaccataa cctgcagtgc cagctcaagt gtaagttaca tgcactggtt ccagcagaag 180 ccaggcactt ctcccaaact ctggatttat agcacatcca acctggcttc tggagtccct 240 gctcgcttca gtggcagtgg atctgggacc tcttactctc tcacaatcag ccgaatggag 300 gctgaagatg ctgccactta ttactgccag caaaggagta gttacccacc cacgttcgga 360 ggggggacca agctggaaat aaaacgtacg gtggctgcac catctgtctt catcttcccg 420 ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 480 tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 540 caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 600 acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 660 ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgttag 708

    <210> 108 <211> 705 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: chimeric monoclonal antibody <400> 108 atggagtttc agacccaggt ctttgtattc gtgttgctct ggttgtctgg tgttgatgga Page 47

    2018200685 30 Jan 2018

    gacattgtga tgacccagtc Sequences 342-31PCT.txt tcaaaaattc atgtccacat cagtaggaga cagggtcagc 120 atcacctgca aggccagtca gaatgttcgt actgctgtag cctggtatca acagaaacca 180 gggcagtctc ctaaagcact gatttacttg gcatccaacc ggcacactgg agtccctgat 240 cgcttcacag gcagtggatc tgggacagat ttcactctca ccattagcaa tgtgcaatct 300 gaagacctgg cagattattt ctgtctgcaa cattggaatt atcctctgac gttcggtgga 360 ggcaccaagc tggaaatcaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca 420 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 540 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 600 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 660 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 705 <210> 109 <211> 723 <212> DNA <213> Artificial <220> <223> Description of artificial sequence: chimeric monoclonal antibody <400> 109 atggattcac aggcccaggt tcttatgtta ctgctgctat gggtatctgg tacctgtggg 60 gacattgtga tgtcacagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact 120 atgagctgca agtccagtca gagcctttta tatagtagca atcaaaagaa ctacttggcc 180 tggtaccagc agaaaccagg gcagtctcct aaactgctga tttactgggc atccactagg 240 gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 300 atcagcagtg tgaaggctga agacctggca gtttattact gtcagcaata ttatagctat 360 ccgctcacgt tcggtgctgg gaccaagctg gagctgaaac gtacggtggc tgcaccatct 420 gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 480 ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 540 caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 600 ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 660 gaagtcaccc atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 720 tag <210> 110 <211> 723 <212> DNA <213> Artificial 723

    Page 48

    Sequences_342-31PCT.txt

    2018200685 30 Jan 2018 <220>

    <223> Description of artificial sequence: chimeric monoclonal antibody <400> 110 atggaatcac agactcaggt cctcatgtcc ctgctgttct gggtatctgg tacctgtggg 60 gacattgtga tgacacagtc tccatcctcc ctgactgtga cagcaggaga gaaggtcact 120 atgagctgca agtccagtca gagtctgtta aacagtggaa atcaaaagaa ctacttgacc 180 tggtaccagc agaaaccagg gcagcctcct aaactgttga tctactgggc atccactagg 240 gaatctgggg tccctgatcg cttcacaggc agtggatctg gaacagattt cactctcacc 300 atcagcagtg tgcaggctga agacctggca gtttattact gtcagaatga ttatagttat 360 ccattcacgt tcggctcggg gacaaagttg gaaataaaac gtacggtggc tgcaccatct 420 gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 480 ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 540 caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 600 ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 660 gaagtcaccc atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 720

    tag 723 <210> 111 <211> 720 <212> DNA <213> Artificial

    <220> <223> Description of artificial sequence: chimeric monoclonal antibody <400> 111 atggattcac aggcccaggt tcttatattg ctgctgctat gggtatctgg tacctgtggg 60 gacattgtga tgtcacagtc tccatcctcc ctggctgtgt cagcaggaga gaaggtcact 120 atgagctgca aatccagtca gagtctgctc aacagtagaa cccgaaagaa ctacttggct 180 tggtaccagc agaaaccagg gcagtctcct aaactgctga tctactgggc atccactagg 240 gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 300 atcagcagtg tgcaggctga agacctggca gtttattact gcaagcaatc ttataatctg 360 tacacgttcg gaggggggac caagctggaa ataaaacgta cggtggctgc accatctgtc 420 ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 480 ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa 540 tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc 600 agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta cgcctgcgaa 660 gtcacccatc agggcctgag ctcgcccgtc acaaagagct Page 49 tcaacagggg agagtgttag 720

    2018200685 30 Jan 2018

    Sequences_3 42 - 31PCT. txt <210> 112 <211> 723 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: chimeric monoclonal antibody <400> 112

    atggattcac aggcccaggt tcttatgtta ctgctgctat gggtatctgg tacctgtggg 60 gacatcgtga tgtcacagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact 120 atgagctgca agtccagtca gagcctttta tatagtagca atcaaaagaa ctacttggcc 180 tggtaccagc agaaaccagg gcagtctcct aaactgctga tttactgggc atccactagg 240 gaatctgggg tccctgatcg cttcacaggc agtggatctg caacagattt cactctgacc 300 atcagcagtg tgcaggctga agaccttgca gattatcact gtggacaggg ttacagctat 360 ccgtacacgt tcggaggggg gaccaagctg gaaataaaac gtacggtggc tgcaccatct 420 gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 480 ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 540 caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 600 ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 660 gaagtcaccc atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 720 tag 723

    <210> 113 <211> 723 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: chimeric monoclonal antibody <400> 113

    atggattcac aggctcaggt tcttatgtta ctgctgctat gggtatctgg tacctgtggg 60 gacattgtga tgtcacagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact 120 atgagctgca agtccagtca gagcctttta tatagtagca atcaaaagaa ctacttggcc 180 tggtaccagc agaaaccagg gcagtctcct aaactgctga tttactgggc atccactagg 240 gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 300 atcagcagtg tgaaggctga agacctggca gtttattact gtcagcaata ttatagctat 360 ccgctcacgt tcggtgctgg gaccaagctg gagctgaaac gtacggtggc tgcaccatct 420 gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 480 ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 540

    Page 50

    Sequences_342- 31PCT. txt

    2018200685 30 Jan 2018

    caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 600 ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 660 gaagtcaccc atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 720 tag 723

    <210> 114 <211> 705 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: chimeric monoclonal antibody <400> 114

    atggaatcac agactctggt cttcatatcc atactgctct ggttatatgg agctgatggg 60 aacattgtaa tgacccaatc tcccaaatcc atgtccatgt cagtaggaga gagggtcacc 120 ttgacctgca aggccagtga gaatgtggtt acttatgttt cctggtatca acagaaacca 180 gagcagtctc ctaaactgct gatatacggg gcatccaacc ggtacactgg ggtccccgat 240 cgcttcacag gcagtggatc tgcaacagat ttcactctca ccatcagcag tgtgaaggct 300 gaagacctgg cagtttatta ctgtcagcaa tattatagct atccgctcac gttcggtgct 360 gggaccaagc tggagctgaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca 420 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 540 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 600 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 660 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 705

    <210> 115 <211> 466 <212> PRT <213> Artificial <220>

    <223> Description of artificial sequence: chimeric monoclonal antibody <400> 115

    Met Glu Trp Thr Trp Val Phe Leu Phe Leu Leu Ser Val Thr Ala Gly 1 5 10 15 Val His Ser Gin Val Gin Leu Gin Gin Ser Gly Ala Glu Leu Met Lys 20 25 30 Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe

    35 40 45

    Page 51

    Sequences_342-31PCT. txt

    2018200685 30 Jan 2018

    Ser Ser Tyr Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu 50 55 60

    Glu Trp Ile Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn 65 70 75 80

    Glu Lys Phe Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn 85 90 95

    Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110

    Tyr Tyr Cys Ala Arg Tyr Asp Tyr Pro Trp Phe Ala Tyr Trp Gly Gln 115 120 125

    Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val 130 135 140

    Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160

    Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 165 170 175

    Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 180 185 190

    Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 195 200 205

    Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 210 215 220

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

    Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 245 250 255

    Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270

    Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 275 280 285

    Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290 295 300

    Page 52

    Sequences_342-31PCT.txt

    2018200685 30 Jan 2018

    Asn Ala Lys 305 Thr Lys Pro 310 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 315 320 Val Val Ser Val Leu 325 Thr Val Leu His Gln Asp Trp 330 Leu Asn Gly Lys 335 Glu Tyr Lys Cys Lys 340 Val Ser Asn Lys Ala Leu Pro 345 Ala Pro Ile Glu 350 Lys Thr Ile 355 Ser Lys Ala Lys Gly Gln Pro Arg Glu 360 Pro Gln Val Tyr 365 Thr Leu Pro 370 Pro Ser Arg Asp 375 Glu Leu Thr Lys Asn 380 Gln Val Ser Leu Thr Cys Leu 385 Val Lys Gly 390 Phe Tyr Pro Ser Asp Ile 395 Ala Val Glu Trp 400 Glu Ser Asn Gly Gln 405 Pro Glu Asn Asn Tyr Lys Thr 410 Thr Pro Pro Val 415 Leu Asp Ser Asp Gly 420 Ser Phe Phe Leu Tyr Ser Lys 425 Leu Thr Val Asp 430 Lys Ser Arg 435 Trp Gln Gln Gly Asn Val Phe Ser Cys 440 Ser Val Met His 445 Glu Ala Leu His Asn 450 Gly Lys 465 <210> 116 <211> 467 <212> PRT <213> Artificial <220> His Tyr 455 Thr Gln Lys Ser Leu 460 Ser Leu Ser Pro <223 > Description of artificial sequence: chimeric monoclonal antibody <400> 116 Met Asp Trp 1 Leu Trp 5 Asn Leu Leu Phe Leu Met Ala 10 Ala Ala Gln Ser 15 Ile Gln Ala Gln Ile 20 Gln Leu Val Gln Ser Gly Pro 25 Glu Leu Lys Lys 30

    Page 53

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    Sequences_342-31PCT.txt

    Pro Gly Glu Thr Val Lys Xie Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45

    Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu 50 55 60

    Lys Trp Met Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala 65 70 75 80

    Glu Glu Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser 85 90 95

    Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr 100 105 110

    Tyr Phe Cys Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly 115 120 125

    Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140

    Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160

    Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175

    Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190

    Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205

    Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220

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

    Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255

    Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270

    Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285

    Page 54

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    Sequences_342-31PCT. txt

    Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300

    His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320

    Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335

    Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350

    Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365

    Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375 380

    Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400

    Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415

    Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430

    Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445

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

    450 455 460 Pro Gly 465 ' Lys <210> 117 <211> 465 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: chimeric monoclonal antibody <400> 117 Met Glu . Trp Ile Trp Ile Phe Leu Phe Ile Leu Ser Gly Thr Ala Gly

    15 10 15

    Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg 20 25 30

    Page 55

    2018200685 30 Jan 2018

    Pro Gly Ala Ser 35

    Thr Asp Tyr Tyr 50

    Glu Trp Ile Gly 65

    Glu Lys Phe Lys

    Thr Ala Tyr Met 100

    Tyr Phe Cys Ala 115

    Thr Thr Leu Thr 130

    Pro Leu Ala Pro 145

    Gly Cys Leu Val

    Asn Ser Gly Ala 180

    Gln Ser Ser Gly 195

    Ser Ser Leu Gly 210

    Ser Asn Thr Lys 225

    Thr His Thr Cys

    Ser Val Phe Leu 260

    Arg Thr Pro Glu 275

    Val Lys

    Ile Asn

    Glu Ile 70

    Gly Lys 85

    Gln Leu

    Arg Ser

    Val Ser

    Ser Ser 150

    Lys Asp 165

    Leu Thr

    Leu Tyr

    Thr Gln

    Val Asp 230

    Pro Pro 245

    Phe Pro

    Val Thr

    Leu

    Trp

    Tyr

    Ala

    Ser

    Tyr

    Ser

    135

    Lys

    Tyr

    Ser

    Ser

    Thr

    215

    Lys

    Cys

    Pro

    Cys

    Sequences_342-31PCT. txt

    Ser Cys Lys Ala Ser Gly Tyr 40 45

    Val Lys Gln Arg Thr Gly Gln 60

    Pro Gly Ser Gly Asn Thr Tyr 75

    Thr Leu Thr Ala Asp Lys Ser 90

    Ser Leu Thr Ser Glu Asp Ser 105 110

    Gly Ala Phe Asp Tyr Trp Gly 120 125

    Ala Ser Thr Lys Gly Pro Ser 140

    Ser Thr Ser Gly Gly Thr Ala 155

    Phe Pro Glu Pro Val Thr Val 170

    Gly Val His Thr Phe Pro Ala 185 190

    Leu Ser Ser Val Val Thr Val 200 205

    Tyr Ile Cys Asn Val Asn His 220

    Lys Val Glu Pro Lys Ser Cys 235

    Pro Ala Pro Glu Leu Leu Gly 250

    Lys Pro Lys Asp Thr Leu Met 265 270

    Val Val Val Asp Val Ser His 280 285

    Thr Phe

    Gly Leu

    Tyr Asn 80

    Ser Ser 95

    Ala Val

    Gln Gly

    Val Phe

    Ala Leu 160

    Ser Trp 175

    Val Leu

    Pro Ser

    Lys Pro

    Asp Lys 240

    Gly Pro 255

    Ile Ser

    Glu Asp

    Page 5 6

    2018200685 30 Jan 2018

    Pro Glu Val Lys Phe 290

    Ala Lys Thr Lys Pro 305

    Val Ser Val Leu Thr 325

    Tyr Lys Cys Lys Val 340

    Thr Ile Ser Lys Ala 355

    Leu Pro Pro Ser Arg 370

    Cys Leu Val Lys Gly 385

    Ser Asn Gly Gln Pro 405

    Asp Ser Asp Gly Ser 420

    Ser Arg Trp Gln Gln 435

    Ala Leu His Asn His 450

    Sequences_342-31PCT.txt

    Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 295 300

    Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 310 315 320

    Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 330 335

    Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 345 350

    Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 360 365

    Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 375 380

    Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 390 395 400

    Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 410 415

    Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 425 430

    Gly Asn Val Phe Ser Cys Ser Val Met His Glu 440 445

    Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 455 460

    Lys

    465 <210> 118 <211> 467 <212> PRT <213> Artificial <220>

    <223> Description of artificial sequence: chimeric monoclonal antibody <400> 118

    Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 15 10 15

    Page 57

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    Sequences_342-31PCT. txt

    Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg 20 25 30

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

    Thr Ser Tyr Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60

    Glu Trp Ile Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn 65 70 75 80

    Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 85 90 95

    Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val 100 105 110

    Tyr Tyr Cys Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly 115 120 125

    Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140

    Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160

    Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175

    Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190

    Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205

    Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220

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

    Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255

    Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270

    Page 58

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    Sequences_342-31PCT. txt

    Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285

    Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300

    His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320

    Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335

    Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350

    Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365

    Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375 380

    Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400

    Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415

    Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430

    Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445

    His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460

    Pro Gly Lys 465 <210> 119 <211> 466 <212> PRT <213> Artificial <220>

    <223> Description of artificial sequence: chimeric monoclonal antibody <400> 119

    Met Glu Trp Arg Ile Phe Leu Phe Ile Leu Ser Gly Thr Ala Gly Val 15 10 15

    Page 59

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    Sequences_342-31PCT.txt

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

    Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 35 40 45

    Asp Tyr Val Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu 50 55 60

    Trp Ile Gly Glu Ile Tyr Pro Gly Ser Gly Ser Thr Tyr Tyr Asn Glu 65 70 75 80

    Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr 85 90 95

    Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 100 105 110

    Phe Cys Ala Arg Gly Val Leu Leu Arg Ala Met Asp Tyr Trp Gly Gln 115 120 125

    Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 130 135 140

    Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160

    Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 165 170 175

    Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 180 185 190

    Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 195 200 205

    Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 210 215 220

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

    Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 245 250 255

    Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270

    Page 60

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    Sequences_342-31PCT.txt

    Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285

    Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300

    Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 305 310 315

    Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330

    Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345

    Lys Thr lie Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365

    Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 370 375 380

    Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala 385 390 395

    Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410

    Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425

    Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445

    Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460

    Ser His Glu

    Glu Val His

    Thr Tyr Arg 320

    Asn Gly Lys 335

    Pro lie Glu 350

    Gln Val Tyr

    Val Ser Leu

    Val Glu Trp 400

    Pro Pro Val 415

    Thr Val Asp 430

    Val Met His

    Leu Ser Pro

    Gly Lys

    465 <210> 120 <211> 469 <212> PRT <213> Artificial <220> <223> Descriptior <400> 120

    monoclonal antibody

    Page 61

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    Sequences_342-31PCT. txt

    Met Asp Trp Ile Trp Ile Met Leu His Leu Leu Ala Ala Ala Thr Gly 15 10 15

    Ile Gln Ser Gln Val His Leu Gln Gln Ser Gly Ser Glu Leu Arg Ser 20 25 30

    Pro Gly Ser Ser Val Lys Leu Ser Cys Lys Asp Phe Asp Ser Glu Val 35 40 45

    Phe Pro Phe Ala Tyr Met Ser Trp Ile Arg Gln Lys Pro Gly His Gly 50 55 60

    Phe Glu Trp Ile Gly Asp Ile Leu Pro Ser Ile Gly Arg Thr Ile Tyr 65 70 75 80

    Gly Glu Lys Phe Glu Asp Lys Ala Thr Leu Asp Ala Asp Thr Val Ser 85 90 95

    Asn Thr Ala Tyr Leu Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala 100 105 110

    Ile Tyr Tyr Cys Ala Arg Gly Glu Gly Tyr Gly Ala Trp Phe Ala Tyr 115 120 125

    Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly 130 135 140

    Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 145 150 155 160

    Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175

    Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190

    Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205

    Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220

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

    Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255

    Page 62

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    Sequences_342-31PCT. txt

    Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270

    Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285

    Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300

    Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 305 310 315 320

    Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335

    Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345 350

    Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365

    Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 370 375 380

    Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395 400

    Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415

    Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430

    Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445

    Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460

    Leu Ser Pro Gly Lys 465 <210> 121 <211> 240 <212> PRT <213> Artificial <220>

    <223> Description of artificial sequence: chimeric monoclonal antibody

    Page 63

    Jan 2018

    Sequences_3 42-31PCT. txt <400> 121

    Met Glu Ser Gln Thr Gln Val Leu Met Ser Leu Leu Phe Trp Val Ser 15 10 15

    O

    ΓΠ Gly Thr Cys Gly Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr 20 25 30 vn

    00 Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser 35 40 45 o

    o

    CM Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln OO 50 55 60 o

    C\| Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80

    Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 85 90 95

    Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr 100 105 110

    Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr 115 120 125

    Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140

    Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160

    Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 165 170 175

    Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 180 185 190

    Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 195 200 205

    Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 210 215 220

    Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 122 <211> 235

    Page 64

    5 30 Jan 2018

    Sequences_342-31PCT.txt <212> PRT <213> Artificial <220>

    sequence: chimeric monoclonal antibody

    Ser Phe Leu Leu Ile Ser Ala Ser 10 15 <223> Description of artificia <400> 122

    Met His Phe Gln Val Gln Ile Phe 1 5

    OO

    Val Ile Met Ser Arg Gly Gln Ile O ²⁰ <N

    OO t—I Met Ser Ala Ser Pro Gly Glu Lys O 35 40 <N

    Ser Ser Val Ser Tyr Met His Trp 50 55

    Val Leu Thr Gln Ser Pro Ala Ile 25 30

    Val Thr Ile Thr Cys Ser Ala Ser 45

    Phe Gln Gln Lys Pro Gly Thr Ser 60

    Pro Lys Leu Trp Ile Tyr Ser Thr 65 70

    Ser Asn Leu Ala Ser Gly Val Pro 75 80

    Ala Arg Phe Ser Gly Ser Gly Ser 95

    Gly Thr Ser Tyr Ser Leu Thr Ile 90 95

    Ser Arg Met Glu Ala Glu Asp Ala 100

    Ala Thr Tyr Tyr Cys Gln Gln Arg 105 110

    Ser Ser Tyr Pro Pro Thr Phe Gly 115 120

    Gly Gly Thr Lys Leu Glu Xie Lys 125

    Arg Thr Val Ala Ala Pro Ser Val 130 135

    Phe Ile Phe Pro Pro Ser Asp Glu 140

    Gln Leu Lys Ser Gly Thr Ala Ser 145 150

    Val Val Cys Leu Leu Asn Asn Phe 155 160

    Tyr Pro Arg Glu Ala Lys Val Gln 165

    Trp Lys Val Asp Asn Ala Leu Gln 170 175

    Ser Gly Asn Ser Gln Glu Ser Val 180

    Thr Glu Gln Asp Ser Lys Asp Ser 185 190

    Thr Tyr Ser Leu Ser Ser Thr Leu 195 200

    Thr Leu Ser Lys Ala Asp Tyr Glu 205

    Lys His Lys Val Tyr Ala Cys Glu 210 215

    Val Thr His Gln Gly Leu Ser Ser 220

    Page 65

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    Sequences_342-31PCT.txt Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 123 <211> 234 <212> PRT <213> Artificial <220>

    <223> Description of artificial sequence: chimeric monoclonal antibody <400> 123

    Met Glu Phe Gln Thr Gln Val Phe Val Phe Val Leu Leu Trp Leu Ser 15 10 15

    O

    04 Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser 20 25 30

    Thr Ser Val Gly Asp Arg Val Ser lie Thr Cys Lys Ala Ser Gln Asn 35 40 45

    Val Arg Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro 50 55 60

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

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

    Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln His Trp 100 105 110

    Asn Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu 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

    Page 66

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    His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro 210 215 220

    Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 124 <211> 240 <212> PRT <213> Artificial <220>

    <223> Description of artificial sequence: chimeric monoclonal antibody <400> 124

    Met 1 Asp Ser Gin Ala Gin Val 5 Leu Met Leu Leu Leu Leu Trp Val Ser 10 15 Gly Thr Cys Gly Asp Ile Val Met Ser Gin Ser Pro Ser Ser Leu Ala 20 25 30 Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gin Ser 35 40 45 Leu Leu Tyr Ser Ser Asn Gin Lys Asn Tyr Leu Ala Trp Tyr Gin Gin 50 55 60 Lys Pro Gly Gin Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr 100 105 110 Tyr Cys Gin Gin Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr 115 120 125 Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 14 0 Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val 165 170 175

    Page 67

    Sequences_342-31PCT.txt

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    Asp Asn Ala Leu 180 Gln Ser Gly Asn Ser Gln 185 Glu Ser Val Thr Glu Gln 190 Asp Ser Lys 195 Asp Ser Thr Tyr Ser 200 Leu Ser Ser Thr Leu 205 Thr Leu Ser Lys Ala Asp 210 Tyr Glu Lys His 215 Lys Val Tyr Ala Cys Glu 220 Val Thr His Gln Gly Leu Ser Ser Pro Val 225 230 <210> 125 <211> 240 <212> PRT <213> Artificial <220> Thr Lys Ser Phe Asn Arg 235 Gly Glu Cys 240 <223> Description of artificial sequence: chimeric <400> 125 monoclonal antibody Met 1 Glu Ser Gln Thr Gln Val 5 Leu Met Ser 10 Leu Leu Phe Trp Val Ser 15 Gly Thr Cys Gly 20 Asp Ile Val Met Thr Gln 25 Ser Pro Ser Ser Leu Thr 30 Val Thr Ala 35 Gly Glu Lys Val Thr 40 Met Ser Cys Lys Ser 45 Ser Gln Ser Leu Leu Asn 50 Ser Gly Asn Gln 55 Lys Asn Tyr Leu Thr Trp 60 Tyr Gln Gln Lys 65 Pro Gly Gln Pro Pro Lys 70 Leu Leu Ile Tyr Trp Ala 75 Ser Thr Arg 80 Glu Ser Gly Val Pro Asp Arg 85 Phe Thr Gly 90 Ser Gly Ser Gly Thr Asp 95 Phe Thr Leu Thr 100 Ile Ser Ser Val Gln Ala 105 Glu Asp Leu Ala Val Tyr 110 Tyr Cys Gln 115 Asn Asp Tyr Ser Tyr 120 Pro Phe Thr Phe Gly 125 Ser Gly Thr Lys Leu Glu 130 Ile Lys Arg Thr 135 Val Ala Ala Pro Ser Val 140 Phe Ile Phe Pro 145 Pro Ser Asp Glu Gln Leu 150 Lys Ser Gly Thr Ala Ser 155 Val Val Cys 160

    Page 68

    Sequences_342-31PCT. txt

    2018200685 30 Jan 2018

    Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 170 Gln Trp Lys 175 Val 165 Asp Asn Ala Leu Gln Ser 180 Gly Asn Ser Gln Glu Ser 185 Val Thr 190 Glu Gln Asp Ser Lys Asp Ser Thr 195 Tyr Ser Leu Ser Ser Thr 200 Leu 205 Thr Leu Ser Lys Ala Asp Tyr Glu Lys 210 His Lys Val Tyr Ala Cys 215 220 Glu Val Thr His Gln Gly Leu Ser Ser Pro 225 230 <210> 126 <211> 239 <212> PRT <213> Artificial <220> Val Thr Lys Ser Phe Asn 235 Arg Gly Glu Cys 240 <223> Description of artificial sequence: chimeric <400> 126 monoclonal antibody Met 1 Asp Ser Gln Ala Gln 5 Val Leu Ile Leu Leu Leu 10 Leu Trp Val 15 Ser Gly Thr Cys Gly Asp Ile 20 Val Met Ser Gln Ser Pro 25 Ser Ser 30 Leu Ala Val Ser Ala Gly Glu Lys 35 Val Thr Met Ser Cys Lys 40 Ser 45 Ser Gln Ser Leu Leu Asn Ser Arg Thr 50 Arg Lys Asn Tyr Leu Ala 55 60 Trp Tyr Gln Gln Lys 65 Pro Gly Gln Ser Pro 70 Lys Leu Leu Ile Tyr Trp 75 Ala Ser Thr Arg 80 Glu Ser Gly Val Pro Asp 85 Arg Phe Thr Gly Ser Gly 90 Ser Gly Thr 95 Asp Phe Thr Leu Thr Ile Ser 100 Ser Val Gln Ala Glu Asp 105 Leu Ala 110 Val Tyr Tyr Cys Lys Gln Ser Tyr 115 Asn Leu Tyr Thr Phe Gly 120 Gly 125 Gly Thr Lys

    Page 69

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    Sequences_342-31PCT. txt

    Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140

    Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160

    Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175

    Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190

    Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205

    Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220

    Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 127 <211> 240 <212> PRT <213> Artificial <220>

    <223> Description of artificial sequence: chimeric monoclonal antibody <400> 127

    Met Asp Ser Gln Ala Gln Val Leu Met Leu Leu Leu Leu Trp Val Ser 15 10 15

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

    Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser 35 40 45

    Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln 50 55 60

    Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80

    Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Ala Thr Asp 85 90 95

    Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr 100 105 110

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    Sequences_342-31PCT. txt

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    His Cys Gly Gln Gly Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr 115 120 125 Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe lie Phe 130 135 140 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 165 170 175 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 180 185 190 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 195 200 205 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 210 215 220 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 128 <211> 240 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: chimeric monoclonal antibody <400> 128 Met Asp Ser Gln Ala Gln Val Leu Met Leu Leu Leu Leu Trp Val Ser 1 5 10 15 Gly Thr Cys Gly Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala 20 25 30 Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser 35 40 45 Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln 50 55 60 Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80

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    Sequences_342-31PCT.txt

    Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 85 90 95

    Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr 100 105 110

    Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr 115 120 125

    Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140

    Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160

    Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 165 170 175

    Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 180 185 190

    Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 195 200 205

    Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 210 215 220

    Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240

    <210> 129 <211> 234 <212> PRT <213> Artificial <220> <223> Description of artificial sequence : chimeric <400> 129 Met Glu Ser Gln Thr Leu Val Phe Ile Ser Ile Leu Leu 1 5 10

    monoclonal antibody

    Trp Leu Tyr 15

    Gly Ala Asp Gly Asn Ile 20

    Met Ser Val Gly Glu Arg 35

    Val Val Thr Tyr Val Ser 50

    Val Met Thr Gln Ser Pro Lys 25

    Val Thr Leu Thr Cys Lys Ala 40 45

    Trp Tyr Gln Gln Lys Pro Glu 55 60

    Ser Met Ser 30

    Ser Glu Asn

    Gln Ser Pro

    Page 72

    Sequences_342-31PCT. txt

    2018200685 30 Jan 2018

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

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

    Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gin Gin Tyr Tyr 100 105 110

    Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 115 120 125

    Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin 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 Gin Trp Lys Val Asp Asn Ala Leu Gin Ser 165 170 175

    Gly Asn Ser Gin Glu Ser Val Thr Glu Gin 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 Gin Gly Leu Ser Ser Pro 210 215 220

    Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 130 <211> 18 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: Oligonucleotide <400> 130 ccaagggcta tggcgttc

    <210> <211> <212> <213> 131 18 DNA Artificial <220>

    Page 73

    2018200685 30 Jan 2018

    Sequences_342-31PCT.txt <223 > Description of artificial sequence: Oligonucleotide <400> 131 ccgaaggtgt acctggtc <210> 132 <211> 117 <212> PRT <213> Artificial <220>

    <223> Description of artificial sequence: Translation of PCR product <400> 132

    Gln 1 Val Gln Leu Gln 5 Gln Ser Gly Ala Glu 10 Leu Met Lys Pro Gly 15 Ala Ser Val Lys Ile 20 Ser Cys Lys Ala Thr 25 Gly Tyr Thr Phe Ser 30 Ser Tyr Trp Ile Glu 35 Trp Val Lys Gln Arg 40 Pro Gly His Gly Leu 45 Glu Trp Ile Gly Glu 50 Ile Leu Pro Gly Ser 55 Gly Ser Thr Asn Tyr 60 Asn Glu Lys Phe Lys 65 Gly Lys Ala Thr Phe 70 Thr Ala Asp Thr Ser 75 Ser Asn Thr Ala Tyr 80 Met Gln Leu Ser Ser 85 Leu Thr Ser Glu Asp 90 Ser Ala Val Tyr Tyr 95 Cys Ala Arg Tyr Asp 100 Tyr Pro Trp Phe Ala 105 Tyr Trp Gly Gln Gly 110 Thr Leu Val Thr Val 115 Ser Ala

    <210> 133 <211> 118 <212> PRT <213> Artificial <220>

    <223> Description of artificial sequence: Translation of PCR product <400> 133

    Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 15 10 15

    Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30

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    Sequences_342-31PCT.txt

    2018200685 30 Jan 2018

    Gly Met Asn Trp Val Lys Gin Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45

    Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe 50 55 60

    Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80

    Leu Gin Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95

    Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gin Gly Thr 105 110

    100 Ser Val Thr Val Ser 115 <210> 134 <211> 116 <2I2> PRT <213> Artificial <220> <223> Description i <400> 134 Gin Val Gin Leu Gin 1 5

    Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30

    Tyr Ile Asn Trp Val Lys Gin Arg Thr Gly Gin Gly Leu Glu Trp Ile 35 40 45

    Gly Glu Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe 50 55 60

    Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80

    Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95

    Ala Arg Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gin Gly Thr Thr Leu 100 105 110

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    Sequences_342-31PCT.txt

    Thr Val Ser Ser 115

    2018200685 30 Jan 2018 <210> 135 <211> 118 <212> PRT <213> Artificial <220>

    <223> Description of artificial sequence: Translation of PCR product <400> 135

    Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 15 10 15

    Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30

    Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45

    Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60

    Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80

    Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95

    Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110

    Thr Leu Thr Val Ser Ser 115 <210> 136 <211> 118 <212> PRT <213> Artificial <220>

    <223> Description of artificial sequence: Translation of PCR product <400> 136

    Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 15 10 15

    Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30

    Page 7 6

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    Val Ile

    Gly Glu 50

    Lys Gly 65

    Met Gln

    Ala Arg

    Ser Val <210>

    <211>

    <212>

    <213>

    <220>

    <223>

    <400>

    Gln Val 1

    Ser Trp Val 35 lie Tyr Pro

    Lys Ala Thr

    Leu Ser Ser 85

    Gly Val Leu 100

    Thr Val Ser 115

    137

    120

    PRT

    Artificial

    Sequences_3 42-31PCT. txt Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile

    40 45

    Gly Ser Gly Ser Thr Tyr Tyr Asn Glu Lys Phe 55 60

    Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 70 75 80

    Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 90 95

    Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr 105 110

    Ser

    Description of artificial sequence: Translation of PCR product

    137

    His Leu Gln Gln Ser Gly Ser Glu Leu Arg Ser Pro Gly Ser 5 10 15

    Ser Val

    Lys Leu Ser Cys Lys Asp Phe Asp Ser Glu Val Phe Pro Phe 20 25 30

    Ala Tyr

    Met Ser Trp Ile Arg Gln Lys Pro Gly His Gly Phe Glu Trp 35 40 45

    Ile Gly 50

    Asp Ile Leu Pro Ser Ile Gly Arg Thr Ile Tyr Gly Glu Lys 55 60

    Phe Glu 65

    Asp Lys Ala Thr Leu Asp Ala Asp Thr Val Ser Asn Thr Ala 70 75 80

    Tyr Leu

    Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr 85 90 95

    Cys Ala

    Arg Gly Glu Gly Tyr Gly Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110

    Gly Thr

    Leu Val Thr Val Ser Ala

    115 120

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    Sequences_342-31PCT.txt

    2018200685 30 Jan 2018 <210> 138 <211> 113 <212? PRT <213> Artificial <220>

    <223> Description of artificial sequence: Translation of PCR product <400> 138

    Asp 1 lie Val Met Thr Gln 5 Ser Pro Ser Ser Leu Thr 10 Val Thr Ala Gly 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 139 <211> 106 <212> PRT <213> . Artificial

    <220>

    <223> Description of artificial sequence: Translation of PCR product <400> 139

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

    Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30

    His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr 35 40 45

    Page 78

    Sequences 342-31PCT.txt

    2018200685 30 Jan 2018

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

    Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu 65 70 75 80

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

    Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 105

    100 <210> 140 <211> 107 <212> PRT <213> Artificial <220> <223> Description i <400> 140 Asp Ile Val Met Thr 1 5

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

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

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

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

    Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln His Trp Asn Tyr Pro Leu 85 90 95

    Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

    100 105 <2I0> 141 <211> 113 <212> PRT <213> Artificial <220> <223=> Description of artificial sequence

    Page 79

    Sequences_342-31PCT. txt

    141

    2018200685 30 Jan 2018 <400>

    Asp Ile 1

    Glu Lys

    Ser Asn

    Ser Pro 50

    Pro Asp 65

    Ile Ser

    Tyr Tyr

    Lys

    Val Met Ser 5 Gln Ser Pro Ser Ser 10 Leu Ala Val Ser Val 15 Gly Val Thr 20 Met Ser Cys Lys Ser 25 Ser Gln Ser Leu Leu 30 Tyr Ser Gln 35 Lys Asn Tyr Leu Ala 40 Trp Tyr Gln Gln Lys 45 Pro Gly Gln Lys Leu Leu Ile Tyr 55 Trp Ala Ser Thr Arg 60 Glu Ser Gly Val Arg Phe Thr Gly 70 Ser Gly Ser Gly Thr 75 Asp Phe Thr Leu Thr 80 Ser Val Lys 85 Ala Glu Asp Leu Ala 90 Val Tyr Tyr Cys Gln 95 Gln Ser Tyr 100 Pro Leu Thr Phe Gly 105 Ala Gly Thr Lys Leu 110 Glu Leu

    <210> <211> <212 > <213>

    <220>

    <223>

    <400>

    Asp lie 1

    142

    113

    PRT

    Artificial

    Description of artificial sequence: Translation of PCR product

    142

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

    Glu Lys

    Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30

    Gly Asn

    Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45

    Pro Pro 50

    Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 55 60

    Pro Asp 65

    Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 70 75 80

    Page 80

    2018200685 30 Jan 2018 lie Ser Ser Val Gln Ala 85

    Sequence s_3 4 2 - 31PCT. txt Glu Asp Leu Ala Val Tyr Tyr Cys

    Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys 100 105

    Lys

    Gln Asn 95

    Leu Glu He 110 <210> 143 <211> 112 <212> PRT <213> Artificial <220>

    <223> Description of artificial sequence: Translation of PCR product <400> 143

    Asp 1 lie Val Met Ser 5 Gln Ser Pro Ser Ser 10 Leu Ala Val Ser Ala 15 Gly Glu Lys Val Thr 20 Met Ser Cys Lys Ser 25 Ser Gln Ser Leu Leu 30 Asn Ser Arg Thr Arg 35 Lys Asn Tyr Leu Ala 40 Trp Tyr Gln Gln Lys 45 Pro Gly Gln Ser Pro 50 Lys Leu Leu He Tyr 55 Trp Ala Ser Thr Arg 60 Glu Ser Gly Val Pro 65 Asp Arg Phe Thr Gly 70 Ser Gly Ser Gly Thr 75 Asp Phe Thr Leu Thr 80 He Ser Ser Val Gln 85 Ala Glu Asp Leu Ala 90 Val Tyr Tyr Cys Lys 95 Gln Ser Tyr Asn Leu 100 Tyr Thr Phe Gly Gly 105 Gly Thr Lys Leu Glu 110 He Lys

    <210> 144 <211> 113 <212> PRT <213> Artificial <220>

    <223> Description of artificial sequence: Translation of PCR product <400> 144

    Asp He Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 15 10 15

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    Glu Lys Val Thr Met Ser Cys 20

    Ser Asn Gln Lys Asn Tyr Leu 35

    Ser Pro Lys Leu Leu Ile Tyr 50 55

    Pro Asp Arg Phe Thr Gly Ser 65 70

    Ile Ser Ser Val Gln Ala Glu 85

    Gly Tyr Ser Tyr Pro Tyr Thr 100

    Sequences_342-31PCT.txt Lys Ser Ser Gln Ser Leu Leu

    25 30

    Ala Trp Tyr Gln Gln Lys Pro 40 45

    Trp Ala Ser Thr Arg Glu Ser 60

    Gly Ser Ala Thr Asp Phe Thr 75

    Asp Leu Ala Asp Tyr His Cys 90

    Phe Gly Gly Gly Thr Lys Leu 105 110

    Tyr

    Gly

    Gly

    Leu

    Gly

    Glu

    Ser

    Gln

    Val

    Thr

    Gln

    Ile

    Lys <210> 145 <211> 113 <212> PRT <213> Artificial <220>

    <223> Description of artificial sequence: Translation of PCR product <400> 145

    Asp 1 Ile Val Met Ser 5 Gln Ser Pro Ser Ser 10 Leu Ala Val Ser Val 15 Gly Glu Lys Val Thr 20 Met Ser Cys Lys Ser 25 Ser Gln Ser Leu Leu 30 Tyr Ser Ser Asn Gln 35 Lys Asn Tyr Leu Ala 40 Trp Tyr Gln Gln Lys 45 Pro Gly Gln Ser Pro 50 Lys Leu Leu Ile Tyr 55 Trp Ala Ser Thr Arg 60 Glu Ser Gly Val Pro 65 Asp Arg Phe Thr Gly 70 Ser Gly Ser Gly Thr 75 Asp Phe Thr Leu Thr 80 Ile Ser Ser Val Lys 85 Ala Glu Asp Leu Ala 90 Val Tyr Tyr Cys Gln 95 Gln Tyr Tyr Ser Tyr 100 Pro Leu Thr Phe Gly 105 Ala Gly Thr Lys Leu 110 Glu Leu

    Page 82

    2018200685 30 Jan 2018

    Lys

    Sequences_342-31PCT.txt <210> 146 <211> 107 <212> PRT <213> Artificial <220>

    <223> Description of artificia <400> 146

    Asn Ile Val Met Thr Gln Ser Pro 1 5 sequence: Translation of PCR product

    Lys Ser Met Ser Met Ser Val Gly 10 15

    Glu Arg Val Thr Leu Thr Cys Lys 20

    Ala Ser Glu Asn Val Val Thr Tyr 25 30

    Val Ser Trp Tyr Gln Gln Lys Pro 35 40

    Glu Gln Ser Pro Lys Leu Leu Ile 45

    Tyr Gly Ala Ser Asn Arg Tyr Thr 50 55

    Gly Val Pro Asp Arg Phe Thr Gly 60

    Ser Gly Ser Ala Thr Asp Phe Thr 65 70

    Leu Thr Ile Ser Ser Val Lys Ala 75 80

    Glu Asp Leu Ala Val Tyr Tyr Cys 85

    Gln Gln Tyr Tyr Ser Tyr Pro Leu 90 95

    Thr Phe Gly Ala Gly Thr Lys Leu 100

    Glu Leu Lys 105 <210> 147 <211> 324 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: codon optimized nucleic acid <400> 147

    cgtacggtgg ccgctcccag cgtgttcatc ttccccccca gcgacgagca gctgaagtcc 60 ggcaccgcca gcgtggtgtg cctgctgaac aacttctacc cccgggaggc caaggtgcag 120 tggaaggtgg acaacgccct gcagagcggc aacagccagg agagcgtcac cgagcaggac 180 agcaaggact ccacctacag cctgagcagc accctgaccc tgagcaaggc cgactacgag 240 aagcacaagg tgtacgcctg cgaggtgacc caccagggcc tgtccagccc cgtgaccaag 300 agcttcaaca ggggcgagtg ctag 324

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    Sequences_342-31PCT.txt

    <210> 148 <211> 107 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: codon optimized protein <400> 148 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

    15 10 15

    Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30

    Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45

    Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60

    Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80

    Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95

    Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 149 <211> 981 <212> DNA <213> Artificial <220>

    <223> Description of artificial sequence: codon optimized nucleic acid <400> 149

    ggcccaagcg tgttccccct ggcccccagc agcaagagca ccagcggcgg cacagccgcc 60 ctgggctgcc tggtgaagga ctacttcccc gagcccgtga ccgtgagctg gaacagcgga 120 gccctgacct ccggcgtgca caccttcccc gccgtgctgc agagcagcgg cctgtacagc 180 ctgagcagcg tggtgaccgt gcccagcagc agcctgggca cccagaccta catctgcaac 240 gtgaaccaca agcccagcaa caccaaggtg gacaagagag tggagcccaa gagctgcgac 300 aagacccaca cctgcccccc ctgcccagcc ccagagctgc tgggcggacc cagcgtgttc 360 ctgttccccc ccaagcccaa ggacaccctg atgatcagca ggacccccga ggtgacctgc 420 gtggtggtgg acgtgagcca cgaggaccca gaggtgaagt tcaactggta cgtggacggc 480

    Page 84

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    Sequences_3 42-31PCT. txt

    gtggaggtgc acaacgccaa gaccaagccc agagaggagc agtacaacag cacctacagg 540 gtggtgtccg tgctgaccgt gctgcaccag gactggctga acggcaagga atacaagtgc 600 aaggtctcca acaaggccct gccagccccc atcgaaaaga ccatcagcaa ggccaagggc 660 cagccacggg agccccaggt gtacaccctg ccccccagcc gggaggagat gaccaagaac 720 caggtgtccc tgacctgtct ggtgaagggc ttctacccca gcgacatcgc cgtggagtgg 780 gagagcaacg gccagcccga gaacaactac aagaccaccc ccccagtgct ggacagcgac 840 ggcagcttct tcctgtacag caagctgacc gtggacaagt ccaggtggca gcagggcaac 900 gtgttcagct gcagcgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 960 agcctgagcc ccggcaagta g 981

    <210> 150 <211> 326 <212> PRT <213> Artificial <220>

    <223> Description of artificial sequence: codon optimized protein <400> 150

    Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 15 10 15

    Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 20 25 30

    Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 35 40 45

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

    Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 65 70 75 80

    Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro 85 90 95

    Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 100 105 110

    Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125

    Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140

    Page 85

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    Sequences_342-31PCT. txt

    Val Ser His Glu Asp Pro Glu Val 145 150

    Val Glu Val His Asn Ala Lys Thr 165

    Ser Thr Tyr Arg Val Val Ser Val 180

    Leu Asn Gly Lys Glu Tyr Lys Cys 195 200

    Ala Pro Ile Glu Lys Thr Ile Ser 210 215

    Lys Phe Asn Trp Tyr Val Asp Gly 155 160

    Lys Pro Arg Glu Glu Gln Tyr Asn 170 175

    Leu Thr Val Leu His Gln Asp Trp 185 190

    Lys Val Ser Asn Lys Ala Leu Pro 205

    Lys Ala Lys Gly Gln Pro Arg Glu 220

    Pro Gln Val Tyr Thr Leu Pro Pro 225 230

    Ser Arg Glu Glu Met Thr Lys Asn 235 240

    Gln Val Ser Leu Thr Cys Leu Val 245

    Lys Gly Phe Tyr Pro Ser Asp Ile 250 255

    Ala Val Glu Trp Glu Ser Asn Gly 260

    Gln Pro Glu Asn Asn Tyr Lys Thr 265 270

    Thr Pro Pro Val Leu Asp Ser Asp 275 280

    Gly Ser Phe Phe Leu Tyr Ser Lys 285

    Leu Thr Val Asp Lys Ser Arg Trp 290 295

    Gln Gln Gly Asn Val Phe Ser Cys 300

    Ser Val Met His Glu Ala Leu His 305 310

    Asn His Tyr Thr Gln Lys Ser Leu 315 320

    Ser Leu Ser Pro Gly Lys 325

    Page 86